JPH11266894A - Production of d-xylose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an industrially advantageous production method of D-xylose by using readily available and inexpensive glucose, molasses, etc., as raw materials. SOLUTION: D-Arabitol is assimilated as much as possible in the process producing D-xylulose from D-arabitol by fermentation. Successively, D-xylulose- containing solution is prepared from D-arabitol by fermentation. Further, D- xylulose-containing solution is isomerized by using an enzyme such as glucose isomerase and D-xylose crystal is separated by crystallization from the resultant isomerization liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to a method for producing D-xylose crystals from fermentable sugars.

[0003]

2. Description of the Related Art

D-xylose reacts quickly with certain amino acids to brown, imparts a natural color to processed foods that require coloring, and enhances their flavor when processing beef, pork, fish, and the like. It is used for various foods such as chikuwa, mirinboshi, ham and sausage because it has properties such as enhancing the preservative effect of foods.

[0005] Further, xylitol, which is a reduced product of D-xylose, is metabolized independently of insulin, so that it is used as a transfusion or shock transfusion when glucose metabolism is abnormal, and has a refreshing sweetness. It is used in various foods because it does not serve as a substrate for oral bacteria.

It is known that D-xylose can be conventionally produced by hydrolyzing xylan from a plant having a high xylan content, such as cottonseed shell, bacas, wood, and coconut shell.

However, these plants not only contain glucose, arabinose, mannose and the like as sugars other than D-xylose, but also lignin, lipids, etc. Because of their various behaviors against chemicals and chemicals, it is extremely difficult to produce high-purity D-xylose from them, and even if it could be produced, much cost was required to remove the impurities.

In addition to the production method by hydrolysis of xylan, a method of producing D-xylose by fermenting glucose through several steps has also been reported.

However, for example, Japanese Unexamined Patent Publication No.
In the method described in Japanese Patent Publication No. 6, D-xylose in the total saccharide contained in the obtained D-xylose-containing solution is 53 to 64.
% In order to obtain D-xylose as crystals therefrom, the D-xylose-containing solution is subjected to chromatographic fractionation prior to its crystallization to obtain a sugar other than D-xylose 17- 22% D-xylulose, 5-
High-purity D-xylose cannot be obtained as crystals unless 15% of D-arabitol and 5 to 10% of other sugars are separated as much as possible. In addition, chromatographic fractionation
Although it is an excellent method for separating a desired component from a mixture, D-xylose is proportional to the number and amount of other components contained in a D-xylose-containing liquid as a raw material for chromatographic fractionation. Separation becomes difficult.

[0010] Further, in general, a large amount of water must be supplied to a chromatographic fraction to recover a target component from a chromatographic column. Get higher. In addition, it has been pointed out that the chromatographic fractionation is problematic in that sugars are isomerized in the chromatographic column depending on the type of resin used and operating conditions.

An object of the present invention is to solve the above-mentioned problems and to provide an industrially advantageous method for producing D-xylose from easily available and inexpensive raw materials such as glucose and molasses.

[0012]

[Means for Solving the Problems]

The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, fermentation has been carried out from D-arabitol to D-arabitol.
When D-arabitol is assimilated as much as possible in the step of producing xylulose, and the D-xylulose-containing solution prepared from D-arabitol by fermentation is further isomerized to D-xylose using an enzyme such as glucose isomerase. From the obtained isomerized solution by crystallization.
The present inventors have found that xylose crystals can be easily separated, and have completed the present invention.

That is, means for solving the problems of the present invention are as follows.

