JPH0892273A - Xylosyl fructoside crystal and its production - Google Patents

Abstract

PURPOSE: To obtain a high-purity xylosyl fructoside(XF) crystal having >=0.3mm average grain diameter and properties of noncaking such as granulated sugar from an aqueous solution of the XF without using an organic solvent such as ethanol. CONSTITUTION: This method for producing a xylosyl fructoside(XF) crystal is to concentrate an aqueous solution of the XF of <=75% purity without containing an organic solvent to a <=90% concentration as high as possible, regulate the initial starting temperature of the resultant concentrate to 60-65 deg.C, then grow the crystal while slowly reducing the temperature of the concentrate to about 30 deg.C and recover the grown XF crystal from the resultant massecuite. The recovery of the XF crystal is carried out by allowing the massecuite to stand until the viscosity thereof is reduced or increasing the temperature of the massecuite from 30 deg.C, reducing the viscosity and separating the XF crystal. The resultant XF crystal is a crystal having >=98% purity, >=0.3mm average grain size, 1/2 water of crystallization and 112.1-112.3 deg.C melting point.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a xylosylfructoside crystal having an anti-caries function, a bifidobacteria growth effect function, and an α-glucosidase inhibitory activity, and a method for producing the same.

[0002]

2. Description of the Related Art Xylosylfructoside (formal name: O-α
-D-Xylopyranosyl- (1 → 2)-β-D-Fructofranoside)
(Abbreviation: XF) is produced from sucrose and xylose by the enzymatic reaction of levan sucrose by S. Hestrin et al., And they are named xylo sucrose (J. Am. Chem. Soc. 77, 6710 (1955)). Further, it is known that XF is also produced by the transfer reaction of the enzyme β-fructofuranosidase. Regarding the industrial production method of XF, a method of producing it by using these enzymes originating from various microorganisms has been proposed (Japanese Patent Publication No. 57-58).
No. 905, No. 4-200386, No. 4-91795, No. 3-27285.
Japanese Patent Publication No. 5-70415).

XF has an anti-cariogenic function as described in JP-B-57-58905 and a function of bifidobacteria growth effect as described in JP-A-62-207286. More recently, it was discovered that it has an inhibitory effect on α-glucosidase (Japanese Patent Application No. 6-
No. 190968) has attracted attention as a sweetener or food material having various functions.

It has been reported that XF crystals were obtained by adding ethanol to an XF solution and gently concentrating (Tooru Taga et al., Carbohydr. Re.
s., 241 (1944) 63-69), but nothing about the properties and size of the crystal has been reported. Also, there is no report as to whether the crystals do not solidify like granulated sugar.

[0005]

DISCLOSURE OF THE INVENTION In order to make XF easier to use, for example, to make it easier to use in processed foods, etc., XF that does not solidify like granulated sugar is used.
Addressed the challenge of providing crystals.

[0006] According to the findings obtained by the experiments of the present inventors, the aqueous solution of XF has a high purity and is simply concentrated as in the usual sugar crystallization method. Crystal nuclei were not generated, and XF crystals could not be obtained by such a method. In addition, the present inventors
According to the method described in oru Taga et al., ethanol was added to a highly pure (95% pure) XF solution and gently concentrated. As a result, a fine white precipitate could be obtained. It was When this precipitate was observed under a microscope, no crystal plane and anisotropy could be confirmed and it was in an amorphous state. Also, this precipitate was separated from the solvent by centrifugation and left at room temperature, but the precipitate had deliquesed overnight.

From the above, even if crystals are formed by the above method using ethanol, such crystals are practically difficult. In addition, the use of ethanol in the crystallization process is costly to produce on an industrial scale, requires additional requirements that ethanol must be recovered and used, and ethanol as a foreign substance in the final crystal product. However, there is a problem in that

Therefore, in the present invention, the average particle size is 0.3 mm.
It is an object of the present invention to produce XF crystals which do not solidify like the above granulated sugar without using an organic solvent such as ethanol.

[0009]

In order to achieve the above-mentioned object, the method for producing an XF crystal of the present invention comprises: (1) an aqueous solution containing no XF organic solvent having a purity of 75% or more as high as 90% or less. Concentrate to a concentration, (2) adjust the initial temperature of the resulting concentrate to 60-65 ° C, (3) then grow the crystals while gradually cooling the temperature of the concentrate to about 30 ° C. , (4) XF grown from the white bottom
In the method for producing an XF crystal of the present invention, which comprises recovering a crystal, the method of recovering a grown XF crystal is performed by leaving the white undercoat until the viscosity of the white undercoat decreases, or Can be separated and collected after the temperature is raised above 30 ° C. to reduce the viscosity under white.

In the XF crystal production method of the present invention, an aqueous solution of XF having a purity of 75% or more and containing no organic solvent can be obtained by chromatographic separation.

Further, the XF crystal of the present invention is an orthorhombic crystal having a purity of 98% or more and an average particle size of 0.3 mm or more and a half of crystal water, which is obtained from an aqueous solution. ,
Its melting point is 112.1 to 112.3 ° C., and its specific light intensity is 5
It is characterized in that it is 4.0 to 54.2.

FIG. 1 shows a method 1 for manufacturing an XF crystal according to the present invention.
It is a flow chart of the whole process showing an example, and this flow chart also includes a flow for obtaining an XF crystal from an XF aqueous solution having a purity of 75% or more according to the present invention.

