Process for the purification of vanillin or its derivatives obtained by a biotechnological process

Abstract

Title: Process for purifying vanillin or its derivatives obtained by a biotechnological process The present invention relates to a process for purifying a fermentation mash (M), obtained by a biotechnological process, comprising biomass and vanillin or its derivatives in aqueous solution, for the preparation of a crystallized vanillin or its derivatives, characterized in that throughout the purification process, the vanillin or its derivatives, in protonated or salified form, remain in aqueous solution.

Description

Title of the invention: Process for the purification of vanillin or its derivatives obtained by a biotechnological process Field of the invention

[0001] The present invention relates to a process for the purification of vanillin or its derivatives obtained by a biotechnological process. Prior art

[0002] Vanillin can be obtained by different methods known to those skilled in the art, and in particular by the following two routes: • A so-called natural route based on a biotechnological process comprising in particular the culture of a microorganism capable of allowing the biotransformation of a fermentation substrate into vanillin. Such a process is known in particular from application EP0885968 in which the fermentation substrate is ferulic acid. US patent 5017388 describes a process in which the fermentation substrate is eugenol and / or isoeugenol. These processes result in the preparation of a vanillin known as natural vanillin. • A so-called synthetic route comprising classic chemical reactions from guaiacol not involving microorganisms. This process results in the preparation of a vanillin called synthetic vanillin.

[0003] Finally, vanillin can also be prepared according to a route described as "bio-based" in which the vanillin is derived from lignin. In particular, mention may be made of documents US 2745796, DE1132113 and the article entitled "Preparation of lignin from wood dust as vanillin source and comparison of different extraction method" by Azadbakht et al in International Journal of Biology and Biotechnology, 2004, vol 1, No 4, pp 535-537.

[0004] Currently, natural vanillin can be purified according to the process described in application EP 2791098, which includes a liquid / liquid extraction step of impurities having a higher pKa than that of vanillin. The yield of this process is good, generally greater than 80%, however, in order to obtain improved qualities with regard to organoleptic properties such as the odor and / or color of vanillin, several additional purification steps are necessary, thus reducing the overall yield of this process. The energy efficiency of this process is also generally degraded due to the use of large quantities of solvents.

[0005] International application WO 2014 / 114590 also describes a process for purifying natural vanillin. This process consists of evaporating the natural vanillin, this evaporation being able to be carried out by distillation or by vacuum evaporation of melted vanillin. This process is likely to produce very pure natural vanillin, with good yield, with a simple to implement device and operating continuously to be compatible with industrial processes. However, such a process could be difficult to implement in practice due to the number and size of equipment required.

[0006] Furthermore, vanillin or its derivatives obtained by a biotechnological process may contain certain impurities having boiling points very close to vanillin or its derivatives. Thus it is necessary to size the equipment appropriately in order to allow efficient separation of vanillin or its derivatives from these products. This generally involves an extension of the residence time in the distillation equipment which can generate new impurities due to the instability at high temperature of vanillin or its derivatives and / or impurities.

[0007] This is why it would be advantageous to have an improved process compared to those proposed in the prior art, particularly in terms of environmental and / or energy impact while improving the overall purification efficiency as well as the yield of vanillin or its derivatives. It is also important that the process for purifying vanillin or its derivatives makes it possible to produce vanillin or its derivatives whose organoleptic properties are preserved, particularly in terms of taste, color and / or odor.

[0008] Brief description

[0009] The present invention relates to a process for purifying a fermentation must (M), obtained by a biotechnological process, comprising biomass and vanillin or its derivatives in aqueous solution, for the preparation of a crystallized vanillin or its derivatives, characterized in that throughout the purification process, the vanillin or its derivatives in protonated or salified form remain in aqueous solution. Description of the figures

[0010] Figures 1 and 2 show diagrammatically different processes for purifying vanillin or its derivatives obtained by a biotechnological process according to the present invention. Detailed description

[0011] In the context of the present invention, and unless otherwise indicated, the expression "between .. .and ..." includes the limits.

