Meihu Ma

The inhibition of fluorescence resonance energy transfer between multicolor quantum dots for rapid and sensitive detection of Staphylococcus aureus.

In this paper, we constructed the fluorescence resonance energy transfer (FRET) system between two multi-color quantum dots (QDs) of two sizes for rapid and sensitive detection of Staphylococcus aureus. In this system, green-emitting QDs conjugated with rabbit anti-S. aureus antibodies were used as energy donors while orange-emitting QDs conjugated with goat-anti-rabbit IgG were used as energy acceptors to form FRET system. Pre-binding of Staphylococcus aureus (S. aureus) on the donor occupied the binding sites and thus blocked resonance energy transfer between two colors QDs, leading to the quenching fluorescence of the acceptor. The fluorescence of acceptor has a linear calibration graph with the concentrations of S. aureus from 52 to 2.6×10(5) CFU mL(-1). The low detection limit was 10 CFU/mL. It was worth mentioning that the detection method of S. aureus had been applied to the analysis of apple juice and milk samples, which could potentially be developed into a sensor in the further study.

Mass Spectrometry and Two-Dimensional Electrophoresis To Characterize the Glycosylation of Hen Egg White Ovomacroglobulin.

Glycosylation of proteins plays an important role in their biological functions, such as allergenicity. Ovomacroglobulin (OVMG) is a glycoprotein from hen egg white, but few studies have been done so far to delineate the glycosylated sites of OVMG. The present study characterized the glycosylation of OVMG using mass spectrometry and two-dimensional electrophoresis. MALDI-TOF-MS showed that the OVMG subunit [M + H](+) ion has a peak at m/z 183297; therefore, the carbohydrate moiety is calculated as 11.5% of the whole OVMG molecule. HPLC-ESI-MS/MS confirmed that of 13 potential N-glycosylation sites of OVMG, 11 sites were glycosylated; 1 site (N(1221)) was found in both glycosylated and nonglycosylated forms. On the two-dimensional electrophoresis gel, a series of OVMG spots horizontally distributed at 170 kDa, with an isoelectric point range of 5.03-6.03, indicating the heterogeneity of glycosylation of OVMG. These results provided important information for understanding of structure, function, and potential allergenic sites of OVMG.

Chemiluminescence evaluation of antioxidant activity and prevention of DNA damage effect of peptides isolated from soluble eggshell membrane protein hydrolysate.

A new kind of soluble eggshell membrane protein (SEP) was prepared from eggshell membrane (ESM). The extraction rate of SEP could rise to 90% by two times, basically accomplishing the complete utilization of the whole ESM. Five proteases were employed as hydrolytic enzyme for the preparation of antioxidative peptides from SEP, and the antioxidative activities of the hydrolysates were investigated using a chemiluminescence method. Among the hydrolysates, alcalase hydrolysates with the highest free radical scavenging activity were further separated into three fractions using Sephadex G-25 gel filtration chromatography of the 4 h hydrolysate (SP1, SP2, and SP3). Among these three fractions, SP2 with an average molecular weight of 618.86 Da possessed the strongest fraction of scavenging activity. The IC50 values of the superoxide radicals, hydroxyl scavenging activities, and protective effect on DNA damage caused by hydroxyl radicals generated were 0.10, 0.18, and 0.95 mg/mL, respectively. These results demonstrate that inexpensive ESM waste could be a new alternative in the production of antioxidative peptides.

