Chitin preparation method based on microbial fermentation

Abstract

The invention discloses a chitin preparation method based on microbial fermentation, and belongs to the technical field of biological thallus fermentation culture. The method comprises the following steps: (1) fermentation with acid-producing bacteria: putting a shrimp shell matrix and the acid-producing bacteria into a closed anaerobic fermentation system, adjusting the pH value to be acidic, and fermenting; (2) protease-producing bacteria fermentation: mixing solid residues obtained after fermentation in the step (1) with protease-producing bacteria, transferring the mixture into a fermentation tank, readjusting the pH value to be neutral, and carrying out aerobic fermentation; and (3) after fermentation post-treatment is completed, drying the fermented material to obtain the feed additive. The method has the beneficial effects that compared with a traditional chemical hydrolysis method, native microbial resources are adopted, exogenous strains are not needed, environmental pollution is avoided, and low-cost extraction of the chitin in the shrimp shells is realized. Experimental results show that the decalcification rate and the deproteinization rate of the method can maintain high chitin extraction efficiency, and meanwhile, the method has great ecological and economic values.

Description

Technical Field

[0001] The present invention relates to the technical field of biological cell fermentation and cultivation, and in particular to a method for preparing chitosan based on microbial fermentation. Background Art

[0002] Chitin, the primary structural component of shrimp shells, is a naturally occurring polymer with high structural stability, comprising approximately 30% to 50% of the shell's dry weight. Its complex molecular structure makes it highly resistant to acids, alkalis, and enzymes, making it difficult to degrade. Chitin has broad application prospects and can be converted into high-value-added chemicals such as chitosan and amino sugars. These derivatives are widely used in medicine, food, environmental protection, fertilizers, and other fields.

[0003] However, processing shrimp shells presents certain technical challenges. Their high hardness and complex chemical composition make the degradation and chitin extraction processes difficult. Traditional processing methods primarily include mechanical pulverization and chemical hydrolysis, but these methods are often associated with high energy consumption, the use of large amounts of chemical reagents, and environmental pollution. Chemical hydrolysis is particularly common among these traditional methods, but its requirement for strong acids or bases not only increases production costs but also poses potential environmental risks. Therefore, microbial fermentation is a more environmentally friendly and economical solution for treating shrimp shells, particularly by utilizing microorganisms to degrade chitin and protein. Microorganisms effectively degrade shrimp shells by breaking down minerals, proteins, and other components, releasing valuable chemicals. Furthermore, compared to traditional chemical methods, microbial fermentation offers lower energy consumption and less environmental pollution, while also achieving a high chitin extraction rate. Using microbial fermentation to process shrimp shells and extract their useful components not only aligns with the concept of sustainable development but also holds significant ecological and economic value.

[0004] Microbial fermentation has significant advantages in processing shrimp shells, but current technologies still have some problems and challenges. The pain point of existing technologies mainly lies in the reliance on exogenous bacterial species. Since protease bacteria and acid-producing bacteria often coexist in the same fermentation system, there is metabolic competition between them. The organic acids (such as lactic acid, acetic acid, etc.) produced by acid-producing bacteria during the fermentation process will cause the acidity of the fermentation liquid to increase, thereby affecting the activity of protease bacteria and inhibiting their degradation of proteins. This metabolic competition not only reduces the efficiency of protein degradation, but may also affect the extraction process of chitin. Therefore, how to overcome this problem and avoid competitive inhibition between strains remains an important challenge in current technology.

[0005] Chinese patent application publication number CN110862465A discloses a method for extracting chitin from Litopenaeus vannamei shells. The method involves fermenting the shrimp shells with Lactobacillus rhamnosus, then inoculating the shells with Bacillus amyloliquefaciens and continuing the fermentation to remove protein from the shrimp shell powder, thereby obtaining chitin represented by Formula I. This patent utilizes Lactobacillus rhamnosus to remove calcium carbonate, and then uses Bacillus amyloliquefaciens to remove protein, thereby extracting chitin from the shrimp shell powder. However, this patent involves complex procedures and does not utilize the shrimp shell's own microorganisms, resulting in limited adaptability. Therefore, further improvements are needed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is how to provide a method for screening microbial fermentation strains using shrimp shells as the main matrix, aiming to efficiently degrade shrimp shells and extract chitin through microbial fermentation.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The present invention provides a method for preparing chitosan based on microbial fermentation, comprising the following steps:

