KR20030097166A - A method for production of Chitosan from Fungus and a method for preventing of turbid of bamboo-shoot by using thereof - Google Patents

Abstract

PURPOSE: A method for producing chitosan from Fungus and a method for preventing turbidness of a bamboo-shoot bottle using the produced chitosan are provided, thereby decreasing the amount of acid and alkali used to decrease the amount of the wastewater and wastes, and mass-producing the chitosan. CONSTITUTION: A method for producing chitosan from Fungus comprises the steps of: culturing Absidia coerulea IFO 5301 or Gongronella butleri IFO 8080 in a medium containing 2.0% of carbon source, 1.0% of organic nitrogen source, 0.5% of inorganic nitrogen source, 0.1% of phosphate source, 0.1% of potassium source, 0.1% of sodium chloride, and 0.01 to 0.05% of metal salt at pH 5.5 to 6.5 and 24 to 30 deg. C for 6 day; and culturing the medium in a fed-batch bioreactor at pH 5.5 to 6.5, 27 deg. C and 600 to 800 rpm for 36 to 48 hours; and isolating and separating chitosan from the cultured Absidia coerulea IFO 5301 or Gongronella butleri IFO 8080. A method for preventing turbidness of a bamboo-shoot bottle comprises adding 10 to 40 mg of the chitosan produced from fungus into a bottle containing boiled bamboo-shoot.

Description

A method for production of Chitosan from Fungus and a method for preventing of turbid of bamboo-shoot by using approx}

The present invention relates to a method for producing chitosan using fungi and a method for preventing clouding of late bamboo shoots using the same. More specifically, the present invention is a chitosan production method that can reduce the secondary water pollution and waste treatment by simplifying the manufacturing process by separating and extracting a large amount of fungi under optimum conditions, and a method for preventing white bamboo shoots using the same It is about.

Chitosan is a straight chain polysaccharide in which D-glucosamine is bound to β-1,4 and is naturally contained in cell walls of bacteria such as Mucor and Phycomtes . Chitosan is a biopolymer used in the production of chelating agents, paper enhancers, viscosity modifiers, adhesives, biodegradable films and fibers in flocculants and metal ions in wastewater treatment. The most widely used and studied biodissolved polymers, particularly chitosan, are flocculants of suspended solids in food processing wastewater. Chitosan is used in wastewater treatment in very wide food processing operations, including egg breakage, vegetables, shrimp, cheese, meat and apple juice processing.

Currently commercially available chitosan is usually derived from crab or shrimp shell chitin by deacetylation using strong alkalis. However, this process is a secondary source of pollution for streams and marine pollutants due to the decay of the load of raw materials, crabs and shrimp shells, the large amount of acid, alkali costs required for the production of chitin and chitosan, and the waste liquor produced during the manufacturing process. It is causing. In addition, the amount of shell waste produced from crustacean processing is gradually increasing, which is a serious problem for fish processing plants because of its very slow biodegradation, which is a serious problem for fish processing plants because of its very slow biodegradation. In order to supplement the problems in the production from such shellfish, biosynthesis of chitin and chitosan by the bio technique using microorganisms and enzymes is urgently needed.

In particular, chitosan is reported to exist in cell walls such as filamentous fungus in addition to crustaceans.The chitosan produced in this way extracts chitosan by cultivation of a large amount of fungi, and thus, processes such as deliming and deacetylation can be omitted as compared to the case of preparation from shellfish shells. Since there is an advantage of obtaining a homogeneous product, it can be said that if a strain and culture conditions containing a large amount of chitosan are found first, it can be utilized as a manufacturing method of chitosan.