(1) Step of preparing an XF aqueous solution having a purity of 75% or more In the crystallization method of XF of the present invention, a crystallizable XF is used.
The purity of the solution is 75% or more. The reason why the purity is set to 75% or more will be clarified by the following experiment. That is,
XF crystals were recovered from an XF solution having a purity of 98.5%, and the crystals were further recovered using molasses after the recovery. The molasses purity after repeating this operation two more times for a total of four times is 75.5.
%Met. Crystallization was carried out by the method of the present invention using this molasses as a raw material.
Although it grew to about 100 to 200 μm, crystals and molasses could not be separated by centrifugation. From this, it can be seen that the crystallizable XF solution of the present invention has a purity of 75% or more.

The XF aqueous solution as a starting material in the present invention is manufactured as follows. As the XF-containing raw material, an XF-containing aqueous solution can be obtained by a known method, for example, a transfer reaction of levansucrase or β-fructofuranosidase (for example, JP-B-5-70415 and JP-B-57-58905). Japanese Patent Laid-Open No. 4-200386, Japanese Patent Laid-Open No. 4-91795, Japanese Patent Laid-Open No. 3-272.
85 method, etc.). In this method, the above-mentioned enzyme may be used, or a culture medium of cells having the above-mentioned enzyme may be used.

However, the XF aqueous solution obtained by these known methods has a purity of 50% or less. Any purification means may be used to obtain an XF aqueous solution having a purity of 75% or more, which is a raw material for the crystallization method of the present invention, but the production amount that can be provided as a food material is a ton unit per day. For the purpose of producing a large amount of crystals on an industrial production scale, a chromatographic separation method using an alkali metal or alkaline earth metal type cation ion exchange resin as a separating agent and water as a separating solvent is advantageous. In addition, JP-A-3
The method for separating and purifying XF described in JP-A-83992 is a chromatographic separation method using a chemically modified silica carrier as a separating agent. The silica carrier is the above-mentioned alkali metal or alkaline earth metal type cation ion exchange resin carrier. Since the particle size thereof is extremely small (for example, the silica carrier of the same publication has a particle size of 15 μm, the cation ion exchange resin carrier used in the present invention has a particle size of 50 to 500 μm). When packed in an industrial-scale ion-exchange resin column having a large-scale column, the pressure loss increases, and the liquid must be passed at an extremely high pressure, which is not suitable for industrial-scale separation and purification in terms of equipment.

The separation device by this chromatographic separation method includes
In particular, as an industrial scale apparatus having a large production scale, various apparatuses such as a pseudo moving bed type equipment and a continuous separation equipment of its improved equipment (for example, Japanese Patent Publication No. 42-15681, Japanese Patent Laid-Open No. 63- 158105, JP-A-2-49
The device used in Japanese Laid-Open Patent Publication No. 159) can be preferably used. However, the XF crystallization method of the present invention can be applied not only to the continuous method but also to a single-column batch operation method.

(2) Step of obtaining first seed crystal In the purification step of (1) above, an XF aqueous solution having a purity of 98% or more is obtained, and this aqueous solution is freeze-dried and left to stand. 2 for freeze-dried products
Since it absorbs moisture after a day and becomes a candy-like form, if it is stored in a refrigerator at about 4 ° C. for about 3 months, extremely minute crystal nuclei are generated while the whole is in a hard candy-like form. On the other hand, 98% or more of XF solution was concentrated to a solid content concentration of about 90%, and this was placed on a glass plate, and a very small amount of the hard candy-shaped one containing the above crystal nuclei was added to this with a spatula. When mixed vigorously, the transparent XF solution on the glass plate turns fine crystals into white and extremely fine XF crystal nuclei are obtained.

(3) Crystallization step of XF XF having a purity of 75% or more obtained in the purification step of (1) above.
An aqueous solution containing no organic solvent is prepared, and the XF aqueous solution is concentrated to a concentration of 90% or less. The initial temperature of the XF concentrate is adjusted to 60 to 65 ° C., and then the crystal is grown while gradually cooling the temperature of the concentrate to about 30 ° C.

The method for growing the crystal as described above is generally called an auxiliary crystal method, and a sugar solution having a certain purity or higher is concentrated to the solubility of the sugar or higher and the sugar to be crystallized is concentrated in the concentrated sugar solution. This is a method in which fine crystals are added, and then the temperature is gradually lowered while gently stirring, and the difference in solubility of sugar depending on temperature is used to grow crystals to a certain size. It is an effective crystallization method for sugar.

Next, it will be explained that the crystallization by the auxiliary crystal method as described above is advantageous for XF. The present inventors measured the solubility of XF crystals due to changes in temperature, and the results are shown in FIG. According to FIG. 2, the solubility of XF is 20-80.
It is clear that the temperature is higher than that of the sucrose shown as the comparative example in any temperature range of ° C, and that the gradient of the change in solubility due to the temperature difference is extremely large. The gradient of the change in solubility due to the temperature difference is very similar to that of nystose shown as a comparative example in FIG. 2, but the solubility is extremely high as compared with nystose.

The present inventors have confirmed that the growth rate of XF crystals is much slower than that of sucrose or 1-kestose. For example, when the purity is about 95%, in the case of sucrose or 1-kestose, the average particle size of the crystal is about 0.5 mm after the fine seed crystal is put.
The time required to grow to a degree is generally about 1-3.
Although it is said to be about time, it was revealed for the first time by the experiments by the present inventors that XF requires at least 10 hours or more. In the case of nystose, JP-A-6-16
As described in Japanese Patent No. 5700, its purity is 9
At 0%, the time required for crystallization is 7 to 10 hours, and it is understood that when the purity is also taken into consideration, it is considerably slower than nystose.

