Microencapsulation of vitamins: A review and meta-analysis of coating materials, release and food fortification.

Vitamins are responsible for providing biological properties to the human body; however, their instability under certain environmental conditions limits their utilization in the food industry. The objective was to conduct a systematic review on the use of biopolymers and lipid bases in microencapsulation processes, assessing their impact on the stability, controlled release, and viability of fortified foods with microencapsulated vitamins. The literature search was conducted between the years 2013-2023, gathering information from databases such as Scopus, PubMed, Web of Science and publishers including Taylor & Francis, Elsevier, Springer and MDPI; a total of 49 articles were compiled The results were classified according to the microencapsulation method, considering the following information: core, coating material, solvent, formulation, process conditions, particle size, efficiency, yield, bioavailability, bioaccessibility, in vitro release, correlation coefficient and references. It has been evidenced that gums are the most frequently employed coatings in the protection of vitamins (14.04%), followed by alginate (10.53%), modified chitosan (9.65%), whey protein (8.77%), lipid bases (8.77%), chitosan (7.89%), modified starch (7.89%), starch (7.02%), gelatin (6.14%), maltodextrin (5.26%), zein (3.51%), pectin (2.63%) and other materials (7.89%). The factors influencing the release of vitamins include pH, modification of the coating material and crosslinking agents; additionally, it was determined that the most fitting mathematical model for release values is Weibull, followed by Zero Order, Higuchi and Korsmeyer-Peppas; finally, foods commonly fortified with microencapsulated vitamins were described, with yogurt, bakery products and gummy candies being notable examples.

Experimental and Computational Analysis of Synthesis Conditions of Hybrid Nanoflowers for Lipase Immobilization.

This work presents a framework for evaluating hybrid nanoflowers using Burkholderia cepacia lipase. It was expanded on previous findings by testing lipase hybrid nanoflowers (hNF-lipase) formation over a wide range of pH values (5-9) and buffer concentrations (10-100 mM). The free enzyme activity was compared with that of hNF-lipase. The analysis, performed by molecular docking, described the effect of lipase interaction with copper ions. The morphological characterization of hNF-lipase was performed using scanning electron microscopy. Fourier Transform Infrared Spectroscopy performed the physical-chemical characterization. The results show that all hNF-lipase activity presented values higher than that of the free enzyme. Activity is higher at pH 7.4 and has the highest buffer concentration of 100 mM. Molecular docking analysis has been used to understand the effect of enzyme protonation on hNF-lipase formation and identify the main the main binding sites of the enzyme with copper ions. The hNF-lipase nanostructures show the shape of flowers in their micrographs from pH 6 to 8. The spectra of the nanoflowers present peaks typical of the amide regions I and II, current in lipase, and areas with P-O vibrations, confirming the presence of the phosphate group. Therefore, hNF-lipase is an efficient biocatalyst with increased catalytic activity, good nanostructure formation, and improved stability.

Glycosylation and charge distribution orchestrates the conformational ensembles of a biotechnologically promissory phytase in different pHs - a computational study.

Phytases [myo-inositol(1,2,3,4,5,6) hexakisphosphate phosphohydrolases] are phytate-specific phosphatases not present in monogastric animals. Nevertheless, they are an essential supplement to feeding such animals and for human special diets. It is crucial, hence, the biotechnological use of phytases with intrinsic stability and activity at the acid pHs from gastric environments. Here we use Metadynamics (METADY) simulations to probe the conformational space of the Aspergillus nidulans phytase and the differential effects of pH and glycosylation in this same space. The results suggest that strategic combinations of pH and glycosylation affect the stability of native-like conformations and alternate these structures from a metastable to a stable profile. Furthermore, the protein segments previously reported as more thermosensitive in phytases from this family present a pivotal role in the conformational changes at different conditions, especially H2, H5-7, L8, L10, L12, and L17. Also, the glycosylations and the pH-dependent charge balance modulate the mobility and interactions at these same regions, with consequences for the surface solvation and active site exposition. Finally, although the glycosylations have stabilized the native structure and improved the substrate docking at all the studied pHs, the data suggest a higher phytate receptivity at catalytic poses for the unglycosylated structure at pH 6.5 and the glycosylated one at pH 4.5. This behavior agrees with the exact change in optimum pH reported for this enzyme, expressed on low or high glycosylating systems. We hope the results and insights presented here will be helpful in future approaches for rational engineering of technologically promising phytases and intelligent planning of their heterologous expression systems and conditions for use.Communicated by Ramaswamy H. Sarma.

