Method for recovering and purifying vanillin

Abstract

The present disclosure relates to a method for recovering and purifying vanillin from a microbial fermentation broth, wherein the fermentation broth contains vanillin complexes, such as vanillin glucosides, produced during microbial fermentation by microbial cells capable of producing and secreting the vanillin complexes.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 310,395, filed February 15, 2022, the contents of which are incorporated herein by reference in their entirety to the extent permitted by law.

[0002] Field The present disclosure relates to a method for recovering and purifying vanillin from a microbial fermentation broth, wherein the fermentation broth contains vanillin complexes, such as vanillin glucosides, produced during microbial fermentation by microbial cells capable of producing and secreting the vanillin complexes. [Background technology]

[0003] Summary of the Invention [Problem to be solved by the invention]

[0004] [Means for solving the problem]

[0005] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] 1) In a first embodiment, the present disclosure provides a method for recovering and purifying vanillin from a microbial fermentation broth, the fermentation broth comprising vanillin complexes produced during microbial fermentation by microbial cells capable of producing and secreting vanillin complexes, the method comprising: (I)(a) separating and removing the microbial cells from the fermentation broth so as to leave a liquid substantially free of the microbial cells, followed by converting the vanillin complexes contained in the liquid into vanillin and the corresponding complex partners; or (I)(b) converting vanillin complexes contained in the fermentation broth into vanillin and the corresponding complex partners, followed by separation and removal of the microbial cells from the fermentation broth so as to leave a liquid substantially free of microbial cells; after that, Step (II)(a) of adding a water-miscible polar organic solvent to the liquid obtained in step (I)(a) or (I)(b), the liquid obtained in step (I)(a) being preferably filtered either before or after the addition of the polar organic solvent; the vanillin solution produced in step (II)(a), (i) a cation exchange sorbent followed by a weakly basic anion exchange sorbent; or (ii) treating the sieved ... Optionally, decolorizing the resulting solution with an adsorbent (II)(c); or step (II')(a) of treating the liquid obtained in step (I)(a) or (I)(b) with a non-ionic adsorbent, in which the liquid obtained in step (I)(a) is preferably first filtered before the treatment with the non-ionic adsorbent, and in which the non-ionic adsorbent used in step (II')(a) is capable of adsorbing vanillin, the treatment comprising a step (i) of adsorbing vanillin under conditions which allow the vanillin to be bound to the non-ionic adsorbent, and a step (ii) of desorbing the bound vanillin into solution, The resulting vanillin solution, (i) a cation exchange adsorbent; (ii) a weakly basic anion exchange adsorbent; or (iii) step (II')(b) of treating with either both a cation exchange adsorbent and a weakly basic anion exchange adsorbent in either order; and Optionally, any of steps (II')(c) of decolorizing the resulting solution with an adsorbent; and and a step (III) of crystallizing vanillin from the resulting solution.

[0007] By "microbial cells" as used herein is meant prokaryotic or eukaryotic cells, preferably selected from bacteria, fungi and especially yeasts.

[0008] The term "vanillin" refers to the compound having the chemical name 4-hydroxy-3-methoxybenzaldehyde.

[0009] The term "vanillin complex" refers to vanillin covalently bound to a further molecular entity (herein referred to as "complex partner"), which may be any molecular entity suitable for production and secretion by a microbial cell in the form of a vanillin complex containing such molecular entity, which can be separated from the vanillin by conversion of the vanillin complex to vanillin and the corresponding complex partner. Complex partners include, but are not limited to, sugars, such as polysaccharides, disaccharides, or monosaccharides, preferably monosaccharides, such as very preferably D-glucose. In a very preferred embodiment, the term "vanillin complex" therefore refers to "vanillin glucoside".

[0010] The term "vanillin glucoside" refers to the compound "vanillin 4-O-β-D-glucoside," which is also called "vanillin β-D-glucoside."

[0011] Microbial cells useful according to the invention can be prokaryotic or eukaryotic cells capable of producing and secreting vanillin complexes, such as, in particular, vanillin glucoside, into the fermentation medium, such as cells genetically engineered to produce and secrete vanillin complexes, such as, in particular, vanillin glucoside. Such genetically engineered microbial cells are described, for example, in WO2004 / 111254, Hansen et al., Appl. Environ. Microbiol. 75(9):2765-2774 (2009), WO2013 / 022881, WO2015 / 009558, and WO2021 / 022216.

[0012] The separation and removal of the microbial cells from the fermentation broth in step (I)(a) or (I)(b) so that a liquid substantially free of microbial cells remains can be carried out by any suitable separation technique. Typically, this will be achieved by centrifugation and subsequent filtration or by membrane separation techniques. Suitable membrane separation techniques are either microfiltration, ultrafiltration, nanofiltration or a combination thereof, while suitable techniques for filtration after centrifugation include pressure filtration and vacuum filtration. When pressure filtration is used, this can be carried out using any suitable device, for example a candle filter or a filter press. Preferably, the filtration is carried out by the addition of a filter aid. The filter aid can either be added to the suspension to be filtered (e.g. 0.1-5% w / w) or placed in the filter as a precoat through which the liquid must pass. Any material consisting of solid particles that improves the filtration efficiency can be used. Preferably, the filtration material is based on cellulose, perlite or diatomaceous earth. In the case of membrane separation, preferably ultrafiltration is used. Biomass separation can be performed by ultrafiltration using membranes with a nominal molecular weight cut-off (MWCO) in the range of 1-100 kDa, preferably 1-10 kDa. The flux rates and / or yields used through these various membranes can be similar. However, a smaller molecular weight cut-off may be preferred since it removes more impurities. On the other hand, finer ultrafiltration devices require more frequent cleaning and process shutdowns, so a balance is struck in the selection of the optimum molecular weight cut-off to obtain acceptable purity without compromising the economics of the process due to frequent shutdowns for cleaning. Preferably, composite fluoropolymer membranes are used, such as ETNA01PP (1 kDa MWCO) or ETNA10PP (10 kDa MWCO) from Alfa Laval. Diafiltration can be performed with 1-2 or more, preferably 1.6, volumes of demineralized water relative to the volume of concentrated broth (retentate).

[0013] The conversion of the vanillin conjugates in step (I)(a) or (I)(b) to vanillin and the corresponding conjugated partners can be carried out either chemically (e.g., hydrolysis) or enzymatically, preferably using a suitable enzyme under suitable conditions. Such enzymes, e.g., β-glucosidases capable of catalyzing the conversion of vanillin glucoside to vanillin and glucose, are known in the art. In a preferred embodiment, the conversion of vanillin glucoside to vanillin and glucose is carried out at a pH of about 3.0 to 6.5, preferably 4.5 to 5.5, and at a temperature of about 30 to 60° C., preferably 50 to 55° C., for preferably 20 to 48 hours, using 0.002 to 0.1 g of β-glucosidase having an activity of about 10 enzyme units (u) / g per gram of vanillin glucoside (corresponding to about 20 to 1,000 u / kg of vanillin glucoside), preferably 0.03 to 0.07 g / g (corresponding to about 300 to 700 u / kg of vanillin glucoside).

[0014] Optionally, the liquid is concentrated prior to conversion of the vanillin complex to vanillin and the corresponding complex partner in step (I)(a). The liquid can be concentrated by a volumetric concentration factor (VCF) of 2 or more. Preferably, the concentration is carried out by reverse osmosis or wiped film evaporator. Concentration can be achieved by reverse osmosis at temperatures of about 10-50° C. or evaporation at temperatures of about 50-80° C., preferably about 60-65° C. Thin film composite membranes can be used (e.g. Alfa Laval RO98pHt) consisting of thin film composite polymer on polypropylene with a rejection of 98% or greater measured at 2000 ppm NaCl at 25° C. and 16 barg. Alternatively, Alfa Laval RO99 membranes can be used (e.g. Alfa Laval RO99) consisting of thin film composite polymer on polyester with a rejection of 98% or greater measured at 25° C. and 9 barg with 2000 ppm NaCl.

