Numerous diagnostic technologies have been developed over the decades to ensure food safety. At first, most diagnostic techniques implied visual methods, such as the visual inspection of food products and their microscopic analysis.

Later on, molecular diagnostic techniques were first introduced in the 1950s, then constantly refined and sophisticated over the following years, up to the introduction of PCR-based techniques (Polymerase Chain Reaction) that revolutionised the field of molecular diagnosis. Thanks to molecular diagnostics, molecular epidemiology, databases of pathogenic genetic profiles, data mining and the use of omics sciences, it is possible to ensure greater precision in diagnosing infectious diseases and greater transparency along the food production chain.

Furthermore, these new techniques allow for a quicker identification of pathogens and prompt implementation of targeted control measures to prevent the diffusion of foodborne infectious diseases. Thus, diagnostic technologies stand out as essential tools to ensure food safety and safeguard public health.

The present article will briefly review the main aspects of these technologies and their impact on food safety. Furthermore, we will evaluate the contribution that the OnFoods project Spoke 03 can offer in terms of research and innovation in this sector.

Molecular diagnostics. Molecular diagnostics is a constantly evolving technology that allows for the identification of specific pathogens. Thanks to the use of omics sciences such as genomics, proteomics and metabolomics, it is possible to identify pathogens with greater precision, as opposed to traditional methods. Genomics is one of the most widely used omics sciences in molecular diagnostics, as it allows for the identification of the DNA sequences of pathogens, which are then compared with those present in reference databases. Thus, it makes it possible to identify the pathogen in question with a higher degree of precision. Proteomics is another omics science often applied to molecular diagnostics to identify the proteins synthesised by pathogens and to use them as a marker for their identification. Metabolomics used to be a little-used omics science in molecular diagnostics, but it has been arousing growing interest in recent years. It allows identifying the metabolites produced by pathogens and to use them as markers for their identification.

Molecular epidemiology. Molecular epidemiology allows researchers to study the distribution and frequency of epidemic outbreaks. This approach is revolutionising the control and prevention of foodborne diseases, as it makes it possible to trace the diffusion of infectious diseases by analysing the genomic sequences of the pathogen at issue. Through molecular epidemiology, health authorities can identify the source of infection that caused a specific foodborne disease outbreak and consequently take the necessary measures to prevent the disease from spreading.

Pathogen genetic profile databases. Databases containing the genetic profiles of pathogens are a fundamental tool for molecular diagnostics. Thanks to these databases, scientists can compare the genomic sequences of isolated pathogens with those present in the database and identify the pathogen in question. The databases are constantly updated with new pathogen genomic sequences, which allows researchers to grow increasingly precise in identifying isolated pathogens.

Data mining. Data mining is yet another crucial tool for food security. Thanks to this technique, scientists can analyse large amounts of data to evaluate the correlations between the presence of pathogens and certain factors, such as production plants or distribution chains. These data can be used to take targeted preventive measures, including more frequent inspections in certain facilities or recommendations to implement specific food handling procedures.

Spoke 3 Work Package 3.2 focuses on reducing the risks associated with food production, processing, storage and sale by developing new advanced strategies to prevent chemical, biological and toxicological risks in food products.

The activities envisaged in Spoke 3 are mainly aimed at developing models to provide forecasts and to describe the effects of uncontrolled or unforeseen processing and storage conditions in terms of food safety. These predictive models will be used to develop new processes and/or optimise the existing ones to mitigate the microbiological risks along the agri-food supply chain.

The Spoke also seeks to reduce the risk of allergens and toxicity in food, promoting alternative products in place of food additives, and developing synergistic combinations with other "green" products, as well as employing innovative techniques to reduce the content of heavy metals and nitrates in some specific food products. Furthermore, it aims to develop a "farm-to-table" approach to mitigate the impact of plant pathogens in terms of crop quality and safety.

The research activities of WP 3.2 also plan to explore the potential of different breeding systems, different breeds and alternative nutritional regimes to ensure the quality and wholesomeness of foods such as milk and dairy products.

Furthermore, WP 3.2 aims to test innovative risk reduction and mitigation measures by conducting "challenge" studies on model foods deliberately contaminated with pathogenic bacteria. It also envisages developing specific fermentation processes based on selected microorganisms (protective cultures), namely, hydrolysed food matrices to be integrated within more traditional food production protocols, thus increasing food safety and shelf life. The inactivation of food pathogens at the production, conservation, retail and consumption stages will also be pursued by applying natural antimicrobial substances (e.g. essential oils) and hydrolysed raw matrices.

