JP2003012702A - Method for producing low molecular chitosan - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a low molecular chitosan from a high molecular chitosan in a good reproducibility. SOLUTION: This method for producing a low molecular chitosan comprises hydrolyzing the high molecular part of an acetylated chitosan having 10-30% degree of acetylation and >=1,000,000 molecular weight with a chitosan- decomposing enzyme, and removing a fraction having <10,000 moleclar weight to produce the low molecular chitosan having >=10,000 and <1,000,000 molecular weight or its salt. The chitosan-decomposing enzyme has <100% hydrolysis activity against a glycol chitin and <=15% hydrolysis activity against the chitosan (50-200 cps viscosity) having <=1% degree of acetylation by setting 100% hydrolyzing activity against the chitosan (100-300 cps viscosity) having 30% degree of acetylation at pH 7.0-7.5, at 37 deg.C temperature for 10-20 min reaction.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a low molecular weight chitosan, a low molecular weight chitosan and a pharmaceutical composition containing the low molecular weight chitosan.

[0002]

2. Description of the Related Art Chitin is a polysaccharide which exists in a very large amount in nature, and is mainly produced on an industrial scale by being extracted from crustaceans such as crab and shrimp and squid soft shell. Chitosan is a polymeric polysaccharide having an amino group, which is obtained by treating chitin with concentrated alkaline water, and is known to have various physiological activities (Gihodo "Chitin.
See Chitosan Handbook, pages 302-376). Among them, chitosan has a strong antibacterial action as a natural product-derived substance, and its safety is extremely higher than that of synthetic antibacterial agents (for example, "Utilization of chitin / chitosan", Special Issue on Business World, Volume 46).
No. 28, pp. 106-111, 1998). In particular, methicillin cephem-resistant Staphylococcus aureus
lin resistants aureus) (hereinafter referred to as "MRSA"), including Staphylococcus aur
eus) (For example, processing technology, Vol. 33, 512-515 1998, Meijo University Faculty of Agriculture 34, 25-34 1998, Textile science 35, 37-39
Page 1993, Research Report, Osaka Prefectural Institute of Public Health No. 29, 7-12 1995, Processing Technology Volume 32, 339-343 1997
, Processing Technology, Vol. 33, p. 530-532, 1998, etc.), Escherichia coli (eg Tokura S e
t al. Macromol. Symp. 120, 1-9, 1997 etc.), Pseudomonas aeruginosa
(For example Loke WK et al. J. Biomed. Mater. Res. 53,
8-17, 2000, Kim HJ et al. J. Biomater. Sci. Pol
ym. Ed. 10, 543-56, 1999, etc.) and the like. By the way, at present, the emergence of resistant bacteria against synthetic antibacterial agents that are effective against these bacteria is a big problem. The antibacterial action of chitosan is not clear, but it is expected that the mechanism is different from that of synthetic antibacterial agents. Therefore, even bacteria resistant to synthetic antibacterial agents may be susceptible to chitosan. However, the effect of chitosan on drug-resistant bacteria other than MRSA has not been studied at all. Moreover, the effect on Serratia marcescens, which is a problem due to nosocomial infections, has not been examined. Furthermore, Streptococcus, which is involved in oral hygiene,
For mutans (Streptococcus mutans), chitosan derivatives (eg Kurita K. et al. Chem. Lett. 3
17-318, 1998, etc.) or squid soft shell-derived chitosan (see, for example, JP-A-7-157434, etc.) has been reported to be effective. However, although the safety of chitosan has been confirmed, it is unknown about its derivatives, so
It is preferable to use chitosan without derivatization. Furthermore, it is said that S. mutans is not effective against chitosan derived from shrimp and crab, and that S. mutans is sensitive only to chitosan derived from squid soft shell. I'm not sure. Since shrimp, crab, etc. can be cultured, chitosan produced from a substance that can be expected to provide a stable supply of raw materials is preferable as a raw material.

Chitosan produced from chitin has a molecular weight of 1
It is a high-molecular-weight polysaccharide of over 1,000,000 and is soluble in water only under acidic conditions. When it is neutral or alkaline, it becomes insoluble in water and is in a state where physiological activity cannot be fully exerted. Therefore, in order to increase the solubility in water, attempts have been made to derivatize high molecular weight chitosan or reduce the molecular weight thereof without impairing the physiological activity of high molecular weight chitosan. The latter method is preferable because derivatization makes it a non-natural product and it is necessary to ensure safety.

[0004] For example, it is known that the antibacterial activity of high molecular weight chitosan is exerted even in low molecular weight chitosan having a molecular weight of 10,000 or more (see, for example, the Journal of the Japanese Society of Agricultural Chemistry, Volume 74, Annual Meeting, Performance No. 2E183α, etc.). ). Furthermore, it has been reported that chitosan having a molecular weight of 10,000 or less has side effects such as skin irritation (see, for example, JP 2000-169327 A), and a method for producing a low molecular weight chitosan having a molecular weight of 10,000 or more and less than 1 million has been investigated. ing. As a method, hydrolysis of polymer chitosan by a chemical or enzymatic method has been studied. As a chemical method, a method of treating with an oxidant (see, for example, Japanese Patent No. 3076212, JP-A-10-158305, etc.), a method of ultrasonic treatment (for example, Chitin and Chitosan Research Vol. 5, pages 75-79 1999, etc.) As an enzymatic method, a method using chitosanase (for example, Patent No.
No. 2763112, JP-A No. 11-322809, etc.), a method using chitinase (see, for example, Journal of the Japanese Society of Agricultural Chemistry, Vol. 74, Annual Meeting Summary, Performance No. 2F313α, etc.) and the like. However, it is unclear whether the molecular weight distribution is obtained by the method of treating with an oxidizing agent, and whether low molecular weight chitosan having the same molecular weight can always be obtained. The ultrasonic treatment method is considered unsuitable for mass production. Further, the method using chitosanase has a problem that the reaction control for always obtaining low molecular weight chitosan having the same molecular weight is complicated. It is not known whether low molecular weight chitosan having a molecular weight of 10,000 or more can be obtained by hydrolysis of high molecular weight chitosan using chitinase.

[0005]

[Problems to be Solved by the Invention] Polymer chitosan derived from shrimp and crab, whose supply is secured in the future, is used as a material, and it is soluble in water even under neutral conditions and its safety is ensured. There is a need for a method by which non-derivative low molecular weight chitosan can be supplied in a state of always having the same molecular weight distribution. Further, the obtained low molecular weight chitosan has antibacterial activity equal to or higher than that of the high molecular weight chitosan, and further S. aureus, E.
It has the same antibacterial activity against synthetic antimicrobial-resistant bacteria such as Escherichia coli and P. aeruginosa as the standard strains.
It is also desirable to have the same effect on S. mutans and other strains.

An object of the present invention is to provide a method for reproducibly producing low molecular weight chitosan from high molecular weight chitosan.

[0007]

[Means for Solving the Problems] The inventors of the present invention efficiently and efficiently obtain a low molecular weight chitosan having desired physical properties and antibacterial activity.
And after diligently studying a method for producing with good reproducibility, the present inventor has already reported that chitosan degrading enzyme derived from Bacillus sp. SANK70699 strain (FERM BP-6666) (JP 2000- The present inventors have succeeded in producing a low molecular weight chitosan having a molecular weight of 10,000 or more and less than 1,000,000 from a polymeric chitosan derived from crab / shrimp by an enzymatic reaction using 312583 gazette), and completed the present invention.

The gist of the present invention is as follows. (1) The degree of acetylation is 10 to 30% and the molecular weight is 100.
A low molecular weight chitosan having a molecular weight of 10,000 or more and less than 1 million or a salt thereof is produced by hydrolyzing 10,000 or more high molecular weight partially acetylated chitosan with a chitosan degrading enzyme and then removing a fraction having a molecular weight of less than 10,000. In the method, the chitosan degrading enzyme has a hydrolysis activity for 30% acetylated chitosan (viscosity 100 to 300 cps) under reaction conditions of pH 7.0 to 7.5 and temperature 37 ° C for 10 to 20 minutes. 10
The above method wherein the hydrolysis activity against glycol chitin is less than 100% and the hydrolysis activity against chitosan having a degree of acetylation of 1% or less (viscosity 50 to 200 cps) is 15% or less when 0% is set. (2) A low molecular weight chitosan or a salt thereof dissolves in 10 g or more in 100 g of water at 0 to 100 ° C. (1)
The method described. (3) The method according to (1) or (2), wherein the low molecular weight chitosan or a salt thereof has antibacterial activity. (4) The chitosan degrading enzyme is Bacillus sp.
s sp.) SANK70699 strain (FERM BP-6666) or Bacillus sp. SANK70799 strain (FERM BP-6667) -derived chitosan degrading enzyme (1) to (3). (5) 10 under conditions of pH 3 to 12 and 70 ° C. or lower
The method according to any one of (1) to (4), wherein the polymer partially acetylated chitosan is hydrolyzed with a chitosan degrading enzyme for minutes to 7 days. (6) Polymer partially acetylated chitosan is dissolved in water under acidic condition to make it weakly alkaline, and then chitosan degrading enzyme is added for hydrolysis to produce low molecular chitosan or its salt. The method according to any one of (1) to (5). (7) Molecular weight of 10,000 or more and less than 1,000,000, 0 to 100 ° C
Low molecular weight chitosan or a salt thereof which dissolves in 10 g or more with respect to 100 g of water. (8) A pharmaceutical composition comprising the low molecular weight chitosan or the salt thereof according to (7). (9) A preventive and / or therapeutic agent for wounds and / or pressure sores, which comprises the low molecular weight chitosan or salt thereof according to (7) as an active ingredient. (10) An antibacterial agent containing the low molecular weight chitosan or salt thereof according to (7) as an active ingredient. (11) An antihyperlipidemic agent containing the low molecular weight chitosan or the salt thereof according to (7) as an active ingredient. (12) An anti-atherosclerotic agent containing the low molecular weight chitosan or the salt thereof according to (7) as an active ingredient. (13) An antidiabetic agent containing the low molecular weight chitosan or salt thereof according to (7) as an active ingredient. (14) An antiobesity agent containing the low molecular weight chitosan or the salt thereof according to (7) as an active ingredient. (15) The degree of acetylation is 10 to 30% and the molecular weight is 100.
The method according to (1), wherein 10,000 or more of the polymer partially acetylated chitosans are derived from crustaceans. (16) pH 3 to 12, under the condition of 70 ℃ or less, 1
It is characterized by hydrolyzing a polymer partially acetylated chitosan having an acetylation degree of 10 to 30% and a molecular weight of 1,000,000 or more with a chitosan degrading enzyme for 0 minutes to 7 days.
5) The method described. (17) Under conditions of pH 6.0 to 7.5 and 37 ° C. to 40 ° C., while shaking at 80 to 170 rpm for 2 to 3 days, the degree of acetylation is 10 to 30% and the molecular weight is 100.
The method according to (16), which comprises hydrolyzing 10,000 or more high molecular weight partially acetylated chitosan with a chitosan degrading enzyme. (18) The degree of acetylation is 10 to 30% and the molecular weight is 100.
More than 10,000 high molecular weight partially acetylated chitosans were previously
Any one of (1) or (15) to (17), characterized by being dissolved in water under the conditions of 2.5 to 3.5 and then adding an alkali to adjust the pH to 6.0 to 7.5. The method described in one. (19) The chitosan degrading enzyme is Bacillus sp.
lus sp.) SANK70699 strain (FERM BP-6666) or Bacillus sp. SANK70799 strain (FERM BP-6667) derived chitosan degrading enzyme, (15)
The method according to any one of (1) to (18). (20) A low molecular weight chitosan having a molecular weight of 10,000 or more and less than 1 million or a salt thereof, which is produced by the method according to any one of (1) or (15) to (19). (21) The low molecular weight chitosan or salt thereof according to (20), which is dissolved in 100 g of water at 0 to 100 ° C. in an amount of 10 g or more. (22) The pH of 1% by mass low molecular weight chitosan salt aqueous solution is pH
The low molecular weight chitosan or the salt thereof according to (20) or (21), which is 4.8 to 6.3. (23) The pH at which a precipitate is deposited when an alkali is added to a 1% by mass low molecular weight chitosan salt aqueous solution is pH 6.4 to 8.2, (20) or (21)
The low-molecular-weight chitosan or a salt thereof described in 1. (24) Low molecular weight chitosan or a salt thereof having the following properties [I] to [IV]; [I] having a molecular weight of 10,000 or more and less than 1 million: [II] with respect to 100 g of water at 0 to 100 ° C. Dissolve 10 g or more: pH of [III] is pH 4.8 to 6.3: [IV] pH at which a precipitate is deposited when an alkali is added to a 1% by mass low molecular weight chitosan salt aqueous solution is pH 6. 4 to 8.2. (25) A pharmaceutical composition comprising the low molecular weight chitosan or a salt thereof according to any one of (20) to (24). (26) An antibacterial agent containing the low molecular weight chitosan or salt thereof according to any one of (20) to (24) as an active ingredient. (27) An antihyperlipidemic agent containing the low molecular weight chitosan or salt thereof according to any one of (20) to (24) as an active ingredient. (28) An anti-atherosclerotic agent containing the low molecular weight chitosan or a salt thereof according to any one of (20) to (24) as an active ingredient. (29) An antiobesity agent containing the low molecular weight chitosan or the salt thereof according to any one of (20) to (24) as an active ingredient. (30) An antidiabetic agent containing the low molecular weight chitosan or salt thereof according to any one of (20) to (24) as an active ingredient. (31) A therapeutic and / or prophylactic agent for a wound and / or a pressure sore, which comprises the low molecular weight chitosan or a salt thereof according to any one of (20) to (24) as an active ingredient. The present invention hydrolyzes a high molecular weight partially acetylated chitosan having a degree of acetylation of 10 to 30% and a molecular weight of 1,000,000 or more with a chitosan degrading enzyme, and then removing a fraction having a molecular weight of less than 10,000 to obtain a molecular weight of less than 10,000. A method for producing low molecular weight chitosan of 10,000 or more and less than 1 million or a salt thereof, wherein the chitosan degrading enzyme has a pH of 7.0 to 7.5 and a temperature of 37 ° C.
100% hydrolytic activity against 30% acetylated chitosan (viscosity 100-300 cps) under reaction condition for ~ 20 minutes
, The hydrolysis activity for glycol chitin is less than 100%, and the hydrolysis activity for chitosan having a degree of acetylation of 1% or less (viscosity 50 to 200 cps) is 15%.
% Of the above method is provided. The molecular weight of the low molecular weight chitosan is preferably 10,000 to 500,000, and more preferably 50,000 to 300,000. The low molecular weight chitosan or a salt thereof may be dissolved in 100 g of water at 0 to 100 ° C. in an amount of 10 g or more. The water may be distilled water, tap water, or well water. Furthermore, the low molecular weight chitosan or its salt is preferably pH 1. 0 to 12.0 under the condition of pH 1.0 to 12.0.
It is preferable that 10 g or more is dissolved in 100 g of water under the condition of 0 to 7.0. In addition, the low molecular weight chitosan or its salt preferably has antibacterial activity.

