Antimicrobial and Antibiofilm Properties of Latvian Honey against Causative Agents of Wound Infections.

Honey is widely used in traditional medicine and modern wound healing biomaterial research as a broad-spectrum antimicrobial, anti-inflammatory and antioxidant agent. The study's objectives were to evaluate the antibacterial activity and polyphenolic profiles of 40 monofloral honey samples collected from beekeepers in the territory of Latvia. The antimicrobial and antifungal activity of Latvian honey samples were compared with commercial Manuka honey and the honey analogue sugar solutions-carbohydrate mixture and tested against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, clinical isolates Extended-Spectrum Beta-Lactamases produced Escherichia coli, Methicillin-resistant Staphylococcus aureus and Candida albicans. Antimicrobial activity was evaluated with the well-diffusion method (80% honey solution w/v) and microdilution method. The honey samples with the highest antimicrobial potential were tested to prevent biofilm development and activity against a preformed biofilm. The principal component analysis of the antimicrobial properties of honey samples vs. polyphenolic profile was performed. Eleven honey samples exhibited antibacterial activity to all investigated bacteria. The antibacterial effect of the samples was most significant on the Gram-positive bacteria compared to the studied Gram-negative bacteria. Latvian honey presents promising potential for use in wound healing biomaterials, opening the possibility of achieving long-term antibacterial effects.

Japanese Quince Seeds as a Promising Rich Source of Proteins and Essential Amino Acids.

The Japanese quince (Chaenomeles japonica) seeds (JQS) are generated in large amounts as a by-product in considerable quantities during industrial quince fruit processing. Due to economic, environmental, and nutritional reasons, the utilization of JQS as a protein source seems to be highly justified. Therefore, three cultivars were studied, 'Rasa', 'Rondo', and 'Darius' of JQS as a protein source during three harvesting years. The total protein content in JQS determined by the Kjeldahl method amounted from 24.6 to 33.2% and was significantly affected (p ≤ 0.05) by the cultivar and harvesting year. The protein isolation was most effective in the pH range of 6-9, and their profile was mainly characterized by a molecular weight of 54.1-57.7 kDa and 70.0-77.6 kDa. The amino acids profile of JQS turned out to be valuable from a nutritional point of view due to the relatively high content of essential and relatively essential amino acids such as histidine (6-9%), leucine (11-12%), lysine (10-16%), phenylalanine (12-16%), glutamine/glutamic acid (7-15%), and tyrosine (9-13%). The initial analysis of JQS indicates that they can be a good source of proteins characterized by a favorable amino acid profile.

Determination of Floral Origin Markers of Latvian Honey by Using IRMS, UHPLC-HRMS, and 1H-NMR.

The economic significance of honey production is crucial; therefore, modern and efficient methods of authentication are needed. During the last decade, various data processing methods and a combination of several instrumental methods have been increasingly used in food analysis. In this study, the chemical composition of monofloral buckwheat (Fagopyrum esculentum), clover (Trifolium repens), heather (Calluna vulgaris), linden (Tilia cordata), rapeseed (Brassica napus), willow (Salix cinerea), and polyfloral honey samples of Latvian origin were investigated using several instrumental analysis methods. The data from light stable isotope ratio mass spectrometry (IRMS), ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS), and nuclear magnetic resonance (NMR) analysis methods were used in combination with multivariate analysis to characterize honey samples originating from Latvia. Results were processed using the principal component analysis (PCA) to study the potential possibilities of evaluating the differences between honey of different floral origins. The results indicate the possibility of strong differentiation of heather and buckwheat honeys, and minor differentiation of linden honey from polyfloral honey types. The main indicators include depleted δ15N values for heather honey protein, elevated concentration levels of rutin for buckwheat honey, and qualitative presence of specific biomarkers within NMR for linden honey.

Rapid determination of pharmaceuticals in wastewater by direct infusion HRMS using target and suspect screening analysis.

A wide-scope screening of active pharmaceutical ingredients (APIs) and their transformation products (TPs) in wastewater can yield valuable insights and pinpoint emerging contaminants that have not been previously reported. Such information is relevant to investigate their occurrence and fate in various environmental compartments. In this study, we explored the applicability of direct infusion high resolution mass spectrometry (DI-HRMS) for comprehensive and rapid detection of APIs and their TPs in wastewater samples. The method was developed using a Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) system and incorporated both wide-scope suspect screening and semi-quantitative determination of selected analytes. The identification strategy was based on the following criteria: narrow accurate mass window (±1.25 ppm) for two most abundant full-MS signals, isotopic pattern fit and additional confirmation on the basis of MS2 spectra at three fragmentation levels. The tentative identification of suspects and target compounds relied on an in-house database containing more than 500 different APIs and TPs. The measured fragment spectra were matched against experimental MS2 patterns obtained from a publicly available spectral library (MassBank of North America) and in-silico generated fragmentation features (from the CFM-ID algorithm). In total, 79 suspects were identified and 24 target compounds were semi-quantified in 72 wastewater samples. The highest detection frequencies in treated wastewater effluents were observed for diclofenac, metoprolol and telmisartan, while hydroxydiclofenac, dextrorphan, and carbamazepine metabolites were the most frequently detected TPs. The obtained API profiles were in accordance with the national consumption statistics and the origin of wastewater samples. The developed method is suitable for rapid screening of APIs in wastewater and can be used as a complementary tool to characterize API emissions from wastewater treatment facilities and to identify problematic compounds that require more rigorous monitoring.

