A process for preparing chitooligosaccharides based on enzymatic chitin hydrolysis

Abstract

The present application relates to chitosan oligosaccharide preparation technical field, disclose a kind of process for preparing chitosan oligosaccharide based on chitin enzymolysis, comprising: screening chitosanase strain, after optimization, fermentation culture is expanded, and chitosanase liquid is prepared;Take shrimp and crab shell raw material, after sterilization, by acid-producing decalcification strain fermentation decalcification, after adjusting pH, access chitin deacetylase strain and protease-producing strain are carried out and deproteinization and deacetylation, water washing and drying are refined chitosan substrate;Substrate is dissolved in dilute acid, add chitosanase liquid, and enzymolysis is carried out at 30~40℃ for 6~10 hours;After enzyme inactivation, plate frame pressure filtration is obtained Coarse enzymatic hydrolysate;Again by ultrafiltration removal macromolecular impurities, nanofiltration desalination and monosaccharide are removed and concentrated, and chitosan oligosaccharide refined liquid is obtained;Concentrated after spray drying is finished product.The present application realizes shrimp and crab shell pretreatment stage acid-alkali zero emission, product total sugar content≥98.0%, white to white-like powder, no oxidation browning, with the advantages of green environmental protection and high purity and high stability.

Description

Technical Field

[0001] This invention relates to the field of chitosan oligosaccharide preparation technology, and in particular to a process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin. Background Technology

[0002] Chitin is a natural polysaccharide widely found in the shells of marine organisms such as shrimp and crab. Chitosan, its deacetylated product, is further degraded to obtain chitosan oligosaccharide, which has good water solubility, high bioavailability, antibacterial, antioxidant, and immunomodulatory activities. It has broad application prospects in the fields of medicine, health products, biological dressings, and drug controlled-release carriers.

[0003] Currently, the industrial production of chitosan oligosaccharides mainly adopts the acid-base chemical method, which involves first extracting chitin from shrimp and crab shells by decalcification with concentrated acid and deproteinization with concentrated alkali, then obtaining chitosan by deacetylation with high temperature and concentrated alkali, and finally obtaining chitosan oligosaccharides through acid degradation or oxidative degradation.

[0004] However, this process consumes a large amount of acid and alkali, has high energy consumption, and generates a large amount of high-salt, high-concentration organic wastewater. With the increasing environmental protection requirements of the country, this highly polluting chemical production technology has been subject to increasingly strict restrictions. Summary of the Invention

[0005] The purpose of this invention is to provide a process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin, which solves the technical problems of serious environmental pollution, high acid and alkali consumption, high wastewater treatment costs, and increasing restrictions on production due to the use of acid-base chemical methods in the prior art. To achieve the above objectives, the present invention provides a process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin, comprising the following steps: Strains capable of producing chitosanase were screened from the natural environment. After strain identification and selection optimization, a target strain with high chitosanase production and high specificity was obtained. The target strain was then fermented on a large scale to prepare a highly active chitosanase solution. Shrimp and crab shells were taken as raw materials, sterilized, and then fermented with acid-producing decalcifying strains to remove calcium. After pH adjustment, chitin-producing deacetylase strains and protease-producing strains were introduced for fermentation to remove protein and deacetylate. After fermentation, the mixture was washed with water and dried to obtain refined chitosan substrate. The refined chitosan substrate was dissolved in a dilute acid solution, and the chitosan enzyme solution was added. The enzymatic hydrolysis reaction was carried out at 30-40°C for 6-10 hours. After enzymatic hydrolysis, the enzyme is inactivated by heating, and the crude enzymatic hydrolysate of chitosan oligosaccharide is obtained by plate and frame filtration. The crude enzymatic hydrolysate of chitosan oligosaccharide is passed through an ultrafiltration membrane to remove macromolecular impurities, and then through a nanofiltration membrane to desalt and remove monosaccharides, thereby achieving fractional concentration of chitosan oligosaccharide to obtain a refined chitosan oligosaccharide solution. The chitosan oligosaccharide solution was concentrated and then spray-dried to obtain the chitosan oligosaccharide product.

