JPH06133791A - Low-molecular dextran - Google Patents

Abstract

PURPOSE:To obtain the subject compound having low viscosity and high anticariogenicity and useful for sweetener, etc., by reacting a high-concentration sucrose solution with dextran synthase under specific condition and reacting the product with a high-concentration sucrose solution and a solution containing dextran synthase. CONSTITUTION:The objective low-molecular dextran having an average molecular weight of about 2,000-100,000 and useful as a sweetener having high anticariogenicity, a raw material for a plasma increasing agent, a raw material for various foods, etc., is produced by culturing Leuconostoc mesenteroides strain NRRL B-512 in a medium, removing the bacterial cells by the centrifugal separation of the culture liquid, adding ethyl alcohol to the culture liquid at a low temperature, separating the precipitate to obtain a dextran synthase, adding this enzyme to a high-concentration sucrose solution and reacting at pH4-8 and 10-40 deg.C to perform the 1st stage synthesis reaction, adding an additional high-concentration sucrose solution and a solution containing dextran synthase to the reaction liquid of the 1st stage reaction and carrying out the 2nd stage reaction of the solutions one or more times.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing low-molecular-weight dextran from sucrose, and more specifically, to a reaction solution obtained by allowing dextran synthase to act on a high-concentration sucrose solution. Add the solution and the solution containing dextran synthase, allow the reaction to proceed again, and if necessary,
The present invention relates to a novel method for producing low-molecular-weight dextran directly from sucrose in high yield, which is characterized by repeating this operation. An object of the present invention is to provide a method for industrially advantageously producing a desired low-molecular-weight dextran directly by using a dextran synthase without requiring a step of hydrolyzing dextran produced in the reaction. . The low-molecular-weight dextran produced by the present invention is dextran sulfate,
It is useful as a raw material for dextran iron and plasma expanders. Furthermore, it can be applied as a raw material for various foods.

[0002]

2. Description of the Related Art Conventionally, a method for producing low-molecular-weight dextran has been described in Leuconost.
oc mesenteroides) and other methods to hydrolyze high-molecular-weight dextran (molecular weight of 1,000,000 or more) produced by fermentation with acid, alkali, or dextran-degrading enzyme, and then physically decompose by ultrasonic waves. However, all of these methods have the drawbacks that dextran having the desired molecular weight cannot be obtained in good yields, or the operation becomes complicated, and improvements have been sought. In addition, Japanese Examined Japanese Government
0-6631 shows a method for directly producing a low-molecular-weight dextran by simultaneously acting a dextran synthase and the same degrading enzyme. However, in this method, two kinds of enzymes having different properties are used at the same time. When the temperature and pH of the reaction solution and the enzyme activity ratio change, the molecular weight of the low-molecular-weight dextran produced tends to change, and strict control of reaction conditions is necessary, which is a problem in practice.

On the other hand, as a method for directly producing low-molecular-weight dextran using only dextran synthase, maltose as an acceptor of glucose is added to the reaction solution in advance, and dextran synthase is added to this solution. Methods of making it work are known. However, in this method, when the amount of maltose added is too large, the molecular weight of dextran produced becomes too small, and when the amount of maltose added is too small, a large amount of high molecular weight dextran is produced together with low molecular weight dextran, and low molecular weight dextran is produced. However, there was a problem in terms of practical use because the yield was low.

[0004]

According to the present invention, dextran synthase is added to a high-concentration sucrose solution at pH 4-8, 10-4.
It is characterized by comprising at least once a first step of acting at 0 ° C., and a second step of reacting the reaction solution obtained by the first step with a new high-concentration sucrose solution and a solution containing dextran synthase. It is a method for producing low molecular weight dextran.

The characteristics of the present invention are that the production of high-molecular-weight dextran is suppressed to a low level by increasing the concentration of sucrose used in the reaction, and that the reaction is carried out sequentially, and the liquid in which the reaction step of the first step is completed. In addition, a solution containing a high concentration of sucrose and an appropriate amount of dextran synthase is newly added, the reaction of the second step is performed, and these steps are repeated as necessary. That is, by carrying out these reactions in a stepwise manner, the high-molecular-weight dextran that was initially produced in a small amount was relatively decreased, and as a result, the low-molecular-weight dextran aimed at by the present invention had a high yield (vs. sucrose). Per theoretical yield of 70% or more).

