JPH0568580A - Higher chitosan oligosaccharide and its production - Google Patents

Abstract

PURPOSE:To obtain the subject oligosaccharide useful as medicines, cosmetics, etc., in high yield by carrying out chitosan hydrolyzing reaction with a chitosan hydrolase in an ultrafilter, then removing the produced chitosan oligosaccharide to the outside of the ultrafilter, supplying a chitosan solution and repeating the reaction. CONSTITUTION:Chitosan hydrolyzing reaction with an enzyme having the chitosan hydrolyzing activity is carried out in an ultrafilter having a membrane permeability regulated so that >=40wt.% higher chitosan oligosaccharide which is a penta- to octadecasaccharide can be contained in a mixture of the chitosan oligosaccharide permeating the membrane to produce a chitosan oligosaccharide mixture containing the chitosan oligosaccharide. The resultant chitosan oligosaccharide mixture is then removed to the outside of the ultrafilter. A chitosan solution corresponding to the amount of the removed mixture is fed into the ultrafilter. The chitosan hydrolyzing reaction, removal of the chitosan oligosaccharide mixture and feed of the chitosan solution are then continuously carried out to afford the objective higher chitosan oligosaccharide.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing higher chitosan oligosaccharides and higher chitin oligosaccharides, and more specifically, among oligosaccharides that are partial degradation products of chitosan or chitin,
The present invention relates to a method for producing an oligosaccharide having a relatively high degree of polymerization.

[0002]

2. Description of the Related Art In general, chitosan oligosaccharide, which is a partial decomposition product of chitosan, is desired to be developed for use in food additives, cosmetics, pharmaceuticals, etc. In recent years, it has been found to have various physiological activities such as antibacterial activity, antitumor activity, immunostimulatory activity, plant elicitor activity, etc., and their added value and demand are increasing.

Further, higher chitin oligosaccharides, which are N-acetylated products of such higher chitosan oligosaccharides, have physiological activities similar to those of the above-mentioned higher chitosan oligosaccharides, and also have excellent lectin inhibitory properties, and chitinase and lysozyme. It is very useful as a substrate for

[0004]

[Problems to be Solved by the Invention]
A method for efficiently producing higher chitosan oligosaccharides and higher chitin oligosaccharides that are higher than sugar has not been established. Especially 7
Currently, it is difficult to prepare higher chitosan oligosaccharides and higher chitin oligosaccharides that are higher than sugars and are not currently on the market.

The production of chitosan oligosaccharides by enzymatic degradation of chitosanase is recognized as effective because chitosan is easily dissolved in dilute acid, and there are a few reports in fact [M.
Izume and A. Ohtakara, Agri
c. Biol. Chem, 51, 1189 (1987).
/ Yoshiyoshi Miyoshi et al., The 5th Chitin-Chitosan Symposium Proceedings p90 (1991)], the yield of chitosan oligosaccharides with five or more sugars is low.

[0006] In addition, there is a report that a higher sugar chain such as hexasaccharide, heptose, etc. is produced from a disaccharide by utilizing the glycosyl transfer reaction of lysozyme [Usui Taiichi et al., 3rd Chitin-Chitosan Symposium Abstracts] p30 (1988)] The disaccharide chitin oligosaccharide has a problem in terms of cost because it is currently expensive.

Further, when the chitosan decomposition reaction by chitosanase is carried out by a so-called batch method using only an enzymatic reaction, mainly 2 to 5 sugars are produced, and the productivity of higher chitosan oligosaccharides having 5 or more sugars is low.

The present invention has been made in order to solve all the above problems, and it is a higher chitosan oligosaccharide,
The challenge is to enable large-scale continuous production of high-grade chitin oligosaccharides.

