Cell-immobilized beads with excellent conversion activity and method for preparing the same

Abstract

The present invention relates to cell-immobilized beads and a method for preparing the same, and more specifically, to cell-immobilized beads in which cells contained in the immobilized beads have excellent conversion activity and in which the conversion activity is maintained even during dispensing and storage, a method for preparing the cell-immobilized beads, and use of the conversion activity of the beads.

Description

Technical Field

[0001] The present invention relates to cell-immobilized beads and a method for preparing the same, and more particularly, to cell-immobilized beads in which the conversion activity of cells contained in the immobilized beads is excellent and the conversion activity is maintained even during dispensing and storage, a method for preparing cell-immobilized beads, and uses of the conversion activity of the beads. Background Art

[0002] Psicose has attracted increasing attention as a dietary sweetener, but it belongs to rare sugars and is a monosaccharide that rarely exists in nature. Therefore, in order to apply it to the food industry, it is necessary to develop a technology for efficiently producing psicose. As a conventional method for producing psicose, mainly a production method through a chemical synthesis process and a biological production method using microbial enzymes are known. For recent food materials, an environmentally friendly biological production method is more preferable than a chemical production method, and thus many studies have been conducted on the use of microorganisms in the production method of psicose.

[0003] Psicose 3-epimerase is an enzyme that converts fructose to produce psicose, which is possessed by microorganisms that produce psicose. It is dissolved extracellularly, and production methods through enzyme immobilization methods and production methods through immobilization of cell-containing carriers are being studied industrially. However, among them, a production method using a cell-immobilized carrier by a more economical method is mainly applied industrially, without a process of obtaining an enzyme from cells. As a cell-immobilized carrier for producing psicose, alginic acid suitable for producing food raw materials is used. Alginic acid is applied as a food additive in various fields such as lactic acid bacteria capsules, beverages, medical tissue engineering materials, and drug delivery systems. Since alginic acid easily forms a hydrogel, it is often used as a carrier (bead), especially for the immobilization of enzymes and cells. In addition, alginate beads using alginic acid or its salts are swellable and stretchable porous materials that randomly form β-1,4 bonds, so that cells or enzymes can be stably immobilized, which is advantageous.

[0004] However, when exposed to an environment in which microorganisms can grow in a state containing a large amount of moisture, the cell-immobilized beads of alginic acid will rot after a few hours, and thus, due to the weakening of the binding force of the beads, the activity of psicose 3-epimerase in the cells also rapidly decreases as the cells are released from the carrier, which reduces the psicose production rate in the reaction process of converting fructose to produce psicose.

[0005] In addition, in the state where alginic acid or its salt is impregnated in an aqueous solution of beads, the characteristics of porosity are well maintained, but there are problems in storing and distributing outside the aqueous solution without special chemicals and prevention of contamination. That is, the cell-immobilized alginate carrier containing water exists in a fresh water state as it was during preparation, so it is not easy to store during mass production, and there may be strict limitations such as high costs and liquid storage during distribution.

[0006] Furthermore, since the pH suitable for the activity of most enzymes and cells is mainly distributed around pH 7, they have the characteristics of swelling and decomposition due to the weakening of the binding force of alginate beads around pH 7, as well as the weakening of the binding force at high temperatures, so their industrial applications are limited.

[0007] To solve these problems, a method for preparing dry beads with excellent storage and distribution while maintaining conversion activity is needed by establishing a production process and production conditions for removing water contained in an alginate carrier on which biocatalysts such as cells and enzymes are immobilized. Summary of the Invention

[0008] Technical Problem

[0009] An object of the present invention is to provide dry beads for producing allulose, in which microbial cells of a Microbacterium strain having allulose conversion activity for producing allulose from fructose are immobilized on alginic acid or its salt, and a method for preparing dry beads for producing allulose.

[0010] Another object of the present invention is to provide reconstituted beads obtained by reconstituting dry beads for producing allulose from fructose, and a composition for producing allulose using the reconstituted beads, or a method for producing an allulose-containing product from a fructose-containing substrate using the reconstituted beads.

[0011] Yet another object of the present invention is to provide a composition for producing allulose including cell-immobilized beads in a dry form, or a method for preparing an allulose-containing product from a fructose-containing substrate using the composition.

[0012] Technical Solution

[0013] An embodiment of the present invention provides dry beads in which microbial cells of a Microbacterium strain having allulose conversion activity for producing allulose from fructose are immobilized on a carrier and dried to have a low water content, and a method for preparing dry beads, reconstituted beads obtained by reconstituting the dry beads and a method for preparing the reconstituted beads, a composition for producing allulose including the dry beads and / or the reconstituted beads, and a method for producing allulose.

[0014] The dried beads containing microbial cells immobilized on a carrier according to the present invention have a low water content, as well as excellent storage stability and thermal stability.

[0015] Furthermore, the recovery rate of the physical properties of the reconstituted beads obtained by reconstituting the dried beads is excellent, so they exhibit a level similar to the physical properties of the beads before drying, and have a relatively equivalent or similar level of allulose conversion reaction activity. The dried beads reduce the volume of the beads, and increase the production amount of allulose by increasing the column packing amount per unit volume. By preparing dried beads with high storage stability, the problems in the prior art, such as the weak binding force and decomposition of the beads due to microbial contamination of the undried beads in the hydrated state during storage, resulting in the release of cells outside the beads and a decrease in conversion activity, are solved, thus having the advantage of being able to stably provide a high production amount of allulose for a long time.

[0016] One embodiment of the present invention relates to dried beads for producing allulose, which contain alginic acid or alginate as a carrier, and microbial cells of a Microbacterium genus microorganism that produces allulose epimerase immobilized on the carrier, and the water content of the dried beads is 14% or less.

[0017] Another embodiment of the present invention relates to reconstituted beads obtained by reconstituting the dried beads using water or a fructose solution.

[0018] The water content of the dried beads according to the present invention is 14% or less, and based on the undried beads with a water content of 90% or more by volume or weight of 100%, the volume or weight of the dried beads obtained after drying can be 50% or less.

[0019] One embodiment of the present invention relates to dried beads, in which microbial cells of a Microbacterium genus strain having allulose conversion activity to produce allulose from fructose are immobilized on a carrier and dried, so that the water content of the dried beads is low.

[0020] Specifically, the dried beads according to the present invention may have at least one or more of the following properties:

[0021] (i) The water content is 14% or less,

[0022] (ii) Based on the weight of the undried beads of 100%, the weight after drying is 35% or less,

[0023] (iii) The bulk density is 0.6 Kg / L to 0.8 Kg / L,

[0024] (iv) Based on the 100% conversion activity of the freshly produced dried beads, after storage at a temperature of 25 °C for 38 weeks after production, the allulose conversion activity is 60% or more,

[0025] (v) Based on the 100% conversion activity of the freshly produced dry beads, after storage at a temperature of 60 °C for 38 weeks after production, the allulose conversion activity is 50% or more.

[0026] (vi) Based on the reaction column filling rate (volume %) of the undried beads with a water content of 90% or more based on 100%, the reaction column filling rate (volume %) is 35% or less, and

[0027] (vii) The average particle diameter of the reconstituted beads recovered from the dry beads is 1.1 mm to 1.9 mm.

[0028] A further embodiment of the present invention relates to reconstituted beads, wherein the dry beads are reconstituted using water or a fructose solution. Specifically, the reconstituted beads can have at least one or more of the following properties:

[0029] (i) The water content is 90% or more.

[0030] (ii) Based on the reaction column filling rate (volume %) of the undried beads with a water content of 90% or more based on 100%, the reaction column filling rate (volume %) is 35% or more.

[0031] (iii) The average particle diameter of the beads is 1.1 mm to 1.9 mm.

[0032] (iv) Based on the average particle diameter of 100% of the dry beads, the average particle diameter of the reconstituted beads is 120% to 190%.

