Analysis of several methods for the extraction of high quality DNA from acetic acid bacteria in wine and vinegar for characterization by PCR-based methods

https://doi.org/10.1016/j.ijfoodmicro.2008.09.008Get rights and content

Abstract

Acetic acid bacteria (AAB) are fastidious microorganisms with poor recovery in culture. Culture-independent methods are currently under examination. Good DNA extraction is a strict requirement of these methods. We compared five methods for extracting the DNA of AAB directly from wine and vinegar samples. Four matrices (white wine, red wine, superficial vinegar and submerged vinegar) contaminated with two AAB strains belonging to Acetobacter pasteurianus and Gluconacetobacter hansenii were assayed. To improve the yield and quality of the extracted DNA, a sample treatment (washing with polyvinyl pyrrolidone or NaCl) was also tested. DNA quality was measured by amplification of the 16S rRNA gene with conventional PCR. DNA recovery rate was assessed by real-time PCR. DNA amplification was always successful with the Wizard method though DNA recovery was poor. A CTAB-based method and NucleoSpin protocol extracted the highest DNA recoveries from wine and vinegar samples. Both of these methods require treatment to recover suitable DNA for amplification with maximum recovery. Both may therefore be good solutions for DNA extraction in wine and vinegar samples. DNA extraction of Ga hansenii was more effective than that of A. pasteurianus. The fastest and cheapest method we evaluated (the Thermal shock protocol) produced the worst results both for DNA amplification and DNA recovery.

Introduction

Acetic acid bacteria (AAB) are important microorganisms in the food industry because of their ability to oxidize sugars and alcohols. While their activity sometimes causes food spoilage (in wines, beer, grapes and other fruits, etc.), they are also the main actors in the production of vinegar. Correct and fast enumeration and identification protocols are needed to suitably control the process. One of the biggest hurdles when studying AAB is their cultivation, growth and maintenance in pure culture, especially when isolated from high ethanol and/or acetic acid sources such as wine and vinegar (Entani et al., 1985, Sievers et al., 1992, Gullo et al., 2006). These extreme media are responsible for the limited culture recovery of a significant number of microbiota (Sievers et al., 1992, Sokollek et al., 1998, Millet and Lonvaud-Funel, 2000, Trcek, 2005). AAB are considered “fastidious microorganisms”, mainly because of their inability to recover a part of the population that, though viable, in adverse conditions enters into a non-culturable state (McDougald et al., 1998, Fleet, 1999, Millet and Lonvaud-Funel, 2000, Giraffa and Neviani, 2001). With AAB, it has been reported that conventional plate counts are considerably lower than the fluorescence optical counts of microbial cells from industrial acetators (Mesa et al., 2003, Baena-Ruano et al., 2006).

To overcome the disadvantages of culture-dependent methods, therefore, in recent years several molecular techniques based on PCR have been successfully used to detect bacteria without cultivation in foods (reviewed by Fleet, 1999, Giraffa, 2004). However, the validity and robustness of the results obtained from such molecular techniques depend on the efficient recovery of bacterial DNA.

DNA extraction is usually affected by factors such as incomplete cell lysis, DNA sorption to a particular material, coextraction of enzymatic inhibitors and degradation or damage of DNA (Miller et al., 1999). Clearly, the application of a suitable DNA extraction protocol for a specific sample is essential for correct microbial diversity estimation. The DNA extraction method must be simple, quick and efficient. Safety, cost and DNA quality must also be considered. DNA quality is critical because the efficiency of PCR amplification can be reduced by inhibitors from the matrix. DNA extraction has therefore been highlighted as a limitation of culture-independent methods (Abriouel et al., 2006, Cankar et al., 2006).

With wine and vinegar, which are complex matrices, the presence of polysaccharides and a range of polyphenolics (including tannins) can interfere with enzymatic reactions and even degrade the DNA during direct DNA extraction (Do and Adams, 1991, Demeke and Adams, 1992, Wilson, 1997). No standardized DNA extraction method is yet available for wine and vinegar and all described methods involve chemical and enzymatic lysis. These methods often combine detergents, physical disruption, solvent extraction and enzymatic lysis to obtain crude extracts of nucleic acid (Sokollek et al., 1998, Nanda et al., 2001, González et al., 2004, Gullo et al., 2006, Haruta et al., 2006, Ndoye et al., 2006).

