Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/02—Oxygen as only ring hetero atoms
  • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the present invention relates to means for recovering residues from the distillation of fermentation products and in particular a process for the production of compounds of economic interest from these residues from the distillation of fermentation products. This production process also includes steps suitable for the extraction of these compounds.
• the invention relates in particular to the recovery of distillation residues from fermentation products from the food industry.
• these residues or by-products are used as substrates for the culture of microorganisms capable of developing and producing molecules of economic interest; these compounds are in particular molecules with high added value such as flavors, colors, enzymes, amino acids, lipids, carbohydrates, biologically active products in the therapeutic, food-processing or agricultural fields, etc.
• Patent FR 2 705 971 describes a process for the production of R- ⁇ -decalactone comprising the culture of microorganisms of the genus Sporidiobolus or Fusu um in a culture medium containing a precursor of R- ⁇ -decalactone chosen from ricinoleic acid, lesberolic acid or the salts or esters with C1 to C3 alcohols of these acids.
• the extraction of the R- ⁇ -decalactone produced is carried out during the culture, with an extraction solvent immiscible with water.
• the inventors have, in the context of this invention, observed that residual products from the distillation of wine, and in particular Cognac wine (these residual products being designated in this specific case by the expression "vinasse”), are susceptible to constitute suitable substrates for the culture of microorganisms, with the aim of producing molecules of interest economic.
• the present patent application therefore relates to a process for the preparation of compounds of economic interest from substrates constituted by or comprising residues from the distillation of fermentation products and the use of microorganisms for the implementation of this process.
• the present application also relates to a process for extracting the compounds produced, said process being advantageously associated with the production process.
• the extraction steps proposed according to the invention can follow the phase or phases of production of the compounds or be integrated into this phase.
• the conditions chosen for the extraction are determined as a function of their influence on the production of the compounds, for example on the production yield.
• the invention relates to a process for the preparation of compounds of economic interest (called “compounds”), comprising the steps of: a) culturing under aerobic fermentation conditions, at least one microorganism chosen for its ability to synthesize said said compounds, in the presence of a substrate comprising a residue from the distillation of fermentation products, b) recovery of the compounds produced.
• the subject of the invention is in particular a process for the production of odorous volatile compounds (or flavors).
• the invention provides a process which allows the formation of natural flavors.
• fermentation can be carried out in an aerobic, anaerobic or mixed environment.
• the substrate used for carrying out the process comprises, for example, a residue resulting from the distillation of fermentation products from plant organs (roots, tubers, etc.) or more generally from parts of plants.
• the substrate placed work includes a residue from the distillation of wine and, for example, a residue from the distillation of Cognac or Armagnac wine. This residue is called "vinasse”.
• Vinasses are the liquid by-product obtained from the still after distillation of the wine. Cognac is produced on lees and generates large volumes of vinasse: around two thirds of the volume of distilled wine. The vinasses no longer contain alcohol, and almost no more aromatic compounds, these being passed through the distillate. However, they contain a certain number of substances, and in particular non-negligible quantities of organic acids (7.55 to 10.05 g / l).
• the average composition of the Cognac distillery vinasses is as follows:
• Citric and malic acid 0.05 g / l
• Glycerol for vinasse about 7g / l normal
• COD is the Chemical Oxygen Demand, that is to say the quantity of oxygen necessary to oxidize the organic matter contained in the effluents.
• BOD 5 is the Biological Oxygen Demand necessary, for 5 days, for microorganisms contained in water to oxidize part of the materials carbonaceous.
• the tartaric acid of Cognac distillery vinasses is in the form of potassium bitartrate (KHC 4 H 4 O 6 ).
• the substrate is chosen from the distillation residues of fermentation products of fruit, beets, sugar cane, cereals, in particular malt, barley, wheat , corn or rice.
• the distillation residues of fermentation products from apple juice are the distillation residues of calvados
• the distillation residues of fermentation products from sugar cane are the distillation residues of rum, etc.
• the substrates thus defined taken in isolation or as a mixture, can be used to produce various compounds such as volatile odorous compounds (or flavors). Other compounds such as proteins, amino acids, lipids, carbohydrates, nucleosides, alcohols or derivatives of these compounds can be produced simultaneously with these flavors obtained according to the invention.
• the substrates described can thus, depending on the microorganism chosen for the implementation of the process, generally allow the production of biologically active compounds.
• the substrates defined in the context of the invention can be adapted for the culture of various microorganisms and in particular bacteria, yeasts, or fungi.