Bamboo shoots have high nutritional value and have been used for a long time in medicinal use. They are rich in protein and contain minerals such as calcium, iron and phosphorus, and vitamins A and B. In particular, the composition of late bamboo shoots is 93% moisture, crude protein 2.8%, sugar 2.4%, fiber 0.9%, fat 0.2%, ash 0.7%, amino acid content of about 1600mg (/ 100g bamboo shoot), among which tyrosine and asparagine , Valine and glutamic acid make up about 70% of the total content. In addition, bamboo shoots are relatively high in fiber separated by the sixth nutrient, which is effective in promoting intestinal exercise function, relieving constipation, and eliminating stool, so that the value added if developed as a health food or traditional processed food using the same.

Bamboo shoots are easily decayed at room temperature and have difficulty in storage. In addition, even if stored at low temperatures, the hardening of the soft bamboo shoots, such as hard tissue changes occurs, not only become inadequate for processing, but also due to the decomposition of nutrients loss and intrinsic umami taste is reduced. Therefore, the development of processed foods using this is urgently required.

In view of the above, the present inventors selected chitosan-producing strains to investigate the optimum culture conditions, and the cells cultured under the optimum conditions were washed with ethanol overnight after washing with water several times and then lyophilized to produce dry cells. Alkali-insoluble materials (AIM) were obtained by alkali extraction of the dried cells, and after acid treatment, the pH was adjusted to 8.5 and left at 4 ° C. for 12 hours, followed by filtration and washing with water. After lyophilization to produce fungal chitosan (FCS), the fungus chitosan was added to the late bamboo shoots to investigate the anti-clouding effect was completed.

Accordingly, an object of the present invention is to provide a chitosan production method that can simplify the production process and reduce secondary water pollution and waste treatment by cultivating and extracting the fungus in large quantities under optimum conditions.

Another object of the present invention is to provide a method of preventing cloudy turbidity of late bamboo shoots using fungus chitosan produced using fungus.

The object of the present invention is to select the chitosan-producing strains to investigate the optimum culture conditions, and the cells cultured under the optimum conditions after washing with ethanol overnight after washing several times and then lyophilized to produce dried cells, the drying Alkali-insoluble materials (AIM) were obtained by alkali extraction of the cells, and after acid treatment, the pH was adjusted to 8.5 and left at 4 ° C. for 12 hours. The precipitate was filtered, washed with water, neutralized, and lyophilized. It was achieved by producing fungal chitosan (FCS) and adding the fungus chitosan to late bamboo shoots to investigate the anti-clouding effect.

Hereinafter, the configuration of the present invention.

1 is a schematic configuration diagram of a batch bioreactor used in the production of chitosan using the fungus of the present invention.

Figure 2 shows the dry cell weight according to the initial pH of Absidia coerulea IFO 5301, Gongronella butleri IFO 8080, Mucor tuberculisporus IFO 9256 and Rhizopus delemer IFO 4775 strain.

Figure 3 shows the results of alkali insolubles according to the initial pH of Absidia coerulea IFO 5301, Gongronella butleri IFO 8080, Mucor tuberculisporus IFO 9256 and Rhizopus delemer IFO 4775 strain.

Figure 4 shows the results of fungal chitosan according to the initial pH of Absidia coerulea IFO 5301, Gongronella butleri IFO 8080, Mucor tuberculisporus IFO 9256 and Rhizopus delemer IFO 4775 strain.

5 shows the effect of temperature on dry cell weight production by Absidia coerulea IFO 5301.

Figure 6 shows the effect of incubation time on dry cell weight production by Absidia coerulea IFO 5301.

7 is a flowchart briefly illustrating a process of separating and extracting the fungus chitosan of the present invention.

8 is a flow chart briefly illustrating a process of producing a late bamboo shoot bottle of the present invention.

9 is a graph showing the cloudiness prevention effect of late bamboo shoots according to the concentration of the fungus chitosan.

The present invention comprises the steps of preparing a chitosan producing strain; Culturing the chitosan producing strain under optimal conditions; Separating and extracting the fungus chitosan from the cells obtained in the step and lyophilizing; Preparing a late bamboo shoot bottle; The fungus chitosan obtained in the above step is added to the late bamboo shoot blight consisting of the step of investigating the anti-clouding effect.