[0009] (1) Fermentation with acid-producing bacteria

[0010] The shrimp shell substrate and acid-producing bacteria are placed in a closed anaerobic fermentation system, the pH is adjusted to acidic, and fermentation is carried out;

[0011] (2) Fermentation with protease-producing bacteria

[0012] The solid residue after fermentation (1) and the protease-producing bacteria are mixed and transferred to a fermentation tank, the pH is readjusted, and aerobic fermentation is carried out;

[0013] (3) Post-fermentation treatment

[0014] After the fermentation is completed, the fermented material is dried to obtain the product.

[0015] Preferably, in (1), the fermentation temperature is 35-45°C and the fermentation time is 1-9 days.

[0016] Preferably, in (1), the pH is adjusted to 5.0-6.5.

[0017] Preferably, in (1), the acid-producing bacteria include Lactobacillus fermentum ATCC11739.

[0018] Preferably, in (1), the culture medium used for fermentation is composed of: 10 g / L peptone, 8 g / L beef extract powder, 4 g / L yeast extract powder, 10 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1 g / L Tween 80, and 15 g / L agar powder.

[0019] Preferably, in (1), the material-liquid ratio of the shrimp shell matrix to the culture medium is 1 g: (8-12) mL; and the inoculation amount of the acid-producing bacteria is 2-10%.

[0020] Preferably, in (2), the protease-producing bacteria include Bacillus cereus ATCC13824.

[0021] Preferably, in (2), the temperature of aerobic fermentation is 32-42° C., the time is 1-9 days, and the pH is readjusted to 6.0-8.0.

[0022] Preferably, in (2), the culture medium used for aerobic fermentation is composed of: 10 g / L peptone, 5 g / L beef extract, 10 g / L sucrose, 4 g / L disodium hydrogen phosphate, and 2 g / L magnesium sulfate.

[0023] Preferably, in (2), the solid residue to culture medium ratio is 1 g: (8-12) mL; and the inoculation amount of protease-producing bacteria is 2-10%.

[0024] Preferably, in (3), the drying temperature is 60-70° C., and the drying time is 22-26 h.

[0025] The beneficial effects of the present invention are:

[0026] 1. The present invention provides a chitosan preparation process using naturally fermented isolated strains. By simulating natural fermentation to separate strains from shrimp shells, the strains are isolated and the native strains do not require sterilization or additional oxygenation, thereby simplifying the production process, ensuring product purity, and improving resource utilization efficiency.

[0027] 2. The present invention utilizes time-controlled two-step fermentation and targeted bacterial isolation technology, making the fermentation process more efficient and achieving better strain screening results. By utilizing the microbial resources inherent in shrimp shells, the process can better adapt to environmental conditions and maintain stable degradation activity over long-term use.

[0028] 3. The present invention adopts staged regulation of environmental parameters to achieve spatiotemporal separation and efficient enrichment of bacterial communities. By regulating parameters such as temperature, pH value and dissolved oxygen content during the fermentation process, spatiotemporal separation of different microbial groups can be achieved. In different fermentation stages, the growth environment of acid-producing bacteria and protease-producing bacteria can be selectively optimized so that they can play the greatest role in different stages. For example, in the initial stage, by increasing the temperature and controlling the pH value, the activity of acid-producing bacteria is promoted, so that they decompose the protein in the shrimp shell and release organic acids; in the later stage, by adjusting the pH value or increasing the oxygen supply, the activity of acid-producing bacteria is inhibited, and the growth of protease-producing bacteria is promoted, thereby optimizing the decomposition effect of protein and chitin.

