Polyphasic identification of Penicillia and Aspergilli isolated from Italian grana cheese

"Available online 18 January 2018"Penicillium and Aspergillus genera, both including mycotoxin producing species, were reported as associated to cheese and cheese working environment, but never studied in an extensive way in Italian grana cheese (Grana Padano and Parmigiano Reggiano). The aim of this work was to address the identification of Aspergilli and Penicillia associated to grana cheese in order to lay down the basis for risk assessment and safe processing for a high quality production. One hundred and four strains belonging to Aspergillus and Penicillium genera were obtained from cheese crust and from ripening room air (with the latter largely dominant), and identified following a polyphasic approach, strongly required for the identification at the species level. Morphological observation was used along with molecular techniques, RAPD-PCR fingerprinting and calmodulin gene sequencing (CaM), the former aimed to limit as much as possible the latter sequencing effort. Seventy four percent of the strains were assigned to Penicillium subgenus Penicillium, section Fasciculata. Main mycotoxin producing species identified were A. flavus, P. crustosum and P. verrucosum, while the dominant species in both air and cheese crust was P. solitum, which has never been so far reported as mycotoxigenic. Results obtained in this study confirmed that mycotoxin contamination is a possible issue to face during grana cheese making.The present work was supported by the following institutions: Consorzio del Formaggio Parmigiano Reggiano, Consorzio per la tutela del Formaggio Grana Padano, Fondazione Romeo ed Enrica Invernizzi, the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/ 2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Simone Decontardi carried out this work within the PhD school “Agrisystem” of the Universita Cattolica del Sacro Cuore (Italy).info:eu-repo/semantics/publishedVersio

Polyphasic identification of Penicillium spp. isolated from Spanish semi-hard ripened cheeses

P. 409-444Fifteen samples of semi-hard ripened cheeses, both spoiled (10) and unspoiled (5), and obtained from cheese factories located in Northwest of Spain, were analysed by a dilution plating technique and direct sampling. A total of 32 isolates were identified at species level by a polyphasic approach (phenotypic characterization, partial extrolite analysis and molecular identification). Most isolates (65.6%) belonged to the species P. commune; other species found were P. solitum, P. chrysogenum, P. nordicum, P. expansum and P. cvjetkovicii. All of the P. commune isolates were able to produce cyclopiazonic acid, while the P. nordicum and the P. expansum isolates were producers of ochratoxin A and patulin respectively. Despite this, the role of P. commune as beneficial fungi in cheese ripening should be investigated. Molecular identification based on BenA sequence analysis was able to identify the majority of isolates. The three mycotoxins investigated can be considered key for identification. The polyphasic approach seems to be a very valuable tool for identification of isolates of this complex genusS

Active Whey Protein Edible Films and Coatings Incorporating Lactobacillus buchneri for Penicillium nordicum Control in Cheese

Fungal contamination of food is responsible for health issues and food waste. In this work, the incorporation of a lactic acid bacteria (LAB) with antifungal properties (Lactobacillus buchneri UTAD104) into whey protein-based films and coatings was tested for the control of an ochratoxigenic fungi (Penicillium nordicum) in a cheese matrix. The incorporation of L. buchneri cells resulted in thicker films with less luminosity than control films and colour alteration. Nevertheless, cells inclusion did not alter moisture content, water vapour permeability, mechanical properties, hydrophobicity and chemical structure of the films. Whey protein films were able to maintain the viability of L. buchneri UTAD104 cells in 105 CFU/mL after 30 days of storage at 25 \textdegreeC. When applied in cheese, films and coatings containing L. buchneri cells prevented fungal contamination for at least 30 days, while control cheeses with films and coatings either without LAB or with Lactobacillus casei UM3 (a strain without antifungal ability) showed fungal contamination during that period. Ochratoxin A was not found in cheeses treated with films and coatings containing L. buchneri UTAD104. Results showed that the inclusion of a LAB with antifungal properties in edible films and coatings can help to reduce or eliminate P. nordicum contamination in cheeses.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Ana Guimarães received support through grant SFRH/BD/103245/2014 from the Portuguese FCT.info:eu-repo/semantics/publishedVersio

Risks to human and animal health related to the presence of deoxynivalenol and its acetylated and modified forms in food and feed

