Chemical Profiles, In Vitro Antioxidant and Antifungal Activity of Four Different Lavandula angustifolia L. EOs.

Lavandula angustifolia L., known as lavender, is an economically important Lamiaceae due to the production of essential oils (EOs) for the food, cosmetic, pharmaceutical and medical industries. The purpose of this study was to determine the chemical composition of EOs isolated from four inflorescences of L. angustifolia L. collected in different geographical areas: central-southern Italy (LaCC, LaPE, LaPS) and southern France (LaPRV). The essential oils, obtained by steam distillation from plants at the full flowering stage, were analyzed using gas chromatography coupled with mass spectrometry (GC-MS). More than 70 components identified in each sample showed significant variability among the main constituents. The four EOs analyzed contained the following as main component: linalool (from 30.02% to 39.73%), borneol (13.65% in LaPE and 16.83% in La PS), linalyl acetate (24.34% in LaCC and 31.07% in LaPRV). The EOs were also evaluated for their in vitro antifungal activity against two white rot fungi (Phanerochaete chrysosporium and Trametes cingulata) as potential natural biodeteriogens in the artworks field, and against Sclerotium rolfsii, Botrytis cinerea and Fusarium verticilloides responsible for significant crop yield losses in tropical and subtropical areas. The results confirm a concentration-dependent toxicity pattern, where the fungal species show different sensitivity to the four EOs. The in vitro antioxidant activity by DPPH assay showed better scavenging activity on LaCC (IC50 26.26 mg/mL) and LaPRV (IC50 33.53 mg/mL), followed by LaPE (IC50 48.00 mg/mL) and LaPS (IC50 49.63 mg/mL). The potential application of EOs as a green method to control biodeterioration phenomena on a work of art on wood timber dated 1876 was evaluated.

Lavender Essential Oil Modulates Hepatic Cholesterol Metabolism in HepG2 Cells.

Cholesterol is an essential lipid that guarantees several biological processes in eukaryotic cells. Its metabolism is regulated by a complex protein network that could be significantly influenced by numerous exogenous sources, such as essential oils (EOs). For instance, it has been speculated that monoterpenoid and sesquiterpenoid compounds contained in lavender essential oil (LEO) may exert important hypocholesterolemic activities. However, the molecular mechanisms by which LEO influences cholesterol homeostasis are not characterized. In this work, we evaluated the ability of LEO to regulate the protein network that controls cholesterol metabolism in the HepG2 cell line. The main findings indicate that LEO administration increases intracellular cholesterol content. Concurrently, LEO affects the expression of proteins involved in cholesterol uptake, biosynthesis, and trafficking. These effects are partially mediated by terpinene-4-ol, one of the most abundant compounds in LEO. These results demonstrate that LEO modulates cholesterol metabolism in hepatic cells.

Effects of EOs vs. Antibiotics on E. coli Strains Isolated from Drinking Waters of Grazing Animals in the Upper Molise Region, Italy.

The health and safety of grazing animals was the subject of microbiological monitoring on natural source of drinking waters in the upper Molise region, Italy. Surface water samples, on spring-summer season, were collected and submitted to analyses using sterile membrane filtration, cultural medium, and incubation. The level of environmental microbial contamination (Total viable microbial count, yeasts and fungi) and faecal presence (Total and faecal coliforms, E. coli, and Salmonellae spp.) were carried out. By the selective microbiological screening, twenty-three E. coli strains from drinking waters were isolated and submitted to further studies to evaluate antibiotic resistance by antibiograms vs. three animal and two diffuse human antibiotics. Furthermore, after a fine chemical characterization by GC and GC-MS, three Essential Oils (EOs) of aromatic plants (Timus vulgaris, Melaleuca alternifolia, Cinnamomun verum) aromatograms were performed and results statistically compared. The effects of EOs vs. antibiotics on E. coli strains isolated from drinking waters showed a total absence of microbial resistance. In our experimental conditions, even if some suggestions will be further adopted for better managements of grazing animals, because the health and safety represent a guarantee for both animals and humans.

Biodeterioration of carbographic ribbon: Isolation, identification of causal agents and forensic implications.

