Advertisement

Applied Microbiology and Biotechnology

, Volume 104, Issue 3, pp 1175–1186 | Cite as

Diversity of volatile organic compound production from leucine and citrate in Enterococcus faecium

  • Matilde D’Angelo
  • Gabriela P. Martino
  • Victor S. Blancato
  • Martín Espariz
  • Axel Hartke
  • Nicolas Sauvageot
  • Abdellah Benachour
  • Sergio H. Alarcón
  • Christian MagniEmail author
Applied genetics and molecular biotechnology
  • 98 Downloads

Abstract

Enterococcus faecium is frequently isolated from fermented food; in particular, they positively contribute to the aroma compound generation in traditional cheese. Citrate fermentation is a desirable property in these bacteria, but this feature is not uniformly distributed among E. faecium strains. In the present study, three selected E. faecium strains, IQ110 (cit), GM70 (cit+ type I), and Com12 (cit+ type II), were analyzed in their production of aroma compounds in milk. End products and volatile organic compounds (VOCs) were determined by solid-phase micro-extraction combined with gas chromatography mass spectrometry (SPME-GC-MS). Principal component analysis (PCA) of aroma compound profiles revealed a different VOC composition for the three strains. In addition, resting cell experiments of E. faecium performed in the presence of leucine, citrate, or pyruvate as aroma compound precursors allowed us to determine metabolic differences between the studied strains. GM70 (cit+ type I) showed an active citrate metabolism, with increased levels of diacetyl and acetoin generation relative to Com12 or to citrate defective IQ110 strains. In addition, in the experimental conditions tested, a defective citrate-fermenting phenotype for the Com12 strain was found, while its leucine degradation and pyruvate metabolism were conserved. In conclusion, rational selection of E. faecium strains could be performed based on genotypic and phenotypic analyses. This would result in a performing strain, such as GM70, that could positively contribute to flavor, with typical notes of diacetyl, acetoin, 3-methyl butanal, and 3-methyl butanol in an adjuvant culture.

Keywords

Enterococcus faecium Aroma compound Leucine Citrate Rational strain selection 

Notes

Acknowledgments

We are especially grateful to M.S. Gilmore for providing us E. faecium Com12. We would like to thank the Agencia Nacional de Promoción Científica y Tecnológica, Consejo Nacional de Investigaciones Cientifícas y Técnicas, and Universidad de Rosario for the financial support. MD and GPM are CONICET fellows, and VSB, ME, SHA, and CM are researchers of the same institution.

Funding information

This study was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 11220150100855) and Agencia Nacional de Promoción Científica y Tecnológica (ANPyCT, PICT2014-1513 and 3482 and PICT2015-2361).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_10277_MOESM1_ESM.pdf (1 mb)
ESM 1 (PDF 1065 kb)
253_2019_10277_MOESM2_ESM.xlsx (411 kb)
ESM 2 (XLSX 410 kb)

