Implementation of a food safety management system: a practical guide for micro-companies / Implementación de un sistema de gestión de seguridad alimentaria: una guía práctica para microempresas

Food system is continuously exposed to several safety hazards throughout manufacturing processes. Consequently, the understanding of these mechanisms, the likelihood of their occurrence, and the consequences involves the implementation of an appropriate risk assessment program. Hazard Analysis and Safety Critical Control Points (HACCP) is a powerful tool for self-control system which is fundamental to adapt the requirements of each sector and size of a company but however, is currently limited it at industrial level. In fact, as a result to its flexible application, the recommendations at European level for companies with less than 10 workers focused on a food safety management system (FSMS) based on the principles of good hygiene and manufacturing practices. Therefore, in order to help companies implementing self-monitoring systems, several reference guides have been created under the supervision of health authorities. For this purpose, the aim of these study was to create an abbreviated guide as an introduction to self-control in food safety for spanish micro-enterprises. It is presented as a summary of each prerequisite focused solely on understanding the meaning and documentation necessary to implement it. To carry out the research, European legislation and the main guides at national level, particularly from autonomous communities were took into account. Starting from this information, was provided a classification of the main plans related to prerequisites for hygiene and traceability fulfilling. Moreover, indications concerning the documentation which should be prepared for each one of the prerequisites was detailed. (AU)

El sistema alimentario está continuamente expuesto a varios riesgos de seguridad a lo largo de los procesos de fabricación. En consecuencia, la comprensión de estos mecanismos, la probabilidad de su ocurrencia y las consecuencias implica la implementación de un programa adecuado de evaluación de riesgos. El Análisis de Peligros y Puntos Críticos de Control de Seguridad (APPCC) es una poderosa herramienta para el sistema de autocontrol que es fundamental para adaptar los requisitos de cada sector y tamaño de una empresa pero que, sin embargo, actualmente está limitado a nivel industrial. De hecho, como consecuencia de su aplicación flexible, las recomendaciones a nivel europeo para empresas de menos de 10 trabajadores se centraron en un sistema de gestión de la seguridad alimentaria (SGSA) basado en los principios de buenas prácticas de higiene y fabricación. Por ello, con el fin de ayudar a las empresas a implementar sistemas de autocontrol, se han elaborado varias guías de referencia bajo la supervisión de las autoridades sanitarias. Para ello, el objetivo de este estudio fue crear una guía abreviada como introducción al autocontrol en seguridad alimentaria para microempresas españolas. Se presenta como un resumen de cada requisito previo enfocado únicamente en comprender el significado y la documentación necesaria para implementarlo. Para realizar la investigación se tuvo en cuenta la legislación europea y las principales guías a nivel nacional, en particular de las comunidades autónomas. A partir de esta información, se proporcionó una clasificación de los principales planes relacionados con los requisitos previos para el cumplimiento de la higiene y la trazabilidad. Además, se detallaron indicaciones sobre la documentación que se debe preparar para cada uno de los requisitos previos. (AU)

The importance of transcriptomics and proteomics for studying molecular mechanisms of mycotoxin exposure: A review.

This review aims to highlight recent advances where transcriptomics and proteomics have been used as a key tool to understand molecular toxicity of mycotoxins. The most studied mycotoxin by using transcriptomic approach is deoxynivalenol (DON), followed by aflatoxins (AFs) and zearalenone (ZEA). Instead, proteomics mostly focuses on AFs but also in this case, mildly to ZEA and DON. However, in both omics approaches, fewer studies investigated the toxicological effect of emerging mycotoxins, patulin, ochratoxin A, T-2 toxin, alternariol and amino-14,16-dimethyloctadecan-3-ol. The study of changes in the expression of genes involved in immune system are the most common purposes for transcriptomics whereas cellular processes in proteomics field. Concerning the techniques used to perform the experiments, RT-qPCR is the most employed in gene expression analysis whereas liquid chromatography coupled with mass spectrometry is the master technique for proteomics assays. The gathered data have reported that the interest in using these omic approaches has increased in the last five years. However, in vitro models take precedence over the in vivo and ex vivo ones. Therefore, there is a need to enhance the use of in vivo models and alternative methods to better understand mycotoxins mode of action on animal and human health.

In vitro blood brain barrier exposure to mycotoxins and carotenoids pumpkin extract alters mitochondrial gene expression and oxidative stress.

