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.

Orally administered multispecies probiotic formulations to prevent uro-genital infections: a randomized placebo-controlled pilot study.

PURPOSE: The aim of this study was to evaluate in the vagina of 60 pre-menopausal women the detection of orally administered multispecies probiotic formulations showing anti-microbial properties in test in vitro. METHODS: A randomized, double-blind, three-arm parallel pilot study was carried out on 60 pre-menopausal women. Subjects were randomly divided in three groups (F_1, F_2, F_3). Each group received a daily oral administration of probiotic mixtures (for 14 days and at the day 21, 7 days after the wash-out) containing: Lactobacillus acidophilus and Lactobacillus reuteri (F_1), or Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis (F_2), or placebo (F_3), respectively. Vaginal swabs were collected at four experimental times, at t0 and at t7, t14 and t21 days, and analyzed by qPCR. At the same time, the anti-microbial activity of the probiotic formulations was verified by assays in vitro against microorganisms as Escherichia coli and Candida albicans. RESULTS: L. acidophilus and L. reuteri as well as L. plantarum, L. rhamnosus and B. lactis were significantly increased on 7 days in the groups administered with F_1 and F_2, respectively, compared to group F_3. A similar significant trend was observed on 21 days, 7 days after the wash-out. F_1 and F_2 showed coherent anti-microbial properties. CONCLUSION: Both probiotic formulations F_1 and F_2, chosen because of their anti-microbial activity against pathogens responsible for vaginal dysbiosis and infections, led to vaginal detection and enhancement of the amount of species of formulates when orally administered. This work provides the basis for further clinical investigations of the F_1 and F_2 capacity to prevent or treat uro-genital infections.

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.

A Fast and Simple DNA Mini-barcoding and RPA Assay Coupled with Lateral Flow Assay for Fresh and Canned Mackerel Authentication.

Nowadays, food authentication is more and more required given its relevance in terms of quality and safety. The seafood market is heavily affected by mislabelling and fraudulent substitutions/adulterations, especially for processed food products such as canned food items, due to the loss of morphological features. This study aims to develop new assays based on DNA to identify fresh mackerel (Scomber spp.) and commercial products. A new primer pair was de novo designed on the 5S rRNA gene and non-transcribed spacer (NTS), identifying a DNA mini-barcoding region suitable for species identification of processed commercial products. Moreover, to offer a fast and low-cost analysis, a new assay based on recombinase polymerase amplification (RPA) was developed for the identification of fresh 'Sgombro' (Scomber scombrus) and 'Lanzardo o Occhione' (Scomber japonicus and Scomber colias), coupled with the lateral flow visualisation for the most expensive species (Scomber scombrus) identification. This innovative portable assay has great potential for supply chain traceability in the seafood market. Supplementary Information: The online version contains supplementary material available at 10.1007/s12161-022-02429-6.

Check Your Shopping Cart: DNA Barcoding and Mini-Barcoding for Food Authentication.

The molecular approach of DNA barcoding for the characterization and traceability of food products has come into common use in many European countries. However, it is important to address and solve technical and scientific issues such as the efficiency of the barcode sequences and DNA extraction methods to be able to analyze all the products that the food sector offers. The goal of this study is to collect the most defrauded and common food products and identify better workflows for species identification. A total of 212 specimens were collected in collaboration with 38 companies belonging to 5 different fields: seafood, botanicals, agrifood, spices, and probiotics. For all the typologies of specimens, the most suitable workflow was defined, and three species-specific primer pairs for fish were also designed. Results showed that 21.2% of the analyzed products were defrauded. A total of 88.2% of specimens were correctly identified by DNA barcoding analysis. Botanicals (28.8%) have the highest number of non-conformances, followed by spices (28.5%), agrifood (23.5%), seafood (11.4%), and probiotics (7.7%). DNA barcoding and mini-barcoding are confirmed as fast and reliable methods for ensuring quality and safety in the food field.

DNA-Based Herbal Teas' Authentication: An ITS2 and psbA-trnH Multi-Marker DNA Metabarcoding Approach.

