Commercial wine yeast nitrogen requirement influences the production of secondary metabolites (aroma, hydroxytyrosol, melatonin and other bioactives) during alcoholic fermentation.

During alcoholic fermentation, Saccharomyces cerevisiae synthesizes different compounds, which are crucial for product quality: volatile compounds with sensory impact, and bioactive compounds such as melatonin (MEL) and hydroxytyrosol (HT), linked to health benefits. As many of these compounds are related with yeast's nitrogen metabolism, their production have been studied in four different commercial strains with different nitrogen requirement (Red Fruit, Uvaferm VRB, Lalvin Rhone 2323 and Lalvin QA23) being, Uvaferm UVR the higher nitrogen demander strain. All strains produced the secondary metabolites, notably Uvaferm UVR produced the highest HT concentration, despite its low growth. Uvaferm UVR emerged also as a significant producer of MEL, indicating a potential role in fermentation related stress. Moreover, Uvaferm UVR shows the highest total concentrations of volatile compounds. Multivariate analysis revealed distinct clustering based on nitrogen requirements of the strains, highlighting the strain-dependent metabolic responses.

A consortium of different Saccharomyces species enhances the content of bioactive tryptophan-derived compounds in wine fermentations.

In recent years, the presence of molecules derived from aromatic amino acids in wines has been increasingly demonstrated to have a significant influence on wine quality and stability. In addition, interactions between different yeast species have been observed to influence these final properties. In this study, a screening of 81 yeast strains from different environments was carried out to establish a consortium that would promote the improvement of indolic compound levels in wine. Two strains, Saccharomyces uvarum and Saccharomyces eubayanus, with robust fermentative capacity were selected to be combined with a Saccharomyces cerevisiae strain with a predisposition towards the production of indolic compounds. Fermentation dynamics were studied in pure cultures, co-inoculations and sequential inoculations, analysing strain interactions and end-of-fermentation characteristics. Fermentations showing significant interactions were further analyzed for the resulting indolic compounds and aroma profile, with the aim of observing potential interactions and synergies resulting from the combination of different strains in the final wine. Sequential inoculation of S. cerevisiae after S. uvarum or S. eubayanus was observed to increase indolic compound levels, particularly serotonin and 3-indoleacetic acid. This study is the first to demonstrate how the formation of microbial consortia can serve as a useful strategy to enhance compounds with interesting properties in wine, paving the way for future studies and combinations.

Bioaccumulation and biochemical responses in the peppery furrow shell Scrobicularia plana exposed to a pharmaceutical cocktail at sub-lethal concentrations.

Pharmaceutical drugs in the aquatic medium may pose significant risk to non-target organisms. In this study, the potential toxicity of a mixture of three compounds commonly detected in marine waters (ibuprofen, ciprofloxacin and flumequine) was assessed, by studying bioaccumulation, oxidative stress and neurotoxicity parameters (catalase CAT, superoxide dismutase SOD, glutathione reductase GR, glutathione S-transferase GST, lipid peroxidation LPO, glutathione peroxidase GPX, metallothionein MT and acetylcholinesterase AChE) in the clam Scrobicularia plana. Temporal evolution of selected endpoints was evaluated throughout an exposure period (1, 7 and 21 days) followed by a depuration phase. The accumulation of all drugs was fast, however clams showed the ability to control the internal content of drugs, keeping their concentration constant throughout the exposure and reducing their content after 7 days of depuration. The induction of biochemical alterations (SOD, CAT, LPO, MT, AChE) was observed in gills and digestive gland probably related to an imbalance in the redox state of clams as a consequence of the exposure to the drug mixture. These alterations were also maintained at the end of the depuration week when the high levels of SOD, CAT, GST and LPO indicated the persistence of oxidative stress and damage to lipids despite the fact that clams were no longer exposed to the mixture.

Monitoring of pharmaceuticals in aquatic biota (Procambarus clarkii) of the Doñana National Park (Spain).

In this work the presence of different pharmaceuticals at Doñana National Park (Spain) and their main entry sources (input source or entry points) have been stated over the 2011-2016 years period. Twenty-three selected pharmaceuticals (corresponding to eight therapeutic families) were evaluated in crayfish and water samples from Doñana National Park (Spain) (six sampling points selected in order to cover different possible pollution sources into and surrounding the Park). The multiresidue determination was carried out using enzymatic-microwave assisted extraction prior to high performance liquid chromatography mass spectrometry detection. Sulphonamides (sulfadiazine, sulfamerazine, sulfamethazine, and sulfamethoxazole); trimethoprim, an antibiotic that is frequently co-administered with sulfamethoxazole; amphenicols (chloramphenicol, florfenicol and thiamphenicol); fluoroquinolones (ciprofloxacin, enrofloxacin, flumequine, danofloxacin, gatifloxacin, norfloxacin, marbofloxacin and grepafloxacin); penicillins (amoxicillin); tetracyclines (chlortetracycline and oxytetracycline); non-steroidal anti-inflammatory drugs (salicylic acid and ibuprofen); beta-blocker drugs (atenolol); and antiepileptics (carbamazepine) were analysed. Ciprofloxacin, ibuprofen, salicylic acid, flumequine, and carbamazepine were detected and/or quantified at some of the selected sampling points. A clear ecotoxicological risk to the ecosystem was demonstrated from the occurrence of ciprofloxacin in samples obtained after the punctual and massive presence of people inside the Park. Furthermore, flumequine and carbamazepine have been detected in Procambarus clarkii specimens in concentrations around 30 ng g-1 and 14 ng g-1, respectively, and their occurrence in the specimens could indicate the persistence of the discharge sources. The main source of pharmaceuticals into the Park might be the livestock farming activities, and the influence of urban wastewaters from surrounding villages does not seem to be very important.