Therefore, as described above, the solubility of XF in water is extremely higher than that of other sugars, the difference in solubility depending on temperature is larger than that of other sugars, and the crystallization rate of XF is different. Based on these facts, which are considerably slower than those of sugar, the method of crystallization of XF from water containing no organic solvent is the decoction method of continuously concentrating and growing crystals while supplying sugar solution. It is found that the auxiliary crystal method is suitable.
Actually, when the decoction method is carried out, the growth rate of the crystals is extremely slow, so that it is extremely difficult to control the concentration and the supply rate of the sugar solution, and the crystallization is not successful.

Next, the reason for concentrating the above XF aqueous solution to a concentration as high as possible (90% or less) will be described.

In the case of precipitating XF crystals from an XF aqueous solution by the assisted crystallization method, nucleation of XF does not occur only by ordinary concentration, so it is necessary to increase the initial concentration by concentration, and to increase the initial concentration as much as possible. This is preferable from the viewpoint of increasing the recovery rate of XF crystals. However, X
The present inventor has for the first time confirmed that when the F aqueous solution is concentrated and the solid content concentration exceeds 90%, the viscosity of the liquid increases sharply and becomes candy-like, making it difficult to add and mix the seed crystals. Therefore, the initial concentration is limited to 90%, preferably 9%.
The highest possible concentration of 0% or less is set to 88% to 90%. Of course, the initial concentration may be lower than this range from the viewpoint of increasing the recovery rate of the XF crystals.

Next, the initial temperature of the XF concentrate is set to 60
The reason for adjusting the temperature to ˜65 ° C. will be described.

Since the auxiliary crystallization method is a method of crystallization by utilizing the difference in solubility caused by the temperature difference, in the crystallization method of XF, the starting temperature and the temperature drop rate of the XF concentrated liquid are large. Is an important factor in First, regarding the initial temperature, from the solubility of XF, 60 ℃, 70 ℃, 8
The solid content concentration of the saturated solution at 0 ° C. was determined, and then the supersaturation degree of the XF concentrated liquid having a solid content concentration of 80%, 85%, 88%, 90% was determined. The results are shown in Table 1 below.

[0027]

[Table 1] According to Table 1 above, when the initial temperature is 60 to 65 ° C., the supersaturation degree is 1.1 to 1, judging from the supersaturation degree of the XF concentrate.
Since the range is 1.2, this temperature range is preferable as the initial temperature for the reason described below. When a fine seed crystal is added to the XF concentrated solution having a supersaturation degree in the range of 1.1 to 1.2, the crystal grows without disappearing. However, if the initial temperature of the XF concentrate exceeds 65 ° C, X
The supersaturation degree of the F concentrate becomes less than 1.1, the growth rate of the crystal becomes slow, and the productivity of the XF crystal becomes poor.
When the degree of supersaturation is 1 or less, XF crystals are not generated and even seed crystals disappear. On the other hand, on the contrary, if the initial temperature of the XF concentrate is less than 60 ° C, the degree of supersaturation increases,
Since the temperature of the XF concentrate is low, the growth rate of the crystal becomes slow, and the viscosity of the XF concentrate increases and the diffusion rate becomes slow, resulting in a slow growth rate of the crystal.
Therefore, for the above reason, the initial temperature of the XF concentrate is set to 60 to 65 ° C.

Next, the temperature of the XF concentrate is gradually increased to about 3
The conditions for growing crystals while cooling to 0 ° C. will be described. While gently stirring the XF concentrated liquid having an initial temperature of 60 to 65 ° C., the liquid temperature is linearly lowered to 30 ° C. according to the growth rate of the crystal, and the crystal is grown while keeping the supersaturation constant. As a result of repeated trial and error experiments by the present inventors, the conditions shown in Table 2 below are the optimum conditions for growing the average grain size of crystals to about 0.5 mm. is there.

[0029]

[Table 2] When the liquid temperature was linearly cooled down to 30 ° C. in the cooling time shown in Table 2 above for gradually cooling the XF concentrate, the size of the XF crystals was the average particle size and the purity of the mother liquor was When it is more than 85%, it is almost 0.5 mm and the mother liquor purity is
When it is less than 0.1%, the grain size grows to 0.3 to 0.5 mm, and the target crystal grain size is obtained.

(4) XF crystal recovery step Immediately after the completion of the cooling step, the degree of supersaturation of the honey is still 1 or more, and the white bottom (the mixture of the crystal and the honey) has a high viscosity, and so-called stickiness causes the honey to be separated. It is bad, and even if centrifugal separation is performed in this state, the depletion is poor. Therefore, it is necessary to set the standing time shown in Table 2 above after the cooling step and to stand while gently stirring during this period. Also, as another method for improving the syrup property, a method of raising the temperature of the under-white before syrup, for example, a method of raising the syrup temperature to about 35 ° C. without leaving it for a long time, improves the syrup property. However, there is an inconvenience that the recovery rate is slightly reduced (the recovery rate is about 5% or less when calculated from the solubility). In this way, it is sufficient to decide whether to perform the syrup with a standing time or to perform the syrup with increasing the temperature in consideration of the equipment cost of the crystal recovery apparatus.

A centrifuge can be used for the XF crystal recovery apparatus, and XF crystals and molasses can be separated by the same method as the sucrose (granulated sugar) syrup operation. The separated crystals are dried at a temperature below 90 ° C, preferably below 80 ° C. The reason for using such a drying temperature is XF
The crystal has 1/2 mol of water of crystallization as water of crystallization, but when the temperature of crystallization exceeds 100 ° C., the water of crystallization abruptly separates from the XF crystal, and the crystal structure collapses when about half of the water of crystallization separates.
This is because it becomes candy-like.

The XF crystal obtained by the method of the present invention is a stable substance at ordinary temperature, is a substance which is hard to be solidified by a usual storage method, and is excellent in handleability. Therefore, the XF crystal of the present invention can be used with the same feeling as granulated sugar and can be used as any food material.

