Application of Raman spectroscopy for the determination of proteins denaturation and amino acids decomposition temperature.

Raman spectroscopy was employed to study the thermal denaturation of three different proteins, bovine serum albumin (BSA), lysozyme, ovalbumin; and the decomposition temperature of three amino acids, l-glutamine, l-cysteine, and l-alanine, all of them as lyophilized powders. All the Raman bands observed in the spectra obtained were recorded and analyzed at preset heating temperatures. The results obtained for either protein denaturation temperature TD and amino acid decomposition temperatures TM-dc, were compared with those measured by differential scanning calorimetry (DSC). The DSC and Raman results were additionally corroborated with a thermogravimetric analysis (TGA) for the case of proteins. This exercise indicated almost complete coincidence in the determination of these transition temperatures between the three techniques, evidencing the applicability of Raman spectroscopy in the study of denaturation and decomposition temperatures of proteins and amino acids.

Determination of the denaturation temperature of the Spike protein S1 of SARS-CoV-2 (2019 nCoV) by Raman spectroscopy.

In the present work the temperature response of the constitutive S1 segment of the SARS-CoV-2 Spike Glycoprotein (GPS) has been studied. The intensity of the Raman bands remained almost constant before reaching a temperature of 133 °C. At this temperature a significant reduction of peak intensities was observed. Above 144 °C the spectra ceased to show any recognizable feature as that of the GPS S1, indicating that it had transformed after the denaturation process that it was subjected. The GPS S1 change is irreversible. Hence, Raman Spectroscopy (RS) provides a precision method to determine the denaturation temperature (TD) of dry powder GPS S1. The ability of RS was calibrated through the reproduction of TD of other well studied proteins as well as those of the decomposition temperature of some amino acids (AA). Through this study we established a TD of 139 ± 3 °C for powder GPS S1 of SARS-CoV-2.

Development and characterization of structured water-in-oil emulsions with ethyl cellulose oleogels.

The food industry confronts an enormous challenge to develop stable margarine-type water-in-vegetable oil (W/O) emulsion-based table spreads with reduced concentration of saturated fat and without trans fats. In the present work, we developed a gelled W/O emulsion (Gelled-W/O-E) containing 20% of water using a mixture of a conventional W/O emulsion (W/O-E) stabilized with glycerol monostearate (GMS), and an ethyl cellulose (EC) oleogel. The mechanical, microstructure and stability of the resulting gelled emulsion (Gelled-W/O-E) was compared with control systems consisting of conventional W/O emulsions (W/O-E) and EC-GMS oleogels (EC-GMS-O; no water added) formulated using the same GMS (0.5% and 1.0%) and EC (7%) concentration as in the Gelled-W/O-E. The Gelled-W/O-E showed higher elasticity and emulsion stability in comparison with the control systems. This in spite the EC and GMS concentrations used were below the minimal concentration required to develop a gel, and the tentatively lower solid content in the Gelled-W/O-E than in the EC-GMS-O because the presence of water. We observed that by increasing the GMS concentration in the Gelled-W/O-E, the water droplet size decreased as gel elasticity and W/O emulsion stability significantly increased. We associated this behavior to a synergistic GMS-EC interaction that kept the GMS at the water-oil droplet interface. These results showed the role of water droplets as active fillers in determining the rheological properties of the Gelled-W/O-E, and that the GMS efficiency as emulsifier increased in the presence of EC in the oil phase. After comparing the microstructural properties of commercial margarine spreads with those of the Gelled-W/O-E, we concluded that the structured W/O emulsion is a novel way to achieve similar functionality to margarine spreads, without the use of saturated and trans-fats.

Rheological properties of ethyl cellulose-monoglyceride-candelilla wax oleogel vis-a-vis edible shortenings.

