A method for extracting chitin from glutathione fermentation residue

Abstract

This invention relates to the field of chitin extraction technology and discloses a method for extracting chitin from glutathione fermentation residue. The method includes: sequentially pressing, dehydrating, pulverizing, and vacuum drying the glutathione fermentation residue; adding alkali solution and reacting at a constant temperature to remove proteins, followed by washing to obtain deproteinized residue; adding concentrated sodium hydroxide solution and heating to carry out a deacetylation reaction, with concentrated sodium hydroxide solution added periodically during the reaction, followed by washing to obtain the deacetylated product; sequentially adding a decolorizing agent for decolorization and dilute hydrochloric acid solution to remove impurities, followed by washing with water, vacuum drying, and pulverizing to obtain the finished chitin product. By employing gradient alkali treatment and a constant-temperature alkali replenishment process, the degree of deacetylation of chitin is improved and main chain degradation is reduced; the multi-stage purification steps combining physical and chemical methods improve the purity of the finished product; simultaneously, protein byproducts are recovered and waste liquid is recycled, improving resource conversion rate and reducing waste liquid discharge.

Description

Technical Field

[0001] This invention relates to the field of chitin extraction technology, specifically a method for extracting chitin from glutathione fermentation residue. Background Technology

[0002] Glutathione is an important bioactive polypeptide, currently mainly produced industrially through large-scale fermentation using microorganisms such as yeast. During the extraction of glutathione, a large amount of fermentation residue is generated. This residue mainly consists of microbial cell walls and residual intracellular material, rich in organic macromolecules such as proteins, glucans, and chitin. However, most production enterprises currently treat glutathione fermentation residue in a relatively simple way, typically directly landfilling it as solid waste or simply drying it before selling it as inexpensive primary feed. This treatment method not only results in a significant waste of high-value-added components in the residue but also, due to its high water content and rich organic matter, the accumulated residue is prone to spoilage, increasing the environmental burden on enterprises.

[0003] Chitin has broad application prospects in medicine, agriculture, and chemical industries. Traditional industrial production of chitin mainly uses seafood waste such as shrimp and crab shells as raw materials, while the technical route of extracting chitin from waste mycelia of microorganisms such as fungi or yeast still has significant limitations. Chitin inside the cell wall of microorganisms typically forms a dense three-dimensional cross-linked structure with proteins, dextran, and other components through hydrogen bonds and covalent bonds, making it difficult for conventional extraction reagents to effectively penetrate the cell wall. To disrupt this structure and isolate chitin, existing technologies usually rely on a single high-concentration strong acid or strong alkali, followed by a prolonged and vigorous reaction at high temperatures. This treatment method easily causes the glycosidic bonds on the chitin polysaccharide backbone to break and degrade, resulting in a significant decrease in the molecular weight of the extracted chitin, uncontrollable deacetylation, and severely reduced physicochemical properties of the final product.