[0006] The method involves screening chitosanase-producing strains from the natural environment, obtaining high-yield and highly specific target strains of chitosanase through strain identification and optimization, and then expanding the fermentation culture of the target strains to prepare highly active chitosanase solutions. The specific steps include: Using powdered chitosan as the sole carbon source, the chitosanase-producing strains were initially screened using the clear zone method based on the ratio of the clear zone produced by the strain to the colony diameter, D / d. The initially screened strains were subjected to shake-flask fermentation for secondary screening. High-yield strains were further screened by enzyme activity assay. The high-yield strains were then subjected to morphological observation, physiological and biochemical identification, and 16S rDNA sequence analysis to complete the classification and identification of the strains. Using monoclonal selection and / or genetic engineering, the identified strains were optimized under shake-flask conditions to obtain target strains with high chitosanase production and strong enzymatic hydrolysis specificity. The optimized target strain was inoculated into the fermentation medium and fermented for 48–72 hours at a temperature of 28–37°C and a pH of 6.5–7.5. The fermentation supernatant containing chitosanase was collected by centrifugation or filtration to obtain the highly active chitosanase solution.

[0007] The method involves taking shrimp and crab shells as raw materials, sterilizing them, and then sequentially fermenting them with acid-producing decalcifying strains for decalcification, adjusting the pH, and then inoculating them with chitin-producing deacetylase strains and protease-producing strains for deproteinization and deacetylation. After fermentation, the mixture is washed with water and dried to obtain a refined chitosan substrate. The specific steps of the method include: The shrimp and crab shell raw materials are washed and crushed to obtain coarse shrimp and crab shell powder; The coarse powder of shrimp and crab shells is sterilized; Acid-producing decalcifying strains were inoculated into sterilized shrimp and crab shell powder at an inoculation rate of 1% to 5% of the dry weight of the raw material. The fermentation temperature was controlled at 30 to 35°C and the moisture content at 40% to 70%. Solid-state fermentation was carried out for 24 to 48 hours. Adjust the pH of the fermentation system to 6.0-7.5, inoculate with chitin deacetylase-producing strains and protease-producing strains, with an inoculation amount of 1%-3% of the dry weight of the raw materials, control the fermentation temperature at 25-35℃, and continue solid-state fermentation for 24-48 hours. After fermentation, the mixture was washed with water until neutral and dried at 40–60°C to obtain a refined chitosan substrate with a degree of deacetylation of 70%–95%.

[0008] The method specifically includes dissolving the refined chitosan substrate in a dilute acid solution, adding the chitosan enzyme solution, and performing an enzymatic hydrolysis reaction at 30–40°C for 6–10 hours. The refined chitosan substrate was added to an acetic acid solution with a mass concentration of 1% to 3% to prepare a chitosan solution with a mass concentration of 1% to 5%, and the pH was adjusted to 4.0 to 6.5. Add the chitosan enzyme solution to the chitosan solution, with an enzyme addition amount of 5-50 U / g substrate, control the enzymatic hydrolysis temperature at 30-40℃, and stir for 6-10 hours at a stirring speed of 50-200 rpm.

[0009] The method specifically includes the following steps: after enzymatic hydrolysis, the enzyme is inactivated by heating, and the crude chitosan oligosaccharide hydrolysate is obtained by plate and frame filtration. After enzymatic hydrolysis, heat to 85–95°C and hold for 10–20 minutes to inactivate the enzyme. After cooling to 40-60℃, insoluble impurities are removed by plate and frame filtration, and the collected filtrate is the crude enzymatic hydrolysate of chitosan oligosaccharide.

