On the formation and stability of cellulose-based emulsions in alkaline systems: Effect of the solvent quality.

With amphiphilic properties, cellulose molecules are expected to adsorb at the O/W interface and be capable of stabilizing emulsions. The effect of solvent quality on the formation and stability of cellulose-based O/W emulsions was evaluated in different alkaline systems: NaOH, NaOH-urea and tetrabutylammonium hydroxide (TBAH). The optimal solvency conditions for cellulose adsorption at the O/W interface were found for the alkaline solvent with an intermediate polarity (NaOH-urea), which is in line with the favorable conditions for adsorption of an amphiphilic polymer. A very good solvency (in TBAH) and the interfacial activity of the cation lead to lack of stability because of low cellulose adsorption. However, to achieve long-term stability and prevent oil separation in NaOH-urea systems, further reduction in cellulose's solvency was needed, which was achieved by a change in the pH of the emulsions, inducing the regeneration of cellulose at the surface of the oil droplets (in-situ regeneration).

Lignin Extraction from Waste Pine Sawdust Using a Biomass Derived Binary Solvent System.

Lignocellulosic biomass fractionation is typically performed using methods that are somehow harsh to the environment, such as in the case of kraft pulping. In recent years, the development of new sustainable and environmentally friendly alternatives has grown significantly. Among the developed systems, bio-based solvents emerge as promising alternatives for biomass processing. Therefore, in the present work, the bio-based and renewable chemicals, levulinic acid (LA) and formic acid (FA), were combined to fractionate lignocellulosic waste (i.e., maritime pine sawdust) and isolate lignin. Different parameters, such as LA:FA ratio, temperature, and extraction time, were optimized to boost the yield and purity of extracted lignin. The LA:FA ratio was found to be crucial regarding the superior lignin extraction from the waste biomass. Moreover, the increase in temperature and extraction time enhances the amount of extracted residue but compromises the lignin purity and reduces its molecular weight. The electron microscopy images revealed that biomass samples suffer significant structural and morphological changes, which further suggests the suitability of the newly developed bio-fractionation process. The same was concluded by the FTIR analysis, in which no remaining lignin was detected in the cellulose-rich fraction. Overall, the novel combination of bio-sourced FA and LA has shown to be a very promising system for lignin extraction with high purity from biomass waste, thus contributing to extend the opportunities of lignin manipulation and valorization into novel added-value biomaterials.

New deep eutectic solvent assisted extraction of highly pure lignin from maritime pine sawdust (Pinus pinaster Ait.).

Lignocellulosic biomass is a renewable and sustainable feedstock, mainly composed of cellulose, hemicellulose, and lignin. Lignin, as the most abundant natural aromatic polymer occurring on Earth, has great potential to produce value-added products. However, the isolation of highly pure lignin from biomass requires the use of efficient methods during lignocellulose fractionation. Therefore, in this work, novel acidic deep eutectic solvents (DESs) were prepared, characterized and screened for lignin extraction from maritime pine wood (Pinus pinaster Ait.) sawdust. The use of cosolvents and the development of new DES were also evaluated regarding their extraction and selectivity performance. The results show that an 1 h extraction process at 175 °C, using a novel DES composed of lactic acid, tartaric acid and choline chloride, named Lact:Tart:ChCl, in a molar ratio of 4:1:1, allows the recovery of 95 wt% of the total lignin present in pine biomass with a purity of 89 wt%. Such superior extraction of lignin with remarkable purity using a "green" solvent system makes this process highly appealing for future large-scale applications.

Cellulose-stabilized oil-in-water emulsions: Structural features, microrheology, and stability.

Cellulose-based oil-in-water (O/W) emulsions were studied by diffusing wave spectroscopy (DWS) regarding the effect of the cellulose concentration and mixing rate on the average droplet size, microrheological features and stability. Furthermore, the microstructure of these emulsions was imaged by cryo-scanning electron microscopy (cryo-SEM). The micrographs showed that cellulose was effectively adsorbed at the oil-water interface, resembling a film-like shell that protected the oil droplets from coalescing. The non-adsorbed cellulose that was observed in the continuous aqueous medium, contributed to the enhancement of the viscosity of the medium, leading to an improvement in the stability of the overall system. Generally, the higher the cellulose concentration and mixing rate, the smaller the emulsion droplets formed, and the higher was their stability. The combination of both techniques, DWS and cryo-SEM, revealed a very appealing and robust methodology for the characterization and design of novel emulsion-based formulations.

