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Riboflavin-overproducing strains of Lactobacillus fermentum for riboflavin-enriched bread

  • Applied genetics and molecular biotechnology
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Abstract

Lactobacillus fermentum isolated from sourdough was able to produce riboflavin. Spontaneous roseoflavin-resistant 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 riboflavin-producing 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.

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References

  • Abbas CA, Sibirny AA (2011) Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers. Microbiol Mol Biol Rev 75:321–360

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Brandt MJ (2014) Starter cultures for cereal based foods. Food Microbiol 37:41–43

    Google Scholar 

  • Burge SW, Daub J, Eberhardt R, Tate J, Barquist L, Nawrocki EP, Eddy SR, Gardner PP, Bateman A (2013) Rfam 11.0: 10 years of RNA families. Nucleic Acids Res 41:226–232

    Article  CAS  Google Scholar 

  • Burgess C, O’ Connell-Motherway M, Sybesma W, Hugenholtz J, van Sinderen D (2004) Riboflavin production in Lactococcus lactis: potential for in situ production of vitamin-enriched foods? Appl Environ Microbiol 70:5769–5777

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Burgess CM, Smid EJ, Rutten G, van Sinderen D (2006) A general method for selection of riboflavin-overproducing food grade micro-organisms. Microb Cell Factories 5:24

    Article  CAS  Google Scholar 

  • Burgess CM, Smid EJ, van Sinderen D (2009) Bacterial vitamin B2, B11 and B12 overproduction: an overview. Int J Food Microbiol 133:1–7

    Article  PubMed  CAS  Google Scholar 

  • Capozzi V, Menga V, Digesu AM, De Vita P, van Sinderen D, Cattivelli L, Fares C, Spano G (2011) Biotechnological production of vitamin B2-enriched bread and pasta. J Agric Food Chem 59:8013–8020

    Article  PubMed  CAS  Google Scholar 

  • Capozzi V, Russo P, Dueñas MT, López P, Spano G (2012) Lactic acid bacteria producing B-group vitamins: a great potential for functional cereals products. Appl Microbiol Biotechnol 96:1383–1394

    Article  PubMed  CAS  Google Scholar 

  • Catzeddu P, Mura E, Parente E, Sanna M, Farris GA (2006) Molecular characterization of lactic acid bacteria from sourdough breads produced in Sardinia (Italy) and multivariate statistical analyses of results. Syst Appl Microbiol 29:138–144

    Article  PubMed  CAS  Google Scholar 

  • Coquard D, Huecas M, Ott M, van Dijl JM, van Loon AP, Hohmann HP (1997) Molecular cloning and characterisation of the ribC gene from Bacillus subtilis: a point mutation in ribC results in riboflavin overproduction. Mol Gen Genet 254:81–84

    Article  PubMed  CAS  Google Scholar 

  • Corsetti A, Settanni L (2007) Lactobacilli in sourdough fermentation. Food Res Int 40:539–558

    Article  CAS  Google Scholar 

  • Corsetti A, Lavermicocca P, Morea M, Baruzzi F, Tosti N, Gobbetti M (2001) Phenotypic and molecular identification and clustering of lactic acid bacteria and yeasts from wheat (species Triticum durum and Triticum aestivum) sourdoughs of Southern Italy. Int J Food Microbiol 64:95–104

    Article  PubMed  CAS  Google Scholar 

  • De Vuyst L, Vrancken G, Ravyts F, Rimaux T, Weckx S (2009) Biodiversity, ecological determinants, and metabolic exploitation of sourdough microbiota. Food Microbiol 26:666–675

    Article  PubMed  CAS  Google Scholar 

  • De Vuyst L, Van Kerrebroeck S, Harth H, Huys G, Daniel HM, Weckx S (2014) Microbial ecology of sourdough fermentations: diverse or uniform? Food Microbiol 37:11–29

    Google Scholar 

  • Edwars U, Rogall T, Blocker H, Emde M, Bottger EC (1989) Isolation and direct complete nucleotide determination of entire genes: characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853

    Article  Google Scholar 

  • European Food Information Council (2006) Mini guide 06/2006, http://www.eufic.org/article/en/expid/miniguide-vitamins/#3. Accessed

  • European Food Safety Authority (2007) Introduction of a qualified presumption of safety (QPS) approach for assessment of selected microorganisms referred to EFSA. EFSA J 587:1–16

    Google Scholar 

  • Fabian E, Bogner M, Kickinger A, Wagner KH, Elmadfa I (2012) Vitamin status in elderly people in relation to the use of nutritional supplements. J Nutr Health Aging 16:206–212

    Article  PubMed  CAS  Google Scholar 

  • Flynn A, Moreiras O, Stehle P, Fletcher RJ, Müller DJ, Rolland V (2003) Vitamins and minerals: a model for safe addition to foods. Eur J Nutr 42:118–130

    Article  PubMed  CAS  Google Scholar 

  • Gelfand MS, Mironov AA, Jomantas J, Kozlov YI, Perumov DA (1999) A conserved RNA structure element involved in the regulation of bacterial riboflavin synthesis genes. Trends Genet 15:439–442

    Article  PubMed  CAS  Google Scholar 

  • Guyot JP (2012) Cereal-based fermented foods in developing countries: ancient foods for modern research. Int J Food Sci Technol 47:1109–1114

    Article  CAS  Google Scholar 

  • Jakobsen J (2008) Optimisation of the determination of thiamin, 2-(1-hydroxyethyl)thiamin, and riboflavin in food samples by use of HPLC. Food Chem 106:1209–1217

