Processing method for biosynthesizing chitosan by using bacteria
Through microbial culture of Enterobacter closure and enzyme dissociation combined with solvent extraction, the problem of alkali waste liquid pollution in microbial fermentation method is solved, low-cost and environmentally friendly chitosan production is achieved, and the application field of chitosan is expanded.
Patent Information
- Application Number
- CN202510597869.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-09
- Publication Date
- 2025-08-15
AI Technical Summary
The existing methods for preparing chitosan by microbial fermentation methods mainly refer to the shrimp and crab shell method, resulting in the production of more alkali waste liquid, which is unfriendly in the environment and is relatively expensive.
Microbial culture was carried out using Enterobacter cloaca, glycolysis was dissociated with specific enzymes and extracted chitosan by solvents, combining enzyme stabilizers and controlling reaction conditions, reducing chemical consumption and environmental impact.
It has achieved low-cost and environmentally friendly chitosan production, reduced the use of chemicals, reduced the environmental burden, and provided a rich source of raw materials. Chitosan can be used in food additives, drug carriers, etc.
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbial fermentation, in particular to a processing method for biosynthesizing chitosan using bacteria. Background Art
[0002] Chitosan is a natural cationic polymer obtained by deacetylation of chitin. It is widely used in food, chemical, pharmaceutical and agricultural fields. However, due to its large molecular weight, it can only be dissolved in certain acidic solutions, which limits its application. Chitosan oligosaccharides, also known as chitosan oligosaccharides, are the product of chitosan degradation by chitosanase. They have the characteristics of low molecular weight, high solubility, and are more easily absorbed by organisms. Some special functions of chitosan can only be manifested by degrading it into chitosan oligosaccharides. Studies have shown that chitosan oligosaccharides have high efficiency in antibacterial, antioxidant, lipid-lowering, blood pressure-lowering, infection prevention, arthritis control and anti-tumor effects. It is an oligosaccharide with great development potential.
[0003] The main degradation processes of chitosan include shrimp and crab shell extraction and microbial fermentation. The shrimp and crab shell extraction method uses the crustaceans of marine arthropods such as shrimp and crabs, the crustaceans of insects, the shells and bones of mollusks to extract chitin, and then deacetylates it with a strong alkaline solution to obtain chitosan. This process consumes a large amount of strong acid and strong alkali, and there is a great pressure on the environmental protection. Compared with the shrimp and crab shell extraction method, the microbial fermentation method has the advantages of stable product quality, small differences, low ash content, and less acid required during the extraction process. At present, the microbial fermentation method for preparing chitosan reported in the literature is mainly filamentous fungi, but its extraction method mainly draws on the shrimp and crab shell method. In order to remove the protein in the mycelium, it will also produce a lot of alkaline waste liquid, which is not environmentally friendly. Therefore, a processing method using bacterial biosynthesis of chitosan is proposed to solve the above problems. Summary of the Invention
[0004] (1) Technical problems solved
[0005] In response to the shortcomings of the existing technology, the present invention provides a processing method for synthesizing chitosan using bacteria, which has the advantages of low chemical consumption, environmentally friendly process, low cost, and easy promotion of production. It solves the problem that the microbial fermentation method for preparing chitosan, which is currently reported in the literature, mainly uses filamentous fungi, but its extraction method mainly draws on the shrimp and crab shell method, and in order to remove the protein in the mycelium, it also produces a large amount of alkaline waste liquid, which is not environmentally friendly.
[0006] (2) Technical solution
[0007] To achieve the above object, the present invention provides the following technical solution: a processing method for biosynthesizing chitosan using bacteria, comprising the following steps:
[0008] S1. Microbial culture: culturing Enterobacter cloacae to a growth state;
[0009] S2, glycolysis, using specific enzymes to separate the sugars in bacteria;
[0010] S3, chitosan extraction, using a solvent to extract chitosan from the glycolysis solution;
[0011] S4, chitosan drying, drying the extracted chitosan to form powder or granules.
[0012] Preferably, the S1 uses a special culture medium for culturing Enterobacter cloacae to improve bacterial growth and glycolysis efficiency.
[0013] Preferably, the culture medium is one of nutrient agar medium, tryptone soy broth medium or LB medium.
[0014] Preferably, the specific steps of culturing Enterobacter cloacae in S1 are as follows:
[0015] S1.1. Prepare the plate culture medium: Heat the sodium chloride sucrose agar medium to dissolve and cool, add 3-5 ml of clindamycin solution to the sodium chloride sucrose agar medium and mix well.
[0016] S1.2. Pour 20 ml of the mixed culture medium solution into a culture dish and gently shake the dish to evenly distribute the culture medium solution on the bottom of the dish. Once the culture medium solution solidifies, the plate culture medium is obtained.
[0017] S1.3. Preparation of activated sludge mixture: Weigh 15 g of activated sludge soil sample and place it in a conical flask containing 100 ml of sterile water and glass beads. Shake to mix evenly. Use a 1 ml sterile pipette to draw 1 ml of soil suspension from the sample and add it to a large test tube containing 9 ml of sterile water and mix thoroughly to prepare the activated sludge mixture.