To optimize the detection of AAB from wine and vinegar samples and minimize these limitations, we evaluated five nucleic acid extraction methods on the basis of DNA PCR amplification quality and recovery. Amplification of 16S rRNA gene was assessed by conventional PCR and taken as an indicator of good DNA extraction. Real-time quantitative PCR was used to evaluate the reaction efficiency by quantifying DNA recovery.

Section snippets

Bacterial strain and media

The reference strains used in this study were Acetobacter pasteurianus (LMG 1262T) and Gluconacetobacter hansenii (DSMZ 5602T). These strains were grown in 30 ml of Glucose medium (GY: 1% yeast extract; 1% glucose, w/v) (Cultimed, Barcelona, Spain) for 3–5 days at 30 °C. Each day the number of cells per ml was determined by microscopic counting until the population was 108 cell ml 1, which was the cell size used for DNA extraction. 1 ml volume of the growth medium was centrifuged and the pellet

PCR inhibition assay

To estimate the quality of the DNA obtained from both species before and after treatments with the extraction protocols, we used a PCR system based on amplification of 16S rRNA gene and visualization onto agarose gel (Table 2). To validate the methods, we tested four matrices (white and red wines, commercial and traditional vinegars) using GY medium as control. Our results were similar for both species. Amplification was generally good when DNA extracted from cells grown in GY medium was used.

Discussion

Ideally, DNA extraction methods should be simple, quick and efficient. Choosing an extraction method often involves a trade-off between cost (materials and labor), the optimal yield of DNA and the removal of substances that could influence the PCR reaction (Cankar et al., 2006). It is necessary, therefore, to carefully validate the suitability of a method for specific samples. The main criteria for this validation are based on quantitative and qualitative analysis.

Today the direct lysis method

Acknowledgments

We are grateful for financial assistance from the Spanish Government (Project AGL 2004-07494-C02-02/ALI) and from the European Commission (Project WINEGAR, Cooperative Research under the Sixth Framework Programme of the European Community, 2005–2007). We also thank the Language Service of the Rovira i Virgili University for revising the manuscript.

References (38)

  • McDougaldD. et al.

    Nonculturability: adaptation or debilitation

    FEMS Microbiology Ecology

    (1998)
  • NdoyeB. et al.

    Thermoresistant properties of acetic acid bacteria isolated from tropical products of Sub-Saharan Africa and destined to industrial vinegar

    Enzyme and Microbial Technology

    (2006)
  • PrietoC. et al.

    Application of molecular methods for analysing the distribution and diversity of acetic acid bacteria in Chilean vineyards

    International Journal of Food Microbiology

    (2007)
  • SibataniA.

    Precipitation and counting of minute quantities of labelled nucleic acids as cetytrimethylammonium salt

    Analytical Biochemistry

    (1970)
  • SieversM. et al.

    Acetobacter europaeus sp. nov., a main component of industrial vinegar fermenters in Central Europe

    Systematic and Applied Microbiology

    (1992)
  • TrcekJ.

    Quick identification of acetic acid bacteria based on nucleotide sequences of the 16S–23S rDNA internal transcribed spacer region and of the PQQ-dependent alcohol dehydrogenase gene

    Systematic and Applied Microbiology

    (2005)
  • CankarK. et al.

    Critical points of DNA quantification by real-time PCR-effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    BMC Biotechnology

    (2006)
  • DemekeT. et al.

    The effects of plant polysaccharides and buffer additives on PCR

    BioTechniques

    (1992)
  • Cited by (44)

    View all citing articles on Scopus
    1

    (Guillamón) Current address: Departamento de Biotecnología de los alimentos, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), P.O. Box 73, E-46100 Burjassot, Valencia, Spain.

    View full text