[0010] The method involves passing the crude chitosan oligosaccharide enzymatic hydrolysate through an ultrafiltration membrane to remove macromolecular impurities, followed by nanofiltration to desalt and remove monosaccharides, thereby achieving fractional concentration of the chitosan oligosaccharide to obtain a refined chitosan oligosaccharide solution. Specifically, the method includes: The crude enzymatic hydrolysate of chitosan oligosaccharide was passed through an ultrafiltration membrane with a molecular weight cutoff of 3000–10000 Da, under an operating pressure of 0.2–0.6 MPa and a temperature of 20–40 °C, and the permeate was collected. The ultrafiltration permeate is passed through a nanofiltration membrane with a molecular weight cutoff of 200–500 Da, and the operating pressure is 0.5–2.0 MPa, the temperature is 20–40 °C, and the pH is 4.0–6.0. ​​The nanofiltration retentate is then collected. During nanofiltration, pure water is added to the retentate for percolation washing until the conductivity of the permeate drops below a set threshold, thus obtaining the chitosan oligosaccharide purified solution.

[0011] The method of concentrating the chitosan oligosaccharide purified solution and then spray-drying it to obtain the chitosan oligosaccharide product specifically includes: The chitosan oligosaccharide purified solution was concentrated to a solid content of 20% to 40% under vacuum conditions of -0.06 to -0.09 MPa and temperature of 40 to 60°C. The concentrated liquid is spray-dried with an inlet air temperature of 140-160℃ and an outlet air temperature of 70-80℃. The actual heating time of the material is ≤5 seconds, and the dried powder is collected. After cooling the dried powder, vacuum packing or nitrogen-filled packaging is used to obtain the chitosan oligosaccharide product.

[0012] The resulting chitosan oligosaccharide product has a yield of 50% to 55% based on refined chitosan substrate, a total sugar content of ≥98.0% based on glucosamine, water-insoluble matter ≤0.2%, residue on ignition ≤0.5%, arsenic content ≤1ppm, and the product appearance is a white to off-white powder without oxidative browning.

[0013] This invention discloses a process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin. This process completely replaces traditional strong acid and strong alkali treatment with a multi-stage fermentation method using a complex of microorganisms—acid-producing decalcifying bacteria, chitin deacetylase-producing bacteria, and protease-producing bacteria—achieving zero acid and alkali emissions during the shrimp and crab shell pretreatment stage. Low-temperature enzymatic hydrolysis at 30–40°C combined with precise time control of 6–10 hours avoids oxidative browning of low-molecular-weight chitosan oligosaccharides, ensuring the product's appearance quality and chemical stability. Combined with ultrafiltration-nanofiltration dual-membrane fractionation purification and low-temperature spray drying, the resulting product has a total sugar content ≥98.0%, is a white to off-white powder without browning, and has a yield of 50%–55%. It possesses the advantages of being environmentally friendly, high-purity, and highly stable, making it suitable for large-scale industrial production. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0015] Figure 1 This is a flowchart of the process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin in this invention. Detailed Implementation

[0016] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0017] This invention provides a process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin, comprising the following steps: S1: Screening strains with chitosanase production capacity from the natural environment, obtaining high-yield and high-specificity target strains of chitosanase after strain identification and breeding optimization, and then expanding the fermentation culture of the target strains to prepare highly active chitosanase solution.

[0018] Specifically, S11: Using powdered chitosan as the sole carbon source, the chitosanase-producing strains were initially screened using the clear zone method based on the ratio of the clear zone produced by the strain to the colony diameter, D / d. S12: The initial screening strains were subjected to shake-flask fermentation for secondary screening. High-yield strains were further screened by enzyme activity assay. The high-yield strains were then subjected to morphological observation, physiological and biochemical identification and 16S rDNA sequence analysis to complete the classification and identification of the strains. S13: Using monoclonal selection and / or genetic engineering, the performance of the identified strains was optimized under shake-flask conditions to obtain target strains with high chitosanase production and strong enzymatic hydrolysis specificity. S14: Inoculate the optimized target strain into the fermentation medium and ferment for 48–72 hours at a temperature of 28–37℃ and a pH of 6.5–7.5. Collect the fermentation supernatant containing chitosanase by centrifugation or filtration to obtain the highly active chitosanase solution.