[0029] 4. The present invention utilizes spatiotemporal separation and efficient enrichment of the two bacterial communities. Through phased environmental regulation, competition between acid-producing bacteria and protease bacteria is avoided, thereby improving the degradation efficiency of the entire fermentation process. This method not only reduces harmful metabolic competition in the fermentation broth but also maximizes the metabolic products of the bacterial communities at different stages, thereby improving the extraction rate of chitin. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram of screening and culturing the acid-producing strains from the raw materials in Example 1 of the present invention;

[0031] Figure 2 This is a diagram of screening and culturing the protease-producing strains produced by self-fermentation of raw materials in Example 1 of the present invention;

[0032] Figure 3 This is a diagram showing the effect of screening acid-producing strains in Example 1 of the present invention;

[0033] Figure 4 This is a growth curve of the acid-producing strain in Example 1 of the present invention;

[0034] Figure 5 This is a diagram showing the effect of screening protease-producing strains in Example 1 of the present invention;

[0035] Figure 6 This is a growth curve of the protease-producing strain in Example 1 of the present invention;

[0036] Figure 7 A comparison diagram of the chitosan raw material (top) and the product (bottom) in Example 2 of the present invention;

[0037] Figure 8 This is a schematic diagram of the process for preparing chitosan in Example 2 of the present invention. DETAILED DESCRIPTION

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0039] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0040] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples were repeated three times and the results were averaged.

[0041] The present invention will be further described below with reference to specific embodiments.

[0042] The shrimp shell raw materials used in the following examples were all sourced from Hefei, Anhui.

[0043] Example 1:

[0044] Isolation and analysis of microorganisms in shrimp shells

[0045] 1. Raw material self-fermentation and optimization processing

[0046] The shrimp shells were grouped and cultured to simulate the natural fermentation process. The acid-producing group was set as follows: 5g shrimp shell, 0g carbon source (glucose), 0g nitrogen source (peptone); 5g shrimp shell, 5g carbon source (glucose), 3g nitrogen source (peptone); 5g shrimp shell, 10g carbon source (glucose), 3g nitrogen source (peptone); 0g shrimp shell, 5g carbon source (glucose), 3g nitrogen source (peptone). The four groups were controlled with each other. After expansion and purification, the strain to be optimized was identified by the size of the transparent circle in a culture medium containing CaCO3.

[0047] The protease-producing groups were set as shrimp shell 5g, carbon source (sucrose) 0g, nitrogen source (peptone) 0g, shrimp shell 5g, carbon source (sucrose) 5g, nitrogen source (peptone) 0g, shrimp shell 5g, carbon source (sucrose) 10g, nitrogen source (peptone) 0g, shrimp shell 0g, carbon source (sucrose) 5g, nitrogen source (peptone) 0g. The four groups were controlled with each other. After expansion and purification, the strain to be optimized was identified by the size of the transparent circle in a casein-containing culture medium.

[0048] 2. Isolation and Identification of Strains

[0049] (1) Prepare the culture medium, acid-producing bacteria culture medium: peptone 10g / L, beef extract powder 8g / L, yeast extract powder 4g / L, glucose 10g / L, dipotassium hydrogen phosphate 2g / L, diammonium hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween 80 1g / L, agar powder 15g / L,

[0050] Protease-producing bacteria culture medium: peptone 10g / L, beef extract 5g / L, sucrose 10g / L, disodium hydrogen phosphate 4g / L, magnesium sulfate 2g / L.

[0051] After evenly melting, dispense into test tubes, filling 1 / 3 of the test tube height, add stoppers and sterilize. After sterilization, place the culture medium in the test tube into an inclined surface and cool.

[0052] (2) After the culture medium in the test tube cools down to form a slant, select high-yield colonies on the screening plate, streak inoculate on the slant, and culture at 37°C for about 5 days. This is the slant culture, which is ready for use.

[0053] (3) Utilization of isolated strains

[0054] 16S-F(5'-AGCAGTAGGGAATCTTCCA-3'SEQ ID NO:1)

[0055] and 16S-R (5'-ATTTCACCGCTACACATG-3' SEQ ID NO: 2)

[0056] Amplification of 16S rDNA of isolated acid-producing strains, and

[0057] 16S-F(5'-AGAGTTTGATCCTGGCTCAG-3'SEQ ID NO:3)

[0058] and 16S-R(5'-GGTTACCTTGTTACGACTT-3'SEQ ID NO:4)

[0059] The 16S rDNA of the isolated protease-producing strain was amplified.

