GB2406855A - Production of xylitol from a carbon source other than xylose and xylulose - Google Patents

Abstract

A process for the production of xylitol from a carbon source comprising conversion of the carbon source to a raw bioconversion product containing xylitol, optional chromatographic fractionationation of the raw bioconversion product to obtain a solution enriched in xylitol and crystallisation of the raw bioconversion product or the solution enriched in xylitol to obtain crystalline xylitol and a crystallisation run-off. The carbon source is other than xylose, xylulose, mixtures of xylose and xylulose and polymers and oligomers containing xylose or xylulose as major components. Preferably the carbon source is a six-carbon sugar, especially glucose.The conversion of the carbon source may be via the ribulose-5-phospate pathway, the xylitol-1-phosphate pathway or the arabitol pathway and may be carried out in the presence of xylitol phosphate dehydrogenase activity. The conversion may be carried out using a microbial host. The chromatographic fractionation may be carried out using a cationic exchange resin.

Description

RECOVERY OF XYLITOL

FIELD OF THE INVENT1011

The present invention is related to the field of xylitol production. Especially, the invention relates to a process of producing xylitol by conversion of S a suitable carbon source, such as glucose to xylitol. The conversion is typically carried out as microbial or enzymatic conversion. The invention also relates to the recovery of xylitol from raw bioconversion prclucts obtained from said mi- Grobial or enzymatic conversion, using crystallization with optional chroma- tographic fractionation.

BACKGROUND OF TElE INVENTION

Xylitol is a five-carbon sugar alcohol having considerable value as a special sweetener. Xylitol is a natural constituent of many fruits and vegeta bles, and although the level found in natural products is usually less than 1%, it has always been a natural part of modem manes diet. The human body pro- duces 5 to 15 grams of xylitol per day in normal metabolism, Xylitol Is approximately as sweet as sucrose, noncaloric, noncariogenic and =riostatic. Islumerous in viva, in vitro and clinical studies have demonstrated that xylitol has the ability to inhibit the formation of new caries and to be a beneficial factor in the improvement of oral health. Xylitol can also be safely consumed by diabetics. Furthermore, xylitol does not typically have an unpleasant aftertaste like other sugar substitutes. In crystalline applications, it provides in the mouth a pleasant cooling effect, which is nearly 40% greater than that of sorbitol.

The unique properties of xylitol bring new value to sugar-free foods as well as pharmaceutical and oral hygiene products. Despite the advantages of xylitol, the utilization of xylitol on a commercial scale has been limited due to its relatively high cost because of complicated nultistep prowesses involved in its production on a commercial scale.

Xylitol is generally prepared frorr biomasserived xylan-containing material, especially hemicellulose hydrolysates, by various chemical and en- zymatic methods. For instance, it is known to produce xylitol by hydrolyzing the xylan-hemicellulose portion of birch trees and other hardwood material to ob fain D-xylose. C)-xylose is converted to xylitol by catalytic hydrogenation. Xyli- tol is purified by crystallization to a Purim over 99%.

Currently, it has been proposed that xylitol can also be produced by microbial fermentation of various carbohydrate substrates, such as pentose and hexose sugars, typically xylose, glucose, t,-arabitol and D- xylulose. Xylitol is produced and accumulated in the fermentation broth, from which xylitol has then been recovered by various methods. However, the availability of xylose and D-xylulose for the fermentation is limited, because the preparation of xy- tose and D-xylulose is laborious and requires multistep processes. On the other hand, when other carbon sources than xylose or D-xylulose are used for the fermentation, numerous by-products, besides the desired xylitol, are formed in the fermentation broth during the fermentation step. At least the fol- loNing by-products, impurities or remaining fermentation nutrients can gener- ally be found in the fermentation broths: glucose, maltose and malto oligosacchandes, acetoin, butanediol. xylulose, ribulose, ribitol, acetic acid, formic acid, pyruvic acid, succinic acid, propionic acid, lactic acid, peptone, glycerol etc. Advantageously, said nondesired products should be removed before the final step of crystallizing the desired xylitol. However, due to the dif- ferent nature of these nonesired compounds, the removal thereof from fer mentation broths has been complicated.

EP 1 075 795 (Ajinomoto KK) discloses a process for producing xyli- tol by fermentation of a xylitol-producing microorganism in an aqueous fermentation medium' In the fermentation step of this process, xylitol is typically pro- duced from glucose through a D-arabitol intermediate. Xylitol is then recovered from the fermentation broth by removing the solid matter from the fermentation medium, desalting the resulting fermentation broth by means of a cation- exchange resin and an anion exchange resin, subjecting the resulting desalted solution to chromatography using a strongly acid cation-exchange resin, which is typically in the Ca2+ form, to separate xylitol from other sugar alcohols and sugars. Alternatively, the desalting step may be carried out in two stages, whereby the first stage comprises ion- exclusion chromatography with a strongly acid cation-exchange resin, which is recited to be in the Nan form or N113 form, and the second stage comprises desalting using a cation-exchange resin and an anion-exchange resin. As a final step, the xylitol fraction which is obtained from the chrornatographic steps and which is high in xylitol content is subjected to crystallization to obtain crystalline xylitol. The crystallization is car ried out using cooling crystallization to provide a xylitol purity of 99.1%. EP 672 161 B1(Xyrofin Oy) discloses a process for the production of xylitol in a single fermentation using a recombinant host, comprising (a) constructing within a microbial host a metabolic pathway which leads to the synthesis of xylitol as an end product from a carbon source other than O-xyloso, Dxylulose, matures of D-xylose and D-xylulose, and polymers and oligomers containing D-xylose or D-xylulose as major connponents, (b) growing said recombinant host under conditions that provide for the synthesis of said xylitol using said pathway on said carbon source and (c) recovering said xy itol produced in step (b). In one embodiment the process, arabitol is an intermediate In said pathway and glu cose is used as said carbon source It is proposed that xylitol produced in the fermentation broth can be purified using chromatographic steps disclosed in US 5 081 026 (Suomen Xyrofln Oy, Heikkila et al.) and crystallization. In US 5 081 026, the chromatographic separation typically utilizes a cation exchange resin in an alkali metal form or in an alkaline earth metal form on a divinyl ben zene cross-linked sulfonated polystyrene support. The crystallization is carried out using cooling crystallization to provide a xylitol purity of 99.4% starting from xylitol purity of 82.5%.