[0033] (v) Based on 100% of the undried beads, the substrate feed flow rate (mL / minute) for maintaining the allulose conversion rate at 25% or more by adding a 50% by weight fructose solution is 110 to 300, and

[0034] (vi) Based on the allulose production amount of the beads before drying by 100%, the allulose production amount of the allulose conversion product obtained from the fructose-containing raw material is about 110% or more.

[0035] Specifically, the moisture content or water content of the dry beads according to the embodiments of the present invention can be 14% or less, 12% or less, 10% or less, 9.9% or less, 9.5% or less, 9.0% or less, 8.9% or less, 8.7% or less, 8.5% or less, 8% or less. The lower limit value of the water content can be 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more, and the range of the water content can be a combined range of the upper limit value and the lower limit value, such as 1% to 14%, 2% to 12%, 3% to 10%, 3% to 8%, 4% to 8%, or 5% to 8%.

[0036] For the dried beads, based on 100% by weight of the undried beads, for example, undried beads with a water content of more than 90%, the weight of the dried beads can be 35% by weight or less, 30% by weight or less, 25% by weight or less, 20% by weight or less, 17% by weight or less, 15% by weight or less, 12% by weight or less, 10% by weight or less, or 8% by weight or less, preferably 30% by weight or less, 25% by weight or less, 20% by weight or less, 17% by weight or less, 15% by weight or less, 12% by weight or less, 10% by weight or less, or 8% by weight or less. Specifically, among 100 g of the beads before drying prepared by recovering from the solution, the weight after drying can be 50 g or less, for example, 10 g or less. By measuring the weight before drying and the weight after drying, the change in the weight (mass) of the dried beads is expressed as a relative percentage value, and the weights before and after drying have the same measurement unit.

[0037] The dried beads according to the present invention have an excellent recovery rate of the physical properties of the beads after restoration, and the allulose conversion reaction efficiency shows a level similar to that of the beads before drying.

[0038] Furthermore, based on 100% of the undried beads, for example, the reaction column filling rate (volume %) of undried beads with a water content of more than 90%, the reaction column filling rate (volume %) of the dried beads can be 35% or less, 30% or less, 25% or less, 20% or less, 17% or less, 15% or less, 13% or less, or 11% or less, for example, 1.0% to 35%, 1.0% to 30%, 1.0% to 25%, 1.0% to 20%, 1.0% to 20%, 1.0% to 17%, 1.0% to 15%, 1.0% to 13%, 1.0% to 11%, 3.0% to 35%, 3.0% to 30%, 3.0% to 25%, 3.0% to 20%, 3.0% to 20%, 3.0% to 17%, 3.0% to 15%, 3.0% to 13%, 3.0% to 11%, 5.0% to 35%, 5.0% to 30%, 5.0% to 25%, 5.0% to 20%, 5.0% to 20%, 5.0% to 17%, 5.0% to 15%, 5.0% to 13%, 5.0% to 11%, 5.5% to 35%, 5.5% to 30%, 5.5% to 25%, 5.5% to 20%, 5.5% to 20%, 5.5% to 17%, 5.5% to 15%, 5.5% to 13%, or 5.5% to 11%.

[0039] Specifically, among 100 mL of the beads before drying prepared by recovering from the solution, the volume after drying can be 35 mL or less, for example, 15 mL or less. By measuring the volume before drying and the volume after drying, the change in the volume of the dried beads is expressed as a relative percentage value, and the volumes before and after drying have the same measurement unit.

[0040] For the dried beads, the water content of beads of the same weight decreases, resulting in a reduced volume and an increased density. The dried beads according to the present invention have a reduced volume and weight as compared to the beads before drying, which makes them easy to store and distribute. They have an excellent physical property recovery rate, so that the dried beads exhibit a level of physical properties similar to that of the beads before drying and have a relatively equivalent or similar level of allulose conversion reaction activity. The dried beads reduce the volume of the beads and increase the production amount of allulose by increasing the column packing amount per unit volume. By preparing dried beads with high storage stability, the problems considered to be problems in the prior art, such as the weak binding force and decomposition of the beads due to microbial contamination during storage of the undried beads in the water-containing state, resulting in the release of cells outside the beads and a decrease in the conversion activity, are solved, and thus have the advantage of being able to stably provide a high allulose production amount for a long time.

[0041] The dried beads according to the present invention have excellent storage stability and thermal stability, and thus have the advantage of maximizing the retention of the allulose conversion activity of the beads before drying. When preparing the dried beads, it is important to have physical properties and activities close to those of the beads before drying.

[0042] Specifically, based on the 100% conversion activity of the dried beads just after production, after preparing the dried beads and storing them at a temperature of 25°C for 38 hours, the allulose conversion activity of the dried beads can be 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 91% or more, 92% or more, 93% or more, or 94% or more. Based on the 100% conversion activity of the dried beads just after production, after preparing the dried beads and storing them at a temperature of 60°C for 38 weeks, the enzyme conversion activity of the dried beads can be 50% or more, 60% or more, 70% or more, or 75% or more.

[0043] An example of the measurement of storage stability is carried out under the condition that the dried beads are stored in a fructose-containing substrate solution. The stored beads are filled in a reaction column, and a substrate containing fructose with 50°Bx (%), adjusted to a temperature of 50°C and a pH value of 6.5 to 7.2, is made to flow through the reaction column filled with the beads, and an allulose conversion reaction is carried out at a flow rate maintaining an allulose conversion rate of 25% or more, and the allulose contained in the obtained conversion reaction product is quantified. The measurement method of the allulose conversion activity is the same as that of the beads before storage, and can be expressed as a percentage of the relative conversion activity based on the 100% conversion activity of allulose of the beads before storage. The fructose-containing substrate can be a fructose-containing raw material with a solid content of 40 - 60% by weight and a fructose purity of 30 - 99% (weight / weight (w / w)).

[0044] Furthermore, the drying beads according to the present invention can provide a higher conversion rate and production amount and last for a longer time in the production of allulose from a fructose-containing substrate. Specifically, for the drying beads, based on 100% of the allulose production amount of the beads before drying, by using the reconstituted beads reconstituted with a fructose solution, the allulose content of the allulose conversion reaction product obtained from a fructose-containing raw material with a solid content of 40% to 60% by weight and a fructose purity of 30% to 99% (w / w) can be about 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, or 180% or more, preferably 150% or more, 160% or more, 170% or more, or 180% or more, for example, 101% to 300%, 105% to 300%, 110% to 300%, 120% to 300%, 130% to 300%, or 140% to 300%. Preferably, the microbial cells used in the beads can be cells heat-treated at a temperature of 30°C to 70°C, and based on 100% of the allulose production amount of the beads before drying, the allulose content of the allulose conversion reaction product of the reconstituted beads reconstituted with a fructose solution can be about 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, and the upper limit value can be 205% or less, 210% or less, 220% or less, 230% or less, 250% or less, 260% or less, 270% or less, 280% or less, 290% or less, 300% or less, and the range can be a combined value of the lower limit value and the upper limit value.

[0045] For the reconstituted beads, the substrate feed flow rate (mL / minute) of the reconstituted beads that maintains the allulose conversion rate at 25% or more by adding a fructose solution can be 0.15 or more, 0.17 or more, 0.20 or more, 0.22 or more, or 0.24 or more, for example, 0.15 to 0.5, 0.17 to 0.5, 0.20 to 0.5, 0.22 to 0.5, or 0.24 to 0.5.

[0046] When reconstituting the drying beads with water or a fructose solution according to an embodiment of the present invention, for example, when filling the drying beads into a reaction column for producing allulose, the drying beads can be reconstituted into their original spherical shape in water or a fructose solution.

[0047] Specifically, based on 100% of the reaction column filling rate (volume%) of the non-dried beads, the reaction column filling rate (volume%) of the reconstituted beads obtained by reconstituting the drying beads according to the present invention can be 35% or more, 40% or more, or 45% or more.

