JP2944723B2 - Method for producing chitin / chitosan oligomer having 2,5-anhydromannitol group having narrow molecular weight distribution - Google Patents

Description

[Background of the Invention] <Industrial application field> The present invention has a narrower molecular weight distribution than chitin or chitosan.
Chitin / chitosan oligomer having a 2,5-anhydromannitol group having a narrow molecular weight distribution capable of producing chitin oligosaccharide or chitosan oligosaccharide which is a mixture of chitin or chitosan oligomer having a 2,5-anhydromannitol group A method for producing the same.

<Prior Art> In recent years, the diet has been enriched, and crabs and shrimps have been imported from overseas, and chitin and chitosan have been produced in large amounts from the chitin of the crust. These chitins and chitosans are themselves being developed as pesticides, artificial skins, life-related substances, and the like, and chitin oligosaccharides or chitosan oligosaccharides with higher added value have been attracting attention.

It is known that such a chitin oligosaccharide or chitosan oligosaccharide can be generally produced by enzymatically decomposing chitin with chitinase and chitosan with chitosanase.

In addition, chitin and chitosan are partially hydrolyzed with concentrated hydrochloric acid to form N-acetylglucosamine or N-acetylglucosamine.
It is known to produce acetyl-chitopentaose and chitopentaose.

On the other hand, a method of producing glucosamine and the like by adding nitrous acid to chitosan and reacting at a temperature of 20 to 25 ° C. using the Bans-Like method for quantifying amino nitrogen (US Pat.
922,260) are also known.

<Problem to be Solved by the Invention> However, the method of decomposing with an enzyme requires a large-scale facility for mass production because the reaction concentration is low. Further, since the concentration of the product is as low as 0.001%, it has to be concentrated, and in order to obtain an oligosaccharide having a further appropriate molecular weight, much labor is required for fractionation. And the end Since a structure having the structure described above is produced, there is a concern that when heated by a concentration operation, a sterilization operation, or the like, a Maillard reaction occurs to cause coloring.

In the method of partially hydrolyzing with concentrated hydrochloric acid, since the same product as the above enzyme is obtained, coloring as described above,
The problem of ring opening cannot be solved.

Furthermore, the method of reacting nitrous acid with chitosan may be due to the use of high molecular weight chitosan, or the amount of nitrous acid used is also added in an equimolar amount to 1 mol of amino group of chitosan. Therefore, it is decomposed to glucosamine, which is a monosaccharide, or the reaction is carried out for a long time at a relatively high temperature of 20 ° C or higher. A product other than chitosan oligosaccharide is generated by a recombination reaction of the product.

Therefore, since the product obtained by the above reaction may contain a recombined glycoside compound or the like in which the expression of toxicity is a concern in addition to the glucosamine, the product may be used as a food additive or pharmaceutical. Has a problem.

[Summary of the Invention] <Summary> The present inventor has conducted intensive studies in view of the above problems, and as a result,
A 2,5-anhydromannitol group obtained by using a relatively low molecular weight chitin / chitosan as a raw material and performing a reaction with nitrous acid under relatively mild conditions for a relatively short time and then reducing it That chitin / chitosan oligomers having a specific molecular weight can be produced with a constant molecular weight, and chitin / chitosan oligomers having a 2,5-anhydromannitol group having a relatively narrow molecular weight distribution can be produced. Based on the above, the present invention has been completed.

That is, the method for producing a chitin-chitosan oligomer having a 2,5-anhydromannitol group having a narrow molecular weight distribution according to the present invention comprises reacting chitin or chitosan with nitrous acid, and then reducing the reaction to 2,5-anhydromannitol. A method for producing a chitin / chitosan oligomer having a mannitol group, wherein the chitin or chitosan has an average molecular weight of 5
It is characterized by using not more than 000 chitin or chitosan and performing the reaction under the following conditions (1) to (5).

(1) Reaction temperature: -5 to 10 ° C (2) Hydrogen ion concentration: 1 to 6 (3) Reaction time: 0.5 to 10 hours (4) Amino group in nitrous acid / chitin / chitosan: 0.01 to 1 (mol) (Ratio) (5) Solubilizer / water: 0.1 to 20% by volume <Effect> Such a method of the present invention provides a relatively high molecular weight chitin.
Using chitosan as a raw material, reacting it with nitrite under specific conditions that are relatively mild, and then reducing it, a chitin chitosan having a 2,5-anhydromannitol group having a relatively narrow molecular weight distribution is obtained. Oligomer can be obtained, chitin of specific molecular weight with high medicinal effect
Because of the high concentration of chitosan oligomer,
It is also highly effective when used as a food additive or pharmaceutical,
It is extremely useful.