[0060] The PCR mixture consisted of 1 μL 16S-F, 1 μL 16S-R, 1 μL template DNA, 12.5 μL GC buffer I (2×), 4 μL dNTPs, 0.25 μL Ex Taq DNA polymerase, and sterile water was added to 25 μL. The amplification procedure was performed at 94°C for 5 minutes, followed by 30 cycles at 94°C for 45 seconds, 57°C for 30 seconds, and a final extension step at 72°C for 10 minutes. The amplified fragments were purified, sequenced, and deposited in the NCBI database. Sequences were aligned using BLAST analysis to confirm the strains screened. The acid-producing strain was Lactobacillus fermentum, and the protease-producing strain was Bacillus cereus.

[0061] Take one loop of each isolated bacterial strain, inoculate it into 50 mL of culture medium, and culture it at 37°C. Take samples every 2 hours and measure the optical density (OD value) at 600 nm using a visible spectrophotometer. The culture time is used as the horizontal axis, and the OD value is used as the horizontal axis. 600 The value is the vertical axis, and the growth curve is drawn.

[0062] Example 2:

[0063] A method for preparing chitosan based on microbial fermentation comprises the following steps:

[0064] (1) Fermentation with acid-producing bacteria

[0065] First, Lactobacillus fermentum ATCC11739 (commercially available) was inoculated into an acid-producing culture medium at a 6.5% inoculum rate. Then, 100g of crushed shrimp shell substrate and the acid-producing culture medium were mixed at a material-liquid ratio of 1g:10mL, ensuring uniform wetting of the substrate. After mixing, the mixture was placed in a closed anaerobic fermentation system, adjusted to a temperature of 39°C and an initial pH of 6.0. The fermentation process lasted for 3 days. (The composition of the acid-producing culture medium was the same as described in Example 1.)

[0066] This step is to decalcify the shrimp shell matrix, and the decalcification rate is measured:

[0067] The raw shrimp shells were treated chemically by soaking them in 1.25 mol / L HCl (solid-to-liquid ratio of 1:10) for 1 hour, filtering the residue, and then washing it with deionized water multiple times and filtering it. The filtrates were mixed and diluted to 250 mL, and the calcium content (M1) in the filtrate was determined using disodium ethylenediaminetetraacetic acid (EDTA) complexometric titration. Next, the fermented shrimp shell broth was filtered, the residue was washed with deionized water multiple times and filtered, and the filtrates were mixed and diluted to 250 mL, and the calcium content (M2) in the filtrate was determined. Using uninoculated shrimp shell fermentation broth as a control, the same procedure was used to determine the surface calcium content (M0) of the raw shrimp shells.

[0068] but

[0069] Decalcification rate (%) = (M2-M0) / (M1-M0) × 100%

[0070] Where: the total calcium content of the original shrimp shell is M1, g;

[0071] The calcium removed from the fermented shrimp shells was M2, g;

[0072] The calcium content on the surface of the original shrimp shell is M0, g.

[0073] (2) Fermentation with protease-producing bacteria

[0074] Bacillus cereus ATCC13824 (commercially available) was inoculated into a protease-producing culture medium at an inoculum rate of 7.2%. The solid residue after fermentation (1) was then mixed with the protease-producing culture medium at a material-liquid ratio of 1 g:10 mL. The mixture was transferred to a fermenter, the temperature in the fermenter was adjusted to 39°C, and the ventilation rate was set to 0.2 vvm. Aerobic fermentation was carried out for 7 days. (The composition of the protease-producing culture medium was the same as that described in Example 1.)

