Method for preparing food-grade glucosamine by deacidification of chitin enzymatic hydrolysate through electrodialysis

Abstract

The application provides a method for preparing food-grade glucosamine by deacidifying chitin enzymatic hydrolysate through electrodialysis. The method is as follows: step 1: obtain desalted shell powder; step 2: obtain chitin powder; step 3: obtain acidic enzymatic hydrolysate containing glucosamine; step 4: obtain deacidified glucosamine solution; step 5: obtain crude glucosamine crystals; and step 7: obtain food-grade glucosamine finished product. The application solves the pain points of “glucosamine easy to degrade and low purity” in the traditional preparation process of food-grade glucosamine from chitin by constructing an integrated process system of “tea saponin assisted deproteinization + glycyrrhizic acid dipotassium-myo-inositol synergistic enzymolysis + selective ion membrane electrodialysis deacidification”.

Description

Technical Field

[0001] This invention relates to the technical field of functional sugar preparation, specifically to a method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate. Background Technology

[0002] Glucosamine is a key derivative of chitin found in the shells of crustaceans and has significant functional value in food. While the acid hydrolysis method is a mature technology in the traditional chitin-to-glucosamine production process, it suffers from problems such as harsh reaction conditions, easy oxidation and degradation of the sugar ring, and the generation of large amounts of acidic wastewater. Furthermore, subsequent deacidification requires a large amount of alkali solution, which can easily introduce impurities that affect food-grade purity requirements.

[0003] Enzymatic hydrolysis, as a mild conversion technology, can degrade chitin into a mixture containing glucosamine under normal temperature and pressure. However, the enzymatic hydrolysis process requires maintaining an acidic environment to ensure enzyme activity, resulting in a large amount of residual organic acids (such as citric acid and acetic acid) in the product solution, directly affecting the purity and taste of glucosamine. Existing deacidification methods, such as resin exchange, have drawbacks such as limited adsorption capacity and frequent regeneration, while alkali neutralization easily generates salt byproducts, making it difficult to meet the strict limits on impurity ions (such as sodium and chloride ions) for food-grade products. At the same time, traditional processes also have problems such as large amounts of papain used in the deproteinization step and easy residue, easy non-specific degradation of glucosamine during enzymatic hydrolysis, and short duration of chitosanase activity leading to insufficient conversion rate, further restricting the production efficiency and quality of food-grade glucosamine. Therefore, developing an efficient, green deacidification and purification technology that can simultaneously solve the problems of deproteinization residue, glucosamine degradation, and insufficient enzyme activity has become the core requirement for the preparation of food-grade glucosamine. Summary of the Invention

[0004] Technical problem to be solved: In view of the above-mentioned technical problems, the purpose of this invention is to provide a method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate. By innovatively constructing an integrated process system of "tea saponin-assisted deproteinization + dipotassium glycyrrhizate-inositol synergistic enzymatic hydrolysis + selective ion membrane electrodialysis deacidification", the pain points of "easy degradation and low purity of glucosamine" in the traditional process of preparing food-grade glucosamine from chitin are solved.