Medicinal plants have been widely used in traditional medicine due to their therapeutic properties. Although they are mostly used as herbal infusion and tincture, employment as ingredients of food supplements is increasing. However, fraud and adulteration are widespread issues. In our study, we aimed at evaluating DNA metabarcoding as a tool to identify product composition. In order to accomplish this, we analyzed fifteen commercial products with DNA metabarcoding, using two barcode regions: psbA-trnH and ITS2. Results showed that on average, 70% (44-100) of the declared ingredients have been identified. The ITS2 marker appears to identify more species (n = 60) than psbA-trnH (n = 35), with an ingredients' identification rate of 52% versus 45%, respectively. Some species are identified only by one marker rather than the other. Additionally, in order to evaluate the quantitative ability of high-throughput sequencing (HTS) to compare the plant component to the corresponding assigned sequences, in the laboratory, we created six mock mixtures of plants starting both from biomass and gDNA. Our analysis also supports the application of DNA metabarcoding for a relative quantitative analysis. These results move towards the application of HTS analysis for studying the composition of herbal teas for medicinal plants' traceability and quality control.

Anticholesterolemic Activity of Three Vegetal Extracts (Artichoke, Caigua, and Fenugreek) and Their Unique Blend.

Hepatic-related diseases, in particular hyperlipidemia and hypercholesterolemia, are a thorn on the side of the national health institutes around the globe. Indeed, liver lipid and cholesterol dysregulation could lead to atherosclerotic plaque formation and cardiovascular diseases. Currently, statin administration and monacolin K consumption are the main therapies proposed to counter this alarming connection, but relevant side effects are known. To overcome this issue, safe nutraceutical formulations and/or vegetal extracts, endowed with anticholesterolemic activity, could be instrumental in hypercholesterolemia prevention and treatment. In the present work, the anticholesterolemic efficacy of three vegetal extracts used in traditional medicine (artichoke, caigua, and fenugreek), their unique blend (ACFB), and the monacolin K-containing red yeast extract (RYR), was investigated with an in vitro approach based on hepatic cell line HepG2. The impact on cholesterol of the three extracts, their blend, and RYR were investigated by determining hepatocyte total and free cholesterol and bile acids biosynthesis. According to our results, the anticholesterolemic activity of the vegetal extracts was confirmed, and a novel choleretic activity of caigua extract was evidenced. ACFB showed to be safer than RYR while showing a similar effect on total and free cholesterol and bile acids synthesis compared to it. The anticholesterolemic activity of the blend was obtained with lower vegetal extract concentrations compared with the single vegetal extract, potentially indicating an additive effect between the extracts. In conclusion, the vegetal extracts and their blend, ACFB, are safe and are endowed with anticholesterolemic activity, potentially providing complementary therapies to the statin-based ones for hyperlipidemia and hypercholesterolemia-related complications.

The hidden 'plant side' of insect novel foods: A DNA-based assessment.

In the context of novel foods, a category for which the market demand is increasing worldwide, the consumption of edible insects and related insect-based products is expected to grow in the next years. Insects represent an important source of energy for the human diet but there is a lack of scientific knowledge about their processing to ensure safe food items to the consumer. In this study we adopted a combined DNA-based approach to verify the identity of the declared species in five categories of commercial insect-based products (mt COI DNA barcoding) and to characterize plant declared ingredients or contaminants (nu ITS2 DNA metabarcoding) with particular attention to putative elements of allergenic concern belonging, for example to the insect rearing substrate. Moreover, the same approach has been used to assess its sensitivity to cases of contamination and counterfeits to insect flour with low cost (and potentially allergenic) vegetable flours like wheat and soybean. Results show the success of insect DNA barcoding authentication even for highly processed products. Furthermore, the DNA metabarcoding analysis revealed a high efficacy as a screening method to identify both plant ingredients and vegetal traces belonging to insect farming or possible adulteration events, also acting as an early warning strategy for the occurrence of allergens of human concern. This approach could support the development of new risk assessment procedures for novel foods by regulatory authorities to ensure their quality, safety, and acceptance which will become more required in order to face the challenge of feeding the world population in the next decades.

