Skip to main content
SpringerLink
Log in

Dynamics of lactic acid bacteria populations in Rioja wines by PCR-DGGE, comparison with culture-dependent methods

  • Applied microbial and cell physiology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Lactic acid bacteria populations of red wine samples from industrial fermentations, including two different vinification methods were studied. For this investigation, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis was employed to supplement previous results that were obtained by culture-dependent methods. PCR-DGGE was aimed to study two targeted genes, 16S ribosomal DNA (rDNA) and rpoB, and the results were useful to evaluate the microbial populations in wine samples. Moreover, an improvement of a detection limit determined so far for DGGE analysis was obtained with the method described in this study, what made possible to identify lactic acid bacteria populations below 101 colony-forming unit/mL. The species Oenococcus oeni was the most frequently detected bacterium, but identifications close to species Oenococcus kitaharae and Lactococcus lactis that are not often found in wine were firstly identified in samples of this research. PCR-DGGE allowed to detect 9 out of 11 lactic acid bacteria species identified in this study (nine by PCR-16S rDNA/DGGE and four by PCR-rpoB/DGGE), while five species were detected using the modified de Man, Rogosa and Sharpe agar. Therefore, the two methods were demonstrated to be complementary. This finding suggests that analysis of the lactic acid bacteria population structure in wine should be carried out using both culture-dependent and culture-independent techniques with more than one primer pair.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Alexandre H, Costello PJ, Remize F, Guzzo J, Guilloux-Benatier M (2004) Saccharomyces cerevisiae–Oenococcus oeni interactions in wine: current knowledge and perspectives. Int J Food Microbiol 93:141–154. doi:10.1016/j.ijfoodmicro.2003.10.103

    Article  PubMed  CAS  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic Local Alignment Search Tool. J Mol Biol 215:403–410

    PubMed  CAS  Google Scholar 

  • Andorrà I, Landi S, Mas A, Guillamón JM, Esteve-Zarzoso B (2008) Effect of oenological practices on microbial populations using culture-independent techniques. Food Microbiol 25:849–856. doi:10.1016/j.fm.2008.05.005

    Article  PubMed  Google Scholar 

  • Andorrà I, Landi S, Mas A, Esteve-Zarzoso B, Guillamón JM (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 

  • Bae S, Fleet GH, Heard GM (2006) Lactic acid bacteria associated with wine grapes from several Australian vineyards. J Appl Microbiol 100:712–727

    Article  PubMed  CAS  Google Scholar 

  • Bester L, Cameron M, du Toit M, Witthuhn RC (2010) PCR and DGGE detection limits for wine spoilage microbes. S Afr J Enol Vitic 31:26–33

    CAS  Google Scholar 

  • Case RJ, Boucher Y, Dahllöf I, Holmström C, Doolittle WF, Kjelleberg S (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

    Article  PubMed  CAS  Google Scholar 

  • Cocolin L, Mills D (2003) Wine yeast inhibition by sulphur dioxide: a comparison of culture-dependent and independent methods. Am J Enol Vitic 54:125–130

    CAS  Google Scholar 

  • Cocolin L, Campolongo S, Alessandria V, Dolci P, Rantsiou K (2011a) 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 

  • Cocolin L, Dolci P, Rantsiou K (2011b) Biodiversity and dynamics of meat fermentations: the contribution of molecular methods for a better comprehension of a complex ecosystem. Meat Sci 89:296–302. doi:10.1016/j.meatsci.2011.04.011

    Article  PubMed  CAS  Google Scholar 

  • de Revel G, Martin N, Pripis-Nicolau L, Lonvaud-Funel A, Bertrand A (1999) Contribution to the knowledge of malolactic fermentation influence on wine aroma. J Agric Food Chem 47:4003–4008

    Article  PubMed  Google Scholar 

  • 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 

  • Divol B, Lonvaud-Funel A (2005) Evidence for viable but nonculturable yeasts in Botrytis-affected wine. J Appl Microbiol 99:85–93. doi:10.1111/j.1365-2672.2005.02578.x

    Article  PubMed  CAS  Google Scholar 

  • Dobson CM, Deneer H, Lee S, Hemmingsen S, Glaze S, Ziola B (2002) Phylogenetic analysis of the genus Pediococcus, including Pediococcus claussenii sp. nov., a novel lactic acid bacterium isolated from beer. Int J Syst Evol Microbiol 52:2003–2010. doi:10.1099/ijs.0.02191-0

    Article  PubMed  CAS  Google Scholar 

  • Dols-Lafargue M, Lee HY, Le Marrec C, Heyraud A, Chambat G, Lonvaud-Funel A (2008) Characterization of gtf, a glucosyltransferase gene in the genomes of Pediococcus parvulus and Oenococcus oeni, two bacterial species commonly found in wine. Appl Environ Microbiol 74:4079–4090. doi:10.1128/AEM.00673-08

    Article  PubMed  CAS  Google Scholar 

  • du Toit M, Engelbrecht L, Lerm E, Krieger-Weber S (2011) Lactobacillus: the next generation of malolactic fermentation starter cultures—an overview. Food Bioproc Technol 4:876–906. doi:10.1007/s11947-010-0448-8

