Differential viscosity-average molecular weight chitosan from shrimp and crab shell, complex, and preparation method and application thereof

Abstract

The patent discloses a differentiated viscosity average molecular weight chitosan from shrimp and crab shells, a compound and a preparation method and application thereof, and comprises the following steps: mixing shrimp and crab shell powder with a desalination solution, then performing microwave heating by using a depigmentation solution, repeatedly freezing and thawing by using a deproteinization solution and a urea solution, and obtaining three-deacetylated chitin after drying treatment; mixing the three-deacetylated chitin with a deacetylation solution, obtaining a deacetylation product by ultrasonic treatment, and obtaining a macromolecular weight chitosan by washing with pure water and drying treatment; dissolving the macromolecular weight chitosan in dilute acetic acid, adding a hydrogen peroxide solution, and obtaining a differentiated viscosity average molecular weight chitosan powder after post-treatment. The technical problems of difficulty in accurately controlling the viscosity average molecular weight distribution, poor uniformity of product physicochemical properties and complex compound preparation process in the prior art are solved, and an innovative path is provided for efficient utilization of marine resources and ecological protection.

Description

Technical Field

[0001] This invention relates to the field of chitosan preparation technology, specifically to a chitosan with differentiated viscosity-average molecular weight derived from shrimp and crab shells, a complex thereof, its preparation method and application, particularly to International Patent Classification C08B37 / 08 (chitosan; chondroitin sulfate; hyaluronic acid; derivatives thereof). Background Technology

[0002] Chitosan (CS) is a linear polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine, formed by the chemical or enzymatic deacetylation of chitin or chitin. As a natural biopolymer, the molecular weight and degree of deacetylation of chitosan are core parameters determining its performance and applications, directly influencing its solubility, reactivity, and biocompatibility at the structural level. High molecular weight chitosan (HMWCS, greater than 300 kDa) has long molecular chains, strong intermolecular interactions, poor solubility in weak acids, and a tendency to entangle into viscous systems. Low molecular weight chitosan (LMWCS, less than 200 kDa), on the other hand, exhibits superior solubility and flowability, is easily absorbed and utilized, and can be used as a drug delivery carrier to improve bioavailability. Furthermore, unlike high molecular weight chitosan, low molecular weight chitosan composites still suffer from disadvantages such as weak mechanical strength, poor resistance to degradation, low functional group density, and difficulty in modification.

[0003] Deacetylation degree, as another indicator, reflects the content of free amino groups in chitosan molecules, thus affecting the charge-carrying properties of chitosan. For example, high-deacetylation degree chitosan (HDACS, greater than 80%) and low-deacetylation degree chitosan (LDACS) can protonate more free amino groups under acidic conditions, easily forming "strongly positively charged" molecules. Therefore, regulating the acetylation value of chitosan is beneficial for promoting its functionalization in various life processes to achieve "mild regulation" or "low-irritation protection." Environmental researchers often use low molecular weight, low-acetylation degree chitosan to prepare natural impedance coatings with low swelling, low porosity, and high corrosion resistance. The food industry selects chitosans of different molecular weights and acetylation degrees as mixed raw materials, and through different system ratios, changes the density distribution of functional groups on the chitosan molecular chain, improving the reactivity with multiple raw materials, and thus achieving functional compatibility between systems. In short, the processing, regulation, and design of chitosan raw materials have created positive conditions for realizing and expanding the functionalization of chitosan-based biomaterials. Similarly, in the field of medical device manufacturing, precisely controlled chitosan raw materials can achieve "customized design" for key properties when preparing chitosan-based composite materials, such as the viscoelasticity and crosslinking design of the composite material, in order to meet the special needs of different medical scenarios.

[0004] Shrimp and crab shells are a common waste product in aquaculture, rich in chitin, and chitosan is an important derivative of chitin. However, in existing technologies, chitosan preparation generally uses the chitin-containing shells of shrimp, crabs, or insects as raw materials. The process mainly includes a series of steps such as decalcification with dilute acid → deproteinization by cooking with dilute alkali under heterogeneous conditions → oxidative decolorization → deacetylation by cooking with concentrated alkali under heterogeneous conditions → filtration → washing → drying. However, the vigorous reaction of concentrated alkali under cooking conditions leads to uncontrollable damage to the main chain of high molecular weight chitosan. Therefore, how to extract chitosan with differentiated viscosity-average molecular weight from shrimp and crab shells, and how to use this differentiated viscosity-average molecular weight chitosan for post-processing to obtain complexes for efficient utilization in the field of biomedical materials, thereby realizing the product transformation of marine waste, is an urgent problem to be solved. Summary of the Invention

[0005] The purpose of this invention is to extract chitosan with differentiated viscosity-average molecular weight from shrimp and crab shells, solving the technical problems in existing technologies such as the difficulty in precisely controlling the viscosity-average molecular weight distribution, poor uniformity of physicochemical properties, and complex complex preparation processes when preparing chitosan from biomass raw materials. It also aims to obtain polysaccharide derivatives with specific viscosity-average molecular weight ranges and excellent microstructures, providing an innovative path for the efficient utilization and ecological protection of marine resources. This will enable the prepared chitosan and its complexes to serve as high-quality natural polymer matrix materials, widely applicable in fields such as biomedical materials, environmental adsorbents, and fine chemical raw materials.

[0006] To solve this technical problem:

[0007] This patent provides a differentiated viscosity-average molecular weight chitosan derived from shrimp and crab shells, which is prepared by the following method:

[0008] Step A: Mix shrimp and crab shell powder with desalination solution, wash with pure water to remove mineral salts from the shrimp and crab shells; then microwave heat with depigmentation solution, repeatedly freeze and thaw with deproteinization solution and urea solution, and dry to obtain tri-de-chitosan;

[0009] Step B: Mix the tri-dechitosan with the deacetylation solution, sonicate to obtain the deacetylated product, wash with pure water and dry to obtain high molecular weight chitosan.

[0010] Step C: Dissolve high molecular weight chitosan in dilute acetic acid, then add hydrogen peroxide solution, and after post-processing, obtain chitosan powder with differentiated viscosity-average molecular weight.

[0011] Furthermore, the differentiated viscosity-average molecular weight chitosan is composed of high molecular weight chitosan, medium molecular weight chitosan, and low molecular weight chitosan.

[0012] Furthermore, high molecular weight chitosan has a molecular weight greater than 1.40 × 10⁻⁶. 6 Da; Medium molecular weight chitosan has a molecular weight between 1.00 × 10⁻⁶. 6 Up to 1.40×10 6 Between Da; the molecular weight of low molecular weight chitosan is less than 1.00 × 10⁻⁶. 6 Da.

[0013] Furthermore, in step A, the mass-to-volume ratio of shrimp and crab shell powder to desalting solution is 1:(10~12), 1:(12~14), or 1:(14~15), and the desalting solution is hydrochloric acid solution with a concentration of 8~10%.

[0014] Furthermore, the concentration of the decolorizing solution is 3-5%, and the decolorizing solution includes hydrogen peroxide solution or potassium permanganate solution; the microwave heating temperature is 60-65℃, and the microwave heating time is 2-4 hours.

[0015] Furthermore, the deproteinizing solution includes a strong alkaline solution and a urea solution. The strong alkaline solution includes one or more combinations of sodium hydroxide solution, potassium hydroxide solution, or sodium hydroxide solution, and the number of freeze-thaw cycles is 2 to 3.

[0016] Furthermore, the drying temperature is 60~70℃, and the drying time is 2~4 hours.

[0017] Furthermore, in step B, the mass-to-volume ratio of tridechitoxin to the deacetylation solution is 1:(10~12), 1:(12~15), or 1:(15~20), and the deacetylation solution is a sodium hydroxide solution with a concentration of 50~60%.

[0018] Furthermore, in step C, the mass-to-volume ratio of high molecular weight chitosan to dilute acetic acid is 1:50.

[0019] Furthermore, the mass-to-volume ratio of high molecular weight chitosan to hydrogen peroxide solution is 1:1, and the concentration of hydrogen peroxide solution is 30-40%.

[0020] Further post-treatment includes water bath treatment, dialysis treatment, and drying treatment; the rejection rate for dialysis treatment is 2000~4000 Da.

[0021] Another aspect of this patent provides the application of chitosan with differentiated viscosity-average molecular weight derived from shrimp and crab shells in biomedical materials or environmental adsorption materials.

[0022] Furthermore, applications in biomedical materials include medical dressings; medical dressings are in powder or gel form and are used for wound healing.