[0009]

The present invention has been made as a method for producing higher chitosan oligosaccharides and higher chitin oligosaccharides by solving these problems, and is characterized by the method for producing higher chitosan oligosaccharides. , A chitosan degradation reaction with an enzyme having a chitosan degrading activity is performed in an ultrafilter with the maximum membrane permeability adjusted to produce a chitosan oligosaccharide mixture containing higher chitosan oligosaccharides, and then the chitosan oligosaccharide mixture is It is removed to the outside of the ultrafilter, and then the removed chitosan solution is supplied into the ultrafilter, and then the chitosan decomposition reaction, the removal of the chitosan oligosaccharide, and the supply of the chitosan solution are continuously performed. To produce high-grade chitosan oligosaccharides.

Further, a characteristic of the method for producing a higher chitin oligosaccharide is that the chitosan oligosaccharide produced by the above-mentioned method for producing a higher chitosan oligosaccharide is N-acetylated to form a poorly water-soluble high grade 8-18 sugar. The purpose is to produce chitin oligosaccharides.

Here, the chitosan used as a substrate for the enzyme reaction may be any soluble chitosan solution,
In particular, by using low molecular weight chitosan having a molecular weight of 5,000 to 200,000, it is possible to prepare a high concentration chitosan solution having a low viscosity, and there is an advantage that the yield of higher chitosan oligosaccharide is significantly increased.

The buffer used is preferably an acetic acid-sodium acetate buffer, but it is not particularly limited.

Further, the pH of the chitosan solution is not particularly limited as long as it is around the optimum pH of the enzyme.

Although the concentration of chitosan is not limited,
A high concentration of 3% or more is preferable.

Further, the type of the ultrafiltration device is not limited, and the material of the ultrafiltration membrane is not limited. The point is that a membrane that does not allow the enzyme to permeate but allows the chitosan oligosaccharide to permeate may be used.

Further, the enzyme is preferably chitosanase, but any enzyme having a good degradability of chitosan with a deacetylation degree of 50 to 100% may be used. For example, lysozyme, chitinase or cellulase may be used, and the kind thereof is particularly preferable. Not limited.

It should be noted that almost no change with time was observed in the composition ratio of the chitosan oligosaccharide obtained by the production method of the present invention. Also, the permeation rate of the ultrafiltration membrane gradually decreases, but this is due to the soiling of the membrane rather than the lowering of the enzyme activity, which can be eliminated by washing the membrane.

[0018]

EXAMPLES Examples of the present invention will be described below. Example 1 5% low molecular weight chitosan [deacetylation degree of about 90%, molecular weight 1.6 × 10 ^{4 to} 1.7 × 10 ⁵ / Kyowa Yushi Co., Ltd.]
Bacillus sp. Was added to 400 ml of the solution (pH 5.2). PI-
68 ml (6 U / ml) of a crude chitosanase solution derived from 7S (Microtechnology Research Institute No. 9843) was added, and the enzyme reaction was carried out at 37 ° C. in an ultrafilter. The crude chitosanase solution used was obtained by concentrating the culture filtrate with an ultrafilter.

Next, the produced oligosaccharide was quickly removed from the reaction system, and a 5% low molecular weight chitosan solution corresponding to the reduced amount was added.

The ultrafilter is a Minitan Ultrafiltatio manufactured by Japan Millipore Limited.
n System, the ultrafiltration membrane has a molecular weight cutoff of 1
0000, filtration area 600 cm ² , pressure 20 psi
Adjusted to.

The operation time was 80 hours after the permeated liquid started to come out.

The analysis of chitosan oligosaccharide was performed by HPLC. When the composition ratio of the chitosan oligosaccharide contained in the dried product (chitosan degradation product) of the vortex membrane liquid at each time was determined by HPLC, it was almost constant and no change with time was observed. Further, even if the pH of the permeable membrane liquid was alkaline, almost no precipitation occurred.

It was observed that the membrane permeation rate gradually decreased, and the production amount of chitosan oligosaccharides also gradually decreased correspondingly. By the way, the reduction rate from the 18th hour to the 80th hour is 2
It was 6%.

Further, HPLC is used as a column for TSKge.
Lamide-80 (4.6 x 25 cm), using acetonitrile / 50 mM phosphoric acid (4: 6) as the eluent. The flow rate was adjusted to 0.7 ml / min.