[0018] S1.4. Culture medium coating: Pipette 0.2 ml of the activated sludge mixture and drop it on the center of the surface of the culture medium on the plate. Use a sterile glass spreading rod to gently spread the activated sludge mixture outward in concentric circles to ensure uniform distribution.
[0019] Preferably, the cooling temperature in S1.1 is to 65° C., and the shaking time in S1.3 is 15 to 20 minutes.
[0020] Preferably, the specific enzymes in S2 are α-amylase and β-amylase to improve the glycolysis efficiency.
[0021] Preferably, the solvent in S3 is ethanol or propanol.
[0022] Preferably, the specific steps of extracting chitosan from the glycolysis solution using a solvent in S3 are as follows:
[0023] S3.1. Filter or centrifuge the glycolysis solution to remove suspended particles;
[0024] S3.2, dissolving the chitosan in the glycolysis solution using a solvent;
[0025] S3.3, separating chitosan from the solvent by evaporation or extraction of the solvent;
[0026] S3.4. Use water or other solvents to wash the chitosan to remove residual solvents or impurities.
[0027] (3) Beneficial effects
[0028] Compared with the prior art, the present invention provides a processing method for biosynthesizing chitosan using bacteria, which has the following beneficial effects:
[0029] The processing method of synthesizing chitosan by bacterial biosynthesis has the advantages of low chemical consumption, environmentally friendly process, low cost, and easy promotion of production. The chitosan produced by Enterobacter cloacae has good biodegradability, which means that it can be naturally decomposed by the human body after use, reducing the burden on the environment. Moreover, compared with other bacteria, Enterobacter cloacae may have a lower biosynthesis cost, thereby reducing the production cost of chitosan. At the same time, Enterobacter cloacae can grow in a variety of environments, such as soil, water and other biomass resources, which provides a rich source of raw materials for the production of chitosan. In addition, the chitosan synthesized by Enterobacter cloacae can be combined with other compounds or agents to form composite materials or drugs with different functions and uses. For example, it can be used as a food additive, drug carrier or biomaterial. DETAILED DESCRIPTION
[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 are within the scope of protection of the present invention.
[0031] A method for biosynthesizing chitosan using bacteria comprises the following steps:
[0032] S1. Microbial culture: culturing Enterobacter cloacae to a growth state;
[0033] S2, glycolysis, using specific enzymes to separate the sugars in bacteria;
[0034] S3, chitosan extraction, using a solvent to extract chitosan from the glycolysis solution;
[0035] S4, chitosan drying, drying the extracted chitosan to form powder or granules.
[0036] Specifically, S1 uses a special culture medium for the cultivation of Enterobacter cloacae to improve the growth and glycolysis efficiency of the bacteria, and the culture medium is one of nutrient agar medium, tryptone soy broth medium or LB medium.
[0037] Furthermore, the specific steps of S1 for culturing Enterobacter cloacae are as follows:
[0038] S1.1. Prepare the plate culture medium: Heat the sodium chloride sucrose agar medium to dissolve and cool, add 3-5 ml of clindamycin solution to the sodium chloride sucrose agar medium and mix well.
[0039] S1.2. Pour 20 ml of the mixed culture medium solution into a culture dish and gently shake the dish to evenly distribute the culture medium solution on the bottom of the dish. Once the culture medium solution solidifies, the plate culture medium is obtained.
[0040] S1.3. Preparation of activated sludge mixture: Weigh 15 g of activated sludge soil sample and place it in a conical flask containing 100 ml of sterile water and glass beads. Shake to mix evenly. Use a 1 ml sterile pipette to draw 1 ml of soil suspension from the sample and add it to a large test tube containing 9 ml of sterile water and mix thoroughly to prepare the activated sludge mixture.
[0041] S1.4. Culture medium coating: Pipette 0.2 ml of the activated sludge mixture and drop it on the center of the surface of the culture medium on the plate. Use a sterile glass spreading rod to gently spread the activated sludge mixture outward in concentric circles to ensure uniform distribution.
[0042] Furthermore, the temperature is cooled to 65° C. in S1.1, and the shaking time in S1.3 is 15 to 20 minutes.
[0043] Specifically, the specific enzymes in S2 are α-amylase and β-amylase to improve the efficiency of glycolytic dissociation.
[0044] Furthermore, in the process of extracting chitosan, the following methods are used to ensure that the enzymes used have optimal activity and stability to improve the extraction efficiency and purity: 1) Use enzyme stabilizers: Use enzyme stabilizers to improve the stability of the enzyme; 2) Control temperature and pH: During the addition and reaction of the enzyme, control the temperature and pH to maintain the optimal activity of the enzyme; 3) Use appropriate enzyme concentration: Use appropriate enzyme concentration according to the bacterial species and extraction conditions to improve the activity of the enzyme; 4) Use buffer: Use buffer to reduce enzyme degradation and inactivation; 5) Optimize reaction time and temperature: Optimize reaction time and temperature to improve enzyme activity.