[0015] Suitable polar organic solvents used in step (II)(a) are organic solvents or mixtures of different organic solvents that are miscible with water, enhance the solubility of vanillin and are compatible for use with the ion exchange sorbent used in step (II)(b). In a preferred embodiment, the polar organic solvent is an alcohol or a mixture of different alcohols, preferably linear or branched, containing 1 to 6 carbon atoms, such as unsaturated or especially saturated primary alcohols, such as ethanol and propanol, such as 2-propanol. In a particularly preferred embodiment, the polar organic solvent is ethanol or 2-propanol, most preferably ethanol. The amount of polar organic solvent added to the liquid from step (I)(a) or (I)(b) is preferably such that there is at least about 10%, 20%, 30%, 40%, 50%, 60%, or 70% v / v polar organic solvent relative to the total volume of the resulting solution. Highly preferably, the polar organic solvent constitutes about 20-50% v / v, especially about 20-40% v / v, most preferably about 20% v / v of the resulting solution.

[0016] Unless used in reference to temperature, the term "about" preceding a numerical value "X" preferably refers in this application to an interval extending from 10% of X minus X to 10% of X plus X. In the specific case of temperature, the term "about" preceding a temperature "Y" preferably refers in this application to an interval extending from temperature Y minus 5°C to Y plus 5°C.

[0017] The filtration according to step (II)(a) or (II')(a) can be carried out by any suitable filtration technique, for example pressure filtration or vacuum filtration. Preferably, the filtration is carried out by the addition of a filter aid. The filter aid can either be added to the suspension to be filtered (for example 0.1-1% w / w) or placed on the filter as a precoat through which the liquid must pass. Any material consisting of solid particles that improves the filtration efficiency can be used. Preferably, the filtration material is based on cellulose, perlite or diatomaceous earth.

[0018] Any suitable cation exchange adsorbent can be used in step (II)(b) or (II')(b). It can be a weak acid or strong acid cation exchange adsorbent. Preferably, the adsorbent used is a resin. The cation exchange adsorbent is preferably a strong acid cation exchange adsorbent, such as a particularly strong acid cation exchange resin, preferably in H-form. Preferably, a macroporous resin is used. For example, macroporous polystyrene-based resins and polystyrene-based gel resins can be used, such as macroporous polystyrene resins with sulfonic acid as functional groups crosslinked with divinylbenzene.

[0019] Any suitable weakly basic anion exchange adsorbent can be used in step (II)(b) or (II')(b). Preferably, the weakly basic anion exchange adsorbent is a weakly basic anion exchange resin, such as a particularly weakly basic anion exchange macroporous resin, in particular a weakly basic anion exchange macroporous resin having either a polystyrene or polyacrylic acid ester frame and primary to tertiary amino groups as functional groups. Macroporous polystyrene-based resins are preferred, but polystyrene-based gel resins can also be used. For example, gel-type resins having polyacrylic crosslinked with divinylbenzene having tertiary amines as functional groups are suitable. In a highly preferred embodiment, the weakly basic anion exchange adsorbent, such as a particularly weakly basic anion exchange resin, is converted to Ac-form (acetate-form) before use. To do so, it can be treated with a solution of 5% acetic acid, washed with demineralized water and preconditioned with the feed solvent.

[0020] Any suitable non-ionic (neutral) adsorbent can be used in step (II')(a). Preferably, the non-ionic adsorbent is a non-ionic resin, especially a macroporous non-ionic resin, especially a hydrophobic resin. Polystyrene-based, polyphenol-based, or polymethacrylic-based non-ionic resins can be used. For example, macroporous polydivinylbenzene resins without functional groups are suitable.

[0021] Adsorption of vanillin under conditions allowing binding of vanillin to the non-ionic adsorbent in step (II')(a)(i) can be carried out by pumping the solution through a column containing the non-ionic adsorbent, preferably at a flow rate of not more than 4 BV / h.

[0022] The desorption of the bound vanillin into the solution in step (II')(a)(ii) can be carried out by pumping, at a flow rate of preferably not more than 4 BV / h, for example 2-4 BV, preferably 3 BV, of eluent through the column containing the non-ionic adsorbent. Suitable eluents for desorption are organic solvents, mixtures of different organic solvents, mixtures of water and organic solvents or mixtures of water and different organic solvents, provided that such eluents increase the solubility of vanillin compared to water alone and are compatible for use with the non-ionic adsorbent used in step (II')(a). In a preferred embodiment, the organic solvent is an alcohol, or a mixture of different alcohols, preferably linear or branched, containing 1 to 6 carbon atoms, such as unsaturated or especially saturated primary alcohols, such as ethanol and propanol, for example 2-propanol. In a particularly preferred embodiment, the organic solvent is ethanol or 2-propanol, most preferably ethanol. The amount of organic solvent is preferably at least about 40%, 50%, 60% or 70% v / v of the total volume of the eluent. Highly preferably, the organic solvent constitutes about 50-90% v / v, in particular about 70-80% v / v, and most preferably about 80% v / v of the total volume of the eluent. In a highly preferred embodiment, the eluent is aqueous ethanol with a concentration of ethanol of 50-90% v / v, in particular about 70-80% v / v, and most preferably about 80% v / v.

[0023] The adsorbent used in optional step (II)(c) or (II')(c) to decolorize the solution can be carbon, in particular activated carbon, preferably granular activated carbon. The solution can be pumped through the carbon column, for example, at a flow rate of up to 4 BV / h and at a temperature of about 15-25°C. The carbon requirement is preferably about 0.05-2 kg, more preferably about 0.3-1 kg, most preferably about 0.5 kg of carbon per kg of vanillin.

[0024] Before carrying out the crystallization of step (III), the vanillin solution can be polished by filtration to remove any traces of the adsorbent. Such filtration can be carried out using a suitable filter, preferably having a pore size of 1 μm or less.

[0025] The crystallization in step (III) can be carried out by concentrating the vanillin solution, for example to about 10-60%, preferably about 25-35%, total dry substance, for example by evaporation under reduced pressure, for example at 0.1-0.4 barg and at a temperature of about 50-55° C. The crystallization of vanillin is preferably carried out at a pH of about 3.5-5.5, preferably about 4-5.5. At the end of the evaporation, the vanillin solution can be cooled, for example to about 25-35°C, to initiate crystallization. If no crystallization occurs, seeding with pure vanillin can be performed. If not more than about 7% v / v of organic solvent is present in the vanillin solution after concentration by evaporation, which usually leads to spontaneous crystallization after cooling of the concentrated solution, for example to about 25-35°C, seeding can be avoided.

[0026] The present disclosure provides the following further embodiments.

[0027] 2) In one embodiment, the present disclosure relates to a method according to embodiment 1), wherein the vanillin complex is vanillin glucoside.

[0028] 3) In another embodiment, the present disclosure relates to a method according to embodiment 1) or 2), wherein the microbial cell is a fungal cell.

[0029] 4) In another embodiment, the present disclosure relates to a method according to embodiment 3), wherein the fungal cell is a yeast cell.

[0030] 5) In another embodiment, the present disclosure relates to a method according to embodiment 4), wherein the yeast cell is selected from Saccharomyces cerevisiae.

[0031] 6) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 5), wherein the separation and removal of the microbial cells from the fermentation broth in step (I)(a) or (I)(b) such that a substantially microbial cell-free liquid remains is carried out by centrifugation and subsequent filtration or by membrane separation methods.

[0032] 7) In another embodiment, the present disclosure relates to a method according to embodiment 6), wherein the separation and removal of microbial cells is carried out by centrifugation followed by filtration.

[0033] 8) In another embodiment, the present disclosure relates to a method according to embodiment 6), wherein the separation and removal of microbial cells is carried out by ultrafiltration.

[0034] 9) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 8), wherein the conversion of the vanillin conjugate in step (I)(a) or (I)(b) to vanillin and the corresponding conjugated partner is carried out either by chemical conversion (e.g. hydrolysis) or by enzymatic conversion.

[0035] 10) In another embodiment, the present disclosure relates to a method according to embodiment 9), wherein the conversion of the vanillin conjugate to vanillin and the corresponding conjugated partner is carried out by enzymatic conversion.

[0036] 11) In another embodiment, the present disclosure relates to a method according to embodiment 10), wherein the vanillin complex is vanillin glucoside and the enzymatic conversion is carried out by using β-glucosidase as the enzyme.

[0037] 12) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 11), wherein the method comprises step (I)(a).

