JPH0516834B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing a polysaccharide, and more particularly to a method for producing a polysaccharide using grass fiber as a starting material.

(Prior Art) Conventionally, as a method for saccharification of cellulose, an enzymatic method in which cellulose is decomposed by cellulase is known (see Japanese Patent Publication No. 57-3357 and Japanese Patent Application Laid-open No. 57-146592).

The final product obtained by such an enzymatic method is a monosaccharide such as glucose. Such monosaccharides such as glucose are used as pharmaceuticals, nutritional supplements, and the like.

However, for monosaccharides such as glucose, for example, B
No treatment results for type hepatitis were observed.

On the other hand, dextran, which is a polysaccharide in which glucose is linked in a linear chain, is known as a plasma expander or a pharmaceutical preparation.

For this reason, the medicinal efficacy of polysaccharides such as dextran is attracting attention.

By the way, such dextran is usually produced from sucrose using microorganisms.

(Problem to be Solved by the Invention) This is because by using sucrose as a starting material in this way, the production efficiency of dextran can be improved.

On the other hand, it is also conceivable to produce polysaccharides such as dextran by hydrolyzing natural cellulose that forms plant fibers such as rice straw in which glucose is linked in a linear chain.

However, when natural cellulose is used as a starting material, natural cellulose is generally highly crystalline and highly resistant to hydrolysis. It is necessary to improve hydrolysis.

Furthermore, when cellulose treated in this way is treated with enzymes such as cellulase, JP-A-57-
As described in Publication No. 146592, etc., it is ultimately converted into monosaccharides such as glucose.

For this reason, natural cellulose has not been used as a starting material in conventional methods for producing polysaccharides.

Therefore, an object of the present invention is to provide a method for producing polysaccharides, which uses fibers from grasses, such as rice straw, as a starting material, which have not been used in the past, to produce polysaccharides without pretreatment. be.

(Means for Solving the Problem) As a result of repeated studies to achieve the above object, the present inventors immersed rice straw in a culture solution and naturally fermented it while irradiating it with sunlight. The present invention was achieved based on the discovery that it is possible to easily obtain the following.

That is, the present invention involves immersing grass fibers in a culture solution containing ammonia nitrogen, nitrate nitrogen, soluble phosphoric acid, and soluble potassium in the presence of fermentation bacteria attached to the grass plant. Fermentation is performed while irradiating sunlight, and then the pH value of the culture solution is 7 to 7.
The method for producing a polysaccharide is characterized in that the fermentation is stopped when a temperature of 9.2 is reached.

In the present invention having such a configuration, if the grass fiber is rice straw, the yield of the obtained polysaccharide can be increased.

(Function) According to the present invention, fibers from grasses, such as rice straw, are fermented without pretreatment, and the culture solution is
When the pH value reaches 7 to 9.2, by stopping the fermentation, the molecular weight in terms of dextran decreases.
A product whose main component is a water-soluble polysaccharide having an amount of 70×10 ³ or more and containing a secondary amine component can be obtained.

Furthermore, since the fermentation proceeds under sunlight irradiation, it is possible to prevent the grass fibers from rotting during fermentation due to various bacteria adhering to the grass fibers.

However, the obtained polysaccharide has a therapeutic effect on hepatitis B.

(Structure of the Invention) As the starting material of the present invention, grass fiber is used. Such grass fibers include:
Rice straw, reed, wheat straw, etc. can be used. In particular, when rice straw is used as a starting material, the yield of polysaccharide can be significantly improved.

Such grass fibers are immersed in a culture solution.

At this time, it is preferable to form the grass fiber into chips of about 5 to 30 mm, so that the raw material can be easily immersed in the culture solution.

The culture solution used also contains ammonia nitrogen, nitrate nitrogen, soluble phosphoric acid, and soluble potassium.

Such a culture solution contains ammonia nitrogen 1.0
wt%, nitrate nitrogen 5.5 wt%, soluble phosphoric acid 6.0
% by weight, and 0.01 to 0.5% by weight of a basal culture solution consisting of 19.0% by weight of soluble potassium.

The immersion ratio between the culture solution and the grass fiber is
For good fermentation, it is preferable to immerse and ferment at a ratio of 15:1 to 60:1.

In the present invention, fermentation is carried out while irradiating sunlight onto grass fibers immersed in a culture solution.

It is preferable to carry out such fermentation in a culture tank that is sealed with a material that transmits sunlight, such as a lid made of a transparent vinyl sheet, to prevent contamination with bacteria during fermentation.

If you try to ferment the grass fiber without exposing it to sunlight, the bacteria attached to the grass fiber will cause it to rot, making it difficult to obtain the final target polysaccharide. I can't.

As described above, since the present invention requires sunlight irradiation, it is preferable to use a container with a shallow bottom and a wide mouth as the culture tank.