Assessment of pharmaceutical mixture (ibuprofen, ciprofloxacin and flumequine) effects to the crayfish Procambarus clarkii: A multilevel analysis (biochemical, transcriptional and proteomic approaches).

The knowledge about the effects of pharmaceuticals on aquatic organisms has been increasing in the last decade. However, due to the variety of compounds presents in the aquatic medium, exposure scenarios and exposed organisms, there are still many gaps in the knowledge on how mixtures of such bioactive compounds affect exposed non target organisms. The crayfish Procambarus clarkii was used to analyze the toxicity effects of mixtures of ciprofloxacin, flumequine and ibuprofen at low and high concentrations (10 and 100 µg/L) over 21 days of exposure and to assess the recovery capacity of the organism after a depuration phase following exposure during additional 7 days in clean water. The crayfish accumulated the three compounds throughout the entire exposure in the hepatopancreas. The exposure to the mixture altered the abundance of proteins associated with different cells functions such as biotransformation and detoxification processes (i.e. catalase and glutathione transferase), carbohydrate metabolism and immune responses. Additionally changes in expression of genes encoding antioxidant enzymes and in activity of the corresponding enzymes (i.e. superoxide dismutase, glutathione peroxidase and glutathione transferase) were reported. Alterations at different levels of biological organization did not run in parallel under all circumstances and can be related to changes in the redox status of the target tissue. No differences were observed between control and exposed organisms for most of selected endpoints after a week of depuration, indicating that exposure to the drug mixture did not produce permanent damage in the hepatopancreas of P. clarkii.

Simultaneous determination of metformin and glimepiride in human serum by ultra high performance liquid chromatography quadrupole time of flight mass spectrometry detection.

This work proposes a simple method for the simultaneous determination of two antidiabetic compounds, metformin and glimepiride, by ultra-high performance liquid chromatography using quadrupole time of flight mass spectrometry detection with electrospray ionization. The method was validated and shown to be linear, selective, accurate and precise. The chromatographic separation was performed using a BEH C18 column (50 mm x 2.1 mm, 1.7 µm particle size). The mobile phase was composed by 0.05% (v/v) aqueous formic acid solution and acetonitrile using a gradient elution program, at a flow rate of 0.3 mL/min. Linearity range for metformin was 1.7-100 µg·L-1, using propranolol as internal standard and 3.3-100 µg·L-1 for glimepiride using glibenclamide as internal standard. Limits of detection and lower limits of quantitation were 0.4 µg·L-1 and 1.7 µg·L-1 for metformin and 0.7 and 3.3 µg·L-1 for glimepiride, respectively. The method was used for the simultaneous determination of these compounds in human serum samples with lower limits of detection and quantitation than serum levels reported in previous works that allows its applicability in therapy drug monitoring.

Multiresidue determination of 21 pharmaceuticals in crayfish (Procambarus clarkii) using enzymatic microwave-assisted liquid extraction and ultrahigh-performance liquid chromatography-triple quadrupole mass spectrometry analysis.

In this paper, a multiresidue enzymatic-microwave assisted extraction prior to ultrahigh performance liquid chromatography and triple quadrupole mass spectrometry analysis has been developed for the determination of 21 pharmaceuticals in crayfish (Procambarus Clarkii) samples. The analysed compounds corresponding to 6 therapeutic families were: fluoroquinolones (ciprofloxacin, danofloxacin, enrofloxacin, flumequine, gatifloxacin, grepafloxacin, marbofloxacin and norfloxacin); tetracyclines (chlortetracycline and oxytetracycline); sulphonamides (sulfamethoxazole, sulfadiazine, sulfamethazine, sulfamerazine); penicillins (amoxicillin); anfenicols (chloramphenicol, thiamphenicol and florfenicol); non-steroidal anti-inflammatory drugs (ibuprofen and salicylic acid) and trimethoprim an antibiotic that is frequently co-administered with sulfamethoxazole. The main factors affecting the extraction efficiency were optimized for 0.5 g of lyophilized tissue. The enzymatic microwave extraction was carried out using an extraction time of 5 min with 5 mL of an acetonitrile: water (1:1, v/v) mixture, 50 µL of Proteinase-K solution and 5 µL of formic acid at 50 W. After centrifugation, the liquid extract was evaporated and the residue was reconstituted with 1 mL of 0.1% (v/v) formic acid. Chromatographic and MS parameters, in both positive and negative ionization modes, were also optimized. The mobile phase used consisted on a mixture of 0.1% (v/v) formic acid aqueous solution and acetonitrile in gradient elution mode at a 0.4 mL min-1 flow rate. The proposed method was validated and recoveries over 70% were obtained for all the analytes with detection limits in the 0.6-12 ng g-1 range. The proposed method was successfully applied to crayfish specimens from Doñana National Park, Spain.