[Specific description of the invention]

[I] Raw materials (1) Chitin / chitosan Generally, chitin / chitosan is a crustacean such as shrimp and crab,
Beetles, insects such as crickets, shiitake mushrooms, chitin contained as a component of the cell wall of filamentous fungi is treated with dilute hydrochloric acid to remove calcium carbonate, and treated with an alkaline solution for a short time to remove proteins and the like, Alternatively, chitosan obtained by heating and deacetylating this in the presence of concentrated alkali is known.

However, chitin and chitosan used in the method for producing a chitin / chitosan oligomer having a 2,5-anhydromannitol group having a relatively narrow molecular weight distribution according to the present invention may be obtained by using these chitin / chitosan as raw materials. In general, the average molecular weight of these natural chitins is several million, and the average molecular weight of chitosan is a high molecular weight of about 200,000. The reaction cannot be carried out sufficiently unless the reaction is carried out under severe conditions.

Therefore, the chitin / chitosan oligomer obtained by reacting under such severe conditions has various molecular weights in which high molecular weight and low molecular weight are mixed because the reaction easily occurs from the surface of the raw material particles. Only a mixture containing Therefore, it has been difficult to obtain a chitin / chitosan oligomer having a uniform molecular weight.

Therefore, in the present invention, only those having a very low molecular weight which is a special class among these natural chitin / chitosans are selected and used, or these high molecular weight natural chitin / chitosan are hydrolyzed to some extent. It is important to use chitin / chitosan having a uniform molecular weight.

That is, chitin or chitosan used as a raw material in the method for producing a chitin / chitosan oligomer having a 2,5-anhydromannitol group having a narrow molecular weight distribution of the present invention has an average molecular weight of 50,000 or less, preferably 2 to 5%.
10,000, particularly preferably 30,000 to 40,000 are used. Specifically, materials such as crab shell chitin decomposition products are used.

In the method for producing a chitin or chitosan oligomer having a 2,5-anhydromannitol group having a narrow molecular weight distribution according to the present invention, a reaction is caused by alcoholizing an amino group in chitin or chitosan with nitrous acid. And those containing an amino group in chitin are used. However, since natural chitin originally contains units having about 8 to 12% of amino groups, the reaction is also carried out using these units in the present invention. Alternatively, before performing the reaction of the present invention,
Chitin or chitosan obtained by deacetylating some or all of these natural chitins is used.

When performing the reaction of the present invention, these chitin or chitosan are solubilized to make the reaction proceed smoothly. Therefore, those previously cut into fine powder, especially
It is preferred to use flakes with a 15-30 mesh pass, preferably a 30 mesh pass.

(2) Nitrite As the nitrite used in the present invention, nitrite may be used as it is, but in order to allow the reaction to proceed gently, it is necessary to use a nitrite capable of obtaining nitrite in situ. preferable.

Examples of such nitrites include sodium nitrite, potassium nitrite, zinc nitrite, ammonium nitrite, calcium nitrite, barium nitrite, and magnesium nitrite. Of these, alkali metal nitrites, especially nitrite Sodium nitrate and potassium nitrite are preferred.

Since these nitrites are used to deaminate and alcoholate amino groups in chitin or chitosan, they are used in the range of 0.01 to 1 molar equivalent, preferably 0.1 to 0.6 molar equivalent, based on the amino group. The molecular weight of the chitin oligomer or chitosan oligomer produced can be controlled depending on the amount thereof. the above
If the amount is less than 0.01 molar equivalent, the reaction hardly occurs.If the amount exceeds 1 molar equivalent, monosaccharides such as N-acetylglucosamine and glucosamine are easily generated in large amounts, so that the yield of chitin oligosaccharide or chitosan oligosaccharide is reduced. I do.

[II] Reaction (1) Solubilization In producing a chitin / chitosan oligomer having a 2,5-anhydromannitol group having a narrow molecular weight distribution according to the present invention, in order to facilitate reaction of chitin and chitosan as raw materials, These chitin and chitosan are mixed with a solubilizing agent and solubilized in an aqueous solution.

(Solubilizing agent) Examples of the solubilizing agent used for solubilizing the chitin and chitosan include formic acid, acetic acid, butyric acid, oxalic acid, tartaric acid, succinic acid, lactic acid, ascorbic acid, propionic acid,
Adipic acid, organic acids having 1 to 10, preferably 2 to 7 carbon atoms, such as benzoic acid, hydrochloric acid, hydrobromic acid, mineral acids such as nitric acid and the like, among which acetic acid, oxalic acid, tartaric acid, It is best to use lactic acid, especially acetic acid.

