Characterization of ß-lactoglobulin adsorption on silica membrane pore surfaces and its impact on membrane emulsification processes.

Protein adsorption plays a key role in membrane fouling in liquid processing, but the specific underlying molecular mechanisms of ß-lactoglobulin adsorption on ceramic silica surfaces in premix membrane emulsification have not been investigated yet. In this study, we aimed to elucidate the ß-lactoglobulin adsorption and its effect on the premix membrane emulsification of ß-lactoglobulin-stabilized oil-in-water emulsions. In particular, the conformation, molecular interactions, layer thickness, surface energy of the adsorbed ß-lactoglobulin and resulting droplet size distribution are investigated in relation to the solvent properties (aggregation state of ß-lactoglobulin) and the treatment of the silica surface (hydrophilization). The ß-lactoglobulin adsorption is driven by attractive electrostatic interactions between positively charged amino acid residues, i.e., lysin and negatively charged silanol groups, and is stabilized by hydrophobic interactions. The strong negative charges of the treated silica surfaces result in a high apparent layer thickness of ß-lactoglobulin. Although the conformation of the adsorbed ß-lactoglobulin layer varies with membrane treatment and the solvent properties, the ß-lactoglobulin adsorption offsets the effect of hydrophilization of the membrane so that the surface energies after ß-lactoglobulin adsorption are comparable. The resulting droplet size distribution of oil-in-water emulsions produced by premix membrane emulsification are similar for treated and untreated silica surfaces.

The microstructure of the starch from the underutilized seed of jaboticaba (Plinia cauliflora).

This work presents a starch extracted from jaboticaba seeds. The extraction yielded 22.65 ± 0.63% of a slightly beige powder (a* 1.92 ± 0.03, b* 10.82 ± 0.17 and L* 92.27 ± 0.24). The starch presented low protein content (1.19% ± 0.11) and phenolic compounds (0.58 ± 0.02 GAE. g) as contaminants. The starch granules showed small, smooth, irregular shapes and sizes between 6.1 and 9.6 µm. The starch presented a high content of amylose (34.50%±0.90) and a predominance of intermediate chain length (B1-chains 51%), followed by A-chains (26%) in the amylopectin. The SEC-MALS-DRI showed the starch had a low molecular weight (5.3·106 g·mol-1) and amylose/amylopectin content compatible with a Cc-type starch, confirmed in the X-ray diffractogram. Thermal studies showed a low onset temperature (T0 = 66.4 ± 0.46 °C) and gelatinization enthalpy (ΔH = 9.1 ± 1.19 J g-1) but a high-temperature range (ΔT = 14.1 ± 0.52 °C). The jaboticaba starch proved to be a promising material for food and non-food applications.

Influence of Levan on the Thermally Induced Gel Formation of ß-Lactoglobulin.

In this study, the influence of levan on the phase behavior and the thermally induced gelation of the mixed ß-lactoglobulin-levan gels as a function of polymer content, molecular weight and ionic strength was characterized. For this purpose, rheology was used to study the mechanical properties of the gels and the water binding of the network structure was investigated by time domain nuclear magnetic resonance. Phase behavior and network type were analyzed by optical observation and electron microscopy. Levan enhanced the aggregation and gel formation of ß-lg due to segregative forces between the polymer species. Segregation was caused by the excluded volume effect and was more pronounced at lower ionic strength, higher levan contents and higher levan molecular weights. The presence of levan increased the water binding of the gel networks. However, this effect decreased with increasing levan content. At high ionic strength and high levan content, phase separated gels were formed. While segregative forces enhanced network formation, and therefore, increased the gel strength of mixed gels at low ionic strength, levan had also antagonistic effects on the network formation at high ionic strength and high polymer contents.

Structure and adsorption behavior of high hydrostatic pressure-treated ß-lactoglobulin.

HYPOTHESIS: High hydrostatic pressure treatment causes structural changes in interfacial-active ß-lactoglobulin (ß-lg). We hypothesized that the pressure-induced structural changes affect the intra- and intermolecular interactions which determine the interfacial activity of ß-lg. The conducted experimental and numerical investigations could contribute to the mechanistic understanding of the adsorption behavior of proteins in food-related emulsions. EXPERIMENTS: We treated ß-lg in water at pH 7 with high hydrostatic pressures up to 600 MPa for 10 min at 20 °C. The secondary structure was characterized with Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD), the surface hydrophobicity and charge with fluorescence-spectroscopy and ζ-potential, and the quaternary structure with membrane-osmometry, analytical ultracentrifugation (AUC) and mass spectrometry (MS). Experimental analyses were supported through molecular dynamic (MD) simulations. The adsorption behavior was investigated with pendant drop analysis. FINDINGS: MD simulation revealed a pressure-induced molten globule state of ß-lg, confirmed by an unfolding of ß-sheets with FTIR, a stabilization of α-helices with CD and loss in tertiary structure induced by an increase in surface hydrophobicity. Membrane-osmometry, AUC and MS indicated the formation of non-covalently linked dimers that migrated slower through the water phase, adsorbed more quickly due to hydrophobic interactions with the oil, and lowered the interfacial tension more strongly than reference ß-lg.

