Impact of Hydro-Alcoholic Solvents on the Oil and Phenolics Extraction from Walnut (Juglans regia L.) Press-Cake and the Self-Emulsification of Extracts.

The objective was to evaluate the performance of four hydro-alcoholic solvents to simultaneously extract oil and more polar molecules as phenolics, among others, to produce complex extracts that eventually could self-emulsify after solvent removal. Walnut press-cake was selected as the sourcing material. Extractions were performed as a semi-continuous operation up to a solvent-to-solid ratio of 28, with a fractional collection of the effluent. Among the solvents, labelled by their alcohol content EtOH 58, EtOH 86, iPro 60 and iPro 90 for ethanol (EtOH) and isopropanol (iPro), iPro 90 allowed to reach an oil extraction efficiency of 97% while the recovery for the other solvents was in the range of 30-40%. For both alcohols, the increase of the solvent hydration negatively influenced the oil extraction but positively increased the recovery of phenolics that reached 17.6 mg GAE/gcake when EtOH 58 was used. Several fractions contained enough surface-active material and oil to self-assemble as emulsions. IPro 90 and EtOH 86 showed better performances in the sense that most extracts were able to emulsify, though extraction kinetics pointed out differences. The most hydrated solvents behaved equally, with extraction yields in the same range and a similar but limited emulsifying capacity of only few fractions.

Improvement of the oxidative stability of camelina oil by enrichment with phospholipid-quercetin formulations.

Camelina oil (Coil) contains 50-60% of polyunsaturated fatty acids which are susceptible to oxidation. In this work, addition of phospholipids (0-20 mg/g) was assessed to improve the solubility of quercetin in Coil and enhance its oxidative stability. Results showed that the solubility of quercetin in Coil increased up to 7.7-fold by phospholipid addition. The solubility of quercetin in Coil was correlated to the phospholipid concentration and reached a maximum value of 1 298 µg/g. The addition of phospholipid-quercetin formulations increased the Coil stability, measured at 60 °C, from 24 h up to 115 h. Coil saturated with only quercetin (168 µg/g) did not significantly increase Coil stability, whereas phospholipids alone extended the oxidation lag time up to 40 h. This work successfully developed a solvent-free method for improving the solubility of quercetin in Coil and enhance its oxidative stability.

O/W Pickering emulsions stabilized by cocoa powder: Role of the emulsification process and of composition parameters.

We fabricated oil-in-water emulsions stabilized by delipidated commercial cocoa powder. The emulsions were characterized in terms of droplets and particles size distribution and interfacial coverage by cocoa powder by developing methods to separate droplets from adsorbed and unadsorbed cocoa particles. Three different processes were compared for their ability to produce fine and stable emulsions: rotor/stator turbulent mixing, sonication and microfluidization. Among those techniques, microfluidization was the most performing one. In this case, micron-sized emulsions with narrow size distributions could be obtained with >90 wt% of the powder insoluble material anchored to the interfaces, and they were still stable after 90 days. It was demonstrated that the mixing process did not generate finer cocoa particles but provoked disentanglement of the large primary particles, providing them an open, expanded structure that facilitated emulsification. It was also shown that the finer insoluble fraction of the powder and the soluble fraction had no significant impact on emulsification and on kinetic stability. In the poor particles regime, the oil-water interfacial area varied linearly with the amount of adsorbed powder, suggesting that the final droplet size was controlled by the so-called limited coalescence process, as already observed in conventional Pickering emulsions stabilized by spherical solid particles.

Monodisperse Oil-in-Water Emulsions Stabilized by Proteins: How To Master the Average Droplet Size and Stability, While Minimizing the Amount of Proteins.

Hexadecane-in-water emulsions were fabricated by means of a microfluidizer using two types of protein stabilizers, sodium caseinate (NaCAS) and ß-lactoglobulin (BLG). A study of the dependence of the mean droplet diameter and protein coverage on protein concentration was performed. At low protein concentrations, the emulsions were monodispersed and their mean droplet size was governed by the so-called limited-coalescence process. In this regime, the interfacial coverage was constant and was deduced from the linear evolution of the total interfacial area as a function of the amount of adsorbed proteins. In emulsions based on NaCAS, almost all of the initial protein contents were adsorbed at the interfaces. Emulsions formulated at very low protein content underwent unlimited coalescence after prolonged storage or when submitted to centrifugation. Additional NaCAS was incorporated in the continuous phase, right after the emulsification process, as a means of ensuring kinetic stability. The interfacial coverage increased after protein addition. Other strategies including acidification and salt addition were also probed to gain stability. Instead, in emulsions based on BLG, only partial adsorption of the initial protein content was observed. The corresponding emulsions remained kinetically stable against coalescence, and no further addition of protein was required after emulsification. Our approach allows to obtain monodisperse, kinetically stable emulsions and to master their average droplet size, while minimizing the amount of proteins.