[0005] Technical solution: A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate, comprising the following steps: Step 1: Collect shrimp and crab shells, remove internal organs, meat and other impurities, wash them clean, and then crush them under high pressure and ultrasonically to obtain shell powder raw materials with a particle size of 50-100μm. Under the assistance of 150-200W ultrasound, soak the shell powder in 20-25% citric acid solution for 10-15 minutes to remove inorganic salts such as calcium carbonate. Wash the filter residue until neutral to obtain desalted shell powder. Step 2: Place the desalted chitin powder in a papain system and add tea saponin. Enzymatically hydrolyze the chitin powder at 33-38℃ and 250-300W ultrasonic conditions for 15-25 minutes (tea saponin can destroy the spatial structure of the protein, assist papain in efficient deproteinization, reduce the amount of protease used and reduce residue). After the reaction, wash the filter residue until neutral, add hydrogen peroxide solution for oxidation and decolorization, centrifuge and then vacuum dry at 60-70℃ to obtain chitin powder. Step 3: Dissolve chitin powder in citrate buffer at pH 4.0-5.0 to prepare a suspension with a mass concentration of 5-10%. Add chitosanase and enzyme activator, and simultaneously add dipotassium glycyrrhizate and inositol (wherein, dipotassium glycyrrhizate stabilizes the glucosamine structure by forming a weak interaction with glucosamine molecules, inhibiting the non-specific degradation of glucosamine by chitosanase, and inositol forms a stable complex with chitosanase, maintaining the optimal conformation of the enzyme to prolong the duration of activity). React in a constant temperature shaker at 45-55℃ and 150r / min for 8-12h, and then raise the temperature to 80℃ and keep it at 10min to inactivate the enzyme. Step 4: Remove undegraded solid residue using plate and frame filtration, and collect the filtrate, which is the acidic enzymatic hydrolysate containing glucosamine; Step 5: Deacidify using an electrodialysis device. Pump the acidic enzymatic hydrolysate containing glucosamine into the deacidification chamber, controlling the feed temperature at 25-40℃ and the operating pressure at 0.1-0.3MPa. Connect the DC power supply and adjust the voltage to 15-30V and the current density to 50-100mA / cm². 2 The feed solution is circulated within the membrane stack for treatment. The pH and conductivity of the feed solution in the deacidification chamber are monitored in real time. When the pH rises to 6.0-7.0 and the conductivity drops to less than 10% of the initial value, electrodialysis is stopped and the deacidified glucosamine feed solution is collected. Step 6: Add 0.5-1.0% modified activated carbon to the deacidified glucosamine solution, stir and decolorize at 50-60℃ for 30-60 min, remove activated carbon by plate and frame filtration, and vacuum concentrate to a glucosamine concentration of 150-200 g / L at a temperature of 60-70℃ and a vacuum degree of -0.08~-0.09 MPa; cool the concentrate to 5-10℃ and let it stand for 12-24 h to crystallize, and centrifuge to obtain crude glucosamine crystals; Step 7: Dissolve the crude glucosamine crystals in deionized water to a mass concentration of 40-50%, repeat the concentration and crystallization step once, and vacuum dry at 50-60℃ for 4-6 hours to obtain the food-grade glucosamine product.

[0006] Furthermore, in step 1, the high-pressure pulverization conditions are 1.6-2 MPa for 1-5 min, and the ultrasonic pulverization conditions are 350-400 W for 5-8 min.

[0007] Furthermore, the papain system in step 2 is a papain reaction system with deionized water as solvent, a papain concentration of 0.8-1.2% of the mass of desalted shell powder, and a pH adjusted to 6.0-7.0.

[0008] Furthermore, in step 2, the amount of tea saponin added is 0.1-0.2% of the desalted shell powder.

[0009] Furthermore, in step 3, the chitosanase activity is >50 units / mg protein; the enzyme activator is calcium chloride.

[0010] Furthermore, in step 3, the amount of chitosanase added is 1-3% of the chitin powder, the amount of enzyme activator added is 0.1-0.3% of the chitin powder, the amount of dipotassium glycyrrhizate added is 0.05-0.1% of the chitin powder, and the amount of inositol added is 0.03-0.05% of the chitin powder.

[0011] Furthermore, in step 5, the electrodialysis device adopts a three-compartment electrodialysis membrane stack, which consists of an anode chamber, a deacidification chamber, and an electrode water chamber, with adjacent chambers separated by ion exchange membranes; wherein, an anion exchange membrane is used between the deacidification chamber and the anode chamber (allowing organic acid ions to pass through while retaining glucosamine cations), and a cation exchange membrane is used between the deacidification chamber and the electrode water chamber (blocking organic acid ions and glucosamine from passing through); the electrodes are titanium-coated ruthenium electrodes, and the electrode water is a 0.5 mol / L sodium sulfate solution.

[0012] Furthermore, the modified activated carbon in step 6 is granular activated carbon that has been activated by hydrochloric acid. The hydrochloric acid activation conditions are as follows: the activated carbon is placed in a hydrochloric acid solution with a mass concentration of 10-15% and soaked at a constant temperature of 80-90℃ for 2-3 hours, during which the stirring rate is 50-80 r / min. After soaking, it is washed with deionized water until neutral and dried at 105-110℃ for 3-4 hours.