The "low-molecular-weight dextran" in the present invention refers to one having an average molecular weight of about 4,000 to 100,000 (meaning a weight-average molecular weight; the same applies hereinafter).

The dextran synthase used in the present invention is well known, and any dextran synthase that exhibits activity under the reaction conditions for low-molecular-weight dextran synthesis, for example, exhibits enzyme activity in the range of pH 4 to 8 and temperature of 10 to 40 ° C. It can be used. Preferred examples of such dextran synthase include Leuconostoc mesenteroides (Leuconost).
oc mesenteroides) known products.

As a dextran synthase, Leuconostoc m.
In the case of using the product produced by Essenteroides), the dextran synthase is generally prepared as follows. That is, as a dextran synthase producing strain, Leuconostoc mesenteroides (Leu
It is sufficient to use a carbon source, a nitrogen source and an inorganic salt that can be assimilated by this bacterium in the medium using conostoc mesenteroides), but usually 1 to 5% of sucrose is an essential component, and yeast extract, peptone , Soybean powder, organic and inorganic nitrogen additives such as ammonium sulfate, and magnesium, calcium, phosphates, etc. are added as required. The culturing temperature may be in the range where the bacterium grows, but in general, the range of 20 to 30 ° C. is suitable. PH
Is in the range of 5 to 8, culture is carried out with or without slight aeration, and a culture time of about 10 to 30 hours is suitable.

Next, the fermentation-completed culture solution containing dextran synthase is sterilized by centrifugation or membrane separation before use. In addition, if necessary, it is also possible to fractionate with ethyl alcohol, acetone, ammonium sulfate or the like, and partially purify and use it. Using the dextran synthase thus prepared, a low-molecular-weight dextran is directly synthesized from sucrose.

The high-concentration sucrose solution used in this reaction is preferably 30 to 70% w / v. Thirty
If it is lower than% w / v, the amount of high-molecular-weight dextran produced increases, and as a result, the yield of the target low-molecular-weight dextran decreases. On the other hand, if it is more than 70% by weight, the reaction rate decreases and the reaction takes a long time, which is not preferable.

The reaction conditions in the first step are pH 4 to 8 and temperature 1
It is necessary that the reaction time is 0 to 40 ° C. and the reaction time is 5 to 48 hours.
Otherwise, the low molecular weight dextran targeted by the present invention cannot be obtained. Within the above range, the reaction time can be adjusted by the amount of enzyme used and the like.

The reaction conditions in the second step are pH 4 to 8 and temperature 1
The reaction time can be adjusted depending on the amount of enzyme used and the like.

The molecular weight of the low molecular weight dextran obtained by the production method of the present invention is determined by the concentration of sucrose newly added in the second step and the number of repetitions of the second step. That is, the higher the concentration of sucrose newly added in the second step, the smaller the molecular weight of the low-molecular-weight dextran obtained, and the larger the number of repetitions of the second step, the higher the molecular weight of the low-molecular-weight dextran obtained. Therefore, in the present invention, the concentration of sucrose and the number of repetitions may be selected according to the molecular weight of the target low molecular weight dextran. In addition, the reaction of the second step, in which a solution containing a high concentration of sucrose and dextran synthase is newly added to the reaction solution after the reaction of the first step in which the dextran synthase is allowed to act on the high-concentration sucrose solution is performed. If you want to do
The amount of the newly added sucrose solution is appropriately about half to 30 times the solid content of the original reaction liquid, and the concentration of the newly added sucrose solution is the same as that of the original reaction liquid. It is not necessary to use the above-mentioned concentration, that is, 30 to 70% w / v.

After the completion of the reaction, a commonly used method may be used to obtain the desired low-molecular-weight dextran. For example, ethyl alcohol is added to the reaction solution, and the desired molecular weight depends on the concentration. The low-molecular-weight dextran may be prepared by fractionation. Also, the reaction solution contains fructose, leucrose and unreacted sucrose,
It is also possible to apply desalting, decoloring, etc. to the reaction liquid as necessary without separating them, and apply it as it is to a material for food.

[0015]

INDUSTRIAL APPLICABILITY In the present invention, a method for directly producing a target low-molecular-weight dextran by allowing a dextran synthase to act alone on a high-concentration sucrose solution to suppress the production of high-molecular-weight dextran to a low level. Is provided,
The temperature and pH of the reaction solution as well as the enzyme activity can be easily controlled, and compared to conventional methods, the low molecular weight dextran having an average molecular weight of 4,000 to 100,000 can be easily and efficiently compared to the conventional method. Can be obtained in high yield.

