Method for extracting chitin from insect larvae in one step
The one-step method using anionic surfactants and oxidizing agents effectively addresses the inefficiencies of current chitin extraction from insect larvae, achieving high purity and low residual impurities.
Patent Information
- Application Number
- CN202510475732.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-16
- Publication Date
- 2025-07-15
AI Technical Summary
The existing chitin extraction process is complicated, has serious chemical contamination, long treatment cycle, and there is a problem of incomplete removal of protein and fat.
The deprotein, degreasing and demineralization of insect larvae are synchronized by heating and stirring treatment. The specific steps include adding the insect larvae to the mixed solution of anionic surfactant and oxidizing agent, heating and stirring, filtering and drying.
It realizes efficient extraction of chitin in insect larvae, simplifies the process flow, reduces chemical pollution, reduces processing time and reagent costs, and improves the purity and quality of the product.
Smart Images
Figure CN120309761A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of chitin extraction, and specifically relates to a method for extracting chitin from insect larvae in one step. Background Art
[0002] Chitin is a natural polysaccharide mainly composed of N-acetylglucosamine units linked by β-1,4 glycosidic bonds. It is one of the most abundant biopolymers on earth and is widely present in the exoskeletons of crustaceans, insect exoskeletons, and fungal cell walls. Due to its good biocompatibility and biodegradability, chitin has a wide range of applications in the fields of medicine, agriculture, food industry, and environmental protection. In medicine, chitin is used to prepare wound dressings, drug carriers, and tissue engineering scaffolds; in agriculture, it is used as a natural pesticide and soil conditioner; in food processing, it can be used as a thickener and preservative; in environmental protection, it can be used for dye and heavy metal adsorption.
[0003] Insects are an important source of chitin. Compared with shrimp and crab shells, insects have the characteristics of low heavy metal content and few mineral salt impurities. Especially for larvae, due to their easy large-scale cultivation, they have stable output quality. Taking fly maggots as an example, fly maggots are common insect larvae with a short growth cycle, low requirements for the cultivation environment, and can use organic waste as feed, which conforms to the concept of circular economy. Many insects and their by-products (such as insect pupal shells, stumps, molted shells, etc.) are the same as fly maggots, with high protein and fat content. Therefore, for the extraction of chitin from insects and their by-products, deproteinization and defatting are important steps.
[0004] The alkali method is the most traditional method for insect deproteinization, usually using sodium hydroxide to remove proteins at high temperature. The alkali method has good protein removal effect, but it causes serious environmental pollution and also damages the structure of chitin. Biological deproteinization methods include enzymatic hydrolysis and microbial fermentation, which have the advantages of mildness and no pollution, but have a long reaction cycle, incomplete protein removal, and high costs. The emerging deep eutectic solvent extraction method is a process that uses a eutectic solvent composed of a hydrogen bond donor (polyol, urea, and carboxylic acid) and a hydrogen bond acceptor (such as quaternary ammonium salt) to simultaneously remove proteins and minerals in the raw material to obtain chitin. Although deep eutectic solvents have low toxicity and can be recycled, there are still problems with insufficient impurity removal rate, and their recovery process increases the process cost.
[0005] The defatting process of insects widely uses organic solvents, including petroleum ether, isopropanol, ether, ethanol, and n-hexane, etc. Organic solvents have high defatting efficiency, wide application range, and easy removal of residues, but they generally have environmental pollution and biological toxicity, and their volatility and flammability increase the health and safety risks in applications. Summary of the Invention
[0006] The object of the present invention is to provide an efficient extraction method for simultaneously deproteinizing, defatting, and demineralizing in one step from insect larvae, aiming at the problems in the existing chitin extraction process, such as cumbersome process steps, serious chemical pollution, and long treatment cycle. The one-step efficient extraction is achieved through the synergistic effect of an anionic surfactant and an oxidant.
[0007] To achieve the above object, the technical solution adopted by the present invention is: a method for extracting chitin from insect larvae in one step, which is to immerse the insect larvae in a mixed reaction system of an anionic surfactant and an oxidant, and simultaneously complete deproteinization, defatting, and pigment removal through a single heating and stirring treatment. Specifically, it includes: adding the insect larvae into a mixed solution containing an anionic surfactant and an oxidant, heating and stirring, filtering, collecting the solid, washing, and drying to obtain chitin.
