Chitosan based on hermetia illucens pupa shells as well as preparation method and application of chitosan

Abstract

The invention relates to the technical field of high-value utilization of biomass resources, in particular to chitosan based on hermetia illucens pupa shells as well as a preparation method and application of the chitosan. According to the method, hermetia illucens pupa shells are used as raw materials, and the method sequentially comprises the steps of raw material pretreatment, composite organic acid decalcification, gradient alkali deproteinization, oxidant depigmentation and ultrasonic-assisted alkali method deacetylation. The composite organic acid is composed of citric acid and malic acid according to a specific molar ratio, the gradient alkali deproteinization adopts multi-stage treatment with increasing concentration and temperature, and the deacetylation is carried out under the condition of concentrated alkali under the assistance of ultrasonic waves. Aiming at the characteristic of tight protein-pigment crosslinking of the hermetia illucens shells, the invention provides a set of efficient and green synergistic process, and the prepared chitosan has the advantages of high deacetylation degree, low ash content, good color, good molecular weight and the like, and can be widely applied to the fields of medical dressings, water treatment adsorbents and the like.

Description

Technical Field

[0001] This invention relates to the field of high-value utilization technology of biomass resources, specifically to a chitosan based on the black soldier fly pupa shell, its preparation method, and its application. Background Technology

[0002] Chitin is the second most abundant natural high-molecular-weight polysaccharide, widely found in the exoskeletons of insects and crustaceans. Its deacetylated product, chitosan, possesses excellent biocompatibility, biodegradability, and antibacterial activity, and is widely used in medicine, food, and environmental protection. Currently, chitin production mainly relies on byproducts of aquatic product processing such as shrimp and crab. However, these raw materials are significantly limited by season and region, and may carry pollutants such as heavy metals. In recent years, the black soldier fly, a high-value-added insect, has produced a large number of pupal shells in large-scale farming, containing approximately 20%–30% chitin, and holds promise as a novel, high-quality raw material for chitosan production.

[0003] However, the structure of black soldier fly pupa shells differs significantly from that of traditional shrimp and crab shells. Their proteins and pigments are tightly cross-linked, and the pigment content is high. This leads to a series of interconnected and complex challenges when directly applying existing processes. For example, while existing technologies, such as patent CN118373927A, disclose a method for extracting chitin and its derivatives from black soldier fly pupa shells, this process still has significant drawbacks, making it difficult to achieve stable preparation of high-quality chitosan. First, in the decalcification stage, strong acids or high-viscosity eutectic solvents are used. Strong acids easily lead to the hydrolysis of chitin molecular chains, while eutectic solvents affect the thoroughness of decalcification due to inefficient mass transfer. Second, protein removal is a one-step process, which cannot effectively break down the dense protein-pigment cross-linked structure in the black soldier fly shell, resulting in protein and pigment residues. These shortcomings of the preceding steps... The defects directly affect the purity of the intermediate product chitin, leading to higher ash content and compromised molecular weight integrity in the final chitosan product. Furthermore, in the critical deacetylation process, this method relies solely on high-temperature concentrated alkali treatment, lacking effective mass transfer enhancement methods. Residual impurities and molecular chain damage from the preceding process further restrict the uniformity and sufficiency of the deacetylation reaction, resulting in a deacetylation degree of only 60-80% for the prepared chitosan. Moreover, it neglects ash control and molecular weight retention. Therefore, existing technologies struggle to achieve a balanced and stable preparation of chitosan with a deacetylation degree greater than 85%, low ash content, and high molecular weight.

[0004] Therefore, there is an urgent need to develop an efficient and green extraction and preparation process for black soldier fly pupa shells to achieve stable preparation of chitosan with high deacetylation degree, low ash content, excellent color and good molecular weight under mild conditions, so as to promote the high-value utilization of this biomass resource. Summary of the Invention

[0005] In view of this, the present invention provides a chitosan based on black soldier fly larvae shells, its preparation method, and its application. The method includes: mild decalcification using a citric acid-malic acid composite system with a specific ratio; deep deproteinization using a gradient alkali treatment process with progressively increasing concentration and temperature; and efficient deacetylation under ultrasonic assistance. These steps work synergistically to achieve efficient conversion of the black soldier fly larvae shell raw material. The chitosan prepared by the method of the present invention has a high degree of deacetylation, low ash content, good color, and good molecular weight, exhibiting excellent overall quality.

