JP2005137225A - Chitin and chitosan derived from dermatophyte - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide chitin and chitosan in which a specific function is enhanced. <P>SOLUTION: Chitin or chitosan is obtained from dermatophyte. A biological material comprises the chitin or the chitosan. A method for culturing the dermatophyte is also provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to chitin or chitosan obtained from dermatophytes, biomaterials containing the same, and a method for culturing dermatophytes.

  In recent years, effective use of chitin and chitosan has been applied to various fields such as artificial skin as a biomaterial, preservatives, molding agents, soil conditioners, wastewater treatment agents for various foods. In particular, in the field of artificial biomaterials, functional roles such as in vivo digestibility, immunoadjuvant activity, lymphocyte cell separation activity, drug release function, anticoagulant activity, and antibacterial activity of this substance have been recognized. Application in the field can be considered. However, stable supply of chitin / chitosan as a material pursuing individual functionality is not considered.

Sources of chitin and chitosan are known as components of lower animals such as crustaceans, insects, shellfish and fungi, and the outer skeleton and supporting tissues of filamentous fungi. To date, chitin and chitosan as natural resources are not very demanding, and effective use as unused resources has not been considered. However, as the biological and chemical properties of this substance become clear, the demand for its functional polymer material has increased. Ingredients to meet these requirements include crabs and insects such as maggots and some filamentous fungi, especially fungi, such as the genus Mucor, which have been produced by fermentation. I started. However, as mentioned earlier, there are no reports regarding the source of this substance with improved functionality.
Takayuki Motoyama, Hiroyuki Horiuchi, Masamichi Takagi: Chemistry and Biology, Vol.31, No.12, 807-816 (1993) Yoshio Oshima, Kenichi Nishino, Yoshio Yonekura, Motoaki Maeda, Junko Horie, Kyoko Nonomura, Saburo Kishimoto, Shunji Wakabayashi, Shigetaro Somatsu: Burns, 12, 31 (1986)

  The present invention has been made under the above technical background, and an object of the present invention is to provide chitin and chitosan having enhanced specific functions among various functions of chitin and chitosan.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that chitin and chitosan obtained from dermatophytes are more antibacterial, biodegradable, skin than chitin and chitosan derived from other organisms. It was found that the adhesiveness was excellent. In addition, the inventors have found that culturing dermatophytes under specific conditions increases the chitin content in the cell wall, and based on these findings, the present invention has been completed.

  That is, the present invention is chitin or chitosan obtained from dermatophytes.

  Moreover, this invention is a biomaterial containing said chitin or chitosan.

  Furthermore, the present invention provides a method for cultivating dermatophytes, comprising culturing dermatophytes in a medium containing keratin and peptone while supplying a mixed gas containing nitrogen and carbon dioxide into the medium. is there.

  Hereinafter, the present invention will be described in detail.

  The chitin and chitosan of the present invention are obtained from dermatophytes.

Dermatophytes are filamentous fungi that have an affinity for the skin of various animals and cause various skin diseases in the head, body, feet, etc., for example, the genus Trichophyton ( Trichophyton ), Epidermophyton ( It is a filamentous fungus belonging to the genus Epidermophyton ) and Microsporum . Dermatophytes that can be used in the present invention are not particularly limited, and examples thereof include Microsporum audouini , Microsporum canis , Trichophyton tonsarans , Trichophyton violaceum ( Trichophyton violaceum). ), Trichophyton mentagrophytes (Trichophyton mentagrophytes), Trichophyton Ferugineumu (Trichophyton ferrugineum), Trichophyton Sereburihorume (Trichophyton cerebriforme), Trichophyton outlet Li cam (Trichophyton concentricum), Trichophyton Fabihorume (Trichophyton faviforme), Trichophyton Nodohorumansu (Trichophyton nodoformans), Trichophyton rubrum (Trichophyton rubrum), epi Epidermophyton phytone-Furokkosamu (Epdermophyton fl occosum ) etc. can be used. Among these dermatophytes, Trichophyton rubrum is preferably used.

  The chitin or chitosan of this invention can be obtained by the general method employ | adopted when obtaining chitin or chitosan from a filamentous fungus. That is, chitin can be obtained by crushing the cells, collecting the cell wall fraction, treating it with acid and alkali, and collecting the insoluble matter. Chitosan can be obtained by treating this chitin with a strong alkali.

