Ruijin Yang

Investigation of the Protein−Protein Aggregation of Egg White Proteins under Pulsed Electric Fields

Egg whites were exposed to pulsed electric fields (PEFs) to investigate the protein-protein aggregation. No insoluble protein aggregate was found when egg whites were exposed to PEFs at 25, 30, and 35 kV/cm for 400 micros. However, atomic force microscopy showed that the sizes of the protein particles increased. Native polyacrylamide gel electrophoresis (PAGE) demonstrated the existence of aggregates under PEFs at 35 kV/cm for 400 micros. Sodium dodecyl sulfate (SDS)-PAGE in the presence and absence of 2-mercaptoethanol further indicated that sulfhydryl-disulfide interchange reactions occurred under PEFs. Differential scanning calorimetry scans showed 400 micros of PEF treatment at 35 kV/cm denatured 16.5% proteins. Insoluble egg white protein aggregates were induced by PEF (35 kV/cm, 800 micros) and heat (60 degrees C, 3.5 min) treatments. Disulfide bonds were the dominant binding forces in the formation of protein aggregates. However, the weakly noncovalent bonds play a much more important role in the protein aggregation forming in heat treatment (60 degrees C, 3.5 min) than that in PEF treatment (35 kV/cm, 800 micros).

The effect of pulsed electric fields on the inactivation and structure of lysozyme

The effect of pulsed electric fields (PEF) on the activity and structure of lysozyme selected as a model enzyme was investigated. The inactivation of lysozyme in phosphate buffer was a function of electric field strength, treatment time, electrical conductivity, and enzyme concentration. No significant (p>0.05) change in the activity of PEF-treated lysozyme was found after storage for 12, 24 and 48h at 4°C. The effect of PEF on tertiary structure of lysozyme was demonstrated by second-derivative UV spectra and intrinsic fluorescence. The results indicated that the unfolding of tertiary structure was induced by PEF treatment at 35kV/cm for 1200μs, and more tyrosine residues were buried inside the protein after PEF treatment, accompanied by the exposure of more tryptophan residues. CD spectra suggested that the inactivation of lysozyme by PEF was closely related to the loss of α-helix of secondary structure.

Enzymatic production of lactulose and 1-lactulose: current state and perspectives

Lactulose, a synthetic ketose disaccharide, has been widely used in food and pharmaceutical industries as prebiotic food additives and drugs against constipation and hepatic encephalopathy. Lactulose has, so far, been produced chemically using lactose on a commercial scale. The key problems associated with such chemical process are the cost of removal of the catalyst and colored by-products and the product safety. Enzymatic production of lactulose is safe, environment-friendly, and simpler in comparison to the chemical method. As a promising alternative to the chemical method, enzymatic conversion of lactose into lactulose by β-galactosidase or cellobiose 2-epimerase has recently gained a great deal of attention. This could be considered as a possible route for whey surplus because lactose is the major component of cheese whey. Herein, we summarize recent advances on the enzymatic synthesis of lactulose with emphasis on the selectivity of biocatalysts and their catalytic efficiency in free and immobilized forms. The production of 1-lactulose by enzymatic bioconversion of lactose has also been discussed. Furthermore, future research needs of β-galactosidase and cellobiose 2-epimerase for the enzymatic synthesis of lactulose and 1-lactulose are reviewed.

Purification and characterization of inulin fructotransferase (DFA III-forming) from Arthrobacter aurescens SK 8.001

The soil bacterium Arthrobacter aurescens SK 8.001 produces inulin fructotransferase (IFTase), and liquid chromatography-mass spectrometry (LC-MS) and carbon-13 nuclear magnetic resonance (13C NMR) analysis demonstrated that the main product of the enzyme was difructose anhydride III (DFA III). The IFTase was purified by ethanol precipitation, DEAE Sepharose Fast Flow, and Superdex 200 10/300 GL gel chromatography. Its molecular mass was estimated to be 40 kDa by SDS-PAGE and 35 kDa by gel filtration. The enzyme showed maximum activity at pH 5.5 and 60-70 °C, and retained 86.5% of its initial activity after incubation at 60 °C for 4 h. Chemical modification results suggested that a tryptophan residue is essential to enzyme activity. The N-terminal amino acid sequence was determined as AEGAKASPLNSPNVYDVT. The kinetic values, Km and Vmax, were estimated to be 0.52 mM and 0.3 μmol/ml min. Nystose was observed to be the smallest substrate for the produced IFTase. This IFTase provides a promising way to utilize inulin for the production of DFA III.

Production of 1-lactulose and lactulose using commercial β-galactosidase from Kluyveromyces lactis in the presence of fructose.

Production of 1-lactulose and lactulose using commercial β-galactosidase DSM Maxilact® 5000 in the presence of fructose was investigated. Experiments were performed at 40 °C and pH 7.5. Lactose starting concentration was constantly 250 g/l. A novel transgalactosylation product 1-lactulose was detected besides lactulose. Effects of fructose concentration, reaction time and enzyme concentration on transgalactosylation reactions were discussed. In all reactions, the yield ratio 1-lactulose:lactulose was close to 3:1 due to the regioselectivity of β-galactosidase. The maximum production of 1-lactulose and lactulose was approximately 22 and 8 g/l, respectively, when fructose concentration was increased to 100 g/l. Lactose hydrolysis was significantly retarded since fructose strongly attracted water molecules. Higher enzyme concentration can accelerate transgalactosylation reactions without affecting the maximum production of transgalactosylation products. Fructose was a more preferred galactosyl acceptor than lactose, since the synthesis of galactooligosacchairdes was found to be absolutely inhibited in the presence of fructose.

