Showing papers by "Paweł Pomastowski published in 2020"

Interactions of Whey Proteins with Metal Ions.

Abstract: Whey proteins tend to interact with metal ions, which have implications in different fields related to human life quality. There are two impacts of such interactions: they can provide opportunities for applications in food and nutraceuticals, but may lead to analytical challenges related to their study and outcomes for food processing, storage, and food interactions. Moreover, interactions of whey proteins with metal ions are complicated, requiring deep understanding, leading to consequences, such as metalloproteins, metallocomplexes, nanoparticles, or aggregates, creating a biologically active system. To understand the phenomena of metal-protein interactions, it is important to develop analytical approaches combined with studies of changes in the biological activity and to analyze the impact of such interactions on different fields. The aim of this review was to discuss chemistry of β-lactoglobulin, α-lactalbumin, and lactotransferrin, their interactions with different metal ions, analytical techniques used to study them and the implications for food and nutraceuticals.

Zinc Oxide Nanocomposites-Extracellular Synthesis, Physicochemical Characterization and Antibacterial Potential

Abstract: This research presents, for the first time, the potential of the Lactobacillus paracasei LC20 isolated from sweet whey as a novel, effective and accessible source for post-cultured ZnO nanocomposites synthesis. The obtained nanocomposites were subjected to comprehensive characterization by a broad spectrum of instrumental techniques. Results of spectroscopic and microscopic analysis confirmed the hexagonal crystalline structure of ZnO in the nanometer size. The dispersion stability of the obtained nanocomposites was determined based on the zeta potential (ZP) measurements-the average ZP value was found to be -29.15 ± 1.05 mV in the 7-9 pH range. The ZnO nanocomposites (NCs) demonstrated thermal stability up to 130 °C based on the results of thermogravimetric TGA/DTG) analysis. The organic deposit on the nanoparticle surface was recorded by spectroscopic analysis in the infrared range (FT-IR). Results of the spectrometric study exhibited nanostructure-assisted laser desorption/ionization effects and also pointed out the presence of organic deposits and, what is more, allowed us to identify the specific amino acids and peptides present on the ZnO NCs surfaces. In this context, mass spectrometry (MS) data confirmed the nano-ZnO formation mechanism. Moreover, fluorescence data showed an increase in fluorescence signal in the presence of nanocomposites designed for potential use as, e.g., biosensors. Despite ZnO NCs' luminescent properties, they can also act as promising antiseptic agents against clinically relevant pathogens. Therefore, a pilot study on the antibacterial activity of biologically synthesized ZnO NCs was carried out against four strains (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa) by using MIC (minimal inhibitory concentration). Additionally, the colony forming units (CFU) assay was performed and quantified for all bacterial cells as the percentage of viable cells in comparison to a control sample (untreated culture) The nanocomposites were effective among three pathogens with MIC values in the range of 86.25-172.5 μg/mL and showed potential as a new type of, e.g., medical path or ointment formulation.

Synthesis and Antibacterial Activity of (AgCl, Ag)NPs/Diatomite Hybrid Composite

Abstract: In the present research, hybrid (AgCl, Ag)NPs/diatomite composites were synthesized by direct impregnation with aqueous silver nitrate solutions. The silver chloride nanoparticles (AgCl-NPs) were formed as an effect of the exchange reaction when silver interacted with the diatomite mineral impurity halite. Nanoparticles of metallic silver (AgNPs) were created by the reduction of silver ions under the influence of hydrogen peroxide. The content of silver chloride nanoparticles in the (AgCl, Ag)NPs/diatomite composite was limited by the content of the halite in the used diatomite. Samples of natural diatomite and synthesized (AgCl, Ag)NPs/diatomite composites were examined by using scanning electron microscopy, transmission electron microscopy, X-ray powder diffraction, infrared spectroscopy and thermogravimetric analysis. Moreover, the antibacterial potential of synthesized composites was also studied using the MIC (minimal inhibitory concentration) method against the most common drug-resistant microorganisms in the medical field: Gram-positive Staphylococcus aureus and Gram-negative Klebsiella pneumoniae. The obtained hybrid (AgCl, AgNPs)/diatomite composites were shown to have antimicrobial potential. However, widespread use requires further study by using various microorganisms and additional cytotoxic studies on eukaryotic systems, e.g., cell lines and animal models.

Mechanistic Chromatographic Column Characterization for the Analysis of Flavonoids Using Quantitative Structure-Retention Relationships Based on Density Functional Theory.