2100 ml of the whole-membrane permeate obtained in the above 1st to 10th hours was concentrated by an evaporator and freeze-dried to obtain 108 g of a solid chitosan oligosaccharide mixture. The reducing sugar produced by the Schals method is 330 (mg.D-glucosamine /
g).

When analyzed by HPLC, disaccharide: 2.0
%, 3 sugars: 13.3%, 4 sugars: 13.1%, 5 sugars: 1
8.7%, 6-sugar: 18.0%, 7-sugar: 14.1%, 8
Sugar: 9.7%, 9 sugar: 5.5%, 10 sugar: 4.1%, 1
1 sugar: 1.5% was contained, and the content of higher chitosan oligosaccharides having 5 or more sugars was 71.6%. The analysis result is shown in FIG. In FIG. 1, the numbers 2 to 11 near each peak in the chart indicate disaccharides, ... 11 respectively.
The peak of sugar is shown.

The Bacillus sp. Having the ability to produce the chitosanase used in Example 1 above was used. PI-7S (Microtechnology Research Institute, No. 9843) is a strain collected by the present inventors, and its mycological properties are as follows. (A) Morphological properties (a) Morphology of bacilli (b) Spores Elliptic, swelling (c) Motile (d) Gram stain indeterminate (B) Growth condition in each medium (a) Meat broth agar medium When cultured at 37 ° C for 24 to 96 hours, colonies with projecting circles on the entire periphery were formed, and became swelling over time. The color was almost cloudy after 24 hours of culture, but became pale yellow or yellow after 48 hours of culture. The growth condition was recognized as positive. (B) Broth liquid agitation medium Aerobic and cultured at 37 ° C. for 24 hours,
It became cloudy. The growth condition was recognized as positive. (C) Growth under anaerobic growth (D) Physiological properties (a) Catalase production + (b) VP reaction + W (c) pH in VP broth pH 4.8 (d) Gas production from glucose Glucose + arabinose-xylose-mannitol + (f) Liquefaction of gelatin + (g) Decomposition of starch + (h) Decomposition of tyrosine- (i) Utilization of citric acid- (j) ) Egg yolk reaction- (k) Reduction of nitrate- (l) Indole production- (m) Growth at pH 5.7 + (n) Growth in the presence of 5% NaCl- (o) Growth in the presence of 7% NaCl- ( p) Growth at 50 ° C.-In the above, + means positive, − means negative, and + W means weakly positive. About this strain, 1
It has already been deposited with the Institute of Microbial Science and Technology of the Agency of Industrial Science on 28th March.

Example 2 The chitosan oligosaccharide mixture obtained in Example 1 above was dissolved in 6500 ml of methanol to obtain 35 g of a methanol insoluble substance which was not dissolved by the operation. When analyzed by HPLC, trisaccharide: 3.3%, tetrasaccharide: 4.3%, pentasaccharide: 9.0
%, 6 sugar: 12.6%, 7 sugar: 14.3%, 8 sugar: 1
4.9%, 9 sugar: 14.0%, 10 sugar: 13.1%, 1
It contained 1 sugar: 8.3% and 12 sugars: 5.9%, and the content of higher chitosan oligosaccharides having 5 or more sugars was 92.3%. The analysis result is shown in FIG. In FIG. 2, the numbers 3 to 12 attached near each peak in the chart indicate peaks of trisaccharide, ... Thus, it was confirmed that the content of the higher chitosan oligosaccharide in the chitosan oligosaccharide mixture was improved by using the methanol insoluble substance.

Example 3 As an enzyme, Bacillus sp. PI-7S (Microtech Lab
No. 843) -derived crude chitosanase 68 ml (9 U / m
Chitosan oligosaccharide production was carried out in the same manner as in Example 1 using 1). The membrane permeate from the 1st hour to the 10th hour was concentrated with an evaporator and then freeze-dried to obtain 119 g of a chitosan oligosaccharide mixture. The reducing sugar produced by the Schals method is 380 (mg.D-glucosamine /
g).