Enzymatic synthesis and identification of oligosaccharides obtained by transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis.

The enzymatic transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis (KlβGal) leading to the formation of oligosaccharides was investigated in detail. The reaction mixture was analyzed by high performance liquid chromatography with differential refraction detector (HPLC-RI) and two main transgalactosylation products were discovered. To elucidate their overall structures, the products were isolated and purified using preparative liquid chromatography and analyzed by LC/MS, one-dimensional (1D) and two-dimensional (2D) NMR studies. Allo-lactulose(β-d-galactopyranosyl-(1→1)-d-fructose) with two main isomers in D(2)O was identified to be the major transgalactosylation product while lactulose(β-d-galactopyranosyl-(1→4)-d-fructose) turned out to be the minor one, indicating that KlβGal was regioselective with respect to the primary C-1 hydroxyl group of fructose. The maximum yields of allo-lactulose and lactulose were 47.5 and 15.4g/l, respectively, at 66.5% lactose conversion (200g/l initial lactose concentration).

Influence of pulsed electric field treatments on the volatile compounds of milk in comparison with pasteurized processing.

Effects of pulsed electric field (PEF) treatments on the volatile profiles of milk were studied and compared with pasteurized treatment of high temperature short time (HTST) (75 °C, 15 s). Volatile compounds were extracted by solid-phase micro-extraction (SPME) and identified by gas chromatography/mass spectrometry (GC-MS) and gas chromatography-olfactometry (GC-O). A total of 37 volatile compounds were determined by GC-MS, and 19 volatile compounds were considered to be major contributors to the characteristic flavor of milk samples. PEF treatment resulted in an increase in aldehydes. Milk treated with PEF at 30 kV/cm showed the highest content of pentanal, hexanal, and nonanal, while heptanal and decanal contents were lower than in pasteurized milk, but higher than in raw milk. All the methyl ketones detected in PEF milk were lower than in pasteurized milk. No significant differences in acids (acetic acid, butanoic acid, hexanoic acid, octanoic acid, and decanoic acid), lactones, and alcohols were observed between pasteurized and PEF-treated samples; however, 2(5H)-furanone was only detected in PEF-treated milk. Although GC-MS results showed that there were some volatile differences between pasteurized and PEF-treated milk, GC-O data showed no significant difference between the 2 samples.

Characterization of natural low-methoxyl pectin from sunflower head extracted by sodium citrate and purified by ultrafiltration

Sunflower head pectin was extracted by 0.6% (w/v) sodium citrate under 85°C, 3.5h with a solid to liquid ratio of 1:40, giving the maximum uronic acid yield of 16.90% (w/w). The extract was purified by an ultrafiltration membrane with the molecular weight cut-off of 8000 Da and dried by spray-drying to obtain SFHP(A) powders with particle diameters of 2-5 μm. In comparison to the SFHP(B) extracted by ammonium oxalate and isolated by alcohol, SFHP(A) had a close DM (22.56%) but a considerably lower DAm (3.42%). HPSEC-MALLS analysis suggested that SFHP(A) has undergo a deeper degradation resulting in smaller Mw and Mn. HPAEC-PAD showed that SFHP(A) contains more neutral sugars since the degraded RG fragments have been retained during ultrafiltration. Finally, pectin aggregation in aqueous was investigated by FEG-SEM, which reveals that pectin network structure is constructed by microfilaments via coiling, intertwining and crosslinking.

Pulsed electric field (PEF)-induced aggregation between lysozyme, ovalbumin and ovotransferrin in multi-protein system.

The aggregation of multi-proteins is of great interest in food processing and a good understanding of the formation of aggregates during PEF processing is needed for the application of the process to pasteurize protein-based foods. The aggregates formation of a multi-protein system (containing ovalbumin, ovotransferrin and lysozyme) was studied through turbidity, size exclusion chromatography and SDS-PAGE patterns for interaction studies and binding forces. Results from size exclusion chromatography indicated that there was no soluble aggregates formed during PEF processing. The existence of lysozyme was important to form insoluble aggregates in the chosen ovalbumin solution. The results of SDS-PAGE patterns indicated that lysozyme was prone to precipitate, and was relatively the higher component of aggregates. Citric acid could be effective in inhibiting lysozyme from interacting with other proteins during PEF processing. Blocking the free sulphydryl by N-ethylmaleimide (NEM) did not affect aggregation inhibition.

Combined Effects of Heat and PEF on Microbial Inactivation and Quality of Liquid Egg Whites

Combined effects of heat and pulsed electric fields treatment (20 and 40oC, 0-800 µs at 30 kV/cm) on microbial inactivation inoculated in egg whites were studied. Pulsed electric fields treatment time and processing temperature had profound effects on microbial inactivation. Pulsed electric fields treatment with a bipolar pulse (2 µs wide), an intensity of 30 kV/cm, a frequency of 100 Hz, the processing temperature of 40 oC and treatment time for 800 µs, was sufficient to achieve pasteurization conditions using S. enteritidis , E. coli and S. aureus, common spoilage and pathogenic micro-organisms in egg products. This treatment produced a non-significant (p>0.05) increase in foaming capacity and stability and an increase (p<0.05) in emulsifying capacity and stability. Surface free sulfhydryls and hydrophobicity of egg white proteins increased with the increment of the PEF treatment time due to the partial unfolding of egg white proteins. Almost 50 % of trypsin inhibitory activity of ovomucoid in liquid egg white was decreased when the treatment time extended to 800 µs. These results suggested that combined treatment of heat and pulsed electric fields could be applied to process liquid egg whites to get desired products.