[0012] In the present invention, the expression "natural vanillin" designates a vanillin obtained by a biotechnological process. Thus, a process for preparing natural vanillin designates here a biotechnological process comprising the culture of a microorganism capable of allowing the transformation of a fermentation substrate into vanillin. The microorganism may be of wild origin or be a genetically modified microorganism (GMM) obtained by molecular biology. Very preferably Specifically, it may be a ferulic acid fermentation process, such as that described in patent application EP 0885968. In a particular aspect, vanillin may be produced by a glucose or protocatechuic acid fermentation process as described in patent application WO 2013 / 022881.

[0013] In the context of the present invention, the term “vanillin derivative” refers to any compound which can be derived from vanillin and in particular to vanillin in salified form or to glucovanillin.

[0014] In the context of the present invention, the term “vanillic alcohol derivative” refers to any compound which can be derived from vanillic alcohol and in particular to vanillic alcohol in salified form or to the glucoside of vanillic alcohol.

[0015] In the context of the present invention, the term “crystallization” refers to a process in which a substance passes into the solid state by a physical process, such as in particular lowering the temperature.

[0016] In the context of the present invention, the term “precipitation” refers to a process in which a substance passes into the solid state by a chemical transformation, such as in particular by protonation thanks to a change in pH.

[0017] In the context of the present invention, the term “vanillin” or “vanillin in protonated form” designates 4-hydroxy-3-methoxybenzaldehyde.

[0018] [Chem.l] OH .To. (Ws

[0019] The term “vanillin in salified form” refers to the salts of 4-hydroxy-3-methoxybenzaldehyde whose formula is defined below, in which the Metal is chosen from sodium, potassium.

[0020] [Chem.2] Put* it

[0021] The present invention relates to a process for purifying a fermentation must (M), obtained by a biotechnological process, comprising biomass and vanillin or its derivatives in aqueous solution, for the preparation of a crystallized vanillin or its derivatives, characterized in that, throughout the purification process, the vanillin or its derivatives in protonated or salified form remain in aqueous solution.

[0022] According to the present invention, the method for purifying a fermentation must (M) comprises a step (a) of separating the biomass from the biomass of a fermentation must (M) from an aqueous stream (Al) comprising vanillin or its derivatives.

[0023] This step is a step of separating a solid phase: the biomass, from a liquid phase. The liquid phase obtained at the end of the biomass separation step is an aqueous stream (Al) comprising vanillin or its derivatives, in salified or protonated form.

[0024] According to one aspect, the biomass separation step can be carried out by filtration such as frontal filtration or tangential filtration, in particular membrane filtration, such as microfiltration, ultrafiltration, nanofiltration, or reverse osmosis. The membrane filtration can be carried out by concentration or diafiltration. Advantageously, when the biomass separation step is frontal filtration, it is possible to add adjuvants in order to improve the efficiency of the filtration.

[0025] In order to improve the efficiency of solid / liquid separation it is possible to carry out several solid / liquid separation stages, in particular to remove the smallest solid particles.

[0026] According to a particular aspect, the step of separating the biomass can be carried out by one or more microfiltration membranes, in particular having a cutting limit of approximately 0.2 μm, followed by one or more ultrafiltrations having a cutting limit smaller than that of the microfiltration. In this configuration, the vanillin or its derivatives are solubilized in the aqueous phase and the biomass is retained by the membranes. Advantageously, ultrafiltration also allows the separation of molecules solubilized in the fermentation must.

[0027] In order to improve the yield of vanillin or its derivatives in the aqueous phase, a solvent, preferably water, is added during the filtration step. Generally, the amount of solvent added is between 0.5 and 5 equivalents by volume of fermentation must.

[0028] According to one aspect, microfiltration, ultrafiltration or diafiltration can be coupled to a nanofiltration or reverse osmosis step. Indeed, nanofiltration makes it possible to increase the vanillin concentration in the retentate of the nanofiltration step while the water passes through the membrane (permeate of the nanofiltration step). The permeate of this nanofiltration step can advantageously be recycled. Generally, reverse osmosis has a cutting limit less than or equal to 100 Da. Generally, nanofiltration has a cutting limit less than or equal to 400 Da. Generally, nanofiltration has a cutting limit greater than or equal to 100 Da, for example a cutting limit between 100 and 250 Da. Step (aO)

[0029] Prior to this step of separating the biomass from the aqueous solution of vanillin or its derivatives, the fermentation must may be stabilized. Thus, the process of the present invention may optionally comprise a step (aO) of stabilizing the fermentation must (M). Step (a) of the process according to the present invention may be carried out on a stabilized fermentation must (M).