• the defined method is characterized in that the culture step uses at least one yeast of the genus Sporobolomyces odorus capable of producing aromas, under culture conditions allowing the growth of said strain S. odorus and the production of aromas from a substrate comprising vinasse.
• Sporobolomyces odorus is a strain of yeast known for its ability to produce aromas such as lactone, in particular ⁇ - decalactone.
• the subject of the invention is therefore a process for the preparation of flavorings comprising the steps of a) cultivation under aerobic fermentation conditions of Sporobolomyces odorus in the presence of a substrate comprising vinasse, the pH of the medium being between 4 and 7, preferably equal to about 6, the reaction temperature being compatible with the growth of S. odorus. b) recovery of the aromas produced.
• the process thus defined is advantageously carried out with stirring between 50 and 1000 rpm, preferably between 100 and 700 rpm and with aeration from 0.10 to 10 vvm, preferably from 0.10 to 5 vvm.
• the temperature of the culture step can vary depending on the strain (s) of microorganisms used.
• the culture is carried out at a temperature between 10 and 50 ° C, preferably between 20 and 40 ° C, advantageously close to 24 ° C; this temperature is regulated to be kept essentially constant.
• the recovery of the aromas produced can be carried out continuously by solid-liquid extraction.
• the stages of recovery of the compounds produced comprises a stage by which the medium constituted by the culture defined above carried out in aqueous phase, is brought into contact with a medium in lipid phase, for a time sufficient to allow the absorption in the lipid phase of all or part of the compounds products, the medium in the lipid phase being solid at room temperature.
• the invention proposes in this regard the use of lipid media comprising or consisting of vegetable fats or mixtures of vegetable fats.
• lipid media comprising or consisting of vegetable fats or mixtures of vegetable fats.
• HNCH hydrogenated coconut oil
• TTT Fluka Chemie AG
• triolein 20%
• a vegetable fat suitable for carrying out the invention has the property of being odorless, slightly oxidizable in particular to avoid the formation of the "rancid" odor and insoluble at low temperature (notably below 0 ° C. at -20 ° C) in ethanol.
• the invention has demonstrated that an appropriate extraction system is advantageously based on a phase balance between the aqueous phase and the lipid phase, in order to improve both the viability of the microorganisms and the production of compounds, in particular aromas.
• the aromas produced are essentially dissolved in the culture medium which is an aqueous phase. Their partition coefficients being more favorable for a lipid phase, the separation of the aromas from the fermentation medium can be carried out by solid-liquid extraction using a solid oil at room temperature (or vegetable fat). The aromas produced are thus recovered and concentrated in the solid oil in which they are absorbed. It is also possible to obtain the aromas in an alcoholate, that is to say, in solution in ethanol. To do this, the solid oil containing the odorous compounds is dissolved in ethanol. The mixture is then advantageously cooled to between -5 and -20 ° C, for example in melting ice, to separate the solid oil from the alcoholate which contains the aromas in solution. The latter can then be distilled to purify the odorous compounds.
• Vegetable fat can be made up of a mixture of vegetable fat.
• the substrate used can be optionally enriched to promote the growth of the microorganism strains and / or to promote the production of the desired compounds.
• an enrichment in carbohydrate compounds and in particular in glucose will be used.
• the enrichment may alternatively or in addition take into account all the compounds participating in the formation of aromas, such as organic or mineral compounds, in particular metals, for example zinc, magnesium or manganese.
• the method may also include the use, during the culture step, of a precursor capable of promoting the production of the desired compounds, in particular of a precursor used for bioconversion, by the microorganism (s) ) used.
• the method is characterized in that the stage of culture of Sporobolomyces odorus is carried out in the presence of selected substrates and ricinoleic acid or derivatives of ricinoleic acid, for example an ester, which can be assimilated by the microorganism (s).
• the microorganism chosen is S. odorus and the precursor is an ester of ricinoleic acid such as methyl ricinoleate.
• the inventors have observed that the implementation of the process leads to a particularly high level of production of the desired compounds, when the incorporation of the precursor and in particular of methyl ricinoleate in the culture medium is carried out in fractionated form throughout the culture stage.
• This fractionation makes it possible to increase the production of desired compounds present in the aqueous phase.
• this fractionation can take account of stages of extraction of the produced compounds interspersed with the contributions in precursors.