References

  1. Afzal MI, Delaunay S, Paris C, Borges F, Revol-Junelles AM, Cailliez-Grimal C (2012) Identification of metabolic pathways involved in the biosynthesis of flavor compound 3-methylbutanal from leucine catabolism by Carnobacterium maltaromaticum LMA 28. Int J Food Microbiol 157(3):332–339.  https://doi.org/10.1016/j.ijfoodmicro.2012.05.010 CrossRefPubMedGoogle Scholar
  2. Arias CA, Murray BE (2012) The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol 10(4):266–278.  https://doi.org/10.1038/nrmicro2761 CrossRefGoogle Scholar
  3. Barbosa J, Borges S, Teixeira P (2014) Selection of potential probiotic Enterococcus faecium isolated from Portuguese fermented food. Int J Food Microbiol 191:144–148.  https://doi.org/10.1016/j.ijfoodmicro.2014.09.009 CrossRefPubMedGoogle Scholar
  4. Blancato VS, Magni C, Lolkema JS (2006) Functional characterization and Me2+ ion specificity of a Ca2+-citrate transporter from Enterococcus faecalis. FEBS J 273(22):5121–5130.  https://doi.org/10.1111/j.1742-4658.2006.05509.x CrossRefPubMedGoogle Scholar
  5. Blancato VS, Repizo GD, Suarez CA, Magni C (2008) Transcriptional regulation of the citrate gene cluster of Enterococcus faecalis involves the GntR family transcriptional activator CitO. J Bacteriol 190(22):7419–7430.  https://doi.org/10.1128/JB.01704-07 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Blancato VS, Pagliai FA, Magni C, Gonzalez CF, Lorca GL (2016) Functional analysis of the citrate activator CitO from Enterococcus faecalis implicates a divalent metal in ligand binding. Front Microbiol 7:101.  https://doi.org/10.3389/fmicb.2016.00101 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Broadbent JR, Gummalla S, Hughes JE, Johnson ME, Rankin SA, Drake MA (2004) Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in Cheddar cheese. Appl Environ Microbiol 70(8):4814–4820.  https://doi.org/10.1128/AEM.70.8.4814-4820.2004 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chammas GI, Saliba R, Corrieu G, Beal C (2006) Characterisation of lactic acid bacteria isolated from fermented milk “laban”. Int J Food Microbiol 110(1):52–61.  https://doi.org/10.1016/j.ijfoodmicro.2006.01.043 CrossRefPubMedGoogle Scholar
  9. Conrad FD, Owen CM, Patterson J (2004) Solid phase microextraction (SPME) combined with gas-chromatography and olfactometry-mass spectrometry for characterization of cheese aroma compounds. Lebensm Wiss Technol 37:139–154CrossRefGoogle Scholar
  10. Curioni PMG, Bosset JO (2002) Key odorants in various cheese types as determined by gas chromatography-olfactometry. Int Dairy J 12(12):959–984CrossRefGoogle Scholar
  11. de Cadinanos LP, Garcia-Cayuela T, Yvon M, Martinez-Cuesta MC, Pelaez C, Requena T (2013) Inactivation of the panE gene in Lactococcus lactis enhances formation of cheese aroma compounds. Appl Environ Microbiol 79(11):3503–3506.  https://doi.org/10.1128/AEM.00279-13 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Di Rienzo JA, Casanoves F, Balzarini MG, Gonzalez L, Tablada M, Robledo CW (2008) InfoStat, versión 2008. FCA, Universidad Nacional de Córdoba, CórdobaGoogle Scholar
  13. Dudley E, Steele J (2001) Lactococcus lactis LM0230 contains a single aminotransferase involved in aspartate biosynthesis, which is essential for growth in milk. Microbiology 147(Pt 1):215–224.  https://doi.org/10.1099/00221287-147-1-215 CrossRefPubMedGoogle Scholar
  14. Espariz M, Repizo G, Blancato V, Mortera P, Alarcon S, Magni C (2011) Identification of malic and soluble oxaloacetate decarboxylase enzymes in Enterococcus faecalis. FEBS J 278(12):2140–2151.  https://doi.org/10.1111/j.1742-4658.2011.08131.x CrossRefPubMedGoogle Scholar
  15. Espariz M, Zuljan FA, Esteban L, Magni C (2016) Taxonomic identity resolution of highly phylogenetically related strains and selection of phylogenetic markers by using genome-scale methods: the Bacillus pumilus group case. PLoS One 11(9):e0163098.  https://doi.org/10.1371/journal.pone.0163098 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Foulquie Moreno MR, Sarantinopoulos P, Tsakalidou E, De Vuyst L (2006) The role and application of enterococci in food and health. Int J Food Microbiol 106(1):1–24.  https://doi.org/10.1016/j.ijfoodmicro.2005.06.026 CrossRefPubMedGoogle Scholar
