Identification of Key Parameters Inducing Microbial Modulation during Backslopped Kombucha Fermentation.

The aim of this study was to assess the impact of production parameters on the reproducibility of kombucha fermentation over several production cycles based on backslopping. Six conditions with varying oxygen accessibility (specific interface surface) and initial acidity (through the inoculation rate) of the cultures were carried out and compared to an original kombucha consortium and a synthetic consortium assembled from yeasts and bacteria isolated from the original culture. Output parameters monitored were microbial populations, biofilm weight, key physico-chemical parameters and metabolites. Results highlighted the existence of phases in microbial dynamics as backslopping cycles progressed. The transitions between phases occurred faster for the synthetic consortium compared to the original kombucha. This led to microbial dynamics and fermentative kinetics that were reproducible over several cycles but that could also deviate and shift abruptly to different behaviors. These changes were mainly induced by an increase in the Saccharomyces cerevisiae population, associated with an intensification of sucrose hydrolysis, sugar consumption and an increase in ethanol content, without any significant acceleration in the rate of acidification. The study suggests that the reproducibility of kombucha fermentations relies on high biodiversity to slow down the modulations of microbial dynamics induced by the sustained rhythm of backslopping cycles.

Influence of pH on Oenococcus oeni metabolism: Can the slowdown of citrate consumption improve its acid tolerance?

Oenococcus oeni is the lactic acid bacteria most suited to carry out malolactic fermentation in wine, converting L-malic acid into L-lactic acid and carbon dioxide, thereby deacidifying wines. Indeed, wine is a harsh environment for microbial growth, partly because of its low pH. By metabolizing citrate, O. oeni maintains its homeostasis under acid conditions. Indeed, citrate consumption activates the proton motive force, helps to maintain intracellular pH, and enhances bacterial growth when it is co-metabolized with sugars. In addition, citrate metabolism is responsible for diacetyl production, an aromatic compound which bestows a buttery character to wine. However, an inhibitory effect of citrate on O. oeni growth at low pH has been highlighted in recent years. In order to understand how citrate metabolism can be linked to the acid tolerance of this bacterium, consumption of citrate was investigated in eleven O. oeni strains. In addition, malate and sugar consumptions were also monitored, as they can be impacted by citrate metabolism. This experiment highlighted the huge diversity of metabolisms between strains depending on their origin. It also showed the capacity of O. oeni to de novo metabolize certain end-products such as L-lactate and mannitol, a phenomenon never before demonstrated. It also enabled drawing hypotheses concerning the two positive effects that the slowing down of citrate metabolism could have on biomass production and malolactic fermentation occurring under low pH conditions.

Experimental evolution forcing Oenococcus oeni acid tolerance highlights critical role of the citrate locus.

Oenococcus oeni is the main lactic acid bacterium associated with malolactic fermentation (MLF) of wines. MLF plays an important role in determining the final quality of wines. Nevertheless, due to the stressful conditions inherent to wine and especially acidity, MLF may be delayed. This study aimed to explore by adaptive evolution improvements in the acid tolerance of starters but also to gain a better understanding of the mechanisms involved in adaptation toward acidity. Four independent populations of the O. oeni ATCC BAA-1163 strain were propagated (approximately 560 generations) in a temporally varying environment, consisting in a gradual pH decrease from pH 5.3 to pH 2.9. Whole genome sequence comparison of these populations revealed that more than 45% of the substituted mutations occurred in only five loci for the evolved populations. One of these five fixed mutations affects mae, the first gene of the citrate operon. When grown in an acidic medium supplemented with citrate, a significantly higher bacterial biomass was produced with the evolved populations compared to the parental strain. Furthermore, the evolved populations slowed down their citrate consumption at low pH without impacting malolactic performance.

Citrate metabolism in lactic acid bacteria: is there a beneficial effect for Oenococcus oeni in wine?