First, in a method for producing D-xylose, 1) a first step of converting fermentable saccharides into a syrup rich in D-arabitol by aerobic fermentation, and 2) a syrup rich in D-arabitol A second step of producing a syrup rich in D-xylulose by fermenting under aerobic conditions until the amount of D-arabitol contained in the total carbohydrate becomes 3% by weight or less, 3) A syrup rich in D-xylulose A fourth step of separating into D-xylose crystals and a mother liquor by crystallizing the syrups rich in D-xylose by crystallizing the syrups rich in D-xylose. A method of producing D-xylose. Secondly, in a method for producing D-xylose, 1) a first step of converting fermentable saccharides into a syrup rich in D-arabitol by aerobic fermentation, and 2) aerobic converting a syrup rich in D-arabitol. A second step of fermenting D-xylulose in a syrup rich in D-xylulose by fermenting under the conditions until the amount of D-arabitol contained in the total carbohydrates becomes 3% by weight or less. A third step of converting into a syrup rich in D-xylose by isomerization, 4) a syrup mainly consisting of D-xylose and D-xylulose, and a syrup mainly consisting of D-xylose and D by chromatographic fractionation of the syrup rich in D-xylose; A fourth step of separating into a syrup composed of a sugar other than xylulose, 5) a syrup mainly composed of D-xylose and D-xylulose Characterized by through fifth step of separating the D- xylose crystals and a mother liquor by crystallizing a flop of each step sequentially, a method for producing a D- xylose. Third, D used in the third step
The syrup rich in xylulose is the mother liquor from which the D-xylose crystals have been separated or the mother liquor and D obtained in the second step;
-The method for producing D-xylose according to the first or second aspect, wherein the mixture is a mixture with a syrup rich in xylulose.

In the first step of the present invention, the fermentable sugars include glucose, sucrose, fructose,
Molasses or mixtures thereof can be used.

In the aerobic fermentation, a known microorganism which mainly produces D-arabitol using fermentable carbohydrate as a main carbon source, for example, Yamadazyma farinosa (Yamadazyma farinosa)
inosa, Yamadazyma ohmer
i), Candida valida, Candida polymorpha, Candida krusei, Candida famata, Candida palmioleophila, Candida palmioleophila, Zygossaccarmyces Ruxie (Zygosaccharomyces rouxii), Zygosaccharomyces bailii,
Hansenula misoβ, Hansenula saturnus, Debaryomyces sake, Debaryomyces miso var.1 and other Rhodotorula yeast such as Rhodotorula graminis Used.

Aerobic fermentation using these yeasts usually has a pH of 5.0 to 6.0, a temperature of 30 to 40 ° C., and a sugar concentration of the culture solution of 10 to 45% by weight, preferably 20 to 40% by weight. At this time, soybean protein, yeast extract, corn steep liquor, and the like are added as available nitrogen sources for yeast. If necessary, various organic substances and inorganic substances necessary for the growth of yeast can be added to the culture solution.

The fermentation time is about 6 to 8 days, and usually ends when glucose is completely consumed.

The fermented culture solution is subjected to centrifugation, filtration, etc. to separate the cells or to adjust the pH to about 6.0, followed by heat sterilization to kill the yeast cells. After purifying according to activated carbon treatment, ion exchange resin purification, adsorption chromatography, ultrafiltration, etc., the mixture is subjected to the second step.

In the second step of the present invention, in order to generate D-xylulose from D-arabitol, Acetobacter suboxydans ATCC-6 belonging to the genus Acetobacter is required.
21. Gluconobacter suboxidans belonging to the genus Gluconobacter
Boxydans) IFO3172 and acetic acid bacteria such as Gluconobacter roseus IAM1840 are used.

Fermentation using these bacteria is usually carried out at pH
It is carried out at 3.5 to 6.0, at a temperature of 30 to 40 ° C., and at a sugar concentration of the culture solution of 10 to 45% by weight, preferably 30 to 40% by weight.

At this time, if the various nutrients added in the first step are left in the culture solution, the culture can be carried out only by inoculating acetic acid bacteria without adding other nutrients. It is possible to add a nitrogen compound such as soy protein, yeast extract, corn steep liquor and the like, and, if necessary, various organic and inorganic substances as a new nitrogen source that can be used by the cells.