[0006] The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a method for preparing chitosan based on black soldier fly larvae shells, comprising the following steps: S1. The black soldier fly pupa shells are washed, dried, crushed, and sieved to obtain raw material powder; S2. Mix the above raw material powder with the composite organic acid solution, and wash until neutral and dry after reaction; the composite organic acid solution includes citric acid and malic acid. Specifically, the black soldier fly larvae release a large amount of calcium ions in step S2. Citric acid, due to its three carboxyl groups, has a strong chelating ability and can form a stable and soluble complex with calcium ions. Malic acid not only contributes to the chelating effect, but also forms a buffer when combined with citric acid, which helps maintain the stability of the pH of the reaction system and prevents the dissolved calcium salts and other impurities from redepositing on the surface or in the pores of the material. At the same time, this composite acid system can form a mild and continuous acidic environment, which can effectively chelate and dissolve calcium carbonate and prevent the glycosidic bonds in the chitin molecular chain from being destroyed by acid hydrolysis.

[0007] S3. The material obtained in step S2 is subjected to at least three stages of reaction with alkaline solutions of increasing concentration. After each stage of reaction, the mixture is washed until neutral and then dried to obtain chitin. Specifically, the black soldier fly pupa shell contains both soluble and insoluble proteins, with pigments deeply bound. Step S3 employs a gradient process to first remove most of the loose and soluble proteins under mild conditions, initially loosening the protein-pigment complex structure. Subsequently, conditions are gradually increased to specifically break the remaining stubborn cross-links. Furthermore, the dark pigment of the black soldier fly is physically embedded and chemically cross-linked by a dense, insoluble protein shell. If this protein layer is not effectively removed, subsequent oxidants will be unable to effectively penetrate and contact the pigment molecules. However, after the chitin undergoes gradient alkali treatment according to this invention, more pigment is exposed on the material surface, which facilitates subsequent depigmentation.

[0008] S4. Mix the chitin obtained in step S3 with sodium hydroxide solution, and react with ultrasound. After the reaction is complete, wash until neutral and dry to obtain chitosan.

[0009] Specifically, after the decalcification in step S2 and the deproteinization in step S3, the chitin from black soldier fly larvae has a porous structure. High-viscosity concentrated alkali solution can easily clog these micropores, forming a passivation layer and hindering the reaction. Ultrasonic energy can continuously clean the surface pores, prevent interface passivation, and ensure the renewal of the reaction interface, so that the concentrated alkali solution in step S4 can continuously penetrate into the interior of the particles and be fully utilized in the deacetylation stage.

[0010] Based on the above scheme, preferably, in step S2, the concentration of citric acid in the composite organic acid solution is 0.015-0.035 M, and the concentration of malic acid is 0.0225-0.0525 M.

[0011] Based on the above scheme, preferably, the molar ratio of citric acid to malic acid is 2:3.

[0012] Based on the above scheme, preferably, step S3 includes at least three-stage reactions: A 1%-2% sodium hydroxide solution is used, and the reaction is carried out at 40-50℃ for 1-2 hours; its main function is to remove free and loosely bound proteins. A 3%-4% sodium hydroxide solution is used, and the reaction is carried out at 60-80℃ for 1-2 hours. Its main function is to break the tightly bound proteins and initially loosen the protein-pigment cross-links, hydrolyze those proteins that are tightly bound to chitin through non-covalent bonds such as hydrogen bonds and hydrophobic interactions, and begin to break the chemical bonds between pigments and proteins. A 4%-6% sodium hydroxide solution is used, and the reaction is carried out at 80-90℃ for 0.5-2 hours. The main purpose is to thoroughly remove stubborn residual protein and pigment cross-linking fragments. Since most of the easily damaged structures have been removed, this stage uses a relatively short time to target stubborn impurities and minimizes damage to the overall structure of chitin.

[0013] Based on the above scheme, preferably, after step S3 and before step S4, step S3-1 is also included: The material obtained in step S3 is mixed with an oxidant, and after reaction, it is washed until neutral and dried; the oxidant is selected from at least one of potassium permanganate, hydrogen peroxide, and sodium hypochlorite.

[0014] Based on the above scheme, preferably, in step S3-1, the mass concentration of the oxidant is 10%-20%; the solid-liquid ratio of the material obtained in step S3 to the oxidant is 1g:(30-40)mL.

[0015] More preferably, in step S3-1, the reaction temperature of the material obtained in step S3 with the oxidant is 60-90℃, and the reaction time is 2-5 hours.