(5) Comparison between XF crystallization method from XF aqueous solution and XF crystallization method from XF ethanol solution The XF crystallization method from the XF aqueous solution of the present invention is an XF crystallization method from XF ethanol solution. Next, an experimental example showing that it is superior to the above is shown as experimental example 1.

[Experimental Example 1] A purity of 92.4% and a solid content of 8
1.36 kg of 8% XF solution was prepared. The solid content of this solution is 1.2 kg, of which XF is 1.11 k.
g included. This solution was further concentrated with a rotary evaporator to a solid content concentration of about 90%. 15 to this
Add 0 ml of ethanol, mix well, and keep the liquid temperature at 60 ° C.
Then, 1 ml of the XF fine crystal liquid was added, and the liquid temperature was linearly lowered to 30 ° C. over 10 hours while gently rotating. Then, it was left at room temperature while gently rotating. The contents were taken out 24 hours, 36 hours, and 48 hours after the introduction of the XF fine crystals, and the crystal grain size and the crystal ratio (crystallization rate or recovery rate) were determined. The crystal grain size was determined by microscopic observation. The crystallization rate was calculated by measuring the purity of the honey after 24 hours and 36 hours, and was calculated from the weight of the honey and separated crystals with a bucket centrifuge after 48 hours. The separation of the honey and the XF crystals after 24 hours and 36 hours was performed by Advantic Co., Ltd. Place the mixture of XF crystals and honey on the filter paper of a special centrifuge tube that is divided into upper and lower parts by the second filter paper.
Centrifuge at 00G. Since the honey passes through the filter paper and collects in the lower part, the purity of the honey was analyzed by a high performance liquid chromatogram (HPLC).

As a control, 1.36 kg of an XF solution having the same purity of 92.4% and a solid content of 88% was prepared, and the liquid temperature was set to 60.
C., add 1 ml of XF fine crystal liquid to it, slowly lower the liquid temperature to 30 ° C. linearly over 10 hours while gently rotating, and then let stand for 14 hours while gently rotating at room temperature, and then perform bucket-type centrifugation. Separated the nectar and crystals with a separator. The recovery was determined from the weight of the XF crystals after separating the XF crystals, and the average particle size was determined by microscopic observation. This method is a method according to the present invention. The experimental results obtained are shown in Table 3 below. The results in Table 3 are average values of three times for both the ethanol-added and control aqueous solutions.

[0036]

[Table 3] From Table 3 above, it is understood that the crystal growth rate is much slower in the case of crystallizing by adding ethanol than in the case of crystallizing from water, which requires at least 3 times longer time. Regarding the recovery rate, none of the three cases showed a higher value when ethanol was added than when it was an aqueous solution.

The use of alcohols such as ethanol and methanol in the process of crystallizing sugars is a technique often used in laboratory scale tests. In general, alcohols such as ethanol and methanol have a stronger affinity for water than sugars, so they are used for the purpose of depriving the affinity between water and sugars and facilitating the precipitation of sugar crystals. It is generally practiced to add alcohol to the solution to lower the liquid viscosity, increase the diffusion rate, increase the growth rate of sugar crystals, and facilitate separation of sugar crystals and honey after crystallization.

However, according to the above Table 3 of Experimental Example 1, although the liquid supersaturation was higher when the XF concentrated liquid was added with ethanol (other conditions were the conditions of the present invention), the crystal It can be seen that the growth rate is much slower, which is different from the effect of the above-mentioned conventional addition of alcohol. This can be said to be a factor that ethanol eventually inhibits the growth of XF crystals. As described above, the use of ethanol is meaningless for efficiently producing XF crystals, and Experimental Example 1 shows that the crystallization method from an XF aqueous solution containing no organic solvent of the present invention is highly superior. More obvious.

(6) Properties of XF crystal Crystal shape FIG. 3 shows a photograph of the XF crystal of the present invention obtained from an aqueous solution. This crystal is orthorhombic.

Identification of substance The ¹³ C-NMR spectrum of the XF crystal of the present invention is shown in FIG. this
¹³ C-NMR shows that this crystal is xylosylfructoside. Further, the molecular weight as a result of mass spectrum analysis is 312.

Elemental analysis values C = 41.05%, H = 6.41%, O = 52.54%
Met.

Theoretical elemental analysis values for the anhydrous XF, 1/2 mol of water of crystallization, and 1 mol of water of crystallization are: Anhydride: C = 42.31%, H = 6.46
%, O = 51.23% 1/2 water of crystallization: C = 41.12%, H = 6.59
%, O = 52.29% 1 mol of water of crystallization: C = 40.00%, H = 6.71
%, O = 53.29%. Therefore, from the above measurement results, the elemental analysis value of the obtained XF crystal is the closest to the theoretical value of 1/2 mol of water of crystallization.

Moisture value measured by the Karl Fischer method The water content of the XF crystal measured by this method was 2.90%.
When the water of crystallization of XF crystal is 1/2 mol, the theoretical value is 2.80%. Therefore, the water of crystallization of the XF crystal of the present invention was determined to be 1/2 mol based on the results of the above elemental analysis and the results of the measured water content.

Physical Properties Melting Point 112.1 to 112.3 ° C. However, this value is the result of measurement by the following method and is considered to change depending on the heating rate. The melting point is measured by
The melting point was measured by a melting point measuring device EP-5K manufactured by METTLER CORPORATION. X
Fill about 1 mg of F crystals into a dedicated glass capillary,
The temperature was raised from room temperature to 80 ° C at a rate of 1 ° C / min, from 80 ° C to 100 ° C at a rate of 0.5 ° C / min, and from 100 ° C at a rate of 0.2 ° C / min. Three points were measured, but all had the same results.

