Green preparation method of chitin based on shrimp shells
Through facultative anaerobic microbial fermentation and enzymatic treatment, the problems of high energy consumption and incomplete protein removal of microbial fermentation are solved, low-energy consumption and efficient chitin preparation are achieved, and the purity of chitin and the value of biomedical application are improved. .
Patent Information
- Application Number
- CN202411523599.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2025-01-10
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing microbial fermentation method has high energy consumption in the preparation of chitin and has poor effect on removing shrimp shell protein, which affects the purity of chitin and biomedical applications.
Facultative anaerobic microorganisms are used for fermentation, and through enzymatic lysis and fermentation steps, proteins and amino acids in shrimp shells are used for microbial fermentation, reducing energy consumption and cost, and chitin is isolated through enzymatic lysis and chemical precipitation.
It significantly reduces fermentation energy consumption and cost, improves resource utilization efficiency, achieves more thorough protein removal, and improves the purity of chitin and the value of biomedical applications.
Smart Images
Figure CN119286958A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of chitosan preparation, and in particular to a green preparation method of chitosan based on shrimp shells. Background Art
[0002] Chitosan is the second largest biosynthesized polysaccharide on earth after cellulose, and it is also the only cationic polysaccharide existing in nature. It is an important raw material for the pharmaceutical, food, chemical and other industries. Shrimp shells are abundant in sources and contain relatively rich chitosan, protein, calcium, fat and other substances. They are an important raw material for preparing chitosan. The methods for preparing chitosan from shrimp shells include acid-base method, enzymatic method and microbial fermentation method. Compared with the acid-base method, the enzymatic method combined with the chemical method has less effect on the main chain structure of chitosan, but the method is not ideal for removing protein from shrimp and crab shells, and the residual protein will have an adverse effect on the processing and application of chitosan. The reaction conditions of the microbial fermentation method are milder, will not affect the molecular structure of chitosan, and have less pollution to the environment.
[0003] Microbial fermentation mainly uses organic acids and proteases produced by fungal or bacterial fermentation to remove ash and protein from shrimp and crab shells, thereby preparing chitin with higher purity. The fermentation method can also produce by-products such as organic calcium, peptides and amino acids. Due to the chemical bond between protein and chitin, it is difficult to completely remove the protein from shrimp and crab shells. Some people are allergic to protein in seafood, so more thorough removal of protein is particularly important for the application of chitin in the biomedical field, which is also a challenge that chitin research needs to face.
[0004] The existing microbial fermentation methods basically require aerobic fermentation, which has high energy consumption and is not ideal for processing shrimp shell protein.
[0005] In summary, the existing technology obviously has inconveniences and defects in practical use, so it is necessary to improve it. Summary of the invention
[0006] In view of the above-mentioned defects, the purpose of the present invention is to provide a green preparation method of chitosan based on shrimp shells, which utilizes facultative anaerobic microorganisms for fermentation, does not require sterilization and oxygenation, significantly reduces energy consumption, saves costs, and makes full use of components such as protein and amino acids for microbial fermentation, thereby reducing fermentation costs and improving resource utilization efficiency.
[0007] In order to achieve the above object, the present invention provides a green preparation method of chitosan based on shrimp shells, comprising the following steps:
[0008] Step 1: Prepare the ingredients
[0009] The shrimp shells are crushed, sieved, and added with purified water. After 2 to 3 hours, water is added until the shrimp shell powder is covered to obtain the shrimp shell powder raw material.
[0010] Step 2: Enzymatic treatment
[0011] The shrimp shell powder raw material is enzymolyzed with 0.2% lipase for 0.8-1.2 hours, and then deproteinized with 0.2% protease for 0.8-1.2 hours. After the enzymolysis, the enzyme is inactivated by heating to 50-52° C. for 10-12 minutes to obtain an enzymolysis solution.
[0012] Step 3: Fermentation
[0013] Add culture medium components, add purified water to twice the amount of the enzymatic solution, mix well, adjust the pH to 6-7, inoculate lactic acid bacteria in the logarithmic growth phase, the inoculation amount of lactic acid bacteria is 8-10%, ferment at 20-30°C for 72 hours to obtain fermentation liquid.