Elucidating the Protonation State of the γ-Secretase Catalytic Dyad.

γ-Secretase (GS) is an intramembrane aspartyl protease that participates in the sequential cleavage of C99 to generate different isoforms of the amyloid-ß (Aß) peptides that are associated with the development of Alzheimer's disease. Due to its importance in the proteolytic processing of C99 by GS, we performed pH replica exchange molecular dynamics (pH-REMD) simulations of GS in its apo and substrate-bound forms to sample the protonation states of the catalytic dyad. We found that the catalytic dyad is deprotonated at physiological pH in our apo form, but the presence of the substrate at the active site displaces its monoprotonated state toward physiological pH. Our results show that Asp257 acts as the general base and Asp385 as the general acid during the cleavage mechanism. We identified different amino acids such as Lys265, Arg269, and the PAL motif interacting with the catalytic dyad and promoting changes in its acid-base behavior. Finally, we also found a significant pKa shift of Glu280 related to the internalization of TM6-CT in the GS-apo form. Our study provides critical mechanistic insight into the GS mechanism and the basis for future research on the genesis of Aß peptides and the development of Alzheimer's disease.

Changes in Protonation Sites of 3-Styryl Derivatives of 7-(dialkylamino)-aza-coumarin Dyes Induced by Cucurbit[7]uril.

The incorporation of a guest, with different basic sites, into an organized system (host), such as macrocycles, could stabilize, detect, or promote the formation of a certain protomer. In this context, this work aimed to study the influence of cucurbit[7]uril (CB7) on dyes such as 7-(dimethylamino)-aza-coumarins, which have more than one basic site along their molecular structure. For this, three 3-styryl derivatives of 7-(dialkylamino)-aza-coumarin dyes (SAC1-3) were synthesized and characterized by NMR, ESI-HRMS and IR. The spectral behaviour of the SACs in the absence and presence of CB7 was studied. The results showed large shifts in the UV-vis spectrum in acid medium: a hypsochromic shift of ≈5400 cm-1 (SAC1-2) and ≈3500 cm-1 (SAC3) in the absence of CB7 and a bathochromic shift of ≈4500 cm-1 (SAC1-3) in the presence of CB7. The new absorptions at long and short wavelengths were assigned to the corresponding protomers by computational calculations at the density functional theory (DFT) level. Additionally, the binding mode was corroborated by molecular dynamics simulations. Findings revealed that in the presence of CB7 the heterocyclic nitrogen was preferably protonated instead of the dialkylamino group. Namely, CB7 induces a change in the protonation preference at the basic sites of the SACs, as consequence of the molecular recognition by the macrocycle.

Competitive protein adsorption on charge regulating silica-like surfaces: the role of protonation equilibrium.

We develop a molecular thermodynamic theory to study the interaction of some proteins with a charge regulating silica-like surface under a wide range of conditions, including pH, salt concentration and protein concentration. Proteins are modeled using their three dimensional structure from crystallographic data and the average experimental pKa of amino acid residues. As model systems, we study single-protein and binary solutions of cytochrome c, green fluorescent protein, lysozyme and myoglobin. Our results show that protonation equilibrium plays a critical role in the interactions of proteins with these type of surfaces. The terminal hydroxyl groups on the surface display considerable extent of charge regulation; protein residues with titratable side chains increase protonation according to changes in the local environment and the drop in pH near the surface. This behavior defines protein-surface interactions and leads to the emergence of several phenomena: (i) a complex non-ideal surface charge behavior; (ii) a non-monotonic adsorption of proteins as a function of pH; and (iii) the presence of two spatial regions, a protein-rich and a protein-depleted layer, that occur simultaneously at different distances from the surface when pH is slightly above the isoelectric point of the protein. In binary mixtures, protein adsorption and surface-protein interactions cannot be predicted from single-protein solution considerations.