[0048] According to an embodiment of the present invention, the dried beads need a process of restoring them to their original shape before being used in the reaction for the conversion of fructose to allulose. The restoration method can be to add the dried beads to water or the reaction substrate containing fructose to restore them to their original shape. Specifically, after putting 10 g of dried beads into a beaker, 200 mL of water is added at room temperature, and then stirred at a speed of 100 rpm for more than 30 minutes to restore the shape to near the original spherical shape.

[0049] According to an embodiment of the present invention, the dried beads before restoration are placed in a reaction column, and then the dried beads are hydrated to restore them to their original shape by a method of adding or circulating the substrate for the conversion reaction for producing allulose at a supply flow rate of 0.2 to 0.5 SV at a reaction temperature of 50 °C.

[0050] The average particle size of the restored beads obtained by restoring the dried beads can be 1.1 mm to 1.9 mm, 1.15 mm to 1.9 mm, 1.2 mm to 1.9 mm, 1.25 mm to 1.9 mm, 1.3 mm to 1.9 mm, 1.1 mm to 1.8 mm, 1.15 mm to 1.8 mm, 1.2 mm to 1.8 mm, 1.25 mm to 1.8 mm, 1.3 mm to 1.8 mm, 1.1 mm to 1.7 mm, 1.15 mm to 1.7 mm, 1.2 mm to 1.7 mm, 1.25 mm to 1.7 mm, 1.3 mm to 1.7 mm, 1.1 mm to 1.6 mm, 1.15 mm to 1.6 mm, 1.2 mm to 1.6 mm, 1.25 mm to 1.6 mm, or 1.3 mm to 1.6 mm.

[0051] Based on the average particle size (mm) of 100% of the dried beads, the average particle size (mm) of the restored beads restored from the dried beads can be 120% or more, or 130% or more. For example, 120% or more to 190%, 130% or more to 190%, 120% or more to 180%, 130% or more to 180%, 120% or more to 170%, 130% or more to 170%, 120% or more to 160%, or 130% or more to 160%.

[0052] Specifically, based on 100% of the initial reaction column filling volume, the volume can be restored to 40% or more. For example, 40% to 70%. Therefore, compared with the non-dried beads, the reaction column filling rate increases, and therefore, compared with the non-dried beads, the productivity per unit volume provided is higher.

[0053] According to an embodiment of the present invention, a method for preparing dried beads is provided, in which cells of a Microbacterium strain having allulose conversion activity to produce allulose are immobilized on a carrier and dried, so that the water content is low.

[0054] The characteristics of the drying beads are the same as those described above for the drying beads.

[0055] More specifically, the method for preparing the drying beads includes a bead formation step of immobilizing cells on a carrier and a drying step, and optionally, one or more additional steps selected from a heat treatment step for the cells used in bead formation, low-temperature curing of the beads, and a bead coating step may be carried out.

[0056] In the preparation method according to the present invention, the bead formation step can be carried out by dropping a mixed solution containing psicose-producing cells or enzymes and alginic acid or its salt as a carrier into a reaction solution containing a chloride of a divalent cation.

[0057] After the bead formation step, the method may include one or more treatment steps selected from the following steps: a step of curing the beads containing cells or enzymes, a step of washing the chloride of the divalent cation, and a step of treating the cells- or enzyme-containing beads with a fructose-containing substrate. The additional steps carried out after bead formation can be carried out by methods known to those skilled in the art and are not particularly limited.

[0058] In an example of the bead formation step, microbial cells of a strain, a culture containing an enzyme produced by the strain, or a lysate of the strain are added to an aqueous sodium alginate solution in an amount of 1 to 2 times the volume of the microbial cells of the strain, the culture containing the enzyme produced by the strain, or the lysate of the strain, and mixed, and then the resulting mixed solution is dropped into a calcium ion solution of about 0.2 M using an injection pump and a vacuum pump, thereby enabling the production of beads. The enzyme can be purified from the strain, the strain culture, or the strain lysate by conventional methods such as dialysis, precipitation, adsorption, electrophoresis, affinity chromatography, ion exchange chromatography, etc.

[0059] In the present invention, alginic acid or its salt is used as a carrier, and the alginate is not particularly limited, and examples thereof include sodium alginate, potassium alginate, magnesium alginate, ammonium alginate, etc. For example, alginic acid, sodium alginate, or potassium alginate salt as a carrier may have a viscosity of 2,000 cps to 50,000 cps. In addition, considering the formation of caking and the convenience of the preparation method, the concentration of the alginate solution in the bead formation step is 1 wt% to 10 wt%, preferably 1 wt% to 8 wt%, more preferably 2 wt% to 5 wt%, but not limited thereto.

[0060] The microbial cells producing psicose can be a wild-type strain producing psicose epimerase or a recombinant strain into which a gene encoding psicose has been introduced. Alternatively, a strain producing psicose epimerase or a recombinant strain into which a gene encoding psicose epimerase has been introduced can be obtained, and it can be the cells of the strain or an enzyme obtained from the strain.

[0061] The microbial cells to be immobilized on the beads can be cells that have been heat-treated at a temperature of 30°C to 70°C, or 30°C to 65°C, 40°C to 65°C, 50°C to 63°C, for example, 60°C, and the heat treatment can be carried out for 0.1 hour to 2 hours. The cells that produce allulose can be cells recovered from a culture medium or cells obtained by heat-treating a culture solution. Compared with the cells before heat treatment, the heat-treated cells themselves (the allulose conversion activity measured with cells instead of beads) have a relative allulose conversion activity of more than 101%, more than 105%, more than 110%, for example, 101% to 160%. In addition, when measured at 25°C before heat treatment for a cell-containing mixture having a cell concentration of 2% (w / w) and 2% (w / w) alginic acid, the viscosity of the mixed solution of cells and alginic acid or its salt in the bead formation step can be 90% or less, for example, 4,000 cps to 6,000 cps, compared with the mixed solution containing non-heat-treated cells.

[0062] In an example of the heat treatment method, the immobilized beads mixed with alginic acid and cells are dried at 20°C to 70°C to remove the water content of the beads to 14% or less, so that dry beads having a volume or weight of about 50% or less based on the initial beads before drying can be prepared.

[0063] In a specific embodiment of the present invention, the strain that produces allulose epimerase can be a strain that can produce allulose epimerase at a high yield and has high stability. Preferably, the strain can be a Microbacterium strain, for example, Microbacterium foliorum, Microbacterium oxydans, or Microbacterium phyllosphaerae, but is not limited thereto.

[0064] In the method for preparing dry beads for allulose production, it is preferably not to perform a freezing process, which can cause a decrease in cell activity during the freezing process and a rapid increase in production cost when a large amount of freeze-drying process is carried out. Specifically, the dry beads are dried using air or wind, and can be dried by a method of circulating air for several hours using air at a temperature of 20°C to 70°C, for example, 40°C to 50°C.

[0065] Specifically, in the bead formation step, the step of treating the beads with a divalent metal ion is carried out separately, or the step of coating the beads with a swelling inhibitor can be carried out after the step of treating the beads with a divalent metal ion.

[0066] In the step of treating with a divalent metal, it can be selected from Mn 2+ 、Zn 2+ 、Co2+ , Mg 2+ , Ni 2+ , Fe 2+ and Cu 2+ One or more divalent metal ion-treated beads. Specifically, this step can be carried out by loading beads containing an enzyme or cells onto an aqueous solution containing divalent metal ions or a substrate solution containing fructose, or by pouring an aqueous solution containing divalent metal ions or a substrate solution containing fructose onto a column filled with beads. The content of divalent metal ions in the aqueous solution containing divalent metal ions or the substrate solution containing fructose can be 1 mM to 15 mM, but in order to give the beads a sufficient compression effect, it is preferably treated with 5 mM to 10 mM.