In the present invention, it is possible to easily determine whether or not the prescribed fermentation is being performed and to determine when to stop the fermentation by checking the pH value of the culture solution.

In other words, the initial Hz value of the culture solution is slightly alkaline, but if the specified fermentation is carried out, the culture solution's Hz value will be slightly alkaline.
After the PH value suddenly decreases and becomes strongly acidic at about PH4, the PH value gradually increases.

Then, when the pH value of the culture solution reaches 7 to 9.2, fermentation is stopped.

Here, if the pH value of the culture solution is less than 7 or exceeds 9.2, the yield of polysaccharide will decrease. This is presumed to be due to insufficient or excessive hydrolysis of cellulose, which forms the grass fiber.

In such a production method of the present invention, the fermentation time required for fermentation varies depending on the temperature etc., but is about 120 minutes.
~240 days.

Such shortening of the fermentation time can be achieved by introducing protozoa, preferably Monas, into the culture solution and maintaining the fermentation temperature of the mixture at 10 to 30°C. This is thought to be due to the proliferation of protozoa or Monas, which promotes cellulose hydrolysis.

Here, "monas" is a general term for fermentation bacteria such as Bacillus subtilis and pseudomonas that live in the soil and puddles.
DETERMINATIVE BACTERIOLOGY 8TH
"MONAS" listed on page 1195 of "EDITION" is also included.

In addition, such fermenting bacteria such as Bacillus subtilis and Pseudomonas are usually attached to gramineous plants such as rice straw, and it is possible to carry out the prescribed fermentation without adding Monas.

After completion of the fermentation, the fermented product obtained is filtered to remove residual fiber components, and the resulting filtrate is centrifuged to remove precipitates, and then ultrafiltrated to remove impurities. Removes metal ions.

The purified product thus obtained is stored after being sterilized at high temperature and high pressure.

Such purified products are soluble in water but insoluble in ethanol and methanol.

Furthermore, when the molecular weight of the purified product is measured by high performance liquid chromatography, large peaks appear at the positions where the respective peaks of dextran with a molecular weight of 70×10 ³ and dextran with a molecular weight of 500×10 ³ are expressed.

Furthermore, when the infrared absorption spectrum of the purified product is measured, absorptions peculiar to the secondary amine component appearing near 1560 cm ^-1 and 3350 cm ^-1 are also observed.

Therefore, from the above analysis results, the product obtained in the present invention is mainly composed of a water-soluble polysaccharide containing a secondary amine component and having a molecular weight of 70×10 ³ or more in terms of dextran.

Further, according to clinical experiments of the product obtained according to the present invention, it was found that it exhibits a therapeutic effect on hepatitis B.

In addition, in the residual fiber after fermentation, the lignin component in the fiber has been considerably decomposed, and the residual fiber can be used as feed or fertilizer.

(Example) The present invention will be explained in further detail with reference to Examples.

Example 1 A basal culture solution consisting of 1.0% by weight of ammonia nitrogen, 5.5% by weight of nitrate nitrogen, 6.0% by weight of soluble phosphoric acid, and 19.0% by weight of soluble potassium was placed in a culture tank.
Inject 1000 g of culture solution containing 0.1% by weight, add 4 g of Monas liquid, and mix uniformly.

The "Monas liquid" used in this example is a liquid containing a large amount of fermenting bacteria such as Bacillus subtilis and Pseudomonas, and as described later, when the fermented product obtained after the completion of fermentation is filtered, A portion of the resulting filtrate can be used.

Next, 40 g of dried reed chips cut to a length of about 15 mm are added to the culture solution and thoroughly immersed, and then the culture tank is sealed with a transparent vinyl sheet.

The culture tank was placed outdoors to allow sunlight to pass through the transparent vinyl sheet and enter the culture tank, and natural fermentation was carried out at a temperature in the range of 10 to 30°C.

The PH value of the culture solution during fermentation was measured, and the results are shown in Figure 1.

As is clear from Figure 1, the culture solution, which was slightly alkaline at the start of the fermentation, became approximately alkaline after the start of the fermentation.
After 20 days, the pH value drops rapidly to about 4, and then rises slowly. The pH value of the culture solution reached approximately 8.7 on the 192nd day from the start of fermentation, and fermentation was terminated.

The obtained fermented product was filtered to remove fibers, and the resulting filtrate was centrifuged to remove precipitates, and then ultrafiltrated to remove metal ions as impurities.

Further, the obtained product was sterilized under high temperature and high pressure at a pressure of 1.2 Kg/cm ² and a temperature of 121° C. for 60 minutes.

The purified product thus obtained is soluble in water but insoluble in ethanol and methanol.

