Person: Moreno Martín, Gustavo
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First Name
Gustavo
Last Name
Moreno Martín
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Químicas
Department
Química Analítica
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Item Biotransformation of selenium by lactic acid bacteria: formation of seleno-nanoparticles and seleno-amino acids(Frontiers in Bioengineering and Biotechnology, 2020) Martínez, Fernando Gabriel; Moreno Martín, Gustavo; Pescuma, Micaela; Madrid Albarrán, María Yolanda; Mozzi, FernandaSelenium (Se) is an essential micronutrient for the majority of living organisms, and it has been identified as selenocysteine in the active site of several selenoproteins such as glutathione peroxidase, thioredoxin reductase, and deiodinases. Se deficiency in humans is associated with viral infections, thyroid dysfunction, different types of cancer, and aging. In several European countries as well as in Argentina, Se intake is below the recommended dietary Intake (RDI). Some lactic acid bacteria (LAB) can accumulate and bio-transform selenite (toxic) into Se-nanoparticles (SeNPs) and Se-amino acids (non-toxic). The microbial growth, Se metabolite distribution, and the glutathione reductase (involved in selenite reduction) activity of Se-enriched LAB were studied in this work. The ninety-six assayed strains, belonging to the genera Lactococcus, Weissella, Leuconostoc, Lactobacillus, Enterococcus, and Fructobacillus could grow in the presence of 5 ppm sodium selenite. From the total, eight strains could remove more than 80% of the added Se from the culture medium. These bacteria accumulated intracellularly between 1.2 and 2.5 ppm of the added Se, from which F. tropaeoli CRL 2034 contained the highest intracellular amount. These strains produced only the seleno-amino acid SeCys as observed by LC-ICP-MS and confirmed by LC-ESI-MS/MS. The intracellular SeCys concentrations were between 0.015 and 0.880 ppm; Lb. brevis CRL 2051 (0.873 ppm), Lb. plantarum CRL 2030 (0.867 ppm), and F. tropaeoli CRL 2034 (0.625 ppm) were the strains that showed the highest concentrations. Glutathione reductase activity values were higher when the strains were grown in the presence of Se except for the F. tropaeoli CRL 2034 strain, which showed an opposite behavior. The cellular morphology of the strains was not affected by the presence of Se in the culture medium; interestingly, all the strains were able to form spherical SeNPs as determined by transmission electron microscopy (TEM). Only two Enterococcus strains produced the volatile Se compounds dimethyl-diselenide identified by GC-MS. Our results show that Lb. brevis CRL 2051, Lb. plantarum CRL 2030, and F. tropaeoli CRL 2034 could be used for the development of nutraceuticals or as starter cultures for the bio-enrichment of fermented fruit beverages with SeCys and SeNPs.Item Insights into the accumulation and transformation of Ch-SeNPs by Raphanus sativus and Brassica juncea: effect on essential elements uptake(Science of the Total Environment, 2020) Moreno Martín, Gustavo; Sanz Landaluce, Jon; León González, María Eugenia De; Madrid Albarrán, María Yolanda; ElsevierSelenium (Se) at very low doses has important functions for humans. Unfortunately, the low levels of Se in soils in various regions of the world have implemented the agronomic biofortification of crops by applying Se-enriched fertilizers (mainly based on selenate). Lately, the use of nanofertilizers is growing in interest as their low size reduces the amount of chemicals and minimizes nutrient losses in comparison with conventional bulk fertilizers. However, the knowledge on their fate and environmental impact is still scarce. This study aims to evaluate the biotransformation of chitosan-modified Se nanoparticles (Ch-SeNPs) as well as their effect on the metabolism of essential metals (Fe, Cu, Zn and Mo) when applied to hydroponic cultivation of R. sativus and B. juncea. In house-synthesized Ch-SeNPs were characterized in both synthesis and hydroponic culture media by transmission electron microscopy (TEM), dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The composition of one-tenth strength Hoagland's solution did not affect the size, shape and concentration in number of particles per mL of Ch-SeNPs. The