[0067] The step of coating the beads with a swelling inhibitor can be carried out by immersing or adding the beads to a solution containing a swelling inhibitor. The swelling inhibitor can be at least one selected from chitosan, chitin, polyethylene glycol (PEG), polyethyleneimine (PEI), chitosan oligosaccharide, and polylysine. When chitosan oligosaccharide is used as the swelling inhibitor, the weight average molecular weight can be in the range of 700 to 9,000, but there is no particular limitation. The concentration of the swelling inhibitor solution in the coating step can be 0.1 wt% to 10 wt%, preferably 0.1 wt% to 5 wt%, but is not limited thereto.

[0068] Another embodiment of the present invention provides a composition for producing allulose containing dried beads containing microbial cells and a carrier, or a method for producing allulose using dried beads with a fructose-containing substrate.

[0069] Preferably, the method for producing allulose according to the present invention can be carried out by filling a column with beads containing an enzyme or bacterial cells and flowing a fructose-containing substrate solution through, and those skilled in the art can easily select and carry out appropriate operations according to the enzyme, cell, or immobilized carrier used.

[0070] In a specific embodiment of the present invention, when a fructose solution of a certain concentration is supplied to a packed column filled with cells containing allulose epimerase, an epimerization reaction is carried out by the immobilized cells, and fructose is converted to allulose. The converted allulose can be used as pure allulose after being separated and purified by a separation column.

[0071] The immobilized reactor used herein refers to a reactor in which a reaction for producing allulose occurs through cells or enzymes immobilized on a carrier, or through a column filled with cells or enzymes immobilized on a carrier. That is, immobilization means that a material with biological activity, in this case allulose epimerase or glucose epimerase, or cells containing them, is immobilized on a carrier.

[0072] As used herein, operational stability refers to the ability of a bioreactor to operate while maintaining an appropriate level of productivity relative to its initial activity for the continuous production of a target product such as allulose, typically expressed as an operating cycle. When producing allulose using the compression ratio according to the present invention, for example, the allulose content of the reactants obtained from a fructose-containing substrate at a concentration of 40 to 50 degrees Brix can be provided in an amount of more than 20% by weight for more than 15 days. In addition, a column filled with dry beads is supplied at a flow rate having a maximum conversion rate of 90 to 100 of the reaction product obtained from the substrate supplied under temperature conditions of 50 °C, and operational stability can be ensured under conditions where it is supplied at a constant flow rate until the allulose content decreases to less than 20% by weight.

[0073] In the method for producing allulose, based on the total amount of the reactants, the concentration of fructose used as a substrate for effective allulose production can be 40% to 75% (weight / volume (w / v)), for example 50% to 75% (w / v). When the concentration of fructose is lower than the above range, the economic efficiency decreases, while when the concentration is higher than the above range, fructose does not dissolve well. Therefore, the concentration of fructose is preferably within the above range. Fructose can be used in the form of a buffer solution or a solution dissolved in water (such as distilled water).

[0074] In the method for preparing allulose, the reaction can be carried out under conditions of pH 6 to 9.5, for example pH 7 to 9, pH 7 to 8, or pH 8 to 9. In addition, the reaction can be carried out under temperature conditions of above 30 °C, for example above 40 °C.

[0075] When the temperature rises above 80 °C, browning of fructose as a substrate may occur. Therefore, the reaction can be carried out under conditions of 40 °C to 80 °C, for example 50 °C to 75 °C, 60 °C to 75 °C, or 68 °C to 75 °C.

[0076] In addition, the longer the reaction time, the higher the allulose conversion rate. For example, the reaction time can be appropriately adjusted considering industrial and economic aspects, and conditions that maximize the efficiency of converting fructose to allulose can be selected.

[0077] The allulose obtained from fructose by the method of the present invention can be purified by conventional methods, and such determination is within the ordinary technical scope of those skilled in the art. For example, purification can be carried out by one or more methods selected from centrifugation, filtration, crystallization, ion exchange chromatography, and combinations thereof.

[0078] Beneficial Effects

[0079] Compared with the beads before drying, the dried beads, reconstituted beads, and production of allulose using the dried beads and reconstituted beads according to embodiments of the present invention are reduced in volume and weight, making them easy to store and distribute. They increase the column packing amount per unit volume, thus increasing the allulose production amount and having high storage stability. Thereby, the problems of weakening of the bead binding force and decomposition due to microbial contamination during conventional storage, resulting in the outflow of cells outside the beads and a decrease in conversion activity, are solved, and a high allulose production amount can be stably provided for a long time. Description of the Drawings

[0080] Figure 1 Photographs of the beads before drying and the beads obtained after drying are shown, where the immobilized beads are dried according to an embodiment of the present invention.

[0081] Figure 2 A graph showing the water evaporation amounts of the beads obtained before drying in Examples 1 - 3 and the dried beads obtained in Example 2.

[0082] Figure 3 Photographs of the reconstituted beads obtained before and after reconstitution of the dried beads according to an embodiment of the present invention are shown.

[0083] Figure 4 Photographs, stereomicrographs (magnification ×40), and diameters of the beads of dried beads immobilized with cells of Microbacterium oxydans and Microbacterium globiforme according to an embodiment of the present invention are shown.

[0084] Figure 5 Photographs, stereomicrographs (magnification ×40), and diameters of the beads of dried beads immobilized with cells of a recombinant strain of Corynebacterium glutaricum expressing allulose epimerase of Clostridium syndance are shown.

[0085] Figure 6 A graph showing the productivity improvement rate and relative conversion rate of the beads in the allulose conversion reaction by using cell - immobilized beads according to an embodiment of the present invention is shown. Detailed Description of the Invention

[0086] The present invention will be described in more detail with reference to the following examples, but these examples are not intended to limit the scope of the present invention.

[0087] Example 1: Preparation of Cell - Immobilized Beads

[0088] 1-1: Preparation of Microbial Cells

[0089] For the high-concentration culture of Microbacterium foliorum described in Korean Patent No. 10-1944103, each seed medium was prepared according to the composition in Table 1 below, and then autoclaved at 121 °C for more than 15 minutes to prepare each seed medium. Considering price competitiveness and productivity, the composition of each seed medium and the main medium (initial medium) was selected as shown in Table 2 below, and D-allulose 3-epimerase (DPEase) was induced with allulose to increase DPEase activity.

[0090] [Table 1]

[0091]

[0092]

[0093] To prepare the seeds, the Microbacterium foliorum parental strain stored at -70 °C was inoculated into 3 ml of the seed medium. The first-stage seed culture was carried out at 30 °C for 24 hours and then inoculated into 100 ml of the seed medium, and the second-stage seed culture was carried out at 30 °C for 24 hours. Finally, the second-stage seed medium was inoculated into 2 L of the main culture production medium using a 5 L fermenter and cultured at a culture temperature of 30 °C. The air supplied to the fermenter was used in a sterile state using a 0.2 μm air filter, and the culture was carried out according to the fermenter culture conditions in Table 2 below. In Table 2 below, the air supply unit is vvm (volume of air per liquid volume per minute, L / min).

[0094] For each pH-stat supply method, the increase in the carbon source concentration (g / L) in the medium (fermenter) when the carbon source is input once was defined as the f value, and the input time of the carbon source was adjusted so that the f value was 0.25. For the composition of the additional medium, i.e., the feeding solution, 400 g / L of allulose, 40 g / L of yeast extract, and 1 mM of MnCl2 were used. According to the basic principle of pH-stat, when the pH exceeded 6.95, sugar was added to lower the pH, and 9% ammonia water and a sugar solution with a pH in the range of 6.8 - 6.95 were used to adjust the pH. As a result, as shown in the table above, the cell concentration and enzyme activity increased.

