Lactic acid bacteria communities in must, alcoholic and malolactic Tempranillo wine fermentations, by culture-dependent and culture-independent methods


The lactic acid bacteria (LAB) communities from must and through alcoholic (AF) and malolactic fermentations (MLF) of Tempranillo red wines were studied in ten wineries from the Designation of Origin Rioja during three consecutive vintages. A statistical study with data from both methods, PCR-DGGE and plating, was performed. Results showed that the LAB community in the D.O. Rioja was highly determined by the type of fermentation and also by the different stages within the winemaking, while other factors such as year, winery, or sampling subzones had not significant effect on the LAB species distribution. Three microbial families, seven genera, and 25 species were described in this research, and Lactobacillus was the most commonly detected genus before MLF. Curiously, genera and species not frequently detected in wines as Weissella, Fructobacillus, and Oenococcus kitaharae were identified during AF, and no-Oenococcus oeni species were described in some MLF by both methods. For the first time, two new O. oeni allelic groups were determined by 16S rDNA/DGGE being randomly adapted to the wine environment. Further studies targeted to understand the implication of the novel species, and O. oeni allelic groups in Rioja wine fermentations could be really interesting.

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  1. 1.

    Bartowsky EJ (2014) Encyclopedia of food microbiology. Encycl Food Microbiol. doi:10.1016/B978-0-12-384730-0.00357-8

    Google Scholar 

  2. 2.

    Lonvaud-Funel A (2010) Managing wine quality. Manag Wine Qual. doi:10.1533/9781845699987.1.60

    Google Scholar 

  3. 3.

    Renouf V, Gindreau E, Claisse O, Lonvaud-Funel A (2005) Microbial changes during malolatic fermentation in red wine elaboration. J Int Sci Vigne Vin 39:179–190

    CAS  Google Scholar 

  4. 4.

    Petri A, Pfannebecker J, Fröhlich J et al (2013) Fast identification of wine related lactic acid bacteria by multiplex PCR. Food Microbiol 33:48–54. doi:10.1016/

    CAS  Article  Google Scholar 

  5. 5.

    Mesas JM, Rodríguez MC, Alegre MT (2011) Characterization of lactic acid bacteria from musts and wines of three consecutive vintages of Ribeira Sacra. Lett Appl Microbiol 52:258–268. doi:10.1111/j.1472-765X.2010.02991.x

    CAS  Article  Google Scholar 

  6. 6.

    López R, Tenorio C, Gutiérrez ARAR et al (2012) Elaboration of Tempranillo wines at two different pHs. Influence on biogenic amine contents. Food Control 25:583–590. doi:10.1016/j.foodcont.2011.11.029

    Article  Google Scholar 

  7. 7.

    González-Arenzana L, Santamaría P, López R, López-Alfaro I (2013) Indigenous lactic acid bacteria communities in alcoholic and malolactic fermentations of Tempranillo wines elaborated in ten wineries of La Rioja (Spain). Food Res Int 50:438–445. doi:10.1016/j.foodres.2012.11.008

    Article  Google Scholar 

  8. 8.

    Lucena-Padrós H, Jiménes E, Maldonado-Barragán A et al (2015) PCR-DGGE assessment of the bacterial diversity in Spanish-style green table olives fermentations. Int J Food Microbiol 205:47–53. doi:10.1016/j.ijfoodmicro.2015.03.033

    Article  Google Scholar 

  9. 9.

    Bonetta S, Bonetta S, Carraro E et al (2008) Microbiological characterisation of Robiola di Roccaverano cheese using PCR-DGGE. Food Microbiol 25:786–792. doi:10.1016/

    CAS  Article  Google Scholar 

  10. 10.

    Cocolin L, Alessandria V, Dolci P et al (2013) Culture independent methods to assess the diversity and dynamics of microbiota during food fermentation. Int J Food Microbiol 167:29–43. doi:10.1016/j.ijfoodmicro.2013.05.008

    CAS  Article  Google Scholar 

  11. 11.