The present inventors also found that the weight average molecular weight of 4,
It has been found that dextran of 000 to 20,000 has a particularly high cariogenicity, and that dextran having a molecular weight in this range has a low viscosity and is easy to use when used in foods. Therefore, it is possible to use dextran having a molecular weight of 4,000 to 20,000 as an excellent anti-cariogenic sweetener by mixing it with an ordinary sweetener (eg, sucrose, glucose, fructose, etc.). it can. In this case, the mixing ratio of the dextran having a molecular weight of 4,000 to 20,000 and the sweetener is 1: 1 to 1: 1.9.
Is.

Weight average molecular weight 4,000 to 20,000
A test was conducted to show the usefulness of a sweetener containing dextran as an anti-cariogenic agent. Suppression of dextran molecular weight and insoluble glucan (plaque)
Caries bacteria using dextran action average molecular weight 504~476,000 (Streptococcus mutan
The inhibitory effect of s) on insoluble glucan synthase was assayed by the following method.

(Preparation of enzyme) Streptococcu
s mutans NCTC 10449 strain BHI
(Brain Heart Infusion) medium is anaerobically cultured at 37 ° C. and pH 7.0 for 18 hours to remove the bacteria by centrifugation, and ammonium sulfate is added to precipitate the enzyme. This is dissolved in a phosphate buffer of pH 7.0 and dialyzed to obtain a crude enzyme solution of insoluble glucan synthase.

(Assay for Inhibitory Action) When 1% sucrose solution (pH 7.0) was added with dextran of each molecular weight so as to be 1%, crude enzyme solution was added thereto and reacted at 35 ° C. for 20 hours. The absorbance at 660 nm is measured and used as the sample candle level. On the other hand, water was added in place of dextran, and the absorbance at 660 nm when the reaction was carried out in the same manner was used as a control value.
The production inhibition rate of insoluble glucan was calculated by the following formula.

[0020] (Result) Average molecular weight Suppression rate (%) 504 41 1000 59 59 5000 70 11000 81 19600 80 40 000 77 60000 69 69 185000 47 476000 37

[0021]

EXAMPLES The present invention will be described in more detail below with reference to examples. Determining the titer of dextran synthase is 11.
0.1 ml of enzyme solution to 0.9 ml of 1% w / v sucrose solution
And reducing sugars released by reacting at pH 5.2 and 30 ° C. for 10 minutes, Somogyi Nelson (Somogyi.
Nelson method, 1m in 60 minutes under these conditions
The amount of enzyme required to convert g sucrose into dextran was defined as 1 unit. The analysis of dextran produced in the reaction solution in the following examples was performed by high performance liquid chromatography under the following analysis conditions.

(Analysis conditions) Column: Tosoh TSKgel G3000PW _XL (7.
8 _mm ID x 30 _cm ) Eluent: distilled water Flow rate: 0.7 _ml / _min Temperature: 60 ° C Detector: differential refractometer

Manufacturing Example Leuconostoc Mescenteroides (Leucono)
stoc mesenteroides) NRRL B
The −512 strain was inoculated into a medium containing 2% sucrose, 1% yeast extract, 0.5% peptone, 2% dipotassium phosphate and a trace amount of metal salt, and statically cultured at 25 ° C. for 24 hours. After completion of the culture, centrifugation (10000 rpm, 10 minutes) was performed, and after sterilization, 37.5% ethyl alcohol was added little by little to the culture solution at low temperature, and immediately centrifuged (100 rpm).
00 rpm. Then, dextran synthase was obtained as a precipitate.

Examples 1 to 3 The dextran synthase obtained in the production example was added to 30 ml of a 50% w / v sucrose solution so that the amount of the dextran synthase was 60 units per 1 g of sucrose, and the pH was 5.2 at 25 ° C. for 24 hours. The solution was left to stand and the synthesis reaction of the first step of low molecular weight dextran was performed. After the reaction was completed, 5 ml of this reaction solution was taken, and 50m of 20-60% w / v sucrose solution was added in an amount 10 times the solid content of the reaction solution.
1 and dextran synthase were added to sucrose to be added at 60 units per gram, mixed with the reaction solution of the first step, and allowed to stand for 24 hours under the same reaction conditions to obtain a second low molecular weight dextran. The synthetic reaction of the process was performed. Result is,
As shown in Table 1, when the concentration of the sucrose solution newly added during the synthesis reaction of the low-molecular-weight dextran in the second step becomes low, the production amount of the high-molecular-weight dextran increases and It can be seen that the yield decreases.