[0023] This patent also provides a complex comprising sodium β-glycerophosphate and chitosan of differential viscosity-average molecular weight derived from shrimp and crab shells.

[0024] Furthermore, sodium β-glycerophosphate and chitosan with differential viscosity-average molecular weight derived from shrimp and crab shells can undergo a physical cross-linking reaction under ice bath conditions.

[0025] Another aspect of this patent provides the application of a composite material in biomedical materials or environmental adsorption materials.

[0026] Furthermore, applications in biomedical materials include the use of viscoelastic agents, which are biomimetic joint lubricants used to promote the repair of articular cartilage damage and / or treat inflammation.

[0027] This patent also provides a method for preparing a complex, comprising the following steps:

[0028] Step A: Dissolve the chitosan with differentiated viscosity-average molecular weight in hydrochloric acid solution to obtain a chitosan solution;

[0029] Step B: Add the chitosan solution dropwise to the pre-cooled sodium β-glycerophosphate solution while stirring to obtain a mixture;

[0030] Step C: The mixture is freeze-dried under vacuum to obtain the complex.

[0031] Furthermore, in step A, the concentration of hydrochloric acid is 0.1~0.3 M, and the concentration of chitosan solution is 15.0~25.0 mg / mL.

[0032] Furthermore, in step B, the concentration of the pre-cooled sodium β-glycerophosphate solution is 60~100 mg / mL, the volume ratio of sodium β-glycerophosphate solution to chitosan solution is 1:(1.25~2.00), 1:(2.00~3.50), 1:(3.50~5.00), 1:(5.00~8.00), and the stirring time is 35~45 min.

[0033] Furthermore, in step C, the vacuum freeze-drying temperature is -60~-70℃, and the vacuum freeze-drying time is 24~48 h.

[0034] Compared with existing technologies, the present application provides a differentiated viscosity-average molecular weight chitosan derived from shrimp and crab shells, a complex thereof, its preparation method, and its application, which have the following beneficial effects:

[0035] (1) This application uses the alkali-freeze-thaw method and the H2O2 method to extract chitosan raw materials with different molecular weights and acetyl values ​​from the shells of peeled and molted shrimp and crabs. The method is green and environmentally friendly, with low chemical residues, high purity of raw materials, narrow distribution of core parameters, low processing cost, and easy control of reaction conditions. It can achieve large-scale production and is conducive to solving the problem of biological resource utilization of molted shrimp and crab shell waste in aquaculture. At the same time, it is easy to achieve precise control of chitosan molecular weight efficiently.

[0036] (2) The chitosan-β-glycerophosphate sodium complex prepared in this application uses low, medium, high molecular weight or "full molecular weight" chitosan as raw material, which breaks the limitation of traditional single molecular weight and can complete product design according to local conditions, forming products with different viscosity and elasticity.

[0037] (3) Chitosan-β-glycerophosphate sodium complex can also be used in the field of biomedical materials as a viscoelastic agent to promote the repair of articular cartilage damage and / or treat inflammation. Attached Figure Description

[0038] The above description of the present invention and the following detailed embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are merely examples of the claimed technical solutions.

[0039] Figure 1 For the 1.36 x 10 in this patent 6 The characteristic viscosity versus concentration curve of chitosan with different viscosity-average molecular weight (where the horizontal axis represents concentration in g / mL; the vertical axis represents the ratio of characteristic viscosity to concentration).

[0040] Figure 2 The Fourier transform infrared spectrum of chitosan, sodium β-glycerophosphate, and the chitosan-β-glycerophosphate complex in Example 4 of this patent is shown below (the horizontal axis represents wavenumber, and the unit is cm⁻¹). -1 ));

[0041] Figure 3 The shear viscosity curves of different chitosan-β-glycerophosphate sodium complexes in Example 4 of this patent are shown below (where the horizontal axis represents the shear rate, and the unit is (s)). -1 The vertical axis represents viscosity, in mPa·s.

[0042] Figure 4 The following are the dynamic strain scanning curves of different chitosan-β-glycerophosphate sodium complexes in Example 4 of this patent (where the horizontal axis is shear strain γ, in (%); and the vertical axis is energy storage / dissipation modulus, in (mPa)).

[0043] Figure 5The loss factor (tanδ) strain curve of the chitosan-β-glycerophosphate sodium complex in Example 4 of this patent is shown (where the horizontal axis is shear strain in (%) and the vertical axis is loss factor tanδ). Detailed Implementation

[0044] The detailed features and advantages of this application are described below in the specific embodiments. The content of this description is sufficient to enable any person skilled in the art to understand the technical content of this application and implement it accordingly. Based on the specification, claims and drawings disclosed in this specification, a person skilled in the art can easily understand the related objectives and advantages of this application.

[0045] In this specification and claims, several terms will be used, and unless otherwise indicated, these terms will be defined to have the following meanings:

[0046] As used in this patent, both "shrimp shells" and "crab shells" are commercially available and are obtained through manual peeling and natural molting, and are rich in 15-30% chitin (also known as chitin).

[0047] As used in this patent, "differentiated viscosity-average molecular weight chitosan" refers to chitosan prepared through a specific process and having a specific viscosity-average molecular weight distribution. Its molecular weight is designed in a gradient to cover multiple ranges such as high, medium, and low, thereby achieving functional differentiation within a certain range; this product is clearly distinguished from a simple physical mixture of high and low molecular weight chitosan.

[0048] As used in this patent, "high molecular weight chitosan" has a molecular weight greater than 1.40 × 10⁻⁶. 6 Da.

[0049] As used in this patent, "medium molecular weight chitosan" has a molecular weight between 1.00 × 10⁻⁶. 6 Up to 1.40 × 10 6 Between Da and 1.00 × 10 6 Da and 1.40 × 10 6 Da.

[0050] As used in this patent, "low molecular weight chitosan" has a molecular weight of less than 1.00 × 10⁻⁶. 6 Da.

[0051] As used in this patent, the "linear viscoelastic region" (LVR) refers to the range in which stress or strain exhibits a linear relationship in dynamic mechanical analysis (DMA) or rheological testing. It is also the range in which the viscoelastic response of a material conforms to the linear viscoelastic constitutive relationship of the superposition of Hooke's Law (elasticity) and Newton's law of viscosity (viscosity).

[0052] All other terms used herein for special definition are intended to have the general meaning understood by one of ordinary skill in the art, and in particular, meaning that one of ordinary skill in the art, upon reading the claims, specification and drawings of this patent, can directly and without doubt determine how the technical solution of this patent can be implemented.

[0053] Even if there are incomplete descriptions, omissions, or ambiguities in the grammar, words, punctuation, graphics, symbols, etc. of the claims, specification, and drawings of this patent, a person skilled in the art can still arrive at the only correct understanding by reading the claims, specification, and drawings as a whole without extensive reasoning or experimentation, and effectively exclude various incorrect interpretations that are not aimed at achieving the purpose of this patent.

[0054] The "range" disclosed herein is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0055] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0056] Unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0057] Unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0058] Unless otherwise specified, the terms "comprising" and "including" as used herein can be open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included.

[0059] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0060] In this description, unless otherwise stated, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0061] Unless otherwise specified, percentages (%) in this document refer to percentages by mass relative to the composition.

[0062] Unless otherwise stated herein, the sum of the contents of the components in the composition is 100%.

[0063] In this document, unless otherwise stated, “combination of” means a multi-component mixture of the elements, such as two, three, four, and up to the maximum possible multi-component mixture.

[0064] Unless otherwise specified, the term "a" as used in this specification means "at least one".

[0065] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention.

[0067] To make the objectives, technical solutions and advantages of the present invention clearer, the following will further describe in detail the embodiments of the present invention with reference to the accompanying drawings. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0068] (1)Source of reagents and consumables

[0069] Table 1 Sources and catalog numbers of reagents and consumables required for experiments

[0070]

[0071] (2)Source of instruments and equipment

[0072] Table 2 Sources and models of instruments and equipment required for experiments

[0073]

[0074] Example 1

[0075] Prepare chitosan with different viscosity-average molecular weights, and the specific steps are as follows:

[0076] (1)Wash the raw material shrimp shells with pure water, place the washed shrimp shells in an oven for the first drying. The conditions for the first drying are drying at 60 °C for 4 h. Place the dried shrimp shells in a pulverizer for pulverization, and collect them after passing through a 400-mesh sieve, thus obtaining fine shrimp shell powder.