[0008] Further, the insect larvae include one or more of the following: black soldier fly larvae, yellow mealworm, cicada slough, and maggots.
[0009] Further, the anionic surfactant is selected from one or more of the following: sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate, sodium lauryl ether sulfate, and alkylphenol polyoxyethylene ether sulfate.
[0010] Further, the oxidant is selected from one or more of the following: hydrogen peroxide, sodium chlorite, and sodium hypochlorite.
[0011] Further, the insect larvae are undried or dried insect larvae.
[0012] Further, in the mixed solution of the anionic surfactant and the oxidant, the mass concentration of the anionic surfactant is 5-15 wt%, and the mass concentration of the oxidant is 2-10 wt%.
[0013] Further, the mass concentration of the anionic surfactant is 15 wt%, and the mass concentration of the oxidant is 7 wt%.
[0014] Further, the material-liquid ratio of the insect larvae (g) to the mixed solution of the anionic surfactant and the oxidant (mL) is: (0.02-0.1) g / mL.
[0015] Further, the heating temperature is 70°C - 80°C.
[0016] Further, the heating temperature is 80°C.
[0017] Further, the stirring rate is 400-700 rad / min.
[0018] Further, the heating and stirring time is 3-6 h.
[0019] The hydrophobic alkyl chains of anionic surfactants (such as SDS) bind to the fat layer of insect larvae (mainly triglycerides and phospholipids) through hydrophobic interactions, forming a micelle structure (monomers below the CMC value and micelles above the CMC), and encapsulating the lipids in the micelle core. The hydrophilic sulfate or sulfonate head groups of anionic surfactants (such as SDS) strongly interact with water molecules, emulsifying and dispersing the lipids in the solution to achieve efficient degreasing.
[0020] The polar head groups of anionic surfactants (such as SDS) bind to the amino (-NH2) and carboxyl (-COOH) groups of proteins through electrostatic attraction, disrupting the secondary structures such as α-helices and β-sheets of proteins and causing them to denature and precipitate. At the same time, anionic surfactants (such as SDS) penetrate into the hydrophobic core of proteins, breaking hydrogen bonds and van der Waals forces, resulting in the collapse of the protein's spatial conformation. Oxidants oxidize the conjugated double bonds and benzene ring structures in melanin (a macromolecule formed by the polymerization of dopaquinone) through free radical chain reactions, degrading it into small molecule carboxylic acids (such as oxalic acid and maleic acid) to achieve a bleaching effect. Oxidants attack cysteine residues in proteins, oxidizing disulfide bonds (-S-S-) to sulfonic acid groups (-SO3H), completely destroying the tertiary structure of proteins and enhancing the efficiency of protein removal. Taking H2O2 as an example, the oxygen microbubbles (O2) generated by the decomposition of H2O2 can physically damage the dense structure of the chitin-protein complex on the insect cuticle, increasing the penetration depth of the surfactant.
[0021] Anionic surfactants (such as SDS) reduce the surface tension of the solution to 30 - 40 mN / m (72 mN / m for pure water), enabling the reaction solution to fully wet the micropores on the insect cuticle (pore diameter is about 1 - 5 μm), allowing the oxidant to penetrate deep into the cuticle. The hydroxyl radicals (·OH) generated by the decomposition of H2O2 oxidize unsaturated fatty acids (such as oleic acid) in lipids, which are further encapsulated by surfactant micelles to form a more stable emulsion (Zeta potential reaches above -40 mV). The pH of the mixed system is maintained at 4 - 6 (weakly acidic), which not only avoids the hydrolysis of the N-acetyl group of chitin under strong alkaline conditions (the degree of deacetylation increases significantly when pH > 10), but also ensures the activity of the oxidant (the half-life of H2O2 is the longest at pH 3 - 6).
[0022] If only surfactants are used for degreasing and protein removal, subsequent oxidation and bleaching steps are required, increasing the total time consumption by 2 - 3 times. In step-by-step treatment, the surfactant may adsorb on the surface of chitin, requiring additional water washing steps (3 - 5 times), resulting in a water consumption increase of more than 50%. Alkaline protein removal (NaOH treatment) will neutralize the subsequent acidic oxidation conditions (H2O2 decomposes rapidly under alkaline conditions), requiring frequent pH adjustment and increasing the process complexity.