Table 1 Sucrose concentration% w / v High molecular weight dextran Low molecular weight dextran First step Second step Yield% Yield% Average molecular weight Control example 50 20 35.9 52.7 13,000 Example 1 50 30 14.8 73.8 15,600 2 50 40 6.3 6.3 80.2 12,300 3 50 60 2.1 80.2 5,400 High molecular weight dextran; molecular weight of several hundred thousand or more; yield; theoretical yield Calculated as 47.4% sugar

Example 4 To 30 ml of a 50% w / v sucrose solution was added 900 units of the dextran synthase obtained in the preparation example, and the pH was 5.2, 25 ° C.
The mixture was allowed to stand still for 24 hours, and the synthesis reaction of the first step of low molecular weight dextran was performed. After the reaction was completed, 90 ml of a 50% w / v sucrose solution and 2700 units of dextran synthase were added to this reaction mixture and mixed, and the mixture was allowed to stand for 24 hours under the same reaction conditions and the second step of low molecular weight dextran was performed. A synthetic reaction was performed. The low molecular weight dextran obtained under these conditions had an average molecular weight of 6,700 and a yield of 82.3%. The composition was as follows.

Example 5 To 10 ml of the reaction solution obtained in Example 4 (average molecular weight of low-molecular-weight dextran 6,700) was added 1 per solid content of the reaction solution.
Add 0% amount of 30% w / v sucrose solution and 900 units of dextran synthase and mix them to pH 5.2, 2.
The mixture was allowed to stand at 5 ° C for 24 hours, and the low molecular weight dextran synthesis reaction was performed again. The low molecular weight dextran obtained by this reaction had an average molecular weight of 11,700 and a yield of 82.3%.

Example 6 To 1000 g of a 50% w / v sucrose solution, 30,000 units of dextran synthase obtained in the production example was added, and the pH was adjusted to 5.
The reaction was allowed to stand at 2,25 ° C for 24 hours. After the reaction was completed, 3000 g of a 50% w / v sucrose solution and 90,000 units of dextran synthase were newly added to and mixed with this reaction solution, and the mixture was reacted for 24 hours under the same reaction conditions. To this reaction solution, 3780 g of fructose was added and dissolved to obtain about 7.8 kg of an anti-cariogenic sweetener. The composition of the sweetener was approximately as follows.

Example 7 4000 g of a liquid which had been reacted in the same manner as in Example 7 was added to 3060
After concentrating to g, 1660 g of fructose was added to and dissolved in this solution to obtain about 4.7 kg of an anti-cariogenic sweetener. The composition of the sweetener was as follows.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location (C12N 9/46 C12R 1:01)

Claims (8)

[Claims] 1. A first step in which a dextran synthase is allowed to act on a high-concentration sucrose solution at pH 4 to 8 and 10 to 40 ° C., and a new high-concentration sucrose solution in the reaction solution obtained by the first step. A method for producing a low-molecular-weight dextran, which comprises one or more second steps of adding and reacting a solution containing a dextran synthase. 2. The sucrose concentration of the sucrose solution is 30 to 70%.
The method for producing the low-molecular-weight dextran according to claim 1, which is w / v. 3. The method for producing a low molecular dextran according to claim 1, wherein the low molecular dextran is a dextran having a weight average molecular weight of 4,000 to 20,000. 4. Leuconostoc as a dextran synthase.
A method for producing a low-molecular-weight dextran according to claim 1, which uses an enzyme produced by a strain belonging to mesenteroides). 5. A first step in which a dextran synthase is allowed to act on a 45 to 55% w / v sucrose solution at pH 4 to 8 and 10 to 40 ° C., and the reaction solution newly obtained in the reaction solution obtained by the first step. 45 to 55% of 3 to 10 times amount per solid content of liquid
A method for producing a low-molecular-weight dextran, which comprises a second step of adding and reacting a w / v sucrose solution and a solution containing a dextran synthase. 6. A weight average molecular weight of 4,000 to 20,000.
An anti-cariogenic sweetener containing 0 dextran. 7. An anti-cariogenic sweetener whose sweetening component is fructose. 8. A weight average molecular weight of 4,000 to 20,000.
The dextran to fructose ratio of 0 is 1: 1 to 1: 1.
An anti-cariogenic sweetener consisting of 9.