Impact of Oenococcus oeni esterase EstA7 on wine aromatic compounds

Impact of Oenococcus oeni esterase EstA7 on wine aromatic compounds. 10. symposium international d'oenologie de Bordeaux (Oeno 2015).

Tyrosine-containing peptides are precursors of tyramine produced by Lactobacillus plantarum strain IR BL0076 isolated from wine

Abstract Background Biogenic amines are molecules with allergenic properties. They are found in fermented products and are synthesized by lactic acid bacteria through the decarboxylation of amino acids present in the food matrix. The concentration of biogenic amines in fermented foodstuffs is influenced by many environmental factors, and in particular, biogenic amine accumulation depends on the quantity of available precursors. Enological practices which lead to an enrichment in nitrogen compounds therefore favor biogenic amine production in wine. Free amino acids are the only known precursors for the synthesis of biogenic amines, and no direct link has previously been demonstrated between …

Polyphenols in kombucha: Metabolomic analysis of biotransformations during fermentation

Kombucha is a non-alcoholic beverage made of sugared tea that is transformed by a symbiotic consortium of yeasts and bacteria. This beverage is increasingly produced at industrial scale, but its quality standards remain to be defined. Metabolomics analysis was carried out using FT-ICR-MS (Fourier Transform-Ion Cyclotron Resonance-Mass Spectrometry) to understand the chemical transformations induced by the production phases and the type of tea on the non-volatile compounds of kombucha.

Microbial Dynamics between Yeasts and Acetic Acid Bacteria in Kombucha: Impacts on the Chemical Composition of the Beverage.

Kombucha is a traditional low-alcoholic beverage made from sugared tea and transformed by a complex microbial consortium including yeasts and acetic acid bacteria (AAB). To study the microbial interactions and their impact on the chemical composition of the beverage, an experimental design with nine couples associating one yeast strain and one AAB strain isolated from original black tea kombucha was set up. Three yeast strains belonging to the genera Brettanomyces, Hanseniaspora, and Saccharomyces and three strains of Acetobacter and Komagataeibacter species were chosen. Monocultures in sugared tea were analyzed to determine their individual microbial behaviors. Then, cultivation of the ori…

Using CRISPR/Cas platform for Genetic Modification of Commercial Saccharomyces cerevisiae strains

International audience; Traditional wine fermentation is a complex microbial process initiated by various yeast species classified as Saccharomyces and non-Saccharomyces species.To better understand the different interactions occurring within wine fermentations and track a specific yeast population, we wish to obtain GFP-tagged yeast cells that stably expres fluorescence signal without compromising the fermentative capability of the strain.To this end, the CRISPR/Cas system was investigated to genetically modify the commercial Saccharomyces Saccharomyces cerevisiae diploid strain Lalvin EC 1118

Management of Listeria monocytogenes on Surfaces via Relative Air Humidity: Key Role of Cell Envelope

International audience; Although relative air humidity (RH) strongly influences microbial survival, its use for fighting surface pathogens in the food industry has been inadequately considered. We asked whether RH control could destroy Listeria monocytogenes EGDe by envelope damage. The impact of dehydration in phosphate-buffered saline (PBS) at 75%, 68%, 43% and 11% RH on the bacterial envelope was investigated using flow cytometry and atomic force microscopy. Changes after rehydration in the protein secondary structure and peptidoglycan were investigated by infrared spectroscopy. Complementary cultivability measurements were performed by running dehydration–rehydration with combinations o…

An improved protocol for electroporation ofOenococcus oeniATCC BAA-1163 using ethanol as immediate membrane fluidizing agent

Aims:  To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA-1163 strain. Methods and Results:  The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5·8 × 103 per μg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 12·5 kV cm−1, under a resistance of 200 Ω and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB). Conclusions:  An effective protocol to transform O. oeni ATCC BAA-1163 strain by electroporation has been obtained by addition of ethanol to the EP…

Molecular Cloning, Heterologous Expression, and Characterization of Ornithine Decarboxylase from Oenococcus oeni

International audience; Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal ac…

Polyphenols in kombucha: impact of infusion time on extraction and investigation of their behavior during "fermentation"

Kombucha is a non-alcoholic beverage made of sugared tea that is transformed by a symbiotic consortium of yeasts and bacteria. Polyphenols are expected to be responsible of several health benefits attributed to kombucha consumption, among other metabolites. Evidence point at an enhancement of tea polyphenol bioactivity during kombucha elaboration. This study investigated the impact of tea infusion time and of kombucha "fermentation", on the tea polyphenols and the color. Most of the present experiments have been carried out during a 3 rd year Bachelor training by Shane Fennell from Carlow Institute of Technology (Ireland).

Directed evolution of oenococcus oeni to improve acid-tolerance reveals fixed beneficial mutations in the citrate locus

Oenococcus oeni : advances in molecular genetics

International audience

Cyclopropane Fatty Acid Synthase from Oenococcus oeni: Expression in Lactococcus lactis subsp. Cremoris and Biochemical Characterization

Bacterial cyclopropane fatty acid synthases (CFA synthases) catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the double bond of a lipid chain, thereby forming a cyclopropane ring. CFAs contribute to resistance to acidity, dryness, and osmotic imbalance in many bacteria. This work describes the first biochemical characterization of a lactic acid bacterium CFA synthase. We have overexpressed Oenococcus oeni CFA synthase in E. coli in order to purify the enzyme. The optimum cyclopropanation activity was obtained at pH 5.6 and 35.8 °C. The high K(m) (AdoMet) value obtained (2.26 mM) demonstrates the low affinity of O. oeni enzyme toward the L. lactis subsp. cremo…