[0095] [Table 2]

[0096] Cultivation Steps Primary Seed Cultivation Secondary Seed Cultivation Main Cultivation Cultivation Equipment Test Tube Flask 5L Fermenter Cultivation Volume 0.003L 0.1L 2L Inoculum Volume 3%(v / v) 6%(v / v) 5%(v / v) Cultivation Time 24 hours 24 hours 30 - 40 hours RPM 200 200 500-800 Air (vvm) - - 2

[0097] 1-2: Preparation of Cell-Immobilized Beads

[0098] The cultured cells are prepared into beads in the form of cells immobilized on alginic acid, so as to have the characteristics of being able to be used for a long time. Specifically, it is carried out by the following method. The cells after the completion of culturing are centrifuged to recover the cells, and distilled water is mixed to adjust the cell concentration to 4% (w / w), and then it is mixed with 4% (w / w) alginic acid dissolved in water at a ratio of 1:1. Thus, a mixed solution with a final cell concentration of 2% (w / w) and 2% (w / w) alginic acid is prepared.

[0099] The mixed solution is transferred to a silicone tube connected to a slowly operating pump, and the mixed solution is dropped drop by drop through a syringe (inner diameter 0.20 mm to 0.30 mm) connected to the end of the tube, mixed with a 100 mM calcium chloride solution and the solution is stirred to form solidified spherical or oval beads (diameter 1.8 mm to 2.2 mm).

[0100] The prepared beads can be used for the next process, but in order to further increase the internal alginic acid binding force, the prepared beads are stirred while being stored refrigerated for 4 - 6 hours, and replaced with freshly prepared 100 mM calcium chloride solution, and further solidified at a low temperature of 5°C to 15°C or below for about 6 hours. Thus, the immobilized beads in which the final cells are collected are prepared and used.

[0101] 1-3: Coating Treatment of Cell-Immobilized Beads

[0102] The alginic acid beads containing the prepared cells are passed through a mesh sieve to remove the water on the surface. After adding water with a volume twice that of the beads, it is stirred for 10 minutes. This process is repeated 3 times to remove the remaining calcium chloride solution.

[0103] The cell-immobilized beads from which calcium chloride has been removed are added to an aqueous solution of 0.5% (w / v) chitosan oligosaccharide with a volume twice that of the beads, and the mixture is stirred at room temperature for 30 minutes, and chitosan oligosaccharide is coated on the alginic acid beads. The beads coated with chitosan oligosaccharide are passed through a mesh sieve to remove the water on the surface, and then the beads are washed with water with a volume twice that of the beads, and the remaining chitosan oligosaccharide is removed through the sieve.

[0104] Example 2: Drying of cell-immobilized beads

[0105] As a specific illustrative example, the beads prepared, coated and washed in Example 1 are placed flat on a plate so as to be dried well in a drying oven, and then dried by the method of hot air circulation at 45°C (±5°C) for several hours. Drying is continued until the water contained in the beads is reduced to a level of about 14% or less. Figure 1 Photographs of the beads before drying and the beads obtained after drying are shown, in which the immobilized beads are dried according to an embodiment of the present invention.

[0106] The volume, weight, bead diameter, water content, and bead bulk density of the drying beads and the beads before drying were measured separately. The specific measurement methods and results are as follows. The bead measurement results are shown in Table 3 below. The relative values in Table 3 refer to the relative measured values of the volume, weight, diameter, moisture content, and bulk density of the beads after drying based on the value of 100 of the beads before drying.

[0107] (1) Measurement of bead volume change (reactor filling rate)

[0108] To measure the change in volume reduction of the beads before and after drying by a specific method, as shown in the photo of Figure 1 , when weighing 100 mL of the water-removed beads of Examples 1-3 in a graduated cylinder and then measuring the volume after hot air drying, the volume shown in the graduated cylinder is 10 mL, indicating that the volume has decreased by about 10% relative to the 100 volumes before drying.

[0109] (2) Measurement of bead weight change

[0110] Regarding the weight change before and after drying, the weight of the 100 g of the beads before drying recovered from the solution changed by 7.5 g after hot air drying, showing a reduction of about 7.5% relative to the weight before drying.

[0111] (3) Measurement of bead bulk density

[0112] To measure the bulk density of the beads, when filled with 100 mL of the water-removed beads before drying, its weight is 62.2 g, showing a bulk density value of 0.62 kg / L. When filling 100 mL of the drying beads by the same method, its weight is 69.9 g, showing a bulk density of 0.7 kg / L.

[0113] (4) Measurement of bead diameter distribution

[0114] For the measurement of the diameter of the beads obtained before and after hot air drying, when measuring the lengths of 20 beads separately with a measuring instrument (Mitutoyo M530-123), the average diameter of the beads before drying is 2.08 mm, but the average diameter of the beads after drying is 1.05 mm. When setting the average diameter of the beads before drying as 100%, the average diameter of the beads after drying is 50.5% relative to that before drying, indicating a reduction of 49.5%.

[0115] (5) Measurement of bead water content

[0116] For the measurement of water content, using a measuring device (A&D MX-50 Moisture Analyzer), the weight changes of 10 g of beads before drying and 10 g of beads after drying were measured under the condition of infrared irradiation at 125 °C for 30 minutes. The water content of the beads before drying was 95.9% (w / w), but the water content of the beads after drying was 9.93% (w / w). Based on the water content of 100 for the beads before drying, the water content of the beads after drying was approximately 10%.

[0117] [Table 3]

[0118]

[0119] Example 3: Restoration of Dried Beads

[0120] 3-1: Recovery of Beads

[0121] The dried beads obtained in Example 2 need to be restored to their original form before being used in the reaction for converting fructose into allulose. This restoration method can restore them to their original shape by adding the dried beads to 50 °Bx (%) crystalline fructose or water (a reaction substrate containing fructose).

[0122] Specifically, the dried beads before restoration were loaded into a reaction column, and then the conversion reaction substrate for producing allulose (crystalline fructose at 50 °Bx (%) adjusted to a temperature of 50 °C and a pH of 6.5 to 7.2) was fed or circulated at a feed flow rate of 0.2 to 0.5 SV at a reaction temperature of 50 °C. By this method, the dried beads were hydrated and restored to their original shape. Photographs of the state of the dried beads before restoration and the restored beads obtained after restoration are as Figure 3 shown.

[0123] 3-2: Characterization Analysis of Recovered Beads

[0124] As Figure 3 shown in the photographs of the beads before and after restoration, the shape of the beads after restoration was restored from the shape of the dried beads before restoration to spherical or oval beads similar to the beads before drying. Thus, after the restored beads were filled into the reaction column, the reaction with the substrate could proceed smoothly, and the beads were evenly filled in the reaction column, thereby becoming beads suitable for the reaction without the problem of channeling where the reaction substrate flows in a specific direction.

[0125] As shown in Table 4, for the analysis of the prepared beads, when measuring the lengths of 20 beads respectively using a measuring instrument (Mitutoyo M530-123), the average diameter of the beads before restoration was 1.05 mm, but the average diameter of the beads after restoration was 1.43 mm. When setting the average diameter of the beads before drying as 100%, the average diameter of the restored beads was at a level of about 68.8% relative to the beads before drying, indicating a reduction rate of 31.2%.

[0126] When the initial same bead volume was 100 mL, when filling the beads after the drying and hydration restoration steps and the beads before drying without going through the drying step into the reaction column respectively, the filling rate (volume %) of the restored beads after drying in the reaction column was filled as 52 mL, and it was confirmed that the filling rate (volume %) of the restored beads in the reaction column was 52%.

[0127] [Table 4]

[0128] Item Before Drying (Wet Beads) After Drying (Dry Beads) After Recovery Volume Change (ml) 100 - 52 Average Bead Diameter (mm) 2.08 1.05 1.43 Reaction Column Packing Rate (Volume%) 100 - 52

[0129] 3-3: Immersion Treatment of Recovered Beads

[0130] In order to enable the dried beads restored in Example 3-2 to be used in the conversion reaction, a fructose-containing substrate (pH 6.5 - 7.5) with a volume twice that of the bead volume was added, and then the mixture was stirred for 10 minutes. This process was repeated twice, and the inside of the beads was replaced with a 50 Brix (%) crystalline fructose solution.