[0013] The above method produces food-grade glucosamine.

[0014] Furthermore, the purity of the food-grade glucosamine is ≥98%, and the content of impurity ions such as chloride ions and sodium ions is ≤0.1%, which meets the quality requirements for high-purity glucosamine in the fields of food additives and health foods.

[0015] Beneficial effects: 1. This invention solves the pain points of "easy degradation and low purity of glucosamine" in the traditional process of preparing food-grade glucosamine from chitin by innovatively constructing an integrated process system of "tea saponin-assisted deproteinization + dipotassium glycyrrhizate-inositol synergistic enzymatic hydrolysis + selective ion membrane electrodialysis deacidification".

[0016] 2. This invention utilizes tea saponin to assist in deproteinization, improving the thoroughness of deproteinization and reducing subsequent interference. In traditional single papain deproteinization processes, proteins easily form aggregates, and the enzyme can only act on the surface, resulting in a residual deproteinization rate of 0.8%-1.2%. Residual proteins compete with chitosanase for binding sites, leading to a decrease in glucosamine conversion. This invention adds tea saponin, whose hydrophobic groups can break down the hydrophobic core of proteins and disrupt hydrogen and disulfide bonds, dispersing protein aggregates into nanoscale particles. Combined with ultrasonic assistance, the residual deproteinization rate is reduced to 0.22%-0.32%, a 62%-74% reduction compared to Comparative Example 1 (0.85%) without tea saponin. The reduction in residual protein avoids its inhibition of chitosanase activity, laying the foundation for efficient glucosamine conversion. Meanwhile, the denaturing and dispersing effects of tea saponins on proteins can enhance the catalytic efficiency of papain, reducing the amount of papain used from 1.5%-2.0% in traditional processes to 0.8%-1.2%, directly lowering the procurement cost of enzyme preparations. The reduction in the amount of protease used also reduces the amount of residual protease in the subsequent enzymatic hydrolysate, preventing residual protease from degrading glucosamine during the concentration and crystallization stage, thus ensuring the purity of the finished product.

[0017] 3. This invention employs a dipotassium glycyrrhizate-inositol synergistic enzymatic hydrolysis. Dipotassium glycyrrhizate stabilizes the glucosamine structure and inhibits excessive degradation. During the enzymatic hydrolysis process, chitosanase, while degrading chitin, readily exerts non-specific catalysis on the generated glucosamine, resulting in a glucosamine degradation rate of 5%-8%. The addition of dipotassium glycyrrhizate allows its hydroxyl groups to form a weak interaction with glucosamine, encapsulating the glucosamine molecule and shielding easily attacked active sites (such as the C6 hydroxyl group), thus reducing the glucosamine degradation rate to 3.2%-3.5%. Simultaneously, dipotassium glycyrrhizate regulates the microenvironment of the enzymatic hydrolysis system, preventing a sudden drop in pH due to the accumulation of organic acids, maintaining the optimal catalytic pH for chitosanase, and further reducing deamination degradation of glucosamine caused by an acidic environment. This invention incorporates inositol, whose six-membered ring structure can form hydrogen bonds with amino acid residues surrounding the active site of chitosanase, stabilizing the enzyme's three-dimensional conformation, prolonging its half-life, and ensuring sustained stability of enzyme activity. This increases the glucosamine conversion rate from the traditional 82%-85% to 90.2%-93.3%, and the conversion process is smoother, avoiding premature crystallization caused by excessively high local glucosamine concentrations. Inositol prolongs enzyme activity, allowing for more complete chitin degradation and providing more precursors for glucosamine production; dipotassium glycyrrhizate protects newly generated glucosamine from degradation by excess enzymes, achieving a closed loop of "highly efficient production - stable retention."

[0018] 4. This invention utilizes selective ion-exchange membrane electrodialysis for deacidification, efficiently separating organic acids. Ion migration is driven by potential difference, eliminating the need for alkali solutions and preventing salt formation at the source. The finished product contains only 0.04%-0.07% chloride and sodium ions, meeting food-grade impurity control requirements. Furthermore, the titanium-coated ruthenium electrode exhibits high stability during electrodialysis, preventing metal ion impurities generated from electrode dissolution, further ensuring product purity.