[0075] This step deproteinizes the solid residue and the deproteinization rate is determined as follows:

[0076] Weigh 0.3g of sample into a digestion tube. Add 6.4g of a mixed catalyst (copper sulfate and potassium sulfate ground in a 1:10 ratio). Add 10mL of concentrated sulfuric acid in a fume hood, and place in a digestion oven. The oven is set to a two-stage heating cycle: the first stage is 250°C for 60 minutes; the second stage is 420°C for 90 minutes. When the liquid in the digestion tube turns light green during the second stage, heat for an additional 30 minutes. Turn off the digestion oven and let it cool to room temperature. After the solution cools, start the Kjeldahl nitrogen analyzer to determine protein content. The amount of 35% NaOH added is generally four times the amount of H₂SO₄ added, or 40mL. The endpoint is when the liquid in the digestion tube turns brown or dark blue. (If this color does not reach this level, continue adding NaOH.) Distillation is initiated. The ammonia gas produced by distillation is absorbed by a 2% boric acid solution mixed with methyl red and bromocresol green as an indicator. Titrate with a 0.1mol / L standard hydrochloric acid solution, and record the volume of hydrochloric acid consumed. Then protein content (%) = [(V2-V1-V0)×0.1×0.014×6.25] / m×100%

[0077] Where, V2: volume of hydrochloric acid standard solution consumed by the sample, ml;

[0078] V1: volume of hydrochloric acid standard solution consumed by chitin, ml;

[0079] V0: Volume of blank consumed hydrochloric acid standard solution, ml;

[0080] m: mass of the sample, g;

[0081] Deproteinization rate Y = (W1-W2) / W1×100%

[0082] Where: W1: protein content in the sample before fermentation (%);

[0083] W2: protein content in the sample after fermentation (%);

[0084] (3) Post-fermentation treatment

[0085] After the fermentation is completed, the fermented material is dried, the drying temperature is controlled at 65°C, and the drying process is continued for 24 hours to obtain the product.

[0086] In this embodiment, the decalcification rate is 62.8%, the deproteinization rate is 71.1%, the chitosan extraction mass is 46.54 g, and the chitosan purity is 55.9%.

[0087] Example 3:

[0088] The fermentation process was adjusted based on Example 2:

[0089] In the first step, the inoculum amount of the fermentation medium was selected to be 2%, 4%, 6%, 8% and 10% in five gradients for constant temperature fermentation of shrimp shells for decalcification; the second step was the same as in Example 2. The decalcification effect of this example was tested as shown in the following table, and the test results are shown in Table 1:

[0090] Table 1

[0091]

[0092] The more impurities are removed from the raw materials, the higher the purity of the chitosan.

[0093] Example 4:

[0094] The first fermentation step was based on the above-mentioned 4% inoculum constant temperature fermentation conditions, with the fermentation conditions modified to set a temperature gradient of 35°C, 37°C, 39°C, 41°C, and 43°C for constant temperature fermentation of shrimp shells for decalcification; the second fermentation step was the same as in Example 2. The decalcification effect of this example was tested as shown in the following table, and the test results are shown in Table 2:

[0095] Table 2

[0096]

[0097] Example 5:

[0098] The first fermentation step was based on the aforementioned 4% inoculum and constant temperature fermentation conditions at 39°C, with a pH gradient of 5.0, 5.5, 6.0, 6.5, and 7.0 selected for constant temperature fermentation of shrimp shells for decalcification. The second fermentation step was the same as in Example 2. The decalcification effect of this example was tested as shown in the following table, and the test results are shown in Table 3:

[0099] Table 3

[0100]

[0101] Example 6:

[0102] The first fermentation step was based on the above-mentioned constant temperature fermentation conditions of 4% inoculum, pH 5.5, and 39°C, and the fermentation cycles were changed to 1 day, 3 days, 5 days, 7 days, and 9 days for constant temperature fermentation of shrimp shells for decalcification; the second fermentation step was the same as in Example 2. The decalcification effect of this example is tested as shown in the following table, and the test results are shown in Table 4:

[0103] Table 4

[0104]

[0105] Example 7:

[0106] The first step of fermentation conditions was the same as in Example 6. In the second step, the initial inoculum size of the fermentation medium was selected to be 2%, 4%, 6%, 8% and 10% in five gradients for constant temperature fermentation of shrimp shells for protein removal. The deproteinization effect of this example was tested as shown in the following table, and the test results are shown in Table 5:

[0107] Table 5

[0108]

[0109] Example 8:

