Hirofumi Enomoto

Glycation and phosphorylation of α-lactalbumin by dry heating: Effect on protein structure and physiological functions

Alpha-lactalbumin (alpha-LA) was glycated with maltopentaose (MP) through the Maillard reaction (MP-alpha-LA) and subsequently phosphorylated by dry heating in the presence of pyrophosphate to investigate its structure and physiological functions. Glycation occurred effectively, and the sugar content of alpha-LA increased by approximately 22.3% through the Maillard reaction. The phosphorylation of MP-alpha-LA was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorous content of MP-alpha-LA increased by approximately 1.01% by dry heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of alpha-LA increased with an increase in the phosphorylation level. The circular dichroism spectra showed that the change in the secondary structure of the alpha-LA molecule by glycation and subsequent phosphorylation was slight. However, the Trp fluorescence intensity was increased by phosphorylation after glycation. In addition, the differential scanning calorimetry thermograms of alpha-LA showed that the denaturation temperature of MP-alpha-LA was decreased by phosphorylation. These results indicated that molten (partially unfolded) conformations of alpha-LA were formed by dry heating in the presence of pyrophosphate after glycation. The anti-alpha-LA antibody response was significantly reduced by glycation and subsequent phosphorylation. The suppressive effect of alpha-LA on the production of proinflammatory cytokines such as IL-6 and tumor necrosis factor-alpha from THP-1 cells after stimulation with lipopolysaccharide was significantly enhanced by glycation with MP and was further enhanced by phosphorylation after glycation. The Ca phosphate-solubilizing ability of alpha-LA was enhanced by phosphorylation. The apoptotic activity of alpha-LA was reduced by glycation and subsequent phosphorylation. These results suggest that phosphorylation by dry heating in the presence of pyrophosphate after glycation with MP through the Maillard reaction is a useful method for improvement of the physiological functions of alpha-LA.

Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate.

Bovine serum albumin (BSA) was phosphorylated by 2 methods. One is dry-heating in the presence of pyrophosphate, and the other is conjugation with maltopentaose through the Maillard reaction and subsequent dry-heating in the presence of pyrophosphate. The phosphorus content of BSA was increased to approximately 0.45% by dry-heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate, and approximately 0.91% by glycation and subsequent phosphorylation. The circular dichroism spectra showed that the change of secondary structure in the BSA molecule by phosphorylation was mild. However, tryptophan fluorescence intensity of BSA decreased by phosphorylation. The differential scanning calorimetry thermograms of BSA showed a disappearing of the 1st peak and a lowering of the 2nd peak denaturation temperature by phosphorylation. These results indicated molten (partially unfolded) conformations of BSA formed by both phosphorylation methods. The functional properties of BSA such as heat stability and calcium phosphate solubilizing ability were improved by phosphorylation alone and further by phosphorylation after glycation. Transparent gels of BSA with relatively high water-holding capacity were obtained by phosphorylation alone, and the immunogenicity of BSA was reduced significantly by glycation and phosphorylation, respectively.

Improvement of foaming property of egg white protein by phosphorylation through dry-heating in the presence of pyrophosphate.

Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 4 and 85 degrees C for 1 d, and the foaming properties of phosphorylated EWP (PP-EWP) were investigated. The phosphorus content of EWP increased to 0.71% as a result of phosphorylation. To estimate the foaming properties of EWP, the foams were prepared by 2 methods: bubbling of the 0.1% (w/v) protein solution and whipping of the 10% (w/w) protein solution with an electric mixer. The foaming power, which was defined as an initial conductivity of foam from 0.1% (w/v) protein solution, was a little higher in PP-EWP than in native EWP (N-EWP), and the foaming stability of PP-EWP was much higher than that of dry-heated EWP (DH-EWP) and N-EWP. The microscopic observation of foams from the 10% (w/w) solution showed that the foams of PP-EWP were finer and more uniform than those of N- and DH-EWP. Although there were no significant differences in the specific gravity and overrun of the foams between PP- and DH-EWP (P < 0.05), the specific gravity and overrun of the foams from PP-EWP were smaller and higher, respectively, than that of the foams from N-EWP. The drainage volume was smaller in the foams from PP-EWP than in those from N- and DH-EWP. These results demonstrated that phosphorylation of EWP by dry-heating in the presence of pyrophosphate improved the foaming properties, and that it was more effective for the foam stability than for the foam formation.

Characteristics and Enhanced Antioxidant Activity of Egg White Protein Selenized by Dry-Heating in the Presence of Selenite

This study reports a new method for the selenization of food proteins. Egg white protein (EWP) was selenized by dry-heating in the presence of selenite, and the physiochemical and functional properties of the selenized EWP were investigated. Selenization was accelerated with a decrease in pH from 7.0 to 3.0, an increase in heating time from 1 to 5 days, and an increase in incubation temperatures from 25 to 80 °C. The electrophoretic mobility of EWP increased with an increase in the level of selenization. Heat-induced polymerization of EWP was promoted by dry-heating in the presence of selenite. The selenite group was acid-stable and base-labile, which suggests that it was bound to the hydroxyl group of an amino acid and formed an -O-SeHO2 linkage. (77)Se NMR spectral data also suggested that the selenite bond (-O-SeHO2) was bound to EWP. The digestibility of EWP was improved by selenization. The antioxidant activities of EWP, including ABTS(+) free radical scavenging capacity, hydroxyl radical scavenging capacity, reducing power, and the Fe(2+) chelating capacity, were remarkably enhanced by selenization. This finding is the first to describe that EWP can be selenized by dry-heating in the presence of selenite and that the antioxidant activities of EWP are markedly enhanced by selenization. This points to a potentially new method for generating antioxidant food protein and a new method for preparing organic Se.

Distribution Analysis of Anthocyanins, Sugars, and Organic Acids in Strawberry Fruits Using Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry

Anthocyanins, sugars, and organic acids contribute to the appearance, health benefits, and taste of strawberries. However, their spatial distribution in the ripe fruit has been fully unrevealed. Therefore, we performed matrix-assisted laser desorption/ionization, MALDI-IMS, analysis to investigate their spatial distribution in ripe strawberries. The detection sensitivity was improved by using the TM-Sprayer for matrix application. In the receptacle, pelargonidins were distributed in the skin, cortical, and pith tissues, whereas cyanidins and delphinidins were slightly localized in the skin. In the achene, mainly cyanidins were localized in the outside of the skin. Citric acid was mainly distributed in the upper and bottom side of cortical tissue. Although hexose was distributed almost equally throughout the fruits, sucrose was mainly distributed in the upper side of cortical and pith tissues. These results suggest that using the TM-Sprayer in MALDI-IMS was useful for microscopic distribution analysis of anthocyanins, sugars, and organic acids in strawberries.