[0077] (2)Add a hydrochloric acid solution with a concentration of 8% (abbreviated as hydrochloric acid solution) to the shrimp shell powder prepared in the above step (1). The mass-volume ratio of the shrimp shell powder to the hydrochloric acid solution is 1:15; that is, weigh 50 g of the shrimp shell powder obtained in the above step (1), add 750 mL of a hydrochloric acid solution with a concentration of 8%, stir evenly, and place it in a microwave reactor. React at 60 °C for hours to fully remove the mineral salts in the shrimp shell powder, and obtain the first mixture.

[0078] (3) Place the first mixture in step (2) above on a 400-mesh sieve and repeatedly rinse the first wet powder remaining on the sieve with pure water until the pH of the first wet powder is neutral at 6.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the first wet powder can also be 6.5~7.5). Collect the first wet powder with neutral pH using the sieve and place it in an oven for a second drying. The second drying condition is drying at 60℃ for 4 h to obtain dried desalted shrimp shell powder.

[0079] (4) Add a 10% sodium hydroxide solution to the dried desalted shrimp shell powder prepared in step (3) above. The mass-volume ratio of the dried desalted mixed powder to the 10% sodium hydroxide solution is 1:14. After stirring evenly, place it in a microwave reactor and react at 60°C for 4 h to fully remove the impurities in the desalted shrimp shell powder and obtain the second mixture.

[0080] (5) Place the second mixture from step (4) above on a 400-mesh sieve again, and repeatedly rinse the second wet powder remaining on the sieve with pure water until the pH of the second wet powder is neutral at 6.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the second wet powder can also be 6.5~7.5), and collect the second wet powder with neutral pH using the sieve.

[0081] (6) Add a 3% hydrogen peroxide solution to the second wet powder prepared in step (5) above. The mass-volume ratio of the second wet powder to the 3% hydrogen peroxide solution is 1:20. Adjust the pH to 9.0~10.0 using a 10% sodium hydroxide solution. Then place it in a microwave reactor and react at 60°C for 4 h to obtain the third mixture.

[0082] (7) After the third mixture is cooled to room temperature, add an appropriate amount of pure water to dilute it and adjust the pH to 7.5~8.0. Then add urea solution to the system to make the final urea concentration 5%. Stir and mix for 30~40 min to obtain the fourth mixture.

[0083] (8) In order to break the intermolecular forces of the chitosan macromolecular chain and control the degree of breakage, the fourth mixture obtained in step (7) above was frozen at -20°C. One blank control group (not frozen) and four experimental groups with repeated freeze-thaw cycles (Group 1, Group 2, Group 3 and Group 4) were set up. Repeated freeze-thaw cycles refer to two or more freeze-thaw operations. For example, repeated freeze-thaw cycles are: the fourth mixture is frozen at -20°C and then transferred to room temperature. After the fourth mixture is completely thawed, the fourth mixture is frozen at -20°C and then transferred to room temperature again until the fourth mixture is completely thawed.

[0084] Among them, the first group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed twice; the second group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed three times; the third group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed twice; and the fourth group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed three times.

[0085] The mixtures obtained from the control group and the experimental group were placed on a 400-mesh sieve and repeatedly washed under flowing ultrapure water until the pH was neutral at 6.5 (pH = 6.5~7.5 is defined as neutral here; in some other specific embodiments, the repeated washing can also be repeated until the pH is 6.5~7.5). The fourth wet powder with neutral pH was collected and placed in an oven for a fourth drying. The fourth drying condition was drying at 60°C for 4 h to obtain different groups of tri-dechitoxin.

[0086] (9) Add 50% sodium hydroxide solution to the different groups of tri-dechitoxin prepared in step (8) above. The mass-volume ratio of tri-dechitoxin to 50% sodium hydroxide solution is 1:10. After stirring evenly, place in an ultrasonic crusher and continue ultrasonic reaction at 200 W power for 3 h under constant temperature of 75℃ to fully remove acetyl groups and obtain the fifth mixture of different groups, which are the deacetylated products of different groups.

[0087] (10) Place the deacetylated products of different groups obtained in step (9) above on a 2000-mesh sieve and wash them repeatedly under flowing ultrapure water until the pH is neutral 6.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH can also be 6.5~7.5 after repeated washing). Collect the fifth wet powder with neutral pH and place it in an oven for the fifth drying. The fifth drying condition is drying at 60°C for 5 h to obtain powdered high molecular weight chitosan of different groups.

[0088] (11) Weigh out portions of the different groups of high molecular weight chitosan prepared in step (10) above, add 1% dilute acetic acid, and prepare a 2% chitosan / acetic acid solution (w / v) by mass-volume ratio of the different groups of high molecular weight chitosan to the added 1% dilute acetic acid at a concentration of 1:50, so that the high molecular weight chitosan is completely dissolved in 1% dilute acetic acid; then add 30% hydrogen peroxide solution according to the mass-volume ratio of chitosan to 30% hydrogen peroxide solution in the 2% chitosan / acetic acid solution at a concentration of 1:1, and place in a water bath at 60℃ for 5 h to obtain different groups of differential viscosity-average molecular weight chitosan solutions.

[0089] (12) Place the different groups of differentiated viscosity-average molecular weight chitosan solutions obtained in step (11) into dialysis bags with a cutoff of 2000~4000 Da. Then, place the dialysis bags containing the different groups of differentiated viscosity-average molecular weight chitosan solutions into pure water and dialyze at 4°C for 7~9 days until the solution in the dialysis bag is neutral. The dialysis is then completed when the pH value of the solution after dialysis is 6.0~7.5, and the dialysate is obtained. The dialysate is then dried for the sixth time at 60°C for 6 hours to obtain the different groups of differentiated viscosity-average molecular weight chitosan powder.

[0090] Example 2

[0091] (1) Wash the raw shrimp shells with pure water, place the washed shrimp shells in an oven for the first drying, and dry them at 70°C for 2 hours. Then, place the dried shrimp shells in a pulverizer for pulverization and collect them after passing through a 400-mesh sieve to obtain fine shrimp shell powder.

[0092] (2) Add a 10% hydrochloric acid solution (hereinafter referred to as hydrochloric acid solution) to the shrimp shell powder prepared in step (1) above. The mass-volume ratio of the mixed powder to the hydrochloric acid solution is 1:10. That is, weigh 50 g of the shrimp shell powder obtained in step (1) above, add 500 mL of 10% hydrochloric acid solution, stir evenly, place in a microwave reactor, and react at 65°C for 2 h to fully remove the mineral salts in the shrimp shell powder and obtain the first mixture.

[0093] (3) Place the first mixture in step (2) above on a sieve with a 400 mesh screen, and repeatedly rinse the first wet powder remaining on the sieve with pure water until the pH of the first wet powder is neutral 7.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the first wet powder can also be 6.5~7.5). Collect the first wet powder with neutral pH using the sieve and place it in an oven for a second drying. The second drying condition is drying at 70℃ for 2 h to obtain dried desalted shrimp shell powder.

[0094] (4) Add a 10% sodium hydroxide solution to the dried desalted shrimp shell powder prepared in step (3) above. The mass-volume ratio of the dried desalted shrimp shell powder to the 10% sodium hydroxide solution is 1:18. After stirring evenly, place it in a microwave reactor and react at 70°C for 2 h to fully remove the impurities in the desalted shrimp shell powder and obtain the second mixture.

[0095] (5) Place the second mixture from step (4) above on a 400-mesh sieve again, and rinse the second wet powder remaining on the sieve with pure water until the pH of the second wet powder is neutral at 7.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the second wet powder can also be 6.5~7.5), and collect the second wet powder with neutral pH using the sieve.

[0096] (6) Add a 5% hydrogen peroxide solution to the second wet powder prepared in step (5) above. The mass-volume ratio of the second wet powder to the 5% hydrogen peroxide solution is 1:25. Adjust the pH to 9.0~10.0 using a 10% sodium hydroxide solution. Then place it in a microwave reactor and react at 70°C for 2 h to obtain the third mixture.

[0097] (7) After the third mixture is cooled to room temperature, add an appropriate amount of pure water to dilute it and adjust the pH to 7.5~8.0. Then add urea solution to the system to make the final urea concentration 5%. Stir and mix for 30~40 min to obtain the fourth mixture.

[0098] (8) In order to break the intermolecular forces of the chitosan macromolecular chain and control the degree of breakage, the fourth mixture obtained in step (7) above was frozen at -20°C. One blank control group (not frozen) and four experimental groups with repeated freeze-thaw cycles (Group 1, Group 2, Group 3 and Group 4) were set up. Repeated freeze-thaw cycles refer to two or more freeze-thaw operations. For example, repeated freeze-thaw cycles are: the fourth mixture is frozen at -20°C and then transferred to room temperature. After the fourth mixture is completely thawed, the fourth mixture is frozen at -20°C and then transferred to room temperature again until the fourth mixture is completely thawed.