[0004] Meanwhile, the endogenous macromolecular pigments produced during fermentation and metabolism, as well as the inorganic ash remaining in the culture medium, are difficult to completely remove through simple conventional acid and alkali soaking, resulting in a dark color and excessive ash content in the finished chitin product. Furthermore, existing processes generate large amounts of washing wastewater containing high concentrations of organic impurities and waste acids and alkalis after extraction, leading to high treatment costs and ineffective recycling of byproducts. Therefore, overcoming the physical barrier of the dense cell walls of microorganisms, efficiently extracting high-purity chitin while reducing polysaccharide skeleton degradation, and minimizing process wastewater discharge are current technical problems to be solved in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for extracting chitin from glutathione fermentation residue. This method solves the problem that the existing industrial fermentation production of glutathione generates a large amount of fermentation residue, which, if discarded directly, not only wastes resources but also causes environmental pollution. Furthermore, due to the complex structure of microbial mycelia, it is difficult to efficiently extract chitin from them using conventional processing methods.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting chitin from glutathione fermentation residue, comprising the following operations: The glutathione fermentation residue is pressed and dehydrated, then crushed and sieved to obtain residue powder. The residue powder is then dried under vacuum drying conditions to remove moisture and impurities, and cooled for later use. Alkali solution was added to the dried fungal residue powder, the temperature was adjusted and the mixture was stirred at a constant temperature to carry out the reaction. After the reaction was completed, centrifugation was performed to obtain the supernatant and the first precipitate. The first precipitate was collected and washed with deionized water until neutral to obtain deproteinized fungal residue. Concentrated sodium hydroxide solution was added to the deproteinized bacterial residue, the temperature was raised and the mixture was stirred at a constant temperature. During the reaction, the concentrated sodium hydroxide solution was added periodically to maintain the alkalinity of the reaction system. After the reaction was completed, the mixture was cooled, centrifuged and collected to obtain the second precipitate. The second precipitate was washed with deionized water until neutral to obtain the deacetylated product. A decolorizing agent was added to the deacetylated product, and the mixture was stirred at a constant temperature for decolorization. After decolorization, the product was centrifuged to obtain a decolorized waste liquid and a third precipitate. The third precipitate was washed until neutral. Then, dilute hydrochloric acid solution was added to the washed third precipitate and stirred. The product was centrifuged again to collect a fourth precipitate and washed until neutral to obtain crude chitin. The crude chitin was dried and dehydrated under vacuum drying conditions, then pulverized and sieved to obtain the finished chitin product.

[0007] By adopting the above technical solution, this invention utilizes the compositional characteristics of fermentation residue and combines physical and chemical methods to achieve impurity separation and chitin extraction. The specific extraction mechanism and reaction steps are as follows: Step 1: Raw material pretreatment stage. Vacuum drying causes the free water inside the bacterial residue cells to vaporize and precipitate out upon heating. The water escaping process creates a microporous structure on the surface of the bacterial cell walls, increasing the specific surface area of ​​the bacterial residue particles, thereby improving the permeability of subsequent chemical reagents penetrating into the cell walls.

[0008] Step two, the deproteinization reaction stage. Physical cross-linking exists between proteins and carbohydrates in the fermentation residue. In the alkaline solution system, hydrogen bonds and disulfide bonds within protein molecules break; hydroxide ions catalyze the hydrolysis of peptide bonds on protein polypeptide chains, generating water-soluble short-chain polypeptides and amino acid salts. The hydrolysis products dissolve in the liquid phase and are separated from the supernatant, retaining the solid phase components.

[0009] Step 3: Deacetylation reaction stage. The N-acetyl groups on the chitin molecular backbone in the bacterial residue undergo amide bond hydrolysis under high-temperature, concentrated alkaline conditions. The corresponding reaction equation is: R-NHCOCH3 + NaOH → R-NH2 + CH3COONa. The reaction produces a product with partially deacetylated components and sodium acetate. Regularly adding concentrated sodium hydroxide solution stabilizes the hydroxide ion concentration in the reaction system, shifting the chemical equilibrium towards the product and increasing the degree of deacetylation.

[0010] Step four, decolorization and desalination stage. A decolorizing agent is added to oxidize and degrade or adsorb endogenous macromolecular pigments produced during bacterial metabolism. After washing with water, a dilute hydrochloric acid solution is added, where hydrogen ions react with residual inorganic salt ash impurities in the system through an acid-base neutralization reaction. Taking a typical carbonate as an example, the reaction formula is: CaCO3 + 2HCl → CaCl2 + H2O + CO2↑. The inorganic salts are converted into soluble chlorides and discharged with the water washing process, achieving the purification of the target product.

[0011] Step 5, Vacuum Drying Stage. Dehydration is achieved by lowering the vaporization temperature of moisture under vacuum conditions, reducing contact between the material and oxygen, preventing thermal oxidative browning of the decolorized product, and ensuring the stability of the extracted chitin's physicochemical properties.

[0012] Preferably, when pressing and dehydrating the glutathione fermentation residue, the moisture content of the material is controlled to be 40% to 50%; the sieving standard for crushing and sieving is 60 mesh to 100 mesh; the residue powder is dried under vacuum drying conditions at a temperature of 55°C to 65°C, a vacuum degree of 0.07 MPa to 0.09 MPa, and a drying time of 3 hours to 4 hours.

