Influence of nitrogen status in wine alcoholic fermentation

Nitrogen is an essential nutrient for yeast during alcoholic fermentation. Nitrogen is involved in the biosynthesis of protein, amino acids, nucleotides, and other metabolites, including volatile compounds. However, recent studies have called several mechanisms that regulate its role in biosynthesis into question. An initial focus on S. cerevisiae has highlighted that the concept of "preferred" versus "non-preferred" nitrogen sources is extremely variable and strain-dependent. Then, the direct involvement of amino acids consumed in the formation of proteins and volatile compounds has recently been reevaluated. Indeed, studies have highlighted the key role of lipids in nitrogen regulation in…

Membrane fatty acid composition and fluidity are involved in the resistance to freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175

International audience; Determinations of membrane fatty acid composition and fluidity were used together with acidification activity and viability measurements to characterize the physiological state after freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175 cells harvested in the exponential and stationary growth phases. For both strains, lower membrane fluidity was achieved in cells harvested in the stationary growth phase. This change was linked to a lower unsaturated-to-saturated fatty acid ratio for both strains and a higher cyclic-to-saturated fatty acid ratio for L. buchneri R1102 alone. These membrane properties were linked to survival and to maintenance of aci…

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.

Osmotic stress affects the stability of freeze-dried Lactobacillus buchneri R1102 as a result of intracellular betaine accumulation and membrane characteristics

Aims To help cells to better resist the stressful conditions associated with the freeze-drying process during starter production, we investigated the effect of various osmotic conditions on growth, survival and acidification activity of Lactobacillus buchneri R1102, after freeze-drying and during storage for 3 months at 25°C. Methods and Results High survival rates during freeze-drying, but not during storage, were obtained when 0·1 mol l−1 KCl was added at the beginning of fermentation, without any change in membrane properties and betaine accumulation. This condition made it possible to maintain a high acidification rate throughout the process. In contrast, the addition of 0·6 mol l−1 KCl…

Wine microbiome : A dynamic world of microbial interactions

International audience; Most fermented products are generated by a mixture of microbes. These microbial consortia perform various biological activities responsible for the nutritional, hygienic, and aromatic qualities of the product. Wine is no exception. Substantial yeast and bacterial biodiversity is observed on grapes, and in both must and wine. The diverse microorganisms present interact throughout the winemaking process. The interactions modulate the hygienic and sensorial properties of the wine. Many studies have been conducted to elucidate the nature of these interactions, with the aim of establishing better control of the two fermentations occurring during wine processing. However, …

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…

Unsaturated fatty acids from food and in the growth medium improve growth of Bacillus cereus under cold and anaerobic conditions.

International audience; In a chemically defined medium and in Luria broth, cold strongly reduced maximal population density of Bacillus cereus ATCC 14579 in anaerobiosis and caused formation of filaments. In cooked spinach, maximal population density of B. cereus in anaerobiosis was the same at cold and optimal temperatures, with normal cell divisions. The lipid containing fraction of spinach, but not the hydrophilic fraction, restored growth of B. cereus under cold and anaerobiosis when added to the chemically defined medium. This fraction was rich in unsaturated, low melting point fatty acids. Addition of phosphatidylcholine containing unsaturated, low melting point, fatty acids similarly…

Involvement of osmotic cell shrinkage on the proton extrusion rate in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been subjected to hyperosmotic shocks by using permeating (sorbitol, xylitol, glycerol, NaCl) and nonpermeating (PEG 600) solutes. The proton extrusion rate decreased as the osmotic pressure increased, whichever solute was used. However, the total inhibition of the cellular H+ extrusion depended on the solute used. A total inhibition was observed at about 20 MPa with glycerol, xylitol and sorbitol. With PEG 600, a total inhibition of extracellular acidification was obtained at 8.5 MPa. NaCl, with an extracellular pressure of 37.8 MPa (near saturation), did not completely inhibit the extracellular acidification. These results showed that the total inhibition of p…

Yeast–yeast interactions revealed by aromatic profile analysis of Sauvignon Blanc wine fermented by single or co-culture of non-Saccharomyces and Saccharomyces yeasts

International audience; There has been increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. The main reason is that the multistarter fermentation process is thought to simulate indigenous fermentation, thus increasing wine aroma complexity while avoiding the risks linked to natural fermentation. However, multistarter fermentation is characterised by complex and largely unknown interactions between yeasts. Consequently the resulting wine quality is rather unpredictable. In order to better understand the interactions that take place between non-Saccharomyces and Saccharomyces yeasts during alcoholic fermentation, we analysed the vola…

Regulation of stress response in Oenococcus oeni as a function of environmental changes and growth phase

International audience; Oenococcus oeni is a lactic acid bacterium which is able to grow in wine and perform malolactic fermentation. To survive and grow in such a harsh environment as wine, O. oeni uses several mechanisms of resistance including stress protein synthesis. The molecular characterisation of three stress genes hsp18, clpX, trxA encoding for a small heat shock protein, an ATPase regulation component of ClpP protease and a thioredoxin, respectively, allow us to suggest the existence in O. oeni of multiple regulation mechanisms as is the case in Bacillus subtilis. One common feature of these genes is that they are expressed under the control of housekeeping promoters. The express…

Biovi : a research program for reducing chemical input in vine and wine

Decrease of chemical inputs during vine management and winemaking is of great importance from a political and societal point of view. In our ongoing project we propose alternative tools to chemicals in the vineyard and the cellar. We have compared a conventional vineyard protection strategy to an alternative strategy using copper and biocontrol products (Biocontrol) against downy and powdery mildews. Both strategies were compared regarding sanitary quality, berries and/or must enological parameters, and physical, biochemical and biological characteristics (berry surface observation, proteomic, metabolomic, volatilomic, metagenomic analyses). Musts obtained with both strategies were then use…

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).