[0131] After removing the fructose solution on the bead surface, a fructose-containing substrate treated with manganese ions (10 mM MnCl2·4H2O containing 50 Brix crystalline fructose) at least twice the bead volume was added for the full reaction of the beads, and then the mixture was slowly stirred in a 50°C constant temperature water bath for 30 to 60 minutes, and the beads were treated with manganese ions. Then, the fructose-containing substrate on the bead surface was removed through a sieve, and a fructose-containing substrate for allulose conversion (1 mM MnCl2·4H2O containing 50 Brix crystalline fructose) was added in an amount at least twice the bead volume for thoroughly washing the beads, the mixture was stirred for 10 minutes, and the substrate was replaced with a new one. Through such a process, the beads were repeatedly washed at least twice or more to finally prepare the beads for the conversion reaction.

[0132] Example 4: Preparation of Dried Beads (2)

[0133] 4-1: Heat Treatment of Cells

[0134] The Microbacterium strain cultured in Example 1-1 was heat-treated in the cultured state to improve cell activity, thereby performing a process for improving cell activity. Specifically, after completion of the culture in the fermenter, the temperature of the fermenter was gradually increased from 30 °C to 60 °C while stirring the culture solution with a cell concentration of 18.5 (OD600nm) at 150 rpm, and then maintained at 60 °C for 1 hour for cell heat treatment. Subsequently, through the process of cooling from 60 °C to 30 °C, the heat-treated cell culture solution was obtained.

[0135] 4-2: Characterization Analysis of Heat-Treated Cells

[0136] To measure the conversion activity (U / g_cell) values before and after cell heat treatment, a 35°Bx crystalline fructose solution containing MnCl2·4H2O with a final concentration of 1 mM was dissolved in 50 mM PIPES buffer (pH 7.0), and the reaction solution with a final dry cell concentration of 5 mg / ml was adjusted to a volume of 1 mL. Then, the reaction substrate solution containing cells was reacted at 70 °C for 1 hour, and then the supernatant was recovered by centrifugation, and then high-performance liquid chromatography (HPLC) analysis was performed.

[0137] Liquid chromatography analysis was performed using an RID (Refractive Index Detector, Agilent 1260RID) of HPLC (Agilent, USA) equipped with an Aminex HPX-87C column (BIO-RAD). Under the conditions of using water as the mobile phase solvent, a temperature of 80 °C, and a flow rate of 0.6 ml / minute, the cell activity was analyzed by the conversion concentration of allulose in the fructose conversion reaction. In addition, the 5 mg / mL dry cell weight used for measuring cell activity was diluted so that the absorbance of the culture solution became OD600nm 12.5. Then 1 mL was recovered, the supernatant was removed by centrifugation, and then multiplied by the dry cell conversion coefficient of 0.4 for the remaining cells, and the dry cell concentration became 5 mg. 1 mL of the reaction substrate for the conversion reaction was added to the 5 mg of the dry cell concentration from which the supernatant had been removed, and finally the final dry cell concentration was adjusted to 5 mg / mL and used for the reaction.

[0138] The comparison results of the cell concentration change and cell activity before and after cell heat treatment are shown in Table 5 below.

[0139] [Table 5]

[0140]

[0141] As shown in the results of Table 5, the cell concentration after heat treatment was slightly lower than that before heat treatment, indicating that the cell concentration decreased to about 88% compared with that before heat treatment. This is considered to be due to the partial lysis of cells caused by high temperature during the heat treatment of cells at 60°C, resulting in a decrease in cell concentration.

[0142] In addition, assuming that the allulose conversion activity of cells before heat treatment was 100%, the conversion activity was measured by heat-treating the same number of cells as before heat treatment. The result was a 115% increase compared with that before heat treatment. This is considered to be because when cells are heat-treated at 60°C, the cell wall lyses due to high temperature, forming a porous cell wall that allows the substrate to transfer more smoothly into the cytoplasm, thus increasing the allulose conversion activity of the cells.

[0143] The above results confirmed that by heat-treating cells, the decrease in cell concentration and the increase in cell conversion activity actually increased the cell input amount by about 12%. During the preparation of cell-immobilized alginate beads, the cell activity increased by about 15%, and ultimately the productivity during bead production increased by about 25% compared with that before heat treatment.

[0144] 4-3: Preparation of Cell-Immobilized Beads

[0145] The cultured cells were prepared into beads in which the cells were immobilized on alginate to have the characteristics of long-term use.

[0146] Specifically, the heat-treated culture medium was centrifuged to recover the cells, and then it was mixed with distilled water to adjust the cell concentration to 4% (w / w). The 4% (w / w) alginate dissolved in water and the recovered cells were mixed at a weight ratio of 1:1 to prepare a mixed solution with a final cell concentration of 2% (w / w) and 2% (w / w) alginate.

[0147] Compared with the mixed solution using untreated cells in Examples 1-2, the mixed solution tended to have a lower viscosity when using heat-treated cells. The viscosities of the alginate mixtures using the heat-treated cells obtained in this example and the alginate mixtures using the untreated cells obtained in Examples 1-2 were measured at a temperature of 25°C using a Brookfield viscometer. The viscosity measurement results were that the viscosity of the alginate mixture in Examples 1-2 was 5580 cps, and the viscosity of the alginate mixture obtained in this example was 4900 cps, which was 87.8% of that before heat treatment, a decrease of about 12%. This is considered to be because the polysaccharides contained in the cells were partially removed by heat treatment, so the viscosity decreased.

[0148] The viscosities (cps) of the alginate mixtures using heat-treated cells and untreated cells are shown in Table 6 below.

[0149] [Table 6]

[0150] Category Viscosity (cps) Change Rate (%) Alginic Acid Mixed Solution of Example 1-2 5,580 100％ Alginic Acid Mixed Solution of Example 4-2 4,900 87.8％

[0151] The dry beads and the beads before drying were measured for volume, weight, bead diameter, water content, and bead bulk density separately. The specific measurement methods were the same as those in Example 2, and the results are shown in Table 7 below. The relative values shown in Table 7 refer to the relative measured values of the volume, weight, diameter, moisture content, and bulk density of the beads after drying based on the value 100 of the beads before drying.

[0152] [Table 7]

[0153]

[0154] 4-4: Recovery of Dry Beads

[0155] This is the same as the dry bead restoration method in Example 3-1 and the soaking method in Example 3-3. The heat-treated cell-immobilized beads obtained in Restoration Example 4-2 were restored, and the restored dry beads were soaked in a substrate containing fructose (pH 6.5 - 7.5) so that they could be used for the conversion reaction.

[0156] As shown in Table 8, for the analysis of the prepared beads, when measuring the lengths of 20 beads separately using a measuring instrument (Mitutoyo M530-123), the average diameter of the beads before restoration was 1.02 mm, but the diameter of the beads after restoration was 1.41 mm. When calculating the relative size with the average diameter of the beads before drying set as 100%, the average diameter of the beads after drying was 52.7%, and the average diameter of the restored beads was 70.1%.

[0157] When the initial bead volume was 100 mL and the beads after the drying and hydration restoration steps and the beads before the drying step were filled into the reaction column separately, the filling volume of the restored beads after drying was 52 mL, and it was confirmed that the bead volume recovery rate was 48%.

[0158] [Table 8]

[0159] Item Before Drying (Wet Beads) After Drying (Dry Beads) After Recovery Volume Change (mL) 100 - 48 Average Bead Diameter (mm) 2.01 1.02 1.41 Reaction Column Packing Rate (Volume%) 100％ - 48％

[0160] Example 5: Preparation of Dry Beads (3)

[0161] 5-1: Preparation of Cell-Immobilized Beads and Dry Beads

[0162] To ensure that Microbacterium oxydans and Microbacterium foliorum are other species belonging to the same genus besides Microbacterium foliorum, the cells were cultured in the same manner as in Example 1-1, and the cultured cells were centrifuged to recover the cells. The cells of Microbacterium oxydans and Microbacterium foliorum are the same as those described in Korean Patent No. 10-1944104.