[0099] Among them, the first group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed twice; the second group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed three times; the third group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed twice; and the second group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed three times.

[0100] The mixtures obtained from the control group and the experimental group were placed on a 400-mesh sieve and repeatedly washed under flowing ultrapure water until the pH was neutral 7.5 (pH = 6.5~7.5 is defined as neutral here; in some other specific embodiments, the repeated washing can also be to pH 6.5~7.5). The fourth wet powder with neutral pH was collected and placed in an oven for a fourth drying. The fourth drying condition was drying at 70°C for 2 h to obtain different groups of tri-dechitoxin.

[0101] (9) Add 50% sodium hydroxide solution to the different groups of tri-dechitoxin prepared in step (8) above. The mass-volume ratio of tri-dechitoxin to 50% sodium hydroxide solution is 1:20. After stirring evenly, place in an ultrasonic crusher and continue ultrasonic reaction at 300 W power for 2 h under constant temperature of 75℃ to fully remove acetyl groups and obtain the fifth mixture of different groups, which are the deacetylated products of different groups.

[0102] (10) Place the deacetylated products of different groups prepared in step (9) above on a 2000-mesh sieve and wash them repeatedly under flowing ultrapure water until the pH is neutral 7.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH can also be 6.5~7.5 after repeated washing). Collect the fifth wet powder with neutral pH and place it in an oven for the fifth drying. The fifth drying condition is drying at 70°C for 3 h to obtain powdered high molecular weight chitosan of different groups.

[0103] (11) Weigh out portions of the different groups of high molecular weight chitosan prepared in step (10) above, add 2% dilute acetic acid, and prepare a 2% chitosan / acetic acid solution (w / v) by mass-volume ratio of different groups of high molecular weight chitosan to 2% dilute acetic acid of 1:50, so that the high molecular weight chitosan is completely dissolved in 2% dilute acetic acid; then add 30% hydrogen peroxide solution according to the mass-volume ratio of chitosan to 30% hydrogen peroxide solution in the 2% chitosan / acetic acid solution of 1:1, and place in a water bath at 70°C for 2 h to obtain different groups of viscosity-average molecular weight chitosan solutions.

[0104] (12) Place the different groups of differentiated viscosity-average molecular weight chitosan solutions obtained in step (11) into dialysis bags with a cutoff of 2000~4000 Da, and then place the dialysis bags containing the different groups of differentiated viscosity-average molecular weight chitosan solutions into pure water. Dialyze at 4°C for 7~9 days until the solution in the dialysis bag is neutral. The dialysis is then completed when the pH value of the solution after dialysis is 6.0~7.5, and the dialysate is obtained. The dialysate is then dried for the sixth time at 70°C for 3 hours to obtain the different groups of differentiated viscosity-average molecular weight chitosan powder.

[0105] Example 3

[0106] (1) Wash the raw crab shells with pure water, and place the washed crab shells in an oven for the first drying. The first drying conditions are 60℃ for 4 hours. Place the dried crab shells in a pulverizer for pulverization, and collect them after passing through a 400-mesh sieve to obtain fine crab shell powder.

[0107] (2) Add an 8% hydrochloric acid solution (hereinafter referred to as hydrochloric acid solution) to the crab shell powder prepared in step (1) above. The mass-volume ratio of crab shell powder to hydrochloric acid solution is 1:15. That is, weigh 50 g of crab shell powder obtained in step (1) above, add 750 mL of 8% hydrochloric acid solution, stir evenly, place in a microwave reactor, and react at 60°C for 4 h to fully remove the mineral salts in the crab shell powder and obtain the first mixture.

[0108] (3) Place the first mixture in step (2) above on a sieve with a 400 mesh screen, and repeatedly rinse the first wet powder remaining on the sieve with pure water until the pH of the first wet powder is neutral at 6.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the first wet powder can also be 6.5~7.5). Collect the first wet powder with neutral pH using the sieve and place it in an oven for a second drying. The second drying condition is drying at 60℃ for 4 h to obtain dried desalted crab shell powder.

[0109] (4) Add a 10% potassium hydroxide solution to the dried desalted crab shell powder prepared in step (3) above. The mass-volume ratio of the dried desalted crab shell powder to the 10% potassium hydroxide solution is 1:14. After stirring evenly, place it in a microwave reactor and react at 60°C for 4 h to fully remove the impurities in the desalted crab shell powder and obtain the second mixture.

[0110] (5) Place the second mixture from step (4) above on a 400-mesh sieve again, and repeatedly rinse the second wet powder remaining on the sieve with pure water until the pH of the second wet powder is neutral at 6.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the second wet powder can also be 6.5~7.5), and collect the second wet powder with neutral pH using the sieve.

[0111] (6) Add a 3% hydrogen peroxide solution to the second wet powder prepared in step (5) above. The mass-volume ratio of the second wet powder to the 3% hydrogen peroxide solution is 1:20. Adjust the pH to 9.0~10.0 using a 10% sodium hydroxide solution. Then place it in a microwave reactor and react at 60°C for 4 h to obtain the third mixture.

[0112] (7) After the third mixture is cooled to room temperature, add an appropriate amount of pure water to dilute it and adjust the pH to 7.5~8.0. Then add urea solution to the system to make the final urea concentration 5%. Stir and mix for 30~40 min to obtain the fourth mixture.

[0113] (8) In order to break the intermolecular forces of the chitosan macromolecular chain and control the degree of breakage, the fourth mixture obtained in step (7) above was frozen at -20°C. One blank control group (not frozen) and four experimental groups with repeated freeze-thaw cycles (Group 1, Group 2, Group 3 and Group 4) were set up. Repeated freeze-thaw cycles refer to two or more freeze-thaw operations. For example, repeated freeze-thaw cycles are: the fourth mixture is frozen at -20°C and then transferred to room temperature. After the fourth mixture is completely thawed, the fourth mixture is frozen at -20°C and then transferred to room temperature again until the fourth mixture is completely thawed.

[0114] Among them, the first group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed twice; the second group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed three times; the third group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed twice; and the second group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed three times.

[0115] The mixtures obtained from the control group and the experimental group were placed on a 400-mesh sieve and repeatedly washed under flowing ultrapure water until the pH was neutral at 6.5 (pH = 6.5~7.5 is defined as neutral here; in some other specific embodiments, the repeated washing can also be repeated until the pH is 6.5~7.5). The fourth wet powder with neutral pH was collected and placed in an oven for a fourth drying. The fourth drying condition was drying at 60°C for 4 h to obtain different groups of tri-dechitoxin.

[0116] (9) Add 60% sodium hydroxide solution to the different groups of tri-dechitoxin prepared in step (8) above. The mass-volume ratio of tri-dechitoxin to 60% sodium hydroxide solution is 1:10. After stirring evenly, place in an ultrasonic crusher and continue ultrasonic reaction at 200 W power for 3 h under constant temperature of 75℃ to fully remove acetyl groups and obtain the fifth mixture of different groups, which are the deacetylated products of different groups.

[0117] (10) Place the deacetylated products of different groups prepared in step (9) above on a 2000-mesh sieve and wash them repeatedly under flowing ultrapure water until the pH is neutral 6.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH can also be 6.5~7.5 after repeated washing). Collect the fifth wet powder with neutral pH and place it in an oven for the fifth drying. The fifth drying condition is drying at 60°C for 5 h to obtain different groups of powdered high molecular weight chitosan.

[0118] (11) Weigh out portions of the different groups of high molecular weight chitosan prepared in step (10) above, add 1% dilute acetic acid, and prepare a 2% chitosan / acetic acid solution (w / v) by mass-volume ratio of the different groups of high molecular weight chitosan to the added 1% dilute acetic acid at 1:50. The high molecular weight chitosan is completely dissolved in the 1% dilute acetic acid. Then, according to the mass-volume ratio of chitosan to 40% hydrogen peroxide solution in the 2% chitosan / acetic acid solution at 1:1, add 40% hydrogen peroxide solution and place in a water bath at 60°C for 5 h to obtain different groups of differentiated viscosity-average molecular weight chitosan solutions.