As an example of chitosan degrading enzyme, Bacillus sp. SANK 70699 strain (FERM BP-6666)
And Bacillus sp. SANK70799 strain (F
ERM BP-6667: Chitosan degrading enzyme derived from JP-A-2000-312583).

In the above-mentioned method, it is advisable to hydrolyze the partially polymerized acetylated chitosan with a chitosan-degrading enzyme under conditions of pH 3 to 12 and 70 ° C. or lower for 10 minutes to 7 days. A polymer partially acetylated chitosan is dissolved in water under acidic conditions (for example, pH 1 to 6, preferably pH 2 to 4, more preferably pH 2.5 to 3.5), and then dissolved in an alkali solution (for example, sodium carbonate, Add sodium bicarbonate, caustic soda, etc. to make it slightly alkaline (usually pH 5 or above,
(Preferably pH 6-11, more preferably pH 6-7.5)
After that, add chitosan degrading enzyme to hydrolyze,
Low molecular weight chitosan or salts thereof can be produced. Acids used to make acidic conditions include hydrochloric acid, acetic acid,
Examples thereof include trifluoroacetic acid, paratoluenesulfonic acid, acrylic acid, lactic acid, amino acid, citric acid, salicylic acid, glucuronic acid, glycolic acid and the like. This acid reacts with the low molecular weight chitosan to form a low molecular weight chitosan salt. The hydrolysis temperature is usually 70 ° C or lower, preferably 25 ° C.
To 50 ° C, and more preferably 37 to 40 ° C. The hydrolysis time is usually 1 hour or longer, preferably 1 hour to 5 hours.
Days, more preferably 2-3 days.

The present invention has a molecular weight of 10,000 or more and 100 or more.
Provided is a low molecular weight chitosan or a salt thereof, which is soluble in 100 g of water at 0 to 100 ° C. in an amount of less than 10,000.

The present invention further provides a pharmaceutical composition containing the low molecular weight chitosan of the present invention or a salt thereof. The low molecular weight chitosan salt contained in the pharmaceutical composition of the present invention is preferably pharmaceutically acceptable.

The pharmaceutical composition of the present invention can be used as an antibacterial agent for the treatment and / or treatment of wounds and / or pressure sores.

The present invention also provides an antidiabetic agent, an antihyperlipidemic agent, an antiarteriosclerotic agent and an antiobesity agent containing the low molecular weight chitosan of the present invention.

The acetylation degree of chitosan can be calculated from the integral ratio of 3H of the acetyl group obtained by ¹ H-NMR and 7H excluding the hydroxyl groups other than the acetyl group.

The hydrolytic activity of chitosan degrading enzyme is Rand
le-Morgan method (Randle CJM et al. Biochem. J. GL 586-
589, 1955), and can be measured as described in Example 1 below.

Glycol chitin is available from R. Senzyu and S. O.
kimatsu, Nippon Nogeikagaku Kaishi, 23, 432 (1950)
Can be produced as described in.

The present invention will be described in detail below.

In the present specification, "to" indicates a range including the numerical values described before and after it as the minimum value and the maximum value, respectively.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION It is known that when chitin is industrially deacetylated with alkali to produce chitosan, partially acetylated chitosan which is not completely deacetylated is produced (Chitin / Chitosan Handbook p. 707
-709, 1995 Gihodo). It is known that the degree of acetylation of partially acetylated chitosan can be controlled by the alkali concentration, reaction time, etc., 30% from shrimp and crab,
20% and 10% acetylated chitosan are on the market (brand name chitosan 7B, 8B, 9B. All are Katokichi Co., Ltd.)
Made). These partially acetylated chitosans have a molecular weight of 100.
There are more than 10,000 macromolecules, which were dissolved in water under acidic conditions, and the SANK70699 strain (FERM BP-66
By hydrolyzing with the chitosan degrading enzyme produced by 66), low molecular weight chitosan having a molecular weight of 10,000 or more and less than 1 million can be efficiently obtained as various salts.

The substrate is 0.1 to 5% by mass, preferably 0.25.
˜2% by mass, more preferably 0.5 to 1% by mass.

The polymer partially acetylated chitosan is preferably derived from crustaceans such as shrimp and crab. The molecular weight of high molecular weight partially acetylated chitosan can be measured by high performance liquid chromatography using gel permeation chromatography (GPC) (pullulan is a standard substance) (Takiguchi et al., Chitin-Chitosan Research, p.75- 79,199
9). The molecular weights measured by this method are the average molecular weight and the molecular weight distribution.

The degree of acetylation of high molecular weight partially acetylated chitosan is 1 using a toluidine blue solution as an indicator.
It can be measured by performing PVSK colloid titration with a / 400N polyvinyl potassium sulfate solution (Toei K.
et al. Anal. Chem. Acta 83, 59, 1976). The degree of acetylation measured by this method is% of amino groups that are acetylated (for example, 1% acetylation means that one of 100 amino groups is acetylated). ) Is a value that represents.

The enzyme used in the method of the present invention is p
30% acetylated chitosan (viscosity 100-300 c under reaction conditions of H7.0-7.5, temperature 37 ° C for 10-20 minutes).
ps) has a hydrolytic activity of the chitosan degrading enzyme of 1
When it is set to 00%, the hydrolysis activity of the chitosan degrading enzyme for glycol chitin is less than 100%,
Chitosan with an acetylation degree of 1% or less (viscosity 50-200 cp
The hydrolysis activity of the chitosan degrading enzyme against s) is 1
It may be 5% or less. Examples of chitosan degrading enzyme that can be used in the production method of the present invention include bacteria, actinomycetes,
Examples include enzymes produced by filamentous fungi, yeasts and the like.

Examples of enzymes produced by bacteria include the genus Bacillus, Corynebacteriu
m) genus (see Veldkamp, H., Nature, 169, 500 (1952)), Cytophaga genus, Acromobacter hyteropticum (Acromobacter hyteropticum:
Campbell, Jr., LL et al., J. Gen. Microbiol., 5,
894 (1951)), Flavobacterium (Flavobacteriu
genus and Micrococcus
chitosan-degrading enzyme (Campbell,
Jr., LL, et al., J. Gen. Microbiol., 5, 894 (19
51)), and Alteromonas SP (Alter
chitinase A (GenBank Access) produced by omonas sp.
ion No. D13762) and the like, and is preferably a chitosan degrading enzyme produced by the genus Bacillus. Examples of the enzyme produced by the genus Bacillus include Bacillus sp. SANK70699 (FERM BP-6666) strain, Bacillus sp. SANK70799 (FERM BP-6667) strain, and Bacillus alvei (Cody, RM, Cur. Microbiol). ., 19,
201 (1989)), Bacillus circulans (Bacill
us circulans), Bacillus licheniformis (Bacill)
us licheniformis: Cody, RM, Cur. Microbiol., 19,
201 (1989)), Bacillus s.
ubtilis), Bacillus thuringiensis
thuringiensis), Bacillus chitinosporus (Bacillu)
s chitinosporus :: Cody, RM, Cur.Microbiol., 1
9, 201 (1989)), Bacillus pulvifaciens: Cody, RM, Cur. Microbi
ol., 19, 201 (1989)) and Bacillus mecerans: Cody, RM, Cur. Microbiol.,
19,201 (1989)), and more specifically, chitinase A1 (GenBank Access) produced by Bacillus circulans.
ion No.M57601), C1 (GenBank Accession No.D8956)
8) and D (GenBank Accession No. D10594), a chitinase produced by Bacillus licheniformis (GenBank Ac
cession No.U71214), a protein produced by Bacillus subtilis (GenBank Accession No.D26185), and a chitinase produced by Bacillus thuringiensis (GenBank
Accession No.U89796), Bacillus circulans WL-
Chitosan degrading enzyme produced by 12 strains (Tanaka, H., et a
l., J. Gen. Appl. Mcrobiol., 16, 39 (1970)),
Chitosan degrading enzyme produced by Bacillus licheniformis strain X-7u (Takiguchi, Y., et al., Agric. Biol. Che
m, 53, 1539 (1989)) and the like, and more preferably Bacillus sp. SANK70699 (FERM BP-666
6) Stocks and Bacillus SP SANK70799 (FERM BP-6667)
It is a chitosan degrading enzyme produced by the strain. Examples of chitosan-degrading enzymes produced by the genus Cytophaga include Cytophga johnsonae: Stainer, R.
Y., J. Bacteriol., 53, 297 (1947)) and Cytophaga johnsonii: Saundar.
Examples include chitosan degrading enzymes produced by raj, N., et al., Arch., Mcrobiol., 85, 159 (1972)). As a chitosan degrading enzyme produced by Flavobacterium, a chitosan degrading enzyme produced by a Flavobacterium indoltheticum strain (Campbell, Jr., LL, et al., J. Gen. M.
icrobiol., 5, 894 (1951)), Flavobacterium menigosepticu
m) produced endo-N-acetylglucosaminidase F1 (GenBank Acce
ssion No.M80793), F2 (GenBank Accession No.L06
331) and F3 (GenBank Accession No. L06332).