Deoxynivalenol (DON) is a mycotoxin primarily produced by Fusarium fungi, occurring predominantly in cereal grains. Following the request of the European Commission, the CONTAM Panel assessed the risk to animal and human health related to DON, 3-acetyl-DON (3-Ac-DON), 15-acetyl-DON (15-Ac-DON) and DON-3-glucoside in food and feed. A total of 27,537, 13,892, 7,270 and 2,266 analytical data for DON, 3-Ac-DON, 15-Ac-DON and DON-3-glucoside, respectively, in food, feed and unprocessed grains collected from 2007 to 2014 were used. For human exposure, grains and grain-based products were main sources, whereas in farm and companion animals, cereal grains, cereal by-products and forage maize contributed most. DON is rapidly absorbed, distributed, and excreted. Since 3-Ac-DON and 15-Ac-DON are largely deacetylated and DON-3-glucoside cleaved in the intestines the same toxic effects as DON can be expected. The TDI of 1 μg/kg bw per day, that was established for DON based on reduced body weight gain in mice, was therefore used as a group-TDI for the sum of DON, 3-Ac-DON, 15-Ac-DON and DON-3-glucoside. In order to assess acute human health risk, epidemiological data from mycotoxicoses were assessed and a group-ARfD of 8 μg/kg bw per eating occasion was calculated. Estimates of acute dietary exposures were below this dose and did not raise a health concern in humans. The estimated mean chronic dietary exposure was above the group-TDI in infants, toddlers and other children, and at high exposure also in adolescents and adults, indicating a potential health concern. Based on estimated mean dietary concentrations in ruminants, poultry, rabbits, dogs and cats, most farmed fish species and horses, adverse effects are not expected. At the high dietary concentrations, there is a potential risk for chronic adverse effects in pigs and fish and for acute adverse effects in cats and farmed mink

Evaluation in vitro des effets toxiques de contaminants alimentaires sur les cellules dendritiques

Les trichothécènes, mycotoxines produites par différentes espèces de moisissures, sont à l'origine d'intoxications alimentaires graves qui touchent aussi bien l'animal d'élevage que l'homme. Les intoxications alimentaires humaines dues aux mycotoxines sont en général accidentelles. Les maladies décrites à la suite d'intoxications aux trichothécènes sont principalement l'Aleucie Toxique Alimentaire (ATA) rencontrée dans les pays d'Europe de l'Est et les pays en voie de développement, la Stachybotryotoxicose, rencontrée en Europe de l'Ouest et en Amérique du Nord, et " l'Akakabio disease " rencontrée dans le Sud-Est asiatique. L'origine de ses troubles est hématologique. En effet, les trichothécènes sont connues pour leur hématoxicité mais également pour leur immunotoxicité. Une cellule fait le lien entre ces deux toxicités : la cellule dendritique. Les cellules immunitaires sont produites par des cellules souches, au sein de la moelle osseuse. Parmi ces cellules, la cellule dendritique apparaît comme la cellule initiatrice de la réponse immunitaire primaire, capable de stimuler la prolifération des lymphocytes T naïfs. Le but de ce travail est de développer un modèle humain utilisant les cellules dendritiques générées in vitro à partir de monocytes. Ce modèle permettant d'étudier les effets de xénobiotiques sur la maturation des cellules dendritiques. Pour réaliser cette étude, il s'est avéré nécessaire de choisir un modèle cellulaire pertinent et d'optimiser ce modèle pour l'adapter à l'étude des effets immunotoxiques de contaminants de l'alimentation. Ce travail montre l'importance des études réalisées in vitro sur les cellules dendritiques humaines dans la compréhension des effets immunotoxiques de contaminants alimentaires. Il paraît pertinent de proposer que ces études soient réalisées très en amont au cours de l'exploration d'un effet immunotoxique.The trichothecenes, mycotoxins produced by various species of moulds, are at the origin of serious food poisonings which touch as well the livestock as the man. The human food poisonings due to the mycotoxins are in general accidental. The diseases described following intoxications with trichothecenes are mainly Aleucie Toxique Food (ATA) met in the countries of Eastern Europe and the countries in the process of development, Stachybotryotoxicose, met in Western Europe and North America, and Akakabio disease met in the South-East Asia. The origin of its disorders is hematologic. Indeed, the trichothecenes are known for their hematoxicity but also for their immunotoxicity. A cell establishes the link between these two toxicities: the dendritic cell. The immunizing cells are produced by cells stocks, within osseous marrow. Among these cells, the dendritic cell seems the initiating cell of the immunizing answer primary, able to stimulate the proliferation of the lymphocytes T naive. Taking into account the immunotoxicity of the mycotoxins, primarily on the lymphocytes T, it seemed relevant to study the effects of these molecules on the dendritic cells. To make this study, it proved to be necessary to choose a relevant cellular model and to optimize this model to adapt to the study of the adverse effects of contaminants of the food. The model using the CD34+ involves the majority formation of cells of Langerhans (dentritic cells of the skin). These last are implied in the cutaneous reactions, without interest for the study of the effects of food contaminants. This is why the choice was made on dendritic cultures of cells deriving from monocytes, rather than of cells CD34+. The aim of this work is to develop a human model using the dendritic cells generated in vitro from monocytes. This model allowing to study the effects of xenobiotic on maturation of the dendritic cells. This work shows the importance of the studies carried out in vitro on the human dendritic cells in the comprehension of the immunotoxic effects of food contaminants. It appears relevant to propose that these studies are carried out very upstream during the exploration of a immunotoxic effect.BREST-BU Droit-Sciences-Sports (290192103) / SudocSudocFranceF

Innovative in vitro approaches to toxicological investigations of mycotoxins effects