US 5 238 826 (Roquette Freres) discloses a process for the manu facture of D-xylose comprising a multi-step fermentation process starting from a D-glucose syrup. In said fermentation, glucose is converted to xylulose through an arabitol intermediate. Xylulose is then converted to xylose by isom erlsation, followed by hydrogenation of xylose to xylitol. D-xylose is separated from xylulose by chromatographic fractionation using cationic resins, which are typically of the highly acid cationic resin type, preferably in alkali metal form or in alkaline earth metal form, or zeolites, which are typically in NH4+, Na+, K+, Ca2+ or BaZ+ form. The xylose syrup obtained is hydrogenated to xylitol. The xylitol syrup is then crystallized to obtain crystalline xylitol.

US 5,096,820 (Roquette Freres) discloses a process for manufacturing xylitol and xylitol-nch products by enzymatic isomenzation a D xylulose syrup into a syrup containing D-xylose and D-xylulose and then, without extracting the xylose, catalytically hydrogenating this syrup resulting in a xylitol-rich syrup. It is recited that said xylitol-rich syrup is then either dehydrated or subjected to chromatographic treatment or to a treatment of extraction by crystallization. US 5,096,820 also discloses a process for manufacturing xylitol comprising a step of microbiological conversion of D arabitol into D-xylulose by means of microorganisms producing alcohol means of microorganisms producing alcohol dehydrogenase, a step of enzy- matic conversion of D-xylulose into D-xylose by means of glucosisomerase, a step of catalytic hydrogenation of the C)-xylose syrup Into a xylitol- rich syrup and a step of recovery by crystallization of the xylitol from the xylitol-rich syrup and separation of the crystals from their mother liquors.

Regarding the chromatographic step in the above-mentioned U.S. Patent (5, 0gG,820), the patent refers to FR 2,344,514 (Suornen Sokeri Oy).

The latter discloses a chronatographic fractionation method using at least two columns containing sulfonated polystyrene cation exchange resins crosslinked with divinyl benzene, one of said columns containing the resin in an alkaline earth metal form and the other of said columns containing the resin in an Al3.

or Fe3+ fom,.

US 6,340,582 B1 (Ajinomoto Co.) relates to a method for producing xylitol by contacting D-arabitol with a microorganism, which has the ability to convert D-arabitol to xylitol. In one embodiment of the method, said microor- ganism has the ability to metabolize a carbon source to produce NADH (a re- duéd type nicotinamide adenine dinucleotide). In another embodiment of the method, NADtl is added as such to the system. Said microorganism has typi- cally D-a rabito l d ehyd rogenase activity and D-xylu tose red u clase (xylitol dehy drogenase) activity. Xylitol produced in the culture medium is recovered and isolated from the reaction mixture by conventional methods. Specifically, the solid matter may be removed by centrifugation, filtration, or corresponding methods, the resulting liquid fraction may be decoloured and desalted using activated carbon or an ion-exchange resin, and the desired product may be crystallized from the solution.

US 6,348,326 (Mitsubishi Chemical corporation) discloses a method for producing L-nbose from a saccharide raw material, such as glucose via ribitol and C-nbulose intermediates, using fermentation vith microorganisms.

The L-ribose produced in the fernentation broth may be purified by chromatog raphy and/or crystallization. The chromatographic separation is preferably car- ried out with a cation type ion exchanger in alkali metal or alkaline earth mete fonn, for instance with a strong cation exchange resin in Na+ form. The L- ribose fraction obtained from the chromatographic separation is filtered through an ultrafiltration membrane, passed through a column filled with a cation ex change resin and a column filled with an anion exchange resin and crystallized from ethanol to obtain L-ribose cstals with a purity of 99.9%.

H. Onishi and T. Suzuki describe microbial production of xylltol from glucose in Appl. Microbiol., vol. 1B (1969), pp. 1031 to 1035. The process is carried out as a sequential fermentation via D-arabitol and D-xylulose interme- diates. After concentration, xylitol may be recovered from the fermentation broth by extracting with ethyl alcohol, followed by optional treatment with Am" berlite JR-A 400 resin and crystallization from ethyl alcohol.

WO 01/53306 (Xyrofin Oy) discloses methods of manufacturing five- carbon aldo- and keto-sugars and sugar alcohols, such as xylitol, by fermentetion in recombinant hosts Furthermore, said publication discloses recombinant hosts that have been engineered to enhance the production of pentose phase phase pathway intermediates, or the production of one or more of xylitol, [)- arabitol, D-arabinose, D-lyxose, ribKol, D-ribose, D-ribulose, C)-xylose, and/or D-xylulose, and methods of manufacturing the same using such hosts. The desired product, for instance xylitol, is obtained in the fermentation broth. It is generally recited in said reference that the methods of purification of the fer- mentation products are known and include various forms of column chroma- tography (e.g. ion exchange, adsorption, reverse phase etc.) and crystallize. tion.

Acetoin is an especially harmful by-product fondled in the fermenta tion of various carbohydrate substrates to fonn sugars and sugar alcohols, such as xylitol. Therefore acetoin should either be removed or its production should be decreased during [ennentation. EP 525 659 A2 (Mitsubishi Kasei Corporation) discloses a process for controlling the acetoin concentration in a process for manufacturing erythritol. This reference discloses a process for preparing erythritol crystals comprising crystallizing an erythritol-containing liquid obtained by fermentation, wherein the acetoin concentration of the erythritol- containing liquid is controlled to ppm by weight or less, It is recited that the achievement of acetoin control within the abovementioned range re- quires Careful control of conditions in the purification operations following cul turing up to crystallization. In order to effectively remove acetoin by purification and to prevent incorporation of acetoin into crystals during crystallization, the most effective means is evaporative separation. Acetoin can be evaporated off together with water by concentration.

