Periodic Reporting for period 2 - MASTER (Microbiome Applications for Sustainable food systems through Technologies and EnteRprise) Reporting period: 2020-07-02 to 2022-01-01 Summary of the context and overall objectives of the project Microorganisms exist across all ecological niches; from the surfaces we touch, to the foods we eat and also inside of us, with many microorganisms being integral to our health. We discover the make-up and function of microbial communities (microbiomes) using DNA sequencing technologies. Scientists across Europe in the H2020 Innovation Action MASTER (Microbiome Applications for Sustainable food systems through Technologies and EnteRprise) use these technologies to map microbiomes across a range of food chains, acquiring data for the development of safer and more sustainable food systems. MASTER researchers are mining this microbiome data, developing big data management tools, identifying relationships between microbiomes across food chains and generating applications that promote sustainability and contribute to waste management and climate change mitigation. MASTER will yield innovative products and applications such as microbiome products, foods and feeds, services and processes. Research spans multiple food chains and will benefit society by improving food quantity, quality and safety. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far MASTERing plant microbiomes: MASTER is providing microbiome-based solutions to improve production, enhance quality and control disease in fodder crops. Trials with plant growth-promoting, plant biocontrol strains and developed formulations assessed the compatibility and effectiveness of these microorganisms. The development of a microbiome-based diagnostic tool for pathogen detection/prediction of colonisation efficiency is progressing. MASTERing marine microbiomes: Development of microbiome-based solutions continued, to increase the beneficial effects of sustainable aquafeeds. In one case, the effect of probiotic strains on fish growth performance was determined. Furthermore, the fillet quality and sensory values of on-growing Atlantic salmon, fed with and without probiotics, were evaluated. Exciting results contributing to a rapid protocol for pathogen detection in aquaculture systems, using state-of-the-art sequencing, were also generated.MASTERing rumen microbiomes: By rumen microbiome manipulation, MASTER is progressing towards improving ruminant production while reducing methane emissions and providing healthy meat and milk products for the consumer. Thus far, a large number of animals were studied to investigate feed efficiency and methane emissions, providing results linking rumen microbiome and phenotypes. The capacity of selected phenolic compounds to capture hydrogen produced in excess, in a methane inhibition scenario, was shown in vitro. If translated, this has the potential to result in more energy availability for the animal. The benefit of early life rumen microbiome modulation in terms of health and welfare was also determined, whilst developing mathematical models of microbial activity in the rumen, to predict and design further methane control strategies. MASTERing food microbiomes and human gut health: We will provide the food industry with up-to-date microbiome mapping procedures to investigate microbial contaminants along food processing lines, with the aim of enhancing food quality and safety and reducing withholding periods and food waste. Sampling of 114 food processing industries across Europe was completed. Work focused on the characterisation of food waste materials, to develop novel food/feed ingredients. In parallel, novel seafood and meat biopreservation strategies were developed. Microbiome and dietary data continued to be analysed, exploring the diet-gut microbiome relationship, to investigate microbial diversity at strain level and further highlighting the importance of fermented foods as a source of potentially probiotic lactic acid bacteria.MASTERing standards: We created a unified approach to the analysis of food chain microorganisms and standardised validated methods for microbiome processing of samples from different environments and for different types of DNA sequencing. A ready to use bioinformatics workflow for food-chain metagenomic data profiling was developed and implemented. Two databases were created: CuratedFoodMetagenomicDatabase (all publicly available microbiome samples from studies on metagenomes of food/food production) and foodGenVir (at the level of single microbial strains or genes associated with traits of interest). The protocols and databases will be available in an open web portal beyond the end of the project and have the potential to revolutionize food microbiology testing in the future. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) New formulations for the application of arbuscular mycorrhizal fungi and biocontrol bacteria were developed and tested. The potential of ONT technology for pathogen detection in soil was also assessed, as well as technologies for selective enrichment of bacterial gDNA from plants. Progress was made towards optimizing sustainable aquafeeds for aquaculture, including the use of tailored prebiotics and probiotics. Advances were made towards the rapid detection of fish pathogens in aquaculture systems. Potential socio-economic and wider societal implications include a reduction of the dependence on the use of fish meal in aquaculture and more efficient feed utilisation, benefiting natural resource management. Our ruminant studies illustrate the potential to breed animals that emit lower methane levels, while showing the potential of numerous dietary interventions to further reduce methane emissions, increase productivity and ensure nutritious products. The use of phenolic compounds to re-direct hydrogen and provide reduced methane emissions, while providing more energy to the animal, shows great promise to move towards a higher TRL. Modelling developments will contribute to the design of further cutting-edge ruminant nutritional strategies for methane mitigation, maximizing health and animal productivity.MASTER has generated validated procedures to map microbiomes in the food industry, promoting process optimisation, waste reduction and improving food quality and safety. A map of the interconnections between food products, nutrients, microorganisms and the human gut microbiome is being established and will provide dietary recommendations to benefit human health through gut microbiome modulation. MASTER has made significant progress towards the availability of strategies for the biopreservation of seafood and meat, to improve product safety and quality. Procedures for the valorisation brewing waste streams are also at an advanced stage. Development of harmonised protocols, a mock community and automated analytical pipelines within MASTER, especially dedicated to food-related ecosystems will improve the standardisation and potential of sequencing technologies for the food chain. Newly generated MASTER databases provide profiles of food-associated microbiomes, with accompanying data, and strains or genes associated with virulence, pathogenicity, spoilage potential and antimicrobial resistance that will be essential for the longer-term application of sequencing technologies for food testing. These are novel databases and will be freely available to be used by the scientific community and industries.These outcomes will have major implications, enhancing understanding of the microbiomes associated with food chains and addressing key societal challenges including food and nutrition security, health and wellbeing, food waste management and climate change adaptation and mitigation. 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