These solubilizers can be used alone or as a mixture, but are generally used in a mixture with water as a reaction solvent.

These solubilizers are generally used in an amount of 0.5 mol% or more, and preferably equimolar or more, based on chitin and chitosan. , Preferably in an amount of 5 to 10% by volume.

By adjusting the kind and amount of the solubilizer, the hydrogen ion concentration in the reaction described below is adjusted within a specific range.

(2) Reaction Conditions In the method for producing a chitin / chitosan oligomer having a 2,5-anhydromannitol group having a narrow molecular weight distribution according to the present invention, the reaction with nitrous acid is carried out by the following (1) to (5).
It is important to carry out under the following conditions.

(1) Reaction temperature: -5 to 10 ° C, preferably 0 to 8 ° C, particularly preferably 2 to 6 ° C (2) Hydrogen ion concentration: 1 to 6, preferably 2 to 5 (3) Reaction time: 0.5 (10) preferably 0.5 to 3 hours (4) Nitrite / amino group in chitin / chitosan: 0.01 to 1, preferably 0.1 to 0.6 (molar ratio) (5) Solubilizer / water: 0.1 to 20 preferably 0.5 to 0.5 10 (volume ratio) By mixing under such conditions, the reaction becomes
Because of the gentle progress, chitin or chitosan is not hydrolyzed at the glycosidic linkage to which the glucosaminoglucan unit is attached, but rather alcoholates the amino groups in chitin or chitosan by deamination with nitrous acid, Since this alcohol group forms a pseudo-glycol adjacent to the oxygen of the glycosidic bond part of the glucosaminoglucan unit, it is presumed that the following pinacol transfer reaction may be caused.

If the reaction temperature under the above reaction conditions is lower than -5 ° C, the reaction becomes extremely slow. If the reaction temperature is higher than 10 ° C, a hydrolysis reaction occurs, and further, a rearrangement reaction and a recombination occur, and the occurrence of toxicity is a concern. There is a possibility that a bound glycoside compound or the like may be formed. When the hydrogen ion concentration is less than 1, a hydrolysis reaction in which a glycosidic bond is broken is likely to occur, which may cause a transfer reaction or a recombination.
If it exceeds 2,000, nitrous acid will be decomposed, resulting in expiration and difficulty in controlling the molecular weight.

The above reaction is carried out in an aqueous solution under the conditions of the above temperature, hydrogen ion concentration and the like, and the aqueous solution may contain other organic solvents, buffers and the like.

If the reaction time is less than 0.5 hour, the reaction time is insufficient. If the reaction time exceeds 10 hours, not only does the yield not increase, but also side reactions occur, which is not preferable.

After the completion of the reaction, neutralization is performed by adding concentrated ammonia water or alkylamines, or an anion exchange resin is added to adsorb and remove nitrous acid and the like.

[III] Reduction reaction The chitin or chitosan oligomer having a 2,5-anhydromannose group has one of the terminal structural formulas, Or, And the other is Since it is a structure having a 2,5-anhydromannose group, it has high reactivity and has a drawback that it is easy to color. By using 2,5-anhydromannitol as the above, a chitin or chitosan oligomer having reduced reactivity and stable to heat can be obtained.

For such a reduction reaction, any known reducing agent can be selected as long as it can reduce the aldehyde group gently.

Specifically, nickel-based hydrogenation reduction catalysts such as Raney-nickel and Ni-carbon, palladium-based hydrogenation reduction catalysts such as Pd-carbon, diisobutylaluminum hydride, organotin hydrides, and metal hydrogens such as hydrosilane Chloride, lithium aluminum hydride, sodium borohydride, potassium borohydride, lithium borohydride,
Examples thereof include metal hydride complex compounds such as calcium borohydride and zinc borohydride, diborane, alkylborane, and the like. Among these, metal hydride complex compounds, particularly hydrogenation of sodium borohydride and potassium borohydride Optimally, a reducing agent based on a boron compound is used.

Such a reduction reaction is carried out by adding the above reducing agent as it is or by simply removing impurities in the neutralization mixture by filtration or the like, and then adding the reducing agent. It is preferable to carry out continuously by adding the above-mentioned reducing agent.