Comparative proteome analysis of egg yolk plasma proteins during storage

BACKGROUND Physical changes such as chicken egg white thinning and egg yolk flattening occur during storage, implying a decline in egg quality. To reveal the deteriorative process related to chicken egg internal quality, a comparative proteomic method was used in this study to analyze the alterations in egg yolk plasma proteins at different storage times (0, 20 and 40 days) under an ambient temperature of 22 ± 2 °C. RESULTS Using two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, 33 protein spots representing 12 proteins were identified with significant (P < 0.05) alterations in abundance at different storage times. The proteins that showed significant changes in abundance included serum albumin, vitellogenin fragments, IgY chains, ovalbumin, ovoinhibitor, α2 -macroglobulin-like protein 1-like, hemopexin, transthyretin, apolipoprotein A-I and β2 -glycoprotein I precursor. Accelerating degradation for most egg yolk plasma proteins was observed after prolonged storage (from day 20 to day 40). CONCLUSION It is likely that the increased degradation of protease inhibitors such as ovoinhibitor and α2 -macroglobulin-like protein 1-like during prolonged storage lead to an imbalance of protease and antiprotease in egg yolk, which may play a key role in the degradation of egg yolk proteins. These findings will provide an insight into the effects of storage on egg yolk protein changes and give a deeper understanding of the deteriorative process of chicken egg yolk.

Hydroxyapatite nucleation and growth on collagen electrospun fibers controlled with different mineralization conditions and phosvitin

AbstractIn a tenfold-concentrated simulated body fluid, a strategy for rapid deposition of a biomimetic calcium phosphate layer on the scaffolds of electrospun collagen nanofiber membranes was developed. The aim of this study was to explore the effects of mineralization conditions and phosvitin (PV) on hydroxyapatite nucleation and growth. The mineralization model, the pH of the environment, and the deposition time were optimized. Scanning electron microscopy (SEM) images demonstrated that homogeneous and well-crystallized inorganic mineral layers were generated in the dynamic mineralization model system after incubating 3 h at pH 5.7. PV, which possesses the highest level of phosphorylation among egg proteins, was used as a model protein to investigate the contribution of PV in the mineralization process. The morphological structure and composition of the collagen/calcium phosphate composite nanofibers were also characterized by energy dispersive spectroscopy, scanning photoelectron spectrometer, X-ray diffraction (XRD), and Fourier transform infrared spectroscopy. XRD results showed the transformation process of mineralization materials from dicalcium phosphate dihydrate (DCPD) to HA through the changes of characteristic peaks at approximately 11° of DCPD and 31.8° of HA. 1.0 mg/mL. Phosvitin significantly promoted the phase transformation from DCPD to hydroxyapatite. High performance liquid chromatography results indicated that PV induced the mineralization rather than being the part of the hydroxyapatite. The minerals formed on electrospun collagen nanofiber membranes were identified to be from hydroxyapatite. These findings extended the potential application field of PV to biomimetic material.

Calcium binding characteristics and structural changes of phosvitin.

Phosvitin is a unique highly phosphorylated protein that plays a role in the regulation of calcification. We conducted a comprehensive study of the chemical, thermodynamic and structural aspects of the interaction of phosvitin with calcium ions using a calcium ion selective electrode (ISE), isothermal titration calorimetry (ITC), circular dichroism spectrum (CD) and fluorescence spectroscopy, respectively. The results showed that under neutral and alkaline conditions, distinct high affinity and low affinity binding modes existed in the interaction between phosvitin and calcium. The high affinity association constant was approximately 10(4)mol(-1), while the binding sites contained nearly 30mol of calcium per mole of phosvitin. This reaction was driven by enthalpy. The unordered and β-turn conformations of phosvitin increased, while the β-sheet conformation decreased. The main interaction forces were electrostatic force, hydrogen bonds or van der Waals force. The low affinity association constant and binding sites were not constant, as many calcium ions were sequestered by phosvitin. The binding reaction was driven by entropy, and the β-sheet conformation of phosvitin increased while the unordered conformation decreased. The main interaction force was hydrophobic force. However, under acidic conditions, the interaction between phosvitin and calcium was an entropy-driven endothermic reaction, and the main interaction force was weak hydrophobic force. This calcium-binding characteristic of phosvitin may play a specific role in its biological function.