plants were grown inside a box at 25 °C during the months of May–July in 2018. After a week of treatment with Ch-SeNPs, plants were harvested and divided into roots and aerial part. The biotransformation of Ch-SeNPs was evaluated through a process of enzymatic hydrolysis and subsequent analysis by HPLC-ICP-MS and HPLC-ESI-MS/MS. The results confirmed the transformation of Ch-SeNPs to seleno-amino acids: Selenomethionine (SeMet), Semethylselenocysteine (SeMetSeCys) and ɣ-glutamyl-Se-MetSeCys. Moreover, Multiple-way analysis of variance (ANOVA) and principal component analysis (PCA) showed that, regardless the plant species, Ch-SeNPs supplementation affected the absorption of Zn.Item Characterization of AgNPs and AuNPs in sewage sludge by single particle inductively coupled plasma-mass spectrometry(Talanta, 2022) Moreno Martín, Gustavo; Gómez Gómez, Beatriz; León González, María Eugenia De; Madrid Albarrán, María Yolanda; ElsevierThis study develops for the first time an analytical method for the characterization of silver and gold nanoparticles in sewage sludge. The evaluation of the effect of temperature, extracting agent and centrifugation speed and time on the extraction yield was carried out through a multifactorial analysis of variance which allows us to select 289 g, 5 min and 20 mM sodium pyrophosphate tetrabasic as optimal extraction conditions. Under these conditions, the analysis of the extract by single particle inductively coupled plasma-mass spectrometry provided recovery percentages of 70 ± 2% and 56 ± 1% for silver and gold nanoparticles, respectively. Moreover, the complementary results obtained upon analysis of these extracts by transmission electron microscopy and single particle inductively coupled plasma-mass spectrometry showed that the developed method did not modify the original size and shape of these nanoparticles during the extraction procedure. Size detection limits of 23 nm and 16 nm as well as number concentration limits of 3.12 × 109 particles kg−1 and 1.38 × 109 particles kg−1 were obtained for silver and gold nanoparticles, respectively. Moreover, a stability study of silver and gold nanoparticles in sewage sludge for 12 months showed differences between the two nanoparticle types. Although the sizes were not affected during the 12 months, silver nanoparticles underwent an oxidation process from 6 months onwards, which was reflected in an increase in the percentage of ionic silver from 14 ± 1% at 6 months to 24 ± 2% at 12 months. The developed methodology represents a simple, reliable and fast tool for detecting, quantifying and assessing the stability of nanoparticles in an important environmental sample such as sewage sludge.Item Simultaneous determination of the size and concentration of AgNPs in water samples by UV–vis spectrophotometry and chemometrics tools(Talanta, 2018) Moreno Martín, Gustavo; León González, María Eugenia De; Madrid Albarrán, María YolandaThe combination of UV–vis spectrophotometry with a chemometric calibration tool based on partial least squares (PLS) has allowed us the development of a multivariate analytical method that simultaneously estimates the concentration and size of mixtures of silver nanoparticles (AgNPs) in environmental water samples. The method is based on changes in the surface plasmon resonance band (SPRB) of AgNPs when they form aggregated/assembled structures with L-cysteine (L-cys). Measurementts were performed by employed a fixed-time kinetics method that implies that the final spectra (response) are obtained by subtstracting the solutions spectra at fixed times. Optimization of experimental conditions affecting aggregation such as time, temperature, pH and concentration of aggregating substance was performed by experimental design and response surface methodologies (RSM). A multivariate calibration model using AgNPs of known diameter size ((20 ± 3), (41 ± 3), (59 ± 5) and (79 ± 7) nm) within a concentration range between 0.62 and 2.5 mg L−1 was constructed by using a mixture experimental design and PLS. The method was finally applied to estimate size and concentration of AgNPs in AgNPs-spiked river and tap water samples. Water samples were spiked with individual, binary and ternary mixtures of AgNPs of different sizes and by using two types of AgNPs: citrate-coated