The reducing agent is generally added in an amount of 1 mol or more, preferably 1.5 to 3 mol, per 1 mol of the 2,5-anhydromannose group in the chitin oligomer or chitosan oligomer, and the amount of 2,2 in the chitin oligomer or chitosan oligomer is reduced.
The 5-anhydromannose group is reduced to a 2,5-anhydromannitol group.

The reduction reaction is generally carried out at a temperature of 100 ° C. or less, preferably at room temperature or less, generally under normal pressure for several hours.

The reduction is carried out until substantially no 2,5-anhydromanninose groups are present.

[IV] Fractionation Since the raw materials used in the reaction are mixed in the reaction mixture thus obtained, chitin having a 2,5-anhydromannitol group formed from such an aqueous reaction mixture is used. Chitosan is soluble in water so that it can be fractionated for each molecular weight as much as possible, and
The chitin-chitosan oligomer having a 2,5-anhydromannitol group produced by adding a poor solvent of the chitin-chitosan oligomer having a 5-anhydromannitol group gradually or stepwise,
Since it can be precipitated sequentially from chitin or chitosan oligomer having a large molecular weight, if it is separated, it can be divided into various molecular weights. By such a fractionation operation, chitin with a narrower molecular weight distribution can be obtained.
It can be fractionated into chitosan oligomer components.

Examples of the deposition medium that is compatible with the aqueous medium and hardly dissolve the chitin oligomer or chitosan oligomer include alcohols, ketones, ethers, esters, and hydrocarbons.

Specifically, methanol, ethanol, propanol,
C 1-5 carbon atoms such as butanol and ethylene glycol,
Alcohols having 1 to 4, preferably 1 to 5 carbon atoms such as acetone and methyl ethyl ketone, preferably 1 to 4 ketones, 2 to 6 carbon atoms such as ethyl ether, preferably 2 to 4 ethers, ethyl acetate 2 to 1 carbon atoms such as
0, preferably 2 to 5 esters, n-hexane, petroleum ether and other hydrocarbons having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms.

(3) Addition amount The addition amount of the precipitation medium is an amount at which a chitin oligomer or a chitosan oligomer having a higher molecular weight than the aqueous medium is precipitated. Specifically, 1 liter of the aqueous medium is used for the precipitation medium. Is generally used in 0.4 to 1.5 liters, preferably 0.5 to 1 liter.

By sequentially fractionating the chitin oligomer or chitosan oligomer thus precipitated, chitin oligosaccharide or chitosan oligosaccharide can be separated into various polysaccharides having various molecular weights.

[III] Product The structural formula of the chitin / chitosan oligomer thus obtained is as follows: Has a 2,5-anhydromannitol group, and the structural formula at the other end is Or These two ends are either directly or A chain of units of the structural formula having an acetylamino group represented by The chain of the unit of the structural formula having an amino group represented by
000, preferably 0 to 500, particularly preferably 0 to 300
It is a mixture of those that have been bonded. When the ratio of the unit of the structural formula having an acetylamino group / the unit of the structural formula having an amino group exceeds 1, it is a chitin oligomer and 1
If it is less than 1, it is a chitosan oligomer.

When isolated as chitin oligosaccharides or chitosan oligosaccharides having a constant molecular weight obtained by fractionation for each type of polysaccharide in this way, highly effective chitin oligosaccharides or chitosan oligosaccharides having a specific molecular weight can be contained at a high concentration. Therefore, a higher effect can be exhibited.

In addition, chitin / chitosan oligomers having a 2,5-anhydromannitol group at the end are low in reactivity and high in thermal stability, so they are difficult to color, and as such as food additives and pharmaceuticals, or intermediates thereof. It is extremely useful.

(Experimental example)

Example 1 (nitrile / glucosamine residue = molar ratio 0.5) 10 g of flaky chitosan (molecular weight: 40,000) obtained from crab shell chitin decomposition product in a 30-mesh pass was placed in a glass beaker with a stirrer having an internal volume of 500 ml. Acetic acid solution
100 ml (solubiliser / water: 10% by volume) was added little by little under stirring to dissolve, and the mixture was cooled sufficiently to 3 ° C. in an ice water bath.

Then, 14.5 ml of a 10% aqueous solution of sodium nitrite (nitrile / amino group (molar ratio in chitin / chitosan: 0.5)) was added to adjust the hydrogen ion concentration to 3, and the mixture was stirred at 3 ° C. in ice water at 2 ° C. for 2 hours. After reacting for a time, a chitosan oligomer having a 2,5-anhydromannose group was produced.