[0016] Based on the above scheme, preferably, in step S4, the mass concentration of the sodium hydroxide solution is 30%-50%; and the power of the ultrasound is 500-800 W.

[0017] Specifically, insufficient ultrasonic power leads to inadequate cavitation, failing to effectively disrupt the crystalline structure of chitin and hindering the penetration of high-viscosity alkaline solutions to generate sufficient microjets for cleaning pores. Excessive power results in overly strong cavitation, generating numerous useless bubbles and creating a sound barrier, which weakens energy transfer and can also cause mechanical breakage of chitin molecular chains.

[0018] Based on the above scheme, preferably, in step S2, the solid-liquid ratio of the raw material powder to the composite organic acid solution is 1g:(10-15)mL; in step S4, the solid-liquid ratio of the chitin to the sodium hydroxide solution is 1g:(15-20)mL.

[0019] More preferably, in step S2, the reaction time between the raw material powder and the composite organic acid solution is 2-5 hours; in step S4, the reaction temperature between the chitin and the sodium hydroxide solution is 80-100℃, and the reaction time is 2-10 hours.

[0020] In a second aspect, the present invention provides a chitosan obtained by the preparation method described in the first aspect, wherein the degree of deacetylation of the chitosan is not less than 86.5%, and / or the ash content is not more than 0.6%.

[0021] Thirdly, the present invention provides an application of chitosan as described in the second aspect in the fields of medical dressings or water treatment.

[0022] Compared with the prior art, the advantages of the present invention are as follows: (1) This invention innovatively uses black soldier fly pupa shells as raw materials to prepare chitosan, realizing the high-value utilization of waste resources; and the whole process is designed for the characteristics of black soldier fly shells, with significant synergistic effect, and can obtain high-quality chitosan products with high degree of deacetylation (≥86.5%), low ash content (≤0.6%) and high molecular weight.

[0023] (2) The present invention uses a citric acid-malic acid composite system with a specific molar ratio to replace strong inorganic acid decalcification, which has both strong chelation and good buffering performance. It can efficiently decalcify under mild conditions, reduce equipment corrosion and environmental pollution from the source, and conform to the concept of green chemistry. At the same time, the composite acid system can form a mild and continuous acidic environment to protect the integrity of chitin molecular chains.

[0024] (3) This invention uses a gradient alkaline deproteinization process with progressively increasing concentration and temperature to gently and thoroughly break down the protein-pigment cross-linking structure in the black soldier fly pupa shell, thereby protecting the integrity of the chitin molecular chain to the maximum extent while removing impurities. Combined with ultrasonic-assisted deacetylation, the cavitation effect is used to enhance mass transfer, which not only significantly shortens the reaction time and reduces energy consumption, but also effectively improves the overall quality of the final product chitosan.

[0025] (4) The process flow of the present invention is clear, the key parameters are well defined, the operating conditions are highly controllable, and it has good repeatability and stability, making it easy to scale up and realize industrial production. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The infrared spectrum of chitosan obtained in Example 1 of this invention; Figure 2 A comparison diagram of the chitosan obtained from (a) the sample of Example 1 and (b) the sample of Comparative Example 3 of this invention. Detailed Implementation

[0028] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0029] It should be noted that this invention uses black soldier fly larvae shells, a novel waste resource, as raw material, and achieves the preparation of high-quality chitosan through a set of synergistically optimized green processes.

[0030] Its core mechanism is as follows: First, the citric acid-malic acid composite system of this invention can not only gently decalcify, but also form a loose and porous material structure while efficiently removing minerals, thus opening up mass transfer channels for the subsequent penetration of alkaline solution and oxidant; then, the gradient alkali with progressively increasing concentration and temperature can dismantle the dense protein network from the surface to the interior, and the protein-pigment cross-links in the black soldier fly shell are initially loosened. This step not only removes the protein, but its more crucial role is to completely expose the encapsulated dark pigment, thus clearing the way for the subsequent "forced depigmentation" step to achieve high whiteness efficiently and thoroughly.

[0031] Furthermore, the above steps not only deeply remove impurities but also maximize the preservation of the chitin molecular chain integrity. Finally, deacetylation is performed under ultrasonic assistance, utilizing cavitation to enhance mass transfer and break down crystalline regions, achieving a deep and uniform reaction. Each step is interconnected: the preceding step creates the optimal reaction interface and structural basis for the following step, while the following step fully utilizes the optimized material state of the preceding step to achieve a significant increase in efficiency and quality. This synergistic system ultimately yields high-quality chitosan with a degree of deacetylation ≥86.5%, ash content ≤0.6%, and good molecular weight retention under mild conditions, demonstrating a significant overall technical effect that surpasses the simple summation of individual process units.