Food and feed are daily exposed to mycotoxin contamination which effects may be counteracted by antioxidants like carotenoids. Some mycotoxins as well as carotenoids penetrate the blood brain barrier (BBB) inducing alterations related to redox balance in the mitochondria. Therefore, the in vitro BBB model ECV304 was subcultured for 7 days and exposed to beauvericine, enniatins, ochratoxin A, zearalenone (100 nM each), individually and combined, and pumpkin extract (500 nM). Reactive oxygen species were measured by fluorescence using the dichlorofluorescein diacetate probe at 0 h, 2 h and 4 h. Intracellular ROS generation reported was condition dependent. RNA extraction was performed and gene expression was analyzed by qPCR after 2 h exposure. The selected genes were related to the Electron Transport Chain (ETC) and mitochondrial activity. Gene expression reported upregulation for exposures including mycotoxins plus pumpkin extract versus individual mycotoxins. Beauvericin and Beauvericin-Enniatins exposure significantly downregulated Complex I and pumpkin addition reverted the effect upregulating Complex I. Complex IV was the most downregulated structure of the ETC. Thioredoxin Interacting Protein was the most upregulated gene. These data confirm that mitochondrial processes in the BBB could be compromised by mycotoxin exposure and damage could be modulated by dietary antioxidants like carotenoids.

Proteomics evaluation of enniatins acute toxicity in rat liver.

Enniatins (ENs) are emerging mycotoxins produced by Fusarium fungi which are cytotoxic also at low concentrations due to its ionophoric properties. The aim of this study was to evaluate the hepatic toxicity of ENs exposure at different concentrations in Wistar rats through a proteomic approach. Animals were intoxicated by oral gavage with medium (EN A 256, ENA1 353, ENB 540, ENB1 296 µg/mL) and high concentrations (ENA 513, ENA1 706, ENB 1021, ENB1 593 µg/mL) of an ENs mixture and sacrificed after 8 h. Protein extraction was performed using powdered liver. Peptides were analyzed using a liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer. Proteins were filtered by abundance using Mass Professional Profiler software (Agilent Technologies) and 57 were differentially expressed when compared to the control. In terms of abundance, the liver biomarker Carboamoyl-phosphate synthase showed the highest levels in all conditions employed while actin-1 had the lowest. Bioinformatic analysis using DAVID platform reported acetylation, nucleotide phosphate-binding region:NAD and catalytic activity as the most represented terms. Furthermore, metabolism was the most significant and enriched pathway in Reactome overrepresentation. In conclusion, ENs acute exposure caused protein expression changes related to major cellular processes in rats, hinting its involvement in liver disturbance.

In vitro exposure to pumpkin extract induces a protective transcriptomic profile in blood brain barrier electron transport chain

C. maxima (var. Delica) is a variety of pumpkin known for its beneficial effects and its high content in carotenoids (violaxanthin, astaxanthin, antheraxanthin, zeaxanthin, lutein, lycopene and β-carotene), which are natural antioxidants bioavailable to humans through food consumption. Numerous biological effects have been attributed to carotenoids due to their antioxidant activity: improved immune response, anti-inflammatory and anti-tumor properties and reduced risk of cardiovascular and chronic degenerative diseases. They are capable of accumulating in the brain after crossing the blood-brain barrier. Therefore, the aim of this study is to analyze changes in mitochondrial gene expression using an in vitro cell model (ECV304) of the blood brain barrier, after exposure to pumpkin extract. Cells were treated during 24 h at 5 different β-carotene concentrations, as reference extract compound: 1.72×10-4 – 1.72×10-3– 1.72×10-2 – 0.172 – 1.72 nM in DMSO 0.5%. The extracted RNA was used to perform qPCR analysis on 15 mitochondrial related genes: MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-CO1, MT-CO3, MT-ATP6, MT-ATP8, MT-RNR2, MRPL12, OSGIN1, SRXN1, TXNIP, UCP2, and S18 as reference gene. Results demonstrate that dietary carotenoids act at transcriptional level, especially on the genes belonging to the electron transport chain, reporting an overall protective pattern. The findings show a dose dependent differential gene expression pattern by carotenoids exposure, even at low concentrations. (AU)

Toxicity of mycotoxins in vivo on vertebrate organisms: A review.

Mycotoxins are considered to be a major risk factor affecting human and animal health as they are one of the most dangerous contaminants of food and feed. This review aims to compile the research developed up to date on the toxicological effects that mycotoxins can induce on human health, through the examination of a selected number of studies in vivo. AFB1 shows to be currently the most studied mycotoxin in vivo, followed by DON, ZEA and OTA. Scarce data was found for FBs, PAT, CIT, AOH and Fusarium emerging mycotoxins. The majority of them concerned the investigation of immunotoxicity, whereas the rest consisted in the study of genotoxicity, oxidative stress, hepatotoxicity, cytotoxicity, teratogenicity and neurotoxicity. In order to assess the risk, a wide range of different techniques have been employed across the reviewed studies: qPCR, ELISA, IHC, WB, LC-MS/MS, microscopy, enzymatic assays, microarray and RNA-Seq. In the last decade, the attention has been drawn to immunologic and transcriptomic aspects of mycotoxins' action, confirming their toxicity at molecular level. Even though, more in vivo studies are needed to further investigate their mechanism of action on human health.