[0119] (12) Place the different groups of differentiated viscosity-average molecular weight chitosan solutions obtained in step (11) into dialysis bags with a cutoff of 2000~4000 Da, and then place the dialysis bags containing the different groups of differentiated viscosity-average molecular weight chitosan solutions into pure water. Dialyze at 4°C for 7~9 days until the solution in the dialysis bag is neutral. The dialysis is then completed when the pH value of the solution after dialysis is 6.0~7.5, and the dialysate is obtained. The dialysate is then dried for the sixth time at 60°C for 6 hours to obtain the different groups of differentiated viscosity-average molecular weight chitosan powder.

[0120] Example 4

[0121] (1) Wash the raw crab shells with pure water, place the washed crab shells in an oven for the first drying, and dry them at 70°C for 2 hours. Then, place the dried crab shells in a pulverizer for pulverization and collect them after passing through a 400-mesh sieve to obtain fine crab shell powder.

[0122] (2) Add a 10% hydrochloric acid solution (hereinafter referred to as hydrochloric acid solution) to the crab shell powder prepared in step (1) above. The mass-volume ratio of crab shell powder to hydrochloric acid solution is 1:10. That is, weigh 50 g of crab shell powder obtained in step (1) above, add 500 mL of 10% hydrochloric acid solution, stir evenly, place in a microwave reactor, and react at 65°C for 2 h to fully remove the mineral salts in the crab shell powder and obtain the first mixture.

[0123] (3) Place the first mixture in step (2) above on a 400-mesh sieve and repeatedly rinse the first wet powder remaining on the sieve with pure water until the pH of the first wet powder is neutral 7.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the first wet powder can also be 6.5~7.5). Collect the first wet powder with neutral pH using the sieve and place it in an oven for a second drying. The second drying condition is drying at 70℃ for 2 h to obtain dried desalted crab shell powder.

[0124] (4) Add a 10% potassium hydroxide solution to the dried desalted crab shell powder prepared in step (3) above. The mass-volume ratio of the dried desalted crab shell powder to the 10% potassium hydroxide solution is 1:18. After stirring evenly, place it in a microwave reactor and react at 70°C for 2 h to fully remove the impurities in the desalted crab shell powder and obtain the second mixture.

[0125] (5) Place the second mixture from step (4) above on a 400-mesh sieve again, and rinse the second wet powder remaining on the sieve with pure water until the pH of the second wet powder is neutral at 7.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the second wet powder can also be 6.5~7.5), and collect the second wet powder with neutral pH using the sieve.

[0126] (6) Add a 5% hydrogen peroxide solution to the second wet powder prepared in step (5) above. The mass-volume ratio of the second wet powder to the 5% hydrogen peroxide solution is 1:25. Adjust the pH to 9.0~10.0 using a 10% sodium hydroxide solution. Then place it in a microwave reactor and react at 70°C for 2 h to obtain the third mixture.

[0127] (7) After the third mixture is cooled to room temperature, add an appropriate amount of pure water to dilute it and adjust the pH to neutral 7.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the first wet powder can also be 6.5~7.5). Then add urea solution to the system to make the final urea concentration 5%. Stir and mix for 30~40 min to obtain the fourth mixture.

[0128] (8) In order to break the intermolecular forces of the chitosan macromolecular chain and control the degree of breakage, the fourth mixture obtained in step (7) above was frozen at -20°C. One blank control group (not frozen) and four experimental groups with repeated freeze-thaw cycles (Group 1, Group 2, Group 3 and Group 4) were set up. Repeated freeze-thaw cycles refer to two or more freeze-thaw operations. For example, repeated freeze-thaw cycles are: the fourth mixture is frozen at -20°C and then transferred to room temperature. After the fourth mixture is completely thawed, the fourth mixture is frozen at -20°C and then transferred to room temperature again until the fourth mixture is completely thawed.

[0129] Among them, the first group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed twice; the second group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed three times; the third group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed twice; and the second group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed three times.

[0130] The mixtures obtained from the control group and the experimental group were placed on a 400-mesh sieve and repeatedly washed under flowing ultrapure water until the pH reached neutral 7.5 (pH = 6.5~7.5 is defined as neutral here; in some other specific embodiments, repeated washing until the pH can also reach 6.5~7.5) and then the pH reached neutral 7.0. The fourth wet powder with neutral pH was collected and placed in an oven for a fourth drying. The fourth drying condition was drying at 70°C for 2 hours to obtain different groups of tri-dechitoxin.

[0131] (9) Add 60% sodium hydroxide solution to the different groups of tri-dechitoxin prepared in step (8) above. The mass-volume ratio of tri-dechitoxin to 60% sodium hydroxide solution is 1:20. After stirring evenly, place in an ultrasonic crusher and continue ultrasonic reaction at 300 W power for 2 h under constant temperature of 75℃ to fully remove acetyl groups and obtain the fifth mixture of different groups, which are the deacetylated products of different groups.

[0132] (10) Place the deacetylated products of different groups prepared in step (9) above on a 2000-mesh sieve and wash them repeatedly under flowing ultrapure water until the pH is neutral 7.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH can also be 6.5~7.5 after repeated washing). Collect the fifth wet powder with neutral pH and place it in an oven for the fifth drying. The fifth drying condition is drying at 70°C for 3 h to obtain powdered high molecular weight chitosan of different groups.

[0133] (11) Weigh out portions of the different groups of high molecular weight chitosan prepared in step (10) above, add 2% dilute acetic acid, and prepare a 2% chitosan / acetic acid solution (w / v) by mass-volume ratio of the different groups of high molecular weight chitosan to the added 2% dilute acetic acid at 1:50. The high molecular weight chitosan is completely dissolved in the 2% dilute acetic acid. Then, according to the mass-volume ratio of chitosan to 40% hydrogen peroxide solution in the 2% chitosan / acetic acid solution at 1:1, add 40% hydrogen peroxide solution and place in a water bath at 70°C for 2 h to obtain different groups of differentiated viscosity-average molecular weight chitosan solutions.

[0134] (12) Place the different groups of differentiated viscosity-average molecular weight chitosan solutions obtained in step (11) into dialysis bags with a cutoff of 2000~4000 Da, and then place the dialysis bags containing the different groups of differentiated viscosity-average molecular weight chitosan solutions into pure water. Dialyze at 4°C for 7~9 days until the solution in the dialysis bag is neutral. The dialysis is then completed when the pH value of the solution after dialysis is 6.0~7.5, and the dialysate is obtained. The dialysate is then dried for the sixth time at 70°C for 3 hours to obtain the different groups of differentiated viscosity-average molecular weight chitosan powder.

[0135] Example 5

[0136] (1) Mix the raw shrimp shells and crab shells in a mass ratio of 1:1, then wash them with pure water. Place the washed shrimp shells and crab shells in an oven for the first drying. The first drying conditions are 60℃ for 4 hours. Place the dried shrimp shells and crab shells in a pulverizer for pulverization, and collect them after passing through a 400-mesh sieve to obtain a fine mixed powder.

[0137] (2) Add an 8% hydrochloric acid solution (hereinafter referred to as hydrochloric acid solution) to the mixed powder prepared in step (1) above. The mass-volume ratio of the mixed powder to the hydrochloric acid solution is 1:15. That is, weigh 50 g of the mixed powder obtained in step (1) above, add 750 mL of 8% hydrochloric acid solution, stir evenly, place in a microwave reactor, and react at 60°C for 4 h to fully remove the mineral salts in the mixed powder and obtain the first mixture.

[0138] (3) Place the first mixture in step (2) above on a 400-mesh sieve and repeatedly rinse the first wet powder remaining on the sieve with pure water until the pH of the first wet powder is neutral at 6.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the first wet powder can also be 6.5~7.5). Collect the first wet powder with neutral pH using the sieve and place it in an oven for a second drying. The second drying condition is drying at 60°C for 4 h to obtain the dried desalinated mixed powder.

[0139] (4) Add a 10% sodium hydroxide solution to the dry desalted mixed powder prepared in step (3) above. The mass-volume ratio of the dry desalted mixed powder to the 10% sodium hydroxide solution is 1:14. After stirring evenly, place it in a microwave reactor and react at 60°C for 4 h to fully remove the impurities and proteins in the desalted mixed powder, and obtain the second mixture.

[0140] (5) Place the second mixture from step (4) above on a 400-mesh sieve again, and repeatedly rinse the second wet powder remaining on the sieve with pure water until the pH of the second wet powder is neutral at 6.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the second wet powder can also be 6.5~7.5), and collect the second wet powder with neutral pH using the sieve.

[0141] (6) Add a 3% hydrogen peroxide solution to the second wet powder prepared in step (5) above. The mass-volume ratio of the second wet powder to the 3% hydrogen peroxide solution is 1:20. Adjust the pH to 9.0~10.0 using a 10% sodium hydroxide solution. Then place it in a microwave reactor and react at 60°C for 4 h to obtain the third mixture.