[0110] The first step fermentation conditions were the same as those in Example 6. The second step fermentation was based on the above-mentioned 2% inoculum constant temperature fermentation conditions. The fermentation conditions were changed to set a temperature gradient of 33°C, 35°C, 37°C, 39°C and 41°C to perform constant temperature fermentation of shrimp shell deproteinization. The deproteinization effect of this example was tested as shown in the following table, and the test results are shown in Table 6:

[0111] Table 6

[0112]

[0113] Example 9:

[0114] The first step fermentation conditions were the same as those in Example 6. The second step fermentation was based on the above-mentioned 2% inoculum size and constant temperature fermentation conditions of 35°C. The initial pH was selected as 6.0, 6.5, 7.0, 7.5 and 8.0 pH gradients for constant temperature fermentation of shrimp shell deproteinization. The deproteinization effect of this example was tested as shown in the following table, and the test results are shown in Table 7:

[0115] Table 7

[0116]

[0117] Example 10:

[0118] The first step fermentation conditions were the same as those in Example 6. The second step fermentation was based on the above-mentioned 2% inoculum size, pH 6.5, and constant temperature fermentation conditions of 35°C. The fermentation cycles were changed to 1d, 3d, 5d, 7d, and 9d for constant temperature fermentation of shrimp shell deproteinization. The deproteinization effect of this example was tested in the following table, and the test results are shown in Table 8:

[0119] Table 8

[0120]

[0121] The chitosan content of each group of raw shrimp shells was tested and the results are shown in Table 9.

[0122] Table 9

[0123] Group Chitosan production Chitosan purity Remark Example 7 42.09 61.8％ The inoculation amount is 2% Example 8 41.26 63.0％ The temperature is 35℃ Example 9 38.38 67.7％ pH 6.5 Example 10 30.15 86.2％ Fermentation time is 9 days

[0124] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for preparing chitosan based on microbial fermentation, characterized in that: The following steps are involved: (1) Fermentation with acid-producing bacteria The shrimp shell substrate and acid-producing bacteria are placed in a closed anaerobic fermentation system, the pH is adjusted to acidic, and fermentation is carried out; (2) Fermentation with protease-producing bacteria The solid residue after fermentation (1) and the protease-producing bacteria are mixed and transferred to a fermentation tank, the pH is readjusted to neutral, and aerobic fermentation is carried out; (3) Post-fermentation treatment After the fermentation is completed, the fermented material is dried to obtain the product.

2. The method for preparing chitosan according to claim 1, wherein (1), the fermentation temperature is 35-45°C and the fermentation time is 1-9 days.

3. The method for preparing chitosan according to claim 1, wherein (1) Adjust the pH to 5.0-6.

5.

4. The method for preparing chitosan according to claim 1, wherein In (1), the acid-producing bacteria include Lactobacillus fermentum ATCC11739; in (2), the protease-producing bacteria include Bacillus cereus ATCC13824.

5. The method for preparing chitosan according to claim 1, wherein (1) The fermentation medium composition is as follows: peptone 10 g / L, beef extract powder 8 g / L, yeast extract powder 4 g / L, glucose 10 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1 g / L, agar powder 15 g / L.

6. The method for preparing chitosan according to claim 1, wherein (1), the material-liquid ratio of shrimp shell matrix to culture medium is 1 g: (8-12) mL; the inoculation amount of acid-producing bacteria is 2-10%.

7. The method for preparing chitosan according to claim 1, wherein (2) The temperature of aerobic fermentation is 32-42°C, the time is 1-9 days, and the pH is readjusted to 6.0-8.

0.

8. The method for preparing chitosan according to claim 1, wherein (2) The culture medium used for aerobic fermentation was composed of: peptone 10 g / L, beef extract 5 g / L, sucrose 10 g / L, disodium hydrogen phosphate 4 g / L, and magnesium sulfate 2 g / L.

9. The method for preparing chitosan according to claim 1, wherein: (2), the solid residue to culture medium ratio is 1 g: (8-12) mL; the inoculation amount of protease-producing bacteria is 2-10%.

10. The method for preparing chitosan according to claim 1, wherein: (3), the drying temperature is 60-70°C, and the drying time is 22-26 hours.

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