Highly Sensitive Detection of Chemically Modified Thio-Organophosphates by an Enzymatic Biosensing Device: An Automated Robotic Approach.

Pesticides represent some of the most common man-made chemicals in the world. Despite their unquestionable utility in the agricultural field and in the prevention of pest infestation in public areas of cities, pesticides and their biotransformation products are toxic to the environment and hazardous to human health. Esterase-based biosensors represent a viable alternative to the expensive and time-consuming systems currently used for their detection. In this work, we used the esterase-2 from Alicyclobacillus acidocaldarius as bioreceptor for a biosensing device based on an automated robotic approach. Coupling the robotic system with a fluorescence inhibition assay, in only 30 s of enzymatic assay, we accomplished the detection limit of 10 pmol for 11 chemically oxidized thio-organophosphates in solution. In addition, we observed differences in the shape of the inhibition curves determined measuring the decrease of esterase-2 residual activity over time. These differences could be used for the characterization and identification of thio-organophosphate pesticides, leading to a pseudo fingerprinting for each of these compounds. This research represents a starting point to develop technologies for automated screening of toxic compounds in samples from industrial sectors, such as the food industry, and for environmental monitoring.

Non-target and suspected-target screening for potentially hazardous chemicals in food contact materials: investigation of paper straws.

A non-target screening strategy was developed for the safety evaluation of potentially hazardous chemicals in paper food contact materials (FCMs). A tentative list of suspect analytes was generated using publicly available FCM substance inventories, the presence of contaminants in paper straws was confirmed by high-resolution Orbitrap mass spectrometry. Data-independent and data-dependent MS and MS/MS results for candidate compounds were processed using a workflow including peak detection by deconvolution, blank subtraction, retention time alignment, formula assignment and fragmentation spectra search against spectral libraries followed by in silico generated spectra annotation. This workflow allowed for the identification of 74 suspect compounds, of which 40 were assigned a high confidence level of detection. A tentative in silico toxicity evaluation for mutagenic and carcinogenic activities was carried out. Using quantitative structure-activity relationship (QSAR) models it was found that two of the detected compounds tested positive for mutagenicity and three for carcinogenicity.

The Occurrence and Dietary Exposure Assessment of Mycotoxins, Biogenic Amines, and Heavy Metals in Mould-Ripened Blue Cheeses.

The occurrence and dietary exposure assessment of 16 mycotoxins, 6 biogenic amines (BAs), and 13 metallic elements in blue-veined cheeses (n = 46) is reported. Co-occurrence of mycophenolic acid (≤599 µg·kg-1) with roquefortine C (≤5454 µg·kg-1) was observed in 63% of the tested cheeses, while BAs were frequently present at concentrations between 0.2 and 717 mg kg-1. The concentrations of heavy metals in cheeses were very low. Chronic/acute exposure assessment based on consumption data from different European populations indicated that the levels of mycotoxins and heavy metals are safe to consumers, whereas, rather high hazard indexes (HI up to 0.77) were determined for BAs according to the worst-case scenario based on high consumption and 95th percentile occurrence. A more detailed acute dietary intake study indicated that histamine and tyramine were predominant among these BAs, reaching 27 and 41% of the acute oral intake reference doses.

Determination of Picomolar Concentrations of Paraoxon in Human Urine by Fluorescence-Based Enzymatic Assay.

Organophosphate (OP) pesticides are widely used in the agricultural field and in the prevention of pest infestation in private and public areas of cities. Despite their unquestionable utility, several of these compounds demonstrate toxic effects to the environment and human health. In particular, the occurrence of some organophosphate pesticides is correlated to the incidence of nervous system disorders, especially in children. The detection of pesticide residues in the human body represents an important task to preserve human health. In our work we propose the use of esterase-based biosensors as a viable alternative to the expensive and time-consuming systems currently used for their detection in human fluids. Using the esterase-2 activity, coupled with a fluorescence inhibition assay, we are able to detect very low concentration levels of diethyl (4-nitrophenyl) phosphate (paraoxon) in the range of the femtomole (fmol). Method robustness tests indicate the stability of esterase-2 in a diluted solution of 4% human urine, and we are able to accurately determine concentration levels of paraoxon in the range from 0.1 to 2 picomoles (pmol). The system sensitivity for OP detection is calculated at 524 ± 14.15 fmol of paraoxon recognized at 10% of inhibition, with an estimated limit of quantification of 262 ± 8.12 pmol mL-1. These values are comparable with the most recent analysis methods based on mass spectrometry carried out on human samples for pesticide detection. This research represents a starting point to develop cheap and fast testing methods for a rapid screening of toxic substances in human samples.

Development of a Rapid Method for the Determination of Phenolic Antioxidants in Dark Chocolate Using Ultra Performance Liquid Chromatography Coupled to Orbitrap Mass Spectrometry.