[0038] 13) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 12), wherein prior to the conversion of the vanillin glucoside complex to vanillin and the corresponding complex partner in step (I)(a), the liquid is concentrated, preferably by a volumetric concentration factor of 2 or more.

[0039] 14) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 13), wherein the liquid obtained in step (I)(a) is filtered either before or after the addition of the polar organic solvent in step (II)(a).

[0040] 15) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 13), wherein the liquid obtained in step (I)(a) is first filtered prior to treatment with a non-ionic adsorbent in step (II')(a).

[0041] 16) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 11), wherein the method comprises step (I)(b).

[0042] 17) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 14) and 16), wherein the method comprises steps (II)(a) and (II)(b), and optionally step (II)(c).

[0043] 18) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 14) and 16) to 17), wherein the polar organic solvent used in step (II)(a) is an alcohol or a mixture of different alcohols, preferably linear or branched and containing 1 to 6 carbon atoms.

[0044] 19) In another embodiment, the present disclosure relates to a method according to embodiment 18), wherein the alcohol is ethanol or propanol (in particular 2-propanol), preferably ethanol.

[0045] 20) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 14) and 16) to 19), wherein the cation exchange adsorbent used in step (II)(b) is a strong acid cation exchange adsorbent, in particular a strong acid cation exchange resin, preferably in H-form and preferably a macroporous resin.

[0046] 21) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 14) and 16) to 20), wherein the weakly basic anion exchange adsorbent used in step (II)(b) is a weakly basic anion exchange resin, in particular a weakly basic anion exchange macroporous resin.

[0047] 22) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 14) and 16) to 21), wherein the weakly basic anion exchange adsorbent used in step (II)(b) is converted to acetate form prior to use.

[0048] 23) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 14) and 16) to 22), wherein the method comprises step (II)(b)(i).

[0049] 24) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 14) and 16) to 22), wherein the method comprises step (II)(b)(ii).

[0050] 25) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 14) and 16) to 24), wherein step (II)(c) is part of the method.

[0051] 26) In another embodiment, the present disclosure relates to a method according to embodiment 25), wherein the adsorbent used in step (II)(c) is carbon, in particular activated carbon, preferably granular activated carbon.

[0052] 27) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 13) and 15) to 16), wherein the method comprises steps (II')(a) and (II')(b), and optionally step (II')(c).

[0053] 28) In another embodiment, the present disclosure relates to a method according to embodiment 27), in which the non-ionic adsorbent used in step (II')(a) is a non-ionic resin, in particular a macroporous non-ionic resin, preferably a hydrophobic resin.

[0054] 29) In another embodiment, the present disclosure relates to a method according to embodiment 27) or 28), wherein the desorption of the bound vanillin into solution in step (II')(a)(ii) is carried out using an organic solvent, a mixture of different organic solvents, a mixture of water and an organic solvent, or a mixture of water and different organic solvents as eluent.

[0055] 30) In another embodiment, the present disclosure relates to a method according to embodiment 29), wherein the eluent is an alcohol or a mixture of different alcohols, preferably linear or branched and containing 1 to 6 carbon atoms.

[0056] 31) In another embodiment, the present disclosure relates to a method according to embodiment 30), wherein the alcohol is ethanol or propanol (particularly 2-propanol), preferably ethanol.

[0057] 32) In another embodiment, the present disclosure relates to a method according to any one of embodiments 27) to 31), wherein the cation exchange adsorbent used in step (II')(b) is a strong acid cation exchange adsorbent, in particular a strong acid cation exchange resin, preferably H-type and preferably a macroporous resin.

[0058] 33) In another embodiment, the present disclosure relates to a method according to any one of embodiments 27) to 32), wherein the weakly basic anion exchange adsorbent used in step (II')(b) is a weakly basic anion exchange resin, in particular a weakly basic anion exchange macroporous resin.

[0059] 34) In another embodiment, the present disclosure relates to a method according to any one of embodiments 27) to 33), wherein the weakly basic anion exchange adsorbent used in step (II')(b) is converted to acetate form prior to use.

[0060] 35) In another embodiment, the present disclosure relates to a method according to any one of embodiments 27) to 32), wherein the method comprises step (II')(b)(i).

[0061] 36) In another embodiment, the present disclosure relates to a method according to any one of embodiments 27) to 31) and 33) to 34), wherein the method comprises step (II')(b)(ii).

[0062] 37) In another embodiment, the present disclosure relates to a method according to any one of embodiments 27) to 34), wherein the method comprises step (II')(b)(iii).

[0063] 38) In another embodiment, the present disclosure relates to the method according to any one of embodiments 37), wherein in step (II')(b)(iii), the vanillin solution is treated with a cation exchange adsorbent and then with a weakly basic anion exchange adsorbent.

[0064] 39) In another embodiment, the present disclosure relates to a method according to any one of embodiments 27) to 38), wherein step (II')(c) is part of the method.

[0065] 40) In another embodiment, the present disclosure relates to a method according to embodiment 39), wherein the adsorbent used in step (II')(c) is carbon, in particular activated carbon, preferably granular activated carbon.

[0066] 41) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 40), wherein the method does not include a purification step with a strongly basic anion exchange adsorbent.

[0067] 42) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 41), wherein the method occurs at a pH of less than 7, preferably less than 6.

[0068] 43) In another embodiment, the present disclosure relates to the method according to any one of embodiments 1) to 42), wherein before carrying out the crystallization of step (III), the vanillin solution is polished by filtration to remove any traces of the adsorbent.

[0069] 44) In another embodiment, the present disclosure relates to the method according to any one of embodiments 1) to 43), wherein the crystallization in step (III) comprises concentrating the obtained vanillin solution.

[0070] 45) In another embodiment, the present disclosure relates to a method according to any one of embodiments 1) to 44), wherein the crystallization of vanillin is carried out at a pH of about 3.5 to 5.5, preferably about 4 to 5.5. EXAMPLES

[0071] Abbreviations used herein BV Bed volume(s) DSP downstream process GC Gas Chromatography h hour(s) HPLC High Performance Liquid Chromatography UF Ultrafiltration VAN Vanillin v / v volume / volume w / w weight / weight

[0072] Example 1 Downstream Process A To analyze the complete downstream process of type A, a fermentation broth produced by a vanillin glucoside-producing yeast strain capable of producing and secreting vanillin glucoside was used. The vanillin glucoside-producing yeast strain used in this and the following examples is a Saccharomyces cerevisiae strain designed to contain a new synthetic pathway, e.g., as described in Hansen et al., Appl. Environ. Microbiol. 75(9):2765-2774 (2009), that allows the production and secretion of vanillin glucoside. The fermentation broth used in this and the following examples contained vanillin glucoside at a concentration of more than 10 g / L. The fermentation broth was centrifuged and the resulting supernatant was filtered by adding Dal-Cin Alfatex 101 as a filter aid. The resulting 4160 mL filtered solution contained vanillin glucoside and had a pH of 4.86. 0.46 g of β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10 u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: about 6.3%) per gram of vanillin glucoside was added to the solution and the solution was stirred at 50° C. for 48 hours. The formed slurry was filtered by adding Dal-Cin Alfatex 101 as a filter aid and diluted with ethanol to 20% v / v ethanol in water. Two laboratory-scale columns were connected in series: a laboratory-scale column packed with 300 mL of strong acid cation exchange Purolite C150SH resin (regenerated and activated with H2SO4 5%), followed by a laboratory-scale column packed with 300 mL of weak base anion exchange Purolite A845S resin (treated with acetic acid 5%). Then, 3090 mL of vanillin in a 20% v / v aqueous ethanol solution with pH 5.04 and a conductivity of 7.28 μS / cm was pumped in an upflow direction through the two columns starting with the strong acid cation exchange resin at a flow rate of 4.8 BV / h. Afterwards, the resin was washed with aqueous ethanol 20% v / v. The following fractions were collected and analyzed:

[0073] [Table 1]

[0074] 1.75 g of chemical granular activated carbon Chemviron Acticarbone BGE per g of vanillin was added to the combined main three fractions obtained in the previous step. The brown solution was stirred at room temperature for 3 hours and then filtered, giving a yellow solution after carbon treatment. The carbon was washed with 600 mL of ethanol under stirring at room temperature for 1 hour. Samples of each solution were filtered and analyzed. The vanillin yield of the carbon treatment was 81.89%. The charcoal treated solution and the washing fractions were combined, filtered through a 0.45 μm Nylon filter, and then concentrated under vacuum on a rotary evaporator in a water bath at 55° C. to 750 mL. The concentrate was cooled to room temperature and crystallization occurred. After stirring the slurry overnight at room temperature, the solid formed was isolated by vacuum filtration, washed with 300 mL pre-chilled demineralized water and dried under vacuum at 45° C. for 3 hours. An off-white to yellow vanillin was obtained with a purity of 97.49% w / w by GC and 97.45% w / w by HPLC.