Abstract: This work aimed to unravel the retention mechanisms of 30 structurally different flavonoids separated on three chromatographic columns: conventional Kinetex C18 (K-C18), Kinetex F5 (K-F5), and IAM.PC.DD2. Interactions between analytes and chromatographic phases governing the retention were analyzed and mechanistically interpreted via quantum chemical descriptors as compared to the typical 'black box' approach. Statistically significant consensus genetic algorithm-partial least squares (GA-PLS) quantitative structure retention relationship (QSRR) models were built and comprehensively validated. Results showed that for the K-C18 column, hydrophobicity and solvent effects were dominating, whereas electrostatic interactions were less pronounced. Similarly, for the K-F5 column, hydrophobicity, dispersion effects, and electrostatic interactions were found to be governing the retention of flavonoids. Conversely, besides hydrophobic forces and dispersion effects, electrostatic interactions were found to be dominating the IAM.PC.DD2 retention mechanism. As such, the developed approach has a great potential for gaining insights into biological activity upon analysis of interactions between analytes and stationary phases imitating molecular targets, giving rise to an exceptional alternative to existing methods lacking exhaustive interpretations.

Study on Molecular Profiles of Staphylococcus aureus Strains: Spectrometric Approach.

Abstract: Staphylococcus aureus remains a major health problem responsible for many epidemic outbreaks. Therefore, the development of efficient and rapid methods for studying molecular profiles of S. aureus strains for its further typing is in high demand. Among many techniques, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) represents a timely, cost-effective, and reliable strain typing approach, which is still rarely used due to insufficient knowledge about the impact of sample preparation and analysis conditions on the molecular profiles and strain classification efficiency of S. aureus. The aim of this study was to evaluate the effect of the culture conditions and matrix type on the differentiation of molecular profiles of various S. aureus strains via the MALDI TOF MS analysis and different computational methods. The analysis revealed that by changing the culture conditions, matrix type, as well as a statistical method, the differentiation of S. aureus strains can be significantly improved. Therefore, to accelerate the incorporation of the MALDI-based strain typing in routine laboratories, further studies on the standardization and searching of optimal conditions on a larger number of isolates and bacterial species are of great need.

Isolation and Self-Association Studies of Beta-Lactoglobulin.

Abstract: The aim of this study was to investigate isolated β-lactoglobulin (β-LG) from the whey protein isolate (WPI) solution using the column chromatography with SP Sephadex. The physicochemical characterization (self-association, the pH stability in various salt solutions, the identification of oligomeric forms) of the protein obtained have been carried out. The electrophoretically pure β-LG fraction was obtained at pH 4.8. The fraction was characterized by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF MS) technique. The use of the HCCA matrix indicated the presence of oligomeric β-LG forms, while the SA and DHB matrices enabled the differentiation of A and B isoforms in the sample. The impact of sodium chloride, potassium chloride, ammonium sulfate, and sodium citrate in dispersion medium on β-LG electrophoretic stability in solution was also studied. Type of the dispersion medium led to the changes in the isoelectric point of protein. Sodium citrate stabilizes protein in comparison to ammonium sulfate. Additionally, the potential of capillary electrophoresis (CE) with UV detection using bare fused capillary to monitor β-LG oligomerization was discussed. Obtained CE data were further compared by the asymmetric flow field flow fractionation coupled with the multi-angle light scattering detector (AF4-MALS). It was shown that the β-LG is a monomer at pH 3.0, dimer at pH 7.0. At pH 5.0 (near the isoelectric point), oligomers with structures from dimeric to octameric are formed. However, the appearance of the oligomers equilibrium is dependent on the concentration of protein. The higher quantity of protein leads to the formation of the octamer. The far UV circular dichroism (CD) spectra carried out at pH 3.0, 5.0, and 7.0 confirmed that β-sheet conformation is dominant at pH 3.0, 5.0, while at pH 7.0, this conformation is approximately in the same quantity as α-helix and random structures.

Multi-instrumental approach to unravel molecular mechanisms of natural bioactive compounds: Case studies for flavonoids

Abstract: Flavonoids are natural compounds defined as plant secondary metabolites with various beneficial properties for human health that make them perspective drug candidates. However, research in this area should be deepened to collect reasonable amount of data to suggest biochemical mechanism of action that can be a key for evaluation of drug efficacy, side effects. In addition, from analytical point of view, analyte–receptor interaction is an interesting object for study as it includes challenges in sample preparation and analytical measurements. Thus, there is a need in modern instrumental analytical techniques for study of drug–receptor interactions. Combination of instrumental techniques represents multi-instrumental approach and provides an extended set of data to suggest mechanism and to evaluate changes in initial biological activity caused by interaction. Current review is focused on advances and limitations of multi-instrumental approach and analytical techniques for study of flavonoid–receptor interactions and insights into molecular mechanisms of flavonoids.

The Study of Zinc Ions Binding to αS1-, β- and κ-Casein.