  17. Franz CM, Huch M, Abriouel H, Holzapfel W, Galvez A (2011) Enterococci as probiotics and their implications in food safety. Int J Food Microbiol 151(2):125–140.  https://doi.org/10.1016/j.ijfoodmicro.2011.08.014 CrossRefPubMedGoogle Scholar
  18. Gao H, Jiang X, Pogliano K, Aronson AI (2002) The E1β and E2 subunits of the Bacillus subtilis pyruvate dehydrogenase complex are involved in regulation of sporulation. J Bacteriol 184(10):2780–2788CrossRefGoogle Scholar
  19. Giraffa G (2003) Functionality of enterococci in dairy products. Int J Food Microbiol 88(2–3):215–222CrossRefGoogle Scholar
  20. Hanchi H, Mottawea W, Sebei K, Hammami R (2018) The genus Enterococcus: between probiotic potential and safety concerns—an update. Front Microbiol 9:1791.  https://doi.org/10.3389/fmicb.2018.01791 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hazards EPanel oB, Ricci A, Allende A, Bolton D, Chemaly M, Davies R, Girones R, Herman L, Koutsoumanis K, Lindqvist R, Nørrung B, Robertson L, Ru G, Sanaa M, Simmons M, Skandamis P, Snary E, Speybroeck N, Ter Kuile B, Threlfall J, Wahlström H, Cocconcelli PS, Klein G, Prieto Maradona M, Querol A, Peixe L, Suarez JE, Sundh I, Vlak JM, Aguilera-Gómez M, Barizzone F, Brozzi R, Correia S, Heng L, Istace F, Lythgo C, Fernández Escámez PS (2017) Scientific opinion on the update of the list of QPS-recommended biological agents intentionally added to food or feed as notified to EFSA. EFSA J 15(3):e04664.  https://doi.org/10.2903/j.efsa.2017.4664 CrossRefGoogle Scholar
  22. Kieronczyk A, Skeie S, Langsrud T, Le Bars D, Yvon M (2004) The nature of aroma compounds produced in a cheese model by glutamate dehydrogenase positive Lactobacillus INF15D depends on its relative aminotransferase activities towards the different amino acids. Int Dairy J 14(3):227–235.  https://doi.org/10.1016/j.idairyj.2003.07.001 CrossRefGoogle Scholar
  23. Kim MK, Drake SL, Drake MA (2011) Evaluation of key flavor compounds in reduced- and full-fat Cheddar cheeses using sensory studies on model systems. J Sens Stud 26(4):278–290.  https://doi.org/10.1111/j.1745-459X.2011.00343.x CrossRefGoogle Scholar
  24. Lapujade P, Cocaign-Bousquet M, Loubiere P (1998) Glutamate biosynthesis in Lactococcus lactis subsp. lactis NCDO 2118. Appl Environ Microbiol 64(7):2485–2489CrossRefGoogle Scholar
  25. Lebreton F, Willems RJL, Gilmore MS (2014) Enterococcus diversity, origins in nature, and gut colonization. In: Gilmore MS, Clewell DB, Ike Y, Shankar N (eds) Enterococci: from commensals to leading causes of drug resistant infection. BostonGoogle Scholar
  26. Magni C, de Mendoza D, Konings WN, Lolkema JS (1999) Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH. J Bacteriol 181(5):1451–1457CrossRefGoogle Scholar
  27. Marilley L, Casey MG (2004) Flavours of cheese products: metabolic pathways, analytical tools and identification of producing strains. Int J Food Microbiol 90(2):139–159CrossRefGoogle Scholar
  28. Martin MG, Sender PD, Peiru S, de Mendoza D, Magni C (2004) Acid-inducible transcription of the operon encoding the citrate lyase complex of Lactococcus lactis biovar diacetylactis CRL264. J Bacteriol 186(17):5649–5660.  https://doi.org/10.1128/JB.186.17.5649-5660.2004 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Martin MG, Magni C, de Mendoza D, Lopez P (2005) CitI, a transcription factor involved in regulation of citrate metabolism in lactic acid bacteria. J Bacteriol 187(15):5146–5155.  https://doi.org/10.1128/JB.187.15.5146-5155.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Martino GP, Quintana IM, Espariz M, Blancato VS, Gallina Nizo G, Esteban L, Magni C (2016a) Draft genome sequences of four Enterococcus faecium strains isolated from Argentine cheese. Genome Announ 4(1).  https://doi.org/10.1128/genomeA.01576-15