    Cocolin L, Campolongo S, Alessandria V et al (2011) Culture independent analyses and wine fermentation: an overview of achievements 10 years after first application. Ann Microbiol 61:17–23. doi:10.1007/s13213-010-0076-6

    Article  Google Scholar 

  12. 12.

    Case RJ, Boucher Y, Dahllöf I et al (2007) Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies. Appl Environ Microbiol 73:278–288. doi:10.1128/AEM.01177-06

    CAS  Article  Google Scholar 

  13. 13.

    Renouf V, Claisse O, Lonvaud-Funel A (2006) rpoB gene: a target for identification of LAB cocci by PCR-DGGE and melting curves analyses in real time PCR. J Microbiol Methods 67:162–170. doi:10.1016/j.mimet.2006.03.008

    CAS  Article  Google Scholar 

  14. 14.

    González-Arenzana L, López R, Santamaría P, López-Alfaro I (2012) Application of the Different Electrophoresis Techniques to the Detection and Identification of Lactic Acid Bacteria in Wines. In: Ghowsi K (ed) Electrophoresis. Intech, Croatia, pp 137–156

  15. 15.

    Lopez I, Ruiz-Larrea F, Cocolin L et al (2003) Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis. Appl Environ Microbiol 69:6801–6807. doi:10.1128/AEM.69.11.6801-6807.2003

    CAS  Article  Google Scholar 

  16. 16.

    González-Arenzana L, López R, Portu J et al (2014) Molecular analysis of Oenococcus oeni and the relationships among and between commercial and autochthonous strains. J Biosci Bioeng 118:272–276. doi:10.1016/j.jbiosc.2014.02.013

    Article  Google Scholar 

  17. 17.

    Lucore LA, Cullison MA, Jaykus L-AA (2000) Immobilization with metal hydroxides as a means to concentrate food-borne bacteria for detection by cultural and molecular methods. Appl Environ Microbiol 66:1769–1776. doi:10.1128/AEM.66.5.1769-1776.2000

    CAS  Article  Google Scholar 

  18. 18.

    Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    CAS  Article  Google Scholar 

  19. 19.

    Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. doi:10.1093/molbev/mst010

    CAS  Article  Google Scholar 

  20. 20.

    Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–818. doi:10.1093/bioinformatics/14.9.817

    CAS  Article  Google Scholar 

  21. 21.

    Guindon S, Dufayard J-FF, Lefort V et al (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321. doi:10.1093/sysbio/syq010

    CAS  Article  Google Scholar 

  22. 22.

    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0 RID E-9283-2010. Mol Biol Evol 24:1596–1599. doi:10.1093/molbev/msm092

    CAS  Article  Google Scholar 

  23. 23.

    González-Arenzana L, Pérez-Martín F, Palop ML et al (2015) Genomic diversity of Oenococcus oeni populations from Castilla La Mancha and La Rioja Tempranillo red wines. Food Microbiol 49:82–94. doi:10.1016/

    Article  Google Scholar 

  24. 24.

    Lucena-Padrós H, Jiménez E, Maldonado-Barragán A et al (2015) PCR-DGGE assessment of the bacterial diversity in Spanish-style green table-olive fermentations. Int J Food Microbiol 205:47–53. doi:10.1016/j.ijfoodmicro.2015.03.033

    Article  Google Scholar 

  25. 25.

    James JB, Sherman TD, Devereux R (2006) Analysis of bacterial communities in seagrass bed sediments by double-gradient denaturing gradient gel electrophoresis of PCR-Amplified 16S rRna genes. Microb Ecol 52:655–661. doi:10.1007/s00248-006-9075-3

    CAS  Article  Google Scholar 

  26. 26.

    González-Arenzana L, López R, Santamaría P et al (2012) Dynamics of indigenous lactic acid bacteria populations in wine fermentations from La Rioja (Spain) during three vintages. Microb Ecol 62:12–19. doi:10.1007/s00248-011-9911-y

    Article  Google Scholar 

  27. 27.