Lactic acid bacteria (LAB) are Gram positive bacteria frequently used in the food industry for fermentation, mainly transformation of carbohydrates into lactic acid. In addition, these bacteria also have the capacity to metabolize citrate, an organic acid commonly found in food products. Its fermentation leads to the production of 4-carbon compounds such as diacetyl, resulting in a buttery flavor desired in dairy products. Citrate metabolism is known to have several beneficial effects on LAB physiology. Nevertheless, a controversial effect of citrate has been described on the acid tolerance of the wine bacterium Oenococcus oeni. This observation raises questions about the effect of citrate on the capacity of O. oeni to conduct malolactic fermentation in highly acidic wines. This review aims to summarize the current understanding of citrate metabolism in LAB, with a focus on the wine bacterium O. oeni. Metabolism with the related enzymes is detailed, as are the involved genes organized in cit loci. The known systems of cit locus expression regulation are also described. Finally, the beneficial effects of citrate catabolism on LAB physiology are reported and the negative impact observed in O. oeni is discussed.

Oxygen management during kombucha production: Roles of the matrix, microbial activity, and process parameters.

Oxygen plays a key role in kombucha production, since the production of main organic acids, acetic and gluconic acids, is performed through acetic acid bacteria's oxidative metabolism. Oxygen consumption during traditional kombucha production was investigated by comparing kombucha to mono and cocultures in sugared tea of microorganisms isolated from kombucha. Two yeasts, Brettanomyces bruxellensis and Hanseniaspora valbyensis and one acetic acid bacterium Acetobacter indonesiensis were used. Results showed that tea compounds alone were mainly responsible for oxygen depletion during the first 24 h following inoculation. During the first 7 days phase of production in open vessel, the liquid surface was therefore the only access to oxygen for microorganisms, as anaerobic conditions were sustained below this area. During the 5 days second phase of production after bottling, comparison of cultures with different microbial compositions showed that oxygen was efficiently depleted in the head space of the bottles in 3-6 h if the acetic acid bacterium was present. Lower access to oxygen after bottling stimulated ethanol production in B. bruxellensis and H. valbyensis cocultures with or without A. indonesiensis. This study provides insights into the management of oxygen and the roles of the tea and the biofilm during kombucha production.

eGFP Gene Integration in HO: A Metabolomic Impact?

Integrating fluorescent genes including eGFP in the yeast genome is common practice for various applications, including cell visualization and population monitoring. The transformation of a commercial S. cerevisiae strain by integrating a cassette including a gene encoding an EGFP protein in the HO gene was carried out using CRISPR-Cas9 technology. Although this type of integration is often used and described as neutral at the phenotypic level of the cell, we have highlighted that under alcoholic fermentation (in a Chardonnay must), it has an impact on the exometabolome. We observed 41 and 82 unique biomarkers for the S3 and S3GFP strains, respectively, as well as 28 biomarkers whose concentrations varied significantly between the wild-type and the modified strains. These biomarkers were mainly found to correspond to peptides. Despite similar phenotypic growth and fermentation parameters, high-resolution mass spectrometry allowed us to demonstrate, for the first time, that the peptidome is modified when integrating this cassette in the HO gene.

Use of a Minimal Microbial Consortium to Determine the Origin of Kombucha Flavor.

Microbiological, chemical, and sensory analyses were coupled to understand the origins of kombucha organoleptic compounds and their implication in the flavor of the kombucha beverage. By isolating microorganisms from an original kombucha and comparing it to monocultures and cocultures of two yeasts (Brettanomyces bruxellensis and Hanseniaspora valbyensis) and an acetic acid bacterium (Acetobacter indonesiensis), interaction effects were investigated during the two phases of production. 32 volatile compounds identified and quantified by Headspace-Solid Phase-MicroExtraction-Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS) were classified according to their origin from tea or microorganisms. Many esters were associated to H. valbyensis, while alcohols were associated to both yeasts, acetic acid to A. indonesiensis, and saturated fatty acids to all microorganisms. Concentration of metabolites were dependent on microbial activity, yeast composition, and phase of production. Sensory analysis showed that tea type influenced the olfactive perception, although microbial composition remained the strongest factor. Association of B. bruxellensis and A. indonesiensis induced characteristic apple juice aroma.