[0019] 5. In the vacuum concentration stage, the present invention uses gradient heating to avoid caramelization of glucosamine caused by boiling of the liquid; combined with slow cooling, it improves the crystallization rate and increases the crystallization yield, ultimately meeting the requirements for high purity in food-grade products. Detailed Implementation

[0020] This invention proposes a method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0021] The chitosanase used below was purchased from Maclean's Reagents, catalog number: C920565-1U.

[0022] The electrodialysis apparatus used below employs a three-compartment electrodialysis membrane stack, consisting of an anode compartment, a deacidification compartment, and an electrode water compartment, with adjacent compartments separated by ion exchange membranes; wherein, an anion exchange membrane is used between the deacidification compartment and the anode compartment, and a cation exchange membrane is used between the deacidification compartment and the electrode water compartment; the electrodes are titanium-coated ruthenium electrodes, and the electrode water is a 0.5 mol / L sodium sulfate solution.

[0023] Example 1 The modification method of the modified activated carbon is as follows: the activated carbon is placed in a 13% hydrochloric acid solution and soaked at a constant temperature of 80℃ for 2 hours, during which the stirring rate is 60r / min. After soaking, it is washed with deionized water until neutral and dried at 110℃ for 3 hours.

[0024] Example 2 A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Collect shrimp and crab shells, remove internal organs, meat and other impurities, wash them clean, and then crush them under high pressure (2MPa) for 4 minutes and ultrasonically (400W) for 8 minutes to obtain shell powder raw material with a particle size of 50-100μm; under the assistance of 180W ultrasound, soak the shell powder in 25% citric acid solution for 14 minutes to remove inorganic salts such as calcium carbonate, wash the filter residue until neutral, and obtain desalted shell powder; Step 2: Place the desalted chitin powder in a papain system (using deionized water as solvent, the papain concentration is 0.8% of the mass of the desalted chitin powder, and the pH of the system is adjusted to 6.5), and add 0.1% tea saponin; enzymatically hydrolyze for 20 min under ultrasonic conditions of 35℃ and 300W. After the reaction, wash the filter residue until neutral; add hydrogen peroxide solution for oxidation and decolorization, centrifuge, and then vacuum dry at 65℃ to obtain chitin powder; Step 3: Dissolve chitin powder in citrate buffer at pH 4.5 to prepare a suspension with a mass concentration of 8%; add 1% chitosanase and 0.1% calcium chloride, along with 0.05% dipotassium glycyrrhizate and 0.03% inositol; react in a constant temperature shaker at 50℃ and 150r / min for 10h, then raise the temperature to 80℃ and keep it at that temperature for 10min to inactivate the enzyme; Step 4: Remove undegraded solid residue using plate and frame filtration, and collect the filtrate, which is the acidic enzymatic hydrolysate containing glucosamine; Step 5: Deacidify using an electrodialysis device. Pump the acidic enzymatic hydrolysate containing glucosamine into the deacidification chamber; control the feed temperature at 35℃ and the operating pressure at 0.2MPa; connect the DC power supply, adjust the voltage to 30V and the current density to 80mA / cm², so that the feed solution circulates within the membrane stack; monitor the pH and conductivity of the feed solution in the deacidification chamber in real time. When the pH rises to 6.5 and the conductivity drops to less than 10% of the initial value, stop the electrodialysis and collect the deacidified glucosamine feed solution. Step 6: Add 0.5% modified activated carbon to the deacidified glucosamine solution; stir and decolorize at 55℃ for 50 min, remove activated carbon by plate and frame filtration; vacuum concentrate to glucosamine concentration of 200 g / L at 65℃ and -0.08 MPa; cool the concentrate to 8℃ and let it stand for 20 h to crystallize, then centrifuge to obtain crude glucosamine crystals; Step 7: Dissolve the crude glucosamine crystals in deionized water to a mass concentration of 45%, and repeat the concentration and crystallization step once; dry under vacuum at 50°C for 5 hours to obtain the food-grade glucosamine product.