The gelation process, elasticity, and mechanical recovery after shear were studied in mixed oleogels of ethylcellulose (EC), monoglycerides (MG), and candelilla wax (CW). EC oleogels produced without MG showed grainy texture due to incomplete dissolution of crystalline fractions of raw EC in the vegetable oil (150 °C). These fractions were eliminated by dissolving the raw EC/MG mixture in ethanol, evaporating the solvent, dispersing, and dissolving the solid residue in the vegetable oil (150 °C) prior gelation. The EC polymeric network, and MG, and CW crystals had a positive interaction on the elasticity of mixed oleogels. Mixed oleogels produced under static conditions showed a 100 % of elasticity recovery after shearing, a phenomenon associated with an EC interchain hydrogen bonding mediated by hydroxyl groups of MGs. This tentatively resulted from the formation of junction zones of the type EC-[MG]n-EC. The rheological behavior of these olegels was remarkably close to that of commercial shortenings.

Structuration, elastic properties scaling, and mechanical reversibility of candelilla wax oleogels with and without emulsifiers.

The crystal network development, elastic properties scaling behavior, and mechanical reversibility of candelilla wax (CW) oleogels with and without emulsifiers were studied. Saturated monoglycerides (MG) and polyglycerol polyricinoleate (PGPR) were added at 1 or 2 times the critical micelle concentration. Although the micelles of both emulsifiers act as nucleation sites for the mixture of aliphatic acids and alcohols of CW, they did not affect the oleogel's thermodynamic stability. It was established that the crystal network of CW consists of at least two types of crystals, one rich in n-hentriacontane and other rich in aliphatic acids. Both crystals species contributed significantly to the oleogel elasticity. The elastic properties scaling behavior of CW oleogels fitted the fractal model within the weak-link regime. The setting temperature and added emulsifier modified the crystal network fractal dimension. During shearing, oleogels had massive breaking of junction zones, causing the loss of fractality in the crystal network, which in turn decreased the system's elasticity.

Engineering rheological properties of edible oleogels with ethylcellulose and lecithin.

Addition of 1% (w/w) soy lecithin increased the shear moduli 10-fold and gel hardness 20-fold for 10% ethylcellulose (EC) oleogels. Higher lecithin addition levels or addition to gels with a higher EC concentration caused smaller increases. Similar trends were observed in the penetration force of the gels. Gels displayed thermal reversibility and a high temperature plateau at T≈120-130 °C. Large amplitude oscillatory shear rheology demonstrated similar solid-to-fluid transitions indicating that the polymer drives elastic softening and failure of the network. However, EC oleogels differed in their resistance to flow: the addition of unsaturated lecithin promoted a more gradual thickening response compared to gels containing saturated lecithin or only EC (the last two types of gels display strong intra-cycle thickening and thinning, more indicative of brittle failure). The thickening response of EC oleogels containing unsaturated lecithin, resembles more closely that of a model edible fat (lard).

Combined effect of shearing and cooling rate on the rheology of organogels developed by selected gelators.

In this study we investigated the combined effect of shearing and cooling rate in the rheology of organogels developed in high oleic safflower oil by (R)-12-hydroxystearic acid (HSA), its primary amide derivative [(R)-12-hydroxyoctadecanamide, HOA], and the N-octadecyl derivative of HOA [(R)-N-octadecyl-12-hydroxyoctadecanamide, OHOA]. The experimental set up to develop the organogels involved: 1). The use of quiescent (0s-1) or shearing (300, 600, and 1200s-1) conditions during cooling the gelator solutions (2%) just until achieving the gelator's melting temperature (TM) in the vegetable oil, to then continuing the cooling under static conditions until achieving 15°C) The use of cooling rate protocols involving a constant cooling rate of 1°C/min (CR1) or 10°C/min (CR10) in the shearing and static stages, or variable cooling rates in each stage (i.e., VR1-10 or VR10-1). The elasticity of the organogels (G') was measured while cooling under static conditions, once the systems achieved 15°C, and after 60min at this temperature. The rheological results obtained at 15°C showed a cooling rate and molecular weight-dependent effect of shearing on G'. We propose that the molecular relaxation time of gelator molecules, and its increase as molecular weight increases and as temperature decreases, plays an important role on the gelator's susceptibility to go through a shear induced crystallization process. Therefore, high molecular weight molecules like OHOA (551.97Da) would remain stretched by shearing longer times than HSA (300.49Da) and HOA (299.49Da). Thus, when shearing was applied while cooling at the higher cooling rate (i.e., CR10 and VR10-1), the stretched OHOA molecules would lead to the development of mesophase precursors that upon further cooling under quiescent conditions, crystallize developing a well-structured organogel. In contrast, stretched low molecular weight molecules (i.e., HSA and HOA) with shorter relaxation time would dissolve back to the isotropic state during cooling. Additionally, the rheological results of HSA and HOA organogels suggested that the shear induced crystallization process might be dependent on the gelator polarity also. These results show that the application of shear and the extent of its application as temperature decreases until achieving TM, have important implication on the self-assembly of gelator molecules, and therefore in the organization and rheology of the three-dimensional crystal network of the organogel.