[0014] Step 4: Fermentation
[0015] The fermentation liquid is filtered and washed 2 to 3 times, and the separated solid is chitosan.
[0016] According to the green preparation method of chitosan based on shrimp shells of the present invention, in the step 1, when adding purified water and supplementary water, the water level is added to cover the shrimp shell powder.
[0017] According to the green preparation method of chitosan based on shrimp shells of the present invention, in the step 2, the pH value of the entire enzymatic hydrolysis process is controlled under weak alkaline conditions.
[0018] According to the green preparation method of chitosan based on shrimp shells of the present invention, in the step three, the culture medium components include sodium acetate, sucrose, diammonium hydrogen citrate, magnesium sulfate, dipotassium hydrogen phosphate, manganese sulfate and Tween 80.
[0019] According to the green preparation method of chitosan based on shrimp shells of the present invention, in step 4, the calcium in the filtrate after filtering the fermentation liquid is separated by chemical precipitation, and the remaining filtrate is amino acids and proteins.
[0020] According to the green preparation method of chitosan based on shrimp shells of the present invention, during the fermentation process of step three, stirring is performed once every 12 hours.
[0021] The object of the present invention is to provide a green preparation method of chitosan based on shrimp shells, by subjecting shrimp shell powder to enzymatic hydrolysis, inactivation, fermentation and separation steps, making full use of components such as protein and amino acids therein for microbial fermentation, reducing fermentation costs and improving resource utilization efficiency; using facultative anaerobic microorganisms for fermentation, without the need for sterilization and oxygenation, significantly reducing energy consumption and saving costs. In summary, the beneficial effects of the present invention are: it uses facultative anaerobic microorganisms for fermentation, without the need for sterilization and oxygenation, significantly reducing energy consumption and saving costs, making full use of components such as protein and amino acids therein for microbial fermentation, reducing fermentation costs and improving resource utilization efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 is a process flow chart of the present invention; DETAILED DESCRIPTION
[0023] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0024] In the description of the present invention, it should be noted that the terms "upper", "lower", "inside", "outside", "top / bottom" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.
[0025] In the description of the present invention, it should be noted that, unless otherwise clearly stipulated and limited, the terms "installed", "provided with", "mounted / connected", "connected", etc. should be understood in a broad sense. For example, "connected" can be a fixed connection, a detachable connection, or an integral connection. It can be a mechanical connection or an electrical connection. It can be a direct connection or an indirect connection through an intermediate medium. It can be the internal connection of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.
[0026] See also Figure 1 The present invention provides a green preparation method of chitosan based on shrimp shells, comprising the following steps:
[0027] Step 1: Prepare the ingredients
[0028] The shrimp shells are crushed and passed through a 50-mesh sieve. The shrimp shells that cannot pass through are poured back into the grinder for crushing. Pure water is added to just cover the shrimp shell powder for moistening. After 2 to 3 hours, water is added to cover the shrimp shell powder to obtain the shrimp shell powder raw material.
[0029] Step 2: Enzymatic treatment
[0030] The shrimp shell powder raw material is enzymolyzed with 0.2% lipase for 0.8-1.2 hours, and then deproteinized with 0.2% protease for 0.8-1.2 hours. The pH value of the entire enzymolysis process is controlled under weak alkaline conditions. After the enzymolysis, the enzyme is inactivated by heating to 50-52° C. for 10-12 minutes to obtain an enzymolysis solution.
[0031] Step 3: Fermentation
[0032] Sodium acetate, sucrose, diammonium citrate, magnesium sulfate, dipotassium hydrogen phosphate, manganese sulfate, and Tween 80 are added (the amount added is determined according to the amount of fermentation liquid and the formula of MRS liquid culture medium, and the specific amount added is a mature existing technology and is not repeated here), first partially dissolved in water, then purified water is added to twice the amount of the enzymatic solution, mixed evenly, the pH is adjusted to 6-7, and lactic acid bacteria in the logarithmic growth phase (isolated from rotten shrimp shells in this laboratory) are inoculated with an inoculation amount of 8-10%, and a sealed container is fermented at 20-30° C. for 72 hours, wherein stirring is performed once every 12 hours to obtain a fermentation liquid.