[0025] Example 3 A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Place the desalted shell powder into a papain system (using deionized water as solvent, the papain concentration is 0.9% of the mass of the desalted shell powder, and the pH of the system is adjusted to 6.5), and add 0.12% tea saponin at the same time; the rest is the same as step 2 in Example 2; Step 3: Add 1.5% chitosanase and 0.15% calcium chloride, along with 0.06% dipotassium glycyrrhizate and 0.035% inositol; the rest is the same as step 3 in Example 2. Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Add 0.6% modified activated carbon to the deacidified glucosamine solution; the rest is the same as step 6 in Example 2. Step 7: Vacuum dry at 52°C for 5 hours; the rest is the same as step 7 in Example 2.

[0026] Example 4 A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Place the desalted shell powder into a papain system (using deionized water as solvent, the papain concentration is 1.0% of the mass of the desalted shell powder, and the pH of the system is adjusted to 6.5), and add 0.14% tea saponin; the rest is the same as step 2 in Example 2. Step 3: Add 2% chitosanase and 0.2% calcium chloride, along with 0.07% dipotassium glycyrrhizate and 0.04% inositol; the rest is the same as step 3 in Example 2. Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Add 0.7% modified activated carbon to the deacidified glucosamine solution; the rest is the same as step 6 in Example 2. Step 7: Vacuum dry at 54℃ for 5 hours; the rest is the same as step 7 in Example 2.

[0027] Example 5 A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Place the desalted shell powder into a papain system (using deionized water as solvent, the papain concentration is 1.1% of the mass of the desalted shell powder, and the pH of the system is adjusted to 6.5), and add 0.16% tea saponin; the rest is the same as step 2 in Example 2. Step 3: Add 2.5% chitosanase and 0.25% calcium chloride, along with 0.08% dipotassium glycyrrhizate and 0.045% inositol; the rest is the same as step 3 in Example 2. Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Add 0.8% modified activated carbon to the deacidified glucosamine solution; the rest is the same as step 6 in Example 2. Step 7: Vacuum dry at 56°C for 5 hours; the rest is the same as step 7 in Example 2.

[0028] Example 6 A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Place the desalted shell powder in a papain system (using deionized water as solvent, the papain concentration is 1.2% of the mass of the desalted shell powder, and the pH of the system is adjusted to 6.5), and add 0.18% tea saponin; the rest is the same as step 2 in Example 2. Step 3: Add 3% chitosanase and 0.3% calcium chloride, along with 0.09% dipotassium glycyrrhizate and 0.05% inositol; the rest is the same as step 3 in Example 2. Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Add 0.9% modified activated carbon to the deacidified glucosamine solution; the rest is the same as step 6 in Example 2.

[0029] Step 7: Vacuum dry at 58°C for 5 hours; the rest is the same as step 7 in Example 2.

[0030] Example 7 A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Place the desalted shell powder into a papain system (using deionized water as solvent, the papain concentration is 0.9% of the mass of the desalted shell powder, and the pH of the system is adjusted to 6.5), and add 0.2% tea saponin at the same time; the rest is the same as step 2 in Example 2; Step 3: Add 1.8% chitosanase and 0.22% calcium chloride, along with 0.075% dipotassium glycyrrhizate and 0.038% inositol; the rest is the same as step 3 in Example 2. Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Add 1.0% modified activated carbon to the deacidified glucosamine solution; the rest is the same as step 6 in Example 2. Step 7: Vacuum dry at 60℃ for 5 hours; the rest is the same as step 7 in Example 2.

[0031] Example 8 A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Place the desalted shell powder into a papain system (using deionized water as solvent, the papain concentration is 1.1% of the mass of the desalted shell powder, and the pH of the system is adjusted to 6.5), and add 0.15% tea saponin; the rest is the same as step 2 in Example 2. Step 3: Add 2.2% chitosanase and 0.18% calcium chloride, along with 0.065% dipotassium glycyrrhizate and 0.042% inositol; the rest is the same as step 3 in Example 2. Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Add 0.75% modified activated carbon to the deacidified glucosamine solution; the rest is the same as step 6 in Example 2. Step 7: Vacuum dry at 55°C for 5 hours; the rest is the same as step 7 in Example 2.