  The chitin and chitosan of the present invention have properties such as antibacterial properties, biodegradability and absorbability, and skin adhesion, and therefore can be used as biomaterials such as artificial skin and artificial organs. In addition to biomaterials, fibers, films, metal refining carriers, moisturizers, plant growth regulators, deodorizers, fixing agents, paper antibacterial agents, fiber antibacterial agents, pharmaceutical / agrochemical / nutrient delivery carriers, Controlled release carriers, antiviral agents, anticholesterol agents, emulsifying / hygroscopic agents, antithrombotic / blood coagulation inhibitors, cell immunity enhancing agents, metal / plastic surface strengthening agents, food additives, cosmetics, high-performance composite materials, It can also be used as a dye enhancer.

  Dermatophytes may be cultivated by a normal culturing method, but the culturing method of the present invention, that is, a method of culturing while supplying a mixed gas containing nitrogen and carbon dioxide in a medium containing keratin and peptone. Is preferably cultured. Such a culture method increases the chitin content in the cells.

  The amount of keratin contained in the medium is not particularly limited, but is preferably 1 to 5 mg / l. The amount of peptone is not particularly limited, but is preferably 0.5 to 5 g / l. Medium components other than keratin and peptone may be the same as those generally used for culturing filamentous fungi.

  The ratio of nitrogen and carbon dioxide in the mixed gas is not particularly limited, but is preferably carbon dioxide: nitrogen = 10-50%: 50-90%.

  Conditions such as the culture temperature and the culture period may be general conditions employed when obtaining chitin from filamentous fungi. Usually, the culture temperature is preferably 25 to 35 ° C. and the culture period is preferably 15 to 30 hours.

  The culture method of the present invention can increase the chitin content in dermatophytes. Chitin and chitosan obtained from dermatophytes exhibit extremely properties such as antibacterial properties, biodegradability, and skin adhesion, and thus are extremely useful as biomaterials such as artificial skin.

  Hereinafter, the present invention will be described in more detail with reference to examples.

EXAMPLE 1 Various dermatophytes production of chitin by Mucor (Mucor) genus, Rhizopus (Rhizopus) genus, the chitin production filamentous fungi known from Aspergillus (Aspergillus) genus such as a conventional, chitin content in the cell walls 20 On the other hand, in dermatophytes, the chitin content in the cell wall is as low as about 10 to 15%. Therefore, in order to increase the chitin content of dermatophytes, the culture conditions were examined.

First of all, Microsporum audouini (MTU 20001), Microsporum canis (AHU 9577), Trichophyton tonsarans (IFO 5945), Trichophyton tonsurans (IFO 5928), Trichophyton violaceum (OUT 4132), Trichophyton mentagrophytes (IFO 5466,5809), Trichophyton ferrugineum (IFO 5832), Trichophyton cerebriforme (IFO 5930), Trichophyton concentricum (IFO 5972), Trichophyton faviforme (IFO 5934), Trichophyton nodoformans (OUT 4131), Trichophyton rubrum (IFO 5467,6203), Epdermophyton floccosum (MTU 21002 The strain was precultured. These strains are: Fermentation Research Institute (IFO), Osaka University Graduate School of Engineering, Department of Applied Biotechnology (OUT), University of Tokyo School of Medicine (MTU), Hokkaido University Graduate School of Agriculture, Department of Applied Biotechnology ) Can be obtained easily from public conservation agencies. Pre-culture is basic medium A (contains 25g glucose, 2g sodium nitrate, 1g potassium hydrogen phosphate, 0.5g magnesium sulfate, 0.5g potassium chloride, 0.02g ferrous sulfate per liter distilled water), 27 This was carried out by shaking culture at 20 ° C. for 20 hours.