• the inventors have found that a contribution of precursor and in particular of methyl ricinoleate, within the framework of carrying out the process, is advantageously carried out early during the culture step and preferably as soon as it is placed in culture, this contribution being advantageously renewed in quantities, equal or different, at different times later during the culture step.
• the supply of precursor and in particular of methyl ricinoleate can be divided into four supplies, when the duration of the culture is for example close to 3 days.
• the process of the invention is characterized in a particular embodiment, in that the amount of methyl ricinoleate introduced during each supply is between 0.008% (v / v) and 5% (v / v), preferably between 0.008% (v / v) and 0.2% (v / v), advantageously between 0.03% and 0.10% (v / v), preferably close to 0.03% (v / v ) or advantageously around 0.18% (V / V) when the extraction is carried out with vegetable fats.
• the amount of methyl ricinoleate introduced into the culture can be adjusted according to the desired result.
• the amount of methyl ricinoleate introduced during each addition is close to 0.07% (v / v) until a total amount added of between 0.1% (v / v) and 5% (v / v), advantageously between 0.03 and 0.7% (v / v) or 0.1% (V / V) and 0.7 (VA) at the end of culture.
• this quantity is close to 5% (V / V) at the end of the culture.
• the various parameters for optimizing the process will be optimized as a function of the quantity of compounds that one seeks to produce and when these compounds are aromas such as ⁇ -decalactone; these parameters can be chosen so as to produce between 50 mg / l and 1 g / l, for example between 50 mg / l and 700 mg / l, in particular between 50 mg / l and 150 mg / l of ⁇ -decalactone in phase aqueous.
• the total amount and the amount of each fraction of added precursor is determined according to its influence on the bioconversion reaction, the possible toxicity of the compounds produced on the development of the strains of microorganisms and according to the duration of the culture step.
• the method of the invention is thus characterized in that the cultivation step can be carried out for a variable duration and in particular can be carried out for a duration of between a few hours and several days, for example up to 10 days, in particular a minimum duration of 24 hours, in particular a duration between 24 and 72 hours.
• an initial cell density (OD at 620 nm) of the microorganisms provided is used between 0.1 and 20, preferably between 0, 2 and 15 advantageously between 1 and 15.
• the initial optical density of the microorganisms provided for carrying out the culture step is between 5 and 15 or between 5 and 10.
• surfactants such as Tween 20® or anti-foaming agents will advantageously be used.
• the inventors have observed that the production of flavors and in particular of ⁇ -decalactone is improved by the contribution of these surfactants or anti-foaming agents and that the initial cell density (initial biomass) influences this improvement and that the availability of the precursor in the phase aqueous varies the level of ⁇ -decalactone production.
• substrates of the invention can be varied and can in particular be chosen from the following strains, where appropriate after recombination, capable of giving rise to the production of the compounds identified below:
• Cylindrocarpon radicicola steroids Lactococcus lactis aromas Aspergillus enzymes aromas, Bacillus enzymes aromas, aromas
• ATCC under the reference 8661 is used as an example.
• the substrate used for the culture of the microorganisms is sterilized by any suitable means and for example in an autoclave, before the cultivation. If necessary, the substrate can be used without prior sterilization.
• the step of recovering the products formed can be carried out in any suitable manner and in particular the products obtained can be isolated and purified by extraction with a solvent, by distillation, by solid-liquid extraction, by coupled gas-liquid extraction. to one 11
• cryocondensation by supercritical fluids, by membrane processes, in particular perstraction and pervaporation or by chromatographic separation, in particular HPLC, as well as by combinations of these separation techniques.
• the recovery of the compounds can be carried out at the end of the culture step or several times, by interrupting the culture step at determined times for example according to the quantity produced of compounds and in particular to take account of the toxicity of the compounds produced with respect to microorganisms in the culture medium.
• recovery can be carried out in situ, in particular when the compounds produced can have a certain level of toxicity with respect to the microorganisms used.
• the latter can be carried out in the same compartment as that of the production, for example in the same tank, or on the contrary these two stages can be separated spatially.
• the extraction step carried out continuously with the production of the compounds can influence the yield of the reaction.
• a precursor depending on its partition coefficient and its concentration in the lipid phase, its availability in the aqueous phase can be affected, consequently reducing its use by the microorganism (s).
• the extraction can also favor the production of the compounds since the extraction carried out continuously or in a determined manner alternating with the production, reduces the possible toxic activity of the compounds produced with respect to the microorganism (s) of the medium. culture.
• the extraction of the compounds produced by absorption in vegetable fat is followed by a separation of the compounds, in particular of the aromas produced by means of an alcohol.
