New biotic supports for biofilms

Abstract

The present invention pertains to the provision of novel supports for the development of biofilms. The present inventors have indeed shown that it is possible to successfully grow microorganisms in the form of a biofilm by using yeast derivatives as a biotic support. The present invention therefore relates to the use of yeast derivatives as a biofilm support, to methods for preparing a biofilm having yeast derivatives as a support, to biofilms obtained accordingly and to uses thereof.

Description

DescriptionTitle: NEW BIOTIC SUPPORTS FOR BIOFILMSTechnical Field

[0001] The present invention pertains to methods for obtaining biofilms and to biofilms obtained accordingly. The present invention particularly relates to the provision of novel biotic supports for the development of biofilms.Background Art

[0002] Biofilms were the first organized forms of life on Earth around 3.5 billion years ago. They are found in all environments, even the most extreme (pores in glaciers, black smokers in the abyss under high pressure or high salt concentrations, irradiated environments, etc.). These living formations constitute processes of colonization of various environments associated with survival and adaptation processes.

[0003] Biofilms are three-dimensional structures embedded in a matrix of self-produced polymeric substances. Under this lifestyle, bacteria are more resistant to chemical stress (acids, alcohol, detergents, etc.) or physical stress (temperature, pH, flow, etc.). The life cycle of a biofilm is schematically divided into 4 major stages 1 / adhesion to the surface, 2 / growth, 3 / maturation of the biofilm, and 4 / detachment (Douarche, Carine, et al. Reflets de la physique 56 (2018): 20-24).

[0004] A biofilm is therefore a microbial community adhering to a surface and often enclosed in a matrix of exocellular polymers. Certain microbial community organized as biofilms can be pathogenic. Great efforts have therefore been made to develop methods for removing and / or preventing their development in clinical and food manufacturing processes. On the other hand, recent research suggested that biofilms can also be of great interest in several industrial applications.

[0005] One example is malolactic fermentation (MLF) in the oenology field. Mastering the various stages of wine production requires the use of yeast (for alcoholic fermentation) and bacteria (for malolactic fermentation). Oenococcus oeni is the bacterial species most frequently used and best suited to carrying out this second fermentation. Nevertheless, because of the health status of the harvest, the level of acidity, the ethanol content and the nutritional quality, malolactic fermentation is still poorly controlled and is becoming a major concern for winemakers in many parts of the world (France, USA, South America, New Zealand, Australia, etc.). Fermentation is made difficult, particularly due to the high mortality rate of the bacteria, which prevents them from carrying out their metabolism. Current solutions involve a long and costly industrial production and acclimatization process, these pre-acclimatized bacteria are not totally effective, particularly under certain 'difficult' winemaking conditions.

[0006] Strategies aiming at improving the bacteria's resistance to environmental stresses to improve their survival and enable MLF to take place are therefore desirable.

[0007] The use of biofilm cultures in this context has been proposed (see e.g. international publication published under reference WO2016 / 055603) and is an efficient solution to theseproblems. For example, recent studies have shown the existence of O.oeni biofilms on the walls of barrels, which can spontaneously seed wines, and the use of biofilms developed on abiotic supports such as wood or steel to trigger MLF (Bastard, Alexandre, et al. Frontiers in microbiology 7 (2016): 613.; Coelho, Christian, et al. Frontiers in Nutrition 6 (2019): 95.).

[0008] However, the development of microorganisms as biofilms requires favorable and highly regulated conditions. Significant nutritional deficiencies in the wine can e.g. have a strong negative impact on their development.

[0009] There is therefore a need for the development of novel biofilms that would have an increased resistance to external stresses while allowing for the microorganisms to have access to all the nutrients required fortheir proper development and activity.Summary

[0010] The invention is defined by the claims.

[0011] Surprisingly, the inventors have demonstrated that the use of yeast derivatives biotic supports allows for the development of microorganisms such as O. oeni in the form of a biofilm while providing them with the nutrition they need to implant. The inventors also showed that using such yeast-derivatives supported biofilms to carry out malolactic fermentation also makes it possible to multiply the ferment while strengthening the resistance of O. oeni to environmental stresses. The biofilms thereby produced can be directly used to inoculate and ferment fermented brewages such as wine.

[0012] Therefore, according to a first aspect, the present invention pertains to the use of yeast-derivatives as a biofilm support.