[0032] It should be noted that 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 indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0033] In this document, the terms “containing,” “comprising,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0034] In this document, the terms “optional,” “optionally,” or “optional” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0036] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field; among them, citric acid, malic acid, NaOH, hydrogen peroxide, potassium permanganate and sodium hypochlorite are all analytical grade and purchased from Aladdin Reagent Co., Ltd.

[0037] Example 1

[0038] This embodiment provides a method for preparing chitosan based on black soldier fly pupa shells, including the following steps: (1) Raw material pretreatment: Take 100 g of dried black soldier fly pupa shells, clean them with deionized water by ultrasonic cleaning, rinse them with anhydrous ethanol, dry them at 60℃ for 24 hours, pulverize them and pass them through a 60-mesh sieve to obtain sieved powder. (2) Decalcification treatment: The sieved powder was mixed with a composite organic acid solution (0.02 M citric acid and 0.03 M malic acid, molar ratio 2:3) at a solid-liquid ratio of 1:12 (g / mL), and the mixture was shaken at room temperature (25-35℃) for 3 hours. After the reaction, the mixture was filtered, washed with deionized water until neutral, and dried at 60℃ to obtain the decalcified product. (3) Deproteinization treatment: Place the decalcified products in the following order: ① React in 1.5% NaOH solution (solid-liquid ratio 1:18 g / mL) at 45℃ in an air shaker for 1.5 hours, then wash; ② 3.5% NaOH solution (solid-liquid ratio 1:18 g / mL), react in an air shaker at 70℃ for 1.5 hours, then wash; ③ React in 5% NaOH solution (solid-liquid ratio 1:18 g / mL) in an air shaker at 85℃ for 1 hour, then wash. After each reaction, the chitin was washed with deionized water until neutral, and finally dried at 60°C to remove proteins, yielding flake chitin. (4) Depigmentation treatment: Chitosan and 15% hydrogen peroxide solution were mixed at a solid-liquid ratio of 1:35 (g / mL), reacted at 75°C for 3 hours, washed until neutral, and dried to obtain the depigmented product; (5) Deacetylation treatment: The depigmented chitin was mixed with 40% NaOH solution at a solid-liquid ratio of 1:18 (g / mL), placed in a 90℃ water bath, and reacted for 6 hours under the assistance of 600 W ultrasound. After the reaction, the mixture was filtered, washed with deionized water until neutral, and dried at 60℃ to obtain the final product chitosan.

[0039] The final yield of chitosan was 28.5 g. The degree of deacetylation was determined to be 92.1% according to GB / T 38169-2019 standard. The ash content was determined to be 0.3% according to GB / T 35818-2018 method. Its FT-IR spectrum is shown below. Figure 1 As shown, at 1590 cm -1 A distinct -NH2 characteristic absorption peak was observed, confirming a high degree of deacetylation. Using an Ubbelohde viscometer and following the method specified in YY / T 1699-2020, the viscosity-average molecular weight of chitosan was determined to be 65.8 × 10⁻⁶. 4 .

[0040] Example 2

[0041] This embodiment provides a method for preparing chitosan based on black soldier fly pupa shells, including the following steps: (1) Raw material pretreatment: Take 100 g of dried black soldier fly pupa shells, clean them with deionized water by ultrasonic cleaning, rinse them with anhydrous ethanol, dry them at 60℃ for 24 hours, pulverize them and pass them through a 60-mesh sieve to obtain sieved powder. (2) Decalcification treatment: The sieved powder was mixed with a composite organic acid solution (0.015 M citric acid and 0.0225 M malic acid, molar ratio 2:3) at a solid-liquid ratio of 1:10 (g / mL), and the mixture was shaken at room temperature (25-35℃) for 2 hours. After the reaction, the mixture was filtered, washed with deionized water until neutral, and dried at 60℃ to obtain the decalcified product. (3) Deproteinization treatment: Place the decalcified products in the following order: ① React in 1.5% NaOH solution (solid-liquid ratio 1:18 g / mL) at 45℃ in an air shaker for 1.5 hours, then wash; ② 3.5% NaOH solution (solid-liquid ratio 1:18 g / mL), react in an air shaker at 70℃ for 1.5 hours, then wash; ③ React in 5% NaOH solution (solid-liquid ratio 1:18 g / mL) in an air shaker at 85℃ for 1 hour, then wash. After each reaction, the chitin was washed with deionized water until neutral, and finally dried at 60°C to remove proteins, yielding flake chitin. (4) Depigmentation treatment: Chitosan and 15% hydrogen peroxide solution were mixed at a solid-liquid ratio of 1:35 (g / mL), reacted at 75°C for 3 hours, washed until neutral, and dried to obtain the depigmented product; (5) Deacetylation treatment: The depigmented chitin was mixed with 30% NaOH solution at a solid-liquid ratio of 1:15 (g / mL), placed in an 80℃ water bath, and reacted for 2 hours under the assistance of 500 W ultrasound. After the reaction, the mixture was filtered, washed with deionized water until neutral, and dried at 60℃ to obtain the final product chitosan.