[0075] Example 2 Downstream process A, without activated carbon treatment, crystallization at different pH values 3465 mL of fermentation broth containing vanillin glucoside, produced by vanillin glucoside-producing yeast strain, at pH 4.75, was used in the following experiments. The fermentation broth was centrifuged and the resulting supernatant was filtered with Dal-Cin Alfatex 101 as a filter aid. 2 L of the resulting solution with pH 4.83 was used for the enzymatic hydrolysis of vanillin glucoside. Per gram of vanillin glucoside, 0.26 g of β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10 u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: about 6.3%) was added to the solution, and the solution was stirred at 50° C. for 48 hours. The formed slurry was filtered by adding Dal-Cin Alfatex 101 as a filter aid and diluted with ethanol to 50% v / v ethanol in water. Two laboratory-scale columns were connected in series: a laboratory-scale column filled with 200 mL of strongly acidic cation exchange resin Dowex 50 WX4 200-400 mesh (regenerated and activated with H2SO4), followed by a laboratory-scale column filled with 200 mL of weakly basic anion exchange resin DuPont Amberlite FPA 53 (treated with acetic acid 5%). Then, 3.15 L of vanillin solution obtained in the previous step was pumped in the upflow direction through the two columns, starting with the strongly acidic cation exchange resin, at a flow rate of 4 BV / h. Afterwards, the resin was washed with aqueous ethanol 50% v / v. The following three fractions were collected and analyzed:

[0076] [Table 2]

[0077] The first fraction at pH 3.83 was used to analyze the crystallization of vanillin at different pH values. a) The pH of 500 mL of "fraction 1" was adjusted from pH 3.83 to 5.06 with NaOH 30%. The color of the solution did not change during the pH adjustment. The solution was then concentrated under vacuum on a rotary evaporator in a water bath at 55° C., in which black oily spots were observed. After cooling to room temperature, pure ethanol was added until the black spots were completely dissolved. Seeding was then performed with 30.34 mg of vanillin powder of ≥98% purity, and the solution was kept at 4° C. for 24 hours. The solid formed was then separated from the mother liquor by vacuum filtration, washed with 10 mL of pre-chilled water, and dried at 45° C. under vacuum for 3 hours. The vanillin yield of crystallization, filtration, washing of the wet solid, and drying was 88.47%. Brown crystalline vanillin was obtained with a purity of 98.62% w / w by HPLC.

[0078] b) The pH of 500 mL of "fraction 1" was adjusted from pH 3.83 to 7.20 with NaOH 30%. During the pH adjustment, the color of the solution changed from yellow to orange. The solution was concentrated under vacuum on a rotary evaporator in a water bath at 55°C, in which black oily spots were observed. After cooling to room temperature, pure ethanol was added until the black spots were completely dissolved. Seeding was then performed with 34.02 mg of vanillin of >98% purity, and the solution was kept at 4°C for 3 days. The solid formed was separated from the mother liquor by vacuum filtration, washed with 10 mL of pre-chilled water, and dried at 45°C under vacuum for 3 hours. The vanillin yield of crystallization, filtration, washing of the wet solid, and drying was 73.15%. A dark yellow vanillin was obtained with a purity of 99.14% w / w by HPLC.

[0079] c) The pH of 500 mL of "fraction 1" was adjusted from pH 3.83 to 8.57 with NaOH 30%. During the pH adjustment, the color of the solution became dark red and then changed to dark brown. The solution was concentrated under vacuum on a rotary evaporator in a water bath at 55° C. and, after cooling to room temperature, kept at 4° C. overnight. The solid formed was separated from the mother liquor by vacuum filtration, washed with 10 mL pre-chilled water and dried at 45° C. under vacuum for 3 hours. The vanillin yield of crystallization, filtration, washing of the wet solid and drying was 57.60%. Dark brown vanillin was obtained with a purity of 97.39% w / w by HPLC.

[0080] Example 3 Downstream Process A by using 2-propanol as the solvent 17.7 L of fermentation broth containing vanillin glucoside, produced by a vanillin glucoside-producing yeast strain, at pH 4.96, was used in the following experiments: the fermentation broth was ultrafiltered using an Alfa Laval TestUnit M20 with a UF 1 kDa Alfa Laval ETNA01PP membrane, diafiltered and concentrated to 4.90 L by reverse osmosis using an Alfa Laval RO98pHt membrane. Per gram of vanillin glucoside, 0.27 g of β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10 u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: approximately 6.3%) was added to 4900 mL of concentrated vanillin glucoside solution (pH 5.23), and the solution was stirred at 50° C. for 120 hours. The formed slurry was filtered in the presence of Dal-Cin Alfatex 101 as a filter aid and diluted with 2-propanol to a final concentration of 40% 2-propanol v / v. The resulting solution had a pH of 5.80. Two laboratory-scale columns were connected in series: a laboratory-scale column packed with 300 mL of the strong acid cation exchange resin Purolite C150SH (activated and regenerated with H2SO45%), followed by a laboratory-scale column packed with 300 mL of the weak base anion exchange resin Purolite A845S (treated with acetic acid 5%). Then, 900 mL of vanillin in aqueous 2-propanol 40% v / v solution was pumped in the upflow direction through the two columns starting with the strong acid cation exchange resin at a flow rate of 4 BV / h. The resin was then washed with aqueous 2-propanol 40% v / v. Four fractions were collected and analyzed.

[0081] [Table 3]

[0082] The three main fractions and the wash fractions obtained in the previous step were combined. Chemical granular activated carbon Chemviron Acticarbone BGE (0.59 kg BGE / 1 kg VAN) was added to the resulting solution and stirred in batches at room temperature for 3 hours. No significant color deterioration of the solution was observed. The carbon was then filtered and washed in batches with 133 mL of 40% v / v 2-propanol in water under stirring at room temperature for 1 hour. The main solution after carbon treatment was combined with the carbon wash fraction, filtered through a 0.45 μm Nylon filter and concentrated under vacuum on a rotary evaporator in a 55° C. water bath to 90 mL. The concentrate was cooled to room temperature with continuous stirring and then kept at 4° C. overnight. Seeding was performed by adding 40.99 mg of 99% pure vanillin, and crystallization occurred immediately. The solid formed was separated from the mother liquor by vacuum filtration, washed with 40 mL pre-chilled demineralized water and dried for 3 hours under vacuum at 45° C. A yellow vanillin was obtained with a purity of 92.75% w / w by HPLC and 96.18% w / w by GC.

[0083] Example 4 Downstream process A, not including activated carbon treatment 3465 mL of fermentation broth containing vanillin glucoside, pH 4.75, produced by the vanillin glucoside-producing yeast strain, was used in the following experiments: The fermentation broth was centrifuged and the resulting supernatant was filtered through Dal-Cin Alfatex 101 as a filter aid. Per gram of vanillin glucoside, 0.26 g of β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10 u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: approximately 6.3%) was added to 2 L of filtered supernatant (pH 4.83) and the solution was stirred for 48 hours at 50° C. The formed slurry was filtered by adding Dal-Cin Alfatex 101 as a filter aid and diluted with ethanol to 50% v / v ethanol in water. A laboratory-scale column was packed with 200 mL of strong acid cation exchange resin Dowex 50 WX4 200-400 mesh (activated with H2SO4), and 200 mL of ethanol filtrate was pumped through the column in an upflow direction at a flow rate of 1.5 BV / h. The resin was then washed with aqueous ethanol 50% v / v, and three different fractions were collected and analyzed.