[0013] The present invention also pertains to a biofilm, wherein said biofilm is supported by a support comprising yeast derivatives.

[0014] According to a further embodiment, the present invention also pertains to a method for preparing a biofilm, wherein said method comprises inoculating a yeast-derivatives support with microorganisms, and culturing said microorganisms to obtain a biofilm.

[0015] As mentioned above, biofilms are of particular interest for fermenting products in the food industry, in particular in the wine industry.

[0016] Accordingly, the present invention also pertains to a method for preparing a fermented product, wherein said method comprises a step of inoculating said product with the biofilm according to the present invention.Brief Description of the figures

[0017] Figure 1: Scanning electron microscopy images of 6 days-biofilms formed by O. oeni on biological supports Spring Arom and Spring Cell. Magnifications were performed at 4000x and 8000x. Black arrows indicate bacteria.

[0018] Figure 2: Scanning electron microscopy images of malolactic bacterium Oenococcus oeni developing according to a planktonic lifestyle (A) or a biofilm lifestyle (B-F). Biofilm lifestyle showsthat the bacterium uses yeast derivatives as physical support, onto which it first attaches to then produce exopolysaccharides and form a biofilm matrix.

[0019] Figure 3: Malolactic fermentation of 4.1 O. oeni strain in red wine in function of the mode of life and the support used: inactivated yeasts (A) or hulls form (B). Black curves correspond to planktonic bacteria with (open symbols) or without (closed symbols) biological support; grey curves correspond to 6 days-biofilm on biological support. Measurement were replicate three time malic acid concentration data were analysed by Kruskal-Wallis statistical test (P-value < 0.05), * represent statistical differences within the conditions, and rate of malic acid consumption values were analyzed by ANOVA statistical test (P-value < 0.05), different letters indicated statistical differences.Detailed description of the invention

[0020] The present inventors have shown that it is possible to successfully grow microorganisms in the form of a biofilm by using yeast derivatives as a biotic support.

[0021] Therefore, according to a first aspect, the present invention pertains to the use of yeast derivatives as a biofilm support.

[0022] The present invention also pertains to a biofilm, wherein said biofilm’s support comprises yeast derivatives.

[0023] In the context of the present invention, “yeast derivatives” refer to a fraction obtained during the degradation of yeasts by physical or chemical action, for example by plasmolysis, hydrolysis or autolysis of the yeast. Yeast derivatives are all products that can be obtained from whole or fractionated yeast cells, by physical or chemical action. They include yeast extracts, yeast cell walls (also referred to as yeast hulls), mannoproteins and inactivated yeast. They are most often in the form of a powder after grinding or in suspension in a rehydration medium, in the form of yeast cream or pressed yeast.

[0024] According to a specific embodiment, the yeast derivatives according to the present invention are selected from the group consisting of yeast hulls / cell walls, yeast extracts, inactivated yeasts and mixtures thereof. According to a preferred embodiment, the yeast derivatives according to the present invention are selected from yeast hulls / cell walls and inactivated yeasts.

[0025] The yeast derivatives used in the context of the present invention can be obtained from any species of yeast, in particular of the Saccharomyces genus such as Saccharomyces cerevisiae. They can also be obtained from a mixture of various yeast species. The yeast(s) from which the yeast derivates can be obtained is(are) typically selected from brewer's yeasts, oenological yeasts or distillery yeasts. Other types of yeasts that can be used in the context of the present invention include yeast genus from the Kluyveromyces spp, Pichia spp, Metschnikowia spp and Candida spp groups.

[0026] The yeast derivatives can be obtained or prepared according to techniques that are well known by the one skilled in the art. A yeast is a cell schematically composed of an envelope and a cellular content. The envelope is referred to as “cell wall” or “hull”. Yeast cell walls can be produced in different ways, from different types of yeast, optionally as a mix, with various techniques. Yeastcell walls can e.g. be obtained from the lysis (autolysis or heterolysis) of yeast cells, followed by the separation of the soluble and insoluble fractions, for example, by physical means such as centrifugation, and then collecting the insoluble fraction. The insoluble fraction is typically collected by removing the soluble fraction by centrifugation. The insoluble fraction obtained accordingly corresponds to the yeast cell walls. The cell walls correspond to about 25 to 45% of dry weight of the entire yeast cell, an average of about of 35%. On the other hand, the soluble fraction (which is clear in color and of weak turbidity) correspond to the “yeast extract”. Yest cell walls can also be easily obtained from specialized manufacturers.