The standard chitosan used in the present invention was used by grinding C-3646 provided from the US SIGMA company into fine powder (200mesh) as needed.

Hereinafter, the specific method of the present invention will be described step by step with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Preparation of Chitosan Producing Strains

The four strains used in this experiment were distributed as chitosan producing strains such as Gongronella butleri IFO 8080, Absidia coerulea IFO 5301, Rhizopus delemer IFO 4775, and Mucor tuberculisporus IFO 9256.

For preservation of the strains were subcultured every 30 days in PDA medium and stored at 4 ℃ (Table 1).

Medium composition for slant culture (g / ℓ) Composition Contents Potato Powder 200.0 Destros 20.0 Agar 15.0

Example 2: Chitosan Production Strain Culture

Stage 1: Preculture

In pre-culture, 39 g of PDA and 3 g of yeast extract were dissolved in 1,000 ml of deionized water, and each 10 ml was dispensed into each test tube, and then inoculated in PDA slope medium prepared by autoclaving at 121 ° C. for 15 minutes in an incubator at 27 ° C. After 7 to 9 days of incubation, it was again stored in a refrigerator at 4 ℃ or used in the main culture.

Step 2: Main Culture

Medium composition of chitosan producing strains was performed according to the method of Rane (Rane, KD, and DG Hoover. 1993.), glucose 20g, peptone 10g, yeast extract 1.0g, ( 5.0 g of NH ₄ ) ₂ SO ₄ , 1.0 g of K ₂ HPO _4, 1.0 g of NaCl, 0.5 g of MgSO ₄ · 7H ₂ O and 0.1 g of CaCl ₂ · 2H ₂ O were dissolved in 1,000 ml of distilled water and the initial pH was adjusted to 1N-HCl. It was adjusted to 5.0 and 200 ml of medium was dispensed into a 500 ml flat bottom flask, and the culture in the bioreactor was added with 3 liter of liquid medium in a 5 liter reaction tank, followed by autoclaving at 121 ° C. for 15 minutes. After cooling to room temperature, the initial pH was adjusted with 1N HCl, and the main culture was inoculated with 2 ml of the suspension.

In the flask culture of the fungus chitosan producing strain, a rotary shaker (C-SKI-2, Hanyoung Machinery) was used, and the amplitude was 7 cm and the rotation speed was 130 rpm.

The bioreactor used a batch bioreactor (KF-25L; Korea fermenter Co., Ltd.) (Fig. 1).

Experimental Example 1: Investigation of the influence of the media components

1. Influence of carbon source

In the initial pH 6.5, 130 rpm, 27 ℃, 6 days incubation, the concentration of glucose, the carbon source of A. coerulea IFO 5301 at 0.5%, 1%, 2.0% experiments showed the highest yield at 2.0%.

2. Influence of Organic Nitrogen Sources

In the initial pH 6.5, 130 rpm, 27 ℃, 6 days cultivation of the concentration of peptone (0.3%, 0.7%, 1.0%) of the nitrogen source of A. coerulea IFO 5301 showed the highest yield at 1.0% It was.

3. Influence of inorganic nitrogen sources

Inorganic Nitrogen Source (NH ₄ ) ₂ HPO ₄ , NaNO ₃ , NH ₄ Cl, NH ₄ H ₂ PO ₄ , NH ₄ NO ₃ was added at 0.5% concentration in the medium for A. coerulea IFO 5301. After adjusting to, and incubated at 130 rpm, 27 ℃ for 6 days to check the cell mass, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) showed the highest yield.

4. Effect of Ammonium Sulfate Concentration

The amount of ammonium sulfate, which was the most cell production as a nitrogen source in chitosan producing medium, was added to each concentration of 0.1% to 0.5% of the medium, and the cell mass obtained by incubating at 27 ° C for 6 days was examined. The highest yield was obtained when added.