Lastly, WP 3.2 suggests analysing the role of cognitive, emotional and relational factors in determining the perception of risk and other food safety beliefs. Therefore, a series of personalised and effective communication strategies will be developed to promote good food safety practices and involve more and more citizens. Indeed, promoting actions aimed at boosting the stakeholder engagement, such as targeted initiatives for journalists, nutritionists, opinion leaders and influencers, is yet another crucial aspect of Work Package 3.2.

Protective cultures and bio-preservatives to ensure the safety and quality of food, reduce waste and meet public needs.

The agri-food market is entering a new era: consumers are looking for organic and sustainable products, often to be consumed "on the go", with greater attention to the "clean" label attributed to foods manipulated as little as possible, whose ingredient list is either free or very low in additives and preservatives. Furthermore, to meet the needs of both responsible consumers and producers looking for options to offer more transparent and sustainable production chains, it is essential to reduce food waste as a consequence of product deterioration.

Food spoilage is considered a relevant issue affecting food at the production and storage stages. Contaminant microorganisms (deteriorating agents) are among the main causes of food spoilage, as they can colonise food matrices during the primary production, processing or storage phases, altering the organoleptic peculiarities of food and even causing poisoning in some cases.

Protective cultures and bio-preservatives have become tools of interest to most agri-food producers, as they allow them to reduce food spoilage, extend the shelf life of goods and satisfy the consumer needs.

Protective cultures consist of microorganisms selected for their ability to either prevent food deterioration by producing antioxidant compounds or to inhibit the development of pathogens responsible for unwanted contaminations, both of which would undermine the wholesomeness of the product itself.

The most used protective cultures in the food industry involve lactic acid bacteria (LAB), which produce substances capable of preventing or limiting the growth of pathogenic bacteria, moulds and yeasts responsible for food deterioration. As such, protective cultures can help keep the food quality intact, preserving its flavour, texture and nutritional value.

The LABs ability to inhibit the growth of adulterating microorganisms in food makes them very interesting from an industrial standpoint. There are many different mechanisms underlying this biochemical principle.

The metabolism of lactic acid bacteria is characterised by the ability to synthesise compounds with inhibitory power against deteriorating microorganisms, such as organic acids and bacteriocins.

The bio-preservative compounds produced from the typical LAB metabolic activities can include several primary metabolites of carbohydrate fermentation, such as organic acids (e.g. lactic acid, acetic acid, propionic acid), more complex compounds originating from bioconversion or protein synthesis processes, such as bacteriocins, as well as molecules obtained through protein lysis (bioactive peptides). Bio-preservatives can extend the shelf life of food, reducing waste and satisfying the requirements of a public opinion grown increasingly attentive to the quality and safety of food.

Bio-preservatives and protective cultures are applicable to many edible products, including cured meats, fresh meat, fish, cheese, bread, baked goods and many more. However, choosing the most suitable bio-preservative compound and/or protective culture for each product is particularly important to avoid altering the taste or texture of foods.

The selection of lactic acid bacteria (LAB) included in the European Food Safety Authority's (EFSA) Qualified Presumption of Safety (QPS) list as suitable for starter cultures stands out as a promising framework, thanks to their potential to synthesise bacteriocins and antimicrobial compounds.

In recent studies, LABs have been used in the production of milk and dairy products to limit the growth of Listeria monocytogenes, Shigatoxin-producing Escherichia coli, and Salmonella, as well as to inhibit those microorganisms that cause food spoilage in vitro and in model cheeses. LABs can be used either as a single bacterial species or as a pool, depending on their antimicrobial effectiveness.

The LAB_REDSPOIL research activities envisage the characterisation of the microbiota responsible for food spoilage, adopting omics-based approaches and assessing the most dangerous pathogenic bacteria for each food category.

At a later stage, LAB_REDSPOIL will test the specific protective cultures for each product category under laboratory conditions, followed by broader pilot-scale screenings. This second step will be based both on quantitative assessments of the microbiological risk and on "challenge" studies, in which foods will be contaminated with the pathogenic or adulterating microbiota, to identify the best combination of protective cultures and study conditions.

The team will also look into the possibility of increasing storage temperatures (e.g. from 4° to 8°C) and the protective actions taken during temperature abuse or under different processing conditions. Furthermore, researchers will evaluate the use of genetically modified microorganisms with an increased ability to produce antimicrobial compounds. These activities will be carried out both by applying traditional microbiological methods and through omics approaches, including metagenomics and metabolomics.

"The LAB REDSPOIL research project will pay particular attention to the adoption of genetic engineering techniques to generate mutant strains of lactic acid bacteria, edited to boost the production of specific bacteriocins against the hostile microorganism in question. The effectiveness of these microorganisms will be first tested under laboratory conditions and then applied to model foods and on a pilot scale." Daniela Bassi, Pier Sandro Cocconcelli, Viviana Belloso, Vincenzo Castellone.