[0032] Example 9 A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Place the desalted shell powder in a papain system (using deionized water as solvent, the papain concentration is 1.0% of the mass of the desalted shell powder, and the pH of the system is adjusted to 6.5), and add 0.17% tea saponin; the rest is the same as step 2 in Example 2. Step 3: Add 2.8% chitosanase and 0.28% calcium chloride, along with 0.085% dipotassium glycyrrhizate and 0.048% inositol; the rest is the same as step 3 in Example 2. Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Add 0.85% modified activated carbon to the deacidified glucosamine solution; the rest is the same as step 6 in Example 2. Step 7: Vacuum dry at 57°C for 5 hours; the rest is the same as step 7 in Example 2.

[0033] Comparative Example 1 The difference between this comparative example and Example 2 is that tea saponin is not added, as detailed below: A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Place the desalted shell powder in a papain system (using deionized water as solvent, the papain concentration is 0.8% of the mass of the desalted shell powder, and the pH of the system is adjusted to 6.5), without adding tea saponin; the rest is the same as step 2 in Example 2; Step 3: Same as step 3 in Example 2; Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Same as step 6 in Example 2; Step 7: Same as step 7 in Example 2.

[0034] Comparative Example 2 The difference between this comparative example and Example 2 is that dipotassium glycyrrhizate is not added, as detailed below: A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Same as Step 2 in Example 2; Step 3: Dissolve chitin powder in citrate buffer at pH 4.5 to prepare a suspension with a mass concentration of 8%; add 1% chitosanase and 0.1% calcium chloride, add only 0.03% inositol, and do not add dipotassium glycyrrhizate; the rest is the same as step 3 in Example 2. Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Same as step 6 in Example 2; Step 7: Same as step 7 in Example 2.

[0035] Comparative Example 3 The difference between this comparative example and Example 2 is that inositol is not added, as detailed below: A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Same as Step 2 in Example 2; Step 3: Dissolve chitin powder in citrate buffer at pH 4.5 to prepare a suspension with a mass concentration of 8%; add 1% chitosanase and 0.1% calcium chloride, add only 0.05% dipotassium glycyrrhizate, and do not add inositol; the rest is the same as step 3 in Example 2. Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Same as step 6 in Example 2; Step 7: Same as step 7 in Example 2.

[0036] Comparative Example 4 The difference between this comparative example and Example 2 is that the traditional acid hydrolysis method is used instead of enzymatic hydrolysis, as detailed below: A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Same as Step 2 in Example 2; Step 3: Mix chitin powder with 6 mol / L hydrochloric acid at a solid-liquid ratio of 1:10, and reflux hydrolyze at 90℃ for 4 hours; after hydrolysis, neutralize with 40% sodium hydroxide solution to pH 6.5, filter to remove residue, and obtain crude liquid containing glucosamine. Step 4: Same as Example 2; Step 5: Same as in Example 2; Step 6: Same as step 6 in Example 2; Step 7: Same as step 7 in Example 2.

[0037] Comparative Example 5 The difference between this comparative example and Example 2 is that resin exchange deacidification is used instead of electrodialysis deacidification, as detailed below: A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate includes the following steps: Step 1: Same as Example 2; Step 2: Same as Step 2 in Example 2; Step 3: Same as step 3 in Example 2; Step 4: Same as Example 2; Step 5: Pump the acidic enzymatic hydrolysate containing glucosamine into a 732 type cation exchange resin column, controlling the flow rate at 2 BV / h; pump the effluent into a D301 type anion exchange resin column, controlling the flow rate at 2 BV / h; collect the effluent, which is the deacidified glucosamine solution. Step 6: Same as step 6 in Example 2; Step 7: Same as step 7 in Example 2.

