Valerio Mezzasalma

Evaluation of the probiotic properties of new Lactobacillus and Bifidobacterium strains and their in vitro effect

Probiotic ingestion is recommended as a preventive approach to maintain the balance of the intestinal microbiota and to enhance the human well-being. During the whole life of each individual, the gut microbiota composition could be altered by lifestyle, diet, antibiotic therapies and other stress conditions, which may lead to acute and chronic disorders. Hence, probiotics can be administered for the prevention or treatment of some disorders, including lactose malabsorption, acute diarrhoea, irritable bowel syndrome, necrotizing enterocolitis and mild forms of inflammatory bowel disease. The probiotic-mediated effect is an important issue that needs to be addressed in relation to strain-specific probiotic properties. In this work, the probiotic properties of new Lactobacillus and Bifidobacterium strains were screened, and their effects in vitro were evaluated. They were screened for probiotic properties by determining their tolerance to low pH and to bile salts, antibiotic sensitivity, antimicrobial activity and vitamin B8, B9 and B12 production, and by considering their ability to increase the antioxidant potential and to modulate the inflammatory status of systemic-miming cell lines in vitro. Three out of the examined strains presenting the most performant probiotic properties, as Lactobacillus plantarum PBS067, Lactobacillus rhamnosus PBS070 and Bifidobacterium animalis subsp. lactis PBSO75, were evaluated for their effects also on human intestinal HT-29 cell line. The obtained results support the possibility to move to another level of study, that is, the oral administration of these probiotical strains to patients with acute and chronic gut disorders, by in vivo experiments.

DNA Barcoding for Minor Crops and Food Traceability

This outlook paper addresses the problem of the traceability of minor crops. These kinds of cultivations consist in a large number of plants locally distributed with a modest production in terms of cultivated acreage and quantity of final product. Because of globalization, the diffusion of minor crops is increasing due to their benefit for human health or their use as food supplements. Such a phenomenon implies a major risk for species substitution or uncontrolled admixture of manufactured plant products with severe consequences for the health of consumers. The need for a reliable identification system is therefore essential to evaluate the quality and provenance of minor agricultural products. DNA-based techniques can help in achieving this mission. In particular, the DNA barcoding approach has gained a role of primary importance thanks to its universality and versatility. Here, we present the advantages in the use of DNA barcoding for the characterization and traceability of minor crops based on our previous or ongoing studies at the ZooPlantLab (Milan, Italy). We also discuss how DNA barcoding may potentially be transferred from the laboratory to the food supply chain, from field to table.

A Randomized, Double-Blind, Placebo-Controlled Trial: The Efficacy of Multispecies Probiotic Supplementation in Alleviating Symptoms of Irritable Bowel Syndrome Associated with Constipation

Background and Aim. The efficacy of supplementation treatment with two multispecies probiotic formulates on subjects diagnosed with IBS-C and the assessment of their gut microbiota were investigated. Methods. A randomized, double-blind, three-arm parallel group trial was carried out on 150 IBS-C subjects divided into three groups (F_1, F_2, and F_3). Each group received a daily oral administration of probiotic mixtures (for 60 days) F_1 or F_2 or placebo F_3, respectively. Fecal microbiological analyses were performed by species-specific qPCR to assess the different amount of probiotics. Results. The percentage of responders for each symptom was higher in the probiotic groups when compared to placebo group during the treatment period (t60) and was maintained quite similar during the follow-up period (t90). Fecal analysis demonstrated that probiotics of the formulations increased during the times of treatment only in fecal DNA from subjects treated with F_1 and F_2 and not with F_3, and the same level was maintained during the follow-up period. Conclusions. Multispecies probiotic supplementations are effective in IBS-C subjects and induce a different assessment in the composition of intestinal microbiota. This clinical study is registered with the clinical study registration number ISRCTN15032219.

Evaluating the efficacy of restoration plantings through DNA barcoding of frugivorous bird diets.

