Atmospheric plasma processes for microbial inactivation: food applications and stress response in Listeria monocytogenes

This PhD thesis is focused on cold atmospheric plasma treatments (GP) for microbial inactivation in food applications. In fact GP represents a promising emerging technology alternative to the traditional methods for the decontamination of foods. The objectives of this work were to evaluate: - the effects of GP treatments on microbial inactivation in model systems and in real foods; - the stress response in L. monocytogenes following exposure to different GP treatments. As far as the first aspect, inactivation curves were obtained for some target pathogens, i.e. Listeria monocytogenes and Escherichia coli, by exposing microbial cells to GP generated with two different DBD equipments and processing conditions (exposure time, material of the electrodes). Concerning food applications, the effects of different GP treatments on the inactivation of natural microflora and Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli on the surface of Fuji apples, soya sprouts and black pepper were evaluated. In particular the efficacy of the exposure to gas plasma was assessed immediately after treatments and during storage. Moreover, also possible changes in quality parameters such as colour, pH, Aw, moisture content, oxidation, polyphenol-oxidase activity, antioxidant activity were investigated. Since the lack of knowledge of cell targets of GP may limit its application, the possible mechanism of action of GP was studied against 2 strains of Listeria monocytogenes by evaluating modifications in the fatty acids of the cytoplasmic membrane (through GC/MS analysis) and metabolites detected by SPME-GC/MS and 1H-NMR analyses. Moreover, changes induced by different treatments on the expression of selected genes related to general stress response, virulence or to the metabolism were detected with Reverse Transcription-qPCR. In collaboration with the Scripps Research Institute (La Jolla, CA, USA) also proteomic profiles following gas plasma exposure were analysed through Multidimensional Protein Identification Technology (MudPIT) to evaluate possible changes in metabolic processes

Molecular mechanisms and cellular contribution from lung fibrosis to lung cancer development.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing interstitial lung 28 disease (ILD) of unknown etiology, with a median survival of 2-4 years from the time of diagnosis. 29 Although IPF has unknown etiology by definition, there have been identified several risks factors 30 increasing the probability of the onset and progression of the disease in IPF patients such as cigarette 31 smoking and environmental risks factors associated to domestic and occupational exposure. Among 32 them, cigarette smoking together with concomitant emphysema might predispose IPF patients to 33 lung cancer (LC), mostly to non-small cell lung cancer (NSCLC), increasing the risk of lung cancer 34 development. To this purpose, IPF and LC share several cellular and molecular processes driving 35 the progression of both pathologies such as fibroblast transition proliferation and activation, endo- 36 plasmic reticulum stress, oxidative stress, and many genetic and epigenetic markers that predispose 37 the IPF patients to LC development. Nintedanib, a tyrosine-kinase inhibitor, was firstly developed 38 as an anticancer drug and then recognized as an anti-fibrotic agent based on the common target 39 molecular pathway. In this review our aim is to describe the updated studies on common cellular 40 and molecular mechanisms between IPF and lung cancer, whose knowledge might help to find 41 novel therapeutic targets for this disease combination

Satureja montana essential oil and Cotinus coggygria extract to improve microbial quality and the shelf-life of carrot-orange juice.