[0019] In this embodiment, the principle of using the clear zone method for initial screening is as follows: During growth, chitosanase-producing strains secrete chitosanase into the surrounding culture medium, hydrolyzing the colloidal chitosan around the colony to form a clear zone. The larger the ratio of the clear zone to the colony diameter (D / d), the stronger the strain's enzyme-producing ability. Re-screening through shake-flask fermentation further quantifies the enzyme activity of the strain, ensuring that the selected target strains possess stable enzyme-producing genetic characteristics. Optimizing the strain using monoclonal selection and genetic engineering techniques can directionally enhance the strain's enzyme-producing ability and the specificity of chitosanase for hydrolyzing β-1,4 glycosidic bonds, reducing the generation of non-specific hydrolysis byproducts and laying the foundation for a narrow molecular weight distribution of subsequent enzymatic hydrolysis products. Colloidal chitosan in the fermentation medium serves as an inducing substrate, effectively inducing the strain to secrete exocarpic chitosanase. The supernatant collected by centrifugation or filtration is the crude enzyme solution, which can be directly used for subsequent enzymatic hydrolysis reactions without further purification, reducing process costs.

[0020] S2: Take shrimp and crab shell raw materials, sterilize them, and then ferment them sequentially with acid-producing decalcifying strains to remove calcium. After adjusting the pH, inoculate them with chitin-producing deacetylase strains and protease-producing strains to remove protein and deacetylate. After fermentation, wash and dry them to obtain refined chitosan substrate.

[0021] Specifically, S21: Wash and crush the shrimp and crab shell raw materials to obtain coarse shrimp and crab shell powder; S22: Sterilize the coarse powder of shrimp and crab shells; S23: Inoculate the sterilized shrimp and crab shell powder with acid-producing decalcifying strains. The inoculation amount is 1% to 5% of the dry weight of the raw material. Control the fermentation temperature at 30 to 35°C and the moisture content at 40% to 70%. Ferment in solid state for 24 to 48 hours. S24: Adjust the pH of the fermentation system to 6.0-7.5, inoculate with chitin deacetylase-producing strains and protease-producing strains, with an inoculation amount of 1%-3% of the dry weight of the raw materials, control the fermentation temperature at 25-35℃, and continue solid-state fermentation for 24-48 hours. S25: After fermentation, wash with water until neutral, and dry at 40-60℃ to obtain a refined chitosan substrate with a degree of deacetylation of 70%-95%.

[0022] In this embodiment, the core of the staged fermentation strategy in step S2 is to resolve the pH conflict between acid production and decalcification and enzymatic deacetylation. In the first stage, acid-producing decalcifying strains are introduced, and organic acids such as lactic acid and citric acid produced by their metabolism are used to dissolve calcium salt impurities such as calcium carbonate in shrimp and crab shells. The acidic environment in this stage (pH drops to 4.0-5.0) is conducive to the full dissolution of calcium salts. In the second stage, the pH of the system is adjusted to 6.0-7.5 to meet the neutral growth environment requirements of chitin-producing deacetylase strains and protease-producing strains. By controlling pH in stages, decalcification efficiency is ensured while maintaining high activity expression of deacetylase and protease under suitable pH conditions. The protease-producing strains can simultaneously degrade residual protein macromolecules in shrimp and crab shells into soluble amino acids or peptides, achieving multi-effect removal through one-step fermentation. The degree of deacetylation of the obtained purified chitosan substrate reaches 70%–95%, making it completely soluble in 1%–3% dilute acetic acid solution, providing a homogeneous reaction system for subsequent enzymatic hydrolysis. The entire pretreatment process replaces traditional concentrated acid and alkali with microbial metabolism, achieving clean production in the pretreatment stage.