    Article  Google Scholar 

  • Endo A, Okada S (2006) Oenococcus kitaharae sp. nov., a non-acidophilic and non-malolactic-fermenting Oenococcus isolated from a composting distilled shochu residue. Int J Syst Evol Microbiol 56:2345–2348. doi:10.1099/ijs.0.64288-0

    Article  PubMed  CAS  Google Scholar 

  • Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791. doi:10.2307/2408678

    Article  Google Scholar 

  • González-Arenzana L, López R, Santamaría P, Garijo P, Gutiérrez AR, López-Alfaro I, Tenorio C (2010) Comparación de distintos métodos de extracción directa de ADN de vino tinto para el estudio de bacterias lácticas. VI Foro Mundial del Vino, Logroño

    Google Scholar 

  • González-Arenzana L, Santamaría P, López R, Tenorio C, López-Alfaro I (2012a) Ecology of indigenous lactic acid bacteria along different winemaking processes of Tempranillo red wine from La Rioja (Spain). Sci World J. doi:10.1100/2012/796327

    Google Scholar 

  • González-Arenzana L, López R, Santamaría P, Tenorio C, López-Alfaro I (2012b) Dynamics of indigenous lactic acid bacteria populations in wine fermentations from La Rioja (Spain) during three vintages. Microb Ecol 62:1–8. doi:10.1007/s00248-011-9911-y

    Google Scholar 

  • 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 50:538–545. doi:10.1016/j.foodres.2012.11.008

    Article  Google Scholar 

  • Kim JY, Kim D, Park P, Kang H, Ryu EK, Kim SM (2011) Effects of storage temperature and time on the biogenic amine content and microflora in Korean turbid rice wine, Makgeolli. Food Chem 128:87–92. doi:10.1016/j.foodchem.2011.02.081

    Article  CAS  Google Scholar 

  • Liu SQ, Pritchard GG, Hardman MJ, Pilone GJ (1994) Citrulline production and ethyl carbamate (urethane) precursor formation from arginine degradation by wine lactic acid bacteria Leuconostoc oenos and Lactobacillus buchneri. Am J Enol Vitic 45:235–242

    CAS  Google Scholar 

  • Lonvaud-Funel A (1999) Lactic acid bacteria in the quality improvement and depreciation of wine. Anton Leeuw Int J Gen Mol Microbiol 76:317–331

    Article  CAS  Google Scholar 

  • Lonvaud-Funel A (2008) From raisin to wine: activity of a dynamic microbial system. Biofutur 26–29

  • López I, Ruiz-Larrea F, Cocolin L, Orr E, Phister T, Marshall M, VanderGheynst J, Mills DA (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

    Article  PubMed  Google Scholar 

  • López I, López R, Santamaría P, Torres C, Ruiz-Larrea F (2008) Performance of malolactic fermentation by inoculation of selected Lactobacillus plantarum and Oenococcus oeni strains isolated from Rioja red wines. Vitis 47:123–129

    Google Scholar 

  • López-Alfaro I (2004) Detección y Control por Técnicas de la Biología Molecular de Bacterias Lácticas Autóctonas Responsables de la Fermentación maloláctica en Vinos de D.O.Ca. Rioja. Universidad de La Rioja, Logroño

    Google Scholar 

  • Lucena BTL, dos Santos BM, Moreira JLS, Moreira APB, Nunes AC, Azevedo V, Miyoshi A, Thompson FL, de Morais Junior MA (2010) Diversity of lactic acid bacteria of the bioethanol process. BMC Microbiol 10:298. doi:10.1186/1471-2180-10-298

    Article  PubMed  Google Scholar 

  • Lucore L, Cullison M, Jaykus L (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

    Article  PubMed  CAS  Google Scholar 

  • Meroth CB, Walter J, Hertel C, Brandt MJ, Hammes WP (2003) Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis. Appl Environ Microbiol 69:475–482. doi:10.1128/AEM.69.1.475-482.2003

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Millet V, Lonvaud-Funel A (2000) The viable but non-culturable state of wine micro-organisms during storage. Lett Appl Microbiol 30:136–141

    Article  PubMed  CAS  Google Scholar 

  • Mira de Orduña R, Patchett M, Liu S, Pilone G (2001) Growth and arginine metabolism of the wine lactic acid bacteria Lactobacillus buchneri and Oenococcus oeni at different pH values and arginine concentrations RID B-9010-2009. Appl Environ Microbiol 67:1657–1662. doi:10.1128/AEM.67.4.1657-1662.2001

    Article  PubMed  Google Scholar 

  • Muyzer G, Smalla K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology RID H-4002-2011. Anton Leeuw Int J Gen Mol Microbiol 73:127–141. doi:10.1023/A:1000669317571

    Article  CAS  Google Scholar 

  • Ogier J, Casalta E, Farrokh C, Saihi A (2008) Safety assessment of dairy microorganisms: the Leuconostoc genus. Int J Food Microbiol 126:286–290. doi:10.1016/j.ijfoodmicro.2007.08.012