[0042] 25.1 g of chitosan was finally obtained. Its degree of deacetylation was 86.5% according to GB / T 38169-2019 standard. The ash content was 0.4% according to GB / T 35818-2018 method. Within these parameter ranges, this invention can still effectively prepare qualified chitosan. The viscosity-average molecular weight of chitosan was 72.3 × 10⁻⁶ m³ / s, determined using an Ubbelohde viscometer according to YY / T 1699-2020 method. 4 .

[0043] Example 3

[0044] This embodiment provides a method for preparing chitosan based on black soldier fly pupa shells, including the following steps: (1) Raw material pretreatment: Take 100 g of dried black soldier fly pupa shells, clean them with deionized water by ultrasonic cleaning, rinse them with anhydrous ethanol, dry them at 60℃ for 24 hours, pulverize them and pass them through a 60-mesh sieve to obtain sieved powder. (2) Decalcification treatment: The sieved powder was mixed with a composite organic acid solution (0.035 M citric acid and 0.0525 M malic acid, molar ratio 2:3) at a solid-liquid ratio of 1:15 (g / mL), and the mixture was shaken at room temperature (25-35℃) for 5 hours. After the reaction, the mixture was filtered, washed with deionized water until neutral, and dried at 60℃ to obtain the decalcified product. (3) Deproteinization treatment: Place the decalcified products in the following order: ① React in 1.5% NaOH solution (solid-liquid ratio 1:18 g / mL) at 45℃ in an air shaker for 1.5 hours, then wash; ② 3.5% NaOH solution (solid-liquid ratio 1:18 g / mL), react in an air shaker at 70℃ for 1.5 hours, then wash; ③ React in 5% NaOH solution (solid-liquid ratio 1:18 g / mL) in an air shaker at 85℃ for 1 hour, then wash. After each reaction, the chitin was washed with deionized water until neutral, and finally dried at 60°C to obtain flake chitin. (4) Depigmentation treatment: Chitosan and 15% hydrogen peroxide solution were mixed at a solid-liquid ratio of 1:35 (g / mL), reacted at 75°C for 3 hours, washed until neutral, and dried to obtain the depigmented product; (5) Deacetylation treatment: The depigmented chitin was mixed with 50% NaOH solution at a solid-liquid ratio of 1:20 (g / mL), placed in a 100℃ water bath, and reacted for 10 hours under the assistance of 800 W ultrasound. After the reaction, the mixture was filtered, washed with deionized water until neutral, and dried at 60℃ to obtain the final product chitosan.

[0045] 28.8 g of chitosan was finally obtained. Its degree of deacetylation was 94.2% as determined according to GB / T 38169-2019 standard. The ash content was 0.2% as determined according to GB / T 35818-2018 method. The viscosity-average molecular weight of chitosan was 58.1 × 10⁻⁶ as determined using an Ubbelohde viscometer according to YY / T 1699-2020 method. 4 This indicates that the present invention can achieve higher deacetylation degree and purity, but the reaction time is longer.

[0046] Example 4

[0047] The difference between this embodiment and Embodiment 1 lies in step (3): the decalcification products are placed sequentially in: First stage: 1% NaOH solution (solid-liquid ratio 1:18 g / mL), react at 40℃ for 1 hour, then wash; Second stage: 3% NaOH solution (solid-liquid ratio 1:18 g / mL), react at 60℃ for 1 hour, then wash; Third stage: 4% NaOH solution (solid-liquid ratio 1:18 g / mL), react at 80℃ for 0.5 hours, then wash; The remaining steps are the same as in Example 1.