[0013] By employing the above technical solutions and limiting specific moisture content and particle size, particle agglomeration during the dehydration process can be prevented. Setting specific vacuum levels and temperature ranges ensures that the rate of water removal matches the rate of cell wall pore expansion, avoiding irreversible denaturation and solidification of residual proteins due to high temperatures, which is beneficial for improving the reaction conversion rate in subsequent protein removal steps.

[0014] Preferably, the alkaline solution added to the dried fungal residue powder is a sodium hydroxide solution or a potassium hydroxide solution, and the mass-volume concentration of the alkaline solution is 1.5% to 2.5%; the material-liquid ratio of the alkaline solution to the fungal residue powder is 1g:10mL to 1g:15mL; the temperature for constant temperature stirring is 50℃ to 60℃, the stirring speed is 180r / min to 220r / min, and the reaction time is 2.5h to 3h.

[0015] By employing the above technical solution and utilizing an alkaline reaction environment with specific concentrations and temperatures, the dissolution of structural proteins is promoted while inhibiting excessive degradation of polysaccharide backbone macromolecules. This feed-to-liquid ratio and stirring parameters ensure uniform mixing of the solid and liquid phases, reducing mass transfer resistance and improving protein hydrolysis efficiency.

[0016] Preferably, the mass-volume concentration of the concentrated sodium hydroxide solution added to the deproteinized bacterial residue is 10% to 15%; the material-to-liquid ratio of the concentrated sodium hydroxide solution to the deproteinized bacterial residue is 1g:8mL to 1g:12mL; the temperature for heating and constant-temperature stirring is 80°C to 90°C, the stirring speed is 150r / min to 180r / min, and the total reaction time is 5h to 6h.

[0017] By adopting the above technical solution, the combination of alkali concentration and reaction temperature provides activation energy to overcome the steric hindrance effect generated by hydrogen bonds within the chitin molecule, promotes the continuous hydrolysis and removal of acetylamino groups, and breaks the covalent connection between chitin and dextran macromolecules in the cell wall structure.

[0018] Preferably, the concentrated sodium hydroxide solution is replenished at a frequency of once every 1 hour. Before replenishment, the concentrated sodium hydroxide solution is preheated to the same temperature as the current reaction temperature. The amount of each replenishment is 5% to 10% of the initial total volume of the concentrated sodium hydroxide solution.

[0019] By employing the above technical solution and adding alkali solution at the same temperature, fluctuations in local alkali concentration and temperature within the system caused by a single addition of room-temperature reagent are prevented. This operation maintains the thermodynamic stability and concentration uniformity of the reaction system, avoids side reactions, and ensures the reaction rate.

[0020] Preferably, the added decolorizing agent is an activated carbon solution with a mass-volume concentration of 1% to 2%, or a hydrogen peroxide solution with a volume concentration of 3% to 5%.

[0021] By employing the above technical solutions, hydrogen peroxide destroys the chromophores of pigment molecules through redox reactions, while activated carbon relies on its surface porous structure for physical adsorption. Both reagents, at their respective concentrations, can reduce the color of the extract without disrupting the glycosidic backbone.

[0022] Preferably, when the decolorizing agent is an activated carbon solution with a mass-volume concentration of 1% to 2%, the activated carbon solution is prepared by the following method: food-grade powdered activated carbon is passed through a 200-mesh sieve, the sieved food-grade powdered activated carbon is added to deionized water to prepare a solution of the required concentration, stirred and activated for 30 minutes, and then filtered to remove large particulate impurities before use.

[0023] By adopting the above technical solution, large ash particles that may be introduced into the final product are removed through screening and liquid phase activation treatment. During the hydration process, the internal pores of the activated carbon particles are expanded, increasing the surface active adsorption sites and improving the adsorption capacity for macromolecular organic pigments in the system.

[0024] Preferably, the volume concentration of the dilute hydrochloric acid solution added to the washed third precipitate is 0.5% to 1.0%, the ratio of the dilute hydrochloric acid solution to the washed third precipitate is 1g:10mL to 1g:15mL, and the stirring time at room temperature is 15min to 20min.