Development of a DNA Barcoding-Like Approach to Detect Mustard Allergens in Wheat Flours.

The spread of food allergens is a topic of global importance due to its impact on public health. National and International regulations ask food producers and manufacturers to declare product compositions on the label, especially in case of processed raw materials. Wheat flour (Triticum aestivum) can be contaminated by a wide range of species belonging to the Brassicaceae in the field or during grain harvests, storage, and processing. Among them, mustards (Brassica nigra, Brassica juncea and Sinapis alba) are well known allergenic species. Often, food quality laboratories adopt an ELISA approach to detect the presence of mustard species. However, this approach shows cross-reactivity with other non-allergenic species such as Brassica napus (rapeseed). In the last few years, DNA barcoding was proposed as a valid identification method, and it is now commonly used in the authentication of food products. This study aims to set up an easy and rapid DNA-based tool to detect mustard allergenic species. DNA barcoding (matK and ITS2) and chromosome markers (A6, B, C1 genome regions) were selected, and specific primers were validated on incurred reference food matrices. The developed test was proven to be able to distinguish mustard from rapeseed and wheat, overcoming cross-reactivity with Brassica napus.

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

[This corrects the article DOI: 10.1155/2016/4740907.].

Geographical and Cultivar Features Differentiate Grape Microbiota in Northern Italy and Spain Vineyards.

Recent studies have highlighted the role of the grapevine microbiome in addressing a wide panel of features, ranging from the signature of field origin to wine quality. Although the influence of cultivar and vineyard environmental conditions in shaping the grape microbiome have already been ascertained, several aspects related to this topic, deserve to be further investigated. In this study, we selected three international diffused grapevine cultivars (Cabernet Sauvignon, Syrah, and Sauvignon Blanc) at three germplasm collections characterized by different climatic conditions [Northern Italy (NI), Italian Alps (AI), and Northern Spain (NS)]. The soil and grape microbiome was characterized by 16s rRNA High Throughput Sequencing (HTS), and the obtained results showed that all grape samples shared some bacterial taxa, regardless of sampling locality (e.g., Bacillus, Methylobacterium, Sphingomonas, and other genera belonging to Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria). However, some Operational Taxonomic Units (OTUs) could act as geographical signatures and in some cases as cultivar fingerprint. Concerning the origin of the grape microbiome, our study confirms that vineyard soil represents a primary reservoir for grape associated bacteria with almost 60% of genera shared between the soil and grape. At each locality, grapevine cultivars shared a core of bacterial genera belonging to the vineyard soil, as well as from other local biodiversity elements such as arthropods inhabiting or foraging in the vineyard. Finally, a machine learning analysis showed that it was possible to predict the geographical origin and cultivar of grape starting from its microbiome composition with a high accuracy (9 cases out of 12 tested samples). Overall, these findings open new perspectives for the development of more comprehensive and integrated research activities to test which environmental variables have an effective role in shaping the microbiome composition and dynamics of cultivated species over time and space.

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.

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.

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.

Towards a Universal Approach Based on Omics Technologies for the Quality Control of Food.

In the last decades, food science has greatly developed, turning from the consideration of food as mere source of energy to a growing awareness on its importance for health and particularly in reducing the risk of diseases. Such vision led to an increasing attention towards the origin and quality of raw materials as well as their derived food products. The continuous advance in molecular biology allowed setting up efficient and universal omics tools to unequivocally identify the origin of food items and their traceability. In this review, we considered the application of a genomics approach known as DNA barcoding in characterizing the composition of foodstuffs and its traceability along the food supply chain. Moreover, metabolomics analytical strategies based on Nuclear Magnetic Resonance (NMR) and Mass Spectroscopy (MS) were discussed as they also work well in evaluating food quality. The combination of both approaches allows us to define a sort of molecular labelling of food that is easily understandable by the operators involved in the food sector: producers, distributors, and consumers. Current technologies based on digital information systems such as web platforms and smartphone apps can facilitate the adoption of such molecular labelling.