Fluorescence switch biosensor based on quantum dots and gold nanoparticles for discriminative detection of lysozyme

It is important to detect lysozyme (LYZ) in a simple and rapid manner because of its potential application in the treatment of diseases and in the food industry. In this study, it was observed that the strong fluorescence of LYZ-modified Quantum Dots (QDs-LYZ) could be effectively quenched by gold nanoparticle(AuNPs) modified with antibodies against LYZ (anti-LYZ) due to fluorescence resonance energy transfer (FRET) between QDs-LYZ and anti-LYZ-AuNPs. The fluorescence can be reversibly recovered by LYZ (on state) owing to specific competitive interactions between LYZ, QDs-LYZ and anti-LYZ-AuNPs. The interaction of QDs-LYZ with anti-LYZ-AuNPs was studied by absorption, fluorescence spectroscopy and transmission electron microscopy (TEM). Under optimal conditions, LYZ can be detected with a linear range of 50-1000ng/mL and a detection limit (LOD) of 33.43ng/mL. This rapid and selective QD-based sensor was successfully applied for quantitation of LYZ in actual egg products. Furthermore, the strategy described in this report could be followed conveniently to establish similar immunosensors for the rapid detection of other proteins using corresponding antibodies.

The impact of N-glycosylation on conformation and stability of immunoglobulin Y from egg yolk

Immunoglobulin Y (IgY) is a new therapeutic antibody, and its applications in industry are very broad. To provide insight into the effects of N-glycosylation on IgY, its conformation and stability were studied. In this research, IgY was extracted from egg yolk and then digested by peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase. SDS-PAGE and infrared absorption spectrum showed that carbohydrates were distinctly reduced after enzymolysis. The circular dichroism spectrum indicated that the IgY molecule became more flexible and disordered after removal of N-glycan. The fluorescence intensity revealed that Trp residues were buried in a more hydrophobic environment after disposal of N-glycan. Storage stability decreased with the removal of oligosaccharide chains based on size-exclusion chromatography analysis. Deglycosylated IgY exhibited less resistance to guanidine hydrochloride-induced unfolding. After deglycosylation, IgY was more sensitive to pepsin. Therefore, N-glycosylation played an important role in the maintenance of the structure and stability of IgY.

Influence of high-intensity ultrasound on foaming and structural properties of egg white

The effects of high-intensity ultrasound (20 kHz) at varying power pre-treatments (90, 120, 240, 360 and 480 W for 10 min) on foaming and structural properties of egg white were studied in this paper. The highest foaming ability (260.00%) was obtained after 360 W ultrasound treatment which was 4.9-fold to the control group. The lower viscosity and surface tension, and higher protein solubility indicated that the protein was easier to attach to the gas-liquid interface and generate molecular rearrangement. Moreover, the increased free sulfhydryl groups and surface hydrophobicity revealed that the protein adopted a loose structure after ultrasonic processing. SDS-PAGE data proved that subunit of poorly water-soluble ovomucin was declined. Scanning electron microscopy showed different microstructure with smaller aggregates and pore structure compared to non-treated egg white. These results presented more evidence of protein properties under ultrasonic environment, and broadened the application range of ultrasonic technique in food industry.

Hen egg yolk phosvitin stimulates osteoblast differentiation in the absence of ascorbic acid

BACKGROUND Egg yolk phosvitin, one of the most highly phosphorylated extracellular matrix proteins known in nature, has a strong calcium binding and reducing capacity. Here, we investigated the effects of phosvitin on osteoblast differentiation and osteogenic gene expression in cultured mouse osteoblastic MC3T3-E1 cells by using alkaline phosphatase activity analysis, alizarin red S staining and real-time PCR assay. RESULTS Alkaline phosphatase activity and alizarin red S staining analyses demonstrated no significant difference between differentiating MC3T3-E1 cells cultured in the presence of phosvitin and those cultured in the presence of ascorbic acid after 21 days of differentiation. Our real-time PCR assay also indicated the two groups were similar in the expression of the osteogenic gene markers, collagen type I, osteocalcin, runt-related transcription factor 2, and bone morphogenetic protein-2. CONCLUSION Our findings indicate that phosvitin plays a similar role to that of ascorbic acid in osteoblast differentiation and mineralisation.