Impact of different protonation states on virtual screening performance against cruzain.

The cysteine protease cruzain is a Chagas disease target, exploited in computational studies. However, there is no consensus on the protonation states of the active site residues Cys25, His162, and Glu208 at the enzyme's active pH range. We evaluated the impact of different protonation states of these residues on docking calculations. Through a retrospective study with cruzain inhibitors and decoys, we compared the performance of virtual screening using four grids, varying protonation states of Cys25, His162, and Glu208. Based on enrichment factors and ROC plots, docking with the four grids affected compound ranking and the overall charge of top-ranking compounds. Different grids can be complementary and synergistic, increasing the odds of finding different ligands with diverse chemical properties.

pH and non-covalent ligand binding modulate Zika virus NS2B/NS3 protease binding site residues: Discoveries from MD and constant pH MD simulations.

Zika virus (ZIKV) is a global health concern and has been linked to severe neurological pathologies. Although no medication is available yet, many efforts to develop antivirals and host cell binding inhibitors led to attractive drug-like scaffolds, mainly targeting the nonstructural NS2B/NS3 protease (NS2B/NS3pro). NS2B/NS3pro active site has several titratable residues susceptible to pH changes and ligand binding; hence, understanding these residues' protonation is essential to drug design efforts targeting the active site. Here we use in silico methods to probe non-covalent binding and its effect on pKa shifts of the active site residues on a ligand-free protease and with a non-peptidic competitive inhibitor (Ki=13.5 µM). By applying constant pH molecular dynamics, we found that the catalytic residues of the unbound NS2B/NS3pro achieved the protonation needed for the serine protease mechanism over the pH value of 8.5. Nevertheless, the protease in the holo state achieved this same scenario at lower pH values. Also, non-covalent binding affected the catalytic triad (H51, D75, and S135) by stabilizing their distances and interaction network. Thus, NS2B/NS3pro residues configuration for activity might be both pH-dependent and influenced by ligand binding. However, compound presence within the binding site destabilized the NS2B, interfering with the closed and active conformation necessary for substrate binding and catalysis. Our outcomes provide valuable insights into non-covalent inhibitor behavior and its effect on protease active site residues, impacting optimization and design of novel compounds. Communicated by Ramaswamy H. Sarma.

In Silico Characterization of Masitinib Interaction with SARS-CoV-2 Main Protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a global health problem. Despite the current implementation of COVID-19 vaccination schedules, identifying effective antiviral drug treatments for this disease continues to be a priority. A recent study showed that masitinib (MST), a tyrosine kinase inhibitor, blocks the proteolytic activity of SARS-CoV-2 main protease (Mpro ). Although MST is a potential candidate for COVID-19 treatment, a comprehensive analysis of its interaction with Mpro has not been done. In this work, we performed molecular dynamics simulations of the MST-Mpro complex crystal structure. The effect of the protonation states of Mpro H163 residue and MST titratable groups were studied. Furthermore, we identified the MST substituents and Mpro mutations that affect the stability of the complex. Our results provide valuable insights into the design of new MST analogs as potential treatments for COVID-19.

Protonation of Nucleobases in Single- and Double-Stranded DNA.

Single-stranded model oligodeoxyribonucleotides, each containing a single protonatable base-cytosine, adenine, guanine, or 5-methylcytosine-centrally located in a background of non-protonatable thymine residues, were acid-titrated in aqueous solution, with UV monitoring. The basicity of the central base was shown to depend on the type of the central base and its nearest neighbours and to rise with increasing oligonucleotide length and decreasing ionic strength of the solution. More complex model oligonucleotides, each containing a centrally located 5-methylcytosine base, were comparatively evaluated in single-stranded and double-stranded form, by UV spectroscopy and high-field NMR. The N3 protonation of the 5-methylcytosine moiety in the double-stranded case occurred at much lower pH, at which the duplex was already experiencing general dissociation, than in the single-stranded case. The central guanine:5-methylcytosine base pair remained intact up to this point, possibly due to an unusual alternative protonation on O2 of the 5-methylcytosine moiety, already taking place at neutral or weakly basic pH, as indicated by UV spectroscopy, thus suggesting that 5-methylcytosine sites in double-stranded DNA might be protonated to a significant extent under physiological conditions.