This study is part of a comprehensive investigation that was performed in regard to a case of alterations on a carbographic ribbon used in a typewriter that was found and seized by inner security operations of the Arma dei Carabinieri, Italy. Thirty-six coded scripts possessing potentially and criminally liable content were present on the tape; however, only the 6th and 7th scripts exhibited alterations of an uncertain nature. The study included sampling that was performed under sterile conditions of a large surface area of carbographic ribbons. A protocol based on physico-chemical, microbiological, and biomolecular tools was established. Preliminary results revealed the presence of fungal contamination that was primarily located on the inner surface of the 6th and 7th scripts on the black carbographic ribbon. One fungal strain was isolated and identified by universal ITS-PCR primer and rDNA sequencing as Alternaria infectoria strain NIS4. Fungal growth was monitored for 3 weeks in the laboratory under different environmental conditions (temperature, open-closed system, and substrate). The A. infectoria NIS4 strain exhibited the best growth at 28°C under a closed system with RH near saturation. We also noted that the fungal growth was abundant at 15°C. Moreover, this fungus (a potential human pathogen) possessed the ability to colonize the surface of the new carbographic ribbon even when using mineral medium; however, this only occurred in a closed system environment and not in open systems due to rapid desiccation. Under our experimental conditions, the A. infectoria NIS4 strain could degrade gelatin as an organic matter present in trace amounts that are often used as a binder in a carbographic ribbon emulsions. The results revealed that the isolated microorganism was the major biological candidate capable of altering the investigated carbographic ribbon; however, these alterations could only occur under favourable environmental conditions. AIMS: Identify the cause of microbial alterations on a carbographic ribbon in a typewriter used in a hypogean Italian criminal house named "covo." METHODS AND RESULTS: The isolation and identification of biodeteriogens (Alternaria infectoria NIS4) were performed using both culture-dependent and-independent methods, including ITS regions-primed PCR and rDNA techniques. Environmental scanning electron microscopy (ESEM) and optical observations were also performed. Growth tests and biodeterioration simulation tests on carbographic ribbons at the lab scale were performed under different environmental conditions. The A. infectoria NIS4 strain exhibited biodeterioration activity on carbographic ribbons under environmental conditions that were extremely favourable for growth. A high ability to colonize carbographic ribbon surfaces with fast and abundant growth at both 15°C and 28°C under lab-scale conditions at RH near saturation was observed. CONCLUSIONS: In this forensic case study, the ability of the isolated micromycetes A. infectoria NIS4 strain to colonize and induce alterations and degradation in a carbographic ribbon stored under indoor environmental conditions was examined. When favourable conditions change over time, the risk of microbial colonization and the damage produced by the fungal biodeterioration processes on the synthetic material objects has been confirmed. SIGNIFICANCE AND IMPACT OF STUDY: The current study contributes to the knowledge of biodeterioration processes in carbographic ribbon and the responsible agents, and our study provides an example of how environmental microbiology can also aid in forensic studies.

Chemical Profile, In Vitro Biological Activity and Comparison of Essential Oils from Fresh and Dried Flowers of Lavandula angustifolia L.

The chemical composition of essential oils (EOs) from dried and fresh flowers of Lavandula angustifolia L. (lavender), named LA 2019 and LA 2020, respectively, grown in central Italy was analyzed and compared by GC and GC-MS. For both samples, 61 compounds were identified, corresponding to 97.9% and 98.1% of the total essential oils. Explorative data analysis, performed to compare the statistical composition of the samples, resulted in a high level of global similarity (around 93%). The compositions of both samples were characterized by 10 major compounds, with a predominance of Linalool (35.3-36.0%), Borneol (15.6-19.4%) and 1,8-Cineole (11.0-9.0%). The in vitro antibacterial activity assay by disk diffusion tests against Bacillus subtilis PY79 and Escherichia coli DH5α showed inhibition of growth in both indicator strains. In addition, plate counts revealed a bactericidal effect on E. coli, which was particularly noticeable when using oil from the fresh lavender flowers at the highest concentrations. An in vitro antifungal assay showed that the EOs inhibited the growth of Sclerotium rolfsii, a phytopathogenic fungus that causes post-harvest diseases in many fruits and vegetables. The antioxidant activity was also assessed using the ABTS free radical scavenging assay, which showed a different antioxidant activity in both EOs. In addition, the potential application of EOs as a green method to control biodeterioration phenomena on an artistic wood painting (XIX century) was evaluated.

Dry biocleaning of artwork: an innovative methodology for Cultural Heritage recovery?