[0030] In the context of the present invention, the term "stabilization" refers to any method making it possible to avoid degradation, in particular by reduction, of vanillin or its derivatives between the end of fermentation and the purification process.

[0031] According to a first aspect, the stabilization can be carried out by the addition of at least one compound. The compound is preferably chosen from sodium benzoate, ascorbic acid and its salts, potassium, calcium or sodium sorbate, zinc sulfate, propanoic acid, acetic acid or its salts, sodium diacetate. Preferably the amount of compound added is between 0.2 g / L and 6 g / L.

[0032] According to another aspect, the stabilization can be carried out by changing the temperature of the fermentation must. In general, the temperature is controlled so as to reach a temperature between 15°C and 23°C, preferably between 18°C ​​and 21°C.

[0033] According to another aspect, the stabilization can be carried out by changing the pH of the fermentation must. In general, the pH is controlled so as to achieve a pH less than or equal to 7.5, preferably less than or equal to 7, very preferably less than or equal to 6.8. In general, the pH is controlled so as to achieve a pH greater than or equal to 5.0, preferably greater than or equal to 6.

[0034] According to another aspect, the fermentation must may also be pasteurized. In general, the fermentation must is then heated to a temperature between 50°C and 90°C, preferably between 60°C and 80°C. Heating is generally maintained for a period of between 10 min and 120 min, preferably between 15 min and 45 min, for example for 20 min.

[0035] According to another aspect, the fermentation must can be stabilized by ultrasound. In general, ultrasound is emitted into the fermentation must for a period of between 10 min and 120 min.

[0036] According to a particular aspect, these aspects can be carried out separately or jointly, thus, by way of non-limiting example, it is possible to modify the temperature, the pH and to add a compound according to the conditions described above.

[0037] Among these methods, methods without adding compounds are advantageous, without wanting to be bound by any theory, they make it possible to avoid possible deleterious effects on vanillin or its derivatives, particularly in terms of odor or color.

[0038] However, the addition of certain compounds chosen for their compatibility with vanillin or its derivatives and the absence of side effects on vanillin or its derivatives may prove particularly advantageous in facilitating the continuation of the process of purification of vanillin or its derivatives.

[0039] According to one aspect of the present invention, the aqueous stream (Al) comprising vanillin or its derivatives is subjected to at least one filtration step such as frontal filtration or tangential filtration, in particular membrane filtration, such as microfiltration, ultrafiltration, nanofiltration, or reverse osmosis. The membrane filtration can be carried out by concentration or by diafiltration.

[0040] Generally, the filtration can be carried out with a ceramic membrane, preferably having a cutting limit of approximately 0.2 μm.

[0041] The filtrate obtained is an aqueous stream (A2) comprising vanillin or its derivatives, in protonated or salified form, in which impurities have been separated.

[0042] The filtrate can then be subjected to at least one nanofiltration or reverse osmosis step in order to concentrate the aqueous flow (A2) comprising vanillin. Indeed, nanofiltration makes it possible to increase the concentration of vanillin or its derivatives in the retentate of the nanofiltration step while the water passes through the membrane (permeate of the nanofiltration step). The permeate of this nanofiltration step can advantageously be recycled. Generally, reverse osmosis has a cutting limit less than or equal to 100 Da. Generally, nanofiltration has a cutting limit less than or equal to 400 Da. Generally, nanofiltration has a cutting limit greater than or equal to 100 Da, for example a cutting limit between 100 and 250 Da.

[0043] The process of the present invention comprises at least one step of crystallization of vanillin or its derivatives contained in the aqueous stream (A2).

[0044] The crystallization step makes it possible to purify an aqueous stream (A2) comprising vanillin or its derivatives. Preferably, the aqueous stream (A2) has a purity of between 85% and 99%, preferably greater than or equal to 90%, very preferably greater than or equal to 95%. Advantageously, the crystallization step makes it possible to produce a vanillin or its purified derivatives having a purity of greater than or equal to 95%, preferably greater than or equal to 97%, very preferably greater than or equal to 99%. The vanillin or its derivatives having a purity of between 85% and 99% may further comprise at least one other compound chosen from vanillic alcohol or its derivatives, vanillic acid, guaiacol, acetovanillone, 4-((4-hydroxy-3-methoxy-benzyl)oxy)-3-methoxybenzaldehyde and 4-hydroxy-3-(4-hydroxy-3-methoxybenzyl)-5-methoxybenzaldehyde.