Specific illuminance: [α] ²⁰ _D = 54.0-54.2 ° as water-containing crystals
(C = 13, water) Anhydride conversion [α] ²⁰ _D = 55.5 to 55.8 ° (7) The reason why the melting point of XF is considered to change depending on the measuring method, and the drying temperature of the XF crystal is 80 ° C. or lower. The reason why these are desirable will be explained below in Experimental Examples 2 and 3.

[Experimental Example 2] Approximately 10 g of XF crystals having a purity of 99.9% or more were precisely weighed in an aluminum weighing can at 70 ° C and -7.
It was dried at a pressure of 00 mmHg or less for 20 hours, cooled in a desiccator for 3 hours, and the weight loss was measured. as a result,
The weight loss was 0.05%, which was a dry weight loss equivalent to that of granulated sugar. Next, the temperature was set to 100 ° C., and the loss on drying was measured once a day for 15 days continuously under the same reduced pressure condition. FIG. 5 shows the situation of weight loss on drying with respect to the elapsed days. As a result, the XF crystals in the weighing can maintained the crystal structure until the 7th day, but the crystals were melted on the candy on the 8th day (the stage when the loss on drying exceeded 1.5%). However, it was syrup-like and not colored.

[Experimental Example 3] Using a thermobalance, the dry weight loss state of the XF crystal and the thermal change of the XF crystal were observed. The temperature was raised from room temperature to 125 ° C at a rate of 17 ° C per hour. From the temperature of around 80 ℃, the weight loss increases,
The weight was reduced almost linearly up to 125 ° C. Final temperature of 12
The weight loss rate at 5 ° C. was 2.5%.

Further, the differential heat (DTA) of the XF crystal was measured using magnesium chloride as a reference substance. The result is X
FIG. 6 is a graph showing the thermal analysis process of F crystal. As shown in FIG. 6, there is an endothermic reaction at 100 to 106.5 ° C., and the state change of the XF crystal is shown. XF during this time
The weight loss rate of the crystals was 1.5 to 1.7%.

Judging from the above Experimental Examples 2 and 3, the water of crystallization of the XF crystals escaped from a temperature of around 80 ° C., and when about half of the water of crystallization escaped, the crystal structure changed and the crystal structure collapsed. It is thought to melt. From this, it is inferred that the drying temperature of the crystal is preferably 80 ° C. or lower, and the melting point of the crystal changes depending on the measuring method.

[0050]

【Example】

[Example 1] Production of XF by culture and its crude purification process 1: Scopulariopsis HS-0002 was placed on a Petri dish of potato dextrose agar.
( Scopurariopsis sp. HS-0002, FERM BP-3622) was inoculated and cultured at 30 ° C for 1 week.

Process 2: 100 ml of a medium having the following composition was placed in each of two 500 ml Erlenmeyer flasks,
After sterilizing at 121 ° C for 30 minutes in an autoclave, Scopra Liopsis SP HS-00 cultured in Petri dishes
02 was aseptically placed and cultured at 30 ° C. for 24 hours. Further, a jar-famentor having a volume of 10 liters was prepared, and 6 liters of a medium having the following composition was put therein, sterilized at 121 ° C. for 60 minutes, then cooled to 30 ° C., and then cultured in the Erlenmeyer flask having a volume of 500 ml. The culture broth was added aseptically and stirred at 30 ° C. and 300 rpm for 24 hours at an aeration rate of 1 VVm to prepare an inoculum.

Medium composition; unit: weight ratio per liquid volume Sucrose 15% Antifoam oil (Adecan LG109) 500 ppm Corn stapler 4% Potassium dihydrogen phosphate 0.5% Magnesium sulfate 0.03% Urea 04% CaCO ₃ powder 0.1% Process 3: 20 kg sucrose, 300 ml jar mentormentor, antifoam oil (Adecanol LG1
09) 70 ml, corn stapler 6 kg, potassium dihydrogen phosphate 1 kg, magnesium sulfate 60 g, Ca
Add 200 g of CO ₃ powder (each unit of the above medium composition is
(Weight ratio per liquid volume), dissolve with water to make the volume 140
It was made into liter and sterilized at 121 ° C. for 15 minutes.
Next, 20 kg of xylose was dissolved in water to make the volume 60.
After sterilizing to a liter and sterilizing at 121 ° C. for 15 minutes, it was aseptically placed in the above-mentioned 300 liter volume jar-famentor. After adjusting the temperature of the solution in the jarfamenter to 30 ° C. and aseptically inserting 6 liters of the inoculum obtained in the process 2 above, the liquid temperature was 30 ° C., the rotation speed of the stirrer was 300 rpm, and the aeration rate was 1 It culture | cultivated at / 2VVm for 80 hours.

Process 4: After culturing, the solution temperature in the jar-famenter is sterilized by keeping it at 80 ° C. for 5 minutes, then cooled to 60 ° C., and cultured with the cells using a filter press having a filtration area of 1 m ^2. Separated into liquid. Powdered calcium oxide was added to the obtained culture solution to adjust the pH to 7 to 8, calcium chloride was added to this to 0.25%, and deproteinization was carried out at 85 ° C. for 30 minutes with gentle stirring. went. The resulting precipitate was filtered with a filter using a filter paper as a filter material, and then passed through a column filled with 20 liters of a C1 type strongly basic anion resin (Mitsubishi Diaion PA308: trade name) for decolorization. This decolorizing solution is cooled to 4 ° C or lower,
This is an H-type strongly acidic cation resin (Mitsubishi Diaion P
K220) in a column packed with 35 liters and then O
H-type weakly basic anion resin (Mitsubishi Diaion WA3
0: trade name) was passed through a column packed with 35 liters for desalting.