AgNPs (cit-AgNPs) and polyvinylpyrrolidone-coated AgNPs (PVP-AgNPs). A good correspondence was obtained between predicted values and the total amount of AgNPs added with recovery values ranged within 80–160% for the individual mixtures, 68–108% for the binary mixtures and 60–64% for the ternary mixtures of AgNPs. Finally, transmission electron microscopy (TEM) measurements were performed for those cases where discrepancies between the expected and the obtained values were observed. TEM micrographs evidenced the presence of agglomerates or aggregates of AgNPs in some of the mixtures or water tested.Item Determination of size and mass-and number-based concentration of biogenic SeNPs synthesized by lactic acid bacteria by using a multimethod approach(Analytica Chimica Acta, 2017) Moreno Martín, Gustavo; Pescuma, Micaela; Pérez Corona, María Teresa; Mozzi, Fernanda; Madrid Albarrán, María YolandaSelenium nanoparticles (SeNPs) were synthesized by a green technology using lactic acid bacteria (LAB, Lactobacillus acidophilus, L. delbrueckii subsp. bulgaricus and L. reuteri). The exposure of aqueous sodium selenite to LAB led to the synthesis of SeNPs. Characterization of SeNPs by transmission electron microscopy with energy dispersive X-ray spectrum (EDXS) analysis revealed the presence of stable, predominantly monodispersed and spherical SeNPs of an average size of 146 ± 71 nm. Additionally, SeNPs hydrodynamic size was determined by dispersive light scattering (DLS) and nanoparticle tracking analysis (NTA). For this purpose, a methodology based on the use of surfactants in basic medium was developed for isolating SeNPs from the bacterial pellet. The hydrodynamic size values provided by DLS and NTA were 258 ± 4 and 187 ± 56 nm, respectively. NTA measurements of number-based concentration reported values of (4.67±0:30)x 109 SeNPs/mL with a relative standard deviation lower than 5% (n = 3). The quantitative results obtained by NTA were supported by theoretical calculations. Asymmetrical flow field flow fractionation (AF4) on line coupled to the inductively couple plasma mass spectrometry (ICP-MS) and off-line coupled to DLS was further employed to characterize biogenic SeNPs. The distribution of the particle size for the Se-containing peak provide an average size of (247 ± 14) nm. The data obtained by independent techniques were in good agreement and the developed methodologyItem Selenium stress response of the fruit origin strain Fructobacillus tropaeoli CRL 2034(Applied Microbiology and Biotechnology, 2023) Martínez, Fernando Gabriel; Moreno Martín, Gustavo; Mozzi, Fernanda; Madrid Albarrán, María Yolanda; Pescuma, MicaelaThe fruit-origin strain Fructobacillus tropaeoli CRL 2034 can biotransform selenium into seleno-nanoparticles and selenocysteine. The proteomic analysis of F. tropaeoli CRL 2034 exposed to 5 and 100 ppm of Se showed a dose-dependent response since 19 and 77 proteins were deregulated, respectively. In the presence of 5 ppm of Se, the deregulated proteins mainly belonged to the categories of energy production and conversion or had unknown functions, while when cells were grown with 100 ppm of Se, most of the proteins were grouped into amino acid transport and metabolism, nucleotide transport and metabolism, or into unknown functions. However, under both Se conditions, glutathione reductases were overexpressed (1.8–3.1-fold), while mannitol 2-dehydrogenase was downregulated (0.54–0.19-fold), both enzymes related to oxidative stress functions. Mannitol 2-dehydrogenase was the only enzyme found that contained SeCys, and its activity was 1.27-fold increased after 5 ppm of Se exposure. Our results suggest that F. tropaeoli CRL 2034 counteracts Se stress by overexpressing proteins related to oxidative stress resistance and changing the membrane hydrophobicity, which may improve its survival under (food) storage and positively influence its adhesion to intestinal cells. Selenized cells of F. tropaeoli CRL 2034 could be used for producing Se-enriched fermented foods. Graphical Abstract: [Figure not available: see fulltext.] Key points: • Selenized cells of F. tropaeoli showed enhanced resistance to oxidative stress. • SeCys was found in the Fructobacillus mannitol 2-dehydrogenase polypeptide chain. • F. tropaeoli mannitol 2-dehydrogenase activity