US 6 365 383 tRoquette Freres) relates to a method of producing erythritol by fermentation of sugars with erythritol-producing microorganisms. It is also recited that to remove the low quantities of residual glucose or of fer mentation Contaminants such as acetoin, the fernentation broth may be treated by any known technique, for example by transformation into acids by means of treatment in alkaline conditions at a temperature between 100 C and 1 30 C. The acids thus formed are then separated by demineralization. Erythri tol is recovered by concentration and crystallization.

JP 11000187 (Towa) discloses a steam distillation process for puri fying erythritol.

US 980 640 (Xyrofin Oy) discloses a method for recovering a crys tallized organic compound, such as crystalline xylitol from a solution containing said compound, comprising crystallizing said compound by way of nucleation from a supersaturated solution having a high viscosity. A crystalline mass is recovered under conditions typically including continuous intermixing into and from high shear zones and a slow cooling rate of from about 10 to 100 hours effective to promote the nucleation without substantial crystal growth. The crys talline xylitol thus obtained may be recrystallized by conventional methods.

BRIEF DESCRIPTION OF THE IIIVENTION

It is an object of the present invention to provide a method for the production of xylitol by fermentation or by an enzymatic reaction from a suit able carbon source. It is also an object of the invention to provide a process for the recovery of xylitol in a pure Crystalline form and with good yield from fer mantation broths and reaction products thus obtained using less complicated and more straight-forward purification and separation procedures than for in stance in the process of EP 1 075 795 described above.

It was found that fermentation or enzymatic reactions in accordance with the present invention provided a relatively pure raw bioconversion product with less by-products and other nondesired compounds than conventional fermentation processes for the production of xylitol.

Especially, it was found that the impurities present in the raw bio conversion product obtained in accordance with the present invention did not essentially disturb the recovery of xylitol by crystallization or chromatography.

Furthermore, it was found that the recovery of xylitol by chromatography or crystallization was efficient providing xylitol with a high yield and a high purity.

In accordance with an alternative embodiment of the invention, it was found that chromatographic fractionation might be applied to the recovery of xylitol from the raw bioconversion product, whereby chromatographic frac tionation effectively removed ionic substances, such as salts, and non-desired non-ionic compounds, such as hexose sugars and sugar alcohols from the xyli tol product. It was also found that after the chrornatographic fractionation, start- ing from a xylitol purity of about 75%, only one crystallization was required to provide xylitol with good yield and with a purity of even 98%.

The object of the invention was achieved by a method which is characterized by what is stated in the independent claims. The preferred em- bodiments of the invention are disclosed in the dependent claims.

DEFINITIONS RELATING TO THE INVENTION

In connection with the present invention, the expression "conversion of said carbon source to xylitol.' refers to a series of biochemical reactions red suiting in xylitol starting from said capon source.

The expression Raw bioconversion product. refers to the total prod uct obtained from the conversion of said carbon source to xylitol. In addition to the desired xylitol, said raw bioconversion product obtained from the conver sion typically includes the cell mass and other insoluble components, other sugars and sugar alcohols besides the desired xylitol, rests of fermentation nutrients, aétoin and other compounds mentioned above.

The expression Fermentation broths refers to the raw bioconversion product obtained from fermentation.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figure 1 is an illustrative embodiment of the invention and is not meant to limit the scope of the invention in any way.

Figure 1 shouts the results of the hromatographic separation of xylitol from fermentation broth described In Example 1(B). The separation was carried out with a strongly acid cation exchange resin in Ca2+ form.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a process for the produebon of xylitol from a carbon source other than xylose, xylulose, mixtures of xylose and xylulose, and polymers and oligomere containing xylose or xylulose as major comply nents, comprising conversion of said carbon source to xylitol via ribulose-5-phosphate pathway to obtain a raw bioconversion product containing xylitol, optional chromatographic fractionation Of said raw bioconversion product containing xylitol to obtain a solution enriched in xylitol, and crystallization of said raw bioconversion product or optionally said solution enriched in xylitol to obtain crystalline xylitol and a crystallization run- off.

The invention also relates to a process for recovering xylitol from said xylitol-containing solution obtainecl as the raw bioconversion product using crystallization with optional chromatographic fractionation.

In one embodiment of the process of the present invention, the process comprises a further step of subjecting said crystallization run-off to chromatographic fractionation to obtain a solution enriched in xylitol.

In another embodiment of the process of the pmsent invention, the process comprises a further step of subjecting said crystallization run off to further crystallization to obtain crystalline xylitol.

In the conversion step of the present invention, xylitol is produced from the starting carbon source through ribulos - Phosphate pathway. In connection with the present invention, the expression Uribulose-phoshate pathways means the conversion of said carbon source, such as glucose, to xylitol via a metabolic pathway through ribulose-phosphate as an intermedi ate.

The conversion step of the present invention is typically carried out in the presence of xylitol phosphate dehydrogenase activity.

Said conversion may be carried out using a microbe or an enzyme preparation including Pistol phosphate dehydrogenase activity. It is possible to use any known processes with immobilized or free microbial cells, which may include living cells or dead cells. Enzyme preparations can be used in the form of immobilized enzymes or free enzymes in a column reactor or in a stirred tank reactor.

In a preferred embodiment of the invention, said conversion is car ried out by fermentation using a microbial host having xylitol phosphate deny.

cirogenase activity. The microbial host is preferably a genetically modified mi crobial host, the genetic modification of which increases the total activity of xylitol phosphate dehydrogenase during said conversion as compared to said activity in said host prior to being genetically modified, whereby said microbial host in its native form may have xylitol phosphate dehydrogonase activity or g not.

The xylitol phosphate dehydrogenase (XPDH) activity may be as sayed in a medium containing 50mM Trls-HCI having a pH of 7.0 and including 0.2 nnlV1 1sIAI:11. The reaction is started by adding xylulose phosphate at a final concentration of 5 mM. The activity is measured by following the adsorp tion of NADH at 340 nm. The activity is quantified as Ulmg protein for the XPI;)H.