Revisiting the dissolution of cellulose in H3PO4(aq) through cryo-TEM, PTssNMR and DWS.

Cellulose can be dissolved in concentrated acidic aqueous solvents forming extremely viscous solutions, and, in some cases, liquid crystalline phases. In this work, the concentrated phosphoric acid aqueous solvent is revisited implementing a set of advanced techniques, such as cryo-transmission electronic microscopy (cryo-TEM), polarization transfer solid-state nuclear magnetic resonance (PTssNMR), and diffusing wave spectroscopy (DWS). Cryo-TEM images confirm that this solvent system is capable to efficiently dissolve cellulose. No cellulose particles, fibrils, or aggregates are visible. Conversely, PTssNMR revealed a dominant CP signal at 25 °C, characteristic of C-H bond reorientation with correlation time longer than 100 ns and/or order parameter above 0.5, which was ascribed to a transient gel-like network or an anisotropic liquid crystalline phase. Increasing the temperature leads to a gradual transition from CP to INEPT-dominant signal and a loss of birefringence in optical microscopy, suggesting an anisotropic-to-isotropic phase transition. Finally, an excellent agreement between optical microrheology and conventional mechanical rheometry was also obtained.

Legume Beverages from Chickpea and Lupin, as New Milk Alternatives.

Recently, milk consumption has been declining and there is a high demand for non-dairy beverages. However, market offers are mainly cereal and nut-based beverages, which are essentially poor in protein (typically, less than 1.5% against the 3.5% in milk) and are not true milk replacers in that sense. In this work, new beverages from different pulses (i.e., pea, chickpea and lupin) were developed using technologies that enable the incorporation of a high level of seed components, with low or no discharge of by-products. Different processing steps were sequentially tested and discussed for the optimization of the sensorial features and stability of the beverage, considering the current commercial non-dairy beverages trends. The lupin beverage protein contents ranged from 1.8% to 2.4% (w/v) and the chickpea beverage varied between 1.0% and 1.5% (w/v). The "milk" yield obtained for the optimized procedure B was 1221 g/100 g of dry seed and 1247 g/100 g of dry seed, for chickpea beverage and lupin beverage, respectively. Sensory results show that chickpea beverage with cooking water has the best taste. All pulses-based beverages are typical non-Newtonian fluids, similarly to current non-dairy alternative beverages. In this respect, the sprouted chickpea beverage, without the cooking water, presents the most pronounced shear-thinning behavior of all formulations.

Levulinic acid: A novel sustainable solvent for lignin dissolution.

Lignin is a natural, renewable resource with potential to be used in biomaterials. Due to its complex structure, its efficient dissolution is still challenging, which hinders its applicability at large scale. This challenge become harder considering the current need of sustainable and environmentally friendly solvents. To the best of our knowledge, this work reports for the first time the dissolution of kraft lignin in levulinic acid, a "green" solvent, and compares its efficiency with common carboxylic acids and sulfuric acid. It has been found that levulinic acid has a high capacity to dissolve kraft lignin at room temperature (40 wt% solubility), and it efficiency is not compromised when diluting the acid with water (up to 40 wt% water content). The Kamlet-Taft π⁎ parameter of the different acidic solvents was estimated and found to correlate well with their solubility performance. Lignins previously dissolved in levulinic and formic acids were selected to be regenerated and minor differences were found in thermal stability and morphological structure, when compared to native kraft lignin. However, an increase in the content of the carbonyl groups in the regenerated lignin material was observed.

Trichome Density in Relation to Volatiles Emission and 1,8-Cineole Synthase Gene Expression in Thymus albicans Vegetative and Reproductive Organs.

