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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

Centro de Investigación en Sanidad Animal (CISA), Fluorescent proteins are widely used for cell and protein tracking. Most of these proteins show a high signal and need the presence of oxygen to emit fluorescence. Among them, the fluorescent protein mCherry stands up because of its bright signal and fast maturation. Furthermore, the anaerobic cyan-green fluorescent protein Evoglow-Pp1 allows fluorescent detection under anaerobic conditions. In this work, we modified the pNZ:TuR.aFP plasmid, which harbors the gene encoding Evoglow-Pp1 and the promoter of elongation factor Tu from Limosilactobacillus reuteri CECT925, to obtain a plasmid containing the mrfp gene encoding the monomeric mCherry (pNZ:TuR.mCherry). Moreover, both genes were cloned together (pNZ:TuR.aFP.mCherry) developing a chimeric protein; and with a stop codon between them (pNZ:TuR.aFP.STOP.mCherry) resulting in the expression of both Evoglow-Pp1 and mCherry proteins separately under the influence of the same promoter. Lactococcus lactis, Lacticaseibacillus casei, Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus, and L. reuteri strains were transformed with the previously mentioned plasmids, showing an excellent red (pNZ:TuR.mCherry), green (pNZ:TuR.aFP), and red combined with green (pNZ:TuR.aFP.mCherry and pNZ:TuR.aFP.STOP.mCherry) fluorescence signal. Both fluorescence emissions were stable in strains transformed with pNZ:TuR.aFP.STOP.mCherry, while differences in the red or green fluorescence emission were observed in some of the strains harboring pNZ:TuR.aFP.mCherry. Moreover, these plasmids allowed strains differentiation in a complex environment, such as fecal microbiota. Hence, we present the plasmid pNZ:TuR.aFP.STOP.mCherry as a useful tool for the labeling of lactobacilli strains, which would be functional under anoxic conditions, thanks to Evoglow-Pp1, while having the high brightness and good photostability of mCherry. KEY POINTS: • LAB transformed with pNZ:TuR.mCherry expressed the red fluorescent protein mCherry. • LAB transformed with pNZ:TuR.aFP.mCherry developed a fusion of both proteins Evoglow-Pp1 and mCherry. • LAB with pNZ:TuR.aFP.STOP.mCherry expressed both fluorescent proteins separately., This work was supported by RTA2017-00002–00-00 project from the Ministry of Economy and Competitiveness (Spain).