[0023] S3: Dissolve the refined chitosan substrate in a dilute acid solution, add the chitosan enzyme solution, and perform enzymatic hydrolysis at 30-40°C for 6-10 hours.

[0024] Specifically, S31: Add the refined chitosan substrate to an acetic acid solution with a mass concentration of 1% to 3% to prepare a chitosan solution with a mass concentration of 1% to 5%, and adjust the pH to 4.0 to 6.5; S32: Add the chitosan enzyme solution to the chitosan solution, with an enzyme addition amount of 5-50 U / g substrate, control the enzymatic hydrolysis temperature at 30-40℃, stir and hydrolyze for 6-10 hours at a stirring speed of 50-200 rpm.

[0025] In this embodiment, chitosanase maintains high catalytic activity within the range of 30–40°C, sufficient to complete the directed hydrolysis of chitosan to chitosan oligosaccharides within 6–10 hours. However, this temperature is below the critical temperature for the initiation of oxidative browning of chitosan oligosaccharides (approximately 45°C), effectively inhibiting the oxidative free radical chain reaction initiated by the exposure of the reducing end of low molecular weight chitosan oligosaccharide chains during the reaction, thereby avoiding product browning and degradation. The reaction time of 6–10 hours was determined through orthogonal experiments. If the time is too short, the hydrolysis will be insufficient, resulting in a larger product molecular weight and lower yield. If the time is too long, too many monosaccharides may be generated, reducing the purity of the target chitosan oligosaccharide. The amount of chitosanase added is 5–50 U / g substrate. By controlling the ratio of enzyme to substrate, the uniformity and reproducibility of the hydrolysis reaction are ensured. The stirring speed of 50–200 rpm ensures sufficient contact between the enzyme and substrate, while avoiding the effect of excessive shear force on enzyme inactivation.

[0026] S4: After enzymatic hydrolysis, the enzyme is inactivated by heating, and the crude enzymatic hydrolysate of chitosan oligosaccharide is obtained by plate and frame filtration.

[0027] Specifically, S41: After enzymatic hydrolysis, heat to 85-95℃ and incubate for 10-20 minutes to inactivate the enzyme; S42: After cooling to 40-60℃, insoluble impurities are removed by plate and frame filtration, and the collected filtrate is the crude enzymatic hydrolysate of chitosan oligosaccharide.

[0028] In this embodiment, the incubation at 85-95°C for 10-20 minutes in step S4 ensures complete denaturation and inactivation of chitosanase, terminating the enzymatic hydrolysis reaction and preventing incomplete enzyme inactivation from causing further degradation of the product into monosaccharides during subsequent processing, thus affecting product purity. Cooling to 40-60°C after enzyme inactivation aims to adjust the temperature of the hydrolysate to the suitable temperature range for subsequent membrane separation processes, avoiding irreversible thermal damage to the membrane material caused by the direct entry of high-temperature feed into the membrane system. Plate and frame filtration, through the physical retention of the filter cloth, can efficiently remove undissolved solid residues, denatured protein flocs, and other suspended particles remaining in the hydrolysate. The resulting filtrate has good clarity, providing a clean feed liquid for subsequent membrane separation processes, effectively reducing the risk of membrane fouling and extending the service life of the membrane module.

[0029] S5: The crude chitosan oligosaccharide hydrolysate is passed through an ultrafiltration membrane to remove macromolecular impurities, and then through a nanofiltration membrane to desalt and remove monosaccharides, thereby achieving fractional concentration of chitosan oligosaccharides to obtain a refined chitosan oligosaccharide solution.

[0030] Specifically, S51: Pass the crude enzymatic hydrolysate of chitosan oligosaccharide into an ultrafiltration membrane with a molecular weight cutoff of 3000-10000 Da, operating at a pressure of 0.2-0.6 MPa and a temperature of 20-40℃, and collect the permeate; S52: Pass the ultrafiltration permeate through a nanofiltration membrane with a molecular weight cutoff of 200-500 Da, operating at a pressure of 0.5-2.0 MPa, a temperature of 20-40℃, and a pH of 4.0-6.0, and collect the nanofiltration retentate; S53: During nanofiltration, pure water is added to the retentate for percolation washing until the conductivity of the permeate drops below a set threshold to obtain chitosan oligosaccharide purified solution.