    Justé A, Malfliet S, Waud M et al (2014) Bacterial community dynamics during industrial malting, with an emphasis on lactic acid bacteria. Food Microbiol 39:39–46. doi:10.1016/

    Article  Google Scholar 

  28. 28.

    Dicks LMT, Endo A (2009) Taxonomic status of lactic acid bacteria in wine and key characteristics to differentiate species. S Afr J Enol Vitic 30:72–90

    Google Scholar 

  29. 29.

    Pang H, Qin G, Tan Z et al (2011) Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis. Syst Appl Microbiol 34:235–241. doi:10.1016/j.syapm.2010.10.003

    CAS  Article  Google Scholar 

  30. 30.

    Endo A, Irisawa T, Futagawa-Endo Y et al (2011) Fructobacillus tropaeoli sp. nov., a fructophilic lactic acid bacterium isolated from a flower. Int J Syst Evol Microbiol 61:898–902. doi:10.1099/ijs.0.023838-0

    CAS  Article  Google Scholar 

  31. 31.

    Jia R, Chen H, Chen H, Ding W (2016) Effects of fermentation with Lactobacillus rhamnosus GG on product quality and fatty acids of goat milk yogurt. J Dairy Sci 99:221–227. doi:10.3168/jds.2015-10114

    CAS  Article  Google Scholar 

  32. 32.

    García-Ruiz A, Requena T, Peláez C et al (2013) Antimicrobial activity of lacticin 3147 against oenological lactic acid bacteria. Combined effect with other antimicrobial agents. Food Control 32:477–483. doi:10.1016/j.foodcont.2013.01.027

    Article  Google Scholar 

  33. 33.

    Renouf V, Vayssieres LC, Claisse O, Lonvaud-Funel A (2009) Genetic and phenotypic evidence for two groups of Oenococcus oeni strains and their prevalence during winemaking. Appl Microbiol Biotechnol 83:85–97. doi:10.1007/s00253-008-1843-1

    CAS  Article  Google Scholar 

  34. 34.

    Randazzo CL, Ribbera Á, Pitino I et al (2012) Diversity of bacterial population of table olives assessed by PCR-DGGE analysis. Food Microbiol 32:87–96. doi:10.1016/

    CAS  Article  Google Scholar 

  35. 35.

    Bae S, Fleet GH, Heard GM (2006) Lactic acid bacteria associated with wine grapes from several Australian vineyards. J Appl Microbiol 100:712–727. doi:10.1111/j.1365-2672.2006.02890.x

    CAS  Article  Google Scholar 

  36. 36.

    Andorrà I, Landi S, Mas A et al (2010) Effect of fermentation temperature on microbial population evolution using culture-independent and dependent techniques. Food Res Int 43:773–779. doi:10.1016/j.foodres.2009.11.014

    Article  Google Scholar 

  37. 37.

    Rantsiou K, Urso R, Iacumin L et al (2005) Culture-dependent and -independent methods to investigate the microbial ecology of Italian fermented sausages. Appl Environ Microbiol 71:1977–1986. doi:10.1128/AEM.71.4.1977-1986.2005

    CAS  Article  Google Scholar 

  38. 38.

    Renouf V, Claisse O, Miot-Sertier C, Lonvaud-Funel A (2006) Lactic acid bacteria evolution during winemaking: use of rpoB gene as a target for PCR-DGGE analysis. Food Microbiol 23:136–145. doi:10.1016/

    CAS  Article  Google Scholar 

  39. 39.

    Ruiz P, Seseña S, Izquierdo PM, Palop ML (2010) Bacterial biodiversity and dynamics during malolactic fermentation of Tempranillo wines as determined by a culture-independent method (PCR-DGGE). Appl Microbiol Biotechnol 86:1555–1562. doi:10.1007/s00253-010-2492-8

    CAS  Article  Google Scholar 

  40. 40.

