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A novel molecular method for identification of Oenococcus oeni and its specific detection in wine

https://doi.org/10.1016/j.ijfoodmicro.2010.06.006Get rights and content

Abstract

Oenococcus oeni is a species of lactic acid bacteria with economic interest in winemaking. Using both in silico and in vitro analyses, a molecular method was developed that allows the identification of O. oeni isolates and its detection from wine samples. The method is based on the amplification of 16S rRNA gene with universal primers followed by restriction with the endonuclease FseI, generating two fragments of 326 and 1233 bp. Among wine bacteria, the FseI recognition sequence is only found in the 16S rRNA gene of O. oeni, ensuring the specificity of the method. The use of Whatman FTA cards for DNA extraction and purification is an efficient and interesting alternative to current methods, as samples can be easily collected at wineries by a non-specialized technician, stored at room temperature and sent in a mail envelope to the analytical laboratory for processing. The proposed method, with a detection limit between 102 and 103 cfu/mL and a full turnaround time of ca. 8 h, ensures the rapid and reliable detection of O. oeni in wine samples during winemaking surveillance and wine quality control.

Introduction

Lactic acid bacteria (LAB), belonging to genera Lactobacillus, Leuconostoc, Oenococcus and Pediococcus, are present in grape musts with populations that vary from 102 to 104 cfu/mL. Along alcoholic fermentation in winemaking, a natural selection occurs driven by the low pH, the grape sulfiting level and the increase in alcohol concentration and Oenococcus oeni, a heterofermentative coccus, usually becomes the dominant LAB species mainly due to its high tolerance to acidic conditions and ethanol (Costello et al., 1983, Lafon-Lafourcade et al., 1983). After alcoholic fermentation O. oeni populations start to multiply to levels of 106–108 cfu/mL and carry out the malolactic fermentation (MLF), a decarboxylation process driven by the malolactic enzyme (EC 1.1.1.38) that converts l-malic acid into l-lactic acid (Lonvaud et al., 1977). MLF is an important step in the vinification process as it ensures deacidification and microbial stability of red and white wines and its extent will depend on medium conditions (Bousbouras & Kunkee, 1971, Davis et al., 1988, Renouf et al., 2006a, Ribereau-Gayon et al., 2006a). Besides wine deacidification, MLF also enhances organoleptic properties, contributing to wine sensory quality (de Revel et al., 1999, Lonvaud-Funel, 1999).

After malic acid consumption, by O. oeni, the remaining LAB present in wine need to be inactivated, even removed, as due to the pH increase after malic acid catabolism they are able to develop and metabolise other substrates being at the origin of wine depreciation (Ribereau-Gayon et al., 2006b). As a consequence, an accurate control of malic acid and/or total LAB population is required along the winemaking process.

LAB and O. oeni in particular are difficult to study and characterise, as they are quite fastidious implying much time-consuming work often leading to ambiguous results. Several molecular approaches have been applied to detect and/or identify wine LAB, including dot-blot or colony hybridization with specific DNA probes (Lonvaud-Funel et al., 1991), PCR amplification of mle gene with specific primers (Zapparoli et al., 1998), amplification and sequencing of 16S rRNA or rpoB genes (Renouf et al., 2006a, Renouf et al., 2006b, Sato et al., 2001), 16S rRNA- or rpoB-based analysis of restriction fragment length polymorphisms (Claisse et al., 2007, Sato et al., 2000), analysis of wine microbial communities by PCR-DGGE (Lopez et al., 2003, Renouf et al., 2006a, Renouf et al., 2006b, Spano et al., 2007), fluorescent in-situ hybridization (Blasco et al., 2003) and real-time quantitative PCR (Pinzani et al., 2004). Overall, molecular biology techniques are useful for the quick and accurate identification of these microorganisms, opening the road for their use in winemaking surveillance and wine quality control.

The present work concerns the development of a new molecular method for the identification of O. oeni and its specific detection in wine, based on the amplification of 16S rRNA gene followed by restriction with the endonuclease FseI. Among wine bacteria, the FseI recognition sequence is only found in the 16S rRNA gene of O. oeni, thus ensuring the specificity of the method. Furthermore, the DNA extraction and purification procedure rely on the use of Whatman FTA cards, a patented technology for handling and processing of nucleic acids with a validated application in a wide range of fields (forensics, biomedicine, pharmaceutics, genomics, and food testing). The proposed application for wine samples is a novelty and an efficient and interesting alternative to current DNA wine DNA extraction methods, since samples can be easily collected at wineries by a non-specialized technician, stored at room temperature and sent in a mail envelope to the analytical laboratory for direct PCR and amplicon restriction analysis.

Section snippets

Microorganisms, growth conditions and wine samples

Fifteen O. oeni strains, isolated from wines of West Ribatejo region of Portugal at the end of MLF, and belonging to the IBET culture collection were used in this study. The identification of these isolates at species level was confirmed by phenotypic and genomic fingerprinting methods. A commercial malolactic starter from Christian Hansen (Viniflora oenos) and the O. oeni type strain (DSMZ20252T) were also included. All strains were cultured in MTJ broth medium (50% MRS broth, Merck, Germany;

Results and discussion

The cleavage of O. oeni 16S rRNA gene by the endonuclease FseI was already known since the construction of O. oeni PSU-1 physical map (Zé-Zé et al., 1998) and further confirmed with the construction of physical maps for eight O. oeni strains selected as representatives of two genomic divergent groups (Zé-Zé et al., 2000, Zé-Zé et al., 2008).

The specificity of FseI restriction for O. oeni 16S rRNA gene was investigated by in silico comparative sequence analysis of 16S rRNA genes from 22 species

Acknowledgements

This work was partially supported by Fundação para a Ciência e Tecnologia and Agro Medida 8.1 Program Project no. 33. The authors acknowledge Estação Vitivinícola Nacional by providing wine samples and data on microbial counts. A.P. Marques also acknowledges the FCT research grant SFRH/BD/14389/2003.

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