After the reaction, after neutralizing with 15 ml of concentrated aqueous ammonia,
Further, 1.6 g of sodium borohydride (2 times mol with respect to sodium nitrite) was added, and the mixture was stirred at room temperature overnight to carry out a reduction reaction to produce a chitosan oligomer having a 2,5-anhydromannitol group. did.

After completion of the reduction reaction, the reaction solution was filtered to remove insolubles, and the filtrate was concentrated to 100 ml. Then, methanol was added thereto to precipitate the product. At that time, the amount of methanol used was 1: 3 (first fraction), 1: 5 (second fraction), and 1:10 (third fraction) in a concentrated liquid: methanol ratio.

Further, concentration was carried out to dryness, and methanol and acetone were added to precipitate the product. At that time, methanol and acetone were separated at a ratio of 1: 2 (fourth fraction). These precipitates were thoroughly washed with acetone and ether, and dried in a vacuum desiccator.

The solids of the first to fifth fractions obtained by this fractionation were analyzed by high performance liquid chromatography and elemental analysis.

The analysis conditions in the high performance liquid chromatography analysis were as described above.

Column: Asahi Pack GFA-30F Flow rate: 0.3 ml / min Temperature: 50 ° C Mobile phase: 0.5% acetate buffer pH: 4.0 The results are shown in FIGS. 1 (a) to (d).

 The products in each fraction were as shown below.

First fraction (FIG. 1 (a)) Yield: 7.2% by weight Peak a-1: Raw material chitosan Peak a-2: Chitosan oligomer 88.5 mol% Molecular weight: 40,000 to 1,300, (246 sugars to 7 sugars) Peak a- 3: Fraction 2 of sodium acetate salt (Fig. 1 (b)) Yield: 15.2% by weight Peak b-1: Chitosan starting material Peak b-2: 99.7 mol% of chitosan oligomer Molecular weight: 45,000 to 1,300 (277 sugar to 7 Sugar) Peak b-3: sodium acetate third fraction (Fig. 1 (c)) Yield: 8.4% by weight Peak c-1: Chitosan oligomer 100 mol% Molecular weight: 25,000 to 1,000, (154 sugars to 5 sugars) Peak c-2: sodium acetate 4th fraction (FIG. 1 (d)) Yield: 42.2% by weight Peak d-1: 97.4 mol% of chitosan oligomer Molecular weight: 25,000 to 1,300, (154 sugars to 7 sugars) Peak d -2: Chitosan oligomer 2.6 mol% Molecular weight: 1,300 to 900, (7 sugars to 5 sugars) Peak d-3: Comparison of various salts Example 1 A flake-form chitosan of Example 1 (molecular weight: 40,000 was used in place of flaky chitosan having a molecular weight of 200,000, and a precipitate was obtained in the same manner as in Example 1 to obtain a precipitate. Was filtered, washed sufficiently with acetone, dried in a vacuum open, and analyzed by high performance liquid chromatography.

 The result is shown in FIG.

The product was a wide range of chitosan oligosaccharides with molecular weights between 160,000 and 1,500, with a product yield of 0.15 g. This was a yield of 1.5% of theory.

[Brief description of the drawings]

FIGS. 1 (a) to 1 (d) are graphs obtained by fractionating chitosan oligosaccharides produced from low molecular weight chitosan in the examples of the present invention and performing high-performance liquid chromatography analysis, and FIG. FIG. 2 is a diagram drawn by high-performance chromatography analysis of chitin oligosaccharide produced from chitosan of No. 1. Peak a-1: Raw material chitosan Peak a-2: Chitosan oligomer Peak a-3: Sodium acetate peak b-1: Raw material chitosan Peak b-2: Chitosan oligomer peak b-3: Sodium acetate peak c-1: Chitosan Oligomer peak c-2: sodium acetate peak d-1: chitosan oligomer peak d-2: chitosan oligomer peak d-3: various salts

Claims (1)

(57) [Claims] A method for producing a chitin or chitosan oligomer having a 2,5-anhydromannitol group after reacting chitin or chitosan with nitrous acid, wherein said chitin or chitosan has an average molecular weight of 50,000.
Chitin / chitosan having a 2,5-anhydromannitol group having a narrow molecular weight distribution, wherein the following chitin or chitosan is used and the reaction is carried out under the following conditions (1) to (5): Method for producing oligomer. (1) Reaction temperature: -5 to 10 ° C (2) Hydrogen ion concentration: 1 to 6 (3) Reaction time: 0.5 to 10 hours (4) Amino group in nitrous acid / chitin / chitosan: 0.01 to 1 ( (Molar ratio) (5) Solubilizer / water: 0.1-20% by volume