5. Effect of Phosphoric Acid and Potassium Source Concentration

Cause chitosan phosphate and potassium in the composition of the production medium K ₂ HPO ₄ and KH ₂ PO ₄ was added to 0.1~0.3% in concentration of each culture medium 6 days at 27 ℃ A. coerulea IFO result the cell yield obtained by culturing the immature birth 5301 Phosphoric acid and potassium sources showed higher yields of K ₂ HPO ₄ than KH ₂ PO ₄ , and showed the highest yield when the concentration of medium was 0.1%.

6. Effect of sodium chloride concentration

The cell mass obtained by adding 0.1-0.3% sodium chloride to the medium of A. coerulea IFO 5301 was incubated for 6 days at 27 ° C. As a result, when the 0.1% of the medium was added compared to the control group without adding sodium chloride, The highest. However, when added more, the cell yield decreased.

7. Effect of metal salt concentration

Metal salts MgSO ₄ · 7H ₂ O, FeCl ₃ · 6H ₂ O, MnCl ₂ · 4H ₂ O, CaCl ₂ · 2H ₂ O were added to chitosan production medium at a concentration of 0.01 to 0.05%, respectively, to A. coerulea IFO 5301 strain. and then, 27 ℃, at 130 rpm 6 ilgan research gyuncheryang obtained by culturing a result, CaCl ₂ · 2H ₂ O and MgSO ₄ · cell yield from 0.01% and 0.05% concentration of each medium in case of 7H ₂ O inoculated with the High.

The yield of haye cell mass was higher when two metal salts were incubated together than when CaCl ₂ · 2H ₂ O and MgSO ₄ · 7H ₂ O were added alone.

Experimental Example 2 Optimal Initial pH Irradiation

The results of DCW, AIM and FCs of four strains Absidia coerulea IFO 5301, Gongronella butleri IFO 8080, Rhizopus delemar IFO 4775, Mucor tuberculisporus IFO 9256 according to the initial pH of chitosan production medium are shown in Table 2 and FIGS. 2, 3 and 4. .

Yield of DCW, AIM and FCs from Culture Strains at Initial pH Initial pH Strain DCW ¹⁾ mg / 200 mL AIM ²⁾ mg / 200 mL FCs ³⁾ mg / 200 mL 4.5 Absidia coerulea IFO5301 1,238 495 211 Gongronella butleri IFO8080 1,666 446 128 Mucor tuberculisporus IFO9256 1,254 315 16 Rhizopus delemar IFO4775 816 174 14 5.0 Absidia coerulea IFO5301 1,264 506 215 Gongronella butleri IFO8080 1,935 518 148 Muco tuberculisporus IFO9256 968 243 13 Rhizopus delemar IFO4775 530 113 9 5.5 Absidia coerulea IFO5301 1,226 490 209 Gongronella butleri IFO8080 2,044 547 157 Muco tuberculisporus IFO9256 1,128 283 15 Rhizopus delemar IFO4775 580 124 10 6.0 Absidia coerulea IFO5301 1,204 482 205 Gongronella butleri IFO8080 1,726 462 132 Muco tuberculisporus IFO9256 718 180 9 Rhizopus delemar IFO4775 536 114 9 6.5 Absidia coerulea IFO5301 1,514 606 258 Gongronella butleri IFO8080 1,814 485 139 Muco tuberculisporus IFO9256 1,055 265 14 Rhizopus delemar IFO4775 631 135 11

NOTE 1) Dry cell weight;

Fermentation at 27.0 ° C. for 5 days with stirring at 130 rpm

2) Alkali insoluble materials;

Treated with 1N NaOH at 121 ° C. for 15 minutes

3) fungal chitosan;

Treated with 2% acetic acid for 12 hours at 95

Among chitosan producing strains, Absidia coerulea IFO 5301 and Gongronella butleri IFO 8080 showed the highest yield of hay cell according to initial pH. Absidia coerulea IFO 5301 had a dry cell mass of 1,514 mg at initial pH 6.5 and Gongronella butleri IFO 8080 at an initial pH of 5.5. The dry cell mass at 2,044 mg in 200 ml chitosan production medium was the highest (Fig. 2).