Abstract Among the potential contaminants, mycotoxins are of particular concern due to the importance in terms of food and feed safety. The difficulty in establishing a diagnosis for mycotoxicosis relies in the fact that the effects are subclinical, and that multicontamination by various toxins is the most common scenario. The co‐occurrence of these mycotoxins raises questions concerning both food safety and regulation. However, there is still limited knowledge on toxicity data on co‐exposure. The current technical report will describe the activities performed by the fellow in the LUBEM‐Brest University (France). In this context, the work programme offered by the hosting site consisted in vitro toxicological approaches to evaluate the toxicity of mycotoxin mixtures. The aim of this project was to assess human risk to the exposure of two main regulated mycotoxins (ochratoxin A and fumonisin B1) using different innovative cellular models (2D and 3D spheroids). In this framework, these mycotoxins were tested individually and as a combination on intestinal and hepatic cell lines alone or in co‐cultures. Overall, our results show the outstanding potential of using more predictive and realistic approaches for the risk assessment (RA) of mycotoxins. It is of high importance to pursue further toxicological characterisations and exposure evaluations for mycotoxins, in order to determine a more detailed RA. This will serve as a reference to understand multicontamination mechanism of mycotoxins at the cell level and help authority to revise regulation

Fuzzy Model for the Automatic Recognition of Human Dendritic Cells

International audienceBackground and objective: Nowadays, foodborne illness is considered one of the most outgrowing diseases in the world, and studies show that its rate increases sharply each year. Foodborne illness is considered a public health problem which is caused by numerous factors, such as food intoxications, allergies, intolerances, etc. Mycotoxin is one of the food contaminants which is caused by various species of molds (or fungi), which, in turn, causes intoxications that can be chronic or acute. Thus, even low concentrations of Mycotoxin have a severely harmful impact on human health. It is, therefore, necessary to develop an assessment tool for evaluating their impact on the immune response. Recently, researchers have approved a new method of investigation using human dendritic cells, yet the analysis of the geometric properties of these cells is still visual. Moreover, this type of analysis is subjective, time-consuming, and difficult to perform manually. In this paper, we address the automation of this evaluation using image-processing techniques. Methods: Automatic classification approaches of microscopic dendritic cell images are developed to provide a fast and objective evaluation. The first proposed classifier is based on support vector machines (SVM) and Fisher’s linear discriminant analysis (FLD) method. The FLD–SVM classifier does not provide satisfactory results due to the significant confusion between the inhibited cells on one hand, and the other two cell types (mature and immature) on the other hand. Then, another strategy was suggested to enhance dendritic cell recognition results that are emitted from microscopic images. This strategy is mainly based on fuzzy logic which allows us to consider the uncertainties and inaccuracies of the given data. Results: These proposed methods are tested on a real dataset consisting of 421 images of microscopic dendritic cells, where the fuzzy classification scheme efficiently improved the classification results by successfully classifying 96.77% of the dendritic cells. Conclusions: The fuzzy classification-based tools provide cell maturity and inhibition rates which help biologists evaluate severe health impacts caused by food contaminants

Fuzzy Model for the Automatic Recognition of Human Dendritic Cells

Background and objective: Nowadays, foodborne illness is considered one of the most outgrowing diseases in the world, and studies show that its rate increases sharply each year. Foodborne illness is considered a public health problem which is caused by numerous factors, such as food intoxications, allergies, intolerances, etc. Mycotoxin is one of the food contaminants which is caused by various species of molds (or fungi), which, in turn, causes intoxications that can be chronic or acute. Thus, even low concentrations of Mycotoxin have a severely harmful impact on human health. It is, therefore, necessary to develop an assessment tool for evaluating their impact on the immune response. Recently, researchers have approved a new method of investigation using human dendritic cells, yet the analysis of the geometric properties of these cells is still visual. Moreover, this type of analysis is subjective, time-consuming, and difficult to perform manually. In this paper, we address the automation of this evaluation using image-processing techniques. Methods: Automatic classification approaches of microscopic dendritic cell images are developed to provide a fast and objective evaluation. The first proposed classifier is based on support vector machines (SVM) and Fisher’s linear discriminant analysis (FLD) method. The FLD–SVM classifier does not provide satisfactory results due to the significant confusion between the inhibited cells on one hand, and the other two cell types (mature and immature) on the other hand. Then, another strategy was suggested to enhance dendritic cell recognition results that are emitted from microscopic images. This strategy is mainly based on fuzzy logic which allows us to consider the uncertainties and inaccuracies of the given data. Results: These proposed methods are tested on a real dataset consisting of 421 images of microscopic dendritic cells, where the fuzzy classification scheme efficiently improved the classification results by successfully classifying 96.77% of the dendritic cells. Conclusions: The fuzzy classification-based tools provide cell maturity and inhibition rates which help biologists evaluate severe health impacts caused by food contaminants

Automatic Human Dendritic Cells Segmentation Using K-Means Clustering and Chan-Vese Active Contour Model

International audienc