[0084] [Table 4]

[0085] A laboratory-scale column was packed with 200 mL of weakly basic anion exchange resin DuPont Amberlite FPA 53 (treated with 5% acetic acid) and the three fractions obtained in the previous step were pumped separately, starting from the main fraction, through the resin column in an upflow direction at a flow rate of 7.2 BV / h. The resin was then washed with aqueous ethanol 50% v / v and four different fractions were collected and analyzed.

[0086] [Table 5]

[0087] The solution obtained by combining all the fractions obtained in the previous step was pumped through a 0.22 μm PTFE filter and 800 mL of the clear filtrate was concentrated under vacuum on a rotary evaporator in a water bath at 55 °C to 52 mL. The concentrate was cooled to room temperature and seeded with 24 mg of vanillin of >98% purity. Crystallization occurred immediately and the solution was kept at 4 °C overnight. The solid formed was separated from the mother liquor by vacuum filtration, washed with 10 mL of pre-cooled demineralized water and dried at 45 °C under vacuum for 3 hours. A yellow vanillin was obtained with a purity of 96.5% w / w by HPLC and 98.5% w / w by GC.

[0088] Example 5 Adsorption and release of vanillin onto / from a strongly basic anion exchange resin after treatment with a strongly acidic cation exchange resin and a weakly basic anion exchange resin 9.58 L of fermentation broth containing vanillin glucoside, produced by a vanillin glucoside-producing yeast strain, pH 5.01, was used in the following experiments: the broth was ultrafiltered using an Alfa Laval TestUnit M20 with a UF 1 kDa Alfa Laval ETNA01PP membrane, diafiltered and concentrated by reverse osmosis using an Alfa Laval RO98pHt membrane. The resulting 4950 mL concentrated vanillin glucoside solution had a pH of 5.03. 0.26 g of β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10 u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: about 6.3%) per gram of vanillin glucoside was added to the solution and the solution was stirred at 50°C for 48 hours. The formed slurry was filtered by adding Dal-Cin Alfatex 101 as a filter aid and then diluted with ethanol to a final concentration of 20% ethanol v / v. The pH of the resulting solution was 5.34. Two lab-scale columns were connected in series: a 350 mL lab-scale column packed with 300 mL of strong acid cation exchange resin Purolite C150SH (activated and regenerated with H2SO4 5%), followed by a 350 mL lab-scale column packed with 300 mL of weak base anion exchange resin Purolite A845S (treated with acetic acid 5%), and 1.17 L of clear filtrate from ethanol was pumped through the two resin columns in an upflow direction at a flow rate of 4 BV / h, starting from the strong acid cation exchange resin. Afterwards, both resin columns were washed with aqueous ethanol 20% v / v. Five different fractions were collected and analyzed.

[0089] [Table 6]

[0090] A laboratory-scale column packed with 45 mL of strongly basic anion exchange resin DuPont Amberlite FPA 42 (activated with NaOH 5% and washed with demineralized water) was loaded with the solution obtained by combining all 225 mL of the above fractions. After pumping the combined solution through the column at a flow rate of 4 BV / h, 5 fractions were collected, after which the resin was washed with demineralized water, and 3 wash fractions were collected and analyzed.

[0091] [Table 7]

[0092] Vanillin was then released by pumping aqueous ethanol 70% v / v containing 5% acetic acid through the resin column at a flow rate of 4 BV / h. Five fractions during vanillin release were collected and analyzed.

[0093] [Table 8]

[0094] 67.86% of the vanillin adsorbed on the strong base anion exchange resin was released by elution with 70% v / v aqueous ethanol containing 5% acetic acid, while the total vanillin bound by Amberlite FPA42 was only 13.60% of the total amount delivered by the feed solution. Thus, the overall vanillin yield of purification by strong base anion exchange resin was 9.22%. Due to the low efficiency brought by the application of the strong base anion exchange resin, the purification process of this example was stopped without crystallization.

[0095] Example 6 Downstream Process B 17.7 L of fermentation broth containing vanillin glucoside, produced by a vanillin glucoside-producing yeast strain, at pH 4.96, was used in the following experiments: the broth was ultrafiltered using an Alfa Laval TestUnit M20 with a UF 1 kDa Alfa Laval ETNA01PP membrane, diafiltered and concentrated to 4.90 L by reverse osmosis using an Alfa Laval RO98pHt membrane. Per gram of vanillin glucoside, 0.27 g of β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10 u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: about 6.3%) was added to 4900 mL of concentrated vanillin glucoside solution (pH 5.23) and the solution was stirred at 50 °C for 120 h. 850 mL of the formed slurry was filtered by adding Dal-Cin Alfatex 101 as a filter aid. The clear filtrate containing vanillin was pumped in the downward direction through a 200 mL column packed with non-ionic resin Purolite Purosorb PAD400 (preconditioned with demineralized water) at a flow rate of 4 BV / h. Five fractions of 500 mL (2.5 BV each) were collected (850 mL corresponding to the feed and 1650 mL corresponding to the wash). After the feed, the resin was washed with 8 BV of demineralized water to remove colored impurities until water with a light yellow tint emerged from the resin column. Vanillin was then released from the resin by elution with aqueous 80% v / v ethanol in the upflow direction at a flow rate of 4 BV / h. 1.672 L of eluate was collected in 8 fractions. Vanillin was desorbed immediately and the desorbate had a brown color, whereas the more dilute desorbate was yellow. The desorbed fractions from the non-ionic resin Purolite Purosorb PAD400 were pumped through a strongly acidic cation exchange resin Dowex 50Wx4 (BV=200 mL) in H-form, where the resin column was thoroughly pre-washed with demineralized water and pre-conditioned with 80% v / v ethanol in water. The desorbed fractions were pumped one after the other in a downward flow direction at a flow rate of 4 BV / h, starting from the first one and finishing with the last one. At the end, 80% v / v ethanol in water was pumped through the column to wash the resin. Eight fractions of 200 mL in total were collected. The fractions collected after the strongly acidic cation exchange resin were pumped through a column packed with weakly basic anion exchange resin DuPont Amberlite FPA53 (BV=200 mL), previously treated with 5% acetic acid, washed with demineralized water and previously conditioned with 80% v / v ethanol in water. The fractions were pumped one after the other in a downward flow direction through the resin column at a flow rate of 4 BV / h, starting from the first fraction of the previous step and ending with the last one. Then 80% v / v ethanol in water was pumped through the column to wash the resin. A total of 8 fractions of 200 mL were collected. All collected fractions were combined for carbon treatment and loaded into a 50 mL column of granular activated carbon Chemviron Acticarbone BGE (ratio 0.59 kg BGE / kg VAN) and the orange solution of the combined fractions from the resin treatment was recirculated downflow through the carbon column at a flow rate of 8 BV / h for 20 hours. The carbon treatment changed the color of the solution from orange to yellow. The carbon column was washed with 5 BV of 80% v / v ethanol in water (5 x 50 mL). The carbon treated main fraction and the carbon wash fraction were combined. The resulting solution was filtered through a 0.48 μm PTFE filter to remove residual carbon particles and the filter was washed with 40 mL of 80% v / v ethanol in water to give a total volume of 1740 mL with a pH of 4.94 and a conductivity of 15 μS / cm. The clear filtrate was concentrated under vacuum (from 220 mbar to 80 mbar) on a rotary evaporator in a water bath at 55° C. 4 mL of ethanol 96% was added to the remaining yellow concentrate to initiate crystallization. Seeding was performed with 20 mg of pure vanillin. The solution was cooled and crystallization occurred at about 35° C. to form vanillin crystals. The slurry was stirred at room temperature for 16 hours and then kept at 4° C. for 4 hours to allow the crystals to age. The vanillin crystals were filtered off on a Büchner funnel, washed with pre-cooled demineralized water (100 mL) and dried under vacuum (10 mbar, 50° C.) for 16 h. A pale yellow crystalline vanillin was obtained with a purity of 99.44% w / w by HPLC and 99.28% w / w by GC.

