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29 p.-4 fig. ; Lactobacillus fermentum isolated from sourdough was able to produce riboflavin. Spontaneous roseoflavinresistant mutants were obtained by exposing the wild strain (named L. fermentum PBCC11) to increasing concentrations of roseoflavin. Fifteen spontaneous roseoflavin-resistant mutants were isolated, and the level of vitamin B2 was quantified by HPLC. Seven mutant strains produced concentrations of vitamin B2 higher than 1 mg L−1. Interestingly, three mutants were unable to overproduce riboflavin even though they were able to withstand the selective pressure of roseoflavin.Alignment of the rib leader region of PBCC11 and its derivatives showed only point mutations at two neighboring locations of the RFN element. In particular, the highest riboflavinproducing isolates possess an A to G mutation at position 240,while the lowest riboflavin producer carries a T to A substitution at position 236. No mutations were detected in the derivative strains that did not have an overproducing phenotype. The best riboflavin overproducing strain, named L. fermentum PBCC11.5, and its parental strain were used to fortify bread. The effect of two different periods of fermentation on the riboflavin level was compared. Bread produced using the coinoculum yeast and L. fermentum PBCC11.5 led to an approximately twofold increase of final vitamin B2 content. ; This work was founded by the Italian Ministry for Development in the framework of the "Industria 2015 Bando Nuove Tecnologie per il Made in Italy - Realizzazione di una innovativa pasta alimentare funzionale arricchita di componenti bioattivi e probiotici" and by the Spanish Ministry of Economy and Competitiveness grants AGL2012-40084-C03-01, AGL2012-40084-C03-03, and IT886-13 from the Basque Government. ; Peer reviewed

39 p.-7 fig.-2 fig. suppl. ; A critical feature of probiotic microorganisms is their ability to colonize the intestine of the host. Although the microbial potential to adhere to the human gut lumen has been investigated in in vitro models, there is still much to discover about their in vivo behaviour. Zebrafish is a vertebrate model that is being widely used to investigate various biological processes shared with humans. In this work, we report on the use of the zebrafish model to investigate the in vivo colonization ability of previously characterized probiotic lactic acid bacteria. Lactobacillus plantarum Lp90, L. plantarum B2 and Lactobacillus fermentum PBCC11.5 were fluorescently tagged by transfer of the pRCR12 plasmid, which encodes the mCherry protein and which was constructed in this work. The recombinant bacteria were used to infect germ-free zebrafish larvae. After removal of bacteria, the colonization ability of the strains was monitored until 3 days post-infection by using a fluorescence stereomicroscope. The results indicated differential adhesion capabilities among the strains. Interestingly, a displacement of bacteria from the medium to the posterior intestinal tract was observed as a function of time that suggested a transient colonization by probiotics. Based on fluorescence observation, L. plantarum strains exhibited a more robust adhesion capability. In conclusion, the use of pRCR12 plasmid for labelling Lactobacillus strains provides a powerful and very efficient tool to monitor the in vivo colonization in zebrafish larvae and to investigate the adhesion ability of probiotic microorganisms. ; The work performed in Spain was supported at the Centro de Investigaciones Biológicas by grant AGL2012-40084-C03-01 from the Spanish Ministry of Economics and Competitiveness and at AZTI-Tecnalia by the "Departamento de Desarrollo Económico y Competitividad Viceconsergería de Agricultura, Pesca y Politicas Alimentarias" (Project FOODBASK3). The work performed in Italy was supported by the Italian Ministry for Development in the framework of the "Industria 2015 Bando Nuove Tecnologie per il Made in Italy—Realizzazione di una innovativa pasta alimentare funzionale arricchita di componenti bioattivi e probiotici", and by MIUR (PON02_00186_2937475) in the framework of the project named "Protocolli innovativi per lo sviluppo di alimenti funzionali" "(Pro.Ali.Fun.)". ; Peer reviewed