[0163] The cultured cells were centrifuged to recover the cells, and then the recovered cells were treated in the same manner as the method for preparing cell-immobilized beads in Examples 1-2 and the coating method for cell-immobilized beads in Examples 1-3 to prepare beads in a cell-immobilized form. The drying of the beads was carried out in the same manner as in Example 2.

[0164] 5-2: Recovery of Dried Cell-Immobilized Beads

[0165] 10 g of the immobilized beads containing dried cells of Microbacterium oxydans or Microbacterium phyllosphaerae were placed in a beaker, and then 200 mL of water was added at room temperature, and then the mixture was stirred at a speed of 100 rpm for 30 minutes to carry out the restoration process. Figure 4 The process of hydrating with distilled water to restore the bead shape before drying is gradually shown in. For the beads before drying, the dried beads, and the restored dried beads prepared for each strain, 20 beads were respectively selected, and the bead lengths were measured with a measuring device (Mitutoyo M530-123), and the results are shown in Table 9 below. It can be confirmed that the diameters of the beads prepared for each strain showed similar results before and after drying, and the restoration can be carried out at the same diameter level by this restoration method.

[0166] In addition, as Figure 4 shown in the photograph of the restored beads, it can be seen that, similar to the cells of Microbacterium foliorum, it can be restored to a shape close to a sphere.

[0167] [Table 9]

[0168]

[0169] Example 6: Preparation of Dried Beads (4)

[0170] 6-1: Preparation of Cells

[0171] The allulose epimerase-encoding gene (DPE gene; GenBank: EDS06411.1) derived from Clostridium scindens ATCC 35704 was synthesized into a polynucleotide in a modified form optimized for Escherichia coli (named CDPE), and the polynucleotide was inserted into the same restriction enzyme sites of the expression vector pCES208 using restriction enzymes NotI and XbaI (NEB) (J. Microbiol. Biotechnol., 18:639-647, 2008) to prepare a recombinant vector pCES208 / allulose epimerase (pCES_sodCDPE). The recombinant vector (pCES_sodCDPE) plasmid prepared above was transformed into Corynebacterium glutamicum by electroporation to prepare a recombinant Corynebacterium glutamicum strain expressing the CDPE enzyme. The preparation method of the recombinant Corynebacterium glutamicum strain expressing the CDPE enzyme refers to the preparation method described in Korean Patent No. 10-1607633.

[0172] 6-2: Preparation of Cell-Immobilized Beads

[0173] The strain containing the recombinant strain producing allulose epimerase obtained in Example 6-1 was cultured, and then the cells were recovered by centrifugation from the culture broth. The recovered cells were treated in the same manner as the preparation method of the cell-immobilized beads in Example 1-2 and the coating method of the cell-immobilized beads in Example 1-3 to prepare beads in a cell-immobilized form, and the beads were dried in the same manner as in Example 2.

[0174] 6-3: Recovery of Dry Beads

[0175] 10 g of the dried beads prepared in Example 6-2 were placed in a beaker, 200 mL of water was added at room temperature, and then the mixture was stirred at a speed of 100 rpm for 30 minutes for a reconstitution step and allowed to stand for 6 hours. Photographs, stereomicrographs (magnification ×40), and the diameter range of the beads of the dried beads and the reconstituted beads hydrated for 6 hours are as Figure 5 shown. The average diameter of the reconstituted beads rehydrated from the dried beads was 1.05 mm.

[0176] As shown in the Figure 5 photographs, the cell-immobilized beads of Corynebacterium glutamicum could not be reconstituted back into the original bead shape even after 6 hours of hydration, and no further changes were observed even over time.

[0177] As shown in Table 10 below, when measuring the lengths of 20 beads respectively using a measuring instrument (Mitutoyo M530-123), the average diameter of the dry beads before restoration was 0.93 mm, but the diameter of the restored beads was 1.05 mm. When setting the average bead diameter before drying as 100%, the average diameter of the restored beads was at a level of approximately 54% relative to the beads before drying, showing a reduction rate of 46%.

[0178] When the initial bead volume was the same at 100 mL, when filling the beads that had undergone the drying and hydration restoration steps and the beads before drying that had not undergone the drying step into the reaction column respectively, the filling volume of the restored beads after drying was 16 mL, and it was confirmed that the bead volume recovery rate was 16%. As a result, it was confirmed that in Figure 5 the restoration step of the photo, the recovery rate of the dried beads with immobilized Corynebacterium glutamicum cells was low, so the shape of the beads could not be restored to the shape before drying.

[0179] [Table 10]

[0180] Item Before Drying (Wet Beads) After Drying (Dry Beads) After Recovery Volume Change (ml) 100 9.1 16mL Average Bead Diameter (mm) 1.92 0.93 1.05 Reaction Column Packing Rate (Volume%) 100％ - 16％

[0181] Example 7: Conversion reaction using an immobilized biocatalyst

[0182] 7-1: Evaluation of Bead Conversion Activity

[0183] To compare the allulose conversion activities of the dried beads with immobilized cells in Example 2 and the dried beads with immobilized heat-treated cells in Example 3, a productivity comparison experiment was conducted in a reaction column. Coated beads prepared as in Example 1, beads without cell heat treatment and drying processes (bead 1), beads prepared according to Example 2 without heat treatment but with a drying process (bead 2), beads with heat-treated cells prepared according to Example 4-2 immobilized thereon without a drying process (bead 3), and dried beads with heat-treated cells prepared according to Example 4-3 immobilized thereon (bead 4) were prepared.

[0184] The beads 1 were filled into the reaction column as they were. The beads 2, 3, and 4 were restored in substantially the same manner as in Example 3 and filled into the reaction column. A substrate containing 50°Bx (wt%) fructose, adjusted to a temperature of 50 °C and a pH value of 6.5 to 7.2, was fed through the reaction column filled with the beads. An allulose conversion activity and yield experiment was carried out at a flow rate maintaining an allulose conversion rate of 25% or more, and the experimental results are shown in Table 11 below. In the productivity evaluation, in the bead filling volume of the reaction column with the same filled volume, the productivity improvement rate according to the flow rate increase relative to the flow rate of beads 1 was compared with the flow rate of the substrate with an allulose conversion rate of 25% or more from the fructose of the initial reaction substrate. That is, using beads 1 to 4, the bead filling volume (mL) of the reaction column was the same, and the reaction was carried out under the condition that the allulose conversion rate was maintained at 25% or more. Therefore, the productivity was evaluated by the flow rate (mL / min) of the substrate liquid.

[0185] As shown in Table 11, the productivity of the beads (beads 1) without drying treatment was set to 100%, and the beads 2 to 4 were filled into the reaction column in the same volume as beads 1. The fructose-containing substrate was fed according to the reaction conditions. At this time, the results of beads 2 to 4 were obtained.

[0186] As shown in Table 11, although the bead filling volume (mL) of the reaction column was the same for all beads 1 to 4, it was confirmed that due to the improvement of allulose conversion activity by drying and / or heat treatment of the cells, the flow rates maintaining the allulose conversion rate at 25% or more increased in the order of beads 1, 3, 2, and 4. That is, under the condition that the bead filling volume (mL) of the reaction column was the same, a high flow rate maintaining the allulose conversion rate at 25% or more meant a higher allulose conversion rate of the beads filled in the reaction column. Therefore, it was confirmed that beads 1 without cell heat treatment and drying processes showed the lowest allulose conversion activity, the allulose conversion activity of beads 2 without cell heat treatment but with a drying process was higher than that of beads 1, and the dried beads 4 with heat-treated cells had the highest conversion activity.