The precultured strain was cultured in the improved medium B at 25 ° C. for 30 hours with shaking. At this time, a mixed gas of carbon dioxide and nitrogen (CO ₂ 10-50% and N ₂ 50-90%) was bubbled in the medium. In the improved medium B, 3 mg of keratin (manufactured by Sigma) and 1 g of peptone (Eiken Chemical) are added to the basic medium A, adjusted to pH 6.0, and sterilized (110 ° C., 1 atm, 10 minutes). Prepared. The cultured bacteria were collected by filtration, washed twice with a phosphate buffer (pH 6.0), rapidly frozen with liquid nitrogen, and disrupted. The disrupted cells were centrifuged at 12,000 rpm, the cell wall fractions were collected, and the chitin content was measured. As a control, the precultured strain was shake-cultured again for 30 hours under the same conditions as in the preculture, and the chitin content of the cell wall fraction of the cells was measured in the same manner.

  Chitin extraction from the cells was performed as follows. First, 100 ml of 10% NaOH solution was added to 1 g of the cell wall fraction, and the mixture was stirred at 25 ° C. for 48 hours. The precipitate was collected by centrifugation, washed twice with distilled water, treated with 5% acetic acid, and again washed twice with distilled water. Next, the precipitate was degreased with 95% ethanol, dehydrated and dried to obtain chitin.

  The chitin content was calculated from the value obtained by converting chitin into chitosan and then decomposing it into glucosamine, measuring the glucosamine content by the Elson Morgan method. Conversion from chitin to chitosan was carried out as follows. To 0.5 g chitin sample, 4 ml of 3% sodium lauryl sulfate solution (wt / vol) was added, treated at 100 ° C. for 15 minutes, cooled and centrifuged at 3,000 rpm. The precipitate was washed with distilled water, centrifuged again under the same conditions, and the precipitate was treated with 3 ml of KOH solution (diluted in 120 g with 100 ml of distilled water) at 130 ° C. for 1 hour. After cooling, it was centrifuged at 3,000 rpm, the precipitate was washed twice with distilled water, and this precipitate was used as a chitosan extract. The chitosan extract was treated with 6N hydrochloric acid at 110 ° C. for 14 hours in a glass tube sealed with nitrogen gas, and then neutralized with 6 M NaOH solution.

  Table 1 shows the chitin content in the cell wall of the cells cultured by the culture method of the present invention (culture method 2).

As shown in Table 1, the chitin content in the cell walls of dermatophytes was greatly increased by the culture method of the present invention. In particular, the increase in Trichophyton rubrum was significant.

[Example 2] Antibacterial activity of chitosan derived from dermatophytes against various microorganisms It is a well-known fact that chitin and chitosan exhibit antibacterial activity against various microorganisms. However, the antibacterial activity of crustacean chitin and chitosan is known in academic reports, patents, etc. in the reports to date, but there are no microorganisms, especially dermatophyte-derived chitin and chitosan among the fungi. . Therefore, we tried to compare the antibacterial properties of various bacteria, yeast and fungi of crab chitosan and Trichophyton rubrum chitin.

A peptone medium or Sabouraud medium (both manufactured by Kyokuto Pharmaceutical Co., Ltd.) to which a dilute acetic acid (2M) solution of chitosan was added was poured into a sterile small test tube to prepare a test medium. This test medium was inoculated with 3-5 × 10 ⁷ / ml of bacteria, yeast, or dermatophytes pre-cultured in peptone or Sabouraud medium without chitosan. Bacteria were cultured for 18 hours, and fungi were cultured for 48 hours, and then the absorbance (OD 620 nm) was measured. From this absorbance and the absorbance when chitosan was not added, the growth inhibition rate (%) was calculated to evaluate the antibacterial properties of chitosan.

As chitosan, chitosan derived from crab (manufactured by Sigma) and chitosan derived from Trichophyton rubrum (IFO 5467) (extracted in Example 1) were used. The amount of chitosan added was such that the final concentration of chitosan in the medium was 2, 4, 6, 8, or 9 mg / ml.

Antibacterial activity, Staphylococcus aureus (FDA 209P), Staphylococcus epidermidis (ATCC 14990), Streptococcus pyogenes (IID 0688), Enterococcus facecium (IFO 3128), Eschericha coli (NIHJ JL-2), Klebsiella pneumoniae (IID 865), Serratia marusucens (IFO 12648), Salonella typhimurium (IID 971), Pseudomonas aeruginosa (IAM 1095), Haemophillus influenzae (IID 983) 10 bacteria, yeast Candida albicans (ATCC 10261), dermatophyte Microsporum canis ( AHV 9577)
Table 2 shows the growth inhibition rates of crab-derived chitosan and Trichophyton rubrum (IFO 5467) -derived chitosan against various microorganisms.