  31. Martino GP, Quintana IM, Espariz M, Blancato VS, Magni C (2016b) Aroma compounds generation in citrate metabolism of Enterococcus faecium: genetic characterization of type I citrate gene cluster. Int J Food Microbiol 218:27–37.  https://doi.org/10.1016/j.ijfoodmicro.2015.11.004 CrossRefPubMedGoogle Scholar
  32. Martino GP, Espariz M, Gallina Nizo G, Esteban L, Blancato VS, Magni C (2018) Safety assessment and functional properties of four enterococci strains isolated from regional Argentinean cheese. Int J Food Microbiol 277:1–9.  https://doi.org/10.1016/j.ijfoodmicro.2018.04.012 CrossRefPubMedGoogle Scholar
  33. Mortera P, Pudlik A, Magni C, Alarcon S, Lolkema JS (2013) Ca2+-citrate uptake and metabolism in Lactobacillus casei ATCC 334. Appl Environ Microbiol 79(15):4603–4612.  https://doi.org/10.1128/AEM.00925-13 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Ogier JC, Serror P (2008) Safety assessment of dairy microorganisms: the Enterococcus genus. Int J Food Microbiol 126(3):291–301.  https://doi.org/10.1016/j.ijfoodmicro.2007.08.017 CrossRefPubMedGoogle Scholar
  35. Palmer KL, Gilmore MS (2010) Multidrug-resistant enterococci lack CRISPR-cas. MBio 1(4).  https://doi.org/10.1128/mBio.00227-10
  36. Palmer KL, Carniol K, Manson JM, Heiman D, Shea T, Young S, Zeng Q, Gevers D, Feldgarden M, Birren B, Gilmore MS (2010) High-quality draft genome sequences of 28 Enterococcus sp. isolates. J Bacteriol 192(9):2469–2470.  https://doi.org/10.1128/JB.00153-10 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Pudlik AM, Lolkema JS (2012) Rerouting citrate metabolism in Lactococcus lactis to citrate-driven transamination. Appl Environ Microbiol 78(18):6665–6673.  https://doi.org/10.1128/AEM.01811-12 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Repizo GD, Mortera P, Magni C (2011) Disruption of the alsSD operon of Enterococcus faecalis impairs growth on pyruvate at low pH. Microbiology 157(Pt 9):2708–2719.  https://doi.org/10.1099/mic.0.047662-0 CrossRefPubMedGoogle Scholar
  39. Repizo GD, Blancato VS, Mortera P, Lolkema JS, Magni C (2013) Biochemical and genetic characterization of the Enterococcus faecalis oxaloacetate decarboxylase complex. Appl Environ Microbiol 79(9):2882–2890.  https://doi.org/10.1128/AEM.03980-12 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Rijnen L, Bonneau S, Yvon M (1999) Genetic characterization of the major lactococcal aromatic aminotransferase and its involvement in conversion of amino acids to aroma compounds. Appl Environ Microbiol 65(11):4873–4880CrossRefGoogle Scholar
  41. Shmuel Y (2003) Dictionary of food compounds with CD-ROM. Additives, flavors, and ingredients. Chapman and Hall/CRC, New YorkGoogle Scholar
  42. Silbersack J, Jurgen B, Hecker M, Schneidinger B, Schmuck R, Schweder T (2006) An acetoin-regulated expression system of Bacillus subtilis. Appl Microbiol Biotechnol 73(4):895–903.  https://doi.org/10.1007/s00253-006-0549-5 CrossRefPubMedGoogle Scholar
  43. Smit BA, van Hylckama Vlieg JE, Engels WJ, Meijer L, Wouters JT, Smit G (2005) Identification, cloning, and characterization of a Lactococcus lactis branched-chain α-keto acid decarboxylase involved in flavor formation. Appl Environ Microbiol 71(1):303–311.  https://doi.org/10.1128/AEM.71.1.303-311.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Steele J, Broadbent J, Kok J (2013) Perspectives on the contribution of lactic acid bacteria to cheese flavor development. Curr Opin Biotechnol 24(2):135–141.  https://doi.org/10.1016/j.copbio.2012.12.001 CrossRefPubMedGoogle Scholar
  45. Suarez CA, Blancato VS, Poncet S, Deutscher J, Magni C (2011) CcpA represses the expression of the divergent cit operons of Enterococcus faecalis through multiple cre sites. BMC Microbiol 11:227.  https://doi.org/10.1186/1471-2180-11-227 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Tanous C, Kieronczyk A, Helinck S, Chambellon E, Yvon M (2002) Glutamate dehydrogenase activity: a major criterion for the selection of flavour-producing lactic acid bacteria strains. Antonie Van Leeuwenhoek 82(1–4):271–278CrossRefGoogle Scholar