[0038] Performance testing: Glucosamine conversion rate: High performance liquid chromatography (HPLC) (refer to GB / T 30987-2022) was used to detect the ratio of the amount of glucosamine generated in the system after enzymatic hydrolysis to the amount of glucosamine that chitin can theoretically convert, which reflects the efficiency of the enzymatic hydrolysis process; Purity of glucosamine in finished product: The percentage of glucosamine main peak area in the finished product to the total peak area is determined by HPLC (same as above), and must meet the food grade standard (≥98%). Impurity ion content: Chloride ions (Cl) in the finished product were detected using ion chromatography (refer to GB 5009.261-2021). - Sodium ions (Na) + The mass fraction of the substance must meet the food-grade standard (≤0.1%). Deproteinization residual rate: The Kjeldahl nitrogen determination method (refer to GB 5009.5-2016) was used to detect the mass fraction of residual protein in chitin powder, reflecting the effect of the deproteinization process; Deacidification efficiency: The organic acid removal rate of the feed solution before and after electrodialysis (or resin exchange) was measured using a pH meter and conductivity meter (calculated by changes in pH and conductivity) to reflect the efficiency of the deacidification process. The results are shown in Table 1 below: Table 1

[0039] As shown in Table 1 above, the glucosamine conversion rate of all examples was ≥90.2%, with Example 8 having the highest conversion rate (93.3%). This demonstrates that the combined process of tea saponin-assisted deproteinization and dipotassium glycyrrhizate + inositol synergistic enzymatic hydrolysis effectively improves the conversion efficiency of chitin to glucosamine. The purity of all examples was ≥98.5%, and the impurity ion content was ≤0.07%, meeting food-grade standards. Example 8, with the lowest deproteinization residue rate and the highest deacidification efficiency, achieved a finished product purity of 99.0% and impurities of only 0.04%, representing the optimal combination of process parameters. The deproteinization residue rate was ≤0.32%, and the deacidification efficiency was ≥97.1%, significantly superior to traditional processes. In Comparative Example 1, the deproteinization residue rate surged to 0.85%, the glucosamine conversion rate dropped to 85.6%, and the purity decreased to 97.2%. This indicates that tea saponin is crucial for disrupting the spatial structure of proteins and assisting papain in deproteinization, reducing the interference of protein residue on subsequent enzymatic hydrolysis. In Comparative Example 2, the glucosamine conversion rate decreased to 86.3%, and the purity decreased to 97.5%, indicating that dipotassium glycyrrhizate significantly inhibited the non-specific degradation of glucosamine and stabilized its structure. Its absence led to excessive enzymatic degradation of glucosamine, resulting in decreased yield and purity. In Comparative Example 3, the conversion rate decreased to 87.1%, and the purity decreased to 97.8%, demonstrating that inositol can maintain the active conformation of chitosanase and prolong the duration of enzyme activity. Its absence resulted in rapid enzyme activity decay and reduced conversion efficiency. Comparative Example 4 had a conversion rate of only 78.5%, a purity of 95.3% (not meeting food-grade standards), impurity ions of 0.32%, and a deacidification efficiency of 85.6%, indicating that traditional acid hydrolysis suffers from significant drawbacks: "violent reaction, numerous impurities, and poor deacidification." In Comparative Example 5, the deacidification efficiency decreased to 88.4%, with impurity ions of 0.09%, indicating that electrodialysis deacidification is significantly superior to resin exchange in terms of organic acid removal efficiency and can reduce the risk of impurities introduced during resin regeneration.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate, characterized in that, Includes the following steps: Step 1: Collect shrimp and crab shells, remove internal organs, meat and other impurities, wash them clean, and then crush them under high pressure and ultrasonically to obtain shell powder raw materials with a particle size of 50-100μm. Under the assistance of 150-200W ultrasound, soak the shell powder in 20-25% citric acid solution for 10-15 minutes to remove inorganic salts such as calcium carbonate. Wash the filter residue until neutral to obtain desalted shell powder. Step 2: Place the desalted chitin powder in a papain system and add tea saponin. Enzymatically hydrolyze the chitin powder at 33-38℃ and 250-300W ultrasonic conditions for 15-25 minutes. After the reaction, wash the filter residue until neutral, add hydrogen peroxide solution for oxidation and decolorization, centrifuge and then vacuum dry at 60-70℃ to obtain chitin powder. Step 3: Dissolve chitin powder in citrate buffer at pH 4.0-5.0 to prepare a suspension with a mass concentration of 5-10%. Add chitosanase and enzyme activator, along with dipotassium glycyrrhizate and inositol. React in a constant temperature shaker at 45-55℃ and 150r / min for 8-12 hours. Raise the temperature to 80℃ and keep it at that temperature for 10 minutes to inactivate the enzyme. Step 4: Remove undegraded solid residue using plate and frame filtration, and collect the filtrate, which is the acidic enzymatic hydrolysate containing glucosamine; Step 5: Deacidify using an electrodialysis device. Pump the acidic enzymatic hydrolysate containing glucosamine into the deacidification chamber, controlling the feed temperature at 25-40℃ and the operating pressure at 0.1-0.3MPa. Connect the DC power supply and adjust the voltage to 15-30V and the current density to 50-100mA / cm². 2 The feed solution is circulated within the membrane stack for treatment. The pH and conductivity of the feed solution in the deacidification chamber are monitored in real time. When the pH rises to 6.0-7.0 and the conductivity drops to less than 10% of the initial value, electrodialysis is stopped and the deacidified glucosamine feed solution is collected. Step 6: Add 0.5-1.0% modified activated carbon to the deacidified glucosamine solution, stir and decolorize at 50-60℃ for 30-60 min, remove activated carbon by plate and frame filtration, and vacuum concentrate to a glucosamine concentration of 150-200 g / L at a temperature of 60-70℃ and a vacuum degree of -0.08~-0.09 MPa; cool the concentrate to 5-10℃ and let it stand for 12-24 h to crystallize, and centrifuge to obtain crude glucosamine crystals; Step 7: Dissolve the crude glucosamine crystals in deionized water to a mass concentration of 40-50%, repeat the concentration and crystallization step once, and vacuum dry at 50-60℃ for 4-6 hours to obtain the food-grade glucosamine product.