Frugivores are critical components of restoration programs because they are seed dispersers. Thus, knowledge about bird-plant trophic relationships is essential in the evaluation of the efficacy of restoration processes. Traditionally, the diet of frugivores is characterized by microscopically identifying plant residues in droppings, which is time-consuming, requires botanical knowledge, and cannot be used for fragments lacking detectable morphological characteristics (e.g., fragmented seeds and skins). We examined whether DNA barcoding can be used as a universal tool to rapidly characterize the diet of a frugivorous bird, Eurasian blackcap (Sylvia atricapilla). We used the DNA barcoding results to assess restoration efforts and monitor the diversity of potentially dispersed plants in a protected area in northern Italy. We collected 642 Eurasian Blackcap droppings at the restored site during the autumn migration over 3 years. Intact seeds and fragmented plant material were analyzed at 2 plastidial barcode loci (rbcL and trnH-psbA), and the resulting plant identifications were validated by comparison with a reference molecular data set of local flora. At least 17 plant species, including 7 of the 11 newly transplanted taxa, were found. Our results demonstrate the potential for DNA barcoding to be used to monitor the effectiveness of restoration plantings and to obtain information about fruit consumption and dispersal of invasive or unexpected plant species. Such an approach provides valuable information that could be used to study local plant biodiversity and to survey its evolution over time.

Towards a better understanding of Apis mellifera and Varroa destructor microbiomes: introducing ‘phyloh’ as a novel phylogenetic diversity analysis tool

The study of diversity in biological communities is an intriguing field. Huge amount of data are nowadays available (provided by the innovative DNA sequencing techniques), and management, analysis and display of results are not trivial. Here, we propose for the first time the use of phylogenetic entropy as a measure of bacterial diversity in studies of microbial community structure. We then compared our new method (i.e. the web tool phyloh) for partitioning phylogenetic diversity with the traditional approach in diversity analyses of bacteria communities. We tested phyloh to characterize microbiome in the honeybee (Apis mellifera, Insecta: Hymenoptera) and its parasitic mite varroa (Varroa destructor, Arachnida: Parasitiformes). The rationale is that the comparative analysis of honeybee and varroa microbiomes could open new perspectives concerning the role of the parasites on honeybee colonies health. Our results showed a dramatic change of the honeybee microbiome when varroa occurs, suggesting that this parasite is able to influence host microbiome. Among the different approaches used, only the entropy method, in conjunction with phylogenetic constraint as implemented in phyloh, was able to discriminate varroa microbiome from that of parasitized honeybees. In conclusion, we foresee that the use of phylogenetic entropy could become a new standard in the analyses of community structure, in particular to prove the contribution of each biological entity to the overall diversity.

DNA Barcoding Meets Nanotechnology: Development of a Universal Colorimetric Test for Food Authentication

Food trade globalization and the growing demand for selected food varieties have led to the intensification of adulteration cases, especially in the form of species substitution and mixing with cheaper taxa. This phenomenon has huge economic impact and sometimes even public health implications. DNA barcoding represents a well-proven molecular approach to assess the authenticity of food items, although its use is hampered by analytical constraints and timeframes that are often prohibitive for the food market. To address such issues, we have introduced a new technology, named NanoTracer, that allows for rapid and naked-eye molecular traceability of any food and requires limited instrumentation and cost-effective reagents. Moreover, unlike sequencing, this method can be used to identify not only the substitution of a fine ingredient, but also its dilution with cheaper ones.

Evaluating the efficacy of restoration plantings through DNA barcoding characterization of frugivorous bird diets

Frugivores are critical components of restoration programs because they are seed dispersers. Thus, knowledge about bird-plant trophic relationships is essential in the evaluation of the efficacy of restoration processes. Traditionally, the diet of frugivores is characterized by microscopically identifying plant residues in droppings, which is time-consuming, requires botanical knowledge, and cannot be used for fragments lacking detectable morphological characteristics (e.g., fragmented seeds and skins). We examined whether DNA barcoding can be used as a universal tool to rapidly characterize the diet of a frugivorous bird, Eurasian blackcap (Sylvia atricapilla). We used the DNA barcoding results to assess restoration efforts and monitor the diversity of potentially dispersed plants in a protected area in northern Italy. We collected 642 Eurasian Blackcap droppings at the restored site during the autumn migration over 3 years. Intact seeds and fragmented plant material were analyzed at 2 plastidial barcode loci (rbcL and trnH-psbA), and the resulting plant identifications were validated by comparison with a reference molecular data set of local flora. At least 17 plant species, including 7 of the 11 newly transplanted taxa, were found. Our results demonstrate the potential for DNA barcoding to be used to monitor the effectiveness of restoration plantings and to obtain information about fruit consumption and dispersal of invasive or unexpected plant species. Such an approach provides valuable information that could be used to study local plant biodiversity and to survey its evolution over time.