However, the yield of alkali-insoluble substance, which is an intermediate of fungal chitosan according to the initial pH, showed the highest yield among four strains with Absidia coerulea IFO 5301 at the initial pH of 6.5 mg / 200ml, followed by Gongronella butleri IFO 8080. 547 mg / 200 ml at an initial pH of 5.5.

The production of the fungus chitosan was 205-258 mg / 200 ml for Absidia coerulea IFO 5301, which was the highest yield at initial pH 6.5.

Absidia coerulea IFO 5301 produced chitosan better than other strains in the initial pH condition experiments.

Experimental Example 3 investigation of the optimum culture temperature

In the initial pH experiment, Absidia coerulea IFO 5301, which has the highest yield of chitosan, was adjusted to initial pH 6.5 with 1N HCl, and then the culture temperature was changed to 24 ° C, 27 ° C and 30 ° C in order to examine the effect of cell production according to the culture temperature. Was shaken at 130 rpm for 5 days.

Absidia coerulea IFO 5301 produced the most cells at 27 ° C. (FIG. 5).

Experimental Example 4 investigation of the optimum incubation time

2ml of Absidia coerulea IFO 5301 was inoculated into a flask containing 200ml of chitosan production medium with 1N HCl, adjusted to an initial pH of 6.5, and then incubated at an optimum incubation temperature of 27 ℃ and 130rpm in a shaker. Incubated for days, 5 days, 6 days, and 7 days. As a result, the highest yield was obtained at 6 days (FIG. 6).

Experimental Example 5: Investigation of the operating conditions of the batch bioreactor

1. Effect of Bacterial Concentration

Optimal culture conditions of Absidia coerulea IFO 5301 found in the flask culture were applied to a batch bioreactor to produce cells. Operation of the batch bioreactor was performed with air inflow rate of 1.0ℓ / min, agitation speed 600rpm, incubation time 24 hours, medium volume 3ℓ. As a result of checking the cell yield according to the concentration of the suspension, the cell yield was markedly increased up to 70ml. There was little change.

2. Effect of Stirring Speed

In the batch bioreactor, the cell yields of Absidia coerulea IFO 5301 strains were varied at 400 rpm, 600 rpm, 800 rpm, and 1,000 rpm in the batch bioreactor. The yield was highest at 800 rpm.

3. Effect of air inflow

The amount of air introduced into the batch bioreactor was varied from 0.5 to 2.0 l / min, and the yield was the highest at 0.5 l / min.

4. Effect of incubation time

In a batch bioreactor of Absidia coerulea IFO 5301, 70 ml of the suspension was inoculated into a reactor containing 3 liters of chitosan production medium at an incubation temperature of 27 ° C and an initial pH of 6.5. Cells were produced by adjusting the incubation time to 24 hours, 36 hours, and 48 hours at 800 rpm. As a result, the highest yield was obtained when incubated for 36 hours.

As a result of the experiment, it was confirmed that the optimum operating conditions in the production of the cells in the batch bioreactor as shown in Table 3.

Bioreactor Operating Conditions Kinds Optimal condition Seed culture volume 70 mL Medium volume 3L Aeration 0.5 L / min Agitation 600 to 800 rpm Temperature 27 ℃ Initial pH 5.5 to 6.5 Fermentation time 36-48 hours

Example 3 Separation of Alkali Insolubles and Fungus Chitosan

Chitosan was extracted from the cell walls of fungi according to the methods of Kobayashi (Kobayashi, T., Takkiguchi, Y., Shimahara, K. and Sannan, T. 1988.) and Rane (Rane, K.D. and Hoover, D. G. 1993). The cell mass produced in the flask and the bioreactor was washed with water, filtered through a G3 glass filter, treated with ethanol overnight, washed several times, and lyophilized to obtain DCW. Alkali treatment was performed to separate skeletal components such as proteins, which are components of cell walls of cells, and 1N aOH was added at a ratio of 1:40 (w / v) to the cell weight, followed by reaction at 121 ° C. for 15 minutes. After repeated washing with water to neutralize the alkali treatment, it was dehydrated and lyophilized to obtain AIM.