Microbial Interactions in Kombucha through the Lens of Metabolomics.

Kombucha is a fermented beverage obtained through the activity of a complex microbial community of yeasts and bacteria. Exo-metabolomes of kombucha microorganisms were analyzed using FT-ICR-MS to investigate their interactions. A simplified set of microorganisms including two yeasts (Brettanomyces bruxellensis and Hanseniaspora valbyensis) and one acetic acid bacterium (Acetobacter indonesiensis) was used to investigate yeast-yeast and yeast-acetic acid bacterium interactions. A yeast-yeast interaction was characterized by the release and consumption of fatty acids and peptides, possibly in relationship to commensalism. A yeast-acetic acid bacterium interaction was different depending on yeast species. With B. bruxellensis, fatty acids and peptides were mainly produced along with consumption of sucrose, fatty acids and polysaccharides. In opposition, the presence of H. valbyensis induced mainly the decrease of polyphenols, peptides, fatty acids, phenolic acids and putative isopropyl malate and phenylpyruvate and few formulae have been produced. With all three microorganisms, the formulae involved with the yeast-yeast interactions were consumed or not produced in the presence of A. indonesiensis. The impact of the yeasts' presence on A. indonesiensis was consistent regardless of the yeast species with a commensal consumption of compounds associated to the acetic acid bacterium by yeasts. In detail, hydroxystearate from yeasts and dehydroquinate from A. indonesiensis were potentially consumed in all cases of yeast(s)-acetic acid bacterium pairing, highlighting mutualistic behavior.

Evolution in Composition of Kombucha Consortia over Three Consecutive Years in Production Context.

Kombucha is a traditional drink obtained from sugared tea that is transformed by a community of yeasts and bacteria. Its production has become industrialized, and the study of the microbial community's evolution is needed to improve control over the process. This study followed the microbial composition of black and green kombucha tea over three consecutive years in a production facility using a culture-dependent method. Microorganisms were isolated and cultivated using selective agar media. The DNA of isolates was extracted, amplified using 26S and 16S PCR, and sequenced. Identities were obtained after a comparison to the NCBI database. Dekkera/Brettanomyces bruxellensis, Hanseniaspora valbyensis and Saccharomyces cerevisiae were the major yeast species, and the major bacterial genera were Acetobacter and Liquorilactobacillus. Results highlight the persistence of yeast species such as B. bruxellensis detected in 2019. Some yeasts species appeared to be sensitive towards stressful events, such as a hot period in 2019. However, they were resilient and isolated again in 2021, as was the case for H. valbyensis. Dominance of B. bruxellensis was clear in green and black tea kombucha, but proportions in yeasts varied depending on tea type and phase (liquid or biofilm). Composition in acetic acid and lactic acid bacteria showed a higher variability than yeasts with many changes in species over time.

Non-Targeted Metabolomic Analysis of the Kombucha Production Process.

Kombucha is a traditional fermented beverage obtained from the transformation of sugared black tea by a community of yeasts and bacteria. Kombucha production recently became industrialized, but its quality standards remain poorly defined. Metabolomic analyses were applied using FT-ICR-MS to characterize the impacts of production phases and the type of tea on the non-volatile chemical composition of kombucha. Independently from tea type, the first phase of acidification in open vessel was characterized by the release of gluconate and gallate from acetic acid bacteria metabolism and probably from polymeric polyphenols, respectively. The second phase of carbonation in closed vessel induced a consumption or transformation of oleic acid that could be consecutive of oxygen limitation. The first phase had the most impact on molecular diversity, but tea type mainly influenced the global composition in polyphenol profile. Black tea polyphenols were more impacted by microbial activity compared to green tea polyphenols.

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

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 of NaCl (3-0.01%), distilled water, city water and PBS. The main results show that cell membrane permeability and cell envelope were greatly altered during dehydration in PBS at 68% RH followed by rapid rehydration. This damage led cells to recover only 67% of their initial volume after rehydration. Moreover, the most efficient way to destroy cells was dehydration and rehydration in city water. Our study indicates that rehydration of dried, sullied foods on surfaces may improve current cleaning procedures in the food industry.

