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Mycotoxins are toxic metabolites produced by various genera of fungi. Fungi belonging to species of the genera Aspergillus and Penicillium are the main source of ochratoxin A (OTA) in wine. The reduction of OTA in must and wine has been studied using physical, chemical, and biological technologies. In this paper, we report on the capacity of different cultures isolated from Tempranillo winegrapes contaminated with fungus to degrade OTA in tryptic soy broth (TSB) medium at pH 3.5 and 6.5. We studied the interaction between OTA and four isolates during alcoholic fermentation of red wine. This study showed that microorganism isolates reduced the content of OTA in TSB medium and in the wine experiment system. The bacterium Lactobacillus rhamnosus exhibited the greatest mean reduction of OTA in TSB medium at both pH values (55%). During the wine fermentation, the yeast Metschnikowia pulcherrima showed the most reduction of OTA (34%). Liquid chromatography with tandem mass spectrometry was used to identify OTA degradation products, particularly α-OTA (m/z = 257) and the sodium adduct of α-OTA with the loss of the coumarin group (m/z = 170). The presence of OTA in must and wines is a problem for food safety. The use of lactic acid bacteria and yeasts during the winemaking process presents an interesting practical solution.

Cork taint is a musty or moldy off-odor in wine mainly caused by 2,4,6-trichloroanisole (2,4,6-TCA). We examined the role of 14 fungal strains isolated from cork samples in the production of 2,4,6-TCA by O methylation of 2,4,6-trichlorophenol (2,4,6-TCP). The fungal strains isolated belong to the genera Penicillium (four isolates); Trichoderma (two isolates); and Acremonium , Chrysonilia , Cladosporium , Fusarium , Mortierella , Mucor , Paecilomyces , and Verticillium (one isolate each). Eleven of these strains could produce 2,4,6-TCA when they were grown directly on cork in the presence of 2,4,6-TCP. The highest levels of bioconversion were carried out by the Trichoderma and Fusarium strains. One strain of Trichoderma longibrachiatum could also efficiently produce 2,4,6-TCA in liquid medium. However, no detectable levels of 2,4,6-TCA production by this strain could be detected on cork when putative precursors other than 2,4,6-TCP, including several anisoles, dichlorophenols, trichlorophenols, or other highly chlorinated compounds, were tested. Time course expression studies with liquid cultures showed that the formation of 2,4,6-TCA was not affected by a high concentration of glucose (2% or 111 mM) or by ammonium salts at concentrations up to 60 mM. In T. longibrachiatum the O methylation of 2,4,6-TCP was catalyzed by a mycelium-associated S -adenosyl- l -methionine (SAM)-dependent methyltransferase that was strongly induced by 2,4,6-TCP. The reaction was inhibited by S -adenosyl- l -homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings increase our understanding of the mechanism underlying the origin of 2,4,6-TCA on cork, which is poorly understood despite its great economic importance for the wine industry, and they could also help us improve our knowledge about the biodegradation and detoxification processes associated with chlorinated phenols.

Publisher Summary This chapter provides a general description of the most commonly used methods for the preservation of microbial strains. Long-term preservation methods are considered the most appropriate option wherever possible, since they involve stopping the growth of the microbial cells and keeping them in a viable state. This guarantees maximum genetic stability by preventing the appearance of successive generations. Nevertheless, the possibility that the preparation method itself leads to changes cannot be ruled out. There are two preservation methods belonging to this group: freezing and lyophilization. In the first long-term preservation method, the cells are frozen suspended in a liquid medium containing a cryoprotective agent and stored at temperatures below 0oC. As a result, intracellular and extracellular water is maintained in a solid state. The reduced cell metabolism caused by the low temperature and the absence of liquid water prevents growth. Lyophilization is a gentle process in which water is removed from the cells to stop their growth. The genetic stability obtained with this method is high, although not always as high as that seen with freezing, since lyophilization is achieved by sublimation of the ice in the cells. First, the free water in the cells must be frozen and then eliminated by vacuum without increasing the temperature, as this would affect the viability of the microorganism.

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria , Cladosporium , and Aspergillus . The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.