[0027] “Inactivated yeasts” correspond to yeast cells that are no longer living or active so they cannot produce any of the effects of live yeast, such as fermentation. Inactivated yeasts can e.g. be obtained by submitting yeast cells to treatments including thermic, ultrasound or chemical treatments. They can also be easily obtained from specialized manufacturers.

[0028] A “biofilm” is a three-dimensional structure made of a microorganism (typically bacteria and / oryeasts) incorporated in an exopolymeric matrix (polysaccharides, polypeptides, nucleic acids). They develop on (i.e. are attached to) a solid structure corresponding to the “biofilm support”.Generally speaking, the formation of biofilms results from a dynamic and complex process. The biofilm develops in several steps from the initial adhesion to the support to the formation of a three-dimensional structure which can constitute a genuine ecosystem assuring various functions. The “support” is therefore a physical structure, onto which the microorganism attaches to then start the synthesis of the exopolymeric matrix forming the biofilm. A biofilm support is a physical structure and cannot be understood as a mere source of raw materials that are used by the microorganism for incorporation in the biofilm structure. The notion of “support” necessarily implies a physical adhesion of the microorganism, which will then start producing material, such as exopolysaccharides, forming the biofilm matrix. The exopolymeric matrix is a key element of the biofilm and constitutes a genuine physical and biological barrier which protects the bacteria from environmental stresses (antimicrobial agents, acid stresses, thermal stresses, etc.) but also plays the role of filter so as to provide the biofilm with nutrients. The microorganism present in the biofilm benefits from this favorable environment to proliferate and develop a “biofilm phenotype”, which confers a very high resistance to stress and specific functionalities (Rieu et al. Cellular Microbiology 16.12 (2014): 1836-1853).

[0029] In the context of the present invention, the “microorganism” at the origin of the biofilm can be any living unicellular organism, including procaryote and eukaryote unicellular organisms, that can grow in the form of a biofilm and develop a biofilm phenotype. According to a specific embodiment, said microorganism is a fermentative microorganism, i.e. a microorganism that is capable of carrying out fermentation.

[0030] In the context of the present invention, the microorganism is typically selected from the group consisting of bacteria, yeasts and mixture thereof.

[0031] According to a specific embodiment, the microorganism at the origin of the biofilm in the context of the present invention is selected from yeasts, in particular fermentative yeasts. The fermentative yeast may typically be selected from strains belonging to the genus Saccharomyces,Schizosaccharomyces, Brettanomyces, Torulaspora, Candida, Metschnikowia, Kluyveromyces or combinations thereof.

[0032] According to another specific embodiment, the microorganism at the origin of the biofilm in the context of the present invention is selected from bacteria, in particular fermentative bacteria. The fermentative bacteria may typically be selected from lactic bacteria, acetic bacteria and combinations thereof. According to a preferred embodiment, the fermentative bacteria is selected from the group consisting of bacterial strains belonging to the genus Oenococcus, Lactobacillus, Pediococcus, Weissella, Leuconostoc and combinations thereof. According to a more preferred embodiment, the fermentative bacteria are lactic bacteria, preferably Oenococcus oeni.

[0033] The type of microorganism selected will depend on the intended use of the biofilm. The skilled person will know which microorganism to use depending on the application contemplated. For instance, lactic bacteria such as Oenococcus oeni are particularly advantageous for wine fermentation.

[0034] According to a further embodiment, the present invention also pertains to a method for preparing a biofilm as defined above, developing on yeast derivatives as a support.

[0035] The method according to the present invention comprises inoculating microorganisms on yeast derivatives as a support, and culturing said microorganisms under conditions that allow for the growth of said microorganisms in the form of a biofilm, i.e. under conditions that allow for said microorganism to obtain a biofilm phenotype. Such conditions typically include moderate flow conditions, high moist content, pH and temperature adjusted for an optimal growth of the microorganism used, etc. The skilled person is familiar with the conditions to be applied for allowing the growth of a known microorganism as a biofilm.

