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12 Pág. Departamento de Tecnología de Alimentos​​, The genetic engineering of bacteria for food applications has biosafety requirements, including the use of non-antibiotic selectable markers. These can be gene-encoding bacteriocin immunity proteins, such as nisI and pedB, which require the use of promoters to ensure optimal expression. Our aim was to search for promoters for the expression of pediocin (pedB) and nisin (nisI) immunity genes, which could allow the selection of a wide variety of transformed lactic acid bacteria (LAB) and bifidobacteria strains. Eight promoters from LAB or bifidobacteria were initially studied using evoglow-Pp1 as the reporter gene in Lactococcus lactis NZ9000, resulting in the selection of P32, P3N, PTuR and PEF-P, which exhibited a strong constitutive expression. These promoters were further tested for the expression of the food-grade selectable markers pedB and nisI in agar diffusion assays with pediocin and nisin, respectively. The results obtained demonstrated that both the PTuR and PEF-P promoters allowed a good level of expression of nisI and pedB in the LAB and bifidobacteria strains tested. A suitable concentration of nisin or pediocin could be established for the selection of the strains transformed with vectors harbouring the combination of the selected promoters and markers nisI and pedB, and this was successfully applied to different strains of LAB and bifidobacteria. Therefore, PTuR and PEF-P promoters are excellent candidates for the expression of nisI and/or pedB as selectable markers in LAB and bifidobacteria, and they are suitable for use in food grade vectors to allow the selection of genetically engineered strains. KEY POINTS: • Food-grade vectors require non-antibiotic selectable markers such as pedB and nisI. • Eight promoters from LAB or bifidobacteria were initially tested in L. lactis NZ9000. • PTuR and PEF-P efficiently drove the expression of pedB and nisI in LAB and bifidobacteria., Funding This study was funded by the project PID2020-11960RB-I00 from the Spanish Ministry of Science, Innovation and Universities

Among vegetable functional products, soy-based foods stand out for their high content in protein and flavonoids,mainly isoflavones. The fermentation of soy beverages with selected bacterial strains is a way to obtain a product,enriched in isoflavone aglycones, alongside with other isoflavone derivates and other bioactive flavonoids. Two bacterial strains with high activity on the transformation of these compounds, Limosilactobacillus mucosae INIA P508 and Bifidobacterium pseudocatenulatum INIA P815, were tested for the fermentation of soy beverage. Both strains produced high concentration of the aglycones daidzein and genistein, in addition to a significant increase (P < 0.01) of glycitein, O-desmethylangolensin, 6′′ -hydroxy-O-desmethylangolensin, tetrahydrodaidzein and other flavonoid aglycones such as kaempferol, naringenin and luteolin. Two alternatives to conserve the developed functional beverages were explored, cold storage and thermal treatment. Refrigeration did not affect the concentration of isoflavones, even observing an increase in aglycones. Similarly, the thermal treatment and further room temperature storage did not reduce the amounts of daidzein and genistein. Therefore, both strains could be used for the development of soy beverages enriched in bioactive isoflavones and other bioactive flavonoids which would maintain their concentrations in refrigeration or at room temperature after heat treatment.

8 Pág. Departamento de Tecnología de Alimentos​​, Three strains with technological and probiotic properties (Lactococcus lactis INIA 650, Lacticaseibacillus paracasei INIA P272 and Bifidobacterium breve INIA P734) were used in different combinations as adjunct cultures for the manufacture of pasteurized ewe's milk. The three strains showed good viability during cheese manufacture and ripening. Moreover, they all were able to survive after the addition of the cheese samples to an in vitro colonic model. The nisin-producing strain L. lactis INIA 650 was able to produce the antimicrobial peptide nisin not only during cheese manufacture and ripening, but also in the colonic model. None of the strains combinations added to the cheese as adjunct cultures had a negative influence in their physicochemical or sensory characteristics when compared to control cheese. This work shows the suitability of cheese as vehicle for a multi-strain combination with potential dual effect in food and gut., This work was supported by project RTA2017-00002-00-00 from the Spanish Ministry of Science and Innovation

18 Pág. Departamento de Tecnología de Alimentos​​, Limosilactobacillus reuteri INIA P572 is a strain able to produce the antimicrobial compound reuterin in dairy products, exhibiting a protective effect against some food-borne pathogens. In this study, we investigated some probiotic properties of this strain such as resistance to gastrointestinal passage or to colonic conditions, reuterin production in a colonic environment, and immunomodulatory activity, using different in vitro and in vivo models. The results showed a high resistance of this strain to gastrointestinal conditions, as well as capacity to grow and produce reuterin in a human colonic model. Although the in vitro assays using the RAW 264.7 macrophage cell line did not demonstrate direct immunomodulatory properties, the in vivo assays using a Dextran Sulphate Sodium (DSS)-induced colitic mice model showed clear immunomodulatory and protective effects of this strain., This work was supported by project no. RTA2017-00002-00-00 from the Spanish Ministry of Science and Innovation, by the Junta de Andalucía (CTS 164) and Instituto de Salud Carlos III (PI19/01058) with funds from the European Union