Process 5: The desalted solution obtained in the above Process 4 was concentrated under reduced pressure of -700 mmHg (liquid temperature 40 to 50 ° C) to a solid content concentration of about 70%, and Table 4 below was used.
A sugar solution containing XF having the composition of was obtained. The values in Table 4 below are average values of sugar solutions obtained by repeating the method of Example 1 five times.

[0055]

[Table 4] Example 2 Removal of Monosaccharides by Purification Using Chromatographic Separation A pseudo moving bed type chromatographic separation device composed of 10 stainless steel columns each having a volume of 1 liter was used to remove Na.
Type strong acidic cation resin (Dowex Co., XFS-
43279: trade name, particle size 350 μm). The crude sugar solution obtained in Example 1 was passed through this chromatographic separation device to separate into a section containing a large amount of monosaccharides and a section containing XF by the chromatographic separation method. The separation conditions were as follows: each sugar solution shown in Table 5 below was diluted with water to a solid content concentration of 60% as a stock solution, and the passing temperature was 80 ° C and the stock solution flow rate was 2 ml /
Min, eluting water flow rate ml / min, 1 step (time until moving from column to the next column) 200 seconds, 1 cycle (10 steps) 33 minutes 20 seconds. The separated monosaccharide section and XF section are -700 mmHg (liquid temperature 4
The solid content concentration was concentrated to about 70% under reduced pressure (0 to 50 ° C.). The sugar composition of the concentrated XF category is shown in Table 5 below.

[0056]

[Table 5] From Table 5, it can be seen that the monosaccharides in the XF aqueous solution were almost removed by the purification treatment of Example 2.

[Example 3] X having a purity of 75% or more by purification using a chromatographic separation method
Preparation of F A glass column with a diameter of 150 mm was filled with 7 liters of Mitsubishi MCI GEL CK08P (particle size 112.5 ± 37.5 μm), which is a Na-type strongly acidic cation resin, with water. A solution prepared by diluting the monosaccharide-removed XF solution obtained in Example 2 to a solid content concentration of 35% was used as an XF stock solution, and subjected to chromatographic separation by the following treatment using the above column to obtain crystals. A high-purity XF solution used for the purification was obtained.

That is, the temperature inside the column was adjusted to 60 ° C., 140 ml of the above-mentioned XF stock solution whose temperature was adjusted to 60 ° C. was fed onto the resin in 6 minutes, and then the water whose temperature was adjusted to 60 ° C. was used as the eluting water for SV. = 0.2 / Hr at a flow rate for chromatographic separation. In this chromatographic separation, all the separations for one cycle are completed in 53 minutes from the start of feeding the XF stock solution. The chromatographic separation pattern is shown in FIG. 7 with the horizontal axis representing time (minutes) and the vertical axis representing concentration (% by weight).
53 minutes after the start of feeding the XF stock solution, a new XF stock solution was fed and the chromatographic separation was repeated by the same operation thereafter to obtain a highly pure XF solution.

The W ₁ section of FIG. 7 is 1-kestose, XF
₂ (xylosylfructosylfructoside) and sucrose. This category can be used for sweeteners with a bifidobacterial growth effect. R category (recycle category) is a category that includes XF and sucrose,
Since the XF purity of this section was 60 to 70%, it was mixed (recycled) with the XF stock solution and chromatographed again.
The P section (product section) is a section containing high-purity XF, and this section was concentrated and used as a test solution for collecting XF crystals.

The XF purity of the test solution is changed by changing the time at the boundary between the R section and the P section. For example, from 32 minutes to 39 minutes after the start of XF stock solution feed,
When 48 minutes to 48 minutes later are classified as P, the XF purity is 98% or more. Moreover, when the P classification is carried out from 37 minutes to 48 minutes later, the purity becomes 94 to 95%, and after 34 to 35 minutes, it becomes 48%.
After minutes, the purity was about 90%. See also W in FIG.
_{The second} category is a category containing monosaccharides such as xylose, fructose and glucose.

The supply of XF stock solution and water and the fractionation operation of each fraction were carried out by automatic control by a sequencer. R
Table 6 below shows the results of continuous operation for 30 days with the classification being from 32 minutes to 39 minutes and the P classification being from 39 minutes to 48 minutes. Table 6 shows the average value for 24 hours a day.

[0062]

[Table 6] From Table 6 above, it can be seen that a highly pure XF solution can be efficiently obtained by the above method. Since there was almost no pressure loss in the column during the operation of this apparatus, it is understood that this resin can be used as a separating agent and can be applied to a chromatographic separation apparatus such as a simulated moving bed system.

Next, Table 7 below shows the results obtained by changing the fractionation times of the R and P sections. Table 7 also shows the results of Table 6 together, but the purity of the recovered XF solution is reduced by accelerating the fractionation start time of the R section and the P section, but the recycled amount of the R section is reduced. And the amount of XF discharged from the W1 section are reduced, the recovery rate is increased and the amount of XF stock solution treated is increased.

[0064]

[Table 7] Example 4 Crystallization of XF Using the XF solution having a purity of 98.5% obtained in Example 3 above, XF crystals were recovered by the treatment described below.

That is, the XF solution containing 1100 g of solid content was concentrated under reduced pressure using a rotary evaporator (using a 1-liter volume eggplant type flask) so that the solid content concentration became 88 to 90% by weight. The pressure of the rotary evaporator was set to normal pressure, the temperature of the water bath of the heating source was set to 60 ° C, and the temperature of the sugar solution in the flask was raised to 60 ° C. 0.9 ml of fine crystals of XF were added to this. The XF fine crystal liquid was prepared by adding 50 ml of ethanol to 10 g of XF crystal, adding an appropriate amount of glass beads having a diameter of 5 mm, and thoroughly shaking until the crystal grain size became 0.005 to 0.01 mm. It was used.

