Impact of microfluidization on colloidal properties of insoluble pea protein fractions

Microfluidization is a technique commonly used to disrupt and homogenize dispersions such as oil-in-water emulsions or cellular suspensions. In this study, we investigated its ability to alter the physicochemical properties of plant-derived insoluble protein aggregates such as those found in pea protein extracts. Insoluble pea protein dispersions (5% w/w, pH 7) were homogenized at 25–150 MPa for 1–5 cycles. Increasing the homogenization pressure and cycles decreased the particle size (d43) of the unhomogenized insoluble pea proteins from 180 ± 40 μm to 0.2 ± 0.0 μm (at ≥ 125 MPa), leading to more transparent dispersions. Furthermore, the solubility of the insoluble pea proteins increased from 23 ± 1% to 86 ± 4%. Treatments with chaotropic agents, dithiothreitol and urea, revealed that insoluble pea protein aggregates were stabilized not only by disulphide bonds but also by hydrogen bonds and hydrophobic interactions. These molecular interactions were disrupted by microfluidization. The study provides insights into the disruption mechanism of insoluble pea proteins by applying microfluidization and offers a mean to improve their technofunctional properties to facilitate further use in food manufacture.

Evaluation of Chemical Protocols for Inactivating SARS-CoV-2 Infectious Samples

Clinical samples collected in coronavirus disease 19 (COVID-19), patients are commonly manipulated in biosafety level 2 laboratories for molecular diagnostic purposes. Here, we tested French norm NF-EN-14476+A2 derived from European standard EN-14885 to assess the risk of manipulating infectious viruses prior to RNA extraction. SARS-CoV-2 cell-culture supernatant and nasopharyngeal samples (virus-spiked samples and clinical samples collected in COVID-19 patients) were used to measure the reduction of infectivity after 10 min contact with lysis buffer containing various detergents and chaotropic agents. A total of thirteen protocols were evaluated. Two commercially available formulations showed the ability to reduce infectivity by at least 6 log 10, whereas others proved less effective.

RNA Isolation Protocol from Cells and Tissues

The preparation of intact ribonucleic acid is difficult because of the action of nucleases, which are liberated upon tissue homogenisation. In many cells, high concentrations of the ribonucleases are reserved in the secretory granules and upon disruption of the cell, they get mixed with the RNA and lead to its degradation. Guanidinium chloride and thiocyanate are potent chaotropic agents that reduce hydrophobic interactions and disrupt protein tertiary structures, disassociate proteinnucleic acid complexes and disintegrate cellular structures. Guanidinium thiocyanate is especially strong protein denaturant because both the cation and anion disrupt the hydrophobic bonds between the amino acid side chains. RNA usually binds to proteins within a cell and this agent disassociates the nucleoprotein complex, without disrupting RNA structure. Thus RNA can be obtained by using these agents, after homogenisation and low-speed centrifugation and precipitated with ethanol. The protocol below explains the stepwise isolation of total RNA from cells and tissues using TRIzol reagent which is the mono-phasic solution of phenol and guanidine thiocyanate.

Chaotropic effect of trifluoroacetic and perchloric acid on B-cyclodextrin inclusion complexation process with risperidone, olanzapine and their selected impurities

Effective method development together with method`s eco-friendly character are gaining importance in drug analyses nowadays. One of the strategies often applied to improve the efficacy of separation methods, especially in the case of basic ionizable analytes is adding chaotropic salts into the mobile phases. Moreover, the development of the green liquid chromatography method could also be achieved with certain mobile phase additives such as cyclodextrin (CD). The study aims to investigate whether adding chaotropic agents could improve the complexation process by disrupting the analytes' water solvation shell. The model mixture consisted of risperidone, olanzapine, and their related impurities. Method development was aided with experimental design methodology, while optimal separation conditions were selected using Derringer's desirability function. Mathematical models obtained for each of the examined responses enabled the explanation of the single and simultaneous influence of b-CD concentration, chaotropic agents type, and content, as well as the content of acetonitrile in the mobile phase. Retention factors appeared to be the most influenced by acetonitrile content in the mobile phase. The type of chaotropic agent as well as its concentration lead to retention prolongation, but if acetonitrile content in the mobile phase is high, the effect of chaotropic agent becomes negligible. Interaction between analyte and b-CD are relatively weak in comparison to the interaction of analyte form with either chaotropic agent or acetonitrile. Interaction leading to complexation are outperformed by other analyte related interactions in this complicated system, so complexation based retention reduction is not fully exposed. However, increasing b-CD concentration shows a positive effect on the resolution between critical peak pairs. Optimal separation conditions were selected based on 3D plots of Derringer's desirability function. For olanzapine and its impurity, they included the following: acetonitrile content 16% (v/v), trifluoroacetic acid as a chaotropic agent with 0.95% (v/v) content, and 9 mM b-CD concentration. Further, optimal separation conditions for risperidone and its impurity were 25% (v/v) acetonitrile content in the mobile phase, trifluoroacetic acid as chaotrope agent with 0.27% (v/v) content and 5mM b-CD concentration.

Multi-column ultra-high performance liquid chromatography screening with chaotropic agents and computer-assisted separation modeling enables process development of new drug substances

Streamlined workflow for method development and optimization.