[0023] Chitin in the insect cuticle is protonated under weakly acidic conditions (-NH3 +), it has strong electrostatic adsorption with the quaternary ammonium cation (R-N + (CH3)3) of the cationic surfactant (such as cetyltrimethylammonium bromide (CTAB)), forming an insoluble complex (precipitate), clogging the epidermal micropores, and hindering the penetration of reagents. The cationic surfactant combines with negatively charged proteins (isoelectric point pH 4-5) through electrostatic interaction, forming a stable colloidal suspension (particle size > 200 nm), which is difficult to separate by filtration, resulting in a decrease in the protein removal rate of more than 30%. The amino group or quaternary ammonium group of the cationic surfactant is easily oxidized by H2O2 (generating N-oxide), resulting in a decrease in the effective concentration of the oxidant by more than 50%.
[0024] Amphoteric surfactants (such as betaine) are electrically neutral at the isoelectric point (pH 4-6), losing their emulsifying ability (the micelle size increases from 10 nm to 100 nm), while chitin extraction needs to be carried out at pH 4-6, resulting in a 40% decrease in defatting efficiency. Amphoteric surfactants are prone to intramolecular cyclization (such as generating lactones) at high temperatures, losing their surface activity. The tertiary amine group of betaine is easily oxidized by H2O2 to N-oxide, resulting in the destruction of the surfactant structure (degradation rate > 60%), and it is impossible to maintain a stable micelle system.
[0025] In summary, the mixed system of anionic surfactant and oxidant realizes efficient one-step extraction through three major mechanisms: charge complementarity (the negative charge of the surfactant combines with the positive charge of the protein), free radical synergy (the decomposition of the oxidant enhances emulsification), and structure destruction (the composite action disintegrates the cutin layer). However, due to problems such as charge conflict and oxidation inactivation, cationic and amphoteric surfactants cannot meet the physicochemical requirements of chitin extraction, resulting in a significant decline in process performance.
[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0027] 1. Through the synergistic effect of anionic surfactant and oxidant, protein removal, defatting, and pigment removal are completed synchronously in one step, simplifying the traditional multi-step process (such as alkali treatment + oxidative bleaching + organic solvent defatting) into a single-step reaction.
[0028] 2. Anionic surfactants (such as SDS) are used to replace cationic surfactants, amphoteric surfactants, and strong alkalis (such as NaOH). The pH of the reaction system is maintained at a weak acidic level of 4-6, avoiding the discharge of strong alkali wastewater (the traditional alkali method requires neutralization treatment when pH > 12), and there is no need for organic solvent recovery, meeting the requirements of green chemistry.
[0029] 3. The weak acidic reaction conditions (pH value = 4-6) and short-time treatment method (3-6 h) provided by the present invention can significantly reduce the content of proteins and fats.
[0030] 4. The method provided by the present invention is applicable to the treatment of wet / dry raw materials of various insect larvae (such as black soldier fly larvae, maggots, etc.). The liquid-to-solid ratio can be as low as 0.02 g / mL (Example 1), and the reagent cost is reduced by 40%. Compared with the alkali method and the enzymatic hydrolysis method, both the anionic surfactant (SDS) and the oxidant (H2O2) are industrial bulk chemicals, and the unit price is less than 1 / 20 of the enzymatic hydrolysis reagent. Moreover, the energy consumption at the reaction temperature (70 - 80 °C) is only 65% of that of the traditional high-temperature alkali method (95 °C), showing the potential for large-scale production. Description of the Drawings
[0031] Figure 1 This is the chitin extracted from insect larvae in one step in Example 1 of the present invention.
[0032] Figure 2 This is the insect larva raw material in Example 1 of the present invention.
[0033] Figure 3 This is the chitin extracted from insect larvae by the alkali method in Comparative Example 1 of the present invention.
[0034] Figure 4 This is the infrared spectrum diagram of the chitin extracted in Example 4 of the present invention and the infrared spectrum diagram of the chitin standard product. Detailed Description of the Invention
[0035] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the drawings in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.
[0036] The maggot larvae were purchased from: Shanghai Xijiruo Biochemical Co., Ltd.; SDS, hydrogen peroxide, sodium hydroxide, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd., with a purity of 98%.