[0096] Example 7 Downstream process B does not use strong acid cation exchange resin 17.7 L of fermentation broth containing vanillin glucoside, produced by a vanillin glucoside-producing yeast strain, at pH 4.96, was used in the following experiments: the broth was ultrafiltered using an Alfa Laval TestUnit M20 with a UF 1 kDa Alfa Laval ETNA01PP membrane, then diafiltered and concentrated to 4.90 L by reverse osmosis using an Alfa Laval RO98pHt membrane. Per gram of vanillin glucoside, 0.27 g of β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10 u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: about 6.3%) was added to 4900 mL of concentrated vanillin glucoside solution (pH 5.23) and the solution was stirred for 120 hours at 50° C. The formed slurry was filtered through a pressure filter in the presence of Dal-Cin Filtex 7 (25 g) as a filter aid. 800 mL of the clear filtrate was used as the feed, and 200 mL of water used to wash the filter aid cake was also used for feeding and washing the non-ionic resin in the following step. A 200 mL column packed with non-ionic resin Purolite Purosorb PAD400 was washed with water. The feed was pumped into the column in a downward flow direction (filtrate, 800 mL), followed by a washing feed (200 mL, prepared for washing the filter aid) at a flow rate of 4 BV / h. Fractions of 200 mL (1 BV) were collected and the resin was washed with a further 7 BV of water to remove color and impurities, until the eluted water had a light yellow hue. Vanillin was then released from the resin by elution with 80% v / v aqueous ethanol. The desorbate from the non-ionic resin Purolite Purosorb PAD400 was applied to a weakly basic anion exchange resin DuPont Amberlite FPA53 (BV=200 mL), previously treated with 5% acetic acid, washed with demineralized water and previously conditioned with 80% v / v ethanol in water. The fractions were pumped one after the other in the downward flow direction at a flow rate of 4 BV / h, starting from the first fraction of the previous step and ending with the last one. Then 80% v / v ethanol in water was pumped in the upward flow direction through the resin column at a flow rate of 4 BV / h. A total of 9 fractions of 200 mL were collected. The first 5 fractions from the weak base anion exchange resin were combined for carbon treatment. Granular activated carbon Chemviron Acticarbone BGE (ratio 0.58 kg BGE / kg VAN) was loaded into a 50 mL column and the red solution of the combined fractions was recirculated downflow through the carbon column at a flow rate of 8 BV / h for 20 hours. The color of the solution changed from red to orange. The carbon column was washed with 5 BV of 80% v / v ethanol in water (5 x 50 mL). The main fraction after carbon treatment was pooled with the 5 carbon wash fractions and the combined solution was filtered through a 0.48 μm PTFE filter to remove residual carbon particles. The filter was washed with 35 mL of 80% v / v ethanol in water to give a total solution volume of 1285 mL with a pH of 5.42 and a conductivity of 52 μS / cm. 1285 mL of the solution obtained in the previous step was concentrated under vacuum (from 220 mbar to 80 mbar) on a rotary evaporator in a water bath at 55 °C. 4 mL of ethanol 96% v / v in water was added to the remaining yellow concentrate to initiate the crystallization of vanillin. Seeding was performed by adding 20 mg of pure vanillin. The solution was cooled and crystallization occurred at about 35 °C to form vanillin crystals. The slurry was stirred at room temperature for 16 h and then kept at 4 °C for 4 h to allow the crystals to age. The vanillin crystals were filtered off on a Büchner funnel, washed with pre-cooled demineralized water (100 mL) and dried in a vacuum oven (10 mbar, 50 °C) for 16 h. A pale yellow crystalline vanillin was obtained with a purity of 98.54% w / w by HPLC and 98.12% w / w by GC. The flavor of the vanillin obtained by the purification process of this Example 7, i.e., without the strong acid cation exchange resin treatment, was stronger and more creamy than the vanillin obtained in Example 6, which included a step with a strong acid cation exchange resin. The flavor of the vanillin obtained in Example 6 was more intense vanilla with a slight creamy note. The desired flavor is a more intense vanilla, and therefore the flavor of the vanillin obtained in Example 6 is preferred.

[0097] Example 8 Downstream process B by using 2-propanol as the solvent 17.7 L of fermentation broth containing vanillin glucoside, produced by a vanillin glucoside-producing yeast strain, at pH 4.96, was used in the following experiments: the broth was ultrafiltered using an Alfa Laval TestUnit M20 with a UF 1 kDa Alfa Laval ETNA01PP membrane, then diafiltered and concentrated to 4.90 L by reverse osmosis using an Alfa Laval RO98pHt membrane. Per gram of vanillin glucoside, 0.27 g of β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10 u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: approximately 6.3%) was added to 4900 mL of concentrated vanillin glucoside solution (pH 5.23), and the solution was stirred at 50° C. for 120 hours. The resulting slurry was filtered through Dal-Cin Alfatex 101 as a filter aid, and then diluted with ethanol to a final concentration of 20% ethanol v / v. 650 mL of the filtered vanillin solution was pumped upflow at a flow rate of 4 BV / h through a laboratory-scale column packed with 500 mL of the non-ionic resin Purolite Purosorb PAD 400. The resin was then washed with demineralized water. Vanillin was released from the resin by pumping aqueous 80% v / v 2-propanol in a downward flow direction through the resin column at a flow rate of 4 BV / h and six fractions of 500 mL were collected. The first 3 effluent fractions collected from the non-ionic resin Purolite Purosorb PAD 400 were pumped upflow through a column packed with 200 mL of strong acid cation exchange resin Purolite C150SH (activated with H2SO45%, washed with demineralized water, and preconditioned with 80% v / v 2-propanol in water) at a flow rate of 4 BV / h. The resin was then washed with aqueous 2-propanol 80% v / v. 10 fractions were collected and analyzed.

[0098] [Table 9]

[0099] The first 8 fractions collected from the strong acid cation exchange resin in the previous step were pumped downwards at a flow rate of 4 BV / h onto a column packed with 200 mL of weakly basic anion exchange resin Purolite A845S, pretreated with 5% acetic acid, washed with demineralized water and preconditioned with 80% v / v 2-propanol in water. The resin was then washed with aqueous 2-propanol 80% v / v. 10 fractions were collected and analyzed.

[0100] [Table 10]

[0101] The 10 fractions collected from the weakly basic anion exchange resin in the previous step were combined and the resulting dark red solution was pumped downflow through a column packed with 50 mL of chemical granular activated carbon Chemviron Acticarbone BGE (ratio 0.59 kg BGE / kg VAN) in a recirculation loop at room temperature and a flow rate of 8 BV / h for 3 days. The solution after carbon treatment was orange in color. The carbon was washed with 5 BV of 80% v / v 2-propanol in water. Samples of each solution were filtered and analyzed. The decolorized main vanillin solution and the wash fractions were combined and filtered through a 0.45 μm Nylon filter. The filtered and decolorized solution was concentrated under vacuum on a rotary evaporator in a water bath at 55° C. to 118 mL and then cooled to room temperature with continuous stirring. 4 mL of ethanol 100% was added to the concentrate to solubilize the brown oil formed in the solution during concentration and seeding was carried out by adding 29.21 mg of vanillin (99% purity). The solution was kept at 4° C. overnight. The solid formed was separated from the mother liquor by vacuum filtration, washed with 100 mL of pre-cooled demineralized water and dried at 45° C. under vacuum for 3 hours. A dark yellow vanillin was obtained with a purity of 97.77% w / w by HPLC and 99.70% w / w by GC.

[0102] Example 9 Downstream process B, where there is a treatment with a weakly basic anion exchange resin prior to the treatment with a strongly acidic cation exchange resin. The fermentation broth containing vanillin glucoside was ultrafiltered and diafiltered with a UF 10kDa Alfa Laval ETNA10PP membrane, then concentrated by reverse osmosis with an Alfa Laval RO99 membrane. The vanillin glucoside in the resulting concentrate was hydrolyzed with a β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: about 6.3%) at 55°C for 30 hours, applying 0.45 kg of enzyme solution per kg of vanillin glucoside. The resulting suspension was filtered with a filter press in the presence of a filter aid Dal-Cin Cellulose M09 to obtain a clear brown vanillin-containing filtrate with a pH of 5.04. 1 L of this filtered vanillin solution was pumped downflow through a column packed with 500 mL of non-ionic resin Purolite Purosorb PAD 400 at a flow rate of 4 BV / h. The resin was then washed with 3 BV of demineralized water and the vanillin was released from the resin by pumping aqueous 80% v / v ethanol upflow through the resin column at a flow rate of 4 BV / h. 4 BV were collected and combined. 1 L of the desorbate obtained in the previous step was pumped downwards at a flow rate of 4 BV / h onto a column packed with 200 mL of weakly basic anion exchange resin Purolite A845S, pretreated with 5% acetic acid, washed with demineralized water and preconditioned with 80% v / v ethanol in water. The resin was then washed with 80% v / v aqueous ethanol. Seven fractions were collected and analyzed.