Further, the molecular weight of the obtained purified product was measured using high performance liquid chromatography (Hitachi Model 635) under the following conditions, and the measured chart is shown in FIG.

Colum Shodex lonpak s′800p＋s′804＋s′801 Sample 10μ Detector: RI4k×10 ^-5 RLUFS′ Pressure: 25Kg/cm ² Eluent: H ₂ O Flow Rate: 1.0ml/min Chart speed: 5mm/min Colom temp.: 60 ℃RT As is clear from Figure 2, the obtained purified product contains dextran with a molecular weight of 70 × 10 ³ and dextran with a molecular weight of 500
The main peak is expressed at the position where each peak of ×10 ³ dextran is expressed.

Therefore, the purified product obtained in this example mainly contains a polysaccharide having a molecular weight of 70×10 ³ or more in terms of dextran.

In FIG. 2, each peak of dextran with a molecular weight of 70×10 ³ or dextran with a molecular weight of 500×10 ³ is shown as Dextan70K or Dextan500K.

Comparative Example 1 The same procedure as in Example 1 was carried out, except that instead of the transparent vinyl sheet used as the lid of the culture tank in Example 1, a plastic lid that does not transmit sunlight was used to seal the culture tank. I went.

The pH value of the culture solution did not decrease much, the fermentation state was insufficient, and mold was generated, so the subsequent experiments were discontinued.

Example 2 Fermentation was carried out in the same manner as in Example 1, except that dried wheat straw was used instead of dried reeds.

In this example, fermentation was stopped on the 190th day from the start of fermentation, and the pH value of the culture solution was 9.0.

As shown in FIG. 3, the pH value of the culture solution during this period rapidly decreased to about 4.2, and then gradually increased to 9.0.

The obtained fermented product was purified in the same manner as in Example 1, and then measured by high performance liquid chromatography (Hitachi Model 635) under the same conditions as in Example 1. The measured chart is shown in Figure 4.

In this example as well, the obtained purified product has a main peak expressed at the position where the respective peaks of dextran with a molecular weight of 70×10 ³ and dextran with a molecular weight of 500×10 ³ are expressed.

The purified product obtained in this example was soluble in water, but insoluble in ethanol and methanol.

Example 3 Fermentation was carried out in the same manner as in Example 1, except that dried rice straw was used instead of dried reeds.

Such fermentation is carried out after 190 days from the start date of fermentation.
The culture was stopped when the pH value of the culture solution reached 9.2.

The pH value of the culture solution during fermentation is as shown in Figure 5.
After a sudden drop, the PH value gradually increased to 9.2.

The obtained culture was also purified in the same manner as in Example 1, and measured by high performance liquid chromatography (Hitachi Model 635) under the same conditions as in Example 1. The measured chart is shown in Figure 6.

As shown in FIG. 6, in this example as well, the obtained fermentation product had a main peak expressed at the position where the respective peaks of dextran with a molecular weight of 70×10 ³ and dextran with a molecular weight of 500×10 ³ were expressed. It is something.

Moreover, the peak heights corresponding to each dextran shown in FIG. 6 are higher than the peak heights corresponding to each extratran of Example 1 and Example 2 shown in FIGS. 2 and 4.

From this, by using rice straw as a raw material, it is possible to increase the yield of dextran with a molecular weight of 70×10 ³ and a polysaccharide corresponding to dextran with a molecular weight of 500×10 ³ .

Furthermore, the infrared absorption spectrum of the sample measured by high performance liquid chromatography was measured, and the results are shown in FIG.

In Figure 11, near 1560 cm ^-1 and 3350 cm ^-1
Absorption peculiar to the secondary amine component is observed in the vicinity, and the obtained polysaccharide contains the secondary amine component.

Example 4 A clinical experiment was conducted using the purified culture obtained in Example 3.

In the clinical experiment, a 50-year-old male patient with hepatitis B was given an aqueous solution of 100 mg of the purified product obtained in Example 3 extracted with hot water to a concentration of about 1, divided into three doses per day. It was then administered.

After the start of the experiment, the results of the patient's blood tests, etc.
It is shown in Figure 10.

As is clear from FIGS. 7 to 10, after the patient started drinking the aqueous solution of the purified product obtained in Example 3, the symptoms of hepatitis of the patient were rapidly improved.

This patient underwent treatment for 50 days, which would normally require hospitalization for more than 4 months, without any side effects.
I was discharged from the hospital within a day.

Example 5 Regarding the purified culture product obtained in Example 3,
Experiments were conducted using ddy male mice. First, 100 mg/Kg of the purified product was continuously administered to each of 10 mice, and then 20 μ/Kg of carbon tetrachloride, which causes hepatitis, was injected subcutaneously into the abdomen of each mouse. GOT and GPT values were tested to determine the degree of hepatitis.