Natural antimicrobial agentsderived from herbs and plants have been used for millenia in medicine and food preservation. Indeed, essential oils (EO) and plants extracts (PE), in addition to contributing to flavour and taste, can act against food microbiota thus enhancing its stability. Moreover, recently, the consumers’ demand for naturalproducts – free of chemical preservatives - has been strongly increased. Fruit-based juices are susceptible to spoilage microorganisms,mainly yeasts and lactic acid bacteria, which negatively affect their organoleptic and nutritional properties making the product unacceptable to consumers. The aim of this study was to evaluate the effects ofnatural compounds, i.e. EO of Satureja montana and PE of Cotinus coggygria, in combination with mild thermal treatments on shelflife of carrot-orange juice and its microbial stability. In particular, viability of two spoilage microrganims Saccharomyces cerevisiae and Lactobacillus plantarumwas addressed. The EO and PE were preliminarly characterized by gascromatographic analises whichshowedthat S. montana was composed mainly by carvacrol and o-cimene, while the main components of C. coggygria were sulfuretin and fustin. Both S. montana EO and C. coggygriaPE displayed a good antimicrobial activity against S. cerevisiae, with minimum inhibitory concentration (MIC) values of 0.351 mg/ml and 0.094mg/ml, respectively. On the other hand, Lactobacillus plantarum was more resistant thanS. cerevisiae(MIC >0.5 mg/ml). The freshcarrot-orange juice was added with the S. montanaEO or C. coggygriaPE at MIC/4 concentrations for S. cerevisiae. Following its deliberatecontamination with the spoilage microrganisms (4 LogCFU/ml), samples were heat treated (60 °C for 2 or 5 min) and stored at room temperature for 9 days or 4°C for 50 days. Changes insurviving spoilage microbiota, pH, colour and antioxidant activity were monitored over storage. Overall, the results highlighted the possibility of stabilizing a juice with a high pH (5.4)by applying the "Hurdle Technology". The natural compounds enhanced the efficacy of heat treatments in inactivating/damaging the spoilage biota and delaying its recovery over storage. Moreover, no significant changes in the pH and color of the juices were found during storage, while limited variations in the antioxidant activity were detecte

Macroscale coupling between structural and effective connectivity in the mouse brain

Abstract Exploring how the emergent functional connectivity (FC) relates to the underlying anatomy (structural connectivity, SC) is one of the major goals of modern neuroscience. At the macroscale level, no one-to-one correspondence between structural and functional links seems to exist. And we posit that to better understand their coupling, two key aspects should be considered: the directionality of the structural connectome and limitations in explaining networks functions through an undirected measure such as FC. Here, we employed an accurate directed SC of the mouse brain acquired through viral tracers and compared it with single-subject effective connectivity (EC) matrices derived from a dynamic causal model (DCM) applied to whole-brain resting-state fMRI data. We analyzed how SC deviates from EC and quantified their respective couplings by conditioning on the strongest SC links and EC links. We found that when conditioning on the strongest EC links, the obtained coupling follows the unimodal-transmodal functional hierarchy. Whereas the reverse is not true, as there are strong SC links within high-order cortical areas with no corresponding strong EC links. This mismatch is even more clear across networks; only within sensory motor networks did we observe connections that align in terms of both effective and structural strength

Effects of PBS-based packaging on the shelf-life and spoilage profiles of soft cheeses

The intense use and accumulation of non-degradable plastics, which leads to the growing problem of waste disposal, and the lowering in the availability of fossil resources have strongly increased the interest and efforts in using renewable resources to develop biodegradable-based packaging materials. A number of bio-based materials and their innovative applications in food-related packaging have therefore gained much attention over the past several years. The main bio-sourced polymer currently used in food packaging is polylactic acid, but polybutylene succinate (PBS) can be considered a promising polymer from renewable resources for packaging industry for the coming years due to the prediction of a large development of succinic acid production by fermentation of gluco-based resources. The potentialities of PBS in this area are explored within the EU project SUCCIPACK (Grant agreement 289196) which aims to develop sustainable, active, and intelligent food packaging materials based on green PBS that can be flexibly used by packaging and food industries. Aspect that are taken into consideration include the optimization of the synthesis and compounding of polymer and copolymer grades for industrial plastic transformation processes to obtain films, trays and pouches. Also tailored packaging functionalities are obtained through surface treatments to control gas barrier properties and introduce antimicrobials. The performance of the novel packaging materials are assessed for selected food products, including soft cheeses, raw meat, fish products, ready-to-eat vegetables and ready meals for vegetarian. The objective of this preliminary work was to test the effects of PBS packaging material on the shelf-life of two soft cheeses such as ricotta and squacquerone. In particular microbial populations, volatile organic compounds, color and sensory properties were monitored over a 20-days storage at 4\ub0C and compared to control products packaged with the traditional materials. Microbial data of the evolution of main spoilage microorganisms were modelled with the Gompertz equation to calculate the products shelf-life and evaluate possible shifts in microbial population due to the innovative packaging. Volatile microbial metabolites were determined by electronic nose and solid-phase micro-extraction gaschromatography/mass-spectrometry (SPME/GC-MS) analysis. Also the real oxygen permeability of the whole packaged products was measured. The comparison of all the data collected showed that the packaging material and conditions used affected the microbiota and its evolution during storage resulting in significant differences in the volatile metabolites released with a potential impact on cheese sensory attributes