[0036] The biofilm may be prepared by incubating microorganisms in a medium favorable to their growth in the presence of the yeast-derivatives support. The growth time may depend on the species, and, within a species, the strain used. For example, in the case of Oenococcus oeni, the incubation lasts at the most 7 days in modified MRS medium.

[0037] A method for culturing a microorganism such as Oenococcus oeni so as to obtain a biofilm is described in the experimental section below. Yeast derivatives are e.g. inoculated with a bacterial population at a concentration of 106to 108Colony Forming Unit (CFU) / mL, preferably at a concentration of about 107CFU / mL. The method advantageously comprises preventing or eliminating the presence of a supernatant in the culture medium so as to promote the development of a biofilm and prevent the presence of planktonic cells. The culture is then incubated for a period of between 2 to 8 days, preferably 3 to 6 days.

[0038] Such a method can further comprise a step wherein the microorganism is retrieved by means of any method known by the skilled person, preferably sonication.

[0039] As mentioned above, biofilms are of particular interest for fermenting products in the food industry, in particular in the wine industry.

[0040] Accordingly, the present invention also pertains to a method for preparing a fermented product, typically a fermented brewage, wherein said method comprises a step of inoculating said product with the biofilm according to the present invention. Such a method comprises the initiation of fermentation by inoculating the product with a fermentative microorganism, such as bacteria or yeasts, in the form of biofilm.

[0041] The fermentative microorganism in the form of a biofilm is inoculated in a form adherent to the yeast-derivatives support, that is to say still attached to the yeast-derivatives support.

[0042] The fermentative microorganism is typically inoculated at a concentration comprised between 106and 107CFU / mL.

[0043] In the context of the present invention, the fermented product is typically a fermented brewage selected from the group consisting of wines, fruit- and vegetable- based drinks, vinegars, ciders or beers, preferably the fermented brewage is a wine.

[0044] As shown in the international publication published under reference WO2016 / 055603, biofilms made of lactic bacteria are particularly useful for initiating malolactic fermentation in fermented brewages. Thus, the present invention pertains to a method for preparing a fermented brewage, such as wine, comprising the initiation of malolactic fermentation by inoculating said brewage with lactic bacteria, or with a combination of lactic bacteria and fermentative yeasts, in the form of the biofilm supported by a yeast-derivatives support as described herein.

[0045] The invention will now be further illustrated by means of the following non-limiting examples.Examples

[0046] Material and Methods

[0047] Strains and Growth Media

[0048] This study was conducted using a malolactic bacterium Oenococcus oeni , isolated in Burgundy from a white wine (this strain will be referred to as “strain 4.1”). It was grown in MRS modified medium containing: MRS Broth Low pH Condalab® (Madrid, Spain) 50 g / L; fructose 10 g / L; L-malic acid 4 g / L (Bastard et al., 2016). The pH was adjusted to 4.8 (NaOH concentrated solution).20 g / L agar was added to solid MRSm medium. Cultures were incubated at 28 °C. All the assays were performed in triplicate.

[0049] Biofilm formation condition on abiotic surfaces

[0050] To evaluate adhesion properties of strain 4.1 , biofilm development on polystyrene surface was evaluated. One mL of a mid-exponential phase culture (diluted at ODeoonm = 0.05) was added to the wells of a polystyrene 24-well microtiter plate (Cellstar®). After 24 hours at 28°C in anaerobic jars, the medium is gently removed from each well by aspiration. After washing twice with 500pl of physiological water, the adherent cells were resuspended by aspiration / reflux in 1 ml of physiological water. The cells were counted on MRSm agar medium, and the plates were incubated for 7 days at 28°C in anaerobic jars (GasPak EZ). A biological triplicate was performed for each strain.

[0051] Biofilm formation condition on biotic surfaces

[0052] A preculture of O. oeni is produced by inoculating 5 ml of a primary culture in 10 ml of MRSm medium and incubating at 28°C for 24 hours. The bacterial cells are then concentrated in order to inoculate yeast derivatives present in the wells of a microplate. To do this, each well of a 96-well microplate is filled with 40 mg of the yeast derivative under study and 85 pl of bacterial culture, giving a final quantity of 107CFU per well. The plate was placed in a rack filled with water to approximately one third of the height of the plate, then incubated at 28°C for 6 days. After 3 days, 30 pl of MRSm medium was added to each well to keep the system moist and provide nutrients.