1,8-Cineole is the main volatile produced by Thymus albicans Hoffmanns. & Link 1,8-cineole chemotype. To understand the contribution of distinct plant organs to the high 1,8-cineole production, trichome morphology and density, as well as emitted volatiles and transcriptional expression of the 1,8-cineole synthase (CIN) gene were determined separately for T. albicans leaves, bracts, calyx, corolla and inflorescences. Scanning electron microscopy (SEM) and stereoscope microscopy observations showed the highest peltate trichome density in leaves and bracts, significantly distinct from calyx and corolla. T. albicans volatiles were collected by solid phase micro extraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC/MS) and by GC for component identification and quantification, respectively. Of the 23 components identified, 1,8-cineole was the dominant volatile (57-93 %) in all T. albicans plant organs. The relative amounts of emitted volatiles clearly separated vegetative from reproductive organs. Gene expression of CIN was assigned to all organs analyzed and was consistent with the relatively high emission of 1,8-cineole in leaves and bracts. Further studies will be required to analyze monoterpenoid biosynthesis by each type of glandular trichome.

Dissolution of kraft lignin in alkaline solutions.

Lignins are among the most abundant renewable resources on the planet. However, their application is limited by the lack of efficient dissolution and extraction methodologies. In this work, a systematic and quantitative analysis of the dissolution efficiency of different alkaline-based aqueous systems (i.e. lithium hydroxide, LiOH; sodium hydroxide, NaOH; potassium hydroxide, KOH; cuprammonium hydroxide, CuAOH; tetrapropylammonium hydroxide, TPAOH and tetrabutylammonium hydroxide, TBAOH) is reported, for the first time, for kraft lignin. Phase maps were determined for all systems and lignin solubility was found to decrease in the following order: LiOH > NaOH > KOH > CuAOH > TPAOH > TBAOH, thus suggesting that the size of the cation plays an important role on its solubility. The π∗ parameter has an opposite trend to the solubility, supporting the idea that cations of smaller size favor lignin solubility. Dissolution was observed to increase exponentially above pH 9-10 being the LiOH system the most efficient. The soluble and insoluble fractions of lignin in 0.1 M NaOH were collected and analyzed by several techniques. Overall, data suggests a greater amount of simple aromatic compounds, preferentially containing sulfur, in the soluble fraction while the insoluble fraction is very similar to the native kraft lignin.

Silk Fibroin Dissolution in Tetrabutylammonium Hydroxide Aqueous Solution.

Bombyx mori L. silk fibroin (SF) is widely used in different areas due to its ability to form durable and resilient materials with notable mechanical properties. However, in some of these applications the dissolution of SF is required, and this is not often straightforward due to its inability to be dissolved in the majority of common solvents. This work reports a novel approach to dissolve SF using 40 wt % aqueous tetrabutylammonium hydroxide, TBAOH(aq), at mild temperature. A thorough rheological study combined with small-angle X-ray scattering is presented to correlate the SF state in solution with changes in the rheological parameters. The scattering data suggest that the SF conformation in TBAOH(aq) is close to a random coil, possibly having some compact domains linked with flexible random chains. The radius of gyration (Rg) and the molecular weight (Mw) were estimated to be ca. 17.5 nm and 450 kDa, respectively, which are in good agreement with previous works. Nevertheless, a lower Mw value was deduced from rheometry (i.e., 321 kDa) demonstrating a low degree of depolymerization during dissolution in comparison to other harsh processes. The transition from a dilute to a semidilute regime coincides with the estimated critical concentration and is marked by the presence of a shear-thinning behavior in the flow curves, violation of the empirical Cox-Merz rule, and an upward increase in the activation energy. This work paves the way toward the development of advanced high-tech SF-based materials.

Emulsion Formation and Stabilization by Biomolecules: The Leading Role of Cellulose.

Emulsion stabilization by native cellulose has been mainly hampered because of its insolubility in water. Chemical modification is normally needed to obtain water-soluble cellulose derivatives. These modified celluloses have been widely used for a range of applications by the food, cosmetic, pharmaceutic, paint and construction industries. In most cases, the modified celluloses are used as rheology modifiers (thickeners) or as emulsifying agents. In the last decade, the structural features of cellulose have been revisited, with particular focus on its structural anisotropy (amphiphilicity) and the molecular interactions leading to its resistance to dissolution. The amphiphilic behavior of native cellulose is evidenced by its capacity to adsorb at the interface between oil and aqueous solvent solutions, thus being capable of stabilizing emulsions. In this overview, the fundamentals of emulsion formation and stabilization by biomolecules are briefly revisited before different aspects around the emerging role of cellulose as emulsion stabilizer are addressed in detail. Particular focus is given to systems stabilized by native cellulose, either molecularly-dissolved or not (Pickering-like effect).