In said conversion step of the process of the present invention, xyli tol is preferably produced from the starting carbon source through xylitol-1 phosphate pathway. In connection with the present invention, the expression "xylitol-1-phostate pathway" (= xylitol-5-phosphate pathway) means the con version of said carbon source, such as glucose, to capitol through a xylitol-1 phosphate as an intermediate. Microbial hosts that have been genetically modified to be capable of producing increased amounts of xylitol through the xylitol-1-phosphate as an intermediate are especially useful in realizing the microbial conversion Step of the present invention.

In another embodiment of the invention, xylitol is produced from the starting carbon source through arabitol pathway. In connect on with the present invention, the expression "arabitol pathway" means the conversion of said car bon soume, such as glucose to xylitol through ambitol as an intermediate.

The fermentation process and hosts which are useful for the micrm dial conversion of a carbon source to xylitol through xylitol-1-phoshate pathway have been described in WO 01-53306 (Xyrofin Oy). The construction of the genetically modified hosts useful for said microbial conversion is also cle scribed in WO 01-S3306.

The microbial hosts and strains thereof useful in the present inven tion may also be achieved by random chemically induced or spontaneous mutagenesis followed by selection of strains with improved properties.

The range of hosts wherein the microbial conversion of the current invention can be implemented covers bacteria and fungi. Preferably, the con version is carried out with bacteria. In case fungi are used, it is preferably a yeast. Particularly, microbial species with a GRAS status such as yeast Sac charomyces cerevsiae or Gram-positive batenum Bacillus subMlis are sult able as hosts of the current invention. Other suitable hosts are: many species of yeast, e.g. those belonging to genera Saccharomyces, Z:ygosaccharomy ces', Candida or Wuyverornyces (e.g. zygosaGararDmyces nouxii, Candida vtilis or fflayveromyces marxianus or Pichia stipitis), filamentous fungi such as those from genera Aspergi/lus, Penicillium, Tnchoderna, Neurospors, Decor, Fusanum etc. (e.g. Aspergillus Niger, Penicillium roquefoti, Jnchoderrna reese/9, or bacteria such as various species of Escherchia, Coynebactenum, Bacillus, lactic acid bacteria etc. (Eschenschia colt), Coynebacterium glu famicum, Bacillus amyloliquefaciens and Lactobacillus lanais).

A genetically modified Bacillus subtilis strain is an especially pre ferred host in the microbial conversion step of the present invention. Further more, a genetically modified Lactobacillus strain is also useful In the present 1 0 invention.

When the conversion step of the present invention is carried out through arabitol patvvay, said conversion is preferably carried out using a mi crobial host selected from yeasts belonging to the genus Saccharomyces, such as Zygosaccharomyces rouxli. Examples of other yeasts which are useful in this embodiment of the invention are Candida polymorpha, Tonlopss can dida, Pichia fannosa and Torulaspora hansenii. Fungi selected from Dendry phiella saline and SchizophyJJum consomme can also be used.

Said carbon sources for the production of xylitol generally comprise carbon sources other than xylose, xylulose, mixtures of xylose and xylulose, and polymers and oligomers containing xyhee and xylulose as major compo nents. Said xylose and xylulose typically comprise D-xyloso and C)-xylulose.

The carbon source is typically a six-carbon sugar, preferably glucose, and es pecially glucose in D-configuratlon. (glucose is typically used in the forth of glu eose syrup, which is typically derived from starch. Examples of other sugars include fructose and mannose. Furthermore, useful carbon sources within the scope of the current invention are oligosaccherides and polysaccharides that comprise six-carbon sugars, for example sucrose, lactose, maltose, raffinose, inulin, starch etc. These carbon sources may be used individually or in the form of mixtures, such as, for example, inverted sugar or high-fructose syrup.

Other media for carrying out the microbial conversion stage are not limited in any way, and conventional nitrogen sources, phosphorous sources, inorganic ions and organic nutrients can be used. The nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, nitrates and the like. The phosphorous sources include potassium phosphate, sodium phosphate and the like. As inorganic ions, magnesium ions, potassium ions, calcium ions, iron ions, manganese ions, sulfate ions, or the like can be use. Suitable organic nutrients include amino acids, and liver extract, yeast extract, malt extract, peptone, meat extras, corn steep liquor, vitamins and the like.

The cultivation conditions are not in any way limited. For example, microbial conversion may be carried out for 12 to 72 hours within a pH range of S 5 to 8 and within a temperature range of 25 to 40 CC, under aerobic or anaero- bic conditions.

The microbial conversion step of the present invention is preferably carried out using a single host in one ferrnentatlon.

In said conversion step of the present invention. the desired xitol is produced and accumulated in the reaction producVfermentation broth. The conversion of the carbon source, such as glucose to xylitol in the presence of xylitol phosphate dehydrosenase activity is not totally specific, leading into the formation of minor amounts of other sugars and sugar alcohols, such as xylu lose, nbulose, ribitol etc. Ribulose and xylulose are formed as a result of ribu lose 5-phosphate and xylulose 6phosphate dephosphorylation, respect very, by Bacillus subtilis, Ribulose 5-phosphate and xylulose Phosphate are inter- mediates on the pathway to xylitol 1-phosphate, Ribitol is formed from ribulose 5-phosphate via the intermediate ribitol 5-phosphate, The reaction is catalyzed by xylitol phosphate dehydrogenase. Furthermore, the reaction mix ture/fermentation broth may include other nonesired compounds, such as acetoin, and rests of fermentation nutrients, for example.

In a preferred embodiment of the invention using glucose as the carbon source, the fermentation typically provides a xylitol yield higher than 20 %, preferably higher than 25 % and especially higher than 30%, based on the amount of the available glucose, and a xylitol purity higher than 50%, based on the dry substance content (DS).

After the conversionifermentation step, the ceil mass and other insoluble components are removed from the raw bioconversion product or fermentation broth. This step is Gamed out by conventional methods, such as centrifugation, filtering, microfiltration and the like. Furthermore, evaporation may be carried out after filtration.

In one embodiment of the invention, the raw bioconversion product as such is subjected to crystallization.