[0053] After 6 days, the biofilm developed in the wells was either counted (biological triplicate), observed by scanning electron microscopy, or collected for inoculation into wines for MLF.

[0054] To count the number of cells that have developed into a biofilm, the yeast derivative-bacteria combination is sonicated for 2 minutes at 40 kHz in order to remove the bacteria adhering to the biotic support and to reduce the biofilm microcolonies. A series of cascade dilutions at a rate of 1 :10 were carried out in order to carry out a surface enumeration on MRSm agar medium. The plates were incubated for approximately 7 days at 28°C in an anaerobic jar.

[0055] In order to use these biofilms to inoculate the wines, the biofilm was sampled and transferred to 50 mL of wine (targeted inoculation around 5,106CFU / mL) for MLF. L-malic acid consumption was monitored by robot-assisted enzymatic assay (Y15).

[0056] Scanning electron Microscopy

[0057] Cells were fixed on stainless steel by a solution of 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.2 for 1 h at 4°C. The samples were then washed three times with phosphate buffer for 20 min at room temperature. Dehydration was performed by successive immersions in solutions of increasing ethanol content (70, 90, 100%), then three times for 10 min each in successive baths of ethanol-acetone solution (70:30, 50:50, 30:70, 100) and air-dried. Afterward, the samples were coated with a thin carbon layer using a CRESSINGTON 308R and observed with a JEOL JSM 7600F scanning electron microscope (JEOL, Ltd.). SEM was performed at 5 kV and the samples were observed at a working distance of 14.9 mm.

[0058] Microvinifications

[0059] The red wine used for malolactic fermentation assays was a Syrah adjusted at 15% ethanol pH 3.4, malic acid (2.2 g / L) and with a total SO2 of 4 mg / mL.

[0060] Using cryotubes stored at-80°C, two successive precultures were carried out with inoculation at OD600nm=0.05 in MRSm medium. At the end of the exponential phase, 50% of the culture was mixed with 50% of the wine and incubated for 72 h at 28°C. The operation was repeated once, then the wines were inoculated at 5. 106CFU / ml, homogenized and incubated at 20°C. The same experiment is realized with the addition of 40 mg of yeast derivatives (corresponding at a concentration of 47 g / L).

[0061] In parallel, biofilms of strain 4.1 grown for 6 days on Spring Arom™, Spring Cell™ and Spring Ferm™ media were immersed in 50 mL of red wine.

[0062] L-malic acid consumption is monitored over time by enzymatic assay (Food Quality Biosystems) assisted by the Y15 (Biosystems). The population (planktonic or in biofilm) is counted at TO and then regularly during malolactic fermentation.

[0063] To evaluate the biofilm development properties of this strain, 25*25 mm stainless-steel chips were immersed in 20 ml of inoculated MRSm (inoculation at ODeoonm = 0.05). After an incubation of 72 hours, the chips were rinsed twice with physiological saline solution (NaCI 9 g. mL-1), then placed in 15 ml saline for a sonication cycle (1 minute a 40 kHz) to remove the cells in biofilms. The system is vortexed at maximal powerfor2 minutes to tearthe cells out. Then, cells in the solution are counted on a solid MRSm medium (following serial dilutions). It was previously verified that the sonication treatment tears all adhered cells and does not cause cell death.

[0064] Metabolic analyses

[0065] Analyses were performed using ultra-high-pressure liquid chromatography (Dionex Ultimate 3000, Thermo Fisher Scientific, Waltham, MA USA) coupled to a MaXis plus MQ ESI-Q-ToF mass spectrometer (Bruker, Bremen, Germany). Non-polar compounds were analyzed in reverse phase using Acquity BEH C18 1.7 %m, 100 x 2.1 mm column (Waters, Guyancourt, France). The mobile phase was ultrapure water from a Milli-Q system (Merck, Darmstadt, Germany) acidified by the addition of 0.1% (v / v) formic acid (MS grade from Acros Organic, Morris Plains, NJ, USA) for eluent A, 95% (v / v) acetonitrile (MS grade from Biosolve, Dieuze, France) acidified by the addition of 0.1 (v / v) formic acid for eluent B. The elution temperature was 40°C following a gradient: 0-1.10 min 5% (v / v) of eluent B and 95% of eluent B at 6.40 min. The flow rate was 400 pl. min-1. The nebulizer pressure was 2 bar and a nitrogen dry gas flow of 10 L / min was maintained. Ionization was performed by electrospray in positive and negative ion mode. The ion transfer parameters and the injection of calibrant at the beginning of each run was performed according to Evers et al. (2023). To verify the stability of the UHPLC-Q-TOF-MS system, quality controls (mixing of all the samples analyzed in the batch analysis) were analyzed at the beginning, the end and every 10 samples during the batch analysis. All the samples were analyzed randomly. Compass DataAnalysis v4.3 software (Bruker, Bremen, Germany) was used for pre-treatment analysis as developed by Evers et al. (Foods 12.5 (2023): 972).