• a separation of the compounds in particular of the aromas produced by means of an alcohol.
• the invention also relates to the aroma extraction process, implemented independently of the aroma production step.
• This extraction process comprises bringing the medium in aqueous phase containing the flavors into contact with a medium in the lipid phase for a time sufficient to allow the absorption of all or part of the flavors in the lipid phase, this process being characterized in that the medium in the lipid phase is a solid medium at room temperature.
• Figure 1A Relationship between biomass (expressed in g / l of dry matter) and cell density (OD at 620 nm). 13
• Figure 1 B Relationship between the biomass (expressed in g / l of dry matter) and the volume of mycelium.
• Figure 2A State of cultures of S. odorus aged 77 hours on different vinasse-based media.
• Figure 2B Kinetics of growth and development of S. odorus on MT2-malt.
• Figure 2C Growth and development kinetics of S. odorus on MV2-malt.
• Figure 9 Growth and production kinetics of ⁇ -decalactone in S. odorus after fractional additions of methyl ricinoleate.
• the arrow indicates when the precursor was added.
• Figure 10 Growth, ⁇ -decalactone production and bioconversion yield by cultivated S. odorus after fractional additions of methyl ricinoleate.
• the arrow indicates when the precursor was added.
• the arrow indicates when the precursor was added.
• FIG. 13 Impact of a simultaneous supply of Tween 20 and RM on the production of ⁇ -decalactone.
• the arrow indicates when the precursor was added.
• i indicates when the RM additions were made.
• Biomass ⁇ [ ⁇ -decalactone] i indicates the time when the additions of RM were made.
• the Sporobolomyces odorus strain used is the strain deposited under the reference CBS 2636. 1.2 THE LENZITES BETULINA STRAIN
• the strain used is the strain deposited under the reference MIC 38.
• Two culture media were tested in order to determine which one allows the best production of ⁇ -decalactone. These media can be used as control media for testing the substrates.
• the first of these media has the following composition (JOURDAIN, 1985): - 30 g / l of glucose
• the second medium used (FERON, 1996) is formulated as follows: - 1 g / l of glucose
• the Sporobolomyces odorus strain was stored on a petri dish at 4 ° C.
• an MT2 type medium was used. 15 g / l of agarose were added (JOURDAIN, 1985). The medium thus obtained was autoclave 20 minutes at 120 ° C.
• the pH was adjusted to 4.8 using 5 N sodium hydroxide in the case of LMV and HCM N in the case of LMT. 18
• Lenzites betulina was stored at 4 ° C on petri dishes containing a medium prepared as follows:
• BIOMASS PRODUCED 111.1 CASE OF SPOROBOLOMYCES ODORUS
• the odorous volatile compounds must be extracted from the medium.
• the liquid-liquid extraction method with an organic solvent was used with pentane (C 5 H 12 ). This technique is based on the immiscibility of the solvent with the culture medium which is aqueous, and on the superior affinity that aromatic molecules have for it. Thus, the free volatile compounds pass from the aqueous phase to the organic phase.
• the technique known as the internal standard was used. This method is based on the use of pentane containing an aromatic compound to carry out the extraction.
• the chosen compound must have the following characteristics: - be added at a known concentration,
• the internal standard used was geraniol, at a concentration of 30 mg / l.
• the first assays of the organic extracts showed that the quantities of volatile compounds present were insufficient to allow reliable measurements.
• the samples were concentrated approximately 5 times. For this, a very low flow of dry air was used. Upon contact, the highly volatile solvent evaporated while the odorous molecules concentrated in the remaining volume. It is considered that the compounds produced by the microorganisms do not evaporate, or else that they evaporate in the same proportions as the internal standard.
• chromatograph HEWLETT PACKARD HP 5890 Series II, fitted with a capillary column of fused silica impregnated with Carbowax 20M (polymerized polyethylene glycol) 25 m in length and 0.25 mm in diameter.
• Carbowax 20M polymerized polyethylene glycol
• composition of the medium used was optimized to determine whether the vinasse could serve as a substrate for the production of lactone.
• the formulation of the medium has been modified by eliminating certain compounds: - vinasse medium without malt extract
• the malt-free medium can therefore be chosen as a reference medium (MT2-malt) for carrying out growth, production, pH evolution and glucose and lactone concentration kinetics on this medium, whether or not containing vinasse. ( Figures 2B and 2C).