The crystallization may be carried out by any conventional crystalli zation methods, such as evaporative crystallization, cooling crystallization and precipitation crystallization, or a combination thereof. Seeding may be used, if desired. The crystallization is preferably carried out in a water solution, but an alcohol, such as ethanol, or a mixture of water and alcohol can also be used.

The xylitol crystals are recovered for instance by centrifugation or filtering.

In one embodiment of the crystallization process, the crystallization is carried out by evaporation and cooling crystallization. In this embodiment of the invention, the xylitol-containing solution obtained from the Ghromatographic fractionation is first concentrated, e.g. by evaporation, to produce a slightly supersaturated xylitol solution at a temperature of 40 to 70 C. The dry sub- stance content of the xylitol solution istypically 80 to 95% depending on the xylitol purity of the solution, The concentrated solution is subjected to cooling crystallization Before the cooling step, the concentrated solution is typically seeded with xylitol seed crystals. The seeded syrup is then subjected to grad- ual cooling and crystallization at the same time, until the crystallization yield is suitable for effective crystal separation. The crystallization yield is typically be.

tween 50 to 70 % of xylitol and the final temperature is typically 20 to 40 C depending on the purity and the dry substance content of the crystallization mass. The crystallization mass may then be mixed at the final temperature for a period of time, typically 0.S to 20 hours, "hereafter the crystals are collected, for example by centrifugation. Typically crystals are washed during centrifuga tion. A crystal cake is obtained. Optionally the crystal cake can be further puri fied by additional washing' for example with ethanol. The purified crystals are collected for example by centrifuging. Finally the crystals are dried to obtain high purity crystals.

Saict evaporation and cooling crystallization typically provides xylitol crystals with a purity higher than 95%, preferably higher than 98%, in one crys tallization step.

The crystals obtained from the first crystallization may be subjected to one or more recrystallization steps. The recrystallization may be carried out by crystallization methods described above, for instance by evaporation and/or cooling crystallization. In a typical recrystallization process, the crystals ob tained from the first crystallization are first dissolved in water, followed by filter ing, optional treatment with activated carbon, optional ion exchange treatment, concentration e.g. by evaporation, seeding and gradual cooling. The crystals obtained are collected e.g. by centrifuging.

The recrystallization provides crystalline xylitol having a typical pu rity higher than 99 % on RDS.

In the crystallization step, a crystal cake enriched in xylRol and a runoff which is low in xylitol content are obtained.

In one embodiment of the process of the present invention, the run off recovered from the crystallization may be subjected to chromatographic fractionation to obtain a solution enriched in xitol. The run-off may be thus recycled to chromatographic fractionation to obtain a further fraction enriched in xylitol. The chromatographic fractionation is carried out as deseribecl herein after.

In an alternative embodiment of the invention, the raw bioconversion product obtained from the conversion step may be first subjected to chroma tographic fractionation to obtain a solution enriched in xylitol. The solution en riched in xylitol is then subjected to crystallization. In said alternative embodi ment of the invention, the xylitol containing solution obtained as the raw bio conversion product is subjected to chronatographic fractionation to enrich xyli tol and to remove the nonesired compounds, such as nbulose, xylulose and nbitol mentioned above.

In one embodiment of the present invention, the chromatographic fractionation is carried out using a column packing material selected from cation exhange resins. The cation exchange resin may be a strongly add cation exchange resin or a weakly acid cation exchange resin. The resin may be in a monovalent metal form, such as Na+ or K. form The resin may also be in a divalent metal form, such as in Ca2+, 19192+ or Sr2+ forrn. The resin may also be in Al3+ form.

In another embodiment of the invention, the chromatographic fray tionation is earned out using a column packing material selected from anion exchange resins, preferably weakly basic anion exchange resins In an especially preferred embodiment of the invention, the chromate togphic fractionation is canted out using a strong acid cation exchange resin in a divalent metal form, typically in Ca2+forrn.

The chromatographic separation resins Epically have a polystyrene or polyacrylic skeleton. The polystyrene/polyacrylic skeleton is preferably crosslinked with divinylbenzene.

An especially preferred resin is a strongly acid cation exchange resin based on cross-linked styrendivinylbenzene. A suitable cross-linking degree of the resin is 1 to 20 % by weight, preferably 3 to 8 % by weight. The average particle size of the resin is normally 10 to 2000 Am, preferably 100 to 400,um.

Another preferred group of resins is an acrylic weakly acid cation exchange resin with carboxylic functional group cross-linked with from about 1 to about 20 %, preferably from about 3 to about 8 % divinylbenzene.

It is also possible to use zeolites as separation resins in the chroma tographic fractionation step of the present invention.

The eluent used in the chromatographic separation is typically wa ter. However, alcohols, mixtures of an alcohol and water and other aqueous solutions, such as solutions of water with salts are useful. The elusion is pref Drably carried out at a temperature from 10 to 95 C:, more preferably from 30 to 95 C, most preferably from 55 to 85 C.

The chromatography fractionation may be carried out as a batch process or a sin,ulatecl moving bed process, which may be continuous or se quential. In the simulated moving bed process, the chromatographic fractiona 1S tion typically utilizes 3 to 14 columns connected in series. The flow rate in the columns may be in the range of 0.5 to 10 m3 I(him2) of the Gross-sectional area of the column. The columns are provided with feed inlets and product outlets for feeding the feed solution and eluent into the columns and for collecting product fractions from the columns, The chrornatographic fractionation Wpically provides a xylitol fraction having a xylitol content higher than 70 %, typically 80 to 90 %, based on DS.

The xylitol yield in the chromatographic fractionation is typically more than 90%, preferably more than 95%, based on the amount of the available xylitol.

In the chromatographic fractionation, residual fractions can be taken before or after the xylitol fraction, Furthermore, recycle fractions taken from the chromatographic step can be recycled to the chromatographic fractionation or fermentation, for example.

If desired, the xylitol fraction obtained from the chromatographic fractionation can be subjected to an additional step to remove acetoin by con" ventional methods.

The solution enriched in xylitol and obtained from the chroma tographic fractionation is subjected to crystallization. The crystallization is car ried out as described above.