Abstract: The presented studies focused on the specificity binding of particular casein fractions: αS1-, β- and κ-casein (αS1CN, βCN, κCN), with zinc ions. The binding mechanism was determined by kinetic modeling using results of batch sorption. For this goal, models of zero-order kinetics, pseudo-first-order, pseudo-second-order and Weber–Morris intraparticle diffusion were used. The formation of Zn-αS1CN, Zn-βCN and Zn-κCN complexes was additionally monitored using spectroscopic methods such as Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopy, characterizing active functional groups involved in the binding process. Additionally, a mass spectrometry technique—matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)—was used to characterize respective protein fractions and obtained complexes. Spectroscopic and spectrometric studies were carried out both before and after binding the protein with zinc ions. The obtained results showed the difference in Zn-αS1CN, Zn-βCN and Zn-κCN complexes created at separate kinetic stages. On the basis of instrumental studies, a significant influence of acidic (glutamic acid (Glu), aspartic acid (Asp)) and aromatic (tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr)) amino acids on the formation of metal complexes was proven. In turn, spectrometric studies allowed determining the molecular masses of casein isoforms before and after binding to zinc ions.

The Influence of Different Forms of Silver on Selected Pathogenic Bacteria

Abstract: The application of silver nanoparticles as an antibacterial agent is becoming more common. Unfortunately, their effect on microorganisms is still not fully understood. Therefore, this paper attempts to investigate the influence of silver ions, biologically synthesized silver nanoparticles and nanoparticles functionalized with antibiotics on molecular bacteria profiles. The initial stage of research was aimed at the mechanism determination involved in antibiotics sorption onto nanoparticles’ surface. For this purpose, the kinetics study was performed. Next, the functionalized formulations were characterized by Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and a zeta potential study. The results reveal that functionalization is a complex process, but does not significantly affect the stability of biocolloids. Furthermore, the antimicrobial assays, in most cases, have shown no increases in antibacterial activity after nanoparticle functionalization, which suggests that the functionalization process does not always generate the improved antimicrobial effect. Finally, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique was employed to characterize the changes in the molecular profile of bacteria treated with various antibacterial agents. The recorded spectra proved many differences in bacterial lipids and proteins profiles compared to untreated cells. In addition, the statistical analysis of recorded spectra revealed the strain-dependent nature of stress factors on the molecular profile of microorganisms.

Problems with identifying and distinguishing salivary streptococci: a multi-instrumental approach.

Abstract: Aim: The purpose of this study was to create an alternative protocol for the DNA-based identification of salivary microbiota focused on the distinguishing of Streptococcus species. Materials & methods: Salivary bacteria were identified using 16S rDNA sequencing and proteins and lipids profiling using MALDI-TOF/MS as well as FTIR analysis. Results: Most of the isolates belonged to streptococci - mostly the salivarious group indistinguishable by the molecular technique. In turn, MALDI analysis allowed for their fast and reliable classification. Although FTIR spectroscopy demonstrated the correct species classification, the spectra interpretation was time consuming and complicated. Conclusion: MALDI-TOF/MS demonstrated the biggest effectiveness in the identification and discrimination between the salivary streptococci, which could be easily incorporated in the workflow of routine microbiological laboratories.

New Methodology for the Identification of Metabolites of Saccharides and Cyclitols by Off-Line EC-MALDI-TOF-MS

Abstract: A combination of electrochemistry (EC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (off-line EC-MALDI-TOF-MS) was applied for determination of the studied biologically active compounds (D-glucose, D-fructose, D-galactose, D-pinitol, L-chiro-inositol, and myo-inositol) and their possible electrochemical metabolites. In this work, boron-doped diamond electrode (BDD) was used as a working electrode. MALDI-TOF-MS experiments were carried out (both in positive and negative ion modes and using two matrices) to identify the structures of electrochemical products. This was one of the first applications of the EC system for the generation of electrochemical products produced from saccharides and cyclitols. Moreover, exploratory data analysis approaches (correlation networks, hierarchical cluster analysis, weighted plots) were used in order to present differences/similarities between the obtained spectra, regarding the class of analyzed compounds, ionization modes, and used matrices. This work presents the investigation and comparison of fragmentation patterns of sugars, cyclitols, and their respective products generated through the electrochemistry (EC) process.

Silver Nanoparticles: Synthesis, Characteristics, and Application

Abstract: Silver nanoparticles (AgNPs), as a material with attractive properties, has received interest worldwide by the implementation of synthesis particles at nano-size. AgNPs has triggered intense research on the production of nanoparticles using different synthesis methods due actual and future application. The increasing attention of AgNPs resulted in the growth and development of nanoparticles synthesis methods. Recently, various approaches, including chemical and biological, have been used for them preparation. In order to obtain information about size, shape, or optical properties of nanoparticles, a broad range of analytical methods—microscopy, spectroscopy, and spectrometry techniques—have been used. AgNPs exhibit attractive antimicrobial properties against bacteria (Gram-positive and Gram-negative), fungi, and show also selective toxicity toward normal and cancerous cells. This chapter reviewed the methods of AgNPs synthesis, their characteristics, antimicrobial activity, and cytotoxicity against normal and tumor cells.