[0045] Furthermore, the enzyme stabilizer can be β-mercaptoethanol or a protease inhibitor.
[0046] Specifically, the solvent in S3 is ethanol or propanol.
[0047] Furthermore, the specific steps of extracting chitosan from the glycolysis solution using a solvent in S3 are as follows:
[0048] S3.1. Filter or centrifuge the glycolysis solution to remove suspended particles;
[0049] S3.2, dissolving the chitosan in the glycolysis solution using a solvent;
[0050] S3.3, separating chitosan from the solvent by evaporation or extraction of the solvent;
[0051] S3.4. Use water or other solvents to wash the chitosan to remove residual solvents or impurities.
[0052] In summary, the beneficial effects of the present invention are: the processing method of chitosan synthesized by bacteria, the processing method of synthesizing chitosan of the present invention has the advantages of low chemical consumption, environmentally friendly process, low cost, easy promotion and production, etc., the chitosan produced by Enterobacter cloacae has good biodegradability, which means that it can be naturally decomposed by the human body after use, reducing the burden on the environment, and compared with other bacteria, Enterobacter cloacae may have a lower biosynthesis cost, thereby reducing the production cost of chitosan, at the same time, Enterobacter cloacae can grow in a variety of environments, such as soil, water and other biomass resources, which provides a rich source of raw materials for the production of chitosan, in addition, the chitosan synthesized by Enterobacter cloacae can be combined with other compounds or agents to form composite materials or drugs with different functions and uses, for example, it can be used as a food additive, drug carrier or biomaterial, etc., which solves the problem that the microbial fermentation method for preparing chitosan reported in the literature is mainly filamentous fungi, but its extraction method mainly draws on the shrimp and crab shell method, in order to remove protein in the mycelium, it also produces a lot of alkaline waste liquid, which is not environmentally friendly.
[0053] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.
Claims
1. A method for processing chitosan by bacterial biosynthesis, characterized in that: The following steps are involved: S1. Microbial culture: culturing Enterobacter cloacae to a growth state; S2, glycolysis, using specific enzymes to separate the sugars in bacteria; S3, chitosan extraction, using a solvent to extract chitosan from the glycolysis solution; S4, chitosan drying, drying the extracted chitosan to form powder or granules.
2. The method for producing chitosan by bacterial biosynthesis according to claim 1, wherein: The S1 uses a special culture medium for the cultivation of Enterobacter cloacae to improve the growth of bacteria and the efficiency of glycolysis and dissociation.
3. The method for producing chitosan by bacterial biosynthesis according to claim 2, wherein: The culture medium is one of nutrient agar medium, tryptone soy broth medium or LB medium.
4. The method for producing chitosan by bacterial biosynthesis according to claim 1, wherein: The specific steps of culturing Enterobacter cloacae in S1 are as follows: S1.
1. Prepare the plate culture medium: Heat the sodium chloride sucrose agar medium to dissolve and cool, add 3-5 ml of clindamycin solution to the sodium chloride sucrose agar medium and mix well. S1.
2. Pour 20 ml of the mixed culture medium solution into a culture dish and gently shake the dish to evenly distribute the culture medium solution on the bottom of the dish. Once the culture medium solution solidifies, the plate culture medium is obtained. S1.
3. Preparation of activated sludge mixture: Weigh 15 g of activated sludge soil sample and place it in a conical flask containing 100 ml of sterile water and glass beads. Shake to mix evenly. Use a 1 ml sterile pipette to draw 1 ml of soil suspension from the sample and add it to a large test tube containing 9 ml of sterile water and mix thoroughly to prepare the activated sludge mixture. S1.
4. Culture medium coating: Pipette 0.2 ml of the activated sludge mixture and drop it on the center of the surface of the culture medium on the plate. Use a sterile glass spreading rod to gently spread the activated sludge mixture outward in concentric circles to ensure uniform distribution.
5. The method for processing chitosan by bacterial biosynthesis according to claim 4, characterized in that: In the step S1.1, the temperature is cooled to 65° C., and in the step S1.3, the shaking time is 15 to 20 minutes.
6. The method for processing chitosan by bacterial biosynthesis according to claim 1, characterized in that: The specific enzymes in S2 are α-amylase and β-amylase to improve the efficiency of glycolysis.
7. The method for processing chitosan by bacterial biosynthesis according to claim 1, characterized in that: The solvent in S3 is ethanol or propanol.
8. The method for producing chitosan by bacterial biosynthesis according to claim 1, wherein: The specific steps of using a solvent to extract chitosan from the glycolysis solution in S3 are as follows: S3.
1. Filter or centrifuge the glycolysis solution to remove suspended particles; S3.2, dissolving the chitosan in the glycolysis solution using a solvent; S3.3, separating chitosan from the solvent by evaporation or extraction of the solvent; S3.
4. Use water or other solvents to wash the chitosan to remove residual solvents or impurities.