Aims: To quantify the ability of 136 lactic acid bacteria (LAB), isolated from wine, to produce histamine and to identify the bacteria responsible for histamine production in wine. Methods and Results: A qualitative method based on pH changes in a plate assay was used to detect wine strains capable of producing high levels of histamine. Two quantitative, highly sensitive methods were used, an enzymatic method and HPLC, to quantify the histamine produced by LAB. Finally, an improved PCR test was carried out to detect the presence of histidine decarboxylase gene in these bacteria. The species exhibiting the highest frequency of histamine production is Oenococcus oeni. However, the concentration of histamine produced by this species is lower than that produced by strains belonging to species of Lactobacillus and Pediococcus. A correlation of 100% between presence of histidine decarboxylase gene and histamine production was observed. Wines containing histamine were analysed to isolate and characterize the LAB responsible for spoilage. Conclusions: Oenococcus was able to synthesize low concentrations of histamine in wines, while Pediococcus parvulus and Lactobacillus hilgardii have been detected as spoilage, high histamine-producing bacteria in wines. Significance and Impact of the Study: Information regarding histamine-producing LAB isolated from wines can contribute to prevent histamine formation during winemaking and storage.

One hundred and seventy-eight lactobacilli isolated from wine were characterized by a polyphasic approach. Strains were phenotypically identified at genus and species level by classical tests including the analysis of cell morphology, homo/heterofermentative character, sugar fermentation patterns, growth at different temperatures and the optical nature of the isomer of lactic acid produced from glucose. Molecular techniques such as random amplification of polymorphic DNA (RAPD), amplified 16S rDNA restriction analysis (16S-ARDRA), PFGE-RFLP and ribotyping were used to characterize strains, and their potential for identification and/or typing was evaluated. The information obtained with these techniques was processed with the BioNumerics software in order to analyse relationships existing between isolated strains and various reference species of the genus. Then, taxonomic dendrograms were obtained, and this information allowed the proposal of molecular procedures suitable for the identification and typing of these wine micro-organisms. The techniques useful for both identification and typing were RAPD and ribotyping, while 16S-ARDRA was only useful for identification and PFGE-RFLP only for typing purposes. The wine strains were identified asLactobacillus brevis(19 strains),Lactobacillus collinoides(2 strains),Lactobacillus hilgardii(71 strains),Lactobacillus paracasei(13 strains),Lactobacillus pentosus(2 strains),Lactobacillus plantarum(34 strains) andLactobacillus mali(10 strains).

A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising technique for the rapid identification of LAB, allowing positive identification in a few hours (4-16 h).

To determine the effect of three different freezing temperatures on post-freeze-drying survival rates of wine yeasts and lactic acid bacteria (LAB). To know if a similar freeze-drying protocol can be used for both micro-organisms. Cells from liquid culture media were recovered and concentrated in appropriate lyoprotectants. Aliquots of each strain were frozen at −20, −80 and −196°C before vacuum drying. Viable cell counts were done before freezing and after freeze-drying. Survival rates were calculated. Freezing temperatures differently affected yeast and bacteria survival. The highest survival rates were obtained at −20 and −80°C for yeasts, but at −196°C for LAB. Major differences in survival rates were recorded among freeze-dried yeasts, but were less drastic for LAB. Yeasts Pichia membranifaciens, Starmerella bacillaris and Metschnikowia pulcherrima, and LAB Lactobacillus paracasei, Pediococcus parvulus and Lactobacillus mali, were the most tolerant species to freeze-drying, regardless of freezing temperature. Yeast and LAB survival rates differed for each tested freezing temperature. For yeasts, −20°C ensured the highest post-freeze-drying viability and −196°C for LAB. Freezing temperature to freeze-dry cells is a crucial factor for ensuring good wine yeast and LAB survival. These results are important for appropriately preserving micro-organisms and for improving starter production processes.