In the fermentation, the D-
The operation is carried out until the content of arabitol is 5% by weight or less, preferably 3% by weight or less, more preferably 1% by weight or less.
When D-arabitol contained in the fermentation broth at the time of completion of the fermentation exceeds 5% by weight, D-arabitol to be performed later is
It is not preferable because the D-xylose crystal is mixed with D-arabitol as an impurity during the crystallization of xylose, which lowers the purity of the D-xylose crystal.

The obtained culture solution containing D-xylulose is
The cells are removed according to a known method such as centrifugation, and if necessary, purified, and then subjected to the third step.

In the third step of the present invention, in order to isomerize D-xylulose to D-xylose, a commercially available glucose isomerase, for example, "Sweetzyme T" (manufactured by Novo Nordisk) or "Suitase" ( Nagase Seikagaku Corporation) or the like can be used.

In general, isomerization is carried out at a pH of 3.5 to 8.0.
5. The reaction is carried out at a temperature of 30 to 65 ° C and a sugar concentration of the culture solution of 10 to 45% by weight, preferably 30 to 40% by weight.

The D-xylose-rich syrup obtained in the third step is purified according to a known method, concentrated, and crystallized to obtain D-xylose crystals.

The D-xylose-enriched syrup obtained in the third step, if D-xylose and sugars other than D-xylulose have accumulated therein, prior to crystallization of D-xylose Then, by chromatographic fractionation treatment, after separating into a syrup mainly composed of D-xylose and D-xylulose and a syrup mainly composed of sugars other than D-xylose and D-xylulose, mainly D-xylose and D-xylulose Is purified according to a known method and crystallized to obtain D.
Xylose crystals can be obtained.

Chromatographic fractionation is carried out by adding a metal ion such as calcium, aluminum, barium, sodium or strontium to a commercially available cation exchange resin, for example, a strongly acidic cation exchange resin in which sulfonic acid groups are bonded to a crosslinked polymer of styrene-divinylbenzene. D- in the column charged with
It is carried out by passing a xylose-containing liquid.

As the chromatographic fractionation, any of batch type or simulated moving bed type separation and single column type or multi column type column can be adopted.

In order to obtain high-purity D-xylose crystals by crystallization, a first step of producing D-arabitol from fermentable saccharides, a second step of producing D-xylulose from D-arabitol, In the third step of isomerizing isomer into D-xylose, it is preferable to select the conditions for carrying out each step such that the production of crystalline sugars other than D-xylose is as small as possible.

The mother liquor produced as a by-product by separating the D-xylose crystal may be the mother liquor alone or the D-xylose obtained in the second step.
Mixing with a syrup rich in xylulose and subjecting it to the third step to isomerize D-xylulose contained in the mother liquor to D-xylose can improve the productivity of D-xylose.

[0034]

【Example】

Hereinafter, the method of the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to the following examples. In the following examples,% represents% by weight unless otherwise specified.

[0036]

Embodiment 1

(Preparation of preculture solution-1) Anhydrous crystalline glucose 200 g / l, yeast extract 10 g / l, K
100 ml of a medium containing 3 g / l of H ₂ PO ₄ and 0.3 g / l of MgSO ₄ .7H ₂ O was placed in a capacity of 500
ml of Sakaguchi flask, and put in Yamadazyma ohmeri (AT
CC20209) was inoculated with a platinum loop and cultured at 35 ° C. for 2 days to obtain a pre-culture solution-1.

(Preparation of pre-culture solution-2) D-arabitol 50 g / l, yeast extract 2 g / l, KH ₂ P
2 g / l of O ₄ and 0.2 g of MgSO ₄ .7H ₂ O
g / liter of a medium containing 100 ml was placed in a 500 ml Sakaguchi flask, and Gluconobactoer suboxydans (I
One loopful of FO3172) was inoculated and cultured at 30 ° C. for 2 days to obtain a preculture-2.