[0142] (7) After the third mixture is cooled to room temperature, add an appropriate amount of pure water to dilute it and adjust the pH to 7.5~8.0. Then add urea solution to the system to make the final urea concentration 5%. Stir and mix for 3~40 min to obtain the fourth mixture.

[0143] (8) In order to break the intermolecular forces of the chitosan macromolecular chain and control the degree of breakage, the fourth mixture obtained in step (7) above was frozen at -20°C. One blank control group (not frozen) and four experimental groups with repeated freeze-thaw cycles (Group 1, Group 2, Group 3 and Group 4) were set up. Repeated freeze-thaw cycles refer to two or more freeze-thaw operations. For example, repeated freeze-thaw cycles are: the fourth mixture is frozen at -20°C and then transferred to room temperature. After the fourth mixture is completely thawed, the fourth mixture is frozen at -20°C and then transferred to room temperature again until the fourth mixture is completely thawed.

[0144] Among them, the first group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed twice; the second group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed three times; the third group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed twice; and the second group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed three times.

[0145] The mixtures obtained from the control group and the experimental group were placed on a 400-mesh sieve and repeatedly washed under flowing ultrapure water until the pH was neutral at 6.5 (pH = 6.5~7.5 is defined as neutral here; in some other specific embodiments, the repeated washing can also be repeated until the pH is 6.5~7.5). The fourth wet powder with neutral pH was collected and placed in an oven for a fourth drying. The fourth drying condition was drying at 60°C for 4 h to obtain different groups of tri-dechitoxin.

[0146] (9) Add 50% sodium hydroxide solution to the different groups of tri-dechitoxin prepared in step (8) above. The mass-volume ratio of tri-dechitoxin to 50% sodium hydroxide solution is 1:10. After stirring evenly, place in an ultrasonic crusher and continue ultrasonic reaction at 200 W power for 3 h under constant temperature of 75℃ to fully remove acetyl groups and obtain the fifth mixture of different groups, which are the deacetylated products of different groups.

[0147] (10) Place the deacetylated products of different groups prepared in step (9) above on a 2000-mesh sieve and wash them repeatedly under flowing ultrapure water until the pH is neutral 6.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH can also be 6.5~7.5 after repeated washing). Collect the fifth wet powder with neutral pH and place it in an oven for the fifth drying. The fifth drying condition is drying at 60°C for 5 h to obtain powdered high molecular weight chitosan of different groups.

[0148] (11) Weigh out portions of the different groups of high molecular weight chitosan prepared in step (10) above, add 1% dilute acetic acid, and prepare a 2% chitosan / acetic acid solution (w / v) by mass-volume ratio of the different groups of high molecular weight chitosan to the added 1% dilute acetic acid at a concentration of 1:50, so that the high molecular weight chitosan is completely dissolved in 1% dilute acetic acid; then add 30% hydrogen peroxide solution according to the mass-volume ratio of chitosan to 30% hydrogen peroxide solution in the 2% chitosan / acetic acid solution at a concentration of 1:1, and place in a water bath at 60℃ for 5 h to obtain different groups of differential viscosity-average molecular weight chitosan solutions.

[0149] (12) Place the differentiated viscosity-average molecular weight chitosan solutions obtained in step (11) into dialysis bags with a cutoff of 2000~4000 Da. Then place the dialysis bags containing the different groups of differentiated viscosity-average molecular weight chitosan solutions into pure water and dialyze at 4°C for 7~9 days until the solution in the dialysis bag is neutral. The dialysis is then completed when the pH value of the solution after dialysis is 6.0~7.5, and the dialysate is obtained. The dialysate is then dried for the sixth time at 60°C for 6 hours to obtain the different groups of differentiated viscosity-average molecular weight chitosan powders.

[0150] In some other specific embodiments, the raw materials, shrimp shells and crab shells, can be mixed in any proportion, and different groups of chitosan powder with different viscosity-average molecular weights can also be obtained by following the method described in this embodiment.

[0151] Example 6

[0152] (1) Mix the raw shrimp shells and crab shells in a mass ratio of 1:1, then wash them with pure water. Place the washed shrimp shells and crab shells in an oven for the first drying. The first drying conditions are 70℃ for 2 hours. Place the dried shrimp shells and crab shells in a pulverizer for pulverization, and collect them after passing through a 400-mesh sieve to obtain a fine mixed powder.

[0153] (2) Add a 10% hydrochloric acid solution (hereinafter referred to as hydrochloric acid solution) to the mixed powder prepared in step (1) above. The mass-volume ratio of the mixed powder to the hydrochloric acid solution is 1:10. That is, weigh 50 g of shrimp shell powder obtained in step (1) above, add 500 mL of 10% hydrochloric acid solution, stir evenly, place in a microwave reactor, and react at 65°C for 2 h to fully remove the mineral salts in the shrimp shell powder and obtain the first mixture.

[0154] (3) Place the first mixture from step (2) above on a 400-mesh sieve and repeatedly rinse the first wet powder remaining on the sieve with pure water until the pH of the first wet powder is neutral at 7.5 (pH=6.5~7.5 is defined as neutral here; in some other specific embodiments, the pH of the first wet powder can also be 6.5~7.5). Collect the first wet powder with neutral pH using the sieve and place it in an oven for a second drying. The second drying condition is drying at 70°C for 2 hours to obtain the dried desalinated mixed powder.

[0155] (4) Add a 10% sodium hydroxide solution to the dry desalted mixed powder prepared in step (3) above. The mass-volume ratio of the dry desalted mixed powder to the 10% sodium hydroxide solution is 1:18. After stirring evenly, place it in a microwave reactor and react at 70°C for 2 h to fully remove the impurities and proteins in the desalted mixed powder, and obtain the second mixture.

[0156] (5) Place the second mixture from step (4) above on a 400-mesh sieve again, and rinse the second wet powder remaining on the sieve with pure water until the pH of the second wet powder is neutral at 7.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH of the second wet powder can also be 6.5~7.5), and collect the second wet powder with neutral pH using the sieve.

[0157] (6) Add a 5% hydrogen peroxide solution to the second wet powder prepared in step (5) above. The mass-volume ratio of the second wet powder to the 5% hydrogen peroxide solution is 1:25. Adjust the pH to 9.0~10.0 using a 10% sodium hydroxide solution. Then place it in a microwave reactor and react at 70°C for 2 h to obtain the third mixture.

[0158] (7) After the third mixture is cooled to room temperature, add an appropriate amount of pure water to dilute it and adjust the pH to 7.5~8.0. Then add urea solution to the system to make the final urea concentration 5%. Stir and mix for 30~40 min to obtain the fourth mixture.

[0159] (8) In order to break the intermolecular forces of the chitosan macromolecular chain and control the degree of breakage, the fourth mixture obtained in step (7) above was frozen at -20°C. One blank control group (not frozen) and four experimental groups with repeated freeze-thaw cycles (Group 1, Group 2, Group 3 and Group 4) were set up. Repeated freeze-thaw cycles refer to two or more freeze-thaw operations. For example, repeated freeze-thaw cycles are: the fourth mixture is frozen at -20°C and then transferred to room temperature. After the fourth mixture is completely thawed, the fourth mixture is frozen at -20°C and then transferred to room temperature again until the fourth mixture is completely thawed.

[0160] Among them, the first group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed twice; the second group was the fourth mixture frozen for 4 hours and then repeatedly frozen and thawed three times; the third group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed twice; and the second group was the fourth mixture frozen for 8 hours and then repeatedly frozen and thawed three times.

[0161] The mixtures obtained from the control group and the experimental group were placed on a 400-mesh sieve and repeatedly washed under flowing ultrapure water until the pH was neutral 7.5 (pH = 6.5~7.5 is defined as neutral here; in some other specific embodiments, the repeated washing can also be to pH 6.5~7.5). The fourth wet powder with neutral pH was collected and placed in an oven for a fourth drying. The fourth drying condition was drying at 70°C for 2 h to obtain different groups of tri-dechitoxin.

[0162] (9) Add 50% sodium hydroxide solution to the different groups of tri-dechitoxin prepared in step (8) above. The mass-volume ratio of tri-dechitoxin to 50% sodium hydroxide solution is 1:20. After stirring evenly, place in an ultrasonic crusher and continue ultrasonic reaction at 300 W power for 2 h under constant temperature of 75℃ to fully remove acetyl groups and obtain the fifth mixture of different groups, which are the deacetylated products of different groups.