Preferred embodiments of the invention will be described in greater detail by the following examples, which are not construed as limiting the scope of the invention.

In the examples and throughout the specification and claims, the fol lowing definitions have been used: The xylitol yield in the conversion step refers to the percentage (%) amount of the carbon source (glucose) consorted into xylitol during the conver- sion (fermentation), based on the amount of the available glucose.

The xylitol yield in the chromatographic fractionation step refers to the percentage (%) amount of xylitol recovered from the chrornatographic fractionation compared to the amount of xylitol in the solution introduced into the chromatographic fractionation.

The xylitol yield in the crystallization step refers to the percentage (/0) amount of xylitol recovered from the crystallization mass compared to the amount of xylitol in the solution introduced into the crystallization.

The purity of xylitol obtained in the conversion, chromatograptic fractionation and crystallization step, respectively, refers to the purity calcu lated from the dry substance content (DS) of the xylitol product obtained in each step.

DS refers to the dry substance content measured by Karl Fischer ti tration, expressed as % by weight.

RDS refers to the refractometric dry substance content using a cor relation table according to pure xylitol, expressed as % by weight,

Example 1

A. Production of xylitol by fermentation Bacillus subtilis strain (;X7 1pGTK74(LRXPDH)] (constructed in am cordance with Examples 26 and 28 of WO 01/53306) was pre-cultivated in ml of LB-kan medium (including 1 % tryptone, 0.5 % yeast extract, 1 % NaCI; 25 mg/l kanarnycin, manufacturer Sigma) overnight in a shake flask at 37 C with an agitation rate of 200 rpm. The culture was used to inoculate 10 litres of LB-kan in Biostat E fermenter. The culture was grown at 37 C over night. The amount of dissolved oxygen was automatically maintained at or just belovv 10% of saturation. The cells from 20 litres of this culture were harvested by centrifugation (500 x 9) at 4 G and resuspended in 1 litre of cold LB-kan medium. The suspension was used to inoculate 10 litres of fresh medium (LB ken, additionally containing 100 g/l glucose and 3 mg/l chloramphenicol). The fermentation was carried out at 37 C and the amount of dissolved oxygen was controlled as described above. After 12 to 14 days of cultivation' Me femnenta- tion products were analysed by HPLC. The amounts of these products in the culture broths of three independent fermentations are commanded in Table 1'

Table 1,

Fermentation products of B. subtilis GX7 [pGTK74(LRXPDH)] , . . . . . . . Fermentation pro I_ 11 lil Analysed Products, g/l Glucose 18 32 10 Xylulose 4_ _ 1.5 _ 3 _ Ribulose 10 4 10 Xylitol 23 19 30 Ribitol 2* _ 2* 2.

Acetoin 3* 64 2* Acetic acid 4 n.d. 0.5 Lactic acid 0.5 n.d. _ 0.5 n,d. = not determined *= approximate values because of peak overlap The highest xylitol yield in the fermentation was 30% of the con- sumed glucose and the highest xylitol purity was 34% on DS.

B. Chromatographic separation of xylitol from the fermentation broth The starting material for the chromatographic separation was a di- lute culture broth obtained from the fermentation described above and include ing about 30 HO xylitol, 12 % glucose, 12 % ribulose and 3 % xylulose (all based on Rl:)S), phoshates and small amounts of other sugars and polyols.

B1. Pre-treatment of the fermentation broth After the fermentation, a sample of about 10 litres of the dilute fer- mentation broth was heated to 80 C, followed by centrifugation to remove the cells. The broth obtained Mom the centrifugation and having a dry substance concentration of 8 % by weight was concentrarated by evaporation (rotavapor), using a water bath temperature of 50 C. The pH of the soluff'on was adjusted from 6.35 to 5 using dilute HCI solution. Thereafter the solution was filtered (BuGhner funnel) using diatomaceous earth as filtering aim B.2. ChromatographiG separation The chromatography separation was carried out using a small hatch column with an inner diameter of 0.10 en in a pilot scale. The separation parameters are presented in Table 2.

Table 2.

Separation parameters for the chromatographic separation of xylitol

_

Resin type IVlitsubishi Diaion UBK 530@ DVB content of the resin, /0 6 Bead size of the resin, rnm 0.2 for! form of the resin Ca Bed hei ht of the column. m. 1.5 _. . , . Temperature, C 65 Flow rate, ml/min 50 Feed concentration, g (dry substance) 20 1 100 a

__

Feed volume, ml 465 Sample interval, rain 2 The resin (Mitshubishi Diaion USK 530) used for the cl'ron18- tographic separation was a strongly acid cation exchange resin in Ca2+ form with a polystyrene skeleton. A sample of the fermentation broth (which divas pre-treated as described in B.1) was used as the feed in the chromatographic separation. A set of fraction samples vitas collected from the first separation and analysed by HPLC (PbZ+ column) to calculate the out points of the fray tions. The separation profile was GOt into 4 fractions (residue 1, cellulose, residue 2, xylitol).

The chromatography fractionation was carried out using eight feed batches, vrhereby altogether about 250 of a capitol fraction with a yield of 100% (for each batch) was collected. The results of the chromatographic separation are presented in Figure 1. Figure 1 shows that ribulose eluted in the same peak as xylitol, whereby ribulose could not be separated from xylitol by this method. The xylitol fraction had a xylitol content of 75% and a ribulose content of 22%, based on RDS.

C. Crystallization of xylitol after chromatography S C1. Crystallization The feed liquid used for the crystallization included combined xylitol fractions obtained from the chromatographic separation of xylitol described above. The feed liquid was preoncentrated from an RDEi of 1.75 % to an RDS of 50.4 % with a vacuum laboratory evaporator using a water bath with a temperature of 70 C in 7 hours. A syrup with a liquid weight (Iw) of 564 g, an RDS of 50,4 % and 284 9 of dry solids was obtained. The synup was further concentrated to an RIDS of 90.4 % and it was subjected to cooling crystalliza tion in a rotating flask as follows: The syrup vvas cooled to 55 C and had RDS of 90,8 %. Seeding was made with 0.27 g dry seed crystals. The seeded syrup was subjected to cooling according to the following cooling program set forth In Table 3.