[0187] [Table 11]

[0188] Bead Substrate Feed Flow Rate (mL / minute) Productivity Increase Rate (%) Bead 1 0.13 100 Bead 2 0.24 185 Bead 3 0.19 146 Bead 4 0.35 270

[0189] Based on the results in Table 11, looking at the relative productivity values set with the productivity of Bead 1 without undergoing the drying process and the cell heat treatment process being 100, the productivities of Bead 3 and Bead 4 are 146% and 270% respectively, showing a significant increase. The factors for this productivity improvement are that through heat treatment, polysaccharides and the like in the cells are removed, the reactivity of the allulose-converting enzyme contained in the cells with the fructose substrate outside the cells increases, and the heat treatment reduces the viscosity, showing a smoother effect on the fluidity of the fructose substrate in the alginate beads, thereby improving productivity.

[0190] In addition, Bead 4 prepared by drying the alginate beads made from heat-treated cells has its filling rate increased by about 200% at the same filling volume as the undried bead (Bead 3). Finally, when Bead 4 is filled in the same volume as Bead 1 and under the same reaction conditions, the productivity of Bead 4 is increased by 270%.

[0191] 7-2: Analysis of Bead Reaction Stability

[0192] As described in Example 7-1, the dried beads (Bead 4) fixed with the heat-treated cells prepared according to Example 4-3 and the beads (Bead 1) that are unprocessed by cell heat treatment and drying process and are coated beads produced according to Example 1 are processed and filled into the reaction column in the same manner as in Example 7-1. A 0 Brix (%) crystalline fructose solution with the temperature adjusted to 50°C and pH of 6.5 - 7.2 is fed through the reaction column filled with the beads, and the reaction is carried out by setting an initial flow rate such that the conversion rate from fructose to allulose is maintained above 25%. When the value at this time is set to 100%, the reduction rate values of the reaction solution on each reaction day relative to the initial allulose conversion rate are as Figure 6 shown.

[0193] According to the results of the reduction rate relative to the initial allulose conversion rate of Figure 6 this embodiment, when the initial conversion rate of 100% of Bead 1 passes through about 35 days, the reduction relative to the initial conversion rate reaches about 55%. While at an initial conversion rate of 100%, after about 35 days, the stability of heat-treated and dried Bead 4 remains unchanged. It can be seen that the reduction rate relative to the initial conversion rate remains above about 70%. Therefore, it is confirmed that the stability of the beads dried after heat treatment is higher than that of Bead 1.

[0194] This is because when the beads are restored after the bead drying treatment, the compression rate of the beads becomes higher compared to that of Bead 1. According to the reaction temperature of 50°C and the effect of adding Na + ions to adjust the pH of the substrate to reduce the bead binding force, by imparting a relatively high binding force compared to Bead 1 prepared by the conventional method, more favorable results are obtained in terms of bead stability.

[0195] Example 8: Storage Stability of Dried Beads

[0196] The dried beads using the heat-treated cells prepared in Example 4 were stored at storage temperatures of 25°C, 30°C, 37°C, 45°C, and 60°C to evaluate the reduction rate of the conversion activity of the allulose beads according to the number of weeks of storage.

[0197] Specifically, some beads were taken according to the number of weeks of storage of the beads. The temperature of 5 mL of the reaction substrate (50° Brix crystalline fructose solution with pH adjusted to 6.5 - 7.25) was maintained at 60°C in a round flask, 0.5 g of dried beads was added, and the reaction was carried out for 2 hours to obtain the allulose conversion rate. The reduction rate of the allulose conversion activity of the stored dried beads was carried out in the same manner as in Example 7-1. The allulose conversion rate was measured and determined according to the number of weeks of storage.

[0198] According to the storage period and storage temperature of the beads, the reduction rate of the allulose conversion activity of the beads is shown in Table 12 below.

[0199] [Table 12]

[0200]

[0201] As shown in Table 12, by examining the results of the reduction rate according to the number of weeks of storage relative to the conversion rate just after the dried beads were prepared, it was confirmed that the relative activity decreased slightly with the increase in storage temperature, but at a temperature of 37°C or lower, the allulose conversion activity remained above 80% for 38 weeks. Especially at a high temperature of 60°C, the stability of the enzyme remained above 75% even for 38 weeks.

Claims

1. A dry bead for producing allulose, the dry bead comprising alginic acid or an alginate as a carrier, microbial cells of a microorganism of the genus Microbacterium that produces allulose epimerase immobilized on the carrier, and a swelling inhibitor, wherein the water content of the dry bead is 14% or less, Among them, the dry bead is a cell-immobilized bead prepared by dropping a mixed solution of the cells and the carrier into a reaction solution containing a chloride of a divalent cation, wherein the swelling inhibitor is at least one selected from chitosan, chitin, polyethylene glycol (PEG), polyethyleneimine (PEI), chitosan oligosaccharide, and polylysine, wherein the cells are heat-treated cells treated at a temperature of 50°C to 63°C, wherein the dry bead is prepared by hot air drying at a temperature above 40°C and less than 50°C, wherein the heat-treated cells have a relative allulose conversion activity of more than 110% and less than 120% compared to the cells before heat treatment, wherein the microorganism of the genus Microbacterium is Microbacterium foliorum, Microbacterium oxydans, or Microbacterium phyllosphaerae.

2. The drying beads according to claim 1, wherein, Based on the reaction column filling rate of the undried beads with a water content of 90% or more of 100 volume%, the reaction column filling rate of the dry beads is 35 volume% or less.

3. The drying beads according to claim 1, wherein Based on the average particle size of 100% of the dry beads, the average particle size of the restored beads obtained after restoration treatment with a fructose-containing substrate is 120% to 190%.

4. The drying beads according to claim 1, wherein The dry beads are restored with a fructose-containing substrate to obtain restored beads with an average particle size of 1.1 mm to 1.9 mm.

5. The drying beads according to claim 1, wherein, Based on the 100% conversion activity of the dry beads just after production, the dry beads have an allulose conversion activity of 60% or more after being stored at a temperature of 25°C for 38 weeks after production.

6. The drying beads according to claim 1, wherein, Based on the 100% conversion activity of the dry beads just after production, the dry beads have an allulose conversion activity of 50% or more after being stored at a temperature of 60°C for 38 weeks after production.

7. The drying beads according to claim 1, wherein, The average bulk density of the dry beads is 0.6 Kg / L to 0.8 Kg / L.

8. The drying beads according to claim 1, wherein, The cells are heat-treated dead cells obtained by treating a solution containing the cells at a temperature of 50°C to 63°C.

9. The drying beads according to claim 1, wherein, Based on the 100% allulose production amount of the beads without heating and drying treatment of the microbial cells, the allulose production amount of the beads restored from the dry beads with a fructose-containing raw material is a relative allulose production amount of 110% or more.

10. A method for preparing dry beads for the production of allulose, the method comprising the following steps: Cell-immobilized beads are prepared by dropping a mixed solution containing alginic acid or its salt as a carrier and microbial cells of a strain of the genus Microbacterium that produces allulose epimerase into a reaction solution containing a chloride of a divalent cation; the cell-immobilized beads are coated with a swelling inhibitor, and the beads are dried. wherein the swelling inhibitor is at least one selected from chitosan, chitin, polyethylene glycol (PEG), polyethyleneimine (PEI), chitosan oligosaccharide, and polylysine, wherein the cells are cells heat-treated at 50°C to 63°C, wherein the drying treatment is hot air drying and is carried out at a temperature above 40°C and less than 50°C, wherein the heat-treated cells have a relative psicose conversion activity of more than 110% and less than 120% compared to the cells before heat treatment, and wherein the Microbacterium microorganism is Microbacterium foliorum, Microbacterium oxydans, or Microbacterium trichosphaericum.

11. A composition for producing psicose, which contains the dried beads for producing psicose according to any one of claims 1 to 9.

12. A method for preparing reconstituted beads for producing psicose, which comprises the step of adding the dried beads according to any one of claims 1 to 9 to a fructose-containing reaction substrate or water.

13. A reconstituted bead for producing psicose, which is prepared by the method according to claim 12.

14. The reconstituted bead according to claim 13, wherein based on the reaction column filling rate of 100% by volume of the undried beads, the reaction column filling rate of the reconstituted beads is 35% by volume or more.

Images