Development of a new comprehensive method based on one-step liquid-liquid extraction (LLE) combined with ultra-high-performance liquid chromatography-Orbitrap high resolution mass spectrometry (UHPLC-Orbitrap-HRMS) for the determination of eight phenolic compounds in dark chocolate is demonstrated. The study focuses on the optimization of sample preparation, chromatographic separation and Orbitrap-HRMS detection parameters to adjust selectivity and sensitivity of the method. The LLE mixture of formic acid-water-acetonitrile (2:58:40, v/v) and hexane at the component ratio of 1:1 (v/v) was applied for sample preparation. The inter-day and intra-day precision expressed from the relative standard deviations (RSDs) ranged between 5.50-14.2% and 5.60-16.2%. The quantification limits of the target analytes were between 0.1 and 1.0 mg/kg. The method was applied for the analysis of twenty-six samples of dark chocolate with various cocoa solid contents (30-77%) collected from the markets in Latvia. The total levels of the phenolic contents ranged between 330 and 2631 mg/kg. The study demonstrates advantages of the Orbitrap-HRMS method for the study of phenolic compounds in dark chocolate.

Development and optimization of confirmatory liquid chromatography-Orbitrap mass spectrometry method for the determination of 17 anticoccidials in poultry and eggs.

A new sensitive and selective multi-residue method based on liquid chromatography - Orbitrap high resolution mass spectrometry (LC-Orbitrap-HRMS) was developed and validated for the determination of 17 anticoccidials in poultry and eggs. Instrumental parameters were optimized by the means of statistical experimental designs to improve the sensitivity, precision, and repeatability of the method. Further optimization of auto-tuned MS parameters led to an increase of signal intensity by 10% to 99% for 16 out of 17 analytes. The sample preparation procedure included extraction from muscle tissue and egg samples with acetonitrile, followed by preconcentration, reconstitution, and filtration. Validation was performed according to the Commission Decision 2002/657/EC. The occurrence of anticoccidials in eggs and poultry was assessed by using the developed analytical procedure within the Latvian national monitoring program, revealing quantifiable residues for 6 analytes (marker residue of nicarbazin - 4,4'-dinitrocarbanilide (DNC), salinomycin, narasin, toltrazuril, and its two metabolites).

Development of an automated multienzymatic biosensor for risk assessment of pesticide contamination in water and food.

The goal of this research is to better address the problems related to the widespread presence of pesticides in the environment. Despite the unquestionable utility of the pesticides against various pests in the agricultural field, most pesticides and the corresponding pesticide residues are toxic to the environment and hazardous to human health. The recent literature on organophosphate compounds emphasises a clear correlation between their use and the occurrence of disorders in the nervous system, especially in children. The conventional systems for the detection and analysis of these compounds are expensive, time-consuming and require highly specialised operators; moreover, no online automated screening systems are yet available, that would allow the identification and quantification of the presence of these chemicals in samples from industrial sectors such as the food industry. Esterase-based biosensors represent a viable alternative to this problem. In this fellowship programme, we aim to develop a robust and sensitive methodology that enables the screening of toxic compounds using a streamlined process, using an automated robotic system to achieve a continuous monitoring for risk assessment of pesticides.

Determination of pharmaceutical residues in wastewater using high performance liquid chromatography coupled to quadrupole-Orbitrap mass spectrometry.

A multi-class method for the determination of 24 emerging pharmaceutical residues has been developed and validated. The method is based on solid-phase extraction of wastewater samples using Strata-X cartridges followed by high performance liquid chromatography coupled to hybrid quadrupole - Orbitrap high resolution mass spectrometry (HPLC-Q-Orbitrap-HRMS). A single-laboratory validation procedure showed satisfactory analytical performance. The analysis of 21 samples collected at the wastewater treatment plant in Riga revealed the occurrence of 20 compounds of different therapeutic classes. The highest concentration was found for the central nervous system stimulator caffeine - up to 12µgL-1, the analgesic acetaminophen up to 4.2µgL-1, the antibiotic ciprofloxacin in the concentration range of 250-400ngL-1, and the non-steroidal anti-inflammatory drug (NSAID) ibuprofen at 100-325ngL-1.

Amino acids profile and antioxidant activity of different Lupinus angustifolius seeds after solid state and submerged fermentations.

The objective of this study was to investigate the amino acids profile, total phenolic compounds (TPC) content, antioxidant activity after submerged (SmF) and solid state (SSF) fermentations of different Lupinus angustifolius seeds by the Lactobacillus sakei KTU05-6. Additionally, the impact of different lupin seeds as fermentation media for LAB biomass and d/l-lactic acid production was analysed. The d/l ratio for SmF and SSF treated lupin samples varied from 0.15 to 0.45 and from 0.12 to 0.46, 16 respectively. Nutritional analysis highlighted a substantial increase in the TPC content and antioxidant activity up to 31.5-48.8% for SSF treated L. angustifolius samples compared to unfermented. The interaction between analysed factors (lupin variety and fermentation conditions) had a significant influence on essential and nonessential amino acids profile.