[0031] In this embodiment, the first step of ultrafiltration uses a membrane with a molecular weight cutoff of 3000-10000 Da. This pore size allows the effective components of chitosan oligosaccharides with a degree of polymerization of 2-10 to pass through, while retaining incompletely degraded macromolecular chitosan and residual proteins, thus achieving the first step of coarse separation. The second step of nanofiltration uses a membrane with a molecular weight cutoff of 200-500 Da. This pore size can retain chitosan oligosaccharide disaccharides and higher, while allowing inorganic salts and most monosaccharides to pass through and be discharged. Through the combination of two-stage membrane separation and percolation washing, integrated purification of desalting, monosaccharide removal, and narrow distribution fractionation is achieved. During the percolation washing process, as pure water is continuously added, the concentration gradient of small molecule impurities on the cut-off side continues to decrease until the conductivity of the permeate drops to a set threshold, indicating that the salt has been basically removed. The final chitosan oligosaccharide purified solution is a high-purity solution with a concentrated molecular weight distribution.

[0032] S6: Concentrate the chitosan oligosaccharide refined liquid and then spray dry it to obtain the chitosan oligosaccharide product.

[0033] Specifically, S61: The chitosan oligosaccharide purified liquid is concentrated to a solid content of 20% to 40% under vacuum conditions of -0.06 to -0.09 MPa and temperature of 40 to 60°C; S62: Concentrated liquid spray drying, inlet air temperature 140~160℃, outlet air temperature 70~80℃, actual material heating time ≤5 seconds, collect dried powder; S63: After cooling the dried powder, vacuum pack or nitrogen-filled pack to obtain the chitosan oligosaccharide product.

[0034] In this embodiment, the purpose of low-temperature vacuum concentration is to lower the boiling point of water under negative pressure, so that the chitosan oligosaccharide concentrate can be concentrated under mild conditions of 40-60°C, avoiding the adverse effects of high temperature on the thermal stability of chitosan oligosaccharide. Spray drying uses an inlet air temperature of 140-160°C and an outlet air temperature of 70-80°C. The atomizer disperses the concentrate into micron-sized droplets, increasing the specific surface area and heat and mass transfer efficiency, so that the droplets can be dried instantaneously within a few seconds. The actual heating time of the material is ≤5 seconds, which effectively avoids browning or structural damage caused by prolonged residence of chitosan oligosaccharide in high-temperature areas. The resulting dried powder has good flowability, solubility and storage stability. Vacuum packaging or nitrogen-filled packaging can isolate oxygen and moisture, preventing the product from absorbing moisture or oxidizing during storage.

[0035] The chitosan oligosaccharide product obtained by the process of this invention has a product yield of 50% to 55% based on refined chitosan substrate, a total sugar content of ≥98.0% based on glucosamine, water-insoluble matter ≤0.2%, residue on ignition ≤0.5%, arsenic content ≤1ppm, and the product appearance is a white to off-white powder without oxidative browning.

[0036] This invention relates to a process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin. This process completely replaces traditional strong acid and strong alkali treatment with a multi-stage fermentation method using a complex of microorganisms: acid-producing decalcifying bacteria, chitin deacetylase-producing bacteria, and protease-producing bacteria. This achieves zero acid and alkali emissions during the shrimp and crab shell pretreatment stage. Low-temperature enzymatic hydrolysis at 30–40°C combined with precise time control of 6–10 hours avoids oxidative browning of low-molecular-weight chitosan oligosaccharides, ensuring the product's appearance quality and chemical stability. Combined with ultrafiltration-nanofiltration dual-membrane fractionation purification and low-temperature spray drying, the resulting product has a total sugar content ≥98.0%, is a white to off-white powder without browning, and has a yield of 50%–55%. It possesses the advantages of being environmentally friendly, high-purity, and highly stable, making it suitable for large-scale industrial production.