was highest when exposed to selenium.Item Simultaneous determination of the size and concentration of AgNPs in water samples by UV–vis spectrophotometry and chemometrics tools(Talanta, 2018) Moreno Martín, Gustavo; León González, María Eugenia De; Madrid Albarrán, María YolandaThe combination of UV–vis spectrophotometry with a chemometric calibration tool based on partial least squares (PLS) has allowed us the development of a multivariate analytical method that simultaneously estimates the concentration and size of mixtures of silver nanoparticles (AgNPs) in environmental water samples. The method is based on changes in the surface plasmon resonance band (SPRB) of AgNPs when they form aggregated/assembled structures with L-cysteine (L-cys). Measurementts were performed by employed a fixed-time kinetics method that implies that the final spectra (response) are obtained by subtstracting the solutions spectra at fixed times. Optimization of experimental conditions affecting aggregation such as time, temperature, pH and concentration of aggregating substance was performed by experimental design and response surface methodologies (RSM). A multivariate calibration model using AgNPs of known diameter size ((20 ± 3), (41 ± 3), (59 ± 5) and (79 ± 7) nm) within a concentration range between 0.62 and 2.5 mg L−1 was constructed by using a mixture experimental design and PLS. The method was finally applied to estimate size and concentration of AgNPs in AgNPs-spiked river and tap water samples. Water samples were spiked with individual, binary and ternary mixtures of AgNPs of different sizes and by using two types of AgNPs: citrate-coated AgNPs (cit-AgNPs) and polyvinylpyrrolidone-coated AgNPs (PVP-AgNPs). A good correspondence was obtained between predicted values and the total amount of AgNPs added with recovery values ranged within 80–160% for the individual mixtures, 68–108% for the binary mixtures and 60–64% for the ternary mixtures of AgNPs. Finally, transmission electron microscopy (TEM) measurements were performed for those cases where discrepancies between the expected and the obtained values were observed. TEM micrographs evidenced the presence of agglomerates or aggregates of AgNPs in some of the mixtures or water tested.Item Evaluation of the transformation of selenite and selenium nanoparticles to seleno-amino acids produced by Escherichia coli and Staphylococcus aureus by using liquid chromatography -inductively coupled plasma mass spectrometry and single-particle- inductively coupled plasma mass spectrometry and different sample treatments(Spectrochimica Acta Part B: Atomic Spectroscopy, 2023) Moreno Martín, Gustavo; Espada-Bernabé, Elena; Gómez Gómez, Beatriz; León González, María Eugenia De; Madrid Albarrán, María YolandaDue to the scarce knowledge about the impact of selenium nanoparticles (SeNPs) on bacterial populations, the main objective of this work was focused on evaluating the transformations of SeNPs and selenite in Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). For this purpose, an analytical methodology based on bacteria cell wall disruption, carbamidomethylation, enzymatic hydrolysis and high-performance liquid chromatography couple to inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and HPLC- electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) measurements was developed, the latter for unambiguous identification of selenium species. Once bacteria were cultured in the presence of chitosan modified SeNPs (Ch-SeNPs) and selenite at 0, 1 and 2 mg L 1 Se for 24 h, an enzymatic disruption of the bacterial cell wall using lysozym followed by enzymatic hydrolysis with protease was applied. The use of lysozyme to extract selenium speciesprovided a better efficiency in the total selenium content (higher than 96%), compared to a mechanical disruption of the bacterial cell wall. Analysis of the extracts by anionic exchange HPLC-ICP-MS showed a strong influence of incubation time with protease (24, 48 and 72 h) on selenium chromatographic profile. The results showed that selenocysteine (SeCys) was the only Se species identified in both bacteria representing an 80% of total selenium accumulated. The confirmation of the identity of this Se species was carried out after performing a carbamidomethylation process, prior