[0048] The final yield of chitosan was 27.2 g. Its degree of deacetylation was 89.7%, ash content was 0.5%, and viscosity-average molecular weight was 68.5 × 10⁻⁶. 4 This indicates that the process can still be carried out effectively even under relatively mild deproteinization conditions, and the product ash content is within the acceptable range, demonstrating the robustness of the gradient design.

[0049] Example 5

[0050] The difference between this embodiment and Embodiment 1 lies in step (3): the decalcification products are placed sequentially in: First stage: 2% NaOH solution (solid-liquid ratio 1:18 g / mL), react at 50℃ for 2 hours, then wash; Second stage: 4% NaOH solution (solid-liquid ratio 1:18 g / mL), react at 80℃ for 2 hours, then wash; Third stage: 6% NaOH solution (solid-liquid ratio 1:18 g / mL), react at 90℃ for 2 hours, then wash; The remaining steps are the same as in Example 1.

[0051] The final yield of chitosan was 27.8 g. Its degree of deacetylation was 90.8%, ash content was 0.4%, and viscosity-average molecular weight was 61.2 × 10⁻⁶. 4 This demonstrates that even under the most stringent conditions, the method of this invention can still successfully prepare chitosan products with high deacetylation degree, low ash content, and excellent molecular weight retention.

[0052] Example 6

[0053] The difference between this embodiment and Example 1 lies in step (4): Chitosan and 10% potassium permanganate solution are mixed at a solid-liquid ratio of 1:30 (g / mL), reacted at 60°C for 2 hours, filtered after reaction, washed with 1% oxalic acid solution to reduce and remove the generated MnO2, washed with deionized water until neutral, and dried. The remaining steps are the same as in Example 1.

[0054] The final yield of chitosan was 26.5 g. Its degree of deacetylation was 90.5%, ash content was 0.3%, and viscosity-average molecular weight was 63.7 × 10⁻⁶. 4 The product exhibits significant whiteness, demonstrating the effectiveness of potassium permanganate as an oxidant under end-point conditions, particularly in reducing ash content.

[0055] Example 7

[0056] Steps (1) and (5) in this embodiment are the same as in embodiment 1; Step (2) is the same as in Example 2, but under these conditions it is a mild decalcification; Step (3) is the same as in Example 4, but under these conditions, it is a mild deproteinization process; Step (4) is as follows: Chitosan and 20% sodium hypochlorite solution are mixed at a solid-liquid ratio of 1:40 (g / mL), reacted at 90℃ for 5 hours, washed until neutral, and dried.

[0057] The final yield of chitosan was 24.3 g. Its degree of deacetylation was 93.8%, ash content was 0.6%, and viscosity-average molecular weight was 55.9 × 10⁻⁶. 4 Under these combined conditions, the degree of deacetylation is very high, but the ash content is slightly higher due to the relatively mild deproteinization and decalcification. Nevertheless, it still demonstrates the feasibility of the process and the characteristics of the product under different combinations.

[0058] Comparative Example 1

[0059] This comparative example uses a traditional method for preparing chitosan, and differs from Example 1 in that: In step (2), the sieved powder is mixed with 1 M hydrochloric acid solution at a solid-liquid ratio of 1:15 (g / mL) and decalcified at room temperature for 4 hours; In step (3), the protein is removed in one step at 90°C for 4 hours using 4% NaOH solution; In step (5), the acetylation was carried out at 100°C for 8 hours with 50% NaOH solution without ultrasonic assistance.

[0060] The resulting chitosan yielded 24.5 g, with a degree of deacetylation of 75.4%, an ash content of 1.8%, and a viscosity-average molecular weight of 12 × 10⁻⁶. 4 As a comparison with the traditional process (strong acid, one-step strong alkali, no ultrasound), its various indicators were the lowest, and its overall quality was poor, proving the necessity of the innovative process of this invention.

[0061] Comparative Example 2

[0062] The difference between this comparative example and Example 1 is that: only in step (5) deacetylation, ultrasonic assistance is not used, and the reaction is mechanically stirred at 90°C for 6 hours; the remaining steps are exactly the same as in Example 1.

[0063] The resulting chitosan yielded 26.8 g, with a degree of deacetylation of 78.6%, an ash content of 0.5%, and a viscosity-average molecular weight of 15 × 10⁻⁶. 4 This indicates that the lack of ultrasound assistance leads to limited mass transfer, incomplete reaction, and a significant decrease in deacetylation efficiency. Ultrasound is key to improving quality and efficiency.

[0064] Comparative Example 3

[0065] The difference between this comparative example and Example 1 is that only in step (3) the protein removal is a traditional one-step method, using 4% NaOH solution, treated at 90°C for 4 hours at a time, washed until neutral, and dried; the remaining steps are exactly the same as in Example 1.