In addition to chitosan, alkali insolubles were also present, so that chitosan was dissolved in dilute acetic acid to separate chitosan. An alkali insoluble substance was added to the flask in an appropriate amount, and then 2% acetic acid was added in a ratio of 1:50 (w / v). The flask was equipped with a cooling tube, a thermometer, and a stirrer to react at 95 ° C. and 40 ° C. for 12 hours in an oil bath. After acid treatment, the supernatant was recovered by centrifugation at 10,000 rpm for 20 minutes to separate the insoluble and dissolved substances, and the supernatant was recovered by the second and third acid treatment again. The collected supernatant was filtered through a G3 glass filter, and the filtered solution was adjusted to pH 8.5 using a concentrated NaOH solution and left at 4 ° C. for 12 hours to precipitate chitosan. The precipitate was centrifuged and washed with distilled water and neutralized repeatedly to freeze-dry to obtain an FCS (FIG. 7).

Experimental Example 6: Analysis of General Components and Amino Acids of the Fungus Chitosan

a. The dry cells were recovered from the A. coerulea IFO5301 strain to obtain an alkali insoluble substance by alkali treatment, and acid-treated therefrom to produce fungal chitosan. The yield of fungus chitosan was slightly higher at high temperature than at low temperature, but the color was slightly brown compared to the low temperature treatment. The fungus chitosan prepared by treating 2% acetic acid at 40 ° C. for 12 hours with an alkali insoluble substance was treated with FCs-40 and 12 hours at 95 ° C. as an FCs-95.

Chemical Constituents of the Fungus Chitosan Ingredient Fungus Chitosan FCs-40 FCs-95 moisture(%) 6.57 6.58 Ash content (%) 0.25 0.22 Fat(%) 0.32 0.30 nitrogen(%) 6.91 6.71 yield(%) 15.64 16.10 color Creamy-white Brown Amino acid (mg / g) 1.36 1.59

Experimental Example 7 Investigation of Physicochemical Properties of Fungus Chitosan

Table 5 shows the results of analyzing physicochemical properties such as solubility, viscosity, acetylation and molecular weight of the two fungus chitosan.

Physicochemical Properties of Fungus Chitosan Physicochemical Properties Fungus Chitosan FCs-40 FCs-95 Solubility (%) 99.13 99.05 Viscosity (cps) ¹⁾ 2.21 2.23 Acetylation degree (%) ²⁾ 12.7 12.0 Daltons ³⁾ 3.01 × 10 ⁵ 3.12 × 10 ⁵

[week]

1) Measured as 1% solution (using 2% acetic acid as solvent) at 25 ° C

2) measured by the infrared adsorption spectrum

3) intrinsic viscosity method

Experimental Example 8: I.R. of the fungus chitosan. And NMR spectral analysis

The chemical structural formula of the fungus chitosan of the present invention was compared with that of standard chitosan using IR and NMR spectra, and the absorption bands on the IR spectrum were almost identical and the NMR spectrum was also almost identical.

Example 4 Preparation of Late Bamboo Shoot Bottles

Late bamboo shoots were produced in mid-May to mid-June in Hadong, Gyeongnam. Purified salt was used as a solution by dissolving Hanjugeum with purity over 99% and chitosan in 1% acetic acid.

The late bamboo shoots were washed, trimmed and peeled, and then blotted in an appropriate concentration of salt solution. The mixture was quenched in running water and refined, and soaked in water for 30 minutes to remove tyrosine, the causative agent of turbidity. 100 g of the blanched late bamboo shoots were screened, placed in a 225 ml glass bottle, poured with 98 ° C. hot water, degassed for 10 minutes, sterilized for 60 minutes in a 105 ° C. dryer, and cooled at room temperature. The lid was sealed with a sealing film (sealing film) to prepare a late bamboo shoot bottle (Fig. 8).