    Article  CAS  Google Scholar 

  • Kil YV, Mironov VN, Gorishin IY, Kreneva RA, Perumov DA (1992) Riboflavin operon of Bacillus subtilis: unusual symmetric arrangement of the regulatory region. Mol Gen Genet 233:483–486

    Article  PubMed  CAS  Google Scholar 

  • LeBlanc JG, Milani C, de Giori GS, Sesma F, van Sinderen D, Ventura M (2013) Bacteria as vitamin suppliers to their host: a gut microbiota perspective. Curr Opin Biotechnol 2:160–168

    Article  CAS  Google Scholar 

  • Lee ER, Blount KF, Breaker RR (2009) Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression. RNA Biol 6:187–194

    Article  PubMed  CAS  Google Scholar 

  • Mensink GB, Fletcher R, Gurinovic M, Huybrechts I, Lafay L, Serra-Majem L, Szponar L, Tetens I, Verkaik-Kloosterman J, Baka A, Stephen AM (2013) Mapping low intake of micronutrients across Europe. Br J Nutr 110:755–773

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Minervini F, Di Cagno R, Lattanzi A, De Angelis M, Antonielli L, Cardinali G, Cappelle S, Gobbetti M (2012) Lactic acid bacterium and yeast microbiotas of 19 sourdoughs used for traditional/typical Italian breads: interactions between ingredients and microbial species diversity. Appl Environ Microbiol 78:1251–1264

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Ott E, Stolz J, Lehmann M, Mack M (2009) The RFN riboswitch of Bacillus subtilis is a target for the antibiotic roseoflavin produced by Streptomyces davawensis. RNA Biol 6:276–280

    Article  PubMed  CAS  Google Scholar 

  • Powers HJ (2003) Riboflavin (vitamin B2) and health. Am J Clin Nutr 77:1352–1360

    PubMed  CAS  Google Scholar 

  • Ricciardi A, Parente E, Piraino P, Paraggio M, Romano P (2005) Phenotypic characterization of lactic acid bacteria from sourdoughs for Altamura bread produced in Apulia (Southern Italy). Int J Food Microbiol 98:63–72

    Article  PubMed  CAS  Google Scholar 

  • Rivlin RS, Pinto JT (2007) Riboflavin (vitamin B2). In: Zempleni J, Rucker RB, McCormick DB, Suttie JW (eds) Handbook of vitamins, 4th edn. Taylor & Francis Group, Boca Raton, pp 233–253

    Google Scholar 

  • Settanni L, van Sinderen D, Rossi J, Corsetti A (2005) Rapid differentiation and in situ detection of 16 sourdough Lactobacillus species by multiplex PCR. Appl Environ Microbiol 71:3049–3059

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Tavares NR, Moreira PA, Amaral TF (2009) Riboflavin supplementation and biomarkers of cardiovascular disease in the elderly. J Nutr Health Aging 13:441–446

    Article  PubMed  CAS  Google Scholar 

  • Terrade N, Mira de Orduña R (2009) Determination of the essential nutrient requirements of wine-related bacteria from the genera Oenococcus and Lactobacillus. Int J Food Microbiol 133:8–13

    Article  PubMed  CAS  Google Scholar 

  • Terrade N, Noël R, Couillaud R, Mira de Orduña R (2009) A new chemically defined medium for wine lactic acid bacteria. Food Res Int 42:363–367

    Article  CAS  Google Scholar 

  • Turpin W, Humblot C, Guyot JP (2011) Genetic screening of functional properties of lactic acid bacteria in a fermented pearl millet slurry and in the metagenome of fermented starchy foods. Appl Environ Microbiol 77:8722–8734

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS (2002) Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation. Nucleic Acid Res 30:3141–3151

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Vogel RF, Pavlovic M, Ehrmann MA, Wiezer A, Liesegang H, Offschanka S, Voget S, Angelov A, Böcker G, Liebl W (2011) Genomic analysis reveals Lactobacillus sanfranciscensis as stable element in traditional sourdoughs. Microb Cell Factories 10:S6

    Article  Google Scholar 

  • Weckx S, Van der Meulen R, Allemeersch J, Huys G, Vandamme P, Van Hummelen P, De Vuyst L (2010a) Community dynamics of sourdough fermentations as revealed by their metatranscriptome. Appl Environ Microbiol 76:5402–5408

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Weckx S, Van der Meulen R, Maes D, Scheirlinck I, Huys G, Vandamme P, De Vuyst L (2010b) Lactic acid bacteria community dynamics and metabolite production of rye sourdough fermentations share characteristics of wheat and spelt sourdough fermentations. Food Microbiol 27:1000–1008

    Article  PubMed  CAS  Google Scholar 

  • Wegkamp A (2008) Modulation of folate production in lactic acid bacteria. PhD thesis. Wageningen, The Netherlands: Wageningen University

  • Winkler WC, Cohen-Chalamish S, Breaker RR (2002) An mRNA structure that controls gene expression by binding FMN. Proc Natl Acad Sci U S A 99:15908–15913

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Wojcieszyńska D, Hupert-Kocurek K, Guzik U (2012) Flavin-dependent enzymes in cancer prevention. Int J Mol Sci 13:16751–16768

    Article  PubMed Central  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Stephen Elson for the critical reading of the manuscript. 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.

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Correspondence to Giuseppe Spano.

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Russo, P., Capozzi, V., Arena, M.P. et al. Riboflavin-overproducing strains of Lactobacillus fermentum for riboflavin-enriched bread. Appl Microbiol Biotechnol 98, 3691–3700 (2014). https://doi.org/10.1007/s00253-013-5484-7

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