Table 3.

Cooling conditions in the cooling crystallization of xylitol Time, hoursTemperature, DC RDS,'/o Of mother liquor. _ O 55. 90.8 _ _ Seeding 16.5 5789.9 20.5_ 52__ 89.3 - . . 23.5 _ 4586.6 __ _ 41 343. . _B = 4 ml water addled 47.5 35. 84 a _ 65... 35 _ _ 38 Sample and _ _ Centrifiloi Centrifuging was made with a laboratory basket centrifuge Roto Si lenta (5 min / 3500 rpm, 5 ml washing water). A crystal cake with a weight of 123 9 was obtained from 262 g of crystallization mass. Drying of the crystal cake (60 min at 55 C) resulted in a drying loss of 0,32 %. The crystal purity was g8 % on RDS. The crystallization thus provided a xylitol purity of 98% starting from a purity of 75% in one crystallization step By using more effective washing, a crystal purity of over 99 % was reached as shown in the following example C2. The xylitol yield in the direct erysllization was 66.7%, based on the amount of the available xylitol.

C2. Washing of the crystals obtained from the crystallization A small sample of the dried crystals obtained from C.1 above was washed with EtOH (94 %) to remove rests of the mother liquor from the crystal surfaces. The washing was done by adding 1.0 roll EtOH to 0.8S g of the erys- tals and agitating the mixture for about 10 minutes at room temperature. Then the rests of the mother liquor were removed by a filtenog laboratory centrifuge and the crystals obtained were dried for 30 minutes at 50 C. The purity of the crystals was 99.4 % and the melting point of the crystals was 92.9 C.

C3. Recrystallization 9 of the crystals obtained from C1 were dissolved in water to obtain a syrup with an RDS of 26.6 %. Activated carbon (Norit PN39) and Kenite 200 as bodyfeed were added, both in an amount of 0 5 % on DS. The suspension was agitated at 60 DO for 30 minus and filtered through Kenite 200 filter aid. The same procedure was repeated with the addition of 1 % of the same reagents, based on DS. The resulting syrup (am g, an REDS of 14,8 %) was concentrated to an RDS of 87.6 % with a vacuum laboratory evaporator using a water teeth having a tenperture of about 65 C in 4 hours.

The cooling crystallization was started by cooling the syrup to 62 C.

At this stage, the syrup had an RDS of 87.6 %. Seeding was made with 0 06 g dry seed crystals. The cooling crystallization was continued according to the following cooling program set forth in Table 4.

Table 4.

Cooling conditions in the cooling recrystallization of xylitol Time, hours Temperature, C RDS,% 0 62 B7.6 Seeding 0.5 59 __ 1.5 55 2.5 50 Be= . . 3.5 46 _ Cooling finished 4.0 46 Sampling and Centrifugation The centrifugation was carried out with a laboratory basket centri fuge Roto Silente (S min / 3500 rpm, 5 ml washing water). A crystal cake with a weight of 45 g was obtained from 89 9 of crystallization mass. Drying of the crystal cake (60 minutes at 55 TIC) resulted in 0.02 % loss of drying The purity of the crystals was 100 % and the melting point of e crystals was 92.9 C. The xylitol yield in the recrystallization was 58.5%, based on the amount of the available xylitol.

Combined analysis results of crystallisabon tests C1, C2 and C3 are set forth in Table 5 below:

Table 5.

Crystallization results Test C1 and Cobur PH DS Meldng Ca bohydrats contents, % of DS G2 Sample ICUMSA 10-55 Hi. 0 w/w _ Xylitol Rlbub6e Sorbitol | Xylulose.

_1 4000 5.2 90.B 75 24.0 2 11300 4.9 91.6 75 1 0.1 0.9 1.4 3 1 OOQ 5.1 >9 08 1.7 0 0.3 4 25500 4.7 B5.8 50,7 36.2 1.1 2.7 6 >99 92.9 99.4 0.6 0 0 _...... _.

Test C3.

6 90.. _B a7.e; . o. . _ 7 170 5.7 H.1 58 1.0 0. 0.2 A 90 5.6 99.9 92.e 100 O O O g 5.6 25.8 95. 8 42 O 0.3 1 Seeding point 2 Crystallization massin the end 3 Dried crystals 4 Runoff Washed crystals (washing was done with ethanol) 6 Seeding point 7 Crystallization mass in the end 8 Dned crystals 9 Run-off (diluted) D. Direct crystallization of xylitol from the fermentation broth.

This example demonstrates direct crystallization of xylitol from a fermentation broth, which has been supplemented with intermediate crystals recirculated from previous steps of the crystallization process.

The starting material for the direct crystallization test was a cultivation broth obtained from the fermentation in accordance with Example 1A. The cultivation broth had a xylitol content of about 250/0 on RDS. Crystalline xylitol was dissolved into the cultivation broth to adjust the xylitol content thereof to S about 62% on RDS. The crystalline xylitol which was added to the cultivation broth contained intermediate crystals, which had a xylitol purity of more than 99 % and which had been obtained from a traditional crystallization process.

The syrup thus obtained (having a xylitol content of 62% on RDS) was treated with activated carbon in the same way as in Example C3.

The resulting syrup was concentrated by evaporation to an RDS of 92.3% in the same way as in Example C3. Cooling crystallization was then carried out in a rotating flask according to the following cooling program pre sented in Table 6. The seeding was done by adding 0.5 g xylitol seed crystals.

Table 6.

Cooling conditions in the direct crystallization of x!,itol from a fermentation broth Time. hours Temperature, C RDS' % O 57. 92.3 Seeding 46 91.5

-

23 40 90.7 _ _ a_ 38. _ 2g 88.8 42 28 88.0 Sampling and centrifugation The recovery of the crystals (centrifugation) was carried out with a laboratory basket centrifuge Roto Silenta II (3 min / 4500 rpm, 30 ml washing water). A crystal cake with a weight of Z14 9 was obtained from 661 9 of crystallization mass. The crystal cake thus obtained was then subjected to drying (about 1 h at ARC) resulting in a doing loss of 0.4%. The purity of the crystals thus obtained was 99.4% on DS. The xylitol yield in the crystallization was 55. 8% based on the amount of available xylitol. The results of the direct crystallization are presented in the following Table 7.