  47. Tanous C, Chambellon E, Sepulchre AM, Yvon M (2005) The gene encoding the glutamate dehydrogenase in Lactococcus lactis is part of a remnant Tn3 transposon carried by a large plasmid. J Bacteriol 187(14):5019–5022.  https://doi.org/10.1128/JB.187.14.5019-5022.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Tanous C, Chambellon E, Le Bars D, Delespaul G, Yvon M (2006) Glutamate dehydrogenase activity can be transmitted naturally to Lactococcus lactis strains to stimulate amino acid conversion to aroma compounds. Appl Environ Microbiol 72(2):1402–1409.  https://doi.org/10.1128/AEM.72.2.1402-1409.2006 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Torres Manno M, Zuljan F, Alarcon S, Esteban L, Blancato V, Espariz M, Magni C (2018) Genetic and phenotypic features defining industrial relevant Lactococcus lactis, L. cremoris and L. lactis biovar. diacetylactis strains. J Biotechnol 282:25–31.  https://doi.org/10.1016/j.jbiotec.2018.06.345 CrossRefPubMedGoogle Scholar
  50. Torres Manno MA, Pizarro MD, Prunello M, Magni C, Daurelio LD, Espariz M (2019) GeM-Pro: a tool for genome functional mining and microbial profiling. Appl Microbiol Biotechnol 103(7):3123–3134.  https://doi.org/10.1007/s00253-019-09648-8 CrossRefPubMedGoogle Scholar
  51. Ward DE, Ross RP, van der Weijden CC, Snoep JL, Claiborne A (1999) Catabolism of branched-chain alpha-keto acids in Enterococcus faecalis: the bkd gene cluster, enzymes, and metabolic route. J Bacteriol 181(17):5433–5442CrossRefGoogle Scholar
  52. Ward DE, van Der Weijden CC, van Der Merwe MJ, Westerhoff HV, Claiborne A, Snoep JL (2000) Branched-chain alpha-keto acid catabolism via the gene products of the bkd operon in Enterococcus faecalis: a new, secreted metabolite serving as a temporary redox sink. J Bacteriol 182(11):3239–3246CrossRefGoogle Scholar
  53. Yvon M, Rijnen L (2001) Cheese flavour by amino acid catabolism. Int Dairy J 11(4):185–201CrossRefGoogle Scholar
  54. Yvon M, Thirouin S, Rijnen L, Fromentier D, Gripon JC (1997) An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds. Appl Environ Microbiol 63(2):414–419CrossRefGoogle Scholar
  55. Yvon M, Chambellon E, Bolotin A, Roudot-Algaron F (2000) Characterization and role of the branched-chain aminotransferase (BcaT) isolated from Lactococcus lactis subsp. cremoris NCDO 763. Appl Environ Microbiol 66(2):571–577CrossRefGoogle Scholar
  56. Zuljan FA, Repizo GD, Alarcon SH, Magni C (2014) Alpha-acetolactate synthase of Lactococcus lactis contributes to pH homeostasis in acid stress conditions. Int J Food Microbiol 188:99–107.  https://doi.org/10.1016/j.ijfoodmicro.2014.07.017 CrossRefPubMedGoogle Scholar
  57. Zuljan FA, Mortera P, Alarcón SH, Blancato VS, Espariz M, Magni C (2016) Lactic acid bacteria decarboxylation reactions in cheese. Int Dairy J 62:53–62.  https://doi.org/10.1016/j.idairyj.2016.07.007 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Matilde D’Angelo
    • 1
    • 2
  • Gabriela P. Martino
    • 1
    • 3
  • Victor S. Blancato
    • 1
    • 3
    • 4
  • Martín Espariz
    • 1
    • 3
    • 4
  • Axel Hartke
    • 4
  • Nicolas Sauvageot
    • 4
  • Abdellah Benachour
    • 4
  • Sergio H. Alarcón
    • 2
    • 3
  • Christian Magni
    • 1
    • 3
    • 4
    Email author
  1. 1.Laboratorio de Fisiología y Genética de Bacterias Lácticas, Instituto de Biología Molecular y Celular de Rosario (IBR), sede Facultad de Ciencias Bioquímicas y Farmacéuticas (FBioyF)Universidad Nacional de Rosario (UNR), Consejo Nacional de Ciencia y Tecnología (CONICET)RosarioArgentina
  2. 2.Instituto de Química de Rosario (IQUIR), FBioyFUNR-CONICETRosarioArgentina
  3. 3.Laboratorio de Biotecnología e Inocuidad de los Alimentos, FBioyFUNR-Municipalidad de Granadero BaigorriaRosarioArgentina
  4. 4.U2RM Stress/VirulenceNormandie Univ, UNICAENCaenFrance

Personalised recommendations