[0066] Volatilome analysis:

[0067] HS-SPME-GC-MS analyses were performed using an Agilent 8890 gas chromatograph (GC) system coupled with an Agilent 5977B mass spectrometer (MS) (Agilent, Santa Clara, CA, USA) and equipped with Gerstel MPS sampler (Gerstel, Muhlheim an der Ruhr, Germany) for automated HS-SPME sampling. Extraction of volatiles compounds by HS-SPME was performed using a 50 / 30 pm DVB / CAR / PDMS fiber (Supelco, St. Louis, MO, U.S.A.). The samples were incubated 2 min at 50°C before extraction during 30 min and desorption during 2 min at 270°C. Sample were agitated at 250 rpm during 10 s before extraction. Injection was realized in split mode with a ratio 1 .5 / 1 . After eachextraction, fiber was conditioned 10 min at 270°C. The columns used was Agilent HP-5MS (30 m, 0.250 m, 0.25 pm) at flow rate 3 mL / min. The GC oven was set at 30°C for 6 min then increased to 100°C at rate 2.5°C / min, then increased to 105°C at rate 1°C / min and held for 5 min, then increased to 125°C at rate 2.5°C / min, then increased to 300°C at rate 20°C / min held for 4 min (total run time 64.75 min). The MS line transfer temperature was 310°C, MS source temperature was 230°C and MS quadrupole temperature was 150°C. The MS acquisition was performed in electron impact ionization (El) mode at 70 eV in SIM mode. Sample preparation consist to add 5 mL of sample with 5 mL of saturated NaCI solution. Deuterated internals standards are added to sample, t-Butanol-d10 (46.5 mg / L) and Ethyl butanoate-d3 (200 pg / L) for quantification of higher alcohols and esters, respectively. Sample were preparated in 20 mL glass vial and sealed with screw caps. Agilent Mass Hunter Workstation Quantitative Analysis v10.0 was used for data processing. 8 higher alcohols and 18 esters were targeted and quantified.

[0068] Results

[0069] Biofilm development on different supports

[0070] Strain 4.1 , isolated from a Burgundy vineyard during spontaneous malolactic fermentation, was selected for this study. Its adhesion and biofilm development capacities were evaluated on conventionally used supports such as polystyrene or stainless steel. The quantifications of cells adhered for 24 hours on polystyrene and developed in biofilm for 72 hours on steel were 1.17 107CFU / cm2and 2.48 107CFU / cm2respectively, demonstrating the capacity of this strain to develop in biofilm.

[0071] This strain was used to develop biofilms on a biotic support consisting of inactivated yeast (Spring Arom™) or yeast hulls (Spring Cell™) produced by Fermentis.

[0072] The development of a protocol for the development of biofilms on yeast derivatives has led to the design of a product that can be directly inoculated into wine. To develop this product, the biofilm inoculation rate was defined. The quantity of biofilm was monitored over time as a function of the inoculation rate (105, 107or 109CFU for 40 mg of derivatives). On Spring Arom™ support, no difference was observed over time as a function of inoculation rates, whereas on Spring Cell™ support, an initial inoculation at 107CFU significantly improved the quantity of cells developed into biofilm. For Spring Arom™ support, 6.8. 109CFU / mL were counted after 3 days. This quantity decreased to around 109CFU / mL after 6 days and stabilized at 10 and 15 days, with an average of 4.108CFU / mL. A different profile was observed on the Spring Cell™ medium, depending on the inoculation rate. With an inoculation of 105CFU, the quantity of bacteria increases between 3 and 6 days of development, from 3.5 ,108to 1.1 ,109CFU / mL before decreasing and stabilizing at approximately 4. 108CFU / mL. For an inoculation of 107CFU, the number of cells decreased significantly over time, from 1.9 ,109at 3 days to 1.3 ,108CFU / mL at 15 days. In contrast, when inoculated with 109CFU, the number of bacteria increased slightly over time, with a cell count of 5.108at 6 days and 1.2 109CFU / mL at 15 days.