[0066] The resulting chitosan was a deep yellow color, weighing 26.0 g, with a degree of deacetylation of 85.2%, an ash content of 1.5%, and a viscosity-average molecular weight of 10 × 10⁻⁶. 4 This indicates a lack of a gradient strategy. While it can remove proteins, it cannot effectively protect the molecular chains (low viscosity) and thoroughly remove impurities (high ash content, dark color). Furthermore, the one-step high-concentration alkali solution, while forcibly breaking down insoluble proteins and pigment cross-links, inevitably attacks and breaks the chitin molecular chains, resulting in a low product molecular weight. Additionally, the one-step alkali treatment may cause some pigments to carbonize or redeposit inside the material due to overly vigorous reactions, thus negatively impacting the decolorization effect.

[0067] Comparative Example 4

[0068] The difference between this comparative example and Example 1 is that only the decalcification in step (2) is done by the traditional method: using 1 M hydrochloric acid solution, mixing at a solid-liquid ratio of 1:15 (g / mL), and decalcifying at room temperature for 4 hours. The remaining steps are exactly the same as in Example 1.

[0069] The resulting chitosan yielded 27.5 g, with a degree of deacetylation of 88.7%, an ash content of 0.9%, and a viscosity-average molecular weight of 13 × 10⁻⁶. 4 This indicates that even with the same subsequent processes, the initial damage to the molecular chains caused by strong inorganic acids will continue into the final product, resulting in a lower molecular weight and higher ash content.

[0070] Comparative Example 5

[0071] The difference between this comparative example and Example 1 is that the composite organic acid solution in step (2) contains 0.010 M citric acid and 0.015 M malic acid; the remaining steps are exactly the same as in Example 1.

[0072] The resulting chitosan yielded 22.0 g, with a degree of deacetylation of 80.5%, an ash content of 1.2%, and a viscosity-average molecular weight of 17 × 10⁻⁶. 4 Insufficient acidity prevents the complete complexation and dissolution of calcium carbonate in the pupal shell within a reasonable timeframe, resulting in incomplete decalcification. Residual calcium can shield some chitin, hindering the subsequent attack of alkaline solutions and oxidants on internal proteins and pigments, leading to a decrease in protein and color removal rates, ultimately affecting the purity and whiteness of the product.

[0073] Comparative Example 6

[0074] The difference between this comparative example and Example 1 is that the composite organic acid solution in step (2) contains 0.040 M citric acid and 0.060 M malic acid; the remaining steps are exactly the same as in Example 1.

[0075] The resulting chitosan yielded 27.0 g, with a degree of deacetylation of 89.5%, an ash content of 0.8%, and a viscosity-average molecular weight of 13 × 10⁻⁶. 4 High concentrations of acid can exacerbate molecular chain damage and increase the risk of impurity redeposition.

[0076] Comparative Example 7

[0077] The difference between this comparative example and Example 1 is that the composite organic acid solution in step (2) contains 0.035 M citric acid and 0.015 M malic acid, that is, the molar ratio of citric acid to malic acid is 3.5:1.5; the remaining steps are exactly the same as in Example 1.

[0078] The resulting chitosan yielded 27.2 g, with a degree of deacetylation of 90.0%, an ash content of 0.7%, and a viscosity-average molecular weight of 16 × 10⁻⁶. 4 The decalcification efficiency was acceptable, but the molecular weight of the resulting chitosan was lower than that in Example 1, and the ash content increased to 0.7%. If the citric acid ratio is too high: the system becomes too acidic, the buffering capacity weakens, and the effect approaches that of a strong acid. Although the decalcification rate increases slightly, it exacerbates the risk of acid hydrolysis of the chitin molecular chains, which is detrimental to maintaining the final chitosan molecular weight. Simultaneously, the limited solubility of calcium citrate may cause it to precipitate prematurely on the material surface, hindering internal decalcification and increasing ash content.

[0079] Comparative Example 8

[0080] The difference between this comparative example and Example 1 is that the composite organic acid solution in step (2) contains 0.015 M citric acid and 0.035 M malic acid, that is, the molar ratio of citric acid to malic acid is 1.5:3.5; the remaining steps are exactly the same as in Example 1.