An innovative methodology is proposed, based on applied biotechnology to the recovery of altered stonework: the "dry biocleaning", which envisages the use of dehydrated microbial cells without the use of free water or gel-based matrices. This methodology can be particularly useful for the recovery of highly-ornamented stoneworks, which cannot be treated using the conventional cleaning techniques. The experimental plan included initial laboratory tests on Carrara marble samples, inoculated with dehydrated Saccharomyces cerevisiae yeast cells, followed by on-site tests performed on "Quattro Fontane" (The Four Fountains), a travertine monumental complex in Rome (Italy), on altered highly ornamented areas of about 1,000 cm2. The mechanism is based on the spontaneous re-hydration process due to the environmental humidity and on the metabolic fermentative activity of the yeast cells. Evaluation by physical-chemical analyses, after 18 hours of the biocleaning, confirmed a better removal of salts and pollutants, compared to both nebulization treatment and control tests (without cells). The new proposed on-site dry biocleaning technique, adopting viable yeast cells, represents a promising method that can be further investigated and optimized for recovering specific altered Cultural Heritage stoneworks.

Connecting phenome to genome in Pseudomonas stutzeri 5190: an artwork biocleaning bacterium.

Research on biotechnology applications for cultural heritage restoration has shown how microorganisms can be efficient at cleaning particularly complex or ingrained substances through the process called "biocleaning". Bacteria are able to synthesize groups of specific enzymes for the degradation of complex materials present on artwork. Biocleaning has been shown to be less hazardous than some traditional mechanical or chemical techniques for the artwork, to be environmentally-friendly and safe for restorers to use. In order to improve our knowledge of the metabolic mechanisms involved in biocleaning, we analyzed the relationship between the genome and phenome of Pseudomonas stutzeri 5190 in order to identify and confirm the benefits and drawbacks of this bacterium used on on-site artwork as a biocleaning agent. Main phenotype microarray (PM) assays showed that P. stutzeri 5190 was able to use: i) 51 of the 190 carbon sources tested, where 32 were used efficiently, among which there were six amino acids (l-proline, l-alanine, d-alanine, l-glutamic acid, l-asparagine and l-glutamine); ii) 74 of the 95 nitrogen sources tested, where 50 compounds were used efficiently, among which were 28 amino acids and the inorganic nitrate and nitrite compounds, supporting the hypothesis of the strain's ability to remove nitrate salt efflorescence from frescoes. Furthermore, high tolerance to osmotic stress, to basic pH and to toxic compounds was revealed by PM. Putative genes compatible with these phenotypes are described.

Advantages of using microbial technology over traditional chemical technology in removal of black crusts from stone surfaces of historical monuments.

This study compares two cleaning methods, one involving an ammonium carbonate-EDTA mixture and the other involving the sulfate-reducing bacterium Desulfovibrio vulgaris subsp. vulgaris ATCC 29579, for the removal of black crust (containing gypsum) on marble of the Milan Cathedral (Italy). In contrast to the chemical cleaning method, the biological procedure resulted in more homogeneous removal of the surface deposits and preserved the patina noble under the black crust. Whereas both of the treatments converted gypsum to calcite, allowing consolidation, the chemical treatment also formed undesirable sodium sulfate.

Improved methodology for bioremoval of black crusts on historical stone artworks by use of sulfate-reducing bacteria.

An improved methodology to remove black crusts from stone by using Desulfovibrio vulgaris subsp. vulgaris ATCC 29579, a sulfate-reducing bacterium, is presented. The strain removed 98% of the sulfates of the crust in a 45-h treatment. Precipitation of black iron sulfide was avoided using filtration of a medium devoid of iron. Among three cell carriers, Carbogel proved to be superior to both sepiolite and Hydrobiogel-97, as it allowed an easy application of the bacteria, kept the system in a state where microbial activity was maintained, and allowed easy removal of the cells after the treatment.

Art-loving bugs: the resurrection of Spinello Aretino from Pisa's cemetery.

