ORIGINAL ARTICLE Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods F. Nannelli 1,3 , O. Claisse 1 , E. Gindreau 1,2 , G. de Revel 1 , A. Lonvaud-Funel 1 and P.M. Lucas 1 1 UMR 1219 Œnologie, Universite ´ Victor Segalen Bordeaux2, INRA, ISVV, Talence, France 2 SARCO, Laffort Œnologie, Bordeaux, France 3 ENOPROJECT SRL, Greve in Chianti, Florence, Italy Introduction Histamine, tyramine and putrescine are the most signifi- cant biogenic amines (BA) present in wines especially those where malolactic fermentation (MLF) is occurred (Soufleros et al. 1998). Ingestion of wine or other foods containing these amines is implicated in food poisoning incidents (Silla Santos 1996). BA are produced in wines by diverse lactic acid bacteria (LAB), but only by strains carrying specific metabolic pathways that convert precur- sor amino acids into BA: the histidine decarboxylase (HDC) pathway producing histamine, the tyrosine decar- boxylase (TDC) pathway producing tyramine, and two independent pathways producing putrescine: the orni- thine decarboxylase (ODC) and agmatine deiminase (AgDI) pathways (Arena and Manca de Nadra 2001; Lon- vaud-Funel 2001). For a long time BA formation in wine has been mainly associated with non-oenococcal strains, such as spoilage Pediococcus and Lactobacillus strains, whose presence is related with high pH wine conditions. However, the pres- ence of HDC and ODC pathways has been demonstrated in strains of Oenococcus oeni, the most suitable species to perform MLF (Lonvaud-Funel and Joyeux 1994; Marcobal et al. 2004). Arena and Manca de Nadra (2001) reported that Lactobacillus hilgardii X1B isolated from wine was able to produce putrescine from arginine via two pathways. Arginine can be converted to ornithine via the arginine deiminase pathway and then ornithine is decarb- oxylated by the ODC to form putrescine, or arginine is Keywords biogenic amines, lactic acid bacteria, Oenococcus oeni, quantitative PCR, wine. Correspondence Patrick M. Lucas, UMR1219, Universite ´ Victor Segalen Bordeaux 2, INRA, ISVV, 351, Cours de la Libe ´ ration, 33405, Talence, France. E-mail: patrick.lucas@u-bordeaux2.fr 2008 ⁄ 0669: received 18 April 2008, revised 11 July 2008 and accepted 12 August 2008 doi:10.1111/j.1472-765X.2008.02472.x Abstract Aims: To develop rapid methods allowing enumeration of lactic acid bacteria producing biogenic amines in wines and to analyse wine samples by the methods. Methods and Results: Methods based on quantitative PCR targeting bacterial genes involved in histamine, tyramine and putrescine production were devel- oped and applied to detect and quantify the bacteria producing these biogenic amines in wine. Analysis of 102 samples revealed low populations of the tar- geted bacteria in grape must samples, an increased bacteria biomass in wine samples after alcoholic fermentation, reaching the highest population levels (above 10 6 cells ml )1 ) during spontaneous malolactic fermentation. A mini- mum of 10 3 ml )1 producing cells was required for production of more than 1 mg l )1 of biogenic amines. Accumulation of putrescine in wine was corre- lated with the presence of bacteria carrying an ornithine decarboxylation path- way. Trials of winemaking showed that the use of selected bacteria for inducing malolactic fermentation was efficient to limit the proliferation of undesirable bacteria and the production of biogenic amines. Conclusion: Methods using quantitative PCR are efficient to enumerate bio- genic amines-producing cells in wine. Significance and Impact of the Study: The methods can help to better control and to improve winemaking conditions in order to avoid biogenic amine production. Letters in Applied Microbiology ISSN 0266-8254 594 Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 47 (2008) 594–599 ª 2008 The Authors