[0081] The resulting chitosan yielded 23.5 g, with a degree of deacetylation of 82.3%, an ash content of 1.1%, and a viscosity-average molecular weight of 19 × 10⁻⁶. 4 If the malic acid ratio is too high, although the buffering capacity is good, the overall decalcification efficiency will decrease significantly, and it will be impossible to achieve sufficient decalcification within the predetermined time. This will increase the burden on subsequent alkali treatment and ultimately result in the product having excessive ash content.

[0082] Comparative Example 9

[0083] The difference between this comparative example and Example 1 is that the ultrasonic power in step (5) is increased to 1000 W; the remaining steps are exactly the same as in Example 1.

[0084] The resulting chitosan yielded 27.0 g, with a degree of deacetylation of 90.5%, an ash content of 0.4%, and a viscosity-average molecular weight of 9.5 × 10⁻⁶. 4 Excessive ultrasonic cavitation leads to severe mechanical molecular chain breakage, resulting in a sharp decrease in molecular weight.

[0085] Comparative Example 10

[0086] The difference between this comparative example and Example 1 is that the first stage of the gradient alkaline deproteinization in step (3) is: treatment at 4% NaOH and 70°C for 1.5 hours. The second and third stages are the same as in Example 1, and the remaining steps are unchanged.

[0087] The resulting chitin intermediate was dark in color, making subsequent decolorization difficult. The final yield of chitosan was 25.5 g, with a degree of deacetylation of 87.5%, an ash content of 0.8%, and a viscosity-average molecular weight of 18 × 10⁻⁶. 4 This indicates that prematurely applying strong conditions before all loose proteins have been removed may cause these proteins to denature rapidly and coagulate on the surface or in the pores of the material, forming a dense layer. This, in turn, hinders subsequent reactions and results in a high protein residue in the final product.

[0088] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing chitosan based on black soldier fly larvae shells, characterized in that, Includes the following steps: S1. The black soldier fly pupa shells are washed, dried, crushed, and sieved to obtain raw material powder; S2. Mix the above raw material powder with the composite organic acid solution, and wash until neutral and dry after reaction; the composite organic acid solution includes citric acid and malic acid. S3. The material obtained in step S2 is subjected to at least three stages of reaction with alkaline solutions of increasing concentration. After each stage of reaction, the mixture is washed until neutral and then dried to obtain chitin. S4. Mix the chitin obtained in step S3 with an alkaline solution and react with ultrasound. After the reaction is complete, wash until neutral and dry to obtain chitosan.

2. The method for preparing chitosan according to claim 1, characterized in that, In step S2, the concentration of citric acid in the composite organic acid solution is 0.015-0.035 M, and the concentration of malic acid is 0.0225-0.0525 M.

3. The method for preparing chitosan according to claim 2, characterized in that, The molar ratio of citric acid to malic acid is 2:

3.

4. The method for preparing chitosan according to claim 1, characterized in that, At least three-order reactions in step S3 include: A 1%-2% sodium hydroxide solution was used, and the reaction was carried out at 40-50℃ for 1-2 hours. A 3%-4% sodium hydroxide solution was used, and the reaction was carried out at 60-80℃ for 1-2 hours. A sodium hydroxide solution with a mass concentration of 4%-6% was used, and the reaction was carried out at 80-90℃ for 0.5-2 hours.

5. The method for preparing chitosan according to claim 1, characterized in that, After step S3 and before step S4, step S3-1 is also included: The material obtained in step S3 is mixed with an oxidant, and after reaction, it is washed until neutral and dried; the oxidant is selected from at least one of potassium permanganate, hydrogen peroxide, and sodium hypochlorite.

6. The method for preparing chitosan according to claim 5, characterized in that, In step S3-1, the mass concentration of the oxidant is 10%-20%; the solid-liquid ratio of the material obtained in step S3 to the oxidant is 1g:(30-40)mL.

7. The method for preparing chitosan according to claim 1, characterized in that, In step S4, the alkaline solution is a sodium hydroxide solution with a mass concentration of 30%-50%; the power of the ultrasound is 500-800 W.

8. The method for preparing chitosan according to claim 1, characterized in that, In step S2, the solid-liquid ratio of the raw material powder to the composite organic acid solution is 1g:(10-15)mL; in step S4, the solid-liquid ratio of the chitin to the sodium hydroxide solution is 1g:(15-20)mL.

9. A chitosan obtained by the preparation method according to any one of claims 1-8, characterized in that, The degree of deacetylation of the chitosan is not less than 86.5%, and / or the ash content is not more than 0.6%.

10. An application of chitosan as described in claim 9 in the fields of medical dressings or water treatment.

Images