[0033] Step 4: Fermentation
[0034] The fermentation liquid is filtered and washed 2 to 3 times. The separated solid is chitosan. The calcium in the filtrate is separated by chemical precipitation, and the remaining filtrate is amino acids, proteins and other substances.
[0035] To verify the above preparation method, the present invention provides the following examples:
[0036] Example 1
[0037] The shrimp shells are crushed and passed through a 50-mesh sieve. The shrimp shells that cannot pass through are poured back into the grinder for crushing. Pure water is added to just cover the shrimp shell powder for moistening. After 2 hours, water is added to cover the shrimp shell powder to obtain the shrimp shell powder raw material.
[0038] The shrimp shell powder raw material is enzymatically hydrolyzed with 0.2% lipase for 0.8 hours, and then deproteinized with 0.2% protease for 0.8 hours. The pH value of the entire enzymatic hydrolysis process is controlled under weak alkaline conditions. After the enzymatic hydrolysis, it is heated to 50° C. to inactivate the enzyme for 10 minutes to obtain an enzymatic hydrolyzate.
[0039] Sodium acetate, sucrose, diammonium citrate, magnesium sulfate, dipotassium hydrogen phosphate, manganese sulfate, and Tween 80 were added, first partially dissolved in water, then purified water was added to twice the amount of the enzymatic solution, mixed evenly, the pH was adjusted to 6, and lactic acid bacteria in the logarithmic growth phase (isolated from rotten shrimp shells in this laboratory) were inoculated at an inoculation rate of 8%. The container was sealed and fermented at 20°C for 72 hours, with stirring once every 12 hours, to obtain a fermentation liquid.
[0040] The fermentation liquid is filtered and washed twice. The separated solid is chitosan. The calcium in the filtrate is separated by chemical precipitation, and the remaining filtrate is amino acids, proteins and other substances.
[0041] Example 2
[0042] The shrimp shells are crushed and passed through a 50-mesh sieve. The shrimp shells that cannot pass through are poured back into the grinder for crushing. Pure water is added to just cover the shrimp shell powder for moistening. After 2.5 hours, water is added to cover the shrimp shell powder to obtain the shrimp shell powder raw material.
[0043] The shrimp shell powder raw material was enzymolyzed with 0.2% lipase for 1 hour, and then deproteinized with 0.2% protease for 1 hour. The pH value of the entire enzymolysis process was controlled under weak alkaline conditions. After the enzymolysis, the enzyme was inactivated by heating to 51° C. for 11 minutes to obtain an enzymolysis solution.
[0044] Sodium acetate, sucrose, diammonium citrate, magnesium sulfate, dipotassium hydrogen phosphate, manganese sulfate, and Tween 80 were added, first partially dissolved in water, then purified water was added to twice the amount of the enzymatic solution, mixed evenly, the pH was adjusted to 6.5, and lactic acid bacteria in the logarithmic growth phase (isolated from rotten shrimp shells in this laboratory) were inoculated at an inoculation rate of 9%. The mixture was placed in a sealed container and fermented at 25°C for 72 hours, with stirring once every 12 hours, to obtain a fermentation liquid.
[0045] The fermentation liquid is filtered and washed three times. The separated solid is chitosan. The calcium in the filtrate is separated by chemical precipitation, and the remaining filtrate is amino acids, proteins and other substances.
[0046] Example 3
[0047] The shrimp shells are crushed and passed through a 50-mesh sieve. The shrimp shells that cannot pass through are poured back into the grinder for crushing. Pure water is added to just cover the shrimp shell powder for moistening. After 3 hours, water is added to cover the shrimp shell powder to obtain the shrimp shell powder raw material.
[0048] The shrimp shell powder raw material was enzymolyzed with 0.2% lipase for 1.2 hours, and then deproteinized with 0.2% protease for 1.2 hours. The pH value of the entire enzymolysis process was controlled under weak alkaline conditions. After the enzymolysis, the enzyme was inactivated by heating to 52° C. for 12 minutes to obtain an enzymolysis solution.