    Article  PubMed  CAS  Google Scholar 

  • Pérez-Pulido R, Abriouel H, Ben Omar N, Lucas R, Martínez-Canamero M, Galvez A (2006) Safety and potential risks of enterococci isolated from traditional fermented capers. Food Chem Toxicol 44:2070–2077. doi:10.1016/j.fct.2006.07.008

    Article  PubMed  Google Scholar 

  • Phister TG, Rawsthorne H, Joseph CML, Mills DA (2007) Real-time PCR assay for detection and enumeration of Hanseniaspora species from wine and juice RID G-2282-2011. Am J Enol Vitic 58:229–233

    CAS  Google Scholar 

  • Rantsiou K, Urso R, Dolci P, Comi G, Cocolin L (2008) Microflora of Feta cheese from four Greek manufacturers. Int J Food Microbiol 126:36–42. doi:10.1016/j.ijfoodmicro.2008.04.031

    Article  PubMed  CAS  Google Scholar 

  • Reguant C, Carreté R, Constanti M, Bordons A (2005) Population dynamics of Oenococcus oeni strains in a new winery and the effect of SO2 and yeast strain. FEMS Microbiol Lett 246:111–117. doi:10.1016/j.femsle.2005.03.045

    Article  PubMed  CAS  Google Scholar 

  • Renouf V, Favier M (2010) Genetic and physiological characterisation of Oenococcus oeni strains to perform malolactic fermentation in wines. S Afr J Enol Vitic 31:75–81

    Google Scholar 

  • Renouf V, Claisse O, Lonvaud-Funel A (2005a) Understanding the microbial ecosystem on the grape berry surface through numeration and identification of yeast and bacteria. Aust J Grape Wine Res 11:316–327

    Article  Google Scholar 

  • Renouf V, Gindreau E, Claisse O, Lonvaud-Funel A (2005b) Microbial changes during malolactic fermentation in red wine elaboration. J Int Des Sci De La Vigne Et Du Vin 39:179–190

    CAS  Google Scholar 

  • Renouf V, Claisse O, Lonvaud-Funel A (2006a) 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

    Article  PubMed  CAS  Google Scholar 

  • Renouf V, Claisse O, Miot-Sertier C, Lonvaud-Funel A (2006b) 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/j.fm.2005.01.019

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Ribéreau-Gayon P, Dubourdieu D, Donèche B, Lonvaud A (2007) Handbook of enology. Wiley, England

    Google Scholar 

  • Ruiz P, Izquierdo PM, Seseña S, Palop ML (2010) Analysis of lactic acid bacteria populations during spontaneous malolactic fermentation of Tempranillo wines at five wineries during two consecutive vintages. Food Control 21:70–75. doi:10.1016/j.foodcont.2009.04.002

    Article  CAS  Google Scholar 

  • Ruiz-Larrea F, López-Alfaro I, Alegría E, Zarazaga M, Torres C (2001) Aspectos prácticos de la fermentación malolácica. Gobierno de La Rioja, Logroño

    Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method—a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Torriani S, Felis GE, Fracchetti F (2011) Selection criteria and tools for malolactic starters development: an update. Ann Microbiol 61:33–39. doi:10.1007/s13213-010-0072-x

    Article  CAS  Google Scholar 

  • Vanvuuren HJJ, Dicks LMT (1993) Leuconostoc oenos—a review. Am J Enol Vitic 44:99–112

    CAS  Google Scholar 

  • Zhang Z, Ye Z, Yu L, Shi P (2011) Phylogenomic reconstruction of lactic acid bacteria: an update. BMC Evol Biol 11:1. doi:10.1186/1471-2148-11-1

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by funding and predoctoral grant (BOR 6 March 2009) from the Government of La Rioja, the INIA project RTA2007-00104-00-00 and the FEDER of the European Community and was made possible by the collaborating wineries.

Author information

Authors and Affiliations

  1. Instituto de Ciencias de la Vid y del Vino (Gobierno de La Rioja, Universidad de La Rioja, CSIC), C/ Madre de Dios 51, 26006, Logroño, La Rioja, Spain

    Lucía González-Arenzana, Rosa López, Pilar Santamaría & Isabel López-Alfaro

  2. Servicio de Investigación y Desarrollo Tecnológico Agroalimentario, Ctra. de Mendavia-Logroño (NA 134, km. 88), 26071, Logroño, La Rioja, Spain

    Isabel López-Alfaro

Authors
  1. Lucía González-Arenzana
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rosa López
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Pilar Santamaría
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Isabel López-Alfaro
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Isabel López-Alfaro.

Rights and permissions

Reprints and permissions

About this article

Cite this article

González-Arenzana, L., López, R., Santamaría, P. et al. Dynamics of lactic acid bacteria populations in Rioja wines by PCR-DGGE, comparison with culture-dependent methods. Appl Microbiol Biotechnol 97, 6931–6941 (2013). https://doi.org/10.1007/s00253-013-4974-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-013-4974-y

Keywords

Search

Navigation