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 the de novo formation 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 to be the microbiologically active site for cellulose production and spatial optimization of yeast-AAB metabolic interactions. The pellicles then grow in thickness while expanding their layered organization. A comparison with pellicles grown from pure AAB cultures shows differences in consistency and structure that highlight the impact of yeasts on the structure and properties of kombucha pellicles.

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

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, although little data are available on the smell of kombucha. Carbon dioxide, potentially polyphenols, and residual ethanol are involved in the mouthfeel. In this review, after defining the key compounds that shape the characteristic sensory properties of kombucha, the impact of different production parameters is discussed. Water composition is determinant in the extraction of tea compounds along with the tea type and infusion duration and temperature. The type and amount of sweeteners play a role in the sweetness and influences the production kinetics. Similarly, the amount of inoculum and its microbial composition have an effect on the production, but the role of the vessels' geometry and temperature are also essential parameters that can be used to adjust the acidification phase's duration. Despite the amount of research carried out, further investigations of kombucha's sensory characteristics are needed. Such research could lead to a better definition of kombucha's quality and to an improved control over its production process.

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 original kombucha consortium and cocultures in sugared tea were compared to determine the interactive microbial effects during successive phases in open and closed incubation conditions. The results highlight the main impact of yeast metabolism on the product's chemical composition and the secondary impact of bacterial species on the composition in organic acids. The uncovered microbial interactions can be explained by different strategies for the utilization of sucrose. Yeasts and AAB unable to perform efficient sucrose hydrolysis rely on yeasts with high invertase activity to access released monosaccharides. Moreover, the presence of AAB rerouted the metabolism of Saccharomyces cerevisiae towards higher invertase and fermentative activities.

CtsR, the Master Regulator of Stress-Response in Oenococcus oeni, Is a Heat Sensor Interacting With ClpL1.

Oenococcus oeni is a lactic acid bacterium responsible for malolactic fermentation of wine. While many stress response mechanisms implemented by O. oeni during wine adaptation have been described, little is known about their regulation. CtsR is the only regulator of stress response genes identified to date in O. oeni. Extensively characterized in Bacillus subtilis, the CtsR repressor is active as a dimer at 37°C and degraded at higher temperatures by a proteolytic mechanism involving two adapter proteins, McsA and McsB, together with the ClpCP complex. The O. oeni genome does not encode orthologs of these adapter proteins and the regulation of CtsR activity remains unknown. In this study, we investigate CtsR function in O. oeni by using antisense RNA silencing in vivo to modulate ctsR gene expression. Inhibition of ctsR gene expression by asRNA leads to a significant loss in cultivability after heat shock (58%) and acid shock (59%) highlighting the key role of CtsR in the O. oeni stress response. Regulation of CtsR activity was studied using a heterologous expression system to demonstrate that O. oeni CtsR controls expression and stress induction of the O. oeni hsp18 gene when produced in a ctsR-deficient B. subtilis strain. Under heat stress conditions, O. oeni CtsR acts as a temperature sensor and is inactivated at growth temperatures above 33°C. Finally, using an E. coli bacterial two-hybrid system, we showed that CtsR and ClpL1 interact, suggesting a key role for ClpL1 in controlling CtsR activity in O. oeni.

Oenococcus oeni: Queen of the cellar, nightmare of geneticists.

Oenococcus oeni is a wine-associated lactic acid bacterium (LAB) responsible mostly for wine malolactic fermentation (MLF). This fastidious bacterium (auxotrophic for many amino acids and slow growing) possesses remarkable adaptability to harsh physicochemical conditions and can reprogramme its metabolic pathways to enhance its survival in wine. Thus, O. oeni is an instructive bacterial model for investigating stress response mechanisms in LAB. However, the lack of appropriate techniques to modify the O. oeni genome has hampered molecular studies of this species. The application of recent advances in molecular genetics promises to provide a better understanding of the regulation of stress responses in this species in the future.