New solvates of the drug naltrexone: protonation, conformation and interplay of synthons.

Naltrexone [systematic name: (4R,4aS,7aR,12bS)-3-cyclopropylmethyl-4a,9-dihydroxy-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7-one] is an important morphine-related drug used for combating alcoholism and opioid dependence. Of the eight crystal forms of naltrexone known thus far, only one exists in the neutral form and it crystallizes as a monohydrate. We have isolated the naltrexone free base as two new solvate forms, i.e. the ethyl acetate 0.33-solvate, C20H23NO4·0.33C4H8O2, (I), and the diethyl ether hemisolvate, C20H23NO4·0.5C4H10O, (II). While just one solvent molecule is present in the asymmetric unit of each solvate, there are three drug molecules (Z' = 3) in ethyl acetate solvate (I) and two (Z' = 2) in diethyl ether solvate (II). In (I), one of the three crystallographically independent drug molecules is present with its cyclopropyl group disordered over two sets of positions, as is the whole diethyl ether solvent molecule in (II). In all known forms, including the title forms, the naltrexone molecule exhibits the same conformation of the fused rings. The only conformational variability of naltrexone is in the cyclopropylmethyl group. Two conformations can be found around the bond connecting this group to the N-heterocycle, which is directly related to drug protonation. We have calculated, at the B3LYP/6-31G** level of theory, the minimum energy conformations of protonated and neutral naltrexone molecules for a chosen torsion angle about this bond. The lowest energy conformers depend on the protonation state and are in agreement with those found in the solid state. Within the cyclopropylmethyl group, the bond joining the methylene C atom to the cyclopropyl fragment also evidences conformational variability. In the literature, there are two well defined conformations around this bond. A third cyclopropyl conformation around this second bond is observed in the title solvates. Concerning the supramolecular features of the previously reported crystal structures, only one classical hydrogen bond between naltrexone molecules and one C(8) homosynthon is known, pointing to the robustness of this synthon and the difficulty in disrupting it. New R22(7) and C22(10) homosynthons are found in both (I) and (II), suggesting that their occurrence derives from crystallization of the neutral drug from nonpolar solvents.

Effects of the Protonation State of Titratable Residues and the Presence of Water Molecules on Nocodazole Binding to ß-Tubulin.

Regulation of microtubule assembly by antimitotic agents is a potential therapeutic strategy for the treatment of cancer, parasite infections, and neurodegenerative diseases. One of these agents is nocodazole (NZ), which inhibits microtubule polymerization by binding to ß-tubulin. NZ was recently co-crystallized in Gallus gallus tubulin, providing new information about the features of interaction for ligand recognition and stability. In this work, we used state-of-the-art computational approaches to evaluate the protonation effects of titratable residues and the presence of water molecules in the binding of NZ. Analysis of protonation states showed that residue E198 has the largest modification in its pKa value. The resulting E198 pKa value, calculated with pH-REMD methodology (pKa =6.21), was higher than the isolated E amino acid (pKa =4.25), thus being more likely to be found in its protonated state at the binding site. Moreover, we identified an interaction between a water molecule and C239 and G235 as essential for NZ binding. Our results suggest that the protonation state of E198 and the structural water molecules play key roles in the binding of NZ to ß-tubulin.

Population density analysis for determining the protonation state of the catalytic dyad in BACE1-tertiary carbinamine-based inhibitor complex.