New Insights on the Role of Urea on the Dissolution and Thermally-Induced Gelation of Cellulose in Aqueous Alkali.

The gelation of cellulose in alkali solutions is quite relevant, but still a poorly understood process. Moreover, the role of certain additives, such as urea, is not consensual among the community. Therefore, in this work, an unusual set of characterization methods for cellulose solutions, such as cryo-transmission electronic microscopy (cryo-TEM), polarization transfer solid-state nuclear magnetic resonance (PTssNMR) and diffusion wave spectroscopy (DWS) were employed to study the role of urea on the dissolution and gelation processes of cellulose in aqueous alkali. Cryo-TEM reveals that the addition of urea generally reduces the presence of undissolved cellulose fibrils in solution. These results are consistent with PTssNMR data, which show the reduction and in some cases the absence of crystalline portions of cellulose in solution, suggesting a pronounced positive effect of the urea on the dissolution efficiency of cellulose. Both conventional mechanical macrorheology and microrheology (DWS) indicate a significant delay of gelation induced by urea, being absent until ca. 60 °C for a system containing 5 wt % cellulose, while a system without urea gels at a lower temperature. For higher cellulose concentrations, the samples containing urea form gels even at room temperature. It is argued that since urea facilitates cellulose dissolution, the high entanglement of the cellulose chains in solution (above the critical concentration, C*) results in a strong three-dimensional network.

Molecular cloning and functional characterization of a monoterpene synthase isolated from the aromatic wild shrub Thymus albicans.

The essential oil of Thymus albicans Hoffmanns. & Link, a native shrub from the Iberian Peninsula, is mainly composed of monoterpenes. In this study, a 1,8-cineole synthase was isolated from the 1,8-cineole chemotype. A partial sequence that lacked the complete plastid transit peptide but contained an extended C-terminal when compared to other related terpene synthases was generated by PCR and Rapid Amplification of cDNA Ends (RACE). The predicted mature polypeptide was 593 amino acids in length and shared 78% and 77% sequence similarity with the homologue 1,8-cineole synthase from Rosmarinus officinalis and Salvia officinalis, respectively. The putative protein possessed the characteristic conserved motifs of plant monoterpene synthases including the RRx8W and DDxxD motifs and phylogenetic analysis indicated that the amplified 1,8-cineole synthase bears greater sequence similarity with other 1,8-cineole synthases from Lamiaceae family relative to the terpene synthases from the genus Thymus. Functional expression of the recombinant protein in Escherichia coli revealed that in the presence of geranyl diphosphate (GPP) 1,8-cineole was the major product but that its production was too low for robust quantification. Other minor conversion products included α-pinene, ß-pinene, sabinene and ß-myrcene suggesting the isolated 1,8-cineole synthase may be a multi-product enzyme. To our knowledge, this is the first report of a functionally characterized monoterpene synthase from Thymus albicans.

Reference genes to quantify gene expression during oogenesis in a teleost fish.

Understanding the molecular events involved in the acquisition of competence during oogenesis is a key step to determine the secret of 'high quality' eggs for aquaculture. Quantitative real time polymerase chain reaction (qPCR) is the technique of election to determine changes in transcript abundance in such studies, but choosing reference genes for normalization, in particular during oogenesis, remains a challenge. In the present study, transcription of 6 functionally distinct genes, ß actin (ACTB), cathepsin D (CTSD), cathepsin Z (CTSZ), elongation factor 1 α (EEF1A), TATA binding protein (TBP) and tubulin A (TUBA1A) was assessed as normalizers of bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) gene expression in mRNA from Mozambique tilapia oocytes during oogenesis. Reverse transcription was equally efficient and varies little in all samples. Most of the genes considered for reference were stable during early stages of oogenesis but variations were observed during vitellogenesis. A single gene and up to 3 genes were shown to be insufficient for reliable normalization throughout the whole oogenesis. The combination of the genes ACTB, CTSD, EEF1A and CTSZ as reference was found to minimize variation and has the most stable expression pattern between maturation stages.