How Lactobacillus plantarum shapes its transcriptome in response to contrasting habitats

Triplets of Lactobacullus plantarum strains were isolated from nine contrasting habitats. Without any passage through other culture media, isolation and cultivation were on model media that strictly reproduced the chemical and physical conditions and stressors of the habitats of origin. Here, we demonstrated how L. plantarum regulates and shapes its transcriptome in response to contrasting habitats. Firstly, multivariate clustering analysis of transcriptional data (RNA-Seq), complemented with metabolomics and phenomics, grouped the strains according to the habitats of origin. Subsequently, selected strains from each habitat switched to repeated cultivation on MRS medium and transcriptomes homogenized into a unique cluster. Adaptation to this common medium mainly relied on activation of genes for phage- and prophage-related proteins and transposases. Finally, the comparison of growth across model media and with respect to MRS medium showed that 44% of the overall 3112 gene transcripts changed depending on the specific habitat. Regulation and shaping of transcriptomes mainly concerned carbohydrate acquisition, pyruvate catabolism, proteolytic system and amino acid, lipid and inorganic ion transport and metabolism, with contrasting responses for contrasting habitats. Pathways reconstruction demonstrated how the large genome size of L. plantarum imparts transcriptome and metabolic flexibility as the basic mechanism for a nomadic lifestyle

Influence of the electrode material on the decontamination efficacy of dielectric barrier discharge gas plasma treatments towards Listeria monocytogenes and Escherichia coli

The decontamination efficacy of a dielectric barrier discharge (DBD) gas plasma generator equipped with four different interchangeable electrode materials (stainless steel, brass, silver and glass/brass) was tested towards Listeria monocytogenes 56Ly and Escherichia coli NCFB 555. Bacteria were suspended in sterile saline solutions and placed for 20, 40 and 60 min under the discharge generated at atmospheric conditions by using air as the working gas. Significant differences emerged only for L. monocytogenes (after 40 and 60 min). Highest reductions (up to about 8 log CFU/ml) were observed by using the silver and brass electrodes after 60 min. Differences in terms of electrical parameters (voltage, current, active power) and chemical species generated in the liquid medium (nitrites and nitrates concentrations) were not significant, relevant or directly attributable to the decontamination efficacy of the different electrodes. Ozone production was higher for oxidation-resistant electrode materials. The decontamination effect was influenced by silver and brass antibacterial properties. Industrial relevance The results of the present work, focused on the impact of the electrode material on the decontamination performance, introduced a new aspect in the technology panorama that could improve possible industrial application. The selection of the electrode material played a role in terms of decontamination efficacy

Cold plasma treatment for fresh-cut melon stabilization

The aim of this study was to evaluate the effect of gas plasma treatment on fresh-cut melon stability during controlled storage. Plasma treatments of 15 + 15 and 30 + 30 min were conducted on fresh cut melon using a dielectric barrier discharge (DBD) generator. Samples were packed and stored for 4 days at 10 \ub0C and evaluated for qualitative, metabolic and microbiological indexes. Qualitative parameters of fresh-cut melon (titratable acidity, soluble solid content, dry matter, colour, texture) were only weakly affected by plasma treatment. Peroxidase (POD) and pectin methylesterase (PME) activities were slightly inhibited by the treatment up to respectively about 17 and 7%. Tissue metabolic heat production decreased proportionally to the treatment duration, while a partial conversion to anaerobic metabolism was observed. Microbial results showed that a significant increase in microbial shelf-life was achieved following the 15 + 15 min plasma treatment due to a delayed growth of spoilage mesophilic and psychrotrophic microflora. Industrial relevance: The demand for fresh-cut products characterized by high qualitative and nutritional values and an acceptable shelf-life has promoted the research for non-thermal treatments. Fresh-cut melon is considered to be highly perishable and potentially hazardous food because it can support the growth of spoilage microflora and several pathogens. Cold plasma has shown its potentiality as an antimicrobial treatment and has been tested on different food products, but the impact on product quality and metabolism is still scarcely known. The results obtained in this study contributed to deepen the knowledge on the effect of plasma treatment on microbial, qualitative and metabolic aspects of fresh-cut melon