Extension of the Salmonella detection method UNE-EN ISO 6579:2003

Motivation: The food sector is in a continuous fight against infectious diseases, among which the most relevant is the bacteria Salmonella. The disease it causes is called Salmonellosis, and it can affect both animals and humans. Salmonellosis causes big losses in the food sector, since it poses a risk to public health, so it is important control it. For this reason, laboratories carry out routinary  food quality controls to ensure the innocuity of the food analized. There are several methods whose objective is to detect efficiently the presence Salmonella bacteria in food. The one we have used in the laboratory is a conventional method whose reglament is established in UNE-EN ISO 6579:2003. The goal of the project is to expand the detection method of Salmonella ISO 6579, in order to confirm that this detection method can ben used efficiently in the tested products.Methods: The ISO 6579:2003 is an horizontal method that consists of a pre-enrichment phase of the test sample in a nonselective liquid medium, followed by a selective enrichment phase, followed by a final phase in which any possible Salmonella colonies are grown in selective, solid culture media. In this way it is posible to confirm or rule out the presence of colonies, and carry out their characterization by biochemical and serological tests. Eight different foods that have not been validated before in our laboratory were artificially contaminated with 10 UFC Salmonella. The limit of detection and the influence of the presence of other microorganisms were both analized.Results: Biochemical and serological confirmation tests have determined the presence of Salmonella in all food products evaluated for the established detection limit. We have also verified that the presence of accompanying flora does not influence the process of Salmonella detection.Conclusions: Since it has been possible to detect the presence of Salmonella under the established detection limit, and with the presence of accompanying flora, it can be concluded that the ISO 6579 methodology is effective in the evaluated foods. Therefore, these food products have been validated in the laboratory following the UNE-EN ISO 6579:2003 regulations and we have carried out the extension of this validation metho

Transition to the reference standard UNE-EN ISO 6579-1: 2017.

Motivation: Nowadays the determination of the food quality is an indispensable requirement at global level. This work focus on how the presence of microorganisms in food can be detected and verify that they ensure the minimum requirements stablished by the law. To acomplish that, government entities like ENAC (Entidad Nacional de Acreditación y Certificados) promulgate standards developed by the ISO (International Organization for Standarization).One of the most important microorganism related with foodborne diseases is Salmonella. This work aims to adapt the laboratory protocol for the detection of the former microorganism to the new standards.Methods:According to the RD 2073/2005, laboratories must adapt the research assay UNE-EN ISO 6579:2003 ,which was modified in 2017, to accomplish the reference standard for the detection of Salmonella. Those changes are not significant, as result it is not necessary to make a complete validation of our protocol; that will make the transition to the new method much easier.The schedule for this transition will be: (1) Find out the changes between the older and newer standard method, such as culture mediums, incubate periods, and changes at bioquimical and serological tests. (2) Define the confirmation test. (3) Conducting verification tests of the method in parallel with the procedure based on the old standard. (4) Perform an evaluation based on the results of the test. (5) Modification of the internal test procedure.Results and conclusion: Even though a validation has not been carried out, the analysis of a wide variety of foods for the detection of Salmonella has been performed throughout the internship. These verification tests have been carried out according to the old ISO using reference strains such as S. thyphimurium (ATCC 14028) and S. enteritidis (ATCC 13076). The procedure consists of inoculating a food sample with the target sample at a concentration of the microorganism close to the limit of detection obtained by the laboratory in its validation, being this limit below 10 CFU / 25g. Currently the laboratory is in phase no. 1. Our expectation is adapt the procedure to the new ISO before the end of the course

Verification of the method to determine the moisture content in meat products

MotivationThere are various reason why food industries can determine humidity, becoming highly important the issue of growing microorganisms caused by an excess on water in raw materials. The normative ISO 9001:2015 is essential for the implantation of an improved orientated system having guarantees to comply with the requirements of the product or service as well as meeting customer´s expectations.MethodsThe drying methods are the most common ones to asses food moisture content. Although these methods give good results, it is difficult sometimes to remove all the humidity by drying. It is also important to mention that food can decompose at certain temperature so other substances can volatilize apart from water. Thus, the measurement of weight loss of the sample caused by water evaporation is made with a forced air circulation heater at atmospheric pressure, according to ISO 1442:1997.ResultsDuring the evaluation the checking of the procedures requires the application of uniform and consistent technical criteria.Due to the importance of meat and meat products because of its high protein in the human diet and its huge quantify demand that is analyzed in laboratories, it is observed by graphical representation the tendency of the obtained data. The proper temperature is 105ºC.ConclusionsThe method to quantify the moisture content of various meat products is valid obtaining reliable results with the possibility of itsfuture accreditation