[0073] Despite these differences in kinetics, the concentrations of cells in biofilms at 6 days are relatively close between the 2 supports. Scanning electron microscopy observations were used to visualize the oenological products developed (Figure 1). The bacteria-free control showed a very different structure to the yeast derivatives, with well-rounded yeasts for Spring Arom™ and more diffuse substances for the Spring Cell™ support. In both supports, we can clearly observe bacteria adhering to the yeast derivatives, which are covered in a sort of glue, suggesting the presence of an extracellular matrix. These images demonstrate the presence of biofilm and validate the system developed.

[0074] Scanning electron microscopy observations (see Figure 2) show that bacteria firstly physically attach to the yeast derivatives, using them as a physical support, and then start producing exopolysaccharides that are incorporated into matrix. The yeast derivatives therefore play a structural role, rather than a mere exopolysaccharide source that would then be incorporated into the biofilm matrix.

[0075] Bacterial phenotype and malolactic fermentation

[0076] Vinification trials were carried out on red wine (Syrah wine adjusted at 15% ethanol pH 3.4, malic acid (2.2 g / L) and with a total SO2 of 4 mg / mL) in order to assess the performance of the ferment developed (Figure 3). Bacteria developed as biofilms on Spring Arom™ or Spring Cell™ supports and adapted planktonic bacteria were inoculated into the wine at a rate of 5. 106CFU / mL. A planktonic bacteria modality with added support (Spring Arom™ or Spring Cell™) was also included in order to assess the impact of the support on malolactic fermentation. Malic acid consumption was monitored over time. When the Spring Arom™ support was used, the adapted planktonic bacteria completed malolactic fermentation in 30 days (malic acid concentration equal to 0.5 g / L). The fermentation speed increases when yeast derivatives are added. Bacteria developed over 6 days in biofilms also underwent malolactic fermentation, although their malic acid consumption was slightly slower than that of adapted planktonic bacteria: nevertheless, in 27 days the malic acid concentration fell below 1 g / L. Similar results were observed with Spring Cell™ media. Efficient malic acid consumption with the planktonic bacteria (in 30 days malic acid concentration equal to 0.5 g / L), improved by the yeast derivative (in 17 days malic acid concentration less than 0.5 g / L). On the other hand, for cells developed for 6 days in biofilms, malic acid consumption equivalent to that of planktonic bacteria was adapted and quantified.

[0077] Omic footprint linked to the phenotype of ferments and the presence of yeast derivatives

[0078] To assess the impact of the planktonic versus biofilm lifestyle and the presence of yeast derivatives on the metabolome and volatilome produced during vinification, LC-MS and GC-MS analyses were carried out. The three vinification conditions using adapted planktonic bacteria with or without the addition of yeast derivatives (Spring Arom™ or Spring Cell™) and bacteria grown for 6 days in biofilms on these yeast derivatives were analyzed.

[0079] Firstly, a principal component analysis (PCA) based on the LC-MS data was used to discriminate changes in metabolic composition between the three conditions. It is interesting to note that among the 7054 features analyzed, the 3 conditions are very well separated in the PCA. Axis 2 allows an effective separation of the planktonic adapted with yeast derivatives and the biofilm modalities, whatever the support. The planktonic bacteria modality is also isolated on the PCA. This shows that, on the one hand, the addition of yeast derivatives modifies the metabolome and, on the other hand, the biofilm or planktonic lifestyle is also an important component of the resulting metabolome.

[0080] Molecular markers significantly more present in the biofilm modality compared with the planktonic modality with or without support were then sought. In the case of the Spring Arom™ support, a total of 1156 features were analyzed. The heat map highlights the features significantly expressed according to the different biological conditions. Changes were observed in the compounds present in the condition using planktonic bacteria with or without support, although some of these compounds remained common. On the other hand, when bacteria were used in biofilms, a major shift was observed with a significant change in the metabolome. With regard to the Spring Cell™ support, changes in the distribution of the 795 discriminating features were also observed depending on the bacterial lifestyle and the presence or absence of the support. However, it is notable that there is more overlap between the 3 modalities with more common compounds between planktonic bacteria with or without support. There was also a clear overlap between the modalities using planktonic bacteria and bacteria in biofilms. So in this case, a less good separation of the modalities is observed.