During 1391-1392, Spinello Aretino painted a cycle of frescoes in Pisa's cemetery on the theme "Storie dei Santi Martiri Efisio e Potito", highly prized by contemporaries and by Vasari. Twenty years ago, one of these frescoes, "Conversione di S. Efisio e Battaglia" (Conversion of S. Efisio and Battle), because of discolouring and bad damage caused by humidity and atmospheric pollution, was removed from the walls using the "tear-off" technique, consisting in covering the surface with a strong cloth bound to the surface with generous layers of formaldehyde-treated glue. As luck would have it, this large fresco (3.50x7.80 m) was abandoned in a storehouse for more than 20 years. When the curators attempted to remove the cloth, much to their dismay they found that the glue resisted any attempt at digestion, even when treated with concoctions of the most aggressive proteolytic enzymes available on the market. It is likely that during the long storage the glue became slowly cross-linked by the formaldehyde to the point of forming an intricate mass of untreatable proteinaceous material. Thus, although poor Spinello died presumably as a bona fide Christian, his painting was condemned to wear the burka, Muslim-fashion, for the rest of its life. When we recently treated the fresco with a suspension of viable Pseudomonas stutzeri cells, these bacteria, although agnostic, were able to fully digest the hardened glue and restore to life Spinello's glorious painting. We show here how proteomics helped us solve the riddle of how these bacteria acted on the burka-obscured fresco.

Improved combined chemical and biological treatments of olive oil mill wastewaters.

A novel system was investigated, finalized to reduce the impact of highly polluting wastewaters, and based on combined actions of catalytic oxidations and microbial biotechnologies. Olive oil mill wastewaters (COD 10,000-100,000 mg O(2)/L) were oxidized up to 80-90% by stoichiometric amounts of dilute hydrogen peroxide (35%) and in the presence of water soluble iron catalysts, either Fe(II) or Fe(III), at concentrations up to 1% w/w and more, i.e., much larger than those reported for conventional Fenton processes. In the combined action, the mineralization activity of a selected microbial consortium was used to degrade residual volatile and nonvolatile organic compounds into CO(2) and biomass. The results of this search could suggest an improved operational methodology capable to reduce the potential impact of wastewater.

Microbiological aspects of humid husk composting.

The feasibility of composting as disposal practice of husk mixed with olive mill wastewaters (OMW) was studied in the present research. The process was investigated with regards to some reliable and easy to be determined microbial activity parameters such as the ATP content, the activity of a pool of enzymes and the viable counts, keeping in mind the well known bias of this last technique. Two different composting technologies were compared: static pile and reactor by studying the cured composts obtained. Among the bioindicators tested, the composting process trend was described better by the ATP content and the activity of some enzymes. In fact, the ATP content showed an increase during the thermophilic phase in both the pile and the reactor, and decreased at the end of the process, it was also higher in the bioreactor-pile technology than in the pile. With regard to the enzymatic activity, with the reactor technology experiment, two peaks were evidenced in the thermophilic phase and during the curing phase in pile, and a drop during the transfer of material from the reactor to the pile. The quality of the cured product obtained using the reactor technology has been evaluated considering phytoxicity and hygienic features: the stabilised compost resulted to have neither phytoxical effect, nor faecal indicator contaminants.

Antimicrobial furostanol saponins from the seeds of Capsicum annuum L. var. acuminatum.

Three new furostanol saponins named capsicoside E (1), capsicoside F (2), and capsicoside G (5) were obtained from the seeds of Capsicum annuum L. var. acuminatum along with known oligoglycosides (3, 4, and 6-10). On the basis of chemical and spectroscopic analyses, the structures of these new furostanol oligoglycosides were elucidated as 26-O-beta-D-glucopyranosyl-22-O-methyl-5alpha-furost-25(27)-en-2alpha,3beta,22xi,26-tetraol-3-O-beta-D-glucopyranosyl(1-->3)-beta-D-glucopyranosyl(1-->2)-[beta-D-glucopyranosyl(1-->3)]-beta-D-glucopyranosyl(1-->4)-beta-D-galactopyranoside (1), 26-O-beta-D-glucopyranosyl-(25R)-5alpha-furost-20(22)-en-2alpha,3beta,26-triol-3-O-beta-D-glucopyranosyl (1-->3)-beta-D-glucopyranosyl(1-->2)-[beta-D-glucopyranosyl(1-->3)]-beta-D-glucopyranosyl(1-->4)-beta-D-galactopyranoside (2), and 26-O-beta-D-gluco-pyranosyl-(25R)-5alpha-furosta-3beta,22xi,26-triol-3-O-beta-D-glucopyranosyl(1-->3)-beta-D-glucopyranosyl(1-->2)-[beta-D-glucopyranosyl(1-->3)]-beta-D-glucopyranosyl(1-->4)-beta-D-galactopyranoside (5). The isolated saponins showed higher antimicrobial activity against yeasts than against common fungi. Data indicated that the antiyeast activity was related to the combination of the oligosaccharide chain (S1, S2, or S3) with an O-methyl group at R(3) and the presence of a hydroxyl group at the C-2 position.