In vitro probiotic properties and in vivo anti-ageing effects of Lactoplantibacillus plantarum PFA2018AU strain isolated from carrots on Caenorhabditis elegans

Lactic acid bacteria (LAB) share and provide several beneficial effects on human health, such as the release of bioactive metabolites, pathogen competition, and immune stimulation. The two major reservoirs of probiotic microorganisms are the human gastro-intestinal tract and fermented dairy products. However, other sources, such as plant-based foods, represent important alternatives thanks to their large distribution and nutritive value. Here, the probiotic potential of autochthonous Lactiplantibacillus plantarum PFA2018AU, isolated from carrots harvested in Fucino highland, Abruzzo (Italy), was investigated through in vitro and in vivo approaches. The strain was sent to the biobank of Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna in Italy for the purpose of patent procedures under the Budapest Treaty. The isolate showed high survival capability under in vitro simulated gastro-intestinal conditions, antibiotic susceptibility, hydrophobicity, aggregation, and the ability to inhibit the in vitro growth of Salmonella enterica serovar Typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus pathogens. Caenorhabditis elegans was used as the in vivo model in order to analyse prolongevity and anti-ageing effects. L. plantarum PFA2018AU significantly colonised the gut of the worms, extended their lifespan, and stimulated their innate immunity. Overall, these results showed that autochthonous LAB from vegetables, such as carrots, have functional features that can be considered novel probiotic candidates

Hyperbaric exposure and oxidative Stress in occupational activities (HEOxS): the study protocol

Background: Hyperbaric exposure (HE) is proven to be a stressor to several mechanisms in living cells. Even if after homeostasis restoration, harmful effects are expected, in particular a presence of free radicals. These latter are the stimulus to negative phenomenon as inflammation or cancer. In Italy, with 7500 km of sea shores, a large quantity of workers is exposed to HE during occupational activities. A deep knowledge of HE and bodily effects is not well defined; hence a multidisciplinary assessment of risk is needed. To detect one or more indicators of HE a research group is organised, under the INAIL sponsorship. The research project focused on the oxidative stress (OxS) and this paper details on the possible protocol to estimate, with a large amount of techniques on several human liquids, the relationship between OxS and HE. Specific attention will be paid to identify confounding factors and their influence. Methods: Blood and urine will be sampled. Several lab techniques will be performed on samples, both targeted, to measure the level of well-known biomarkers, and untargeted. Regard the formers: products of oxidation of DNA and RNA in urine; inflammation and temperature cytokines and protein carbonyles in blood. Untargeted evaluation will be performed for a metabolomics analysis in urine. Confounding factors: temperature, body fat, fitness, allergies and dietary habits. These factors will be assessed, directly or indirectly, prior and after HE. The final scope of the project is to determine one or more indicators that relates to HE in hits twofold nature: depth and duration. Conclusion: The relationship between OxS and HE is not deeply investigated and literature proposes diverging results. The project aims to define the time dependence of biomarkers related to OxS, to rise knowledge in risk assessment in workers exposed to HE

NMR-Based Metabolomics in Food Quality Control

In this chapter, we report the NMR-based metabolomic approach in food analysis and display its more instructive applications in quality control in order to illustrate the set of problems related to the peculiar data source, the potentiality, and the development features of main interest for chemometricians in this field. © 2013 Elsevier B.V

Hepatocytes entrapped in alginate gel beads and cultured in bioreactor: rapid repolarization and reconstitution of adhesion areas

The maintenance of the differentiated hepatocyte phenotype and its specific physiological properties is known to depend on several factors, such as chemical signals, cell-cell and extracellular matrix molecular interactions, as well as the use of three-dimensional matrices. The entrapment of hepatocytes within Ca-alginate at high cell density and the culture under continuous flow favour the development of three-dimensional organization and promote expression of the differentiated hepatic phenotype. This system could represent an improvement in hepatocyte cultivation for basic studies of liver physiology and metabolism; it could also be applicable in toxicology, hepatocyte transplantation or development of bioartificial organs. This report describes the effect of alginate entrapment and culture in a bioreactor on hepatocyte aggregate formation, with particular attention to the reestablishment of cell polarity, cell junctions and three-dimensional re-organization of the cytoskeleton. Oxygen supply and cell oxygen consumption rate were monitored in order to evaluate possible changes in hepatocyte energy requirement. Our data show that after only 6 h of perfusion in the bioreactor, actin and cytokeratin localize along the adhesion areas of the plasma membrane, in which reconstituted bile canaliculi were also observed. Moreover, the presence of connexin at the level of joined membranes of neighbouring cells suggests the establishment of gap junctions between hepatocytes. After the first 30 min of perfusion the oxygen consumption rate remained constant throughout the experimental period