[0081] The Venn diagrams allow us to compare the compounds specific to each support as a function of lifestyle. Compounds specific to the presence of yeast derivatives and to biofilm lifestyles were researched. Among all the compounds, under planktonic conditions, 96 were induced by the use of Spring Cell™ and 138 by that of Spring Arom™, of which 2 were observed to be common. This proportion of shared common compounds is greater in the biofilm mode. Indeed, of the 214 compounds induced by the use of Spring Cell™ and the 285 induced by the use of Spring Arom™, 64 are common. The biofilm modality shares more compounds between the 2 supports (64 compounds) than the adapted planktonic modality (2 compounds), demonstrating that the lifestyle prevails over the support used.

[0082] In addition, an analysis of the volatilome produced was performed by GC-MS using 20 targeted compounds. Regarding the Spring Arom™ support, a strong discrimination of the 3 modalities (biofilm, planktonic adapted with or without support) was observed. The focus is on the 7 main discriminating molecules, which are alcohols and esters. Concerning alcohols, a higher proportion was detected in the conditions without yeast derivatives, as if, whatever the physiological mode of life, the presence of the derivatives trapped these compounds. With regard to esters, certain molecules (ethyl propanoate, ethyl isobutyrate, ethyl butanoate) are more present in the planktonic mode, with the biofilm acting as a filter. On the other hand, a higher proportion of esters such as ethylacetate or ethyl octanoate was detected in the presence of yeast derivatives (for the 2 phenotypes studied).

[0083] For the Spring Cell™ support, as for the metabolome, the volatilome does not seem to be very discriminating between conditions (mode of life and support presence). Only 3 molecules appear to be discriminating (ethyl acetate, 3 methyl butanol and 2-methylbutanol).

Claims

Claims

1. Use of yeast derivatives as a biofilm support.

2. A biofilm, wherein said biofilm is supported by a support comprising yeast derivatives.

3. The use or biofilm according to claim 1 or 2, wherein said yeast derivatives are selected from the group consisting of yeast cell walls, yeast extracts and inactivated yeasts.

4. The use or biofilm according to any one of claims 1 to 3, wherein said yeast derivatives are obtained from yeasts of the Saccharomyces genus, preferably from Saccharomyces cerevisiae.

5. The use or biofilm according to any one of claims 1 to 3, wherein said biofilm is made of microorganisms selected from bacteria and yeasts, preferably bacteria.

6. The use or biofilm according to claim 5, wherein said bacteria are fermentative bacteria.

7. The use or biofilm according to claim 6, wherein said fermentative bacteria are selected from the group consisting of the genus Oenococcus, Lactobacillus, Pediococcus, Weissella, Leuconostoc and combinations thereof.

8. The use or biofilm according to claim 7, wherein said fermentative bacteria are Oenococcus oeni.

9. A method for preparing a biofilm, wherein said method comprises inoculating a yeast-derivatives support with microorganisms, and culturing said microorganisms to obtain a biofilm.

10. The method according to claim 9, wherein said yeast-derivatives support is inoculated with a microorganism population at a concentration of 106to 108CFU / mL, preferably at a concentration of about 107CFU / mL.

11. The method according to claim 9 or 10, wherein said microorganisms are cultured over a period of between 2 to 8 days, preferably 3 to 6 days.

12. A method for preparing a fermented product, wherein said method comprises a step of inoculating said product with the biofilm according to any one of claims 2 to 8, wherein said biofilm is made of fermentative microorganisms.

13. The method according to claim 12, wherein said fermentative microorganisms are selected from bacteria and yeasts.

14. The method according to claim 12 or 13, wherein said fermented product is a fermented brewage selected from the group consisting of wines, fruit- and vegetable- based drinks, vinegars, ciders or beers, preferably the fermented brewage is a wine.

15. The method according to any one of claims 12 to 14, wherein the fermentative microorganisms are inoculated at a concentration comprised between 106and 107CFU / mL.

Images