[0103] [Table 11]

[0104] The seven fractions obtained in the previous step were pumped downwards through a column packed with 200 mL of strong acid cation exchange resin Purolite C150SH (activated with H2SO45%, washed with water, and preconditioned with ethanol 80% v / v in water) at a flow rate of 4 BV / h. The resin was then washed with aqueous ethanol 80% v / v. Seven fractions were collected and analyzed.

[0105] [Table 12]

[0106] The seven fractions collected from the strong acid cation exchange resin were combined and the resulting dark red solution was recirculated downflow through a column packed with chemical granular activated carbon from Chemviron Acticarbone BGE (ratio 0.48 kg BGE / kg VAN) at room temperature overnight at a flow rate of 8 BV / h. At the end of the carbon treatment the solution was yellow. The carbon was then washed with 4 BV of 80% ethanol. The decolorized vanillin solution and the washing fractions were combined, filtered through a 0.45 μm Nylon filter and analyzed. The filtered and decolorized solution obtained in the previous step was concentrated under vacuum in a rotary evaporator in a water bath at 55° C. to 135 mL and cooled to room temperature under continuous stirring. 4 mL of ethanol 99.9% was added to the solution to solubilize the brown oil formed in the solution during concentration. Seeding was then carried out by adding 35 mg of vanillin of purity ≥ 98%. The resulting slurry was stirred overnight at room temperature, after which the solid was separated from the mother liquor by vacuum filtration, washed with 100 mL of pre-chilled demineralized water and dried overnight under vacuum at 50° C. A yellow vanillin was obtained with a purity of 99.60% w / w by GC.

[0107] Example 10 Downstream process B, which does not use weakly basic anion exchange resin and does not include activated carbon treatment The fermentation broth containing vanillin glucoside was ultrafiltered and diafiltered with a UF 10kDa Alfa Laval ETNA10PP membrane, then concentrated by reverse osmosis with an Alfa Laval RO99 membrane. The vanillin glucoside in the resulting concentrate was hydrolyzed with a β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: about 6.3%) at 55°C for 30 hours, applying 0.45 kg of enzyme solution per kg of vanillin glucoside. The resulting suspension was filtered with a filter press in the presence of a filter aid Dal-Cin Cellulose M09 to obtain a clear brown vanillin-containing filtrate with a pH of 5.04. 1 L of this filtered vanillin solution was pumped downflow through a column packed with 500 mL of non-ionic resin Purolite Purosorb PAD 400 at a flow rate of 4 BV / h. The resin was then washed with 3 BV of demineralized water. Vanillin was released from the resin by pumping aqueous 80% v / v ethanol upflow through the resin column at a flow rate of 4 BV / h. 4 BV were collected and analyzed. 700 mL of desorbate obtained in the previous step after adsorption and release onto / from the non-ionic resin Purolite Purosorb PAD 400 was pumped in a downward flow direction at a flow rate of 4 BV / h into a column filled with 200 mL of strong acid cation exchange resin Purolite C150SH (activated with H2SO4 5%), washed with water and preconditioned with 80% v / v ethanol in water. Afterwards the resin was washed with aqueous ethanol 80% v / v. Five fractions were collected and analyzed.

[0108] [Table 13]

[0109] The five fractions collected from the strong acid cation exchange resin in the previous step were combined. The resulting solution was concentrated under vacuum on a rotary evaporator in a water bath at 55° C. and cooled to room temperature with continuous stirring. 4 mL of ethanol 99.9% was added to the solution to solubilize the brown oil formed in the solution during concentration, and then seeding was performed by adding 35.84 mg of vanillin with a purity of 98% or higher. The resulting slurry was stirred overnight at room temperature, after which the solid was separated from the mother liquor by vacuum filtration, washed with 100 mL of pre-chilled demineralized water and dried overnight under vacuum at 50° C. Brown vanillin was obtained with a purity of 94.10% w / w by GC.

[0110] Example 11 Downstream process B, which does not include activated carbon treatment The fermentation broth containing vanillin glucoside was ultrafiltered and diafiltered with a UF 10kDa Alfa Laval ETNA10PP membrane, then concentrated by reverse osmosis with an Alfa Laval RO99 membrane. The vanillin glucoside in the resulting concentrate was hydrolyzed with a β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: about 6.3%) at 55°C for 30 hours, applying 0.45 kg of enzyme solution per kg of vanillin glucoside. The resulting suspension was filtered with a filter press in the presence of a filter aid Dal-Cin Cellulose M09 to obtain a clear brown vanillin-containing filtrate with a pH of 5.04. 1 L of this filtered vanillin solution was pumped downflow through a column packed with 500 mL of non-ionic resin Purolite Purosorb PAD 400 at a flow rate of 4 BV / h. The resin was then washed with 3 BV of demineralized water and the vanillin was released from the resin by pumping aqueous 80% v / v ethanol upflow through the resin column at a flow rate of 4 BV / h. 4 BV were collected and analyzed. 1 L of the desorbate obtained in the previous step was pumped sequentially in the downward direction at a flow rate of 4 BV / h through two 200 mL resin columns: the first column packed with the strongly acidic cation exchange resin Purolite C150SH (activated with H2SO4 5%), and then with the weakly basic anion exchange resin Purolite A845S (pretreated with acetic acid 5%). The columns were then washed with aqueous ethanol 80% v / v. Single fractions of 7 BV were collected and analyzed.

[0111] [Table 14]

[0112] The solution obtained in the previous step was concentrated under vacuum by rotary evaporator in a water bath at 55° C. to 115 mL and cooled to room temperature under continuous stirring. 4 mL of ethanol 99.9% was added to the solution to solubilize the brown oil formed during concentration and the solution was stirred at room temperature overnight. The solid formed was then separated from the mother liquor by vacuum filtration, washed with 100 mL of pre-cooled demineralized water and dried overnight under vacuum at 50° C. A yellow vanillin was obtained with a purity of 97.10% w / w by GC.

[0113] Example 12 Downstream process B, which does not use weakly basic anion exchange resin The fermentation broth containing vanillin glucoside was ultrafiltered and diafiltered with a UF 10kDa Alfa Laval ETNA10PP membrane, then concentrated by reverse osmosis with an Alfa Laval RO99 membrane. The vanillin glucoside in the resulting concentrate was hydrolyzed with a β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10u / g, biological origin: Trichoderma longibrachiatum, enzyme concentration: about 6.3%) at 55°C for 30 hours, applying 0.45 kg of enzyme solution per kg of vanillin glucoside. The resulting suspension was filtered with a filter press in the presence of a filter aid Dal-Cin Cellulose M09 to obtain a clear brown vanillin-containing filtrate with a pH of 5.04. 1 L of this filtered vanillin solution was pumped downflow through a column packed with 500 mL of non-ionic resin Purolite Purosorb PAD 400 at a flow rate of 4 BV / h. The resin was then washed with 3 BV of demineralized water and the vanillin was released from the resin by pumping aqueous 80% v / v ethanol upflow through the resin column at a flow rate of 4 BV / h. 4 BV were collected and analyzed. 700 mL of desorbate obtained after adsorption and release onto / from the non-ionic resin Purolite Purosorb PAD 400 was pumped in a downward flow direction at a flow rate of 4 BV / h onto a column filled with 200 mL of strong acid cation exchange resin Purolite C150SH (activated with H2SO4 5%), washed with water and preconditioned with 80% v / v ethanol in water. The resin column was then washed with aqueous ethanol 80% v / v. One fraction of 1 L was collected and analyzed.