The enzyme produced by actinomycetes is Streptomyces, Nocardiopsis (No
Examples thereof include chitosan degrading enzymes produced by the genus cardiopsis and the genus Actinomyces. The enzyme produced by the genus Streptomyces includes Streptomyces aerocolonigenes (Streptomyces aer
ocolonigenes: Teruta Nakagami et al., Fermentation Research Institute, Kogakuin, 3
0, 19 (1966)), Streptomyces albidofravus, Streptomyces alboflavus (Streptomyces alboflavus: Teruta Nakagami et al., Kogakuin Fermentation Research Institute Report, 30, 19 (1966)), Stre Streptomyces bob
iliae: Teruta Nakagami et al., Fermentation Research Institute, Kogakuin, 30, 19 (19
66)), Streptomyces cinereolver (Strep
tomyces cinereoruber: Tagawa, K., etal., J. Fermen
t. Bioeng., 71, 230 (1991)), Streptomyces coelicolor, Streptomyces erthraeu
s), Streptomyces griseus
iseus: Reynolds, DM, J. Gen. Microbiol., 11, 15
0 (see 1954)), Streptomyces lividans (Strep
tomyces lividans: Neugebauer, E., et al., Arch.Mic
robiol, 156, 192 (1991)), Streptomyces luteoverticilla
tus: Teruta Nakagami et al., Fermentation Research Institute, Kogakuin, 30, 19 (196
6)), Streptomyces olivaseus (Strept
omyces olivaceus), Streptomyces orientalis: Tominaga, Y., et al.,
Agric. Biol. Chem., 40, 2325 (1976)), Streptomyces parvullus: Teruta Nakagami et al., Fermentation Research Institute of the Institute of Industrial Technology, 30, 19 (1966)), Streptomyces remosus (see Streptomyces rim
ousus: Teruta Nakagami et al., Fermentation Research Institute of Kogakuin, 30, 19 (19
66)), Streptomyce
s rochei: Teruta Nakagami et al., Fermentation Research Institute, Kogakuin, 30, 19
(See (1966)), Streptomyces louver (Streptom
yces ruber), Streptomyces vinaceus (Stre
ptomyces vinacceus), Streptomyces viridochromogenes, Streptomyces erythaeu
s), Streptomyces olivaseoviridis (S
treptomyces olivaceoviridis), Streptomyces
Streptomyces plicatus, Streptomyces thermovio
laceus), Streptomyces
sp.) A1 strain (Jeniaux, C., Compt. rend. Soc. Bio
l., 149, 1307 (1955)), A7 strain (Nakagami Teruta et al.,
Kogakuin Fermentation Research Institute Report, 30, 19 (1966)), S-8
4 strains (Ueno, H., et al., J. Gen. Appl. Mcrobiol., 3
6, 377 (1990)), and Streptomyces No. Examples thereof include chitosan-degrading enzymes produced by 73 strains, and more specifically, Streptomyces arvidoflaves.
HUT 6111 strain (see Daihomei, Hiroshima Women's University Bulletin, 2, 15 (1967)), Streptomyces coelicolor HUT6004 strain (see Daihomei, Hiroshima Women's University Bulletin, 2, 15 (1967)), Streptomyces ersulaeus ATCC 11635 strain (Hara, S., et
al., J. Biochem., 105, 484 (1989)), Streptomyces griseus HUT 6037 strain (see Daihomei, Hiroshima Women's University Bulletin, 2, 15 (1967)), HUT 6038 strain (Ohomei, Hiroshima). Women's University Bulletin, 2, 15 (1967)), Streptomyces
Griseus var (Shota Nakagami et al., Fermentation Research Institute, Kogakuin, 3
0, 19 (1966)), Streptomyces olivaseus HUT 6023 strain (Dahomei, Bulletin of Hiroshima Women's University, 2, 15 (1967)), Streptomyces louver HUT 6014 strain (Dahomei, Hiroshima Women's College bulletin, 2, 2. 15 (1967)), Streptomyces vinaceus HUT 6073 strain (Dahomei, Bulletin of Hiroshima Women's University, 2, 15 (1967)) and Streptomyces viridochromogenes HUT 6076 strain (Daibayo, Hiroshima Women's Bulletin, 2) , 15 (1967)), a chitinase produced by Streptomyces coelicolor (GeneBank Acc
ession No. AL021411), a chitinase produced by Streptomyces erythaeus (Swiss-Prot Accession No.
P14529), chitinase A (GeneBank Accession No. D13775) and C produced by Streptomyces lividans.
(GeneBank Accession No. D12647), Exochitinase produced by Streptomyces olivaseoviridis (GeneBank Accession No. X71080), Chitinase-63 produced by Streptomyces precastus (Gene
Bank Accession No. M82804) and endo-N-acetylglucosaminidase H (GeneBank Accession No. K-2
182), and chitinase (GeneBank Accession No. D produced by Streptomyces thermomovioraceus).
14536) and the like. Examples of the enzyme produced by the genus Nocardiopsis include chitosan degrading enzyme produced by Nocardiopsis albus (Nocerdiopsis albus), and more specifically, Nocardiopsis albus subsp. Prasina OPC- 131 shares (Ts
ujibo, H., et al., Biosci. Biotech. Biochem., 56,
1304 (see 1992)), Nocardiopsis SP S
ANK 60599 strain (FERM BP-6665) and the like can be exemplified. The enzyme produced by Actinomyces genus is Actinomyces violaceus (W
igert, H., Naturwiss., 49, 379 (1962)), and the like.

Enzymes produced by filamentous fungi include Aspergillus genus, Rhizopus genus,
Genus Talaromyces, Trichoderma (Trich
Examples thereof include chitosan-degrading enzymes produced by the genus oderma and Penicillium, and preferably chitosan-degrading enzymes produced by Aspergillus, Rhizopus, Talalomyces, and Trichoderma. Aspergillus nidulans (Aspergillus nidulans), Aspergillu niger (Aspergillu niger), Aspergillus candidus (Sherif, A.
A., et al., Appl. Mcrobiol., Biotechnol., 35, 228
(1991)) and Aspergillus fumigatus (Asperg
illus fumigatus) produced by chitosan degrading enzyme, and more specifically, Aspergillus
Chitinase produced by Nidulans (GenBank Accessio
n No. D87063 and D87895), Aspergillus niger K1
Enzymes produced by 4 strains (Tetsu Okimasa et al., Journal of Japan Society for Agricultural Chemistry, 34,
873 (1960)), Aspergillus fumigatus QM45h
Enzymes produced by Monreal, J., et al., Can. J. Mcrob
iol., 15., 689 (1969)) and the like. Examples of the enzyme produced by Rhizopus include chitosan degrading enzymes produced by Rhizopus niveus and Rhizopus oligosporus, and more specifically, produced by Rhizopus niveus. Chitinase (GenBank Acce
ssion No. D10154), Chitinase I produced by Rhizopus oligosporus (GenBank Accession No. D10157), I
I (GenBank Accession No. D10158) and III (GenBank
Accession No. D87894) and the like. Examples of enzymes produced by the genus Talalomyces include chitosan degrading enzymes produced by Talaromyces emersoii (Talaromyces emersoii). , J., et al., Biotechnol. Le
tt., 13 667 (1991)) and the like.
Enzymes produced by Trichoderma include Trichoderma hamatum, Trichoderma
Examples thereof include chitosan-degrading enzymes produced by Trichoderma harzianum and Trichoderma viride, and more specifically, chitinase (GeGe produced by Trichoderma hamatsuum).
nBank Accession No. Z71415 and U88560), and chitinase (GenBank Accession No. L14614 and X800) produced by Trichoderma harzianum.
06) and an enzyme produced by Trichoderma viride QM 6a strain (Monreal, J., et al., Can. J. Mcrobiol., 15., 6
89 (1969)) and the like. Examples of enzymes produced by the genus Penicillium include chitosan degrading enzymes produced by Penicillium lilacinium, and more specifically, enzymes produced by Penicillium lilacinium QM 4e (Monrea
l, J., et al., Can. J. Mcrobiol., 15., 689 (1969)) and the like.

Examples of the enzyme produced by yeast include chitosan degrading enzyme produced by Saccharomyces cerevisiae, and preferably chitinase (GenBank Accession No. M74070) produced by Saccharomyces cerevisiae and ORF D9481.7
(GenBank Accession No. U28373).

More preferably, the SANK70699 strain (FERM BP-666
6) derived chitosan degrading enzyme and SANK70799 strain (FERM BP-66
It is a chitosan degrading enzyme derived from 67). Such an enzyme may be a chitosan degrading enzyme produced by the SANK70699 strain or the SANK70799 strain itself, a mutant or modified form thereof, or a gene encoding a chitosan degrading enzyme of the SANK70699 strain or the SANK70799 strain. It may be a recombinant protein produced from a transformant obtained by introducing it into a host.

The SANK70699 strain has been internationally deposited on February 26, 1999, at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1-1-3 East, Tsukuba, Ibaraki, Japan, with the deposit number FERM BP-6666. Was attached.

The SANK70799 strain was deposited internationally at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1-1-3 East, Tsukuba, Ibaraki, Japan on February 26, 1999, under the deposit number FERM BP-6667. Was attached.

The SANK60599 strain was internationally deposited on February 26, 1999 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, 1-1-3 East, Tsukuba, Ibaraki, Japan, and the deposit number is FERM BP-6665. Was attached.

The chitosan degrading enzyme produced by the SANK70699 strain has the following properties. 1) Molecular weight of 50,000, 6 by SDS-PAGE method
20,000 and 70,000, 2) shows an isoelectric point pI3.5 by an isoelectric focusing method, 3) uses chitosan 7B (manufactured by Katokichi Co., Ltd.) at pH 4 to p.
It hydrolyzes at H10.5, 4) exerts the hydrolysis activity described in 3) at 80 ° C. or lower, 5) the optimum pH of the hydrolysis activity described in 3) is pH 7.5 and pH 10, 6) 3 The optimum temperature of the hydrolytic activity described above is 65 ° C. at pH 7.5 and 50 ° C. at pH 10. 7) Stable at a temperature of 70 ° C. or lower 8) Stable under pH conditions of pH 5 to pH 10.5 is there. 9) pH 7.5 of enzyme for chitosan 7B, temperature 37
Enzyme pH for other substrates, pH 7.5, temperature 37 ° C, when hydrolyzing activity at 20 ° C for 20 minutes is 100%
Table 1 shows the hydrolytic activity for 20 minutes as a relative value. In the table, chitosan 10B is 1% acetylated chitin,
Chitosan 9B is 10% acetylated chitin, Chitosan 8B
Is 20% acetylated chitin, and chitosan 7B is 30% acetylated chitin (all manufactured by Katokichi Co., Ltd.). CM
-Cellulose is sodium carboxymethyl cellulose (manufactured by Wako Pure Chemical Industries, Ltd.). Glycol chitin was prepared by the method described above.

[0034]

[Table 1]

From Table 1, this enzyme has a higher hydrolysis activity for partially acetylated chitosan (chitosan 9B, 8B, 7B) having an acetylation degree of 10 to 30% than that of glycol chitin, and an acetylation degree of 1% or less. It can be seen that the compound has almost no hydrolytic activity against chitosan (chitosan 10B). It has no hydrolytic activity on sodium carboxymethylcellulose. 10) GlcNAc6 (acetylglucosamine hexamer) (produced by Yaizu Suisan Co., Ltd.) was used as GlcNAc4.
(Acetylglucosamine tetramer) and GlcNAc2 (acetylglucosamine dimer) are hydrolyzed, but GlcN6 (glucosamine hexamer) is not hydrolyzed.

The chitosan-degrading enzyme can be produced by culturing chitosan-degrading enzyme-producing microorganisms such as SANK70699 strain, 70799 strain, and 60599 strain in a medium.
For example, 0.1-5.0% polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.), 0.1-1.0% yeast extract (manufactured by Difco Corporation), 0.01-0.5% phosphoric acid Potassium hydrogen, 0.001-0.05% magnesium sulfate,
16-45 in medium of 0.05-0.5% chitosan 7B
Incubate with shaking at 100 to 250 rpm at 1 ° C for 1 to 10 days.

The following points are common to the enzyme derived from the SANK70799 strain and the enzyme derived from the SANK70699 strain.・ PH stable, alkaline, and active. -In the alkaline region, the maximum relative activity is pH10.・ Temperature dependence at pH 10 ・ Under neutral pH conditions, it is stable (heat resistance) up to at least 50 ° C. -GlcNAc dimer is generated from GlcNAc hexamer. -GlcN hexamer does not decompose. -PH 7.0 to 30% acetylated chitosan
Assuming that the hydrolysis activity of 7.5% at 37 ° C. for 10 to 20 minutes is 100%, the activity for 1% acetylated chitosan is 15% or less.

The purified chitosan degrading enzyme is
It may be crudely purified, or may be used as it is without purification of the culture supernatant. The amount and concentration of enzyme used may be small or large and are not particularly limited.

The low molecular weight chitosan or various salt solutions thereof obtained as described above can be used as they are, but concentrated by ammonium sulfate precipitation, organic solvent precipitation, centrifugation, freeze drying, ultrafiltration, etc. You can also Also, dialysis
Purification, desalting, and molecular weight fractionation can also be performed by ultrafiltration, gel filtration, column chromatography and the like. The low molecular weight chitosan obtained by the production method of the present invention is subjected to dialysis using a dialysis membrane, and then the fraction in the dialysis membrane is collected to obtain low molecular weight chitosan having a molecular weight of 10,000 or more. The molecular weight distribution of the produced low molecular weight chitosan hydrochloride or its salt can be determined by a gel filtration method using dextran as a marker. As described in Examples 2 to 5 below, from chitosan 9B (high molecular weight chitosan 9B), low molecular weight chitosan 9B hydrochloride having a molecular weight of 70,000 to 250,000 mixed therein is almost the same as chitosan 8B (high molecular weight chitosan 8B). Obtained a low molecular weight chitosan 8B hydrochloride having a molecular weight of about 70,000 and a low molecular weight chitosan 7B hydrochloride having a molecular weight of about 55,000 from chitosan 7B (high molecular weight chitosan 7B). Also,
When Examples 2 to 5 were repeated and the molecular weight distribution was measured, almost the same results were obtained in all cases, confirming reproducibility. It is a well-known fact to those skilled in the art that the molecular weight of the exclusion limit (molecular weight fraction) of the dialysis membrane is not always strict, and a sample having molecules larger or smaller than the exclusion limit is a membrane having the exclusion limit. When it is subjected to dialysis using the above, molecules smaller than the exclusion limit may be contained in the fraction in the dialysis membrane. In the present invention,
For the sake of convenience, all the components contained in the fraction remaining in the dialysis membrane after such dialysis are considered to have a molecular weight above the exclusion limit of the membrane. For example, exclusion limit (molecular weight fraction)
All the components contained in the fraction remaining in the membrane after dialysis using 10,000 membranes are considered to have a molecular weight of 10,000 or more.

The low-molecular-weight chitosan prepared in this manner had excellent solubility in water regardless of the salt, and in the case of the hydrochloride, a solution having a maximum concentration of about 10% by mass could be prepared. A 1% by weight aqueous solution of each low molecular weight chitosan salt has a pH of 4.8 to
It shows 6.3, and precipitates (colloids) are deposited when an alkali is added to a 1% by mass aqueous solution of low molecular weight chitosan salts.
The pH was pH 6.4-8.2.

Of the low molecular weight chitosan or various salts thereof prepared here, the physiological activity of the hydrochloride was examined using the antibacterial activity as an index. Minimum inhibitory concentrations of low molecular weight chitosan 9B, 8B and 7B hydrochlorides for antibacterial activity (Minimum Inh
ibitory Concentration (MIC) was approximately 100 μg / ml or less regardless of whether the target bacteria were Gram-positive or negative bacteria.
The Staphylococcus intermedium (Staphylococcus intermedium), which has been unknown in the past
dius), streptococcus Streptococcus pyogenes, S. marcescens, respiratory tract infection Moraxella catarrhali
s) showed excellent antibacterial activity. In addition, various drug-resistant bacteria, namely, new quinolone resistant S. aureus, β-lactamase-producing E. coli, carbapenemase-producing P.
Against aeruginosa, it showed excellent antibacterial activity equivalent to each standard strain. Furthermore, S. intermedi, which was said to have no antibacterial activity in shrimp and crab-derived chitosan
It was effective against us even at low concentrations. It also showed antibacterial activity against all intestinal bacteria. Can be a pathogen,
Enterococcus faeca
lis), Enterococcus faecium (Enterococcu
s faecium), Enterococcu
s hirae), Enterobacter cloacae
ter cloacae), Vibrio parahaemolytics (Vib
rio parahaemolytics), and showed excellent antibacterial activity against Clostridium hystolytisum. The antibacterial activity of these low molecular weight chitosan hydrochlorides was tested using S. aureus, S. mutans, S. intermedius and Bacillus subtilis.
It was compared with the antibacterial activity of polymeric chitosan hydrochloride. It was shown that the activity of chitosan hydrochloride with a low molecular weight of about 2 to 10 times was stronger than that of Gram-positive and negative bacteria. Therefore, it was shown that the low molecular weight chitosan has excellent physiological activity equivalent to or higher than that of the high molecular weight chitosan. From this, it is considered that the antibacterial activity that the high molecular weight chitosan has is also retained in the low molecular weight chitosan of the present invention.

Regarding the low molecular weight chitosan hydrochloride obtained by the method of the present invention, the effect of promoting healing of deficient damage, the effect of suppressing weight gain, the action of lowering serum and hepatic total cholesterol, the action of lowering serum and hepatic triglyceride, and the action in serum
HDL-cholesterol raising effect was observed.

Since it has such excellent physiological activity, for example, a therapeutic agent and / or preventive agent for wounds / decubitus, a coating agent, a therapeutic agent and / or preventive agent for atopic dermatitis, an acne treatment and / or External skin preparations such as preventive agents, dental caries treatment and /
Oral hygiene agents such as preventive agents, intestinal agents such as intestinal bacterium improving agents, medical fields such as nosocomial infection preventive agents, food additives such as food preservatives, fields such as animal / fish feeds, plant activity enhancers The low molecular weight chitosan and salts thereof of the present invention can be used in the agricultural field and the like. In addition, as an anti-obesity agent, 2 as an anti-diabetic agent is used for the prevention and / or treatment of obesity and various diseases caused by obesity (collectively referred to as “obesity”).
For the prevention and / or treatment of type 2 diabetes and its complications (collectively referred to as "diabetes") or as anti-arteriosclerosis agents such as arteriosclerosis, ischemic heart disease, cerebrovascular disorder (collectively " The low molecular weight chitosan and salts thereof of the present invention can be used for the prevention and / or treatment of "arteriosclerosis"). Further, it can be used as an antihyperlipidemic agent and the like for the prevention and / or treatment of hypercholesterolemia and hypertriglyceridemia (collectively referred to as "hyperlipidemic agents"). Although various applications have been described above, the applications of the low molecular weight chitosan and salts thereof of the present invention are not particularly limited to these examples.

The use form of the low molecular weight chitosan of the present invention varies depending on the purpose, but it can take various forms for use in the above-mentioned uses. Although listed below, they are not particularly limited. The low molecular weight chitosan and its salt can be used as a white solid. Further, an aqueous solution or an organic solvent solution having a concentration of 10% by mass can be appropriately diluted and used at an arbitrary concentration. Water or an organic solvent (such as alcohols) can be used as the diluent. Finally, when the pH is more than 6.5, it can be used as a solution, and when it is alkaline, it can be used as a colloidal compound, but depending on the type of salt, it can be used as a solution even if it is weakly alkaline. Furthermore, by coexisting an inorganic substance or an organic substance, it can be used as a solution at any pH. In addition, a low molecular weight chitosan solution (or a solution of a low molecular weight chitosan salt) or a dilute solution may be mixed with a cream base material at an arbitrary concentration of 10% by mass or less or higher to use as a cream. The type of base material is shown in the formulation example, but is not particularly limited thereto. A low molecular weight chitosan solution (or a low molecular weight chitosan salt solution) or a dilute solution can be used as a film having an arbitrary thickness. A film having a plastic-like hardness is obtained by drying a low molecular weight chitosan solution (or a solution of a low molecular weight chitosan salt) in a frame having various thicknesses. In addition, a flexible soft sheet is prepared by mixing a low molecular weight chitosan solution (or a low molecular weight chitosan salt solution) with a solvent such as glycerin at an arbitrary ratio and drying it in a frame having various thicknesses. It is obtained by The degree of flexibility is adjusted by the ratio of adding a solvent such as glycerin. Furthermore, the chitosan methylcellulose composite film is obtained by mixing methylcellulose with a low molecular weight chitosan solution (or a solution of a low molecular weight chitosan salt) and drying in a frame having various thicknesses. When used as a spray formulation, alcohol or a propellant may be added to low molecular weight chitosan (or a salt thereof) or a low molecular weight chitosan solution (or a low molecular weight chitosan salt solution). Examples of alcohols include ethanol, and examples of propellants include freon gas (eg, trichlorofluoromethane, dichlorofluoromethane, dichlorotetrafluoroethane, a mixture thereof), liquefied petroleum gas (eg, propane, butane, etc.). ) Can be illustrated.

The low molecular weight chitosan and salts thereof of the present invention are
It may be used as a mixture with other drugs. Further, the low molecular weight chitosan and the salt thereof of the present invention may be used as a medicine or may be used as a specific health and nutrition food. When used as a medicine or food, the low molecular weight chitosan salt of the present invention is preferably acceptable as a medicine or food. When the low molecular weight chitosan of the present invention or a salt thereof is used as a drug or food, it is administered in various forms other than the above. Examples of the dosage form include oral administration by tablets, capsules, granules, powders, syrups, etc., or parenteral administration by injections (intravenous, intramuscular, subcutaneous), drops, suppositories, etc. You can These various formulations are usually used in the technical field of pharmaceutical formulation such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. It can be formulated with known auxiliary agents to be obtained. In the case of molding into a tablet form, those conventionally known in this field can be widely used as a carrier, for example, lactose,
Excipients such as sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid; water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, Binders such as methylcellulose, potassium phosphate, polyvinylpyrrolidone; dry starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose Disintegrating agents such as; sucrose, stearin, cocoa butter, hydrogenated oils, etc. disintegrating inhibitors; quaternary ammonium salts, absorption promoters such as sodium lauryl sulfate, humectants such as glycerin and starch; starch, lactose, kaolin, Bentonite, adsorbent such as colloidal silicic acid; purified talc, stearates, boric acid powder, lubricants such as polyethylene glycol can be exemplified. Further, the tablet may be a tablet coated with a usual coating as necessary, for example, a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet or a double-layered tablet or a multi-layered tablet. In the case of molding in the form of pills, those conventionally known in this field can be widely used as carriers, for example, excipients such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin and talc; gum arabic powder, Examples thereof include tragacanth powder, a binder such as gelatin and ethanol, and a disintegrating agent such as laminaran agar. In the case of molding in the form of suppositories, various carriers conventionally known in this field can be widely used, and examples thereof include polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semisynthetic glycerides. it can. When prepared as an injection, the solution and suspension are preferably sterilized and isotonic with blood, and when formed into the form of these solutions, emulsions and suspensions, they are used as a diluent. Any of those commonly used in the field can be used, and examples thereof include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case,
A sufficient amount of sodium chloride, glucose, or glycerin for preparing an isotonic solution may be contained in the pharmaceutical preparation, and a usual solubilizing agent, buffer, soothing agent, etc. may be added. Good. Further, if necessary, a colorant, a preservative, a flavoring agent, a flavoring agent, a sweetening agent and the like and other pharmaceuticals may be contained. The amount of the active ingredient compound contained in the above-mentioned pharmaceutical preparation is not particularly limited and is appropriately selected within a wide range, but it is usually 1100% by weight, preferably 1 to 90% by weight in the total composition. is there. The administration method of the above-mentioned pharmaceutical preparation is not particularly limited, and it is determined according to various preparation forms, patient's age, sex and other conditions, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. Further, in the case of an injection, it is intravenously administered alone or mixed with a normal replenisher such as glucose or amino acid, and further, if necessary, it is intramuscularly, intradermally, subcutaneously or intraperitoneally administered alone. In the case of suppositories, it will be administered rectally. The amount used varies depending on symptoms, age, body weight, administration method, dosage form, etc., but usually 10 mg to 2000 mg, preferably 10 mg to 550 mg, more preferably 10 mg to 100 mg per day for an adult, It can be administered once or in several divided doses depending on the symptoms. When the low molecular weight chitosan of the present invention is used as a food, a food material or a food additive, it may take the form of the above-mentioned aqueous solution or the like in addition to the form of the above formulation.

[0046]

EXAMPLES Examples, test examples and formulation examples will be given below, but the scope of the present invention is not limited thereto.

<Example 1> of crude enzyme solution derived from SANK70699 strain
Preparation Suspend 5g of Chitosan 7B (Katoyoshi Co., Ltd.) in 1.0L of water
Then, add concentrated hydrochloric acid to completely dissolve the powder to pH 2.5.
It was Add polypeptone (Wako Pure Chemical Industries, Ltd.) to this chitosan solution.
20 g, yeast extract (manufactured by Difco) 10 g, phosphoric acid water
2 g of elemental potassium and 0.4 g of magnesium sulfate were added, and 1N sodium hydroxide was added.
The pH was adjusted to 6.8 with sodium hydroxide and the total amount was adjusted to 1.5L. Separately charcoal
Dissolve 50 g of sodium acid in 500 ml of water and add both solutions separately to each other.
Sterilized at 1 ° C for 15 minutes. After cooling to room temperature, mix both solutions
And inoculate the alkalophilic bacterium SANK70699 strain at 37 ° C for 3 days.
In the meantime, shaking culture was performed at 170 rpm. After culturing, contains bacterial cells
Insoluble matter was removed by centrifugation, and 3 volumes of ethanol were added to 1 volume of the supernatant under ice cooling.
Add tanol and let stand for 30 minutes. 10,000 rpm for 5 minutes
A precipitate was obtained by separation of the heart, and the precipitate was collected using a dialysis membrane (V
ISKASE SALES CORP.), 10 mM Tris / HCl at pH 7.5
Dialysis was performed against the buffer. After dialysis, add 10,000 solution
Centrifuge at rpm for 10 minutes and filter the supernatant to obtain 500 ml of crude enzyme solution.
It was The activity of the enzyme solution was measured as follows. pH 7.
5 400mM MOPS / Na₂CO ₃100 μl of buffer solution, dissolved in chitosan 7B acetic acid
Mix 160 μl of the solution and 140 μl of the enzyme solution and incubate at 37 ° C for 20 minutes.
Then, add 10 μl of acetylacetone to 0.5 M aqueous sodium carbonate.
Add 400 μl of the solution dissolved in 500 μl to the enzyme reaction
After stopping the reaction, the temperature was kept at 100 ° C. for 20 minutes. Ice cold
Then, 0.8 g of N, N-dimethylaminobenzaldehyde was added to ethanol.
400 ml of mixed solution dissolved in 30 ml of concentrated hydrochloric acid and 30 ml of concentrated hydrochloric acid.
1.2 ml of tanol was added to the reaction solution, and the color developed for 10 minutes at 75 ° C.
I responded. After cooling with ice, the precipitate was centrifuged and the supernatant was adjusted to 530 nm.
Absorbance was measured. 1 μmol gluco per minute
Enzyme activity that produces samin as 1 unit per 1 ml
It was 0.097 units.

<Example 2> Preparation of low molecular weight chitosan 9B hydrochloride and measurement of molecular weight distribution Chitosan 9B (manufactured by Katokichi Co., Ltd.) 5 g was suspended in 800 ml of water,
Concentrated hydrochloric acid was added to adjust the pH to 2.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogencarbonate, water was added to bring the total amount to 1000 ml, 200 ml each was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and the temperature was 40 ° C.
The enzyme reaction was performed by shaking at 80 rpm for 3 days. After completion of the reaction, all the reaction solutions were collected, adjusted to pH 12 or higher with 1% caustic soda water under ice cooling, and then centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The recovered precipitate was suspended in 200 ml of water, concentrated hydrochloric acid was added under ice-cooling to dissolve the solution at pH 2.5, and dialyzed against water using a dialysis membrane with a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. Low molecular weight chitosan 9B hydrochloride was obtained as 3.4 g of white fluffy solid. A 1% concentration aqueous solution of the obtained low molecular weight chitosan 9B hydrochloride was prepared, and the molecular weight distribution was measured by gel filtration. That is, 200 μl of the above aqueous solution was spread on a column (86 × 2 cm) packed with a gel filtration material (Toyopearl HW55 Fine, manufactured by Tosoh Corp.) swollen with 200 mM acetate buffer of pH 4.5 in advance, and 1.75 ml each Fractionated into Add pH6 to 160 μl of each fraction.
100 μl of 0, 400 mM acetate buffer, 120 μl of water, 20 μl of chitosanase (manufactured by Wako Pure Chemical Industries, Ltd.) solution (0.1 unit) were added to give 10
The enzyme reaction was carried out by keeping the temperature at 37 ° C for a minute. The absorbance at 530 nm was measured by the same method as in Example 1. The results are shown in Fig. 1. From this result, it was found that the low molecular weight chitosan 9B hydrochloride had a molecular weight of 70,000 to 250,000 and was mixed in substantially the same amount.

<Example 3> Preparation of low molecular weight chitosan 8B hydrochloride and measurement of molecular weight distribution 5 g of chitosan 8B (manufactured by Katokichi Co., Ltd.) was suspended in 800 ml of water,
Concentrated hydrochloric acid was added to adjust the pH to 2.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogencarbonate, water was added to bring the total amount to 1000 ml, 200 ml each was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and the temperature was 40 ° C.
The enzyme reaction was performed by shaking at 80 rpm for 3 days. After completion of the reaction, all the reaction solutions were collected, adjusted to pH 12 or higher with 1% caustic soda water under ice cooling, and then centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The recovered precipitate was suspended in 200 ml of water, concentrated hydrochloric acid was added under ice-cooling to dissolve the solution at pH 2.5, and dialyzed against water using a dialysis membrane with a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. Low molecular weight chitosan 8B hydrochloride was obtained as 4.0 g of a white fluffy solid. The 1% concentration aqueous solution of the obtained low molecular weight chitosan was prepared, and the molecular weight distribution was measured by the same method as in Example 2. The results are shown in Figure 2. From this result, it was found that the low molecular weight chitosan 8B hydrochloride mainly had a molecular weight of 70,000.

<Example 4> Preparation of low molecular weight chitosan 7B hydrochloride and measurement of molecular weight distribution Chitosan 7B 5 g was suspended in 800 ml of water, and concentrated hydrochloric acid was added to adjust the pH.
The powder was completely dissolved as 2.5. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogen carbonate, add water to bring the total volume to 1000 m.
Then, 200 ml each was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and the mixture was shaken at 40 rpm at 80 rpm for 3 days to carry out the enzyme reaction. After completion of the reaction, all the reaction solutions were collected, adjusted to pH 12 or higher with 1% caustic soda water under ice cooling, and then centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The recovered precipitate was suspended in 200 ml of water, concentrated hydrochloric acid was added under ice-cooling to dissolve the solution at pH 2.5, and dialyzed against water using a dialysis membrane with a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. Low molecular weight chitosan 7B hydrochloride was obtained as 3.4 g of white fluffy solid. The 1% concentration aqueous solution of the obtained low molecular weight chitosan was prepared, and the molecular weight distribution was measured by the same method as in Example 2. The results are shown in Figure 3. From this result, it was found that the low molecular weight chitosan 7B hydrochloride mainly had a molecular weight of 55,000.

Example 5 Determination of Molecular Weight Fraction by Gel Filtration Method 200 μl of each 1% dextran T40, T70, T110, T250 (Pharmacia Co., Ltd.) solution was preliminarily adjusted to pH 4.5.
Gel filtration material swollen with 0 mM acetate buffer (Toyopearl HW55
Fine) was developed on a packed column (86 x 2 cm) and fractionated into 1.75 ml each. Sulfuric acid solution 1.8m with 1 volume of water plus 8 volumes of concentrated sulfuric acid
l, a solution of carbazole 0.25g in ethanol 50ml
60 μl was added to 200 μl of each fraction, and the mixture was incubated at 100 ° C. for 10 minutes. After cooling with ice, the absorbance at 420 nm was measured. The results are shown in FIGS.

Example 6 Preparation of low molecular weight chitosan 9B acetate Chitosan 9B (manufactured by Katokichi Co., Ltd.) 5 g was suspended in 800 ml of water,
Acetic acid was added to adjust the pH to 3.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogencarbonate, water was added to bring the total volume to 1000 ml, 200 ml each was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and the mixture was mixed at 40 ° C. for 3 minutes.
The enzyme reaction was performed by shaking at 80 rpm for a day. After the completion of the reaction, all the reaction solutions were recovered, adjusted to pH 3.5 by adding acetic acid, and then dialyzed against water using a dialysis membrane having a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. Low molecular weight chitosan 9B acetate was obtained as 4.3 g of white fluffy solid.

<Example 7> Preparation of low molecular weight chitosan 8B acetate Chitosan 8B (5 g) was suspended in water (800 ml) and acetic acid was added to adjust the pH to 3.
The powder as 5 was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogen carbonate, add water to bring the total volume to 1000 ml.
And transfer 200 ml each to a 500 ml Erlenmeyer flask, and
5 ml of the enzyme solution prepared in (3) was added, and the mixture was shaken at 80 rpm at 40 ° C. for 3 days to carry out the enzyme reaction. After completion of the reaction, collect all the reaction solutions, add acetic acid to adjust the pH to 3.5, and then add molecular weight fraction 1
Dialysis was performed against water using a dialysis membrane.
After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. 5.4g
Low molecular weight chitosan 8B acetate was obtained as a white fluffy solid.

<Example 8> Preparation of low molecular weight chitosan 9B trifluoroacid salt 5 g of chitosan 9B (manufactured by Katokichi Co., Ltd.) was suspended in 800 ml of water,
Trifluoroacetic acid was added to adjust the pH to 2.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogen carbonate, add water to bring the total volume to 1000 ml, and 200 ml each to 500 ml.
The mixture was transferred to an Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and the mixture was shaken at 40 rpm at 80 rpm for 3 days to carry out the enzyme reaction. After completion of the reaction, all the reaction solutions were collected, trifluoroacetic acid was added to adjust the pH to 2.5, and then dialysis was performed against water using a dialysis membrane with a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. Low molecular weight chitosan 9B trifluoroacetate salt was obtained as 5.2 g of white flocculent solid.

<Example 9> Preparation of low molecular weight chitosan 8B trifluoroacid salt 5 g of chitosan 8B (manufactured by Katokichi Co., Ltd.) was suspended in 800 ml of water,
Trifluoroacetic acid was added to adjust the pH to 2.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogen carbonate, add water to bring the total volume to 1000 ml, and 200 ml each to 500 ml.
The mixture was transferred to an Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and the mixture was shaken at 40 rpm at 80 rpm for 3 days to carry out the enzyme reaction. After completion of the reaction, all the reaction solutions were collected, trifluoroacetic acid was added to adjust the pH to 2.5, and then dialysis was performed against water using a dialysis membrane with a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. 4.8 g of low molecular weight chitosan 8B trifluoroacetate was obtained as a white fluffy solid.

<Example 10> Preparation of low molecular weight chitosan 9B paratoluenesulfonate 5g of chitosan 9B (manufactured by Katokichi Co., Ltd.) was suspended in 800ml of water,
Paratoluenesulfonic acid was added to adjust the pH to 2.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogen carbonate, water was added to bring the total amount to 1000 ml, 200 ml each was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and 80 rpm at 40 ° C. for 3 days. The mixture was shaken at 3 ° C. for enzymatic reaction. After the reaction was completed, all the reaction solutions were collected, adjusted to pH 12 or more with 1% caustic soda water under ice cooling, and then 10,000 rpm1
A precipitate was obtained by 0 minute centrifugation. 200 ml of collected precipitate
Suspended in water and added paratoluenesulfonic acid to pH 2.5.
After that, dialysis was performed against water using a dialysis membrane with a molecular weight fraction of 10,000. After dialysis, the solution is filtered,
The filtrate was freeze dried. Low molecular weight chitosan 9B paratoluenesulfonate was obtained as 6.3 g of white fluffy solid.

<Example 11> Preparation of low molecular weight chitosan 8B paratoluenesulfonate 5g of chitosan 8B (manufactured by Katokichi Co., Ltd.) was suspended in 800ml of water,
Paratoluenesulfonic acid was added to adjust the pH to 2.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogen carbonate, water was added to bring the total amount to 1000 ml, 200 ml each was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and 80 rpm at 40 ° C. for 3 days. The mixture was shaken at 3 ° C. for enzymatic reaction. After the completion of the reaction, all the reaction solutions were collected, paratoluenesulfonic acid was added to adjust the pH to 2.5, and then dialysis was performed against water using a dialysis membrane with a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. 5.6 g of low molecular weight chitosan 8B paratoluenesulfonate was obtained as a white fluffy solid.

<Example 12> Preparation of low molecular weight chitosan 9B acrylate 5 g of chitosan 9B (manufactured by Katokichi Co., Ltd.) was suspended in 800 ml of water,
Acrylic acid was added to adjust the pH to 3.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogen carbonate, water was added to bring the total amount to 1000 ml, 200 ml each was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and 80 rpm at 40 ° C. for 3 days. The mixture was shaken at 3 ° C. for enzymatic reaction. After the completion of the reaction, all the reaction solutions were collected, acrylic acid was added to adjust the pH to 3.5, and then dialyzed against water using a dialysis membrane with a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. Low molecular weight chitosan 9B acrylate was obtained as 4.6 g of white fluffy solid.

<Example 13> Preparation of low molecular weight chitosan 9B lactate 5 g of chitosan 9B (manufactured by Katokichi Co., Ltd.) was suspended in 800 ml of water,
Lactic acid was added to adjust the pH to 3.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogencarbonate, water was added to bring the total volume to 1000 ml, 200 ml each was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and the mixture was mixed at 40 ° C. for 3 minutes.
The enzyme reaction was performed by shaking at 80 rpm for a day. After completion of the reaction, collect all the reaction solutions, and use ice-cooled 1% caustic soda water.
After adjusting the pH to 12 or more, centrifugation was performed at 10,000 rpm for 10 minutes to obtain a precipitate. The recovered precipitate was suspended in 200 ml of water, lactic acid was added to adjust the pH to 3.5, and then dialysis was performed against water using a dialysis membrane with a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. Low molecular weight chitosan 9B lactate was obtained as 4.6 g of white fluffy solid.

<Example 14> Preparation of low molecular weight chitosan 8B lactate 5 g of chitosan 8B (manufactured by Katokichi Co., Ltd.) was suspended in 800 ml of water,
Lactic acid was added to adjust the pH to 3.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogencarbonate, water was added to bring the total volume to 1000 ml, 200 ml each was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and the mixture was mixed at 40 ° C. for 3 minutes.
The enzyme reaction was performed by shaking at 80 rpm for a day. After completion of the reaction, collect all the reaction solutions, and use ice-cooled 1% caustic soda water.
After adjusting the pH to 12 or more, centrifugation was performed at 10,000 rpm for 10 minutes to obtain a precipitate. The recovered precipitate was suspended in 200 ml of water, lactic acid was added to adjust the pH to 3.5, and then dialysis was performed against water using a dialysis membrane with a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. Low molecular weight chitosan 8B lactate was obtained as 3.5 g of white fluffy solid.

<Example 15> Preparation of low molecular weight chitosan 7B lactate 5 g of chitosan 7B (manufactured by Katokichi Co., Ltd.) was suspended in 800 ml of water,
Lactic acid was added to adjust the pH to 3.5 and the powder was completely dissolved. After adjusting the pH to 7.5 with saturated aqueous sodium hydrogencarbonate, water was added to bring the total volume to 1000 ml, 200 ml each was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and the mixture was mixed at 40 ° C. for 3 minutes.
The enzyme reaction was performed by shaking at 80 rpm for a day. After the completion of the reaction, all the reaction solutions were collected, lactic acid was added to adjust the pH to 4.0, and then dialysis was performed against water using a dialysis membrane with a molecular weight fraction of 10,000. After completion of dialysis, the solution was filtered and the filtrate was freeze-dried. Low molecular weight chitosan 7B lactate was obtained as 2.6 g of white fluffy solid.

<Example 16> Solubility of low-molecular-weight chitosan salts in water 100 mg of low-molecular-weight chitosan salts prepared in Examples 2-4 and 6-15 were dissolved in 10 ml of water, and pH was measured. 1N caustic soda water was added little by little to this solution, and the pH at which the solution became cloudy and a colloid was deposited was measured. The results are shown in Table 2.

[0063]

[Table 2]

Example 17 Preparation of Polymer Chitosan Hydrochloride 1 g of chitosan 9B (manufactured by Katokichi Co., Ltd.) was suspended in 200 ml of water, adjusted to pH 2.5 with concentrated hydrochloric acid and completely dissolved. This was dialyzed against water and then freeze-dried to obtain high molecular weight chitosan 9B hydrochloride as 537 mg of a white cotton-like solid.
Polymer chitosan 10B, 8B, 7B hydrochloride was also prepared in the same way.

<Example 18> Preparation of crude enzyme solution derived from SANK70799 strain Chitosan 7B (2.5 g) was suspended in 1.0 L of water, and 10 g of polypeptone (manufactured by Wako Pure Chemical Industries, Ltd.) and yeast extract (Difco KK)
5g, dipotassium hydrogenphosphate 1g, magnesium sulfate 0.2g
Was added and sterilized at 121 ° C. for 15 minutes. The SANK70799 strain was inoculated into this medium and cultured at 37 ° C. for 3 days with shaking at 170 rpm. After completion of the culture, the insoluble matter containing the bacterial cells was removed by centrifugation, 3 volumes of ethanol was added to 1 volume of the supernatant under ice cooling, and the mixture was allowed to stand for 30 minutes. A precipitate was obtained by centrifugation at 10,000 rpm for 5 minutes, and this was dialyzed against a 10 mM Tris / HCl buffer solution having a pH of 7.5 using a dialysis membrane having a molecular weight fraction of 10,000. After dialysis, the solution is 10,000 rp
After centrifugation at m for 10 minutes, the supernatant was filtered to obtain 200 ml of a crude enzyme solution. The activity of the enzyme solution was measured by the method described in Example 1, and the enzyme activity of producing 1 μmol of glucosamine per minute was 1
The unit was 0.143 units per ml.

The chitosan degrading enzyme contained in the obtained crude enzyme solution exhibited the following properties: 1) Chitosan 7B (manufactured by Katokichi Co., Ltd.) was used at a pH of 2.5-2.5.
It hydrolyzes under the conditions of 11.5. 2) It exhibits the hydrolysis activity described in 1) at 100 ° C. or lower. 3) The optimum pH of the hydrolysis activity described in 1) is pH 3 to 7 and pH 10. 4) The optimum temperature for the hydrolysis activity described in 1) is pH 6.5.
Is 80 ° C. and pH 10 is 50 ° C. 5) Stable under a temperature condition of 100 ° C or lower. 6) Stable under pH conditions of pH 4 to 10. 7) Table 3 shows the relative hydrolysis activity for each substrate under conditions of pH 7, 37 ° C., and 10 minutes.

[0067]

[Table 3]

8) GlcNAc6 is hydrolyzed to GlcNAc2,
GlcN6 does not hydrolyze. <Example 19> Preparation of low molecular weight chitosan 9B hydrochloride using an enzyme derived from SANK70799 strain 1 g of chitosan 9B was suspended in 100 ml of water, and concentrated hydrochloric acid was added to adjust the pH.
The powder was completely dissolved as 2.5. After adjusting the pH to 6.0 with saturated aqueous sodium hydrogen carbonate, add water to bring the total volume to 200 ml.
Then, the mixture was transferred to a 500 ml Erlenmeyer flask, 5 ml of the enzyme solution prepared in Example 1 was added, and the mixture was shaken at 40 rpm at 80 rpm for 2 days to carry out the enzyme reaction. After the reaction was completed, it was purified by the method described in Example 2 to give 0.6 g of white flocculent solid low molecular weight chitosan 9B.
The hydrochloride salt was obtained.

The low molecular weight chitosan 9B hydrochloride showed the following properties. 1) 10 g or more was dissolved in 100 g of water. 2) The pH of the aqueous solution dissolved in 1% by mass was 6.1. 3) When alkali is added, the precipitate (colloid) is p
Precipitated at H6.5.

<Example 20> Preparation of low molecular weight chitosan 8B hydrochloride using enzyme derived from SANK70799 strain 1 g of chitosan 8B was hydrolyzed by the method described in Example 19,
Low molecular weight chitosan 8B hydrochloride was obtained as 0.45 g of white flocculent solid.

The low molecular weight chitosan 8B hydrochloride showed the following properties. 1) 10 g or more was dissolved in 100 g of water. 2) The pH of the aqueous solution dissolved in 1% by mass was 6.1. 3) When alkali is added, the precipitate (colloid) is p
Precipitated at H6.7.

<Example 21> Preparation of low molecular weight chitosan 7B hydrochloride using enzyme derived from SANK70799 strain 1 g of chitosan 7B was hydrolyzed by the method described in Example 19 to give 0.12 g of low molecular weight chitosan as white fluffy solid. 7B hydrochloride was obtained. The low molecular weight chitosan 7B hydrochloride showed the following properties. 1) 10 g or more was dissolved in 100 g of water. 2) The pH of the aqueous solution dissolved in 1% by mass was 6.2. 3) When alkali is added, the precipitate (colloid) is p
Precipitated at H6.9.

<Example 22> Degree of acetylation of low molecular weight chitosan hydrochloride 100 mg of low molecular weight chitosan hydrochloride prepared in Examples 2 to 4 and Examples 19 to 21 was dissolved in 10 ml of distilled water, and 10 U of chitosanase was added. Koujunyaku Co., Ltd. was added and stirred at room temperature for 3 hours. The reaction solution was freeze-dried, the dried solid was dissolved in D ₂ O, and ¹ H-NMR was measured. The same procedure was carried out except that 100 mg of the polymer chitosan hydrochloride prepared in Example 17 was dissolved in 50 ml of distilled water and 50 U of chitosanase was used. The degree of acetylation was calculated from the integral ratio of 3H of the acetyl group and 7H excluding the hydroxyl groups other than the acetyl group. The results are shown in Table 4.

[0074]

[Table 4]

<Test Example 1> The test strains SANK numbered are the preserved strains of the applicant,
ATCC and IAM are concession strains, and the others are clinical isolates by the applicant. 1. Staphylococcus aureus 209P JC-1 (SANK 70668) 2. Staphylococcus aureus SANK 71283 (MRSA strain) 3. Staphylococcus aureus NR1SR2SR3-b1 (New quinolone resistant strain) 4. Staphylococcus intermedius SANK 71191 5. Streptococcus pneumoniae 2132 6. Streptococcus pyogenes 12255 7. Streptococcus mutans SANK 72288 8. Bacillus subtilus ATCC 6633 9. Escherichia coli NIHJ JC-2 SANK 70569 10. Escherichia coli 609 (high β-lactamase-producing strain) 11. Serratia marcescens IAM 1184 12. Peudomonas aeruginosa PAO1 13. Peudomonas aeruginosa 10728 (high-producing carbapenemase) 14. Moraxella catarrhalis 11045 Working medium 1 to 3, 4, 8, 9 to 10, 11 and 12 to 13 are Muel
ler Hinton Broth (manufactured by Difco), 5, 6 and 14
Was adjusted to pH 6.5 by adding 5% horse serum (manufactured by Gibco) to Mueller Hinton Broth (manufactured by Difco). 7 is
PH was not adjusted with Todd Hewitt Broth (manufactured by Difco). The bacteria to be used for the preparation test of the sample bacterial solution were cultured in the above medium and diluted with the same medium so that the number of the bacteria would be 2 × 10 ⁶ . Preparation of sample Low molecular weight chitosan 9B, 8B, 7 prepared in Examples 2 to 4
B Each hydrochloride was dissolved in 25 mg distilled sterilized water to make 10 ml. Test method Distilled sterile water was used as a diluent in advance to prepare a multiple dilution series of the sample solution, to which 50 μl of the above medium and 50 μl of the test bacterial solution were added, and statically cultured at 37 ° C for 18 hours to determine the MIC. . The results are shown in Table 5.

[0076]

[Table 5]

<Test Example 2> The test strain SANK number is assigned to the preserved strain of the applicant.
ATCC is a stock for sale. 1. S. aureus 209P JC-1 (SANK 70668) (standard strain) 2. S. intermedius SANK 71191 3. S. mutans SANK 72288 4. B. subtilus ATCC 6633 Working medium 1, 2 and 4 are Mueller Hinton Broth (Difco Corporation)
3) is Todd Hewitt Broth (manufactured by Difco Co., Ltd.), and the pH of each was adjusted to 6.5. Bacteria used in the test preparation of sample bacterial solution were cultured overnight in the above medium and the number of bacteria was 2 x 1
It was diluted with the same medium so as to be 0 ⁶ . Preparation of sample Low molecular weight chitosan 9B, 8B, 7 prepared in Examples 2 to 4
Each hydrochloride salt of B and each of the high molecular weight chitosan 9B, 8B and 7B prepared in Example 17 were dissolved in 20 mg of distilled sterilized water to make 10 ml. Test method Diluted sterilized water was used as a diluent in advance to prepare a multiple dilution series of the sample solution, to which 50 μl of the above medium and 50 μl of the test bacterial solution were added, and the mixture was incubated at 37 ° C. for 18 hours to determine the MIC. The results are shown in Table 6.

[0078]

[Table 6]

<Test Example 3> S. aureus 209P as a test bacterium
The JC-1 strain (SANK 70668) and the P. aeruginosa PAO1 strain were used as test bacteria, and the MICs of the low molecular weight chitosans 7B, 8B and 9B prepared in Examples 19 to 21 were measured in the same manner as in Test Example 2. The results are shown in Table 7.

[0080]

[Table 7]

<Test Example 4> The test strain SANK number is assigned to the preserved strain of the applicant,
ATCC is a stock for sale. 1. Staphylococcus aureus 209P 2. Enterococcus faecalis 681 3. Enterococcus faecalis SANK71989 4. Enterococcus faecium 4288 5. Enterococcus faecium SANK70588 6. Enterococcus hirae SANK74194 7. Escherich coli NIHJ 8. Enterobacter cloacae 846 9. Enterobacter cloacae 963 10. Vibrio parahaemolyticus SANK73093 11. Clostridium histolytisum SANK71784 12. Clostridium histolytisum SANK71684 13. Lactobatillus plantarum SANK72388 14. Lactobatillus acidophilus SANK76175 15. Lactobacillus brevis SANK72079 16. Lactobacillus fermentum SANK76375 The culture mediums 1 to 10 are Mueller Hinton Broth (Difco), 11 and 1
2 is GAM broth medium (Nissui), 13-16 is Todd
Hewit medium (Difco) was used.

Other conditions are the same as in Test Example 1. Low molecular weight chitosan 9B prepared in Examples 2 to 4 as a sample,
Each of 8B and 7B hydrochloride was used. The results are shown in Table 8.

[0083]

[Table 8]

<Test Example 5> Mouse acute toxicity test Low molecular weight chitosan 9B, 8B, 7 prepared in Examples 2 to 4
B Each hydrochloride salt was orally administered to mice at a dose of 2,000 mg / kg body weight, and there was no effect at all.

<Formulation Example 1> 3.0 g of propylene glycol,
A solution of 0.1 g of methyl paraoxybenzoate in 80 ml of purified water at 70 ° C
The mixture was heated to 0 and 5.0 g of low molecular weight chitosan 9B hydrochloride prepared in Example 2 was added and dissolved. Stearic acid 3.0 g, cetal 1.0 g, glyceryl monostearate 6.0 g, liquid paraffin 5.0 g, propyl paraoxybenzoate 0.1 g, and polyoxyl stearate 40 2.5 g were dissolved by heating at 70 ° C. and then Example 2 at the same temperature. The low molecular weight chitosan 9B solution prepared in 1. was added, purified water was added to bring the total amount to 100 g, and the mixture was emulsified.
After the emulsification, the mixture was cooled to room temperature with stirring to obtain 100 g of white 5% by mass low molecular weight chitosan 9B hydrochloride cream.

<Formulation Example 2> Propylene glycol 3.0 g,
A solution of 0.1 g of methyl paraoxybenzoate in 80 ml of purified water at 70 ° C
The mixture was heated to 1, and 1.0 g of low molecular weight chitosan 9B hydrochloride prepared in Example 2 was added and dissolved. Stearic acid 3.0 g, cetal 1.0 g, glyceryl monostearate 6.0 g, liquid paraffin 5.0 g, propyl paraoxybenzoate 0.1 g, and polyoxyl stearate 40 2.5 g were dissolved by heating at 70 ° C. and then Example 2 at the same temperature. The low molecular weight chitosan 9B solution prepared in 1. was added, purified water was added to bring the total amount to 100 g, and the mixture was emulsified.
After emulsification, the mixture was cooled to room temperature with stirring to obtain 100 g of white 1% by mass low molecular weight chitosan 9B hydrochloride cream.

<Formulation Example 3> Propylene glycol 3.0 g,
A solution of 0.1 g of methyl paraoxybenzoate in 80 ml of purified water at 70 ° C
The mixture was heated to 0 and 0.5 g of low molecular weight chitosan 9B hydrochloride prepared in Example 2 was added and dissolved. Stearic acid 3.0 g, cetal 1.0 g, glyceryl monostearate 6.0 g, liquid paraffin 5.0 g, propyl paraoxybenzoate 0.1 g, and polyoxyl stearate 40 2.5 g were dissolved by heating at 70 ° C. and then Example 2 at the same temperature. The low molecular weight chitosan 9B solution prepared in 1. was added, purified water was added to bring the total amount to 100 g, and the mixture was emulsified.
After emulsification, the mixture was cooled to room temperature with stirring to obtain 100 g of white 0.5% by mass low molecular weight chitosan 9B hydrochloride cream.

<Formulation Example 4> 3.0 g of propylene glycol,
A solution of 0.1 g of methyl paraoxybenzoate in 80 ml of purified water at 70 ° C
The mixture was heated to 1, and 0.1 g of low molecular weight chitosan 9B hydrochloride prepared in Example 2 was added and dissolved. Stearic acid 3.0 g, cetal 1.0 g, glyceryl monostearate 6.0 g, liquid paraffin 5.0 g, propyl paraoxybenzoate 0.1 g, polyoxyl stearate 40 2.5 g were dissolved by heating at 70 ° C. A chitosan 9B solution was added, purified water was added to bring the total amount to 100 g, and the mixture was emulsified. After the emulsification, the mixture was cooled to room temperature with stirring to obtain 100 g of white 0.1% by mass low molecular weight chitosan 9B hydrochloride cream.

<Formulation Example 5> 100 mg of the low molecular weight chitosan 9B hydrochloride prepared in Example 2 was dissolved in 1 ml of distilled water, the solution was uniformly applied on a glass plate kept horizontally, and air-dried at room temperature. As a result, a transparent film having a thickness of 0.1 mm was obtained.

<Formulation Example 6> 100 mg of low molecular weight chitosan 9B hydrochloride prepared in Example 2 was dissolved in 1 ml of distilled water, and 0.1 ml of glycerin was further mixed with stirring. After defoaming, it was uniformly applied on a glass plate kept horizontal and dried at 50 ° C. for 3 hours to obtain a rubber-like soft sheet.

<Formulation Example 7> 50 mg of low molecular weight chitosan 9B hydrochloride prepared in Example 2 was dissolved in 2 ml of distilled water.
50 mg of methyl cellulose was added and dissolved. After defoaming,
It was evenly applied onto a glass plate kept horizontal and air-dried at room temperature to obtain a transparent chitosan methylcellulose composite film having a thickness of 0.05 mm.

<Formulation Example 8> Propylene glycol 3.0 g,
A solution of 0.1 g of methyl paraoxybenzoate in 80 ml of purified water at 70 ° C
The mixture was heated to 1, and 1.0 g of low molecular weight chitosan 9B hydrochloride prepared in Example 2 was added and dissolved. Stearic acid 3.0 g, cetal 1.0 g, glyceryl monostearate 6.0 g, liquid paraffin 5.0 g, propyl paraoxybenzoate 0.1 g, polyoxyl stearate 40 2.5 g were dissolved by heating at 70 ° C. A chitosan 9B solution was added, purified water was added to bring the total amount to 100 g, and the mixture was emulsified. After emulsification, the mixture was cooled to room temperature with stirring to obtain a white 1.0% by mass low molecular weight chitosan 9B hydrochloride cream (hereinafter referred to as "1% chitosan ointment") (Lot No. 27). The above method was repeated, except that the addition amounts of the low molecular weight chitosan 9B hydrochloride prepared in Example 2 were changed to 2.0 g and 5.0 g, respectively, and white 1.0% by mass low molecular weight chitosan 9B hydrochloride cream (hereinafter, "2% chitosan ointment" and "5% chitosan ointment" were obtained, respectively.
(Lot No. 28 and Lot No. 29). Also, as a placebo,
With the same formulation as above, we also prepared a cream without the addition of low molecular weight chitosan 9B hydrochloride (hereinafter referred to as "base") (Lot
No.26).

<Test Example 8> 1 Introduction A healing test was conducted for the purpose of predicting the effectiveness of a chitosan external preparation in an experimental lacking damage model.

2 Test Material 2-1 Test Substance and Control Drug 1% chitosan ointment prepared in Preparation Example 8 (Lot No. 27),
2% chitosan ointment (LotNo. 28), 5% chitosan ointment (Lot
t No. 29) and the base material (Lot No. 26) were used. Allanto Rox (registered trademark) ointment (20 mg (2 g of aluminum chlorohydroxy allantoinate in 1 g)
%) And polyoxyethylene hydrogenated castor oil and Macrogol 400) Taiyo Yakuhin Kogyo Co., Ltd. were used as additives. 2-2 Method of administration and dose deficiency: The ointment containing chitosan, the base and the control drug (about 0.1 g / site, about 0.2 g / body) were applied twice a day to the injured site (two places per mouse). 2-3 Animals Used 6-week-old Sprague-Dawley (SD) male rats (Japan SLC, Inc.) were purchased and used. Animals were fed with solid feed (F-2 for mouse and rat breeding, Funabashi Farm Co., Ltd.) and tap water using an environmentally controlled breeding device (CLEA Japan, Inc.) with an average temperature of 21.6-22.0 ° C and an average humidity of 59-72%, and lighting. Under the condition of time 7:00 to 19:00, the animals were acclimatized for 6 days and used for the experiment.

3 Experimental Method 3-1 Rat Skin Defect Injury Model Seven-week-old SD male rats (weight: 157.6-235.9 g) were used in 5 groups, 6 groups. After the hair on the back of the rat was shaved with an electric hair clipper, EBA Everclaim (Tokyo Tanabe Seiyaku Co., Ltd.) was applied, and after 15 minutes, the hair was removed by washing. 70% of the back hair loss area
After disinfecting with ethanol, two symmetrical punched wounds were made at the back midline using a circular punch (internal diameter 15 mm) under anesthesia with pentobarbital sodium (40 mg / kg, ip). Twenty-four hours after the creation of the deficient injury, the group was divided into an untreated group, a base group, a chitosan-containing ointment group and a control drug group, and then the test substance was applied to the wound site for 10 days. Rats were housed in individual cages for the duration of the experiment. The effect was judged by measuring the major axis and the minor axis of the damaged portion using a caliper. For the area change during the treatment process, find the area ratio (%) by the formula of (major axis on measurement day x minor axis / major axis after creation of missing damage x minor axis) x 100, and calculate the treated area from the area ratio (5-15 days ), The number of treatment days was calculated for the complete cure rate when the area ratio was 5% or less.

4 Statistical Processing Experimental results are expressed as mean ± standard deviation, and Du was used to determine the results.
nnett's multiple comparison one-sided test (statistical library Star Ligh
1997, Yukms Co., Ltd.) was used. Danger rate is 5% (P <
0.05) was shown as the significance level.

5 Experimental Results A test was carried out by making two defective lesions on the back of 5 animals per group. The data were deleted from the data and summarized in 9 cases for the 5% chitosan ointment group, 9 cases for the 1% chitosan ointment group, 7 cases for the allantolox ointment group, and 10 cases for the other groups. The complete cure rate is shown in FIG. Untreated (non-applied) group: In the non-applied group, a complete cure was observed on the 11th day after the creation of the defect, and a complete cure rate of 80% was observed on the 15th day of the observation. 1% chitosan ointment group: 1 after missing damage
A complete cure was seen from day 0, and a cure rate of 90% was shown on day 15 of observation. 2% chitosan ointment group: A complete cure was seen from the 10th day after the creation of the defect, and a 100% cure rate was observed on the 13th day of the observation. 5% chitosan ointment group: A complete cure was seen from the 10th day after the creation of the defect, and a 100% cure rate was observed on the 12th day of the observation. Base group: A complete cure was seen from the 10th day after the creation of the missing defect,
On the 15th day of the observation, a complete cure rate of 80% was observed. Allanto Rox ointment group: A complete cure was seen from the 11th day after the creation of the missing damage,
On the 14th day of the observation, the cure rate was 100%. Table 9 shows the number of treatment days. 13.7 ± 1.3 days for untreated group, 12.2 ± 1.3 days for 1% chitosan ointment group, 12.5 ± 1.0 days for 2% chitosan ointment group
The 5% chitosan ointment group significantly reduced the number of treatment days to 11.8 ± 0.8 days. Base group and allanto locks ointment group also 1
The treatment days were shortened to 2.7 ± 1.2 days and 12.6 ± 1.0 days, but no significant difference was observed compared with the untreated group.

[0098]

[Table 9]

6 Discussion A healing test of a chitosan external preparation was carried out using a rat skin defect injury model. Chitosan topical agents (1%, 2%, 5% chitosan-containing ointment) are allantolox ointment (effect:
It was confirmed that there was a superior damage-promoting effect on erosion and ulcers caused by trauma / burns, which is superior to the pharmacological effect: granulation formation / necrotic tissue removal.

<Test Example 9> 1 Purpose It was examined whether or not chitosan has a lipid-lowering effect using a cholesterol-loaded hamster.

2. Test material 2-1 Basic feed and experimental feed (mixed feed) FR-2 standard powdered feed for mice and rats with 0.3% cholesterol and 10% coconut oil (Funabashi Farm Co., Ltd.) was used as the basic feed, and the basic feed was 98g. 2% chitosan aqueous solution (prepared by dissolving 2 g of low molecular weight chitosan 9B hydrochloride prepared in Example 2 in 98 g of purified water) 100
The mixed feed prepared by suspending g and drying with a freeze dryer was used as an experimental feed. 2-2 Administration method The animals were allowed to freely take the basic feed and the experimental feed (mixed feed). 2-3 Animals Used 12 male 14-week-old syrian golden hamsters (Japan SLC, Inc.) were purchased and used. Animals are placed in an aluminum cage, 6 animals each, with an average temperature of 23 ± 2
FR-2 standard powder feed for mice and rats (Funabashi Farm Co., Ltd.) and tap water at a temperature of 55 ° C and an average humidity of 55 ± 10%,
The animals were acclimated and managed for 7 days in a breeding room controlled by alternating light and dark for 12 hours and used for the experiment.

3. Experimental Method 15-week-old syrian golden hamsters were used in two groups each consisting of 6 animals. Divide animals into control group and chitosan group (day 0),
From the next day, the control group was fed with the basic feed and the chitosan group was fed with the experimental feed (mixed diet) for 7 days, and on the 8th day (day 8) after grouping, the animals were fasted from 9 am and hunger at 1 pm Blood was collected from the aorta and the liver was removed. Then serum TG
(Triglyceride), serum TC (total cholesterol),
Liver TG, liver TC and liver weight were measured. On the day of grouping (day0), three days later (day3), and five days later (da)
y5), 8 days later (day 8). Serum TG and serum TC were measured by triglyceride E-Test Wako and cholesterol CII-Test Wako (Wako Pure Chemical Industries, Ltd.), respectively.
Was measured by the enzymatic method. Liver TG and liver TC were measured by the method of Folch et al. (J. Biol. Chem., 226, 494-509 (19
According to 57), liver lipids were extracted, and the TG amount / TC amount of the extract was measured by the same method as for serum.

4. Experimental Results 4-1 Measurement of Body Weight Change Table 10 and FIG. 5 show changes in body weight of individuals in each group. The value on the vertical axis of FIG. 7 is the average value ± standard error (SEM) of 6 animals in each group.

[0104]

[Table 10]

An increase in body weight over time was observed in the control group. On the other hand, the increase in body weight was suppressed in the chitosan administration group. In addition, the weight gain rate was significantly reduced in the chitosan-administered group compared to the control group.
(P <0.01, Student's test) Table 11 shows the feed intake of each group.

[0106]

[Table 11]

The food intake was almost the same in both groups. Therefore, it was shown that the inhibitory effect of weight gain by chitosan was not due to the decrease in food intake. 4-2 Measurement of Liver Weight Table 12 shows the liver weight on day 8 of each group. 6 for each group
The average value ± standard error of the animals is shown in FIG.

[0108]

[Table 12]

There was no significant difference in liver weight between the two groups,
Low molecular weight chitosan did not cause liver hypertrophy. 4-3 Measurement of Serum TC Table 13 shows the serum TC value (mg / dl) of each group. The average value ± standard error of 6 animals in each group is shown in FIG. 7.

[0110]

[Table 13]

The TC value was lower in the chitosan administration group than in the control group. 4-4 Measurement of serum TG Table 14 shows the serum TG value (mg / dl) of each group. The average value ± standard error of 6 animals in each group is shown in FIG.

[0112]

[Table 14]

The TG value was lower in the chitosan administration group than in the control group. 4-5 Serum HDL-cholesterol (HDL-CH)
Table 15 shows the serum HDL-CH value (mg / dl) of each group. The average value ± standard error of 6 animals in each group is shown in FIG.

[0114]

[Table 15]

The HDL-CH value was higher in the chitosan-administered group than in the control group. 4-6 Measurement of liver TC No. of each group Table 16 shows the amount of TC (mg / g) per 1 g of liver. The mean value ± standard error of the amount of TC per 1 g of liver of 6 animals in each group is shown in FIG. 10.

[0116]

[Table 16]

In the chitosan-administered group, the total cholesterol content in the liver was lowered. 4-7 Measurement of Liver TG No. of each group Table 17 shows the TG amount (mg / g) per g of liver. FIG. 11 shows the average value ± standard error of the TG amount per 1 g of liver of 6 animals in each group.

[0118]

[Table 17]

In the chitosan-administered group, the liver triglyceride content was decreased.

[0120]

According to the present invention, low molecular weight chitosan can be reproducibly produced from high molecular weight chitosan.

[Brief description of drawings]

1 shows the molecular weight distribution of low molecular weight chitosan 9B hydrochloride prepared in Example 2. FIG. The fraction showing the maximum reducing sugar amount was set as 100% and displayed as a relative ratio. White circle: Dextran T40 (molecular weight 40,000), White triangle: Dextran T70 (molecular weight 70,000), White square: Dextran T110 (molecular weight 11)
10,000), white rhombus: Dextran T250 (molecular weight 250,000), black circle: low molecular weight chitosan 9B hydrochloride.

FIG. 2 shows the molecular weight distribution of low molecular weight chitosan 8B hydrochloride prepared in Example 3. The fraction showing the maximum reducing sugar amount was set as 100% and displayed as a relative ratio. White circle: Dextran T40 (molecular weight 40,000), White triangle: Dextran T70 (molecular weight 70,000), White square: Dextran T110 (molecular weight 11)
10,000), white rhombus: Dextran T250 (molecular weight 250,000), black circle: low molecular weight chitosan 8B hydrochloride.

FIG. 3 shows the molecular weight distribution of low molecular weight chitosan 7B hydrochloride prepared in Example 4. The fraction showing the maximum reducing sugar amount was set as 100% and displayed as a relative ratio. White circle: Dextran T40 (molecular weight 40,000), White triangle: Dextran T70 (molecular weight 70,000), White square: Dextran T110 (molecular weight 11)
10,000), white rhombus: Dextran T250 (molecular weight 250,000), black circle: low molecular weight chitosan 7B hydrochloride.

FIG. 4 shows the complete cure rate in a rat skin defect injury model.

FIG. 5 shows changes in body weight between a control group and a chitosan group.

FIG. 6 shows the average liver weight (g) of the control group and the chitosan group.

FIG. 7 shows the mean serum TC values of the control group and the chitosan group.

FIG. 8 shows the mean serum TG values of the control group and chitosan group.

FIG. 9 shows the average serum HDL-CH values of the control group and the chitosan group.

FIG. 10 shows the average value of TC amount per 1 g of liver in the control group and the chitosan group.

FIG. 11 shows the average value of TG amount per 1 g of liver in the control group and the chitosan group.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/10 101 A61P 9/10 101 17/02 17/02 31/04 31/04 C12P 19/28 C12P 19/28 F term (reference) 4B064 AF21 CA21 CB07 CC06 CC07 DA01 4C086 AA01 AA02 AA03 AA04 EA23 MA01 MA04 ZA45 ZA70 ZA89 ZB35 ZC33 ZC35 4C090 AA04 BA47 BC27 BD37 CA43 DA23

Claims (31)

[Claims] 1. A high molecular weight partially acetylated chitosan having a degree of acetylation of 10 to 30% and a molecular weight of 1,000,000 or more is hydrolyzed with a chitosan degrading enzyme, and then a fraction having a molecular weight of less than 10,000 is removed to obtain a molecular weight. Is a method for producing low molecular weight chitosan or a salt thereof of 10,000 or more and less than 1 million,
The chitosan degrading enzyme has a pH of 7.0 to 7.5 and a temperature of 3
Under reaction conditions of 7 ° C. for 10 to 20 minutes, the hydrolysis activity for 30% acetylated chitosan (viscosity 100-300 cps) is 100%, the hydrolysis activity for glycol chitin is less than 100%, and Degree of conversion is 1%
The above method, wherein the following hydrolytic activity against chitosan (viscosity 50 to 200 cps) is 15% or less. 2. The low molecular weight chitosan or a salt thereof is 0-10.
The method according to claim 1, wherein 10 g or more is dissolved in 100 g of water at 0 ° C. 3. The method according to claim 1, wherein the low molecular weight chitosan or a salt thereof has antibacterial activity. 4. The chitosan degrading enzyme is Bacillus sp.
(Bacillus sp.) SANK 70699 strain (FERM BP-6666) or Bacillus sp. (Bacillus sp.) SANK 70799 strain (FERM BP-666)
The method according to claim 1, which is a chitosan-degrading enzyme derived from 7). 5. The method according to claim 1, wherein the polymer partially acetylated chitosan is hydrolyzed with a chitosan degrading enzyme under conditions of pH 3 to 12 and 70 ° C. or lower for 10 minutes to 7 days. 6. A low molecular weight chitosan or its salt is hydrolyzed by adding an alkali to a solution prepared by dissolving high molecular weight partially acetylated chitosan in water under acidic conditions to make it weakly alkaline, and then adding a chitosan degrading enzyme. Generate 1 to 5
The method described in any one of. 7. A molecular weight of 10,000 or more and less than 1,000,000, 0 to
A low molecular weight chitosan or a salt thereof which dissolves in 10 g or more in 100 g of water at 100 ° C. 8. A pharmaceutical composition comprising the low molecular weight chitosan or the salt thereof according to claim 7. 9. A preventive and / or therapeutic agent for wounds and / or pressure sores, which comprises the low molecular weight chitosan or the salt thereof according to claim 7 as an active ingredient. 10. An antibacterial agent containing the low molecular weight chitosan or a salt thereof according to claim 7 as an active ingredient. 11. An antihyperlipidemic agent containing the low molecular weight chitosan or a salt thereof according to claim 7 as an active ingredient. 12. An anti-atherosclerotic agent containing the low molecular weight chitosan or the salt thereof according to claim 7 as an active ingredient. 13. An antidiabetic agent containing the low molecular weight chitosan or a salt thereof according to claim 7 as an active ingredient. 14. An antiobesity agent containing the low molecular weight chitosan or a salt thereof according to claim 7 as an active ingredient. 15. The method according to claim 1, wherein the high molecular weight partially acetylated chitosan having a degree of acetylation of 10 to 30% and a molecular weight of 1,000,000 or more is derived from crustaceans. 16. A degree of acetylation of 10 to 30% for 10 minutes to 7 days under the conditions of pH 3 to 12 and 70 ° C. or less.
16. The method according to claim 15, wherein the high molecular weight partially acetylated chitosan having a molecular weight of 1,000,000 or more is hydrolyzed with a chitosan degrading enzyme. 17. A pH of 6.0 to 7.5, 37 ° C. to 4
80 to 170 rp for 2 to 3 days at 0 ° C
The method according to claim 16, wherein the partially acetylated high molecular weight chitosan having a degree of acetylation of 10 to 30% and a molecular weight of 1,000,000 or more is hydrolyzed with a chitosan degrading enzyme while shaking at m. 18. A polymer partially acetylated chitosan having a degree of acetylation of 10 to 30% and a molecular weight of 1,000,000 or more is previously dissolved in water under the condition of pH 2.5 to 3.5, and then alkali is added. Method according to any one of claims 1 or 15 to 17, characterized in that the pH is adjusted to 6.0 to 7.5. 19. The chitosan degrading enzyme is Bacillus sp. SANK70699 strain (FERM BP-6666) or Bacillus sp. SANK70799 strain (FERM BP-66).
67) derived from chitosan degrading enzyme,
The method according to any one of claims 15 to 18. 20. A low molecular weight chitosan having a molecular weight of 10,000 or more and less than 1 million or a salt thereof, which is produced by the method according to claim 1 or any one of claims 15 to 19. 21. 1 to 100 g of water at 0 to 100 ° C.
21. The low molecular weight chitosan or a salt thereof according to claim 20, which is dissolved in an amount of 0 g or more. 22. p of 1 mass% low molecular weight chitosan salt aqueous solution
The low molecular weight chitosan or a salt thereof according to claim 20 or 21, characterized in that H is pH 4.8 to 6.3. 23. The pH at which a precipitate is deposited when an alkali is added to a 1% by mass low molecular weight chitosan salt aqueous solution has a pH of 6.
22. The low-molecular-weight chitosan or a salt thereof according to claim 20, which is 4 to 8.2. 24. A low molecular weight chitosan or a salt thereof having the following properties [I] to [IV]; [I] having a molecular weight of 10,000 or more and less than 1 million: [II] in 100 g of water at 0 to 100 ° C. Dissolve 10 g or more: [III] has a pH of 4.8 to 6.3: [IV] The pH at which a precipitate deposits when an alkali is added to a 1% by mass low molecular weight chitosan salt aqueous solution pH 6.4 to 8.2. 25. A pharmaceutical composition containing the low molecular weight chitosan or a salt thereof according to any one of claims 20 to 24. 26. An antibacterial agent containing the low molecular weight chitosan or a salt thereof according to any one of claims 20 to 24 as an active ingredient. 27. An antihyperlipidemic agent containing the low molecular weight chitosan or a salt thereof according to any one of claims 20 to 24 as an active ingredient. 28. An anti-atherosclerotic agent comprising the low molecular weight chitosan or a salt thereof according to any one of claims 20 to 24 as an active ingredient. 29. An antiobesity agent comprising the low molecular weight chitosan or a salt thereof according to any one of claims 20 to 24 as an active ingredient. 30. An antidiabetic agent containing the low molecular weight chitosan or a salt thereof according to any one of claims 20 to 24 as an active ingredient. 31. A therapeutic and / or prophylactic agent for wounds and / or pressure sores, which comprises the low molecular weight chitosan or a salt thereof according to any one of claims 20 to 24 as an active ingredient.