Phytochemical profile of olive oil mill vegetative waters

Olive (Olea europaea L.) belongs to the Oleaceae family and it has been cultivated for olive oil, fine wood, olive leaf and its fruits from 2000 years ago and is one of the most diffused crops in the Mediterranean basin. Its fruits can be consumed as it is, but traditionally they are employed to produce olive oil, which is one of the cornerstones of the Mediterranean diet. It is a commercial good whose market is projected to reach a revenue of 1 billion of US dollars in 2022 worldwide and is a major contribution to Italian economy (1). The production of olive oil consists in several steps that produce, along the final product, several wastes among which pomace is a wet solid waste composed by peels, pulp residues and seed fragments. In modern implants (decanter multiphase) waste can include a dehydrated pomace containing peels and hazelnuts suitable for production of pellet for stoves and a second by-product, called "paté", consisting in hydrated pulp with a high humidity (75-80%) usually used to produce biogas. In the process for the latter, vegetative water is also produced, which is an aqueous toxic waste usually discarded (2). Olive mill wastes are toxic for both natural beneficial and pathogenic microrganisms. Nowadays, in the optic of a better employment of the natural resources, these approaches are no longer sustainable, and as such several national and international projects have been founded in order to use these waste materials for other applications. The aim of this project, which is the first step of the larger ABASA (Agricultural By-products into valuable Assets for Sustainable Agriculture) project founded by LazioInnova- Regione Lazio, is to characterize the phytochemical composition of patè and vegetative waters fractionated by various stages of membrane filtration in order to identify and quantify molecules with biological activity which could be employed in agriculture as phytopesticides, agents in crop protection, soil biofertilizers or as a source of bioactive compounds. In the present study, both pomace and vegetative waters fractions were analyzed by high-resolution NMR spectroscopy. The matrices have been treated with a multi-solvent extraction protocol, and both the hydro soluble and lyposoluble fractions have been analyzed. Mono and polyunsaturated fatty acid, triglycerides, triterpenes derived from ursolic acid, oleuropein, lingstroside and their aglycones, tyrosol and hydroxytirosol were observed in both matrices, with the major difference between them being the relative amount of triglycerides. Of great interest is the presence of oleuropein and lingstroside in waste waters since they are both seco-iridoids with a known antifungal and antimicrobical activity (3) Carbohydrate profile has been obtained for the same fractions. The presence of pectin and oligogalacturonides (OGs) were identified by anion exchange HPLC-Dionex. OGs are cell wall derived pectic fragments elicitors of plant immune responses (4). This finding highlights the high potential of these by-products as biostimulant of plant defense responses. The evaluation of the fractionation efficiency and the biological activity of the different fractions are in progress

Cellular volume determination of alginate-entrapped hepatocytes by MRI diffusion measurements

Cellular volume of hepatocytes entrapped in alginate gel beads were evaluated under in vivo conditions in samples having different cell densities by applying matemathical models to the diffusion data obtained by magnetic resonance imaging (MRI). The calculated average volume is in good agrement with the values from the literature - being closer to the data relative to living tissue than to isolated cells. The non invasive characteristics of magnetic resonance imaging make this method particularly well suited to obtain information From the intact system

Exploring human breast milk composition by NMR-based metabolomics

Breast milk is a complex fluid evolutionarily adapted to satisfy the nutritional requirements of growing infants. In addition, milk biochemical and immunological components protect newborns against infective agents in the new environment. Human milk oligosaccharides, the third most abundant component of breast milk, are believed to modulate the microbiota composition, thus influencing a wide range of physiological processes of the infant. Human milk also contains a number of other bioactive compounds, the functional role of which has not yet been clearly elucidated. In this scenario, NMR-based metabolic profiling can provide a rapid characterisation of breast milk composition, thus allowing a better understanding of its nutritional properties

Effects of resveratrol on HepG2 cells as revealed by 1H-NMR based metabolic profiling

Background: Resveratrol, a polyphenol found in plant products, has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Several in vitro and in vivo studies have demonstrated the influence of resveratrol on multiple intracellular targets that may regulate metabolic homeostasis. Methods: We analysed the metabolic modifications induced by resveratrol treatment in a human hepatoblastoma line, HepG2 cells, using a H-1-NMR spectroscopy-based metabolomics approach that allows the simultaneous screening of multiple metabolic pathways. Results: Results demonstrated that cells cultured in the presence or absence of resveratrol displayed different metabolic profiles: the treatment induced a decreased utilisation of glucose and amino acids for purposes of energy production and synthesis associated to a decreased release of lactate in the culture medium and an increase in succinate utilisation. At the same time, resveratrol treatment slowed the cell cycle in the S phase without inducing apoptosis, and increased Sirt1 expression, also affecting its intracellular localisation. Conclusions: Our results show that the metabolomic analysis of the exometabolome of resveratrol-treated HepG2 cells indicates a metabolic switch from glucose and amino acid utilisation to fat utilisation for the production of energy, and seem in agreement with an effect mediated via AMPK- and Sirt1-activation. General significance: NMR-based metabolomics has been applied in a hepatocyte cell culture model in relation to resveratrol treatment; such an approach could be transferred to evaluate the effects of nutritional compounds with health impact. (C) 2011 Elsevier B.V. All rights reserved

A 1-H NMR-BASED METABOLOMIC APPROACH REVEALS SIGNIFICANT CHANGES IN BREAST MILK COMPOSITION INDUCED BY ORAL PROBIOTIC SUPPLEMENTATION

Pregnancy and the first two years of life are the most critical time periods for interventions to improve child growth and development. In this scenario, both the microbiota and breast milk which are ‘transmitted’ from mother to infant seem to play a crucial role for the promotion of a healthy growth in newborn. Perinatal supplementation with probiotics has been recently proposed as a possible strategy to manipulate maternal physiology, from gut microbiota to breast milk composition, in order to orchestrate the infant development. In this study, we applied a 1H NMR-based metabolomic approach to characterize the breast milk metabolome and possible changes in breast milk composition induced by oral supplementation with VSL#3 probiotic mixture. Our metabolomic approach revealed a systemic effect of the maternal probiotic supplementation on the production or secretion of human milk oligosaccharides (HMOs) and lipids in the mammary gland. Such an approach may become increasingly useful as a tool to design and evaluate dietary and therapeutic strategies aimed at improving the health status of the mother-infant dyad