[0037] The above-disclosed embodiments are merely one or more preferred embodiments of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that all or part of the processes for implementing the above embodiments and equivalent changes made in accordance with the claims of this application still fall within the scope of this application.

Claims

1. A process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin, characterized in that, Includes the following steps: Strains capable of producing chitosanase were screened from the natural environment. After strain identification and selection optimization, a target strain with high chitosanase production and high specificity was obtained. The target strain was then fermented on a large scale to prepare a highly active chitosanase solution. Shrimp and crab shells were taken as raw materials, sterilized, and then fermented with acid-producing decalcifying strains to remove calcium. After pH adjustment, chitin-producing deacetylase strains and protease-producing strains were introduced for fermentation to remove protein and deacetylate. After fermentation, the mixture was washed with water and dried to obtain refined chitosan substrate. The refined chitosan substrate was dissolved in a dilute acid solution, and the chitosan enzyme solution was added. The enzymatic hydrolysis reaction was carried out at 30-40°C for 6-10 hours. After enzymatic hydrolysis, the enzyme is inactivated by heating, and the crude enzymatic hydrolysate of chitosan oligosaccharide is obtained by plate and frame filtration. The crude enzymatic hydrolysate of chitosan oligosaccharide is passed through an ultrafiltration membrane to remove macromolecular impurities, and then through a nanofiltration membrane to desalt and remove monosaccharides, thereby achieving fractional concentration of chitosan oligosaccharide to obtain a refined chitosan oligosaccharide solution. The chitosan oligosaccharide solution was concentrated and then spray-dried to obtain the chitosan oligosaccharide product.

2. The process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin as described in claim 1, characterized in that, The method involves screening strains capable of producing chitosanase from the natural environment, identifying and optimizing the strains to obtain a high-yield, high-specificity target strain for chitosanase, and then expanding the fermentation culture of the target strain to prepare a highly active chitosanase solution. The specific steps include: Using powdered chitosan as the sole carbon source, the chitosanase-producing strains were initially screened using the clear zone method based on the ratio of the clear zone produced by the strain to the colony diameter, D / d. The initially screened strains were subjected to shake-flask fermentation for secondary screening. High-yield strains were further screened by enzyme activity assay. The high-yield strains were then subjected to morphological observation, physiological and biochemical identification, and 16S rDNA sequence analysis to complete the classification and identification of the strains. Using monoclonal selection and / or genetic engineering, the identified strains were optimized under shake-flask conditions to obtain target strains with high chitosanase production and strong enzymatic hydrolysis specificity. The optimized target strain was inoculated into the fermentation medium and fermented for 48–72 hours at a temperature of 28–37°C and a pH of 6.5–7.

5. The fermentation supernatant containing chitosanase was collected by centrifugation or filtration to obtain the highly active chitosanase solution.

3. The process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin as described in claim 1, characterized in that, Shrimp and crab shells are taken as raw materials, sterilized, and then fermented sequentially with acid-producing decalcifying strains for decalcification. After pH adjustment, chitin-producing deacetylase strains and protease-producing strains are inoculated for deproteinization and deacetylation. After fermentation, the mixture is washed with water and dried to obtain a refined chitosan substrate. The method specifically includes: The shrimp and crab shell raw materials are washed and crushed to obtain coarse shrimp and crab shell powder; The coarse powder of shrimp and crab shells is sterilized; Acid-producing decalcifying strains were inoculated into sterilized shrimp and crab shell powder at an inoculation rate of 1% to 5% of the dry weight of the raw material. The fermentation temperature was controlled at 30 to 35°C and the moisture content at 40% to 70%. Solid-state fermentation was carried out for 24 to 48 hours. Adjust the pH of the fermentation system to 6.0-7.5, inoculate with chitin deacetylase-producing strains and protease-producing strains, with an inoculation amount of 1%-3% of the dry weight of the raw materials, control the fermentation temperature at 25-35℃, and continue solid-state fermentation for 24-48 hours. After fermentation, the mixture was washed with water until neutral and dried at 40–60°C to obtain a refined chitosan substrate with a degree of deacetylation of 70%–95%.