Poisonous or non-poisonous plants? DNA-based tools and applications for accurate identification

Plant exposures are among the most frequently reported cases to poison control centres worldwide. This is a growing condition due to recent societal trends oriented towards the consumption of wild plants as food, cosmetics, or medicine. At least three general causes of plant poisoning can be identified: plant misidentification, introduction of new plant-based supplements and medicines with no controls about their safety, and the lack of regulation for the trading of herbal and phytochemical products. Moreover, an efficient screening for the occurrence of plants poisonous to humans is also desirable at the different stages of the food supply chain: from the raw material to the final transformed product. A rapid diagnosis of intoxication cases is necessary in order to provide the most reliable treatment. However, a precise taxonomic characterization of the ingested species is often challenging. In this review, we provide an overview of the emerging DNA-based tools and technologies to address the issue of poisonous plant identification. Specifically, classic DNA barcoding and its applications using High Resolution Melting (Bar-HRM) ensure high universality and rapid response respectively, whereas High Throughput Sequencing techniques (HTS) provide a complete characterization of plant residues in complex matrices. The pros and cons of each approach have been evaluated with the final aim of proposing a general user’s guide to molecular identification directed to different stakeholder categories interested in the diagnostics of poisonous plants.

Chemical, molecular, and proteomic analyses of moss bag biomonitoring in a petrochemical area of Sardinia (Italy)

In this study, Hypnum cupressiforme moss bags were used to examine the atmospheric deposition of trace elements in the oil refinery region of Sardinia (Italy) compared with surrounding natural zones. The concentrations of 13 elements [arsenic (As), calcium (Ca), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), nickel (Ni), lead (Pb), vanadium (V), and zinc (Zn)] were determined using inductively coupled plasma optical emission spectrometry. A significant accumulation of pollutants was detected using active biomonitoring with moss bags compared with a control site. The most relevant contaminants for all of the tested sites were Cr, Cu, Ni, and Zn. Moreover, the accumulation of Cr and Zn in the refinery industrial areas, IA1 and IA2, was more than five times greater than that detected at the control site. Levels of Cd, Mg, and Pb were also higher at all of the monitored sites compared with the control site. Both genomic and proteomic methods were used to study the response of H. cupressiforme to air pollution. No DNA damage or mutations were detected using the amplified fragment length polymorphisms (AFLP) method. At the protein level, 15 gel spots exhibited differential expression profiles between the moss samples collected at the IA1 site and the control site. Furthermore, among the 14 spots that showed a decrease in protein expression, nine were associated with ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and proteins of the light-harvesting complexes of photosystem (PS) II, three were associated with protein synthesis, and three were stress-related proteins. Thus, some of these proteins may represent good moss biosensors which could be used as pre-alert markers of environmental pollution.

Grape microbiome as a reliable and persistent signature of field origin and environmental conditions in Cannonau wine production

Grape berries harbor a wide range of microbes originating from the vineyard environment, many of which are recognized for their role in the must fermentation process shaping wine quality. To better clarify the contribution of the microbiome of grape fruits during wine fermentation, we used high-throughput sequencing to identify bacterial and fungi communities associated with berries and musts of Cannonau. This is the most important cultivar-wine of Sardinia (Italy) where most vineyards are cultivated without phytochemical treatments. Results suggested that microbiomes of berries collected at four different localities share a core composition characterized by Enterobacteriales, Pseudomonadales, Bacillales, and Rhodospirillales. However, any area seems to enrich berries microbiome with peculiar microbial traits. For example, berries belonging to the biodynamic vineyards of Mamoiada were rich in Bacillales typical of manure (i.e. Lysinibacillus, Bacillus, and Sporosarcina), whereas in the Santadi locality, berries showed soil bacteria such as Pasteurellales and Bacteroidales as well as Rhodospirillales and Lactobacillales which are commonly involved in wine fermentation. In the case of fungi, the most abundant taxa were Dothioraceae, Pleosporaceae, and Saccharomycodaceae, and although the proportion of these families varied among localities, they occurred ubiquitously in all vineyards. During vinification processes performed at the same wine cellar under controlled conditions and without using any yeast starter, more than 50% of bacteria groups of berries reached musts, and each locality had its own private bacteria signature, even if Saccharomyces cerevisiae represented the most abundant fungal species. This work suggests that natural berries microbiome could be influenced by pedoclimatic and anthropologic conditions (e.g., farming management), and the fruits’ microorganisms persist during the fermentation process. For these reasons, a reliable wine genotyping should include the entire holobiont (plant and all its symbionts), and bioprospecting activities on grape microbiota could lead to improved viticulture yields and wine quality.