[0163] (10) Place the deacetylated products of different groups prepared in step (9) above on a 2000-mesh sieve and wash them repeatedly under flowing ultrapure water until the pH is neutral 7.5 (pH=6.5~7.5 is defined as neutral here. In some other specific embodiments, the pH can also be 6.5~7.5 after repeated washing). Collect the fifth wet powder with neutral pH and place it in an oven for the fifth drying. The fifth drying condition is drying at 70°C for 3 h to obtain powdered high molecular weight chitosan of different groups.

[0164] (11) Weigh out portions of the different groups of high molecular weight chitosan prepared in step (10) above, add 2% dilute acetic acid, and prepare a 2% chitosan / acetic acid solution (w / v) by mass-volume ratio of different groups of high molecular weight chitosan to 2% dilute acetic acid of 1:50, so that the high molecular weight chitosan is completely dissolved in 2% dilute acetic acid; then add 30% hydrogen peroxide solution according to the mass-volume ratio of chitosan to 30% hydrogen peroxide solution in the 2% chitosan / acetic acid solution of 1:1, and place in a water bath at 70°C for 2 h to obtain different groups of viscosity-average molecular weight chitosan solutions.

[0165] (12) Place the different groups of differentiated viscosity-average molecular weight chitosan solutions obtained in step (11) into dialysis bags with a cutoff of 2000~4000 Da, and then place the dialysis bags containing the different groups of differentiated viscosity-average molecular weight chitosan solutions into pure water. Dialyze at 4°C for 7~9 days until the solution in the dialysis bag is neutral. The dialysis is then completed when the pH value of the solution after dialysis is 6.0~7.5, and the dialysate is obtained. The dialysate is then dried for the sixth time at 70°C for 3 hours to obtain the different groups of differentiated viscosity-average molecular weight chitosan powder.

[0166] In some other specific embodiments, the raw materials, shrimp shells and crab shells, can be mixed in any proportion, and different groups of chitosan powder with different viscosity-average molecular weights can also be obtained by following the method described in this embodiment.

[0167] Test Example 1

[0168] The basic properties of the chitosan powders with different viscosity-average molecular weights prepared in Examples 1-6 above were characterized by viscosity-average molecular weight and degree of deacetylation. The degree of deacetylation was determined according to the YY / T 1699-2020 standard method, and the viscosity-average molecular weight (Mv) was determined using an Ubbelohde viscometer (0.2 M NaCl / 0.1 M HAC, [η] = KMα, K = 1.81 x 10⁻⁶). -3 (α=0.93).

[0169] The results showed that the degree of deacetylation (DDA) and viscosity-average molecular weight of the chitosan powders of different groups prepared in Examples 2-6 were similar to those of the chitosan powders of different groups prepared in Example 1. Taking the chitosan powders of different groups prepared in Example 1 as an example, the results are shown in Table 3 and... Figure 1 As shown, repeated freeze-thaw cycles can help reduce the molecular weight of chitosan, promote subsequent degradation through physical destruction, and increase degradation efficiency.

[0170] Table 3 Summary of Basic Characterization Results of Differentiated Viscosity-Average Molecular Weight Chitosan Powder

[0171]

[0172] Example 7

[0173] The different groups of chitosan with different viscosity-average molecular weights obtained in Example 1 above were further used to prepare complexes, and the specific steps are as follows:

[0174] (1) Preparation of 0.1×PBS dilution buffer solution: Weigh 0.7801 g of sodium chloride, 0.0406 g of sodium dihydrogen phosphate monohydrate and 0.0902 g of disodium hydrogen phosphate using an analytical balance, add pure water, mix well and make up to 1 L, which is the 0.1x PBS dilution buffer solution.

[0175] (2) Preparation of 0.1 M hydrochloric acid solution: Accurately measure 2.58 mL of 36% concentrated hydrochloric acid solution and add it to 297.42 mL of the above 0.1×PBS dilution buffer. After mixing well, it is 0.1 M hydrochloric acid solution.

[0176] (3) Weigh 0.675 g of chitosan powder with differential viscosity-average molecular weight (viscosity-average molecular weight, degree of deacetylation) after the above basic characterization, add it to a beaker containing 35 mL of 0.1 M hydrochloric acid solution prepared in step (2), carefully place a clean magnetic stir bar (3 mm x 4 mm) into the beaker, place the beaker on the magnetic stir bar, adjust the stir bar speed to 500 rpm, stir for 3~4 h to ensure that the chitosan powder sample is fully dissolved, and obtain a chitosan solution with a final concentration of 22.5 mg / mL, referred to as chitosan solution, and then refrigerate at 4℃ overnight.

[0177] In some other specific embodiments, a chitosan solution with a final concentration of 15.0~25.0 mg / mL can also be prepared according to the above method.

[0178] (4) Preparation of sodium β-glycerophosphate solution: Accurately weigh 1.6 g of commercial sodium β-glycerophosphate reagent, add 15 mL of 0.1×PBS buffer obtained in step (1) to dissolve, then make up to 20 mL, invert and mix thoroughly to obtain a sodium β-glycerophosphate solution with a concentration of 80 mg / mL, and store at 4℃ for later use.

[0179] In some other specific embodiments, a sodium β-glycerophosphate solution with a final concentration of 60-100 mg / mL can also be prepared according to the above method.

[0180] (5) Under ice bath conditions, according to the volume ratio of sodium β-glycerophosphate solution to chitosan solution of 1:3.50, accurately take 10 mL of sodium β-glycerophosphate solution of 80 mg / mL and pre-cooled prepared in step (4) above, and add it dropwise to 35 mL of chitosan solution prepared in step (3) above. Stir while adding dropwise for 35~45 min until no particulate matter is precipitated. The mixture is then stored at 4℃.

[0181] In some other specific embodiments, a chitosan solution with a final concentration of 15.0~25.0 mg / mL prepared in step (3) above, or a sodium β-glycerophosphate solution with a final concentration of 60~100 mg / mL prepared in step (4) above, can also be used to obtain a mixture by adding the sodium β-glycerophosphate solution to the chitosan solution in an ice bath at a volume ratio of 1:(1.25~2.00), 1:(2.00~3.50), 1:(3.50~5.00), or 1:(5.00~8.00).

[0182] (6) The mixture prepared in step (5) above is placed in an ultra-low temperature freeze dryer and freeze-dried at -60°C for 48 h to obtain chitosan-β-glycerophosphate sodium complex powder. In some other specific embodiments, it can also be freeze-dried at -70°C for 24 h to obtain chitosan-β-glycerophosphate sodium complex powder.

[0183] (7) Weigh 1.5 mg of the chitosan-β-glycerophosphate sodium complex powder from step (6) above, mix it with 200 mg of dry potassium bromide powder, and grind them together in an agate mortar. During grinding, the sample and the agate mortar are placed under a drying lamp to ensure dryness, and the sample to be tested is obtained. Then, the sample to be tested is evenly spread in a tableting mold, compressed into a tablet, and measured using a Fourier transform infrared spectrometer with a spectral resolution of 2 cm⁻¹. -1 Measurement range: 400~4000 cm -1 The number of scans was 30.

[0184] The results are as follows Figure 2 As shown, the wavenumber is 1649 cm⁻¹. -1 1593 cm -1 and 1315 cm -1 These peaks represent the three characteristic peaks of chitosan: C=O stretching vibration (amide I band), NH bending vibration (amide II band), and CN stretching oscillation (amide III band). The wavenumber in the β-glycerophosphate sodium spectrum is 1115 cm⁻¹. -1 1077 cm -1 976 cm -1 These are the P=O stretching vibration, POC stretching vibration, and POH stretching vibration, respectively, which correspond to the β-GP characteristic peak.

[0185] Pure chitosan has a density of 3700~3100 cm⁻¹ -1A broad peak region exists within the range, but the peak intensity decreases after cross-linking to form a chitosan-β-glycerophosphate sodium complex. This confirms that under weak acid conditions, the -OH or -NH2 in chitosan can react with the -PO4 in β-glycerophosphate sodium. 3- They bind to each other through hydrogen bonds or electrostatic attraction, masking the vibrations of groups within the region, i.e., they interact with each other.

[0186] 1649 cm -1 As one of the characteristic spectral bands of chitosan, it originates from the carbonyl stretching vibration of the acetylamino group. In chitosan or the chitosan-β-glycerophosphate sodium complex, the C=O vibrational peak remains constant and unchanged, indicating that the interaction between chitosan and β-glycerophosphate sodium is the core reaction and does not involve the C=O bond; while the original 1593 cm⁻¹... -1 The amino group peak shifted after crosslinking and was located at 1543 cm⁻¹. -1 The appearance of a new peak indicates that the chitosan amino groups bind to sodium β-glycerophosphate through hydrogen bonding or protonation, resulting in a change in the vibrational mode.