(Main culture) 3 kg of anhydrous crystalline glucose, 75 g of yeast extract, 600 ml of preculture liquid-1 and 4.5 g of a defoamer (manufactured by NOF CORPORATION, "Tastehome CA-1")
23 ”) was placed in a 30-liter incubator at pH 4.5, at a temperature of 35 ° C., and at 300 rpm.
Culturing was performed for 7 days at rpm and aeration rate of 3.3 liter / min. At this time, all the glucose in the culture solution had been consumed. The culture was analyzed by liquid chromatography to find that D-arabitol was 86.0 / ml of the culture.
mg. The obtained culture solution is sterilized, 800 ml of the pre-culture solution 2 is added, and the temperature is 30 ° C and the number of rotations is 800 r.
The culture was performed for 2 days at pm and an aeration rate of 15 L / min.
At this time, D-arabitol in the culture solution was completely consumed. Next, cells were separated from the culture using a centrifuge, and then 20 g of activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd.,
The mixture was stirred at 40 ° C. for 1 hour together with “Shirasagi”, and the activated carbon was filtered off. Further, the filtrate was 500 ml of a cation ion exchange resin (“IR-120B” manufactured by Organo Corporation) and an anion ion exchange resin (“IRA” manufactured by Organo Corporation)
-410 "). The solution was passed through a column filled with 100 ml, and concentrated to a concentration of 40% at a temperature of 50 ° C or lower to obtain 2952 g of a concentrated solution. D-xylulose in this concentrate was 97.1%.

(Isomerization) Isomerase "Sweetzyme T" immobilized on a jacketed glass column (inner diameter 3 cm, height 50 cm) (Novo Nordisk Co., Ltd.)
Was concentrated at a temperature of 55 ° C. at a rate of 300 ml / h from the top of the column to perform isomerization. At this time, the sugar composition of the isomerized solution was D-xylose 7
0.3%, D-xylulose 26.8%, and other sugars 2.9%. 10 g of activated carbon (“Shirasagi”, manufactured by Takeda Pharmaceutical Co., Ltd.) was added to the obtained isomerized solution, and the temperature was 40 ° C.
After stirring at 1 ° C. for 1 hour, the activated carbon was filtered off, and the filtrate was a cation ion exchange resin (manufactured by Organo Corporation, “IR-120”).
B ") and a column filled with 100 ml of an anion ion exchange resin (" IRA-410 ", manufactured by Organo Co., Ltd.).
Concentrated to 0%.

(Crystallization) The obtained concentrated solution 1 having a concentration of 80%
446 g was put into a 2 liter crystallizer equipped with a stirrer, and gradually cooled from 60 ° C. to 20 ° C. over 16 hours to obtain a slurry containing D-xylose crystals.
In the initial stage of slow cooling, 10 g of D-xylose crystal powder was added as a seed. Next, the slurry containing D-xylose crystals was separated into crystals and a mother liquor by a centrifugal separator, and the crystals were washed with a small amount of water. Table 1 shows the weight, solid weight and sugar composition of the obtained D-xylose crystal and mother liquor.

[0042]

[Table 1]

[0043]

Embodiment 2

Mother liquor 4 obtained in the crystallization step of Example 1
A solution adjusted to a concentration of 40% by adding water to 70 g was used as a raw material for isomerization, and isomerization was performed under the same conditions as in the isomerization step of Example 1. 5 g of activated carbon ("Shirasagi", manufactured by Takeda Pharmaceutical Co., Ltd.) was added to the obtained isomerized solution, and the temperature was 40
After stirring at 1 ° C. for 1 hour, the activated carbon was filtered off, and the filtrate was a cation ion exchange resin (manufactured by Organo Corporation, “IR-120”).
B ") and 50 ml of an anion ion exchange resin (" IRA-410 ", manufactured by Organo Co., Ltd.) were passed through the column, and the mixture was further concentrated to a concentration of 77%.