to enzymatic hydrolysis, and analyzing the extract by reversed phase HPLCESI- MS/MS. These analyses confirmed the presence of SeCys, and no relevant differences were found between the metabolic pathway of both forms of selenium. Moreover, the growth of both bacterial species in the presence of selenite resulted in the formation of biogenic SeNPs. Characterization by TEM before and after their separation from the culture medium showed spherical and monodispersed nanoparticles with an average size (155 ± 19) nm and (172 ± 20) nm for E. coli and S. aureus, respectively. Analysis by spICP-MS showed no significant differences in size with respect to TEM after considering the ionization efficiency of Se.Item In-vivo solid phase microextraction for quantitative analysis of volatile organoselenium compounds in plants(Analytica Chimica Acta, 2019) Moreno Martín, Gustavo; Sanz Landaluce, Jon; León González, María Eugenia De; Madrid Albarrán, María Yolanda; ElsevierA new calibration method based on the use of headspace solid-phase microextraction (HS-SPME) and in-fiber internal standardization, combined with gas chromatography coupled to mass spectrometry (GC/MS) was developed for quantifying Se volatile organic species released by plants exposed to chitosan-modified selenium nanoparticles (Cs-SeNPs). The effect of several parameters affecting extraction and separation of the selected organic species of selenium (dimethylselenium (DMSe), diethylselenium (DESe) and dimethyldiselenium (DMDSe)) and deuterated dimethyl sulphide (d6-DMS) employed as internal standard were studied and optimized using an experimental design. The developed methodology was applied for quantifying the volatile selenium compounds produced over time by the plant species Raphanus sativus and Brassica juncea grown in hydroponic solution containing 5 mg Se L−1 in the form Cs-SeNPs. The procedure employed consisted in two steps. Volatile selenium species released from the plants were first extracted in the SPME fiber located at the headspace of a box with a fixed volume. Subsequently, the internal standard placed in a vial subjected to the same conditions as plants was extracted on the same fiber than the one previously used for extracting selenium compounds. Finally the extracted compounds were separated and analyzed by GC/MS. Results evidenced Cs-SeNPs biotransformation into DMSe and DMDSe by both plants species during growing stage, in amounts of the order of ng. Additionally, the resulting data were submitted to multifactorial ANOVA to evaluate the influence of plant type and time of exposure to Cs-SeNPs on the production of volatile selenium compounds.Item In vivo quantification of volatile organoselenium compounds released by bacteria exposed to selenium with HS-SPME-GC-MS. Effect of selenite and selenium nanoparticles(Talanta, 2021) Moreno Martín, Gustavo; Sanz Landaluce, Jon; León González, María Eugenia De; Madrid Albarrán, María Yolanda; ElsevierQuantification of volatile organoselenium species released by Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), after their growth in the presence of 1 and 2 mg Se⋅L-1 as both selenite and chitosan modified selenium nanoparticles (Ch-SeNPs), was achieved by the application of a method based on headspace solid-phase microextraction (HS-SPME) and in-fiber internal standardization, combined with gas chromatography coupled to mass spectrometry (GC-MS). This method consisted of an initial extraction of the released volatile organoselenium compounds on the SPME fiber, followed by the extraction of internal standard (IS), deuterated dimethyl sulfide (d6-DMS), on the same fiber before its desorption at the injection port of GC-MS. The results showed that the biotransformation of selenite and Ch-SeNPs into volatile organoselenium compounds was dependent on both the type of bacterial species and the chemical form of selenium (Se) administered. In this sense, E. coli was able to biotransform both selenite and Ch-SeNPs into dimethylselenium (DMSe) and dimethyldiselenium (DMDSe) while S. aureus, biotransformed selenite into DMSe and DMDSe and, Ch-SeNPs only into DMDSe. Additionally, the formation of a volatile mixed sulfur/selenium compound, dimethyl selenenyl sulfide (DMSeS), from Se in nanoparticulated form has been detected for the first time.