[0049] Sodium acetate, sucrose, diammonium citrate, magnesium sulfate, dipotassium hydrogen phosphate, manganese sulfate, and Tween 80 were added, first partially dissolved in water, then purified water was added to twice the amount of the enzymatic solution, mixed evenly, the pH was adjusted to 7, and lactic acid bacteria in the logarithmic growth phase (isolated from rotten shrimp shells in this laboratory) were inoculated at an inoculation rate of 10%. The mixture was placed in a sealed container and fermented at 30°C for 72 hours, with stirring once every 12 hours, to obtain a fermentation liquid.
[0050] The fermentation liquid is filtered and washed three times. The separated solid is chitosan. The calcium in the filtrate is separated by chemical precipitation, and the remaining filtrate is amino acids, proteins and other substances.
[0051] The present invention provides a green preparation method of chitosan based on shrimp shells. By subjecting shrimp shell powder to enzymatic hydrolysis, inactivation, fermentation and separation, etc., the components such as protein and amino acids therein are fully utilized for microbial fermentation, thereby reducing the fermentation cost and improving the resource utilization efficiency. The facultative anaerobic microorganisms are used for fermentation, without the need for sterilization and oxygenation, which significantly reduces energy consumption and saves costs. In summary, the beneficial effects of the present invention are as follows: the facultative anaerobic microorganisms are used for fermentation, without the need for sterilization and oxygenation, which significantly reduces energy consumption and saves costs. The components such as protein and amino acids therein are fully utilized for microbial fermentation, thereby reducing the fermentation cost and improving the resource utilization efficiency.
[0052] Of course, the present invention may have many other embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art may make various corresponding changes and modifications based on the present invention, but these corresponding changes and modifications should all fall within the scope of protection of the claims attached to the present invention.
Claims
1. A green preparation method of chitosan based on shrimp shells, characterized in that: The steps include: Step 1: Prepare the ingredients The shrimp shells are crushed, sieved, and purified water is added, and water is added after 2 to 3 hours to cover the shrimp shell powder to obtain a shrimp shell powder raw material; Step 2: Enzymatic treatment The shrimp shell powder raw material is enzymatically hydrolyzed with 0.2% lipase for 0.8 to 1.2 hours, and then deproteinized with 0.2% protease for 0.8 to 1.2 hours, and after the enzymatic hydrolysis, the enzyme is inactivated by heating to 50 to 52° C. for 10 to 12 minutes to obtain an enzymatic solution; Step 3: Fermentation Adding culture medium components, adding purified water to twice the amount of the enzymatic solution, mixing evenly, adjusting the pH to 6-7, inoculating lactic acid bacteria in the logarithmic growth phase, the inoculation amount of lactic acid bacteria is 8-10%, fermenting at 20-30° C. for 72 hours, and obtaining a fermentation liquid; Step 4: Fermentation The fermentation liquid is filtered and washed 2 to 3 times, and the separated solid is chitosan.
2. The green preparation method of chitosan based on shrimp shells according to claim 1, characterized in that: In the step 1, when adding purified water and supplementary water, the water level should be added until the shrimp shell powder is covered.
3. The green preparation method of chitosan based on shrimp shells according to claim 1, characterized in that: In the step 2, the pH value of the entire enzymatic hydrolysis process is controlled under weak alkaline conditions.
4. The green preparation method of chitosan based on shrimp shells according to claim 1, characterized in that: In step 3, the culture medium components include sodium acetate, sucrose, diammonium hydrogen citrate, magnesium sulfate, dipotassium hydrogen phosphate, manganese sulfate and Tween 80.
5. The green preparation method of chitosan based on shrimp shells according to claim 1, characterized in that: In the step 4, the calcium in the filtrate after the fermentation liquid is filtered is separated by chemical precipitation, and the remaining filtrate is amino acids and proteins.
6. The green preparation method of chitosan based on shrimp shells according to claim 1, characterized in that: During the fermentation process of step 3, stirring is performed once every 12 hours.