(Crystallization) The obtained concentrated solution was transferred to a 1-liter crystal can with a stirrer, and gradually cooled from 55 ° C. to 20 ° C. over 16 hours to obtain a slurry containing D-xylose crystals. I got In the initial stage of slow cooling, 10 g of D-xylose crystal powder was added as a seed. Next, the slurry containing D-xylose crystals was separated into crystals and a mother liquor by a centrifugal separator, and the crystals were washed with a small amount of water. Table 2 shows the weight, solid weight, and sugar composition of the obtained D-xylose crystal and mother liquor.

[0046]

[Table 2]

[0047]

Embodiment 3

Anhydrous crystalline glucose was fermented in two steps under the same conditions as in Example 1 and further isomerized to give a sugar composition of 69.8% D-xylose and 27.1% D-xylulose.
%, And an isomerized solution having a concentration of 50%, which is 3.1% of other sugars.

(Chromatographic Fractionation Apparatus) As shown in the schematic diagram of FIG. 1, the chromatographic fractionation apparatus used in this example is a 1-liter jacketed glass tower (with an inner diameter of 3.6 c).
m, 105 cm in length) 6 towers A to F are connected in series,
A raw material charging pump G is connected to an upper portion of the tower A via a preheater K and a valve H, and a water charging pump I is connected via a preheater K and a valve J. In the chromatographic fractionation apparatus, effluent receiving tanks L and M are attached to the lower portion of the column F via valves N and O, and a valve P is attached to the tip side of the valve O. Each of the towers A to F was filled with 1000 ml of an aluminum form of a strongly acidic cation exchange resin (“CR-1310” manufactured by Organo Corporation) per one tower.

(Chromatographic fractionation) While maintaining the columns A to F at 60 ° C., the valves H and P were opened, and the valves J, N and O
Is closed, 300 ml of the 50% concentration isomerized solution
At a rate of 50 ml per minute. Next, close the valve H,
The valve J was opened, and water was supplied from the pump I at a rate of 50 ml / min for 21 minutes. This operation was repeated eight times. On the other hand, when the concentration at the outlet of the valve P became 0.2%, the valve P was closed and the valve N was opened. When the concentration at the outlet of the valve N became 4%, the valve N was closed and the valve O was opened. When the concentration at the outlet of the valve O once increased and decreased again to 4%, the valve O was closed and the valve N was opened. When the concentration at the outlet of the valve N became 0.2%, the valve N was closed and the valve P was opened. This operation was repeated on the effluent side. Weight of the effluent obtained in tank L and tank M,
Table 3 shows the concentration and sugar composition.

[0051]

[Table 3]

(Crystallization) The effluent of the tank M was made 75% in concentration.
The slurry was transferred to a 2-liter crystal can with a stirrer, and gradually cooled from 55 ° C to 20 ° C over 16 hours to obtain a slurry containing D-xylose crystals.
In the initial stage of slow cooling, 10 g of D-xylose crystal powder was added as a seed. Next, the slurry containing D-xylose crystals was separated into crystals and a mother liquor by a centrifugal separator, and the crystals were washed with a small amount of water. Table 4 shows the weight, solid weight and sugar composition of the obtained D-xylose crystal and mother liquor.

[0053]

[Table 4]

[0054]

Embodiment 4

Anhydrous crystalline glucose was fermented in two stages under the same conditions as in Example 1 and further isomerized to give a sugar composition of 68.4% D-xylose and 26.6% D-xylulose.
% And other sugars of 5.0% were prepared at a concentration of 50%.