BACE1 is an aspartyl protease with a very relevant role in medicinal chemistry related to Alzheimer Disease since it has demonstrated to be a promising therapeutic target for inhibition and possible control for the progress of the peptide accumulation characteristic of this pathology. The enzymatic activity of this protein is given by the aspartic dyad, Asp93 and Asp289, which can adopt several protonation states depending on the chemical nature of its inhibitors, this is, monoprotonated, diprotonated and di-deprotonated states. In the present study, the analysis of the population density, for a series of protein-inhibitor molecular dynamics simulations, was carried out to identify the most feasible protonation state adopted by the catalytic dyad in the presence of tertiary carbinamine (TC) transition state analog inhibitors. The results revealed that the monoprotonated Asp289i state, in which the Asp93 and Asp289 residue side chains are deprotonated and protonated on the inner oxygen, respectively, is the most preferred in the presence of TC family inhibitors. This result was obtained after evaluating, for all 9 possible protonation state configurations, the individual and combined population densities of a set of parameters sensitive to protonation state of the Aspartic dyad, using an X-ray experimental BACE1/TC crystallographic structure as reference. This case study demonstrates again the usefulness of the concept of population density as a quantitative tool to establish the most stable system settings, among all possible, by measuring the level of occurrence of simultaneous events obtained from a sampling over time. These results will help to clear the phenomena related to the TCs inhibitory pathway, as well as assist in the design of better TC inhibitors against Alzheimer's protease.

Gas-phase dissociation study of erythrinian alkaloids by electrospray ionization mass spectrometry and computational methods.

Alkaloids from plants of the genus Erythrina display important biological activities, including anxiolytic action. Characterization of these alkaloids by mass spectrometry (MS) has contributed to the construction of a spectral library, has improved understanding of their structures and has supported the proposal of fragmentation mechanisms in light of density functional calculations. In this study, we have used low-resolution and high-resolution MSn analyses to investigate the fragmentation patterns of erythrinian alkaloids; we have employed the B3LYP/6-31+G(d,p) model to obtain their reactive sites. To suggest the fragmentation mechanism of these alkaloids, we have studied their protonation sites by density functional calculation, and we have obtained their molecular electrostatic potential map and their gas-phase basicity values. These analyses have indicated the most basic sites on the basis of the proton affinities of the nitrogen and oxygen atoms. The protonated molecules were generated by two major fragmentations, namely, neutral loss of CH3 OH followed by elimination of H2 O. High-resolution analysis confirmed elimination of NH3 by comparison with the losses of H2 and •CH3 . NH3 was eliminated from compounds that did not bear a substituent on ring C. The benzylic carbocation initiated the dissociation mechanism, and the first reaction involved charge transfer from a lone pair of electrons in the oxygen atoms. The second reaction consisted of ring contraction with loss of a CO molecule. The presence of hydroxy and epoxy groups could change the intensity or the occurrence of the fragmentation pathways. Given that erythrinian alkaloids are applied in therapeutics and are promising leads for the development of new drugs, the present results could aid identification of several analogues of these alkaloids in biological samples and advance pharmacokinetic studies of new plant derivatives based on MSn and MS/MS analyses. Copyright © 2017 John Wiley & Sons, Ltd.

Determination of the protonation state for the catalytic dyad in ß-secretase when bound to hydroxyethylamine transition state analogue inhibitors: A molecular dynamics simulation study.

BACE1 is an aspartyl protease of pharmacological interest for its direct participation in Alzheimer's disease (AD) through ß-amyloid peptide production. Two aspartic acid residues are present in the BACE1 catalytic region which can adopt multiple protonation states depending on the chemical nature of its inhibitors, i.e., monoprotonated, diprotonated and di-deprotonated states. In the present study a series of protein-ligand molecular dynamics (MD) simulations was carried out to identify the most feasible protonation state adopted by the catalytic dyad in the presence of hydroxyethylamine transition state analogue inhibitors. The MD trajectories revealed that the di-deprotonated state is most prefered in the presence of hydroxyethilamine (HEA) family inhibitors. This appears as a result after evaluating, for all 9 protonation state configurations during the simulation time, the deviations of a set of distances and dihedral angles measured on the ligand, protein and protein-ligand complex with reference to an X-ray experimental BACE1/HEA crystallographic structure. These results will help to clarify the phenomena related to the HEAs inhibitory pathway, and improve HEAs databases' virtual screening and ligand design processes targeting ß-secretase protein.