[0045] The crystallization is generally carried out in an alcoholic solution. Preferably, the solvent used for the crystallization may be an alcohol soluble with water, preferably ethanol. The solvent used for the crystallization may be a water / alcohol mixture. In general, the amount of alcohol is between 2% and 40% by weight, preferably between 5% and 35% by weight, and very preferably between 15% and 25% by weight. Generally, during crystallization, the concentration of vanillin or its derivatives, at the start of crystallization, is between 5% and 60% by weight, preferably between 10% and 50% by weight, advantageously between 15% and 35% by weight, and even more preferably between 15% and 25%.Very advantageously, crystallization allows the separation of vanillin or its derivatives from the impurities contained in the aqueous flow (A2), this separation is advantageously carried out without degradation of the vanillin or its derivatives. Crystallization is carried out at a temperature between 0°C and 50°C. The crystallization yield is generally greater than or equal to 80%.

[0046] Advantageously, the process of the present invention does not require any organic solvent except for the crystallization solvent.

[0047] Alternatively, the aqueous stream (A2) may be subjected to a precipitation step. The precipitation of vanillin or its derivatives is generally carried out at a temperature between 0°C and 6°C. During the precipitation, the pH of the aqueous stream (A2) may be adjusted to reach a value between 5 and 8. A precipitate (P) of vanillin is obtained.

[0048] The precipitate (P) can be subjected to at least one crystallization step as described above.

[0049] Vanillin or its derivatives obtained at the end of the precipitation and / or crystallization step generally has a color in ethanolic solution at 10% by weight less than or equal to 150 Hazen, preferably less than or equal to 100 Hazen, and very preferably less than or equal to 50 Hazen.

[0050] According to another aspect of the purification process, the aqueous stream (Al) comprising the Vanillin or its derivatives, in salified or protonated form, is subjected to a liquid-liquid extraction step. During the liquid-liquid extraction step, vanillin or its derivatives in salified form is washed in an aqueous stream (A2). The impurities contained in the aqueous stream (Al) are extracted into organic solution (01).

[0051] Generally, the pH of the aqueous solution is greater than 7, preferably greater than 7.5, very preferably greater than 8.

[0052] Generally, the extraction solvent is chosen for its properties of solubilizing the impurities contained in the aqueous stream (Al), advantageously the solvent can be of biosourced origin. Preferably, the solubility of vanillin or its derivatives in protonated or salified form is better in water than in the extraction solvent. According to one aspect, the chosen solvent is compatible with the standards of the food industry, in particular FEMA GRAS, immiscible with water. Preferably the solvent is chosen from methyl ethyl ketone, methyl isobutyl ketone, ethyl acetate, isopropyl acetate or their mixtures.

[0053] The liquid / liquid extraction step may be a discontinuous liquid-liquid extraction. In order to maximize the amount of vanillin or its derivatives obtained in the aqueous phase (A2), the volume ratio of solvent relative to the aqueous stream (A1) comprising vanillin or its derivatives is between 1:5 and 5:1, preferably between 1:1 and 5:1, preferably between 1.5:1 and 3:1.

[0054] According to another aspect, the liquid / liquid extraction step can be carried out continuously. Generally, the volume ratio of solvent relative to the aqueous stream (Al) comprising vanillin or its derivatives is between 5:1 and 1:2, preferably between 3:1 and 1:1.

[0055] The yield of vanillin or its derivatives from the liquid / liquid extraction step is generally greater than or equal to 95%, preferably greater than or equal to 97%, very preferably greater than or equal to 98%.

[0056] According to a particular aspect, the steps of separating the biomass and of liquid / liquid extraction can be carried out simultaneously. This particular aspect makes it possible to wash the vanillin or its derivatives in protonated or salified form in the aqueous stream (A2) from the fermentation must. The biomass is then separated from the two-phase system. This method is advantageous in that the loss of vanillin or its derivatives in the biomass is reduced. This separation step can in particular be carried out continuously by centrifugation, in particular by using a two- or three-phase centrifuge or a countercurrent extractor.