When analyzed by HPLC, disaccharide: 5.0
%, 3 sugars: 15.7%, 4 sugars: 19.7%, 5 sugars: 2
2.0%, 6-sugar: 18.0%, 7-sugar: 10.1%, 8
Sugars: 4.8%, 9 sugars: 2.9%, 10 sugars: 1.8% were contained, and the content of higher chitosan oligosaccharides with 5 or more sugars was 59.6%.

Example 4 12 g of the chitosan oligosaccharide mixture obtained in Example 1 above
Was dissolved in 120 ml of methanol and 75 ml of distilled water, 43 ml of acetic anhydride was added under ice cooling, and the mixture was allowed to stand for 8 hours. 1000 ml of an ethanol-acetone mixed solution (1: 1) was added, the precipitate was collected, acetic acid was removed, and freeze-dried to obtain 10.2 g of chitin oligosaccharide. Incidentally, I
The product was confirmed to be chitin oligosaccharide by R analysis. The analysis result is shown in FIG.

Next, 300 ml of distilled water was added to obtain 2.2 g as an insoluble matter. Acetone 900 was added to the obtained supernatant.
By adding ml, 4.1 g was obtained as a precipitate.

When analyzed by HPLC, tetrasaccharide: 9.
It contained 7%, pentasaccharide: 60.3%, and hexasaccharide: 30.0%. The analysis result is shown in FIG. In FIG. 3, the numbers 4 to 6 attached near each peak in the chart are peaks of tetrasaccharide, pentasaccharide, and hexasaccharide, respectively.

Incidentally, as the HPLC, TSK gel a
It was performed by using side-80 (4.6 x 25 cm), acetonitrile / water (6: 4) as an eluent. The flow rate was adjusted to 0.7 ml / min.

Comparative Example 68 ml of crude chitosanase (6 U / ml) was added to 2100 ml of 5% low molecular weight chitosan solution (pH 5.2), and the enzyme reaction was carried out by the batch method while stirring at 37 ° C. for 10 hours. After the reaction, after removing the enzyme and undegraded chitosan from the enzyme reaction solution with an ultrafiltration membrane having a molecular weight cut off of 10,000,
After concentration, freeze-drying was performed to obtain 69 g of chitosan oligosaccharide.

When the above oligosaccharide was analyzed by HPLC, disaccharide: 22.9%, trisaccharide: 35.1%, tetrasaccharide: 2
6.5%, 5 sugars: 15.5%, and 6 or more sugars were not contained.

[0037]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, chitosan and higher chitosan oligosaccharides are separated by filtration using an ultrafilter having the maximum membrane permeability adjusted to obtain a chitosan oligosaccharide containing the higher chitosan oligosaccharide. After the mixture is removed from the reaction system, the removed chitosan solution is supplied into the ultrafilter, and this operation is continuously repeated to produce a higher chitosan oligosaccharide. The secondary decomposition of sugar is inhibited, the yield of higher chitosan oligosaccharide obtained outside the ultrafilter is increased, and it is possible to produce a large amount of higher chitosan oligosaccharide.

Particularly, in the present invention, since higher chitosan oligosaccharides having 5 or more sugars, which are difficult to obtain by the conventional batch method and the like, can be produced in a large amount in high yield, 5 or more sugars having various excellent physiological activities are obtained. There is a practical advantage that the higher grade chitosan oligosaccharides can be put to practical use.

Furthermore, a higher chitin oligosaccharide can be obtained by N-acetylating the chitosan oligosaccharide obtained by the above method, and a chitin oligosaccharide having a physiological activity similar to that of chitosan oligosaccharide can be obtained. It has the effect that it can be manufactured in large quantities.

[Brief description of drawings]

FIG. 1 is an HPLC analysis chart of Example 1.

2 is an HPLC analysis chart of Example 2. FIG.

FIG. 3 is an HPLC analysis chart of Example 4.

FIG. 4 is an IR analysis chart of Example 4.