Quantitative Structure-Property Relationship (QSPR) Models for a Local Quantum Descriptor: Investigation of the 4- and 3-Substituted-Cinnamic Acid Esterification.

In this work, the theoretical description of the 4- and 3-substituted-cinnamic acid esterification with different electron donating and electron withdrawing groups was performed at the B3LYP and M06-2X levels, as a two-step process: the O-protonation and the nucleophile attack by ethanol. In parallel, an experimental work devoted to the synthesis and characterization of the substituted-cinnamate esters has also been performed. In order to quantify the substituents effects, quantitative structure-property relationship (QSPR) models based on the atomic charges, Fukui functions and the Frontier Effective-for-Reaction Molecular Orbitals (FERMO) energies were investigated. In fact, the Fukui functions, ƒâºC and ƒ(-)O, indicated poor correlations for each individual step, and in contrast with the general literature, the O-protonation step is affected both by the FERMO energies and the O-charges of the carbonyl group. Since the process was shown to not be totally described by either charge- or frontier-orbitals, it is proposed to be frontier-charge-miscere controlled. Moreover, the observed trend for the experimental reaction yields suggests that the electron withdrawing groups favor the reaction and the same was observed for Step 2, which can thus be pointed out as the determining step.

Effect of methylation on relative energies of tautomers and on the intramolecular proton transfer barriers of protonated nitrosamine: A MR-CISD study.

MR-CISD, MR-CISD+Q, and MR-AQCC calculations have been performed on the minima and transition states (corresponding to intramolecular proton transfer between the protonation sites) of the ground state of protonated nitrosamine and N,N-dimethylnitrosamine. Our highest level results (MR-AQCC/cc-pVTZ) for the smaller system indicate that protonation on the N amino (2a) is practically as favorable as the most favorable protonation on the O atom (1a). They also suggest that protonation on the nitroso N atom (2c) is ∼14.5 kcal/mol less favorable than 1a. Results obtained at the MR-CISD+Q/cc-pVTZ level indicate that the effect of methylation on the relative energies of the tautomers is, in order of importance, 2a > 2c and increases their energies by ∼17.5 and 4.8 kcal/mol, respectively. They also indicate that methylation alters significantly the intramolecular proton transfer barriers. The largest differences between the common geometric parameters of both systems have been found for 2a.

The pH dependence of flavivirus envelope protein structure: insights from molecular dynamics simulations.

The flavivirus membrane fusion is triggered by the acid pH of the endosomes after virus endocytosis. The proposed mechanism involves changes in the protonation state of conserved histidine residues of the E protein present in the viral surface that undergoes a series of structural rearrangements that result in the fusion between the endosome and viral bilayers. We studied the pH dependence of E protein rearrangements of dengue virus type 2, used as a model, in the pH range experimented by the virus along the fusion process. We employed a low computational cost scheme to explore the behavior of the E protein by molecular dynamics (MD) simulations of complete systems that include the protein, the solvent, and ions. The procedure alternates cyclically the update of the ionization states of the protein residues with common MD steps applied to the new ionization configuration. Important pH-dependent protein structure rearrangements consistent with the changes of the protonation states of conserved histidine residues were observed. The involvement of other conserved residues in the flavivirus in the rearrangements was also identified. The results show interesting correlations with a proposed model for the fusion mechanism, as well as the experimentally identified key residues, contributing to a better understanding of the structural changes in protein E that lead to the fusion process.

Spectroscopic behavior of loratadine and desloratadine in different aqueous media conditions studied by means of TD-DFT calculations.

In this study, we explained the influence of the stepwise protonation of two antihistaminic drugs on their experimental absorption spectra. We demonstrated the capability of the TD-CAM-B3LYP method, combined with a polarizable continuum model, to produce good performance for the calculated spectra. The lowest energy transitions and the molecular orbital plots were analyzed in detail. The calculated UV spectra are proposed as potential alternatives to initialize the well-known MCR-ALS algorithm, especially when the spectra of the pure analytes are not available. Moreover, it can be a useful strategy for planning an experimental methodology oriented to multiway analysis when the drug species exhibit acid-base properties.