4. The process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin as described in claim 1, characterized in that, The method specifically includes dissolving the purified chitosan substrate in a dilute acid solution, adding the chitosan enzyme solution, and performing an enzymatic hydrolysis reaction at 30–40°C for 6–10 hours. The refined chitosan substrate was added to an acetic acid solution with a mass concentration of 1% to 3% to prepare a chitosan solution with a mass concentration of 1% to 5%, and the pH was adjusted to 4.0 to 6.

5. Add the chitosan enzyme solution to the chitosan solution, with an enzyme addition amount of 5-50 U / g substrate, control the enzymatic hydrolysis temperature at 30-40℃, and stir for 6-10 hours at a stirring speed of 50-200 rpm.

5. The process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin as described in claim 1, characterized in that, After enzymatic hydrolysis, the enzyme is inactivated by heating, and the crude chitosan oligosaccharide hydrolysate is obtained by plate and frame filtration. The method specifically includes: After enzymatic hydrolysis, heat to 85–95°C and hold for 10–20 minutes to inactivate the enzyme. After cooling to 40-60℃, insoluble impurities are removed by plate and frame filtration, and the collected filtrate is the crude enzymatic hydrolysate of chitosan oligosaccharide.

6. The process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin as described in claim 1, characterized in that, The crude chitosan oligosaccharide enzymatic hydrolysate is sequentially passed through an ultrafiltration membrane to remove macromolecular impurities, followed by nanofiltration membrane desalting and monosaccharide removal, and fractional concentration of the chitosan oligosaccharide to obtain a refined chitosan oligosaccharide solution. The method specifically includes: The crude enzymatic hydrolysate of chitosan oligosaccharide was passed through an ultrafiltration membrane with a molecular weight cutoff of 3000–10000 Da, under an operating pressure of 0.2–0.6 MPa and a temperature of 20–40 °C, and the permeate was collected. The ultrafiltration permeate is passed through a nanofiltration membrane with a molecular weight cutoff of 200–500 Da, and the operating pressure is 0.5–2.0 MPa, the temperature is 20–40 °C, and the pH is 4.0–6.

0. ​​The nanofiltration retentate is then collected. During nanofiltration, pure water is added to the retentate for percolation washing until the conductivity of the permeate drops below a set threshold, thus obtaining the chitosan oligosaccharide purified solution.

7. The process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin as described in claim 1, characterized in that, The chitosan oligosaccharide purified solution is concentrated and then spray-dried to obtain the chitosan oligosaccharide product. The method specifically includes: The chitosan oligosaccharide purified solution was concentrated to a solid content of 20% to 40% under vacuum conditions of -0.06 to -0.09 MPa and temperature of 40 to 60°C. The concentrated liquid is spray-dried with an inlet air temperature of 140-160℃ and an outlet air temperature of 70-80℃. The actual heating time of the material is ≤5 seconds, and the dried powder is collected. After cooling the dried powder, vacuum packing or nitrogen-filled packaging is used to obtain the chitosan oligosaccharide product.

8. The process for preparing chitosan oligosaccharides based on enzymatic hydrolysis of chitin as described in claim 1, characterized in that, The yield of the obtained chitosan oligosaccharide product, based on the refined chitosan substrate, is 50% to 55%, the total sugar content based on glucosamine is ≥98.0%, the water-insoluble matter is ≤0.2%, the residue on ignition is ≤0.5%, the arsenic content is ≤1ppm, and the product appearance is a white to off-white powder without oxidative browning.

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