Listeria monocytogenes ability to survive desiccation: Influence of serotype, origin, virulence, and genotype.

Listeria monocytogenes, a bacterium that is responsible for listeriosis, is a very diverse species. Desiccation resistance has been rarely studied in L. monocytogenes, although it is a stress that is largely encountered by this microorganism in food-processing environments and that could be managed to prevent its presence. The objective of this study was to evaluate the resistance of 30 L. monocytogenes strains to moderate desiccation (75% relative humidity) and evaluate the correlation of such resistance with the strains' virulence, serotype and genotype. The results showed a great heterogeneity of strains regarding their ability to survive (loss of cultivability between 0.4 and 2.0 log). Strains were classified into three groups according to desiccation resistance (sensitive, intermediate, or resistant), and the strain repartition was analyzed relative to serotype, virulence level and environmental origin of the strains. No correlation was found between isolate origin and desiccation resistance. All serotype 1/2b strains were classified into the group of resistant strains. Virulent and hypovirulent strains were distributed among the three groups of desiccation resistance. Finally, a genomic comparison was performed based on 31 genes that were previously identified as being involved in desiccation resistance. The presence of those genes was localized among the genomes of some strains and compared regarding strain-resistance levels. High nucleotide conservation was identified between resistant and desiccation-sensitive strains. In conclusion, the findings regarding the strains of serotype 1/2b indicate potential serotype-specific resistance to desiccation, and thus, to relative humidity fluctuations potentially encountered in food-related environments. The genomic comparison of 31 genes associated to desiccation tolerance did not reveal differences among four strains which have different level of resistance to desiccation.

Control of Relative Air Humidity as a Potential Means to Improve Hygiene on Surfaces: A Preliminary Approach with Listeria monocytogenes.

Relative air humidity fluctuations could potentially affect the development and persistence of pathogenic microorganisms in their environments. This study aimed to characterize the impact of relative air humidity (RH) variations on the survival of Listeria monocytogenes, a bacterium persisting on food processing plant surfaces. To assess conditions leading to the lowest survival rate, four strains of L. monocytogenes (EGDe, CCL500, CCL128, and LO28) were exposed to different RH conditions (75%, 68%, 43% and 11%) with different drying kinetics and then rehydrated either progressively or instantaneously. The main factors that affected the survival of L. monocytogenes were RH level and rehydration kinetics. Lowest survival rates between 1% and 0.001% were obtained after 3 hours of treatment under optimal conditions (68% RH and instantaneous rehydration). The survival rate was decreased under 0.001% after prolonged exposure (16h) of cells under optimal conditions. Application of two successive dehydration and rehydration cycles led to an additional decrease in survival rate. This preliminary study, performed in model conditions with L. monocytogenes, showed that controlled ambient RH fluctuations could offer new possibilities to control foodborne pathogens in food processing environments and improve food safety.

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

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. oeni is still lacking, and deletion or inactivation of the hsp18 gene is not presently practicable. As an alternative approach, with the goal of understanding the biological function of the O. oeni hsp18 gene in vivo, we have developed an expression vector to produce antisense RNA targeting of hsp18 mRNA. Recombinant strains were exposed to multiple stresses inducing hsp18 gene expression: heat shock and acid shock. We showed that antisense attenuation of hsp18 affects O. oeni survival under stress conditions. These results confirm the involvement of Lo18 in heat and acid tolerance of O. oeni. Results of anisotropy experiments also confirm a membrane-protective role for Lo18, as previous observations had already suggested. This study describes a new, efficient tool to demonstrate the use of antisense technology for modulating gene expression in O. oeni.

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. cremoris unsaturated phospholipids. These results explain the partial complementation of the L. lactis subsp. cremoris cfa mutant by the O. oeni cfa gene and suggest a probable substrate specificity of the O. oeni enzyme. The current study reveals an essential hypothesis about the specificity of O. oeni CFA synthase which could play a key function in the acid tolerance mechanisms of this enological bacterium.