Phase behavior, structure and rheology of candelilla wax/fully hydrogenated soybean oil mixtures with and without vegetable oil.

Vegetable oil organogelation is one of the most promising strategies to eliminate trans fatty acids in plastic fats. Organogels prepared with edible wax are stable at refrigerator and room temperature. Some functional properties (i.e., texture) of wax organogels can be improved by adding saturated triacylglycerols. Mixtures of fully hydrogenated soybean oil (FH) and candelilla wax (CW) were studied with and without the addition of high oleic safflower oil (HOSFO). Crystallization and melting behavior, X-ray diffraction, and crystalline microstructure of the mixtures were analyzed. The elastic modulus (G'), and the structural recovery after shear of the organogels were also assessed. Mixtures without HOSFO formed solid dispersions of CW and FH crystals, where up to ~10% CW crystals were incorporated into the FH crystal lattice. The vegetable oil solutions of FH/CW mixtures crystallized from the melt, developed mixed crystal networks composed of FH crystals in the ß polymorph and CW in an orthorhombic subcell packing. As the systems crystallized in the most stable polymorph, only minor microstructural changes were shown along 28days of storage at 25°C. CW and FH crystals showed a synergistic effect on the elasticity of organogels. This was attributed to the large number FH crystals nucleated on the surface of CW crystals. The structural recovery after shear was superior for mixed organogels composed of CW platelets and grainy FH crystals compared to that of CW organogels. A recovery of up to 65.7% the G' of gels formed under static conditions was observed upon shearing.

Comparing the crystallization and rheological behavior of organogels developed by pure and commercial monoglycerides in vegetable oil.

We investigated the crystallization and rheological behavior of organogels developed with commercial (MSGC) and pure (MSGP) monoglycerides in safflower oil solutions (0.5% to 8% wt/wt). The MSGC was composed of 1-mono-stearoyl-glycerol (1-MSG, 37.7%) and 1-mono-palmitoyl-glycerol (1-MPG, 54.0%), and the MSGP essentially by 1-MSG (93.51%). The elastic (G') and loss (G″) moduli of the MSGC and MSGP-oil solutions were measured from 80°C until achieving 5°C, and then during isothermal conditions. The d(G')/d(time) rheograms, where d(G')/d(time) is the difference in G' between subsequent time-temperature conditions during cooling, followed closely the phase transition observed by the monoglycerides (MG). The d(G')/d(time) profile showed that the formation of the inverse lamellar α mesophase provided a limited structure to the vegetable oil. In contrast, the crystallization of the sub-α phase in the MSGC-oil system, and of the sub-α1 and sub-α2 phases in the MSGP-oil system structured the vegetable oil through the uptake and retention of oil within their microstructure. Additionally, smaller crystals formed the three-dimensional crystal structure in the MSGC organogels. This is in comparison with the larger crystal size observed in MSGP organogels. Nevertheless, for a similar MG concentration the MSGC organogels showed higher G' and solid fat content (SFC) than the MSGP organogels, and the differences were greater as the MG concentration increased. We consider that the mixed sub-α structure developed by 1-MSG and 1-MPG in the MSGC-oil systems favored the incorporation and retention of higher amounts of oil, in comparison with the sub-α1 and sub-α2 structures developed just by 1-MSG in the MSGP-oil systems.