• the main source of ricinoleic acid is castor oil in which it represents about 90% of fatty acids and is mainly found in the form of triglycerides.
• carbohydrate is not necessary for effective bioconversion of the ricinoleic acid contained in castor oil.
• a second possibility consists in using methyl ricinoleate, that is to say the ester of ricinoleic acid.
• Table 2 Influence of the amount of methyl ricinoleate (RM) supplied to 60 ml cultures after 24 hours, the measurements were made after 77 hours of culture.
• RM methyl ricinoleate
• Table 3 Influence of an early and fractionated supply of RM on the growth and production of ⁇ -decalactone by S. odorus after 77 hours of culture.
• this mode of supplying the RM has made it possible to minimize the quantities of precursor necessary, and to increase the quantity of ⁇ -decalactone present in the aqueous phase.
• the results obtained after 96 h of culture are presented in Table 4.
• the extraction of the lactone from the culture medium is favored by the RM, compound in which it is more soluble than in the culture medium, - the transfer of the RM would be limited by a reduction in the exchange surface due to its coalescence which increases with the quantity supplied,
• Figures 12 and 13 illustrate the production of biomass and ⁇ -decalactone respectively. With regard to growth, this was not affected during the first 48 hours of culture by the presence of
• cultures of 96 h on media having been supplemented were carried out as follows: medium 0: vinasse medium 1: vinasse + -0.01 g / l of FeS0 - 0.13 g / l of CaCl 2 - 3 g / l of MgS0 4 medium 2: vinasse + 1 g / l of yeast extract.
• medium 0 vinasse medium 1: vinasse + -0.01 g / l of FeS0 - 0.13 g / l of CaCl 2 - 3 g / l of MgS0 4
• medium 2 vinasse + 1 g / l of yeast extract.
• Table 5 Influence of complementation of the vinasse medium on the growth and production of ⁇ -decalactone by S. odorus. The results presented were obtained after 96 h of culture.
• composition of the vinasse is not an element liable to limit the production of ⁇ -decalactone
• toxic effect of ⁇ -decalactone observed beyond 250 mg / l could be at the origin of the limitation of its production by S. odorus, 34
• the solid particles contained in the crude vinasse are separated by centrifugation.
• the pH of the vinasse is adjusted to 6 with 5 M NaOH and antifoam (Struktol®) is added at 0.01% (v / v).
• the substrate thus obtained is introduced into the fermenter where it is sterilized at 120 ° C for 20 minutes at 1.4 bars.
• the fermenter is seeded at an optical density of 0.2 (read at 620 nm) from a preculture.
• Sterile methyl ricinoleate is added at a rate of
• the culture can be carried out continuously.
• the parameters of the reaction can vary according to the following indications given by way of example: 35
• the extraction substance used is an oil, an equilibrium is established between the aqueous and lipid phases. Given the consumption by the cells of RM in the aqueous phase, there is a transfer of the precursor from the lipid phase to the aqueous phase.
• HNCH hydrogenated coconut oil
• TTT solid fat
• the RM being lipid, it also dissolves in the HNCH.
• FIG. 14 show that the growth kinetics of the yeast are identical, whether the additions of RM are 4 ⁇ 0.03, 0.06 or
• the solid fat used (hydrogenated coconut oil) therefore proves to be a good system for extracting ⁇ -decalactone.
• the total lactone production (Table II) is improved by the presence of Struktol ® and to a lesser extent by the supply of Tween 20 ® when the initial cell density is 0.2.
• the influence of these adjuvants on the ⁇ -decalactone differs total production in tests with a cell seeding 4. Indeed, in the latter case, the concentration of the lactone obtained in the presence of Struktol ® (990 mg / L) East 39
• the TTT vegetable fat mixture has been tested as an in situ extraction system for ⁇ -decalactone.
• a culture of initial cell density of 4 provided with TTT (5 g / vial) is added with 0.18% (v / v) of RM, 0.01% (v / v) of Tween 20® and 0.01 (v / v) of Struktol ® .
• Lee SJ, Lin SJ and Chou CC "Growth of and production of ⁇ -décalactone by Sporobolomyces odorus in jar fermentor as affected by pH, aeration and fed-batch technique", Journal of fermentation and bioengineering, 82 (1), 195- 199 (1995).
• Dufossé L.H. et al Strategies to overcome toxicity during favor production by micro-organisms: the case of ⁇ -décalactone from Sporidiobolus salmonicolor. International Symposium on Flavors and Sensory Related Aspects March 6-7, 1997.

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