Next, while gently rotating the rotary evaporator, the temperature of the water bath was changed from 60 ° C to 30 ° C.
The temperature decreased linearly to 10 ° C. over 10 hours. After that, the heating power source of the water bath was turned off, and the mixture was allowed to stand at room temperature for 5 hours while gently rotating. Next, after separating into crystals and grown honey by using a bucket type centrifuge, the obtained crystals were dried at 60 ° C. Repeat this operation 14 times,
Average XF crystal purity 99.9% (range 99.9-100)
%), Average XF recovery rate 58.9% (range 57.4-6)
0.2%), and XF crystals having an average crystal grain size of 450 to 550 μm were obtained. In addition, the obtained packing purity is 96.3%.
(Range 95.5 to 96.8%). The results are shown in Table 8 below.

[0067]

[Table 8] Example 5 Recovery of XF Crystals from Swinging Example 1 Using the syrup having an average purity of 96.3% obtained in Example 4 above, XF crystals were collected under the following conditions. That is,
The XF crystals were collected according to the method of Example 4, but the number of times was 6 times. Of these, 5 times, the treated solid content weight of the syrup subjected to crystallization was 10 times each.
It was carried out at 00 g, the remaining one time at 500 g. X
The amount of fine crystals of F used was a little over 0.8 ml per 1000 g, and the standing time was 7 to 10 hours. Regarding the XF crystals obtained by this operation, the average XF crystal purity was 99.8% (range 99.8 to 99.9%), the average XF.
The F recovery rate was 56.7% (range 55.5 to 58.0%), and the average crystal grain size was 450 to 550 μm. In addition, the obtained packing purity is 92.2% (range 91.4-92.9).
%)Met. The results are shown in Table 9 below.

[0068]

[Table 9] Example 6 Recovery of XF Crystal from Swing Example 2 Using the syrup having an average purity of 92.2% obtained in Example 5 above, an XF crystal was collected under the following conditions. That is,
The XF crystals were recovered according to the above-mentioned Example 4, but the number of times was two times, and the weight of the treated solid content of the syrup subjected to crystallization was 1300 g per time. The amount of fine crystals of XF used was 1.2 ml, the cooling time was 16 hours and 20 hours, and the standing time was 18 hours and 14 hours, for a total of 34 hours. XF obtained by this operation
For crystals, average XF crystal purity 99.4% (range 9
9.3 to 99.5%), average XF recovery rate 53.8% (range 53.6 to 54.0%), average crystal grain size 400 to 50.
It was 0 μm. Incidentally, the obtained tightness purity is 84.0.
% (Range 83.6-84.3%). The results are shown in Table 10 below.

[0069]

[Table 10] [Example 7] Recovery example 3 of XF crystals from syrup Using the syrup having an average purity of 84.0% obtained in Example 6, XF crystals were collected under the following conditions. That is,
The XF crystals were collected according to the above-mentioned Example 4, but the number of times of the XF crystals was once, and the weight of the treated solid content of the syrup subjected to crystallization was 1100 g per time. The amount of fine crystals of XF used was 1.0 ml, the initial temperature was 65 ° C., and the cooling time was 72 hours. After 72 hours, the XF crystals were separated from the syrup by centrifugation to obtain XF crystals. The average XF crystal purity of the obtained XF crystals was 98.8.
%, Average XF recovery rate 43.5%, average crystal grain size 350 to
It was 450 μm. Note that the obtained tightness purity is 7
It was 5.5%. The results are shown in Table 11 below.

[0070]

[Table 11] [Example 8] Purification by chromatographic separation of syrup The syrup having a purity of 75.5% obtained in the above Example 7 containing 600 g as a solid content was rotovapped under reduced pressure (using a 1 liter volume eggplant type flask). ) Was used so that the solid content concentration was about 90%. The pressure of the rotary evaporator is normal pressure, the temperature of the water bath of the heating source is 65 ° C, and the temperature of the sugar solution in the flask is 75 ° C.
Raised to. 0.8 ml of XF fine crystals was added thereto. Next, while gently rotating the rotary evaporator, the temperature of the water bath was linearly decreased from 65 ° C. to 30 ° C. over 72 hours. Then, the heating power source of the water bath was turned off, and the mixture was left standing for 24 hours at room temperature while gently rotating. Regarding the precipitated XF crystal,
As a result of measuring the particle size with a microscope, the particle size is about 10
It was confirmed that the growth was 0 to 200 μm. This X
A bucket-type centrifuge was used to separate the F crystals, and they were separated by a centrifugal force of about 1000 G for 30 minutes.
The crystals and nectar could not be separated.

Therefore, this mixture of XF crystals and honey was dissolved in warm water to a solid concentration of 35% and purified by the chromatographic separation method of Example 3 above. The purity of the obtained purified liquid was 95.3%, and the solid content of the recovered liquid was 380 g. The total amount was concentrated to a solid content concentration of 88 to 90%. XF crystals were recovered by the method of Example 5 described below. The obtained XF crystals had a purity of 99.9% and a particle size of 45.
The recovery rate was 57.2% at 0 to 550 μm.

From the results of this Example 8, it is possible to recover X
It can be seen that the purity range of the F solution is 75% or more.
In addition, according to the method of Example 8, the XF solution in which crystals can be collected can be obtained by repurifying the syrup that has been difficult to collect by the crystallization method by the chromatographic separation method.