[0187] The above conclusions all demonstrate that chitosan and sodium β-glycerophosphate can achieve cross-linking through physical association, effectively avoiding the toxic side effects of conventional chemical modifications and effectively improving biocompatibility.

[0188] (8) The linear viscoelastic region of the chitosan-β-glycerophosphate sodium complex powder prepared in step (6) above was determined using an Anton Paar rheometer. The oscillation mode-amplitude scan was selected, the temperature was set to 37℃, the shear strain range was 0.1~100.0%, and the frequency was 1 Hz. Dynamic strain scanning was performed on the chitosan-β-glycerophosphate sodium complex powder.

[0189] The results are as follows Figure 3 As shown, compared to low viscosity-average molecular weight, in the range of 0.1-1000 s -1 Within the range of shear rates, materials with high viscosity-average molecular weight exhibit higher viscosity performance, such as 5.5 × 10⁻⁶. 5 Da and 1.77×10 6 The difference between Da and Da is nearly two orders of magnitude, while the low viscosity-average molecular weight is such as 5.5 × 10⁻⁶. 5 The variation of Da shear viscosity is approximately linear, which is related to short molecular chains, few cross-linking sites, or a thin composite structure.

[0190] (9) The chitosan-β-glycerophosphate sodium complex powder prepared in step (6) above was subjected to a rheological cone model in an Anton Paar rheometer, and the shear rate-viscosity curve mode was selected, with the shear rate range set to 0.1~1000s. -1 The viscosity curve of the chitosan-β-glycerophosphate sodium complex powder was obtained at a temperature of 37℃.

[0191] The results are as follows Figure 4 As shown, within the shear strain range of 1 to 100%, the viscous modulus G” of the prepared chitosan-β-glycerophosphate sodium complex powders of various molecular weights is always higher than their corresponding elastic modulus G'; that is, the chitosan-β-glycerophosphate sodium complexes prepared with different molecular weights are mostly viscous fluids at body temperature of 37℃, and with viscosity as the core, which meets the property requirements for preparing viscoelastic agents.

[0192] Under dynamic external forces, the loss factor tanδ, which is the ratio of viscous loss to elastic energy storage, is commonly used to directly reflect the viscoelastic properties of a material. tanδ=1 indicates that the material is at the viscoelastic transition point, a key value distinguishing solid-liquid properties. When tanδ>1, the material's properties are predominantly viscous, approaching those of a liquid; conversely, when tanδ<1, the material exhibits solid properties, with elasticity as its primary characteristic.

[0193] like Figure 5 As shown, the loss factor tanδ of the chitosan-β-glycerophosphate sodium complex powders prepared at each viscosity-average molecular weight is greater than 1. This indicates that the chitosan-β-glycerophosphate sodium complex powders at each viscosity-average molecular weight are predominantly viscous, and the smaller the viscosity-average molecular weight (e.g., 5.5 × 10⁻⁶), the lower the viscosity-average molecular weight (e.g., 5.5 × 10⁻⁶). 5 The loss factor of Da is much larger than that of other molecular weights, while 1.02 × 10 6 Da, 1.36×10 6 Da, 1.44×10 6 Da, 1.77×10 6 The value of Da is closer to 1, resulting in a more balanced viscosity and elasticity in the preparation of chitosan-β-glycerophosphate sodium complex powder. It can be used as a viscoelastic agent, which is a biomimetic joint lubricant used to promote the repair of articular cartilage damage and / or treat inflammation.

[0194] Compared with existing technologies, the present application provides a differentiated viscosity-average molecular weight chitosan derived from shrimp and crab shells, a complex thereof, its preparation method, and its application, which have the following beneficial effects:

[0195] (1) This application uses the alkali-freeze-thaw method and the H2O2 method to extract chitosan raw materials with different molecular weights and acetyl values ​​from the shells of peeled and molted shrimp and crabs. The method is green and environmentally friendly, with low chemical residues, high purity of raw materials, narrow distribution of core parameters, low processing cost, and easy control of reaction conditions. It can achieve large-scale production and is conducive to solving the problem of biological resource utilization of molted shrimp and crab shell waste in the aquaculture industry. At the same time, it is easy to achieve precise control and degradation of chitosan molecular weight.

[0196] (2) The chitosan-β-glycerophosphate sodium complex prepared in this application uses low, medium, high molecular weight or "full molecular weight" chitosan as raw material, which breaks the limitation of traditional single molecular weight and can complete product design according to local conditions, forming products with different viscosity and elasticity.

[0197] (3) Chitosan-β-glycerophosphate sodium complex can also be used in the field of biomedical materials as a viscoelastic agent to promote the repair of articular cartilage damage and / or treat inflammation.

[0198] In the foregoing description of exemplary embodiments / specific implementations of this patent, various features of this patent are sometimes combined in a single embodiment / specific implementation or its figures and description, with the aim of simplifying the disclosure and aiding in the understanding of one or more of the various aspects of the invention. However, unless expressly stated otherwise or in obvious technical contradiction or exclusion, the descriptive method of this patent should not be construed as reflecting an intention that the claimed features of the invention are more than expressly stated in each claim. Rather, the inventive aspect reflected in the claims lies in not all the features of a single foregoing disclosed embodiment / specific implementation. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, each claim existing independently as a separate embodiment / specific implementation of this patent.

[0199] The terms and expressions used in this specification are for illustrative purposes and not for limitation. Their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but rather to facilitate the understanding that various modifications may be possible within the scope of this patent claim. Therefore, it should be understood that while this patent has been specifically disclosed through preferred embodiments, exemplary embodiments, and optional features, variations or modifications of the concepts disclosed herein may be adopted by those skilled in the art, and such variations and modifications are therefore considered to be within the scope of this patent as defined by the appended claims. The specific embodiments given in this specification are examples of useful embodiments of this patent, and it will be apparent to those skilled in the art that this patent can be implemented using many variations of the devices, device components, and method steps disclosed herein.

[0200] The foregoing description of specific embodiments fully discloses the general features of this patent, enabling others to easily modify and / or adapt such embodiments for various applications by applying knowledge within the scope of the art, without excessive experimentation or deviation from the general concept of this patent. Therefore, based on the teachings and guidance provided herein, it is intended that such modifications and alterations be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not intended to be limiting; thus, the wording or terminology in this specification will be interpreted by those skilled in the art based on the foregoing teachings and guidance.

[0201] Furthermore, the scope of this patent should not be limited to any of the exemplary embodiments described above, but only to the appended claims and their equivalents.

Claims

1. A method for preparing chitosan with differentiated viscosity-average molecular weight derived from shrimp and crab shells for use in biomedical materials, characterized in that, The differentiated viscosity-average molecular weight chitosan was prepared by the following method: Step A: Mix shrimp and crab shell powder with a desalination solution, wash with pure water to remove mineral salts from the shrimp and crab shells; then add a 3% hydrogen peroxide solution, adjust the pH to 9.0-10.0 using a 10% sodium hydroxide solution, microwave heat treatment, cool, adjust the pH to 7.5-8.0, add urea solution to make the final urea concentration 5%, freeze for 4 or 8 hours, repeat freeze-thaw 2-3 times, and dry to obtain tri-de-chitosan; the mass-to-volume ratio of the shrimp and crab shell powder to the desalination solution is 1:(10-12), 1:(12-14), or 1:(14-15); the microwave heating temperature is 60-65℃, the microwave heating time is 2-4 hours; the drying temperature is 60-70℃, the drying time is 2-4 hours; Step B: The tri-dechitosan is mixed with a 50% sodium hydroxide solution, and after ultrasonic treatment, a deacetylated product is obtained. After washing with pure water and drying, a high molecular weight chitosan is obtained. The mass-volume ratio of the tri-dechitosan to the deacetylated solution is 1:(10~12), 1:(12~15), or 1:(15~20). Step C: Dissolve the high molecular weight chitosan in dilute acetic acid, then add a 30% hydrogen peroxide solution, and obtain differentiated viscosity-average molecular weight chitosan powder after post-processing; the mass-to-volume ratio of the high molecular weight chitosan to the dilute acetic acid is 1:50; the mass-to-volume ratio of the high molecular weight chitosan to the hydrogen peroxide solution is 1:1; the post-processing includes water bath treatment, dialysis treatment, and drying treatment; the rejection ratio of the dialysis treatment is 2000~4000 Da.

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