2. The method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate according to claim 1, characterized in that, In step 1, the high-pressure pulverization conditions are 1.6-2 MPa for 1-5 min, and the ultrasonic pulverization conditions are 350-400 W for 5-8 min.

3. The method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate according to claim 1, characterized in that, The papain system in step 2 is a papain reaction system with deionized water as solvent, a papain concentration of 0.8-1.2% of the mass of desalted shell powder, and a pH adjusted to 6.0-7.

0.

4. The method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate according to claim 1, characterized in that, In step 2, the amount of tea saponin added is 0.1-0.2% of the desalted shell powder.

5. The method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate according to claim 1, characterized in that, In step 3, the chitosanase activity is >50 units / mg protein; the enzyme activator is calcium chloride.

6. The method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate according to claim 1, characterized in that, In step 3, the amount of chitosanase added is 1-3% of the chitin powder, the amount of enzyme activator added is 0.1-0.3% of the chitin powder, the amount of dipotassium glycyrrhizate added is 0.05-0.1% of the chitin powder, and the amount of inositol added is 0.03-0.05% of the chitin powder.

7. The method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate according to claim 1, characterized in that, In step 5, the electrodialysis device uses a three-compartment electrodialysis membrane stack, which consists of an anode compartment, a deacidification compartment, and an electrode water compartment. Adjacent compartments are separated by ion exchange membranes. An anion exchange membrane is used between the deacidification compartment and the anode compartment, and a cation exchange membrane is used between the deacidification compartment and the electrode water compartment. The electrodes are titanium-coated ruthenium electrodes, and the electrode water is a 0.5 mol / L sodium sulfate solution.

8. The method for preparing food-grade glucosamine by electrodialysis deacidification of chitin enzymatic hydrolysate according to claim 1, characterized in that, The modified activated carbon in step 6 is granular activated carbon that has been activated by hydrochloric acid. The hydrochloric acid activation conditions are as follows: the activated carbon is placed in a hydrochloric acid solution with a mass concentration of 10-15% and soaked at a constant temperature of 80-90℃ for 2-3 hours, during which the stirring rate is 50-80 r / min. After soaking, it is washed with deionized water until neutral and dried at 105-110℃ for 3-4 hours.

9. Food-grade glucosamine prepared by the method according to any one of claims 1-8.

10. The food-grade glucosamine according to claim 9, characterized in that, The food-grade glucosamine has a purity of ≥98% and an impurity ion content of ≤0.1% such as chloride ions and sodium ions.

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