[0037] Example 1
[0038] The steps for extracting chitin from insect larvae by the one-step method are as follows: Weigh 20 g of maggots under wet weight (the appearance is shown in Figure 2 ), add 250 mL of a mixed solution containing 15 wt% sodium dodecyl sulfate and 7 wt% hydrogen peroxide, place it in a 70 °C constant temperature water bath, and stir and react at a speed of 600 rad / min for 6 h; the concentrations of sodium dodecyl sulfate and hydrogen peroxide in the mixed solution are 15 wt% and 7 wt% respectively.
[0039] Filter the mixture to obtain the solid, wash it with deionized water, and then freeze-dry to obtain chitin. The appearance style of the extracted chitin is shown in Figure 1 .
[0040] Example 2
[0041] Referring to the extraction steps of Example 1, the difference from Example 1 is that the concentration of sodium dodecyl sulfate in the mixed solution is 9 wt%, and the rest is the same as Example 1.
[0042] Example 3
[0043] Referring to the extraction steps of Example 1, the difference from Example 1 is that the concentration of hydrogen peroxide in the mixed solution is 3 wt%, and the rest is the same as Example 1.
[0044] Example 4
[0045] Referring to the extraction steps of Example 1, the difference from Example 1 is that the temperature of the constant temperature water bath is 80 °C, and the rest is the same as Example 1.
[0046] The infrared spectra of the chitin extracted and the chitin standard are shown in Figure 4 .
[0047] Example 5
[0048] Referring to the extraction steps of Example 1, the difference from Example 1 is that sodium dodecyl sulfate in the mixed solution is replaced by sodium dodecylbenzenesulfonate with the same dosage, and the rest is the same as Example 1.
[0049] Example 6
[0050] Referring to the extraction steps of Example 1, the difference from Example 1 is that sodium dodecyl sulfate in the mixed solution is replaced by sodium lauryl polyoxyethylene ether sulfate with the same dosage, and the rest is the same as Example 1.
[0051] Comparative Example 1
[0052] In this Comparative Example 1, traditional alkaline method was used to treat the fly maggots. The specific steps were as follows: Weigh 20 g of fly maggots under wet weight (appearance is shown in Figure 2 ), add 50 mL of 4% sodium hydroxide solution, place it in a 95 °C constant temperature water bath, and stir and react at a speed of 500 rad / min for 6 h; filter the mixture to obtain the solid, wash it with deionized water, and freeze-dry to obtain chitin. The appearance style of the extracted chitin is shown in Figure 3 .
[0053] Comparative Example 2
[0054] In this Comparative Example 2, a cationic surfactant complex and a hydrogen peroxide mixed system were used. Referring to the extraction steps of Example 1, the difference from Example 1 is that sodium dodecyl sulfate in the mixed solution is replaced by cetyltrimethylammonium bromide with the same dosage, and the rest is the same as Example 1.
[0055] Comparative Example 3
[0056] In Comparative Example 3, a zwitterionic surfactant complexing and hydrogen peroxide mixed system was used. Referring to the extraction steps of Example 1, the difference from Example 1 was that sodium dodecyl sulfate in the mixed solution was replaced by betaine with the same dosage, and the rest was the same as Example 1.
[0057] Comparative Example 4
[0058] In Comparative Example 5, only hydrogen peroxide was used as a single component to extract chitin from insect larvae. Referring to the extraction steps of Example 1, the difference from Example 1 was that only hydrogen peroxide was used in the mixed solution with the same dosage, and the rest was the same as Example 1.
[0059] Performance detection:
[0060] 1. Measuring the protein content in the prepared chitin by the Lowry method: Take 100 mg of dry chitin sample, add 10 mL of 5% sodium hydroxide solution, heat in a water bath at 95 °C for 2 h, centrifuge (12,000 rpm, 10 min), and take the supernatant for standby. Take 1 mL of the sample supernatant, add 5 mL of 3% Na2CO3 - 0.5% NaOH mixed solution, immediately vortex and mix, let stand at room temperature for 10 min, then add 0.5 mL of Folin-Ciocalteu reagent, immediately vortex and mix, and measure the absorbance at 750 nm after standing at room temperature for 30 min.
[0061] Using the BSA concentration (μg / mL) as the abscissa and the absorbance as the ordinate, fit a linear equation (R 2 ≥ 0.99).
[0062] Protein content formula: ;
[0063] where C: protein concentration calculated according to the standard curve (μg / mL), V 提取液 : volume of the extract (5 mL), W: mass of the chitin sample (100 mg = 0.1 g).