For comparison, L, which is known as a drug for hepatitis,
- In the same way, carbon tetrachloride was administered to a level group in which 100 mg/Kg of methionine was administered continuously for 6 days, and to a level group in which no drugs were administered.
20μ/Kg was injected subcutaneously into the mouse abdomen, and GOT and GPT values in the blood were examined in the same manner 24 hours later.

The test results are shown in Figure 12. In FIG. 12, GOT and GPT values in blood are expressed as S-GPT.

As is clear from FIG. 12, the level group A to which the purified culture product obtained in Example 3 was administered in advance,
The degree of onset of hepatitis was milder than in level group C, in which no drug was administered, as well as in level group B, in which L-methionine was administered in advance.

Comparative Example 2 In Example 3, after the PH value reached 9.2,
The fermentation was further continued and the fermentation was stopped when the pH value reached 9.8.

When the obtained fermented product was purified in the same manner as in Example 3 and its molecular weight was measured by high performance liquid chromatography, the peak corresponding to glucose was significantly large, indicating that dextran with a molecular weight of 70×10 ³ and dextran with a molecular weight of 500 It was found that the peak corresponding to ×10 ³ dextran had become significantly smaller.
For this reason, further experiments were discontinued.

Example 6 Natural fermentation was carried out under the same conditions as in Example 1, except that dried rice straw chips were used instead of dried reed chips and Monas liquid was not added.

The pH value of the culture solution during fermentation showed the same change as in Example 3, in which dry rice chips were used for fermentation.
Fermentation was stopped when the pH value of the culture solution reached 9.

However, since this example involved natural fermentation without adding Monas liquid, the number of culture days was approximately 20 days longer than the number of culture days in Example 3.

The obtained culture was purified in the same manner as in Example 1, and measured by high performance liquid chromatography under the same conditions as in Example 1. As a result, similar to the high-performance liquid chromatography chart (Figure 6) of the culture of Example 3, a main peak was found at the position where the peaks of dextran with a molecular weight of 70 x 10 ³ and dextran with a molecular weight of 500 x 10 ³ were expressed. was confirmed to occur.

Comparative Example 3 In Example 3, the fermentation was stopped when the pH value reached 6.5, and the obtained fermented product was purified in the same manner as in Example 3, and the molecular weight was measured by high performance liquid chromatography. .

Peak corresponding to glycose, molecular weight 70×10 ³
It was found that the peaks corresponding to dextran with a molecular weight of 500×10 ³ and dextran with a molecular weight of 500×10 3 were extremely small, and the yield was low. For this reason, further experiments were discontinued.

(Effects of the Invention) According to the present invention, grass fiber is pretreated with a water-soluble polysaccharide having a molecular weight of 70×10 ³ or more in terms of dextran and containing a secondary amine component. You can get it without.

Furthermore, the obtained polysaccharide exhibits a therapeutic effect on hepatitis B and can be used for the treatment of hepatitis B.

In addition, the polysaccharide obtained by the present invention is obtained by fermentation from natural raw materials, and is used in miso, miso,
It can also be used in health foods as a food additive for bread, confectionery, etc.

[Brief explanation of the drawing]

Figure 1 shows the pH of the culture solution showing the fermentation status of Example 1.
Figure 2 is a chart of high performance liquid chromatography of the culture obtained in Example 1. Figure 3 is a graph of the pH value of the culture solution showing the fermentation status of Example 2. The figure is a chart of high-performance liquid chromatography of the culture obtained in Example 2, Figure 5 is a graph of the change in PH value of the culture solution showing the fermentation status of Example 3, and Figure 6 is a chart of the pH value of the culture solution obtained in Example 3. Figure 7 shows a chart of high-performance liquid chromatography of the culture obtained in the clinical experiment in Example 4.
Graph showing changes in GOT, Figure 8 is a graph showing changes in LAP and γ-GTP of patients in the clinical experiment of Example 4, Figure 9 is a graph showing changes in jaundice index and platelet count of patients in the clinical experiment of Example 4. Figure 10 is a graph showing the changes in the patient's hematric value, hemoglobin amount, and red blood cell count in the clinical experiment of Example 4. Figure 11 is the infrared absorption spectrum of the culture obtained in Example 3. FIG. 12 is a graph showing the results of the animal experiment of Example 5.

Claims (1)

[Claims] 1. Graminee plant fibers are immersed in a culture solution containing ammonia nitrogen, nitrate nitrogen, soluble phosphoric acid, and soluble potassium in the presence of fermentation bacteria attached to the grasses. 1. A method for producing a polysaccharide, comprising: fermenting the culture solution while irradiating it with sunlight; and then stopping the fermentation when the pH value of the culture solution reaches 7 to 9.2. 2. The method for producing a polysaccharide according to claim 1, wherein the grass fiber is rice straw.