(Chromatographic fractionation) Using the chromatographic fractionation apparatus described in Example 3, while maintaining the columns A to F at 60 ° C., the valves H and P were opened, and the valves J, N and O were opened. In a closed state, 300 ml of the 50% concentration isomerization solution was flowed at a rate of 50 ml per minute. Next, the valve H is closed, the valve J is opened, and water is pumped from the pump I at a rate of 50 ml / min.
Minutes. This operation was repeated eight times. On the other hand, when the concentration at the outlet of the valve P became 0.2%, the valve P was closed and the valve N was opened. 6% concentration at outlet of valve N
, The valve N was closed and the valve O was opened. When the concentration at the outlet of the valve O once increased and decreased again to 6%, the valve O was closed and the valve N was opened. When the concentration at the outlet of the valve N became 0.2%, the valve N was closed and the valve P was opened. This operation was repeated on the effluent side. Table 5 shows the weight, concentration and sugar composition of the effluent obtained in the tank L and the tank M.

[0057]

[Table 5]

(Crystallization) The effluent of the tank M was made 75% in concentration.
The slurry was transferred to a 2-liter crystal can with a stirrer, and gradually cooled from 55 ° C to 20 ° C over 16 hours to obtain a slurry containing D-xylose crystals.
In the initial stage of slow cooling, 10 g of D-xylose crystal powder was added as a seed. Next, the slurry containing D-xylose crystals was separated into crystals and a mother liquor by a centrifugal separator, and the crystals were washed with a small amount of water. Table 6 shows the weight, solid weight, and sugar composition of the obtained D-xylose crystal and the mother liquor.

[0059]

[Table 6]

[0060]

【The invention's effect】

In the present invention, glucose, sucrose, fructose, molasses and the like, which are easily available, can be used as a raw material for producing D-xylose. Further, the present invention can employ a crystallization method which is simple in operation and does not require special equipment, and can easily produce high-purity D-xylose crystals industrially.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a chromatographic fractionation apparatus.

[Description of Symbols] A to F towers G Feed pump H valve I Water feed pump J valve K Preheater L Outflow liquid receiving tank M Outflow liquid receiving tank N valve O valve P valve

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Mara Yamazaki 4-12-38 Iriyamase, Fuji City, Shizuoka Prefecture (72) Inventor Fumito Yamazaki 2-10-1-303, Fujimidai, Fuji City, Shizuoka Prefecture (72) Invention Naoki Okamoto 186-2 Onemoto, Matsudo, Chiba

Claims (3)

[Claims] 1. A method for producing D-xylose, comprising: 1) a first step of converting fermentable saccharides into a syrup rich in D-arabitol by aerobic fermentation; 2) aerobic converting a syrup rich in D-arabitol. A second step of fermenting D-xylulose in a syrup rich in D-xylulose under fermentation conditions until the amount of D-arabitol contained in the total carbohydrates becomes 3% by weight or less; A fourth step of separating the D-xylose crystal and mother liquor by crystallizing the D-xylose-rich syrup by isomerization with D-xylose;
A method for producing D-xylose, wherein the steps are sequentially performed. 2. A method for producing D-xylose, comprising: 1) a first step of converting fermentable saccharides into a syrup rich in D-arabitol by aerobic fermentation; 2) aerobic converting a syrup rich in D-arabitol. A second step of fermenting D-xylulose in a syrup rich in D-xylulose under fermentation conditions until the amount of D-arabitol contained in the total carbohydrates becomes 3% by weight or less; 3) a syrup composed mainly of D-xylose and D-xylulose, and a syrup composed mainly of D-xylulose by chromatographic fractionation of the syrup rich in D-xylose by isomerization with A fourth step of separating into syrups composed of sugars other than D-xylulose, 5) mainly consisting of D-xylulose and D-xylulose Fifth step, the steps characterized by through sequential method of D- xylose is separated into D- xylose crystals and a mother liquor by crystallizing a drop. 3. The D-xylulose-rich syrup used in the third step is a mother liquor from which D-xylose crystals have been separated or a mixture of the mother liquor and the D-xylulose-rich syrup obtained in the second step. The method for producing D-xylose according to claim 1, wherein the method is provided.