Experimental Example 9: Investigation of turbidity effect according to chitosan concentration

In order to prevent this, the salt concentrations used during the manufacturing process of late bamboo shoots were changed to 1%, 2%, and 3% in order to prevent this. After 60 minutes of blending, the effect of the whitening on storage was observed. As a result, when the salt concentration was 1%, the haze was the lowest. Therefore, the experiment was performed with a salt concentration of 1%. In addition, by selecting the conditions with the best anti-clouding effect according to the blending and adding fungal chitosan by concentrations of 10mg / 225ml, 20mg / 225ml and 40mg / 225ml, respectively, to find out the anti-clouding effect according to room temperature storage. In order to measure the turbidity by taking a sample of the liquid at regular intervals. The control group was made by blending late bamboo shoots for 60 minutes at 1% brine concentration.

As a result of the experiment, the effect of turbidity according to the concentration of fungus chitosan was shown in the order of 40mg / 225ml, 20mg / 225ml, 10mg / 225ml. Rather, it tended to decrease due to the aggregation effect of the fungus chitosan (FIG. 9). Therefore, as can be seen from the above results, it was found that the shelf life can be improved by adding the fungus chitosan to the food manufacturing process.

As described above, the chitosan production method using the fungus of the present invention, as described in the above Examples and Experimental Examples, has a simple manufacturing process and reduces the amount of acid and alkali required during the manufacturing process as compared to the conventional chitosan production method from shellfish. It is possible to reduce secondary environmental pollution by reducing waste and waste generated during the manufacturing process, as well as to produce a large amount of fungus chitosan, and to use the produced chitosan to prevent clouding of late bamboo shoots. will be.

Claims (2)

Absidia coerulea IFO 5301 strain or Gongronella butleri IFO 8080 strain in a medium containing 2.0% carbon source, 1.0% organic nitrogen source, 0.5% inorganic nitrogen source, 0.1% phosphoric acid source, 0.1% potassium source, 0.1% sodium chloride, 0.01-0.05% metal salt After the initial pH was fixed at 5.5 to 6.5 and the temperature was maintained at 24 to 30 ° C. for 6 days, the initial pH was fixed at 5.5 to 6.5 and the temperature was maintained at 27 ° C. at a speed of 600 to 800 rpm in a batch bioreactor. A method for producing chitosan, characterized by producing fungus chitosan by separating and extracting the cells obtained after culturing for 36 to 48 hours with stirring. After rinsing the late bamboo shoots, trimming, peel off the skin, bleeding with salt solution, quench in running water and trim again, soak in water for 30 minutes, and then use 100 g of the prepared blanched bamboo shoots as described above in a 225 ml glass bottle. Pour into hot water at 98 ° C, degassed for 10 minutes, sterilize for 60 minutes in a dryer at 105 ° C, cool at room temperature and seal the lid with a sealing film to make late bamboo shoots. Absidia coerulea IFO 5301 strain or Gongronella butleri IFO 8080 strain in a medium containing 2.0% carbon source, 1.0% organic nitrogen source, 0.5% inorganic nitrogen source, 0.1% phosphoric acid source, 0.1% potassium source, 0.1% sodium chloride, 0.01-0.05% metal salt After the initial pH was fixed at 5.5 to 6.5 and the temperature was maintained at 24 to 30 ° C. for 6 days, the initial pH was fixed at 5.5 to 6.5 and the temperature was maintained at 27 ° C. at a speed of 600 to 800 rpm in a batch bioreactor. A method of preventing late bamboo shoot blighting, characterized in that 10 to 40 mg of the fungus chitosan prepared by separating and extracting the cells obtained after culturing for 36 to 48 hours with stirring.