Quality assurance in a microbiology laboratory

Motivation: Quality control companies are becoming increasingly important in different aspects of biotechnology. According to UNE-EN-ISO 17025: 2017, Innoagral is an accredited laboratory, which is devoted to agri-food, water and cosmetic analysis among others. In order to meet the different quality parameters established, one of the main purposes of this laboratory is to ensure the quality of the results emitted. To do so, a series of activities encompassed in the quality assurance of its tests have been defined, among which are: monthly verifications of the testing methods, manipulation and preparation controls of strains and growth mediums, as well as environment and surface controls and assay blanks. Depending on the type of activity, appropriate intervals are established for each analysis.Methods: The presence investigation of Salmonella spp. and Listeria monocytogenes in foods and surfaces is carried out by chromogenic methods which are based on the UNE-EN-ISO 6579-1: 2017 and UNE-EN-ISO 11290-1: 2018. As they are qualitative microorganism detection methods, the samples are subjected to a preincubation stage prior to the spreading in specific media. Both control strains and growth mediums are subjected to their own quality controls to be used in the verification of different methods; all of them based on UNE- EN ISO 11133:2014.Results: Based on the accuracy and precision criteria which are clearly-defined in the laboratory, the validity of the methods is checked, and a limit of detection of 15 ufc/portion, which has to be fulfilled in each of the verifications of the Salmonela spp. and Listeria monocytogenes, is established

Quality control in agrifood and sanitary industry

Motivation: One of the main areas of Biotechnology are the quality control dedicated companies at different levels, such as the agrifood, cosmetic, public health and water analysis. Innoagral is one of this companies, which works according to UNE-EN-ISO 17025: 2005. The goal of the analysis carried out here is the continuous improvement of the product as well as the fulfillment of specifications to keep the quality parameters established for the different matrices.Methods: Samples provided by different companies are evaluated by physical-chemical and/or microbiological methods, which follows the protocols established by Norms UNE-EN-ISO. The main physical-chemical techniques developed are the measurement of pH, conductivity, biologycal oxygen demand (BOD) and chemical oxygen demand (COD), sugar content by refractometry, total protein extraction (Kjeldahl), saturated fats content and alkalinity by volumetry, total fat content extraction (Soxhlet), humidity and ash by gravimetry and quantity of cations and other compounds measurement by spectrophotometry, alwais under the UNE-EN-ISO norms. The microbiological techniques include the detection and counting of Listeria monocytogenes, Escherichia coli, Legionella spp and Salmonella spp, among other microorganisms. For this, the culture media, sterility sample preparation, enrichment , culture, biochemical confirmation and serological tests are always developed subject to the Norms UNE-EN-ISO.Results: After many analyses the main results obtained indicated that most of the samples of drinking water satisfy the established pH ranges (RD 140/2003) and conductivity ranges (UNE-EN 27888:1994). In the case of wastewater, it usually has a higher content in COD than in BOD. The analysis of soils reveals big differences in the heavy metals proportion depending on the origin and composition of the soil, in which the amount of organic matter directly affects phosphorus levels. At the microbiological level, food analyzed follows the Regulations (CE) 1169/2011 and 2073/2005 that dictate, respectively, the standards of food labeling (with the nutrients proportion obtained in the physico-chemical tests) and the microbiological criteria, mainly for Salmonella (UNE-EN-ISO 6579-1: 2017), L. monocytogenes (UNE-EN-ISO 11290: 2018) and E.coli (UN-EN-ISO 16649: 2015).For those samples that don't reach the minimum quality parameters, the companies must set up the necessary changes to comply with the established standards

Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionarily conserved, but span at least a ∼100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins.This research was supported by FEDER Funds, grant BIO2015-66426-R from the Spanish Ministry of Economy and Competitiveness ( J.M.S.-R.), grant RGP0041/2017 from the Human Frontier Science Program ( J.M.S.-R. and E.A.G.) and National Institutes of Health 1R01AR069137 (E.A.G.), Department of Defence MURI W911NF-16-1-0372 (E.A.G.)

Non-conservation of folding rates in the thioredoxin family reveals degradation of ancestral unassisted-folding

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionarily conserved, but span at least a ~100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins. © 2019 The Author(s).This research was supported by FEDER Funds, grant BIO2015-66426-R from the Spanish Ministry of Economy and Competitiveness ( J.M.S.-R.), grant RGP0041/2017 from the Human Frontier Science Program ( J.M.S.-R. and E.A.G.) and National Institutes of Health 1R01AR069137 (E.A.G.), Department of Defence MURI W911NF-16-1-0372 (E.A.G.

Data from: De novo active sites for resurrected Precambrian enzymes

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering