WO1993014216A1

WO1993014216A1 - Process for producing chitin and chitosan from dermatophyte

Info

Publication number: WO1993014216A1
Application number: PCT/JP1992/000037
Authority: WO
Inventors: Kazunori Nakamura; Chikako Yamada
Original assignee: Nakamura, Tomotaka
Priority date: 1992-01-20
Filing date: 1992-01-20
Publication date: 1993-07-22
Also published as: KR930703296A; AU1168692A

Abstract

A process for producing chitin or chitosan in high yields by culturing a certain dermatophyte belonging to the genus Trichophyton, Epidermophyton or Microphyton in an improved medium prepared by adding keratin and/or peptone to a basal medium essentially comprising inorganic salts under bubbling with a gaseous mixture of CO2 and N2. Each of the chitin and chitosan thus obtained has a high antibacterial activity, can be rapidly decomposed and absorbed into the living organism, does not cause skin inflammation, and has a high adherence as a biomembrane, thus being excellent in functionality.

Description

Description Production method of chitin and chitosan by dermatophytes

The present invention relates to a method for producing chitin and chitosan by a dermatophyte, and in particular, cultivating a certain dermatophyte group under specific conditions to produce a bacterium having a high chitin content, which will be widely used in the future. The present invention relates to a method for extracting chitin or chitosan having various functions suitable for the field on a mass production basis. Background technology

In recent years, the effective use of chitin and chitosan has been diversified in various fields, including application to artificial skin as a biomaterial, as well as preservatives for various foods, molding agents, soil conditioners, wastewater treatment agents, and so on. Therefore, application use is foreseen experimentally. In particular, in the field of human: 1: biomaterials, chitin and chitosan have functional properties such as in vivo digestibility, immunoadjuvant activity, lymphocyte cell separation activity, drug release / release function, anticoagulant activity, and antibacterial activity. It is recognized that it has a role, and is expected to have potential applications in many medical fields in the future. However, the stable supply of chitin / chitosan as a material pursuing individual functionality, which is the premise of its realization, has not been achieved to date.

To send chitin and chitosan to the world as a stable supply material. Chitin and chitosan are known as components of lower animals such as crustaceans, insects, shellfish, and fungi, and the skeletal and supporting tissues of filamentous fungi. This However, chitin and chitosan as natural resources have, however, been less demanding and have remained unused, and no attempt has been made to use them effectively. On the other hand, as the biological and chemical properties of chitin and chitosan become evident in the theoretical aspect, the demand for functional polymer materials is increasing. Insects such as typhoids and insects are considered to be the most promising material sources, and some filamentous fungi, especially zygomycetes, for example, Mucor genus, are produced by fermentation. Has been reached. Even if these sources could solve the quantitative aspects and provide a stable supply of chitin and chitosan in the future, as a source of these materials with various enhanced functions as described above, No research and development has been done so far. Disclosure of the invention

Accordingly, an object of the present invention is to provide a novel method capable of stably supplying chitin and chitosan as highly functional substances on an industrial scale.

Another object of the present invention is to provide a new source of chitin and chitosan having various functions that cannot be realized by the same kind of substances derived from conventional crustaceans and insects. I do.

Further, it is a specific object of the present invention to provide a method for producing highly functional chitin and chitosan at a high yield by specially cultivating a certain dermatophyte. I do.

Dermatophytes are known as fungi that infect and parasitize the surface of the stratum corneum of various animals and humans and cause various skin diseases. Separately, there are three genera: Trichophyton, Microsporum, Microsporum, Epidermophyton, and Epidermophyton. Into two parts. The characteristics of the bacterial strains belonging to these are that they have a strong affinity for the skin of various animals and humans and cause various skin diseases on the head, body, and feet. These seemingly disadvantageous features were studied by considering that the inventor might be able to use them in an advantageous manner, and completed the present invention. In other words, we focused on the fact that dermatophytes having affinity for animals as described above would have a greater affinity for living organisms than chitin derived from other organisms. The chitin produced by filamentous fungi includes the genus Mucor, the genus Rhizopus, the genus Aspergillus, and the like. It is known that exists. On the other hand, it is about 0 to 15% in the dermatophytes of the present invention. (See Example 1 below)

On the other hand, the basic structure of chitin is a kind of mucopolysaccharide consisting of 1,4 1/3 bonds of N-acetyl-D-glucosamine, but even if one or two-dimensional structure is understood, 3 or 4 dimensional structures are not understood. The present invention has focused on the fact that if this spatial structure is chitin derived from a dermatophyte having a spatial structure different from that of chitin derived from other organisms, the behavior in a living body will naturally be different. . The results are evident from examples of various functions described later.

Further, as a method for improving the chitin content per cell wall of the captive filamentous fungi by reducing the aforementioned small amount of filamentous fungi, the chitin content is increased. In the present invention, as a result of diligent studies and repeated studies on culture conditions, the present invention has succeeded in realizing a higher chitin production amount than conventional dermatophytes. Next, these culture conditions will be specifically described in Example 1.

Thus, the present invention has succeeded in making dermatophytes a stable source of chitin by finding the specific culture conditions, and has advanced and widespread due to the spatial characteristics of dermatophytes. Chitin and chitosan have succeeded in imparting the desired functionality. Specifically, the chitin produced by the dermatophytes according to the present invention has antibacterial properties against various microorganisms, biodegradability, in vivo It has been found to have superior properties to conventional crustacean-derived chitin in terms of biological functions such as settability, physical strength, and safety.

Thus, according to the present invention, a biopolymer material, in particular, a new material having a new and excellent functionality, which has not been previously anticipated, can be produced as chitin. . BEST MODE FOR CARRYING OUT THE INVENTION

The perennial filamentous fungi that can be used in the present invention are broadly anamorph-type incomplete mycoplasma, Trichophyton, Epidermophyton and Microcrosporum belonging to the genus Actinomycete, or Telemorph-type. It is a fungal species belonging to the genus Aspergillus, Trichophyton, Epidermophyton and Microsporum belonging to the family Ascomycota. Specifically, for example

[i. Trichoph ton mentagrophytes, 丄 FO 546D, 5809,

Τ.violaceum (OUT 4132),

Τ. Ferrugineum (IFO 5832). 1. cerebriforme (IFO 5930),

Ί. Concentricum (IFO 5972),

T. f avif orme (IFO 5934),

T. nodof orinans (OUT 4131),

T. rubrum (IFO 5467, 6203),

1.rubrum (MTU 19012)

1. tonsurans (IFO 5946),

1. tonsurans (IFO 5928),

Arthoroderma uncinatum (IFO 6893),

Hicrosporum audouini (MTU 20001),

M. canis (AHU 9577),

M. gypseum (Bodin) (IFO 5948),

Epidermophyton f loccosum (MTU 21002)

Various dermatophytes can be advantageously used in the present invention.

All of these bacteria are readily available from public preservation agencies. In the above, IF0 is

For, OUT of the Institute for Fermentation, Osaka, Japan

Faculty of nngineering, Osaka Universit, Suita, Japan (Osaka University Fermentation Engineering)

MTU

Faculty of Medicine, Tokyo University, Tokyo, Japan

No, AHU

Faculty of Agriculture, Hokkaido University, Hokkaido, Japan (Hokkaido University Faculty of Agriculture)

It is sputum. Example 1 Production of chitin by various dermatophytes

This example demonstrates that chitin can be obtained in high yield by culturing the following bacterial species under specific culture conditions.

(1 Arthoroderma uncinatum od93 ノ,

(2 Hicrosporum audouini (MTU 000 丄,

(3 Hicrospor um canis (AHU 9o 77),

(4 Trichophyton tonsurans (IFO 5945),

(5 Trichophyton tonsurans (IFO 5928),

(6 Trichophyton violaceum (OUT 4132),

(7 Trichophyton mentagrophy es (IFO 54b6),

(8 Trichophyton mentagrophy tes (IFO 5809),

(9 Trichoph ton ferrugineum (IFO 5832),

(10 Trichophyton cerebriforme (IFO 5930),

(11 Trichophyton concentricum (IFO 5972),

(12 Trichophyton f a vif orme (IFO 5934),

(13 Trichophyton nodof ormans (OUT 4131),

(14 Trichophy on rubrum (IFO 5467),

(15 Trichophyton rubruin (IFO 6203), and

(16 Epidermophy ton f loccosum (MTU 21002) First, as a control, 1 β of distilled water, 25 g of glucose, 2 g of sodium nitrate, 1 g of monohydrogen phosphate 0.5 _g magnesium sulfate, 0.5 g potassium chloride, 0.02 g sulfur A “basal medium” consisting of ferrous acid was prepared, with chitin shaking cultured for 27 hours at 27 for control production.

Next, for the cultivation according to the present invention, 1 to 5 mg of keratin (manufactured by Sigma) and 0.5 to 5 g of peptone (Eisai Kagaku) were added to this “basal medium”. Add the "Improved medium" to III, adjust it to PH 6.0, sterilize (110 ^nC , 1 atmosphere, 10 minutes), inoculate each of the above strains, and add 10 to 5 0% 00 ₂ 5 0-9 0% of a gas mixture of N ₂ by public ring 2 5. Shaking culture was performed with C for 15 to 30 hours.

These cultures were collected by filtration, washed twice with a phosphate buffer (pH 6.0), and then rapidly frozen in liquid nitrogen to break the cells. The disrupted cells were centrifuged at 12,000 rpm, the cell wall fraction was collected, and the chitin content was measured.

To extract chitin from the cells, add 100mβ of 10% NaOH to 1 g of the cell wall fraction, stir at 48 ° C for 48 hours, and wash the insolubles twice with distilled water. The centrifuged precipitate was treated with 5% acetic acid and washed twice with distilled water again. The precipitate was defatted with 95% ethanol and dehydrated and dried, and was used as chitin. The chitinization of chitin is performed by adding 0.5% of the above chitin to a sample of 3 πιβ 3% (wt / vol) sodium 'rauryl' sulfate at 0.5 ° C and adding 100% at 100 ° C. After 5 minutes of treatment and cooling, centrifugation is performed at 30,000 rpm, the precipitate is washed with distilled water, and centrifuged again under the same conditions to give 3 ηι β ΚΟΗ (100 m β to 120 g). (Diluted with distilled water) at 130 ° C for 1 hour. After cooling, the precipitate obtained by centrifugation at 3,000 rpm was washed twice with distilled water, and this precipitate was used as a chitosan extract. This substance is 110 ° C with 6N HCL After hydrolyzing in a glass tube sealed with nitrogen gas for 14 hours, the solution was neutralized with 6 M NaOH, and the chitin content was calculated from the quantification of dalcosamine by the Erson-Morgan method. The results are shown in Table I. Table I Chitin production from various dermatophytes

(Including cell wall and lichitin) Test strain control

(1) Arthoroderma uncinatura (IFO od93) 16.9 20.5

(2) hicrosporum audouini (MTU 20001 S 13. Z 26.4

(3) Hicrosporum canis (AHU 9577) 15.8 27.6

(4) Trichophyton tonsurans (IFO 5946) 15.9 30.6

(5) Trichophyton tonsurans (IFO 5928) 14.5 29.8

(6) Trichophyton violaceum (OUT 4132) 16.7 28.4

(7) Trichophyton mentagrophytes (IFO 5466) 15.3 27.6

(8) Trichophyton mentagrophytes (IFO 5809) 14.9 33.4

(9) Trichophyton ferrugineum (IFO 5832) 14.2 26.9

(10) Trichophyton cerebrif orme (IFO 5930) 16.4 31.9

(11) Trichophyton concentricum (IFO 5972) 12.1 25.3

(12) Trichophyton f avif orme (IFO 5934) 15.6 29.9

(13) Trichophyton nodof ormans (OUT 4131) 13.5 26.5

(14) Trichophyton rubrum (IFO 5467) 17.1 37.5

(15) Trichophyton rubrum (IFO 6203) 16.3 34.5

(16) Epidermophyton f loccosum (MTU 21002) 11.5 19.4

Shaking culture in "Basic medium" for 30 hours

C0 ₂ 20%, N ₂ 80¾ in "improved medium" (keratin 3 mg, peptone lg) As shown in Table I, a mixture of carbon dioxide and nitrogen gas was used in an improved medium in which keratin and peptone were added to an inorganic base medium, as clearly shown in Table I. Culturing under specific conditions of the present invention using publishing, for example, especially in Trichophyton 'Norebulum (IF05467), has a chitin content of 17.1 << per cell wall in the control. According to the present invention, the result increased to 37.5%, which is about twice or more, was obtained. Example 2 Chitosan extracted from Trichophyton 'Rublum

Antimicrobial properties of various microorganisms

It is generally well known that chitin and chitosan exhibit antibacterial activity against various microorganisms. However, to date, although the antibacterial properties of chitin in crustaceans have been introduced in academic reports and patent documents, chitin from crustaceans has not been reported. What the antibacterial properties are is not known at all. Thus, the present invention attempted to compare the antibacterial properties of Rikiji chitin and Trichophyton-Norebulum-derived chitin with respect to various bacteria, yeasts and fungi.

The test was performed using chitosan derived from Rikiji (Sigma) and chitosan extracted in Example 1 from the dermatophyte Trichophyton 'rubrum (IF0 5467) according to the present invention. went.

Each chitosan is diluted with a dilute acetic acid solution (2 M) to a final concentration of 2, 4, 6, 8, and 9 mg Zm fi, respectively. Into a sterile small test tube To prepare a test medium.

In this test medium, 10 kinds of Gram-positive and -negative bacteria shown in the following table, which had been pre-cultured with peptone and sub-oral liquid medium without chitosan in advance, and T. Candida albicans (ATCC 10261) and dermatophytes. as a bacteria and Mi click Rosuporumu 'force varnish (AHU 9577), it was inoculated at a rate of ^{3 ~ 5 X 10 7 / m} β, bacteria 1 8 hour culture the fungus 48 hours After culturing, the absorbance (0D · 620 nm) of each was measured, and the inhibition rate (%) was calculated from the absorbance of the control with no chitosan added. This antibacterial test was performed according to the method of Muzzarelli et al. (See Huzzarelli et al .: Antimicrobial properties of N_carboxylbutybut chitosan, Antimicrobial Agents and Chemotherapy, Vol. 34, No. 10, PP 2019-2023, 1990). )

Table π

Antibacterial activity of chitosan extracted from Trichophyton 'rubrum

Chitosan from Rikiji T. rubuni Chitosan final concentration (mg / m fi) Final concentration (mg /) 2_ 丄 _6_ _8_ 9 2. 丄 _6_ _j8_ 9 Inhibition rate (%) Inhibition rate (%) %)

0 12.4 79.5 82.3 93.4 8.3 23.4 91.0 96.2 98.6

0 19.3 88.6 91.2 93.9 11.8 29.4 89.6 94.3 97.6 0 0 16.3 63.4 89.1 0 15.9 99.2 100 100 0 0 68.9 78.2 93.6 43.5 58.9 90.3 100 100 0 21.3 54.5 78.3 93.2 13.4 53.5 89.4 97.3 100 0 25.4 60.3 88.3 91.7 19.3 48.3 83.1 93.4 100

0 13.4 19.2 39.4 56.2 11.3 24.9 59.7 67.8 83.2 0 0 0 19.4 32.3 0 16.9 24.8 53.9 73.4 0 23.5 38.9 43.9 62.8 0 19.3 38.9 67.7 81.3 0 19.6 31.3 46.9 72.3 9.8 16.3 42.1 59.1 73.4 0 14.2 33.9 68.9 81.3 11.3 24.9 81.3 93.4 100 0 0 11.3 24.5 31.9 15.5 19.9 38.4 53.4 83.9

Note 1 Details of the strains used in Table II are as follows

S. aureus: Staphylococcus aureus

S. epidermidis: Staphylococcus epidermidis

S. pyogenes: Streptococcus pyogenes

E.facecium: Enterococcus f acecium

E.coli: Eschericha coli

R. pneumoniae: Klebsiella pneumoniae

S.raarsucens: Serratia marusucens

S. typhimurium: Salmonella typhimurium

P. aeruginosa: Pseudomonas aeruginosa

Η · influenzae: llaemophillus influenzae

C. albicans: Candida albicans

M. canis: Microsporum canis As clearly shown in Table II, the antibacterial activity of chitosan extracted from dermatophytes according to the present invention against various microorganisms is higher than that of chitosan derived from Rinji. In particular, the antibacterial activity against Gram-negative bacteria and fungi is extremely remarkably higher than that of chitosan derived from Riki. Example 3 Biodegradation and Absorption of Chitin from Dermatophyte R. ru brum

Biodegradation / absorption is important as a material selection factor for biopolymer materials for medical use. This means that not only the safety of the material to the living body, but also that the degradation products are nontoxic and that it is decomposed and absorbed at an appropriate time are required as selection conditions for medical biomaterials. That is to say. Thus, in this example, the results of a comparative test of the in vivo degradation and absorption of chitin derived from dermatophytes and chitin derived from Rikiji are shown.

The material used was chitin extracted from the dermatophyte T. rubrum (IF0 5467) according to the conditions described in Example 1, and the control was It was a section of 3 mm X 3ππη with commercially available chitin-derived chitin (manufactured by Sigma). These sections were embedded in the back of the guinea pig, and their degradation and absorption capacity were observed until 2, 4, 6, 8, and 10 weeks, respectively. The guinea pigs used were a total of six white female guinea pigs (CLEA Japan). The average weight of the five animals was 269 ± 9.3 g, one week before this experiment using solid feed (CLEA Japan) in a rearing environment at a temperature of 22.5 ° C ± 0.3 ° C and a humidity of 55 ± 5%. The one purchased from Japan was used. Cut under the guinea pig back subcutaneously with a length of 1 cm using a dissecting scissors, bury the above chitin and suture.After rearing for each period, incision is made on the back of the guinea pig to decompose and absorb. Observed. Each operation was performed aseptically. The results are shown in Table 111 below. Table in

Decomposition of chitin subcutaneously in guinea pigs, comparative test of absorption capacity Implantation period (weeks) Decomposition, absorption reaction ¹ Flame reaction ²

Reference invention Reference invention

Two

4 Sat +

6 +

8 + + + + + +

10 + + + + + + + +

“Control” is chitin derived from Rikiji, and “Invention J is chitin derived from dermatophyte T. rubruffl (IF0 5467)”.

Note ¹ Decomposition 吸収 Absorption reactivity is as follows: one is not decomposed, soil is 20% decomposed, + is 40% decomposed, ++ is 60% decomposed, +++ is 80% decomposed, +++ is 100% decomposed Show.

^{2 The} inflammatory response indicates that the soil has a weak inflammatory response, one of which has no inflammatory response.

From the above results, it was found that the degradation reaction of chitin derived from the filamentous fungus in guinea pigs was degraded earlier than the chitin derived from Rhizo. Is recognized. In addition, there is no substantial difference between the in vivo inflammatory response and antigenicity. Example 4 Adhesion of Rat and Guinea Pig Skin to Chitin Derived from Dermatophytes and Chitin Derived from Rikiji

Using a commercially available chitin membrane derived from the Chionoecetes opilio (manufactured by Kanto Kagaku) and the chitin membrane derived from the dermatophyte T. rubrura (IF0 5467) (extracted in Example 1) according to the present invention, A comparative test of adhesion to the back skin was performed. As the animals to be used, female Wistar and Wistar female white guinea pigs and Wistar female rats of 4 weeks of age were purchased. The breeding conditions were as described in Example 3, and the average weight of each animal at the start of this experiment was 120 ± 9.2 g in rats and 235 ± 14.6 g in guinea pigs. The hair on the back of each animal is cut with an electric hair clipper, and after 6 hours of depilation, the skin surface is cut in 1.5 mm squares with dissecting scissors, each chitin membrane is applied, and 2 x 2 cm square filter paper is applied. And closed at each period to observe the adhesion to the skin, the dry state, the ability to form epidermis, and the hemostatic property. Each animal used was subjected to the experiment as a group of 6 animals. The results are shown in Table IV.

Table IV Comparison of Adhesion of Chitin from Dermatophytes and Chionoecetes opilio to Rats and Guinea Pigs, etc. Adhesion Closure — Blood Observation Period (Week) 1 2 3 2 3 1 2 3 1 2 3 Membrane ABABABABABABABABABABABA BAB Use number

Foot 1 * * + + + #-* * + # # one * * + + #-* * # # # 2-+ * # # # one * * # + # * # # # one * * # # # 3 + + # # * # + # * * + # + # 4 氺氺 + # # # one * * # + # * * # # # one * * # + # 5 * # + # * * + + # -* * # # # 6-* + # * * # + # * * + # # # Guinea pig

* + + # + #-* * # # * * # + # * * + #

2 * * + # # #-* * # * * # # # * * + # # # 3 * + + # # # * # * * # # # One * * # # # 4 * * + # # # * # * # + # One * * # # # 5 * + + # # # * # * # + # + # # # 6 * + + # # # * # #-* * # # # * # + # Table IV In the above, membrane A is a chitin membrane derived from the Chionoecetes opilio, and membrane B is a chitin membrane derived from the dermatophyte T. rubrum (IF05467).

The validity marks are # is valid, + is valid, * is slightly valid, and-is invalid. As a result, chitin derived from dermatophytes was remarkably effective earlier than chitin derived from Rikiji in all of the adhesiveness to various animal skins, the drying property, the ability to form epidermis, and the hemostatic property. Is allowed to do so. Industrial Applicability The high-yield, high-functional chitin / chitosan produced by the present invention is expected to have the following wide application range. For example, artificial skin, fiber, film, metal purification carrier, artificial organ moisturizer, plant growth regulator, deodorant, fixing agent, paper antibacterial agent, textile antibacterial agent, delivery of pharmaceuticals, agricultural chemicals, and nutrients Carrier, sustained-release carrier, antiviral agent, anticholesterol agent, emulsifying agent, hygroscopic agent, antithrombotic and anticoagulant, cell immunity enhancer, metal, plastic surface enhancer, food additive Applications include cosmetics, cosmetics, high-performance composite materials, and dye enhancers. The present invention enables stable supply of high quality chitin and chitosan to these applied products.

Claims

Scope of the claim: The method comprises culturing a dermatophyte group under specific conditions to increase the chitin content and producing chitin or chitosan at a higher yield than the cells. A method for producing chitin or chitosan. The dermatophyte group is selected from the group consisting of anamorph-type incomplete mycota, Trichophyton, Epidermophyton, and Microsporum strains belonging to the family Mycobacteriaceae. The method described in Scope 1 of a claim. The dermatophyte group is selected from the group consisting of a telemorphic subpopulation of Komori spores, Trichophyton, Epidermophyton belonging to the family Ascomycota, and Microsporum strains. The method of claim 1 wherein the method is: The specific conditions are that an improved medium consisting of keratin and peptone added to a basic medium consisting essentially of inorganic salts is used, and that C した 2 and N2 The method according to claim 2 or 3, further comprising performing mixed gas publishing. The basal medium is a mixture of distilled water, glucose, sodium nitrate, monohydrogen phosphate, magnesium sulfate, potassium chloride, and ferrous sulfate. The method described in range 4. The basal medium is 25 g of 1 β-equivalent of ribose in distilled water, 2 g of sodium nitrate, 1 g of potassium hydrogen phosphate, 0.5 g of magnesium sulfate, and 0.5 g of potassium chloride. 5. The method according to claim 4, wherein 0.5 g, ferrous sulfate 0.02 g force, fermentation solution, and keratin 1 to 5ing and peptone 0.5 to 5 g are added thereto to obtain an improved medium. 7. The dermatophyte,

(1) Art h or 0 derma uncinatum (IFO 6893),

(2) hjLcrosporum audouini CMTU 20001),

(3) hicrosporum canis (AHU 9577),

(4) Trichophyton tonsurans ClFO 5946),

(5) Trichoph ton tonsurans (IFO 5928),

(6) Trichophyton violaceum (OUT 4132),

(7) Trichophyton mentagrophy tes (IFO 5466),

(8) Trichophyton mentagrophytes (IFO 5809),

(9) Trichophyton ferrugineum ClFO 5832),

(10) Trichophyton cerebrif orme (IFO 5930),

(11) richophyton concentricum (IFO 5972),

(12) Trichophyton f aviforme (IFO 5934).

(13) Trichophyton nodof ormans (OUT 4131)

(14) Trichophyton rubrum (IFO 5467),

(15) Trichophyton rubrum (IFO 6203), and

(16) Epidermophyton f loccosum (MTU 21002)

7. The method according to claim 6, which is one strain selected from the group consisting of: The method according to claim 6, wherein the dermatophyte is Trichophyton rubrum (IFO 5467) or Trichophyton rubrum (IFO 6203). The method described. 8. The method according to claim 7, wherein the bacteria cultured in the improved medium are collected by filtration, the cells are disrupted, a cell wall fraction is collected, and chitin is extracted from the precipitate obtained by centrifuging the collected cells. . 0. The bacteria cultured in the improved medium were collected by filtration, the cells were disrupted, the cell wall fraction was collected, and chitin was extracted from the precipitate obtained by centrifugation. 8. The method according to claim 7, wherein the precipitate after washing is converted into a chitosan extract.