[0057] The concentration (C) of the aqueous stream (A2), comprising vanillin or its derivatives, obtained at the end of the liquid / liquid extraction step is generally between 0.1% by weight and 10% by weight.

[0058] The aqueous stream (A2) can then be subjected to a crystallization step such that described previously.

[0059] Prior to the crystallization step, the aqueous stream (A2) comprising vanillin or its derivatives may be concentrated so that the concentration of vanillin or its derivatives in the aqueous stream is between 5% and 60% by weight.

[0060] Alternatively, the aqueous stream (A2) may be subjected to a precipitation step. The precipitation of vanillin or its derivatives is generally carried out at a temperature between 0°C and 6°C. During the precipitation, the pH of the aqueous stream (A2) may be adjusted to reach a value between 5 and 8. A precipitate (P) of vanillin or its derivatives is obtained.

[0061] The precipitate (P) can be subjected to at least one crystallization step as described above.

[0062] In the context of the present invention, prior to the crystallization and / or precipitation steps, the aqueous stream (A2) can optionally be treated on carbon or on resin, as described in particular in application WO 2011 / 039331.

[0063] Advantageously, the vanillin or its derivatives, obtained at the end of the process according to the present invention, generally has a color in ethanolic solution at 10% by weight less than or equal to 150 Hazen, preferably less than or equal to 100 Hazen, and very preferably less than or equal to 50 Hazen. Furthermore, the vanillin or its derivatives obtained at the end of the purification process according to the present invention has compliant organoleptic properties. Examples

[0064] Either a stabilized fermentation must as defined in Table 1 is subjected to a purification process as described below.

[0065] Table 1: Composition of the fermentation must

[0066] [Tables 1] Biomass 1.5% by weight Vanillin 2% by weight Water 95% by weight Other compounds 1.5% by weight

[0067] The biomass is separated from the aqueous phase by tangential diafiltration on a ceramic membrane with a pore diameter of 0.2 μm. The addition of 2 dia-volumes makes it possible to obtain a vanillin yield of >95%.

[0068] The aqueous solution obtained, separated from the biomass, is salified to pH=8.5 then concentrated by reverse osmosis. This last vanillin solution, in salified and concentrated form, is washed with its equivalent volume of isopropyl acetate in order to eliminate the non-salified organic compounds. Vanillin in salified form is then precipitated by reducing the pH to 5, then filtered and washed with water before being dried and ground.

[0069] The purity of the vanillin obtained is 98% and has good organoleptic properties.

Claims

Claims

1. Process for purifying a fermentation must (M), obtained by a biotechnological process, comprising biomass and vanillin or its derivatives in aqueous solution, for the preparation of a crystallized vanillin or its derivatives, characterized in that throughout the purification process, the vanillin or its derivatives in protonated or salified form remain in aqueous solution and comprising: - A step (a) of separating the biomass from the biomass of a fermentation must (M) from an aqueous stream (Al) comprising vanillin or its derivatives, and - At least one step in which the aqueous stream (Al) is subjected to a filtration step by nanofiltration or reverse osmosis characterized in that the reverse osmosis has a cutting limit less than or equal to 100 Da and the nanofiltration has a cutting limit less than or equal to 400 Da, preferably between 100 and 250 Da.

2. Purification method according to claim 1 further comprising a step (aO) of stabilizing the fermentation must (M).

3. Purification process according to any one of claims 1 to 2 characterized in that the aqueous flow (Al) comprising vanillin or its derivatives is subjected to at least one filtration step such as frontal filtration or tangential filtration, in particular membrane filtration, such as microfiltration, ultrafiltration, nanofiltration, or reverse osmosis to make it possible to obtain an aqueous flow (A2) comprising vanillin or its derivatives, in protonated or salified form.

4. Purification process according to any one of claims 1 to 2, characterized in that the aqueous stream (Al) comprising vanillin or its derivatives, in salified form, is subjected to a liquid-liquid extraction step to enable an aqueous stream (A2) to be obtained comprising vanillin or its derivatives, in protonated or salified form.

5. Purification process according to any one of claims 3 to 4, characterized in that said process comprises at least one step of crystallization of vanillin or its derivatives contained in the aqueous stream (A2).

6. A purification method according to any one of claims 3 to 5. characterized in that said process comprises at least one step of precipitation of vanillin or its derivatives contained in the aqueous stream (A2).

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