Peptidases specific for proline-containing peptides and their unusual peptide-dependent regulation in Oenococcus oeni

International audience; Growth of the lactic acid bacterium (LAB) Oenococcus oeni, which is involved in malolactic fermentation during the winemaking process, is stimulated by peptides originating from yeast. In this study, we investigated the impact of peptides on O. oeni growth, peptidase activity and the expression of genes encoding the studied peptidases. Low levels of PepN activity and very high levels of PepI activity were observed in O. oeni, whereas levels of PepX activity were intermediate. The level of biosynthesis of these O. oeni peptidases was shown to depend on peptides present in the culture medium. These results were confirmed by transcriptional analyses of putative pep gene…

Use of antisense RNA to modulate HSP gene expression in oenococcus oeni

International audience

Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation

International audience; Cyclopropane fatty acid (CFA) synthesis was investigated in Oenococcus oeni. The data obtained demonstrated that acid-grown cells or cells harvested in the stationary growth phase showed changes in fatty acid composition similar to those of ethanol-grown cells. An increase of the CFA content and a decrease of the oleic acid content were observed. The biosynthesis of CFAs from unsaturated fatty acid phospholipids is catalysed by CFA synthases. Quantitative real-time-PCR experiments were performed on the cfa gene of O. oeni, which encodes a putative CFA synthase. The level of cfa transcripts increased when cells were harvested in stationary phase and when cells were gr…

CtsR is the master regulator of stress response gene expression in Oenococcus oeni.

ABSTRACT Although many stress response genes have been characterized in Oenococcus oeni , little is known about the regulation of stress response in this malolactic bacterium. The expression of eubacterial stress genes is controlled both positively and negatively at the transcriptional level. Overall, negative regulation of heat shock genes appears to be more widespread among gram-positive bacteria. We recently identified an ortholog of the ctsR gene in O. oeni . In Bacillus subtilis , CtsR negatively regulates expression of the clp genes, which belong to the class III family of heat shock genes. The ctsR gene of O. oeni is cotranscribed with the downstream clpC gene. Sequence analysis of t…

Microbiological and technological parameters impacting the chemical composition and sensory quality of kombucha

International audience; Kombucha is a beverage made from sugared tea transformed by yeasts and acetic acid bacteria. Being originally homemade, it has become an industrially produced soft drink whose quality standards are poorly defined and whose production process is still not fully controlled. Based on current knowledge in beverages, links between kombucha's chemical composition and sensorial compounds are drawn. Macromolecules create turbidity, whereas uncharacterized tea pigments derivatives participate in the color. Residual sugars bring sweetness and organic acids produced by acetic acid bacteria form its characteristic sour taste. Acetic acid is also part of its aroma profile, althou…

Improving acid tolerance in Oenococcus oeni by Adaptive Evolution

International audience; Oenococcus oeni is a lactic acid bacterium (LAB) mainly responsible for the malolactic fermentation (MLF) inwine. MLF plays an important role in determining the final quality of wines[1]. Even though this LAB is naturally present in musts, wines and oenological environment, spontaneous MLF are usually unpredictable because of the stressful conditions and especially due to acidity [2]. The consequence of the mismanagement ofthis step might lead to the depreciation of wine quality. To obtain a clone more tolerant to acidity, we undertaken to replicate O. oeni in a temporally varying environment (pH 5.3 to 3.2) to improve acid tolerance. To discriminate stress tolerance…

The Antisense RNA Approach: a New Application for In Vivo Investigation of the Stress Response of Oenococcus oeni, a Wine-Associated Lactic Acid Bacterium

ABSTRACT Oenococcus oeni is a wine-associated lactic acid bacterium mostly responsible for malolactic fermentation in wine. In wine, O. oeni grows in an environment hostile to bacterial growth (low pH, low temperature, and ethanol) that induces stress response mechanisms. To survive, O. oeni is known to set up transitional stress response mechanisms through the synthesis of heat stress proteins (HSPs) encoded by the hsp genes, notably a unique small HSP named Lo18. Despite the availability of the genome sequence, characterization of O. oeni genes is limited, and little is known about the in vivo role of Lo18. Due to the lack of genetic tools for O. oeni , an efficient expression vector in O…

Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris

International audience

Shedding Light on the Formation and Structure of Kombucha Biofilm Using Two-Photon Fluorescence Microscopy

Kombucha pellicles are often used as inoculum to produce this beverage and have become a signature feature. This cellulosic biofilm produced by acetic acid bacteria (AAB) involves yeasts, which are also part of the kombucha consortia. The role of microbial interactions in thede novoformation and structure of kombucha pellicles was investigated during the 3 days following inoculation, using two-photon microscopy coupled with fluorescent staining. Aggregated yeast cells appear to serve as scaffolding to which bacterial cellulose accumulates. This initial foundation leads to a layered structure characterized by a top cellulose-rich layer and a biomass-rich sublayer. This sublayer is expected t…

Adaptive evolution to improve acid tolerance in Oenococcus oeni

International audience; Oenococcus oeni is a lactic acid bacterium (LAB) mainly responsible for the malolactic fermentation (MLF) inwine. MLF plays an important role in determining the final quality of wines [1]. Even though this LAB is naturallypresent in musts, wines and oenological environment, spontaneous MLF are usually unpredictable because of thestressful conditions and especially due to acidity [2]. The consequence of the mismanagement of this step might leadto the depreciation of wine quality. To obtain a clone more tolerant to acidity, we undertook a replication of O. oeni until 450 generations in a temporally varying environment (pH 5.3 to 2.9) to improve acid tolerance. To discr…

Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris

ABSTRACT Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S -adenosyl- l -methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the comple…