Claims (11)

[Claims] 1. A chitosan decomposing reaction by an enzyme having a chitosan decomposing activity is carried out in an ultrafilter in which the membrane permeability is adjusted to the maximum to produce a chitosan oligosaccharide mixture containing higher chitosan oligosaccharides, The chitosan oligosaccharide mixture is removed to the outside of the ultrafilter, and then a chitosan solution corresponding to the removed portion is supplied into the ultrafilter, and then the chitosan decomposition reaction, the removal of the chitosan oligosaccharide mixture, And a method for producing a higher chitosan oligosaccharide, which comprises continuously repeating the supply of the chitosan solution to produce a higher chitosan oligosaccharide. 2. Chitosan in an ultrafilter in which the membrane permeability is adjusted so that the mixture of chitosan oligosaccharides that permeate through the membrane can contain 40% by weight or more of higher chitosan oligosaccharides of 5-18 sugars. After degrading chitosan with an enzyme having a degrading activity to produce a chitosan oligosaccharide mixture containing higher chitosan oligosaccharides, the chitosan oligosaccharide mixture is removed to the outside of the ultrafilter and then the removed component is obtained. A chitosan solution is supplied into the ultrafilter, and then the chitosan decomposition reaction, the removal of the chitosan oligosaccharide mixture, and the supply of the chitosan solution are continuously repeated to produce a higher chitosan oligosaccharide. And a method for producing a high-grade chitosan oligosaccharide. 3. A chitosan oligosaccharide mixture that passes through a membrane is produced in an ultrafilter in which the membrane permeability is adjusted so that the reducing sugar is 100 to 600 (mg.D-glucosamine / g). After a chitosan decomposition reaction with an enzyme having a decomposition activity is performed to produce a chitosan oligosaccharide mixture containing higher chitosan oligosaccharides, the chitosan oligosaccharide mixture is removed to the outside of the ultrafilter, and then the removed chitosan solution. Is supplied into the ultrafilter, and then the chitosan decomposition reaction, the removal of the chitosan oligosaccharide mixture, and the supply of the chitosan solution are continuously repeated to produce a higher chitosan oligosaccharide. Method for producing high-grade chitosan oligosaccharide. 4. A solid product obtained by drying the chitosan oligosaccharide mixture thus produced is mixed with 5 to 25 times by weight of methanol to contain 70% by weight or more of a higher chitosan oligosaccharide having 5 to 18 sugars as an insoluble matter. The method for producing a higher chitosan oligosaccharide according to any one of claims 1 to 3, wherein a chitosan oligosaccharide mixture is produced. 5. The degree of deacetylation of chitosan used as a substrate for the enzymatic reaction having chitosan degrading activity is 50.
It is -100%, The manufacturing method of the higher chitosan oligosaccharide in any one of Claim 1 thru | or 4. 6. The chitosan used as a substrate for the enzymatic reaction having the chitosan degrading activity has a molecular weight of 5,000 to 2
The method for producing a higher chitosan oligosaccharide according to any one of claims 1 to 5, which is low molecular weight chitosan of 00000. 7. The method for producing a higher chitosan oligosaccharide according to claim 1, wherein the enzyme having a chitosan degrading activity is chitosanase. 8. The chitosanase is an enzyme derived from a microorganism belonging to the genus Bacillus and has an optimum pH of 4.8 to 6.8.
The method for producing a higher chitosan oligosaccharide according to claim 7, which is a chitosanase having a good degradability with respect to a stable pH 3.3 to 7.4, solubilized chitosan having a deacetylation degree of 50 to 100%. 9. The microorganism belonging to the genus Bacillus is Bacillus sp. The method for producing a higher chitosan oligosaccharide according to claim 8, which is PI-7S (Microtechnology Research Institute, Microbiology No. 9843). 10. A higher chitin oligosaccharide characterized by producing a higher chitin oligosaccharide of 7 to 18 sugars by N-acetylating the chitosan oligosaccharide produced by the method for producing a higher chitosan oligosaccharide according to claim 1. Method for producing sugar. 11. A chitosan oligosaccharide produced by the method for producing a higher chitosan oligosaccharide according to claim 1, N-acetylated, dissolved in water and treated with acetone to obtain 4
A method for producing a higher chitin oligosaccharide, which comprises producing a 7-sugar higher chitin oligosaccharide.