As shown in Table 2, Trichophyton rubrum- derived chitosan had higher antibacterial properties against various microorganisms than crab-derived chitosan. In particular, the antibacterial activity against gram-negative bacteria and fungi was significantly higher than crab-derived chitin.

This antibacterial test was conducted according to the method of Muzzarelli et al (Muzzarelli et al: Antimicrobial properties of N-carboxylbutyl chitosan. Antimicrobial Agents and Chemotherapy. Vol 34, No 10, p 2019-2023. 1990.). is there.

[Example 3] Degradability and antigenicity of chitin derived from dermatophytes in vivo Biodegradability and low antigenicity are important factors for selecting a material for a biomaterial. This is required not only for the safety of the raw material to the living body but also for the selection of the medical biomaterial such that the degradation product is non-toxic and can be decomposed and absorbed at an appropriate time. Therefore, we attempted to compare the degradability and antigenicity of dermatophyte-derived chitin and crab-derived chitin in vivo.

  Using a dissecting scissors, a 1 cm long incision was made subcutaneously along the back of the guinea pig, and a 3 mm × 3 mm chitin section was embedded therein and sutured. 2, 4, 6, 8, or 10 weeks after the implantation of the chitin section, the back of the guinea pig was dissected again, and the degradation of the chitin section and the inflammatory reaction of the back of the guinea pig were observed. All operations were performed aseptically.

Chitin used was chitin derived from Trichophyton rubrum (IFO 5467) and chitin derived from crab (manufactured by sigma).

  The guinea pigs used a total of 8 Hartley white female guinea pigs (Claire Japan), which were purchased one week before the experiment. The average body weight of the five animals was 269 ± 9.3 g, and was fed with solid feed (CLEA Japan) in a breeding environment with a temperature of 22.5 ° C. ± 0.3 ° C. and a humidity of 65 ± 5%.

  Table 3 shows the degradability and inflammatory reaction of each chitin section in the guinea pig subcutaneous part.

As shown in Table 3, degradation was observed in the chitin derived from Trichophyton rubrum earlier than the chitin derived from crab. Moreover, there was no difference between the two in respect of the inflammatory reaction in vivo.

[Example 4] Adhesion of dermatophytes and crab-derived chitin to rat and guinea pig skin parts Commercially available snow crab-derived chitin film (manufactured by Kanto Chemical Co.) and Trichophyton rubrum (IFO 5467) -derived chitin film (extracted in Example 1) ) Was used to compare the adhesion and dryness of rats and guinea pigs to the back skin.

  The hair on the back of guinea pigs and rats is cut with an electric clipper, and after 6 hours of hair removal, the skin surface is cut into 1.5 mm squares with a dissecting scissors, each chitin membrane is applied, and it is closed with 2 x 2 cm square filter paper. did. After 1 to 2 weeks and 3 weeks from the application of the chitin film, the adhesion of the chitin film to the skin and the dry state of the skin on which the chitin film was applied were observed.

  Rats were 4-week-old female Wister rats, and guinea pigs were 4-week-old female Hartley white guinea pigs. Animals were raised under the same conditions as in Example 3. At the start of the experiment, the average body weight of the rats was 120 ± 9.2 g, and the average weight of the guinea pigs was 235 ± 14.6 g. Each animal used was used in an experiment as a group of 6 animals.

  Table 4 shows the evaluation results of the adhesion and drying properties of each chitin film.

As shown in Table 4, with respect to the adhesion to the skin part and dryness, the Trichophyton rubrum- derived chitin had a remarkable effect earlier than the crab-derived chitin.

Claims (4)

  Chitin or chitosan obtained from dermatophytes.   The chitin or chitosan according to claim 1, wherein the dermatophyte is a microorganism belonging to the genus Trichophyton, Epidermophyton, or Microsporum.   A biomaterial containing the chitin or chitosan according to claim 1 or 2.   A method for cultivating dermatophytes, comprising culturing dermatophytes in a medium containing keratin and peptone while supplying a mixed gas containing nitrogen and carbon dioxide into the medium.