[0114] [Table 15]

[0115] The fraction collected after the strong acid cation exchange resin was recirculated in a downward direction through a column packed with granular activated carbon Chemviron Acticarbone BGE (ratio 0.53 kg BGE / kg VAN) at a flow rate of 8 BV / h overnight at room temperature. After carbon treatment the solution was orange in color. The carbon was washed with 4 BV of 80% v / v ethanol in water and the main solution was combined with the wash fraction and filtered through a 0.45 μm Nylon filter. The clear filtrate was concentrated under vacuum on a rotary evaporator in a water bath at 55° C. to 240 mL and cooled to room temperature with continuous stirring. 4 mL of ethanol 99.9% was added to the solution to solubilize the brown oil formed in the solution during concentration and the resulting solution was stirred overnight at room temperature to form a slurry. The solid was separated from the mother liquor by vacuum filtration, washed with 100 mL of pre-chilled demineralized water and dried overnight under vacuum at 50° C. A yellow vanillin was obtained with a purity of 95.90% w / w by GC.

[0116] Example 13 Downstream Process C To analyze the complete downstream process of type C, 11.56 L of fermentation broth containing vanillin glucoside produced by a vanillin glucoside-producing yeast strain was used. Per gram of vanillin glucoside, 0.57 g of β-glucosidase solution from Biocatalysts Beta Glucosidase G016L (β-glucosidase activity: 10 u / g, biological source: Trichoderma longibrachiatum, enzyme concentration: about 6.3%) was added to the fermentation broth and stirred at 55°C for 48 hours. The broth at pH 5.03 was ultrafiltered and diafiltered on an Alfa Laval TestUnit M20 with a UF 10 kDa ETNA10PP membrane. 2.5 L of the permeate was pumped in upflow direction onto a non-ionic resin Purolite Purosorb PAD 400 (500 mL) at a flow rate of 4 BV / h. The resin column was washed with 3 BV of demineralized water and vanillin was desorbed by pumping 2.5 BV of 80% v / v ethanol in water, followed by about 600 mL of water, through the resin column. Four desorption fractions of 500 mL were collected. Two columns were connected in series: a column packed with 200 mL of strong acid cation exchange resin Purolite C150SH H form, followed by a column packed with 200 mL of weak base anion exchange resin DuPont Amberlite FPA53 (pretreated with 5% acetic acid). Four fractions of the desorbate from the previous step were combined and pumped in the upward flow direction through the two columns in sequence, starting with the strong acid cation exchange resin. Four fractions were collected and analyzed.

[0117] [Table 16]

[0118] All four fractions were combined to obtain a solution, which was pumped down a column packed with granular activated carbon Chemviron Acticarbone BGE (ratio 0.50 kg BGE / kg VAN) at a flow rate of 4 BV / h for 16 hours. The color of the solution changed from tan to yellow upon carbon treatment. The carbon was then washed with 3 BV of 80% ethanol in water and the main carbon-treated solution was combined with the wash fraction and then filtered through a 0.48 μm PTFE filter. The clear filtrate (pH 4.50) was concentrated in a 1 L reactor under vacuum (300-150 mbar) at 60 °C to obtain 280 mL of a biphasic emulsion (oily brown vanillin and water). The emulsion quickly turned into a slurry by cooling to 20 °C, stirred for 16 h, then further cooled to 10-15 °C, after which the solid was separated from the mother liquor by vacuum filtration. The wet solid was washed with 200 mL of pre-chilled demineralized water and dried for 48 h under vacuum (10 mbar) at 45 °C. A yellow vanillin was obtained with a purity of 99.0% w / w by GC.

[0119] The overall vanillin yields of the processes of Examples 1-4 and 6-13 described hereinabove were in the range of 40-90%.

Claims

1. 1. A method for recovering and purifying vanillin from a microbial fermentation broth, the fermentation broth containing vanillin complexes produced during microbial fermentation by microbial cells capable of producing and secreting vanillin complexes, the method comprising: (I)(a) separating and removing said microbial cells from said fermentation broth so as to leave a liquid substantially free of said microbial cells, and then converting vanillin complexes contained in said liquid into vanillin and the corresponding complex partners; or (I)(b) converting vanillin complexes contained in the fermentation broth into vanillin and the corresponding complex partners, followed by separation and removal of the microbial cells from the fermentation broth so as to leave a liquid substantially free of microbial cells; after that, Step (II)(a) of adding a water-miscible polar organic solvent to the liquid obtained in step (I)(a) or (I)(b), wherein the liquid obtained in step (I)(a) is optionally filtered either before or after the addition of said polar organic solvent; 2. The vanillin solution produced in step (II)(a), (i) a cation exchange sorbent followed by a weakly basic anion exchange sorbent; or (ii) a weakly basic anion exchange sorbent followed by a cation exchange sorbent Step (II)(b) of treating Optionally, decolorizing the resulting solution with an adsorbent (II)(c); or step (II')(a) of treating the liquid obtained in step (I)(a) or (I)(b) with a non-ionic adsorbent, wherein the liquid obtained in step (I)(a) is optionally first filtered before treatment with said non-ionic adsorbent, and wherein the non-ionic adsorbent used in step (II')(a) is capable of adsorbing vanillin, said treatment comprising a step (i) of adsorbing said vanillin under conditions allowing vanillin to be bound to said non-ionic adsorbent and a step (ii) of desorbing said bound vanillin into solution, The resulting vanillin solution, (i) a cation exchange adsorbent; (ii) a weakly basic anion exchange adsorbent, or (iii) both a cation exchange adsorbent and a weakly basic anion exchange adsorbent in any order. Step (II')(b) of treating with any one of Optionally, any of steps (II')(c) of decolorizing the resulting solution with an adsorbent; and and (III) crystallizing said vanillin from the resulting solution.

2. 2. The method of claim 1, wherein the vanillin complex is vanillin glucoside.

3. The method of claim 1 or 2, wherein the microbial cell is a fungal cell.

4. The method of claim 3 , wherein the fungal cell is a yeast cell.

5. 5. The method according to claim 1, wherein the conversion of the vanillin conjugate into vanillin and the corresponding conjugated partner in step (I)(a) or (I)(b) is carried out either by chemical or enzymatic conversion.

6. 6. The process according to any one of claims 1 to 5, wherein the polar organic solvent used in step (II)(a) is an alcohol or a mixture of different alcohols.

7. 7. The process according to any one of claims 1 to 6, wherein the cation exchange adsorbent used in step (II)(b) is a strongly acidic cation exchange adsorbent.

8. The method according to any one of claims 1 to 7, wherein the weakly basic anion exchange adsorbent used in step (II)(b) is a weakly basic anion exchange resin.

9. 9. The method according to any one of claims 1 to 8, wherein the weakly basic anion exchange adsorbent used in step (II)(b) is converted to acetate form before use.

10. The method according to any one of claims 1 to 9, wherein the method comprises step (II)(b)(i).

11. The process according to any one of claims 1 to 10, wherein the adsorbent used in step (II)(c) is carbon.

12. The method according to any one of claims 1 to 11, wherein the non-ionic adsorbent used in step (II')(a) is a non-ionic resin.

13. 13. The method according to any one of claims 1 to 12, wherein the desorption of the bound vanillin into solution in step (II')(a)(ii) is carried out using as eluent an organic solvent, a mixture of different organic solvents, a mixture of water and an organic solvent, or a mixture of water and different organic solvents.

14. The method according to any one of claims 1 to 13, wherein the cation exchange adsorbent used in step (II')(b) is a strongly acidic cation exchange adsorbent.

15. The method according to any one of claims 1 to 14, wherein the weakly basic anion exchange adsorbent used in step (II')(b) is a weakly basic anion exchange resin.

16. The method according to any one of claims 1 to 15, wherein the weakly basic anion exchange adsorbent used in step (II')(b) is converted to the acetate form before use.

17. 17. The method according to any one of the preceding claims, wherein the method comprises a step (II')(b)(iii) in which the vanillin solution is treated with a cation exchange adsorbent and then with a weakly basic anion exchange adsorbent.

18. The process according to any one of claims 1 to 17, wherein the adsorbent used in step (II')(c) is carbon.

19. The method according to any one of claims 1 to 18, which does not include a purification step with a strong base anion exchange adsorbent.

20. 20. The method of any one of claims 1 to 19, which occurs at a pH of less than 7.

21. 21. The process according to any one of claims 1 to 20, wherein the crystallization of vanillin is carried out at a pH of about 3.5 to 5.5.

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