Determination of reducing power and metal chelating ability of antioxidant peptides: revisited methods.

The purpose of this study was to improve two common tests used for antioxidant capacity measurements, i.e. the reducing power and chelating ability measurements, for appropriate comparisons between the molecules tested and chosen references, as the usual methods are often performed in a qualitative way rather than a quantitative way. After revision, it was then possible to determine an AERC indice (Ascorbate Equivalent Reducing Capacity) and a CECC (Carnosine Equivalent Chelating Capacity) or EECC (EDTA Equivalent Chelating Capacity) indice according to the standard chosen, by analogy to the TEAC indice (Trolox Equivalent Antioxidant Capacity) already used in many reported works to determine the free radical scavenging activity. Thus, the determination of these relative indices enables the comparison of antioxidative capacities obtained in various studies. The adaptation of these two tests to micro-scales and the calculation of AERC, EECC and CECC were performed on model peptides.

CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form.

The cetyltrimethylammonium bromide turbidimetric method (CTM) has been developed to quantify the hyaluronic acid (HA) in complex media to overcome the lack of selectivity and specificity of the standard carbazole method. The objective of this work is to assess the potential application of CTM to determine HA concentration. Factors such as duration of incubation, linearity range, HA size and form (natural linear HA or cross linked HA), pH and ionic environment impact were investigated. The incubation time was set to 10 min and the calibration curve was linear up to 0.6 g L(-1). The quantitative method was relevant whatever the HA size and form, and also for a wide range of conditions. The robustness of the CTM added to its high specificity and simplicity demonstrated that the CTM is a valuable method that would be an interesting substitute to the carbazole assay for HA quantification.

Hydrophilic properties as a new contribution for computer-aided identification of short peptides in complex mixtures.

A new method to predict elementary amino acid (AA) composition of peptides (molar mass <1,000 g/mol) is described. This procedure is based on a computer-aided method using three combined analyses-reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and capillary electrophoresis coupled with mass spectrometry-and using a software calculating all possible amino acid combinations from the mass of any given peptide. The complementarity between HILIC and RPLC was demonstrated. Peptide retention prediction in HILIC was successfully modelled, and the achieved prediction accuracy was as high as r²=0.97. This mathematical model, based on amino acid retention contributions and peptide length, provided the information about peptide hydrophilicity that was not redundant with its hydrophobicity. Correlations between respectively the hydrophobicity coefficients and RPLC retention time, hydrophilicity and HILIC retention time, and electrophoretic mobility and migration time were used for ranking all potential AA combinations corresponding to the given mass. The essential contribution of HILIC in this identification strategy and the need to combine the three models to significantly increase identification capabilities were both shown. Applied to an 18-standard peptide mixture, the identification procedure enabled the actual AA combination determination of the 14 di- to pentapeptides, in addition to an over 98 % reduction of possible combination numbers for the four hexapeptides. This procedure was then applied to the identification of 24 unknown peptides in a rapeseed protein hydrolysate. The effective AA composition was found for ten peptides, whereas for the 14 other peptides, the number of possible combinations was reduced by over 95 % thanks to the association of the three analyses. Finally, as a result of the information provided by the analytical techniques about peptides present in the mixture, the proposed method could become a highly valuable tool to recover bioactive peptides from undefined protein hydrolysates.

Modeling the separation of small peptides by cation-exchange chromatography.

Cation-exchange chromatography was applied for the separation of short synthetic peptide standards, with various charges and hydrophobicities. The methodology to develop simple, reproducible and accurate models, based on physicochemical peptide properties, was described. A multilinear regression method was used for the calculation of the models, and descriptors were chosen according to the observed phenomena. The predictive and interpretative ability of the chromatographic models was evaluated considering cross-validated data (root mean-squared error of calibration, root mean-squared error, root mean-squared error of cross-validation and the Fisher ratio). Hydrophobic coefficients for amino acids were calculated with or without consideration of peptide sequences. A simple model, with only two parameters (charge and hydrophobic coefficient) was built. It enabled an accurate prediction of short peptide elution (up to nine residues). As the model was intended for further characterization of complex mixtures of unknown peptides, some mixtures were analyzed to investigate possible interactions between molecules. Peptides eluted in exactly the same pattern as when injected alone, supporting the use of these models for complex mixtures of small peptides.