FTIR analysis of ß-lactoglobulin at the oil/water-interface.

Knowledge about the critical interfacial concentration of a protein supports our understanding of the kinetic stability of an emulsion. Its determination is currently limited to either invasive or indirect methods. The aim of our study was the determination of the critical interfacial concentration of whey protein ß-lactoglobulin at oil/water-interfaces through fluorescence and pendant drop analysis and the comparison to an in situ Fourier-transform-infrared-spectroscopy (FTIR) method. Exponentially decreasing interfacial tension with increasing ß-lactoglobulin content (0.10-1.00 wt%) in pendant drop analysis could partly be confirmed by fluorescence spectra. A critical interfacial concentration of 0.20-0.31 wt% ß-lactoglobulin (1.80-2.69 mg/m2) in oil/water (5/95)-emulsions was determined via FTIR, analyzing the Amide I/Amide II peak intensity ratio. This was confirmed by the increasing formation of intermolecular ß-sheets, revealed by second derivative spectra. With this FTIR method we expand current options to investigate the interfacial behavior of food proteins by determination of secondary structure elements.

A Comprehensive Brownian Dynamics Approach for the Determination of Non-ideality Parameters from Analytical Ultracentrifugation.

Brownian dynamics (BD) has been applied as a comprehensive tool to model sedimentation and diffusion of nanoparticles in analytical ultracentrifugation (AUC) experiments. In this article, we extend the BD algorithm by considering space-dependent diffusion and solvent compressibility. With this, the changes in the sedimentation and diffusion coefficient from altered solvent properties at increased pressures are accurately taken into account. Moreover, it is demonstrated how the concept of space-dependent diffusion is employed to describe concentration-dependent sedimentation and diffusion coefficients, in particular, through the Gralen coefficient and the second virial coefficient. The influence of thermodynamic nonideality on diffusional properties can be accurately simulated and agree with well-known evaluation tools. BD simulations for sedimentation equilibrium and sedimentation velocity (SV) AUC experiments including effects of hydrodynamic and thermodynamic nonideality are validated by global evaluation in SEDANAL. The interplay of solvent compressibility and retrieved nonideality parameters can be studied utilizing BD. Finally, the second virial coefficient is determined for lysozyme from SV AUC experiments and BD simulations and compared to membrane osmometry. These results are in line with DLVO theory. In summary, BD simulations are established for the validation of nonideal sedimentation in AUC providing a sound basis for the evaluation of complex interactions even in polydisperse systems.

Conformational state and charge determine the interfacial stabilization process of beta-lactoglobulin at preoccupied interfaces.

Amphiphilic properties enable proteins like ß-lactoglobulin to stabilize oil/water-interfaces and provide stability in food-related emulsions. During emulsification, the protein undergoes three stages: (I) migration through bulk phase, (II) adsorption, and (III) interfacial rearrangement at the oil/water-interface - the kinetics of which require further research. Therefore, the aim of our study was the analytical and computational investigation of stage (I) and (II) as a function of the interfacial preoccupation, conformational state and charge of ß-lactoglobulin. For this purpose, the adsorption of ß-lactoglobulin (at pH 7, pH 7 containing 0.1 M NaCl, and pH 9) at increasingly preoccupied oil/water-interfaces has been compared through measuring interfacial tension and ζ-potential and through running molecular dynamics simulations. With increasing interfacial preoccupation, (I) the migration via lag time increased and (II) the adsorption rate decreased. The (II) adsorption rate was highest for ß-lactoglobulin containing NaCl, due to dense packing and electrostatic screening. ß-lactoglobulin at pH 7 reached a lower adsorption rate than the more negatively charged ß-lactoglobulin at pH 9, due to exposure of hydrophobic regions that had a greater effect on adsorption rates than electrostatic repulsion. Our research contributes to a profound understanding of the interfacial stabilization mechanism of proteins at oil/water-interfaces, necessary to characterise and control emulsification processes.

Metabolic engineering of the E. coli L-phenylalanine pathway for the production of D-phenylglycine (D-Phg).

D-phenylglycine (D-Phg) is an important side chain building block for semi-synthetic penicillins and cephalosporins such as ampicillin and cephalexin. To produce d-Phg ultimately from glucose, metabolic engineering was applied. Starting from phenylpyruvate, which is the direct precursor of L-phenylalanine, an artificial D-Phg biosynthesis pathway was created. This three-step route is composed of the enzymes hydroxymandelate synthase (HmaS), hydroxymandelate oxidase (Hmo), and the stereoinverting hydroxyphenylglycine aminotransferase (HpgAT). Together they catalyse the conversion of phenylpyruvate via mandelate and phenylglyoxylate to D-Phg. The corresponding genes were obtained from Amycolatopsis orientalis, Streptomyces coelicolor, and Pseudomonas putida. Combined expression of these activities in E. coli strains optimized for the production of L-phenylalanine resulted in the first completely fermentative production of D-Phg.