Acid sensitivity of neomycin-resistant mutants ofOenococcus oeni: a relationship between reduction of ATPase activity and lack of malolactic activity

Mutants of Oenococcus oeni were isolated as spontaneous neomycin-resistant mutants. Three of these mutants harbored a significantly reduced ATPase activity that represented 50% of that of the wild-type strain. Their growth rates were also impaired at pH 5.3 (46-86% of the wild-type level). However, the profiles of sugar consumption appeared identical to those of the parental strain. At pH 3.2, all the mutant strains failed to grow and a drastic decrease in viability was observed after an acid shock. Surprisingly, all the isolated mutants were devoid of malolactic activity. These results suggest that the ATPase and malolactic activities of O. oeni are linked to each other and play a crucial …

Membrane fluidity of stressed cells of Oenococcus oeni

International audience; The determination of membrane fluidity in whole cells of Oenococcus oeni was achieved by membrane probe 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy measurements. The results demonstrated instantaneous fluidity variations with cells directly stressed during the measure. Heat (42°C) or acid (pH 3.2) shocks decreased the anisotropy values (fluidising effects), whereas an ethanol shock (10% ethanol, v/v) increased the membrane rigidity. The velocities of fluidity variation with non-adapted or adapted cells (incubation in inhibitory growth conditions) were compared. The adaptation of the cells to acid conditions had no effect on the membrane fluidity variation a…

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…

Inoculation of Torulaspora delbrueckii as a bio-protection agent in winemaking

International audience; In oenology, bio-protection consists in adding bacteria, yeasts or a mixture of microorganisms on grape must before fermentation in order to reduce the use of chemical compounds such as sulphites. More particularly, non-Saccharvinyces yeasts are used as a total or partial alternative to sulphites. However, scientific data capable of proving the effectiveness of adding these yeasts on grape must is lacking. This study reports the analysis of antimicrobial and antioxidant effects of one non-Saccharamyces yeast, Torulaspora delbruecicii, inoculated at the beginning of the white winemaldng process in two Burgundian wineries as an alternative to sulphiting. The implantati…

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…

High-throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation

Abstract We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITS-RFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods …

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…

A new approach for selection of Oenococcus oeni strains in order to produce malolactic starters.

The lactic acid bacterium Oenococcus oeni, mainly responsible for malolactic fermentation (MLF), is used in new winery process as starter culture for direct inoculation. The difficulty to master MLF according to the wine led us to search a new approach to select effective O. oeni strains. Biochemical and molecular tests were performed in order to characterize three strains of O. oeni selected for malolactic starter elaboration. Malolactic and ATPase activities that appeared as a great interest in MLF were measured and the expression of a small heat shock protein Lo18 was evaluated by immunoblotting and real-time PCR. These results were correlated with the performances of strains in two red …

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

International audience

Corrigendum to ‘Inoculation of Torulaspora delbrueckii as a bio-protection agent in winemaking’ [Food Research International, Inoculation of Torulaspora delbrueckii as a bio-protection agent in winemaking, Volume: 107 (2018), 451–461]

Absence of malolactic activity is a characteristic of H+-ATPase-deficient mutants of the lactic acid bacterium Oenococcus oeni.

ABSTRACT The lack of malolactic activity in H + -ATPase-deficient mutants of Oenococcus oeni selected previously was analyzed at the molecular level. Western blot experiments revealed a spot at 60 kDa corresponding to the malolactic enzyme only in the parental strain. Moreover, the mleA transcript encoding the malolactic enzyme was not detected by reverse transcription (RT)-PCR analysis of mutants. These results suggest that the malolactic operon was not transcribed in ATPase-deficient mutants. The mleR gene encoding a LysR-type regulatory protein which should be involved in expression of the malolactic genes was described previously for O. oeni . Results obtained in this study show that th…

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…

Effects of oxidoreduction potential combined with acetic acid, NaCl and temperature on the growth, acidification, and membrane properties of Lactobacillus plantarum.

International audience; The effects of oxidoreduction potential (Eh) combined with acetic acid, NaCl and temperature on the growth, acidification, and membrane properties of Lactobacillus plantarum were studied. The culture medium was set at pH 5, and two different Eh values were adjusted using nitrogen (Eh = +350 mV) or hydrogen (Eh = -300 mV) gas. In reducing condition, the growth was slowed and the acidification delayed at 37 degrees C, but not at 10 degrees C. A synergistic inhibitory effect of reducing Eh, acetic acid and NaCl was observed, mainly for delaying the lag phase before acidification. These results may be explained by changes in ATPase activity, membrane fluidity and surface…

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…