[0073]

The XF crystal production method of the present invention can be produced from an XF aqueous solution without using an organic solvent such as ethanol. Therefore, when it is produced on an industrial scale, the production cost is low, and there is no concern that ethanol as a foreign substance will be mixed into the final crystal product.

The XF crystals obtained by the production method of the present invention are highly pure and have the property of not consolidating like granulated sugar having an average particle size of 0.3 mm or more.

Crystallization of XF from the water of the present invention to which no organic solvent such as ethanol is added has a much higher crystal growth rate than crystallization by adding an organic solvent such as ethanol.

[Brief description of drawings]

FIG. 1 is a flowchart of the entire process showing an example of a method for producing an XF crystal according to the present invention.

FIG. 2 is a graph showing changes in solubility of XF crystals of the present invention due to changes in temperature.

FIG. 3 is a micrograph showing a crystal structure of an XF crystal of the present invention.

FIG. 4 shows ¹³ C-NMR of the XF crystal of the present invention.

FIG. 5 is a graph showing a situation in which loss on drying was measured for 15 days under reduced pressure in a desiccator at a temperature of 100 ° C.

FIG. 6 is a graph showing a thermal analysis process of an XF crystal.

FIG. 7 shows a chromatographic separation pattern of XF.

─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] October 26, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 4

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0004

[Correction method] Change

[Correction content]

For XF crystals, an orthorhombic crystal was formed by adding ethanol to the XF solution and concentrating gently.
It was reported that crystals were obtained (Tooru Ta
gaet al. , Carbohydr. Res. , 2
41 (1992) 63-69), but nothing about the properties and size of crystals. Also, there is no report as to whether the crystals do not solidify like granulated sugar.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction content]

The XF crystal of the present invention is a crystal obtained from an aqueous solution, having a purity of 98% or more and an average particle size of 0.3 mm or more, and a half of crystal water having a melting point of 112. 0.1 to 112.3 ° C., specific light intensity is 54.0 to 5
It is characterized in that it is 4.2.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0034

[Correction method] Change

[Correction content]

[Experimental Example 1] A purity of 92.4% and a solid content of 8
1.36 kg of 8% XF solution was prepared. The solid content of this solution is 1.2 kg, of which XF is 1.11 k.
g included. This solution was further concentrated with a rotary evaporator to a solid content concentration of about 90%. 15 to this
Add 0 ml of ethanol, mix well, and keep the liquid temperature at 60 ° C.
Then, 1 ml of the XF fine crystal liquid was added, and the liquid temperature was linearly lowered to 30 ° C. over 10 hours while gently rotating. Then, it was left at room temperature while gently rotating. The contents were taken out 24 hours, 48 hours, and 72 hours after the introduction of the XF fine crystals, and the crystal grain size and the crystal ratio (crystallization rate or recovery rate) were determined. The crystal grain size was determined by microscopic observation. The crystallization rate was calculated by measuring the purity of the honey after 24 hours and 48 hours, and was calculated from the weight of the honey and separated crystals with a bucket centrifuge after 72 hours. Incidentally, the separation of the honey and the XF crystals after 48 hours and 72 hours was performed by No. No. manufactured by Advantic Toyo Co., Ltd. Place the mixture of XF crystals and honey on the filter paper of the special centrifuge tube that is divided into the upper and lower parts by the second filter paper.
It was centrifuged at 1000G. Since the honey passes through the filter paper and collects in the lower part, the purity of the honey was analyzed by a high performance liquid chromatogram (HPLC).

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0039

[Correction method] Change

[Correction content]

(6) Properties of XF crystal Crystal shape FIG. 3 shows a photograph of the XF crystal of the present invention obtained from an aqueous solution.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Yoshiharu Ito 1-3, Kita 4jo Nishi, Chuo-ku, Sapporo-shi, Hokkaido Hokuren Agricultural Cooperative Federation (72) Yutaka Koga Kita 4jo Nishi, Chuo-ku, Sapporo-shi, Hokkaido 1 chome 3 Hokuren Agricultural Cooperative Federation (72) Inventor Yuji Sato 1-3 chome Kita 4 Nishi, Chuo-ku, Sapporo-shi, Hokkaido Hokuren Agricultural Cooperative Federation (72) Inventor Tetsuhiro Hibino Higashi Kamikawa-gun, Hokkaido Kawamachi West 8 North 2

Claims (4)

[Claims] (1) An aqueous solution of xylosylfructoside having a purity of 75% or more containing no organic solvent is concentrated to a concentration as high as possible (90% or less), and (2) the initial temperature of the obtained concentrate is 60. Adjusted to ˜65 ° C., (3) then growing the crystals while gradually cooling the temperature of the concentrate to about 30 ° C., (4) from the obtained white bottom, grown xylosyl fructoside crystals A method for producing xylosylfructoside crystals, which comprises recovering 2. The method for recovering the grown xylosylfructoside crystals is as follows. The white undercoat is left to stand until the viscosity of the white undercoat is decreased, or the temperature of the white undercoat is raised above 30 ° C. The method for producing xylosylfructoside crystals according to claim 1, wherein the crystals are separated and recovered after the viscosity is reduced. 3. The xylosylfructoside according to claim 1, wherein the aqueous solution of xylosylfructoside having a purity of 75% or more and containing no organic solvent is obtained by chromatographic separation. Crystal manufacturing method. 4. An orthorhombic crystal having a purity of 98% or more and an average particle size of 0.3 mm or more and a half of water of crystallization, which is obtained from an aqueous solution and has a melting point of 112.1. ~ 1
A xylosyl fructoside crystal having a specific rotation of 54.3 to 54.2 at 12.3 ° C.