[0064] 2. Measuring the fat content in the chitin by the Soxhlet extraction method: Take 2.0 g of dry chitin sample, crush it through an 80-mesh sieve, wrap it in a filter paper to form a cylinder, and place it in a Soxhlet extraction tube. Add 150 mL of n-hexane to the round-bottom flask, connect the Soxhlet extractor, adjust the water bath temperature to 70 - 80 °C, and reflux for 8 hours (siphon frequency about 12 times / hour). After the extraction, recover the n-hexane, place the round-bottom flask in an oven at 105 °C for 1 hour, cool and weigh.
[0065] Calculating the fat content: , W 空瓶 : mass of the round-bottom flask before extraction (g), W 样品: Chitin mass (2.0 g).
[0066] The content data of protein and fat in chitin are shown in Table 1.
[0067] Table 1.
[0068] .
[0069] The one-step extraction method provided by the present invention significantly improves the extraction efficiency and purity of chitin through the synergistic effect of an anionic surfactant (such as sodium dodecyl sulfate) and an oxidant (such as hydrogen peroxide). In Example 1, after heating and reacting a mixed solution of 15 wt% SDS and 7 wt% H2O2 at 70 °C for 6 hours, the protein residue of the obtained chitin is only 5.5% and the fat residue is 5.4%. This indicates that the method of the present invention can efficiently remove impurities such as protein and fat. Further performance improvement can be achieved through parameter optimization (such as surfactant concentration, temperature, reaction time). For example, in Example 4, when reacting at 80 °C, the protein residue is further reduced to 1.4%, verifying the flexibility and controllability of this process.
[0070] The chitin extracted in Example 1 is shown in Figure 1 , and the chitin extracted in Comparative Example 1 is shown in Figure 3 . Among them Figure 1 The extracted chitin presents a uniform white powder shape, with a fluffy structure and no impurities, indicating that the synergistic effect of the anionic surfactant and the oxidant efficiently removes protein, fat and pigments; while Figure 3 the chitin is yellowish in color and has a dense structure, with obvious residual impurities, reflecting the problem of the destruction of the chitin structure by strong alkali treatment and incomplete deproteinization. By comparison, it can be seen that the one-step method of the present invention not only significantly improves the product purity under mild weak acidic conditions, but also avoids the destruction of the molecular integrity of chitin by traditional processes.
[0071] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for extracting chitin from insect larvae in one step, characterized in that, It includes the following steps: adding insect larvae into a mixed solution containing an anionic surfactant and an oxidant, heating and stirring, filtering, collecting the solid, washing and drying to obtain chitin; The oxidant is selected from one or more of the following: hydrogen peroxide, sodium chlorite, and sodium hypochlorite.
2. The method for one-step extraction of chitin from insect larvae according to claim 1, characterized in that, The anionic surfactant is selected from one or more of the following: sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium fatty alcohol polyoxyethylene ether sulfate, and alkylphenol polyoxyethylene ether sulfate.
3. A method for extracting chitin from insect larvae in one step according to claim 2, characterized in that, The anionic surfactant is selected from one or more of the following: sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, and sodium fatty alcohol polyoxyethylene ether sulfate.
4. A method for extracting chitin from insect larvae in one step according to claim 1, characterized in that, For the mixed solution of the anionic surfactant and the oxidant, the mass concentration of the anionic surfactant is 5 - 15 wt%, and the mass concentration of the oxidant is 2 - 10 wt%.
5. A method for extracting chitin from insect larvae in one step according to claim 4, characterized in that, For the mixed solution of the anionic surfactant and the oxidant, the mass concentration of the anionic surfactant is 15 wt%, and the mass concentration of the oxidant is 7 wt%.
6. A method for extracting chitin from insect larvae in one step according to claim 4, characterized in that The ratio of the insect larvae (g) to the mixed solution (mL) is (0.02 - 0.1) g / mL.
7. A method for one-step extraction of chitin from insect larvae according to claim 1, characterized in that, The method of heating and stirring is to stir at 70°C - 80°C and 400 - 700 rad / min for 3 - 6 h.
8. A method for extracting chitin from insect larvae in one step according to claim 1, characterized in that The heating temperature is 80°C.
9. A method for extracting chitin from insect larvae in one step according to claim 1, characterized in that, The insect larvae include one or more of the following: black soldier fly larvae, yellow mealworms, cicada sloughs, and maggots; The insect larvae are undried or dried insect larvae.