Table 7.

Results from the direct crystallization of xviitol from a fermentation broth Test 4 | Cobur PH DS (RDS) Meltng C:arbohydr contend, % of DS Sample IC:UhlSA 155 % 9/* wlw polot, C Xylltol Rlbulose Ribitol Xylulose _.

1 2000 4.9 94.1 67.3 5.6 1.3 2.1 _ 2 16700 4.2 92.7 61.8 3 1.2 1.4 3 700. 4.7 99.4 95.1 99.4 0 0 0.4 4 26000 4.4 85 49. ._ 5.3._. 1 9 _ 2.1 1 Seeding point Crystallization mass in the end 3 Dried and washed crystals (30 ml wash) 4 Runoff The results of Table 7 show that a culture broth including impurities which are normally present in fermentation broths can be subjected to direct crystallization of xylitol, provided that the xylitol content of the culture broth is at least about 60% on RDS.

In the crystallization tests of Examples C and D above, pH vitas measured from a solution having a concentration of 10 to 55% w/w. The melt ing point was measured with the European Pharrnacopea method. The con tents of carbohydrates were determined by HPLC with resins in Pb2+ form.

It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The in vention and its embodiments are not limited to the examples described above but or ay vary within the scope of the claims.

Claims (27)

1. A process for the production of xylitol from a carbon source other than xylose, xylulose, mixtures of xylose and xylulose, and polymers and Olin Comers containing Hose or xylulose as major components, c h a r a c t e r i z e d by the following steps: conversion of said carbon source to xylitol via ribulose-5-phosphate pathway to obtain a raw bioconversion product containing xylitol, optional chromatographic fractionation of said rave bioconversion product containing xylitol to obtain a solution enriched in xylitol, and crystallization of said raw bioconversion product or optionally said solution enriched in xylitol to obtain crystalline xylitol and a crystallization run off.

2. A process as claimed in claim 1, c h a r a c t e r i z e cl in that the process comprises a further step of subjecting said crystallization run<R to chromatographic fractionation to obtain a solution enriched in litol.

3. A process as claimed in claim 1, e h a r a c t e r i z e d in that the process comprises a further step of subjecting said crystallization runoff to further crystallization to obtain crystalline xylRol.

4.Aprocessasclaimcd in claim 1, oh aracterizedinthat said conversion is carded out in the presence of xylitol phosphate dehydro genase activity.

5. A process as claimed in claim 4, c h a r a c t e r i z e d in that said conversion is carried out by fermentation using a microbial host, which is a genetically modified microbial host, the genetic modification of which in creases the total activity of xylitol phosphate dehydrogenase during said con version as compared to said activity in said host pnor to being genetically modified.

6.Aprocessasclaimedinclaim5,characterize din that said microbial host is selected from bacteria and fungi.

7.Aprocessasclaimedinclaim5,characterie din that said microbial host is a genetically modified Bacillus subtilis strain.

8. A process as claimed in claim 1, c h a r a c t e r i z e d in that said conversion is carried out via xy itol-1-phosphate pathway.

9.Aprocessasclaimedinclaim1,characterize din that said conversion is camed out via arabitol pathway.

10.Aprocessasciaimedinclaim9,characterize din that said conversion is carried out using a microbial host selected from the genus Saccharomyces.

11.Aprocessasclaimedinclaim1,characterizedinthat said carbon source is a six-carbon sugar.

12. A process as claimed in slain 11, e h a ra c t e r I z e d in that said six-carbon sugar is glucose.

13. A process as claimed in any one of claims 1 to 12, c h a r a c t e r i z e d in that the conversion of said carbon source to ylitol is higher than 20%, preferably higher than 25%, based on the amount of the available carbon 1 0 source.

14. A process as claimed in any one of the pcecling Saints, e h a r a c t e r i z e d in that the purity of xylitol obtained from said conversion is higher than 50%, based on DS

15. A process as claimed in claim 1 or 2, characterized in 1S that said chromatographic fractionation is carried out using a column packing material selected from cation exchange resins.

16. A process as claimed in claim 15, c h a r a c t e r i z e d in that said cation exchange resin is a strongly acid cation exchange resin.

17.A process asclairredin 150r16,characterizedinthat said resin is in a monovalent metal form.

18.Aprocessasclain,edinclaim150r16,eharacterizeel in that said resin is in a divalent metal form.

19. A process as claimed in any one of claims 15 18, c h a r a c t e r i z e d in that the resin has a polystyrene skeleton crosslinked with divi nylbenzene.

20. A process as claimed in any one of claims 15 to 19, c h a r a c t e r i z e d in that said chromatographic fractionation provides xylitol with a purity higher than 70 %, based on DS, and/or a xylitol yield higher than 90 %.

21. A process as claimed in claim 1 ore, c h a ra c te ri ze d in that said crystallization comprises evaporation and cooling crystallization to obtain a crystal cake enriched in xylitol and a crystallization runoff low in xylitol content.

22. A process as claimed in claim 21, c h a r a c t e r i z e d in that said evaporation and cooling crystallization provides a xylitol purity higher than 95%, preferably higher than 95%' based on DS.

23. A process as claimed in claim 21, e h a r a e t e r i z e d in that said evaporation and cooling crystallization provides a xylitol yield of 40 to 70 %.

24. A process as claimed in claim 21, c h a r a c t e r i z e d in that said crystallization further comprises one or more recrystallization steps.

25. A process as claimed in claim 24, c h a r a c t e r i z e d in that said one or more recrystallization steps are carried out in water.

26. A process as claimed in claim 25, c h a r a c t e r I z e d in that said one or more recrystallization steps comprise evaporation and cooling crystallization.

27. A process as claimed in claim 24, 25 or 26, c h a r a c t e r i z e d in that said one or more recrystallization steps provide xylitol with pu rity higher than gem, based on DS,