    Ribéreau-Gayon P, Dubourdieu D, Donèche B, Lonvaud-Funel A (2007) Handbook of enology, the microbiology of wine and vinifications. Wiley, Sussex

    Google Scholar 

  41. 41.

    Renouf V, Claisse O, Lonvaud-Funel A (2007) Inventory and monitoring of wine microbial consortia. Appl Microbiol Biotechnol 75:149–164. doi:10.1007/s00253-006-0798-3

    CAS  Article  Google Scholar 

  42. 42.

    Figueiredo AR, Campos F, de Freitas V et al (2008) Effect of phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii. Food Microbiol 25:105–112. doi:10.1016/

    CAS  Article  Google Scholar 

  43. 43.

    Cho G-S, Krauss S, Huch M et al (2011) Development of a quantitative PCR for detection of Lactobacillus plantarum starters during wine malolactic fermentation. J Microbiol Biotechnol 21:1280–1286. doi:10.4014/jmb.1107.07003

    CAS  Article  Google Scholar 

  44. 44.

    Pozo-Bayón MA, G-Alegría E, Polo MC et al (2005) Wine volatile and amino acid composition after malolactic fermentation: effect of Oenococcus oeni and Lactobacillus plantarum starter cultures. J Agric Food Chem 53:8729–8735

    Article  Google Scholar 

  45. 45.

    Renouf V, Strehaiano P, Lonvaud-Funel A (2007) Yeast and bacteria analysis of grape, wine and cellar equipments by PCR-DGGE. J Int Des Sci La Vigne Du Vin 41:51–61

    CAS  Google Scholar 

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This work was supported by funding and predoctoral grant (B.O.R. 6th March, 2009) of the Government of La Rioja, the I.N.I.A. Project RTA2007-00104-00-00 and it was possible thanks to the collaborating wineries.

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Correspondence to Isabel López-Alfaro.

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Online resource 1.

Consensus dendrogram obtained by composite data set combining the results of the LAB species detected with culture-dependent methods and independent methods (PCR-16S rDNA/DGGE and PCR-rpoB/DGGE) showing cophenetic correlation values. Samples were labelled with a number corresponding with the isolation stage (1: must, 2: middle AF, 3: final AF, 4: initial MLF, 5: middle MLF and 6: final MLF), with a capital letter indicative of the winery (from A to J) and Roman numbers placed in parenthesis representing the consecutive isolation years (I, II and III). The identified species were labelled with lower Latin letters placed at the top of each representative band (a: O. oeni, b: O. kitaharae, c: L. citreum, d: L. mesenteroides, e: L. pseudomesenteroides, f: L. fallax, g: F, ficulneus, h: F. tropaeoli, i: W. cibaria, j: W. paramesenteroides, k: Weisella sp., l: W. solis, m: L. brevis, n: L. buchneri, o: L. coryniformis, p: L. mali, q: L. hilgardii, r: L. plantarum, s: L. pentosus, t: L. rhamnosus, u: Lactobacillus sp., v: L. uvarum, w: P. pentosaceus, x: P. parvulus, y: L. lactis). Supplementary material 1 (DOC 85 kb)

Online resource 2.

Phylogram for the 110 sequences identified as species belonging to genus Oenococcus, obtained from 16S rDNA PCR/DGGE. Each sequence is referred with the most accurate identification and the identity percentage (%), with the given accession number and with a code that means the isolation stage (from 1 to 6), winery (from A to J) and year (I, II or III). The evolutionary distances are in the units of the number of base substitutions per site. Supplementary material 2 (DOC 28 kb)

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González-Arenzana, L., Santamaría, P., Gutiérrez, A.R. et al. Lactic acid bacteria communities in must, alcoholic and malolactic Tempranillo wine fermentations, by culture-dependent and culture-independent methods. Eur Food Res Technol 243, 41–48 (2017).

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  • Lactic acid bacteria
  • DGGE
  • O. oeni alleles
  • Distribution
  • Ecology