[0025] By employing the above technical solution, low-concentration acid solution neutralizes inorganic salt ash in a short time. These process parameters ensure the dissolution of inorganic salt impurities while avoiding acid-catalyzed degradation and chain breakage of chitin polymers caused by excessively high acid concentrations and prolonged processing times.

[0026] Preferably, the supernatant is concentrated and spray-dried to prepare a feed-grade protein additive; the decolorizing waste liquid is neutralized by acid and alkali and then returned to be used to prepare the alkaline solution and recycled.

[0027] By adopting the above technical solution, free peptides and amino acid synthesis derivatives in liquid phase byproducts can be recovered. The recovered and neutralized waste liquid is directly used to prepare reagents for preceding processes, reducing process water consumption and waste liquid discharge, and improving the overall resource conversion and utilization rate.

[0028] Preferably, when the crude chitin is dried and dehydrated under vacuum drying conditions, the temperature is 50°C to 60°C, the vacuum degree is 0.06MPa to 0.08MPa, and the drying time is 2h to 3h; the screening standard for pulverizing and sieving is 100 mesh to 120 mesh.

[0029] By employing the above technical solution, the critical vaporization temperature of water is lowered using vacuum negative pressure, enabling dehydration to be completed at a lower temperature while maintaining the polysaccharide polymer conformation. The particle size separation process ensures that the finished powder achieves a uniform particle size distribution.

[0030] This invention provides a method for extracting chitin from glutathione fermentation residue. It has the following beneficial effects: 1. This invention employs a gradient alkali treatment and isothermal alkali replenishment process, which improves the extraction quality and degree of deacetylation of chitin. The process first uses a low-concentration alkali solution to remove most of the structural proteins, followed by a concentrated sodium hydroxide solution for deacetylation. This avoids the degradation of the chitin polysaccharide backbone caused by directly using a high-concentration strong alkali. Simultaneously, during the deacetylation reaction, a preheated concentrated alkali solution is added periodically and quantitatively to maintain the stability of hydroxide ion concentration and temperature within the reaction system, promoting the continuous hydrolysis of amide bonds and thus improving the physicochemical properties of the target product.

[0031] 2. This invention improves the purity of the chitin product by combining multi-stage purification steps. In the raw material pretreatment stage, vacuum drying dehydration is used, causing moisture to precipitate from the surface of the bacterial residue and forming a microporous structure, thus improving the permeability of subsequent chemical reagents. In the decolorization and desalination stage, hydrogen peroxide or activated carbon is added to remove pigments produced by fermentation metabolism, and low-concentration dilute hydrochloric acid is used at room temperature to neutralize inorganic salt ash. This combination of physical and chemical treatment removes impurities while preventing excessive thermal browning or acid-catalyzed degradation of the product during purification.

[0032] 3. This invention realizes the comprehensive utilization of fermentation by-products, reduces the amount of waste liquid discharged from the process system, concentrates and spray-dries the supernatant obtained by centrifugation after deproteinization reaction, and recovers the hydrolyzed short-chain peptides and amino acid components to prepare feed-grade protein additives, thereby improving the overall economic added value of glutathione fermentation residue; at the same time, the decolorization waste liquid is directly reused for the preparation of the alkaline solution required for the previous extraction process after acid-base neutralization treatment, which reduces the consumption of fresh water in the process and alleviates the problem of difficult waste liquid treatment in the traditional chitin extraction process. Attached Figure Description

[0033] Figure 1 This is a graph showing the evolution trajectory of chitin extraction rate at each process node in Test Example 1 of the present invention. Figure 2 This is a graph showing the evolution trajectory of chitin purity at each process node in Test Example 1 of the present invention. Figure 3 This is a comparison of the Fourier transform infrared spectra of the chitin (chitosan) products prepared in Example 1 and Comparative Example 1 of the present invention. Detailed Implementation

[0034] The technical solutions in 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 scope of protection of the present invention.

[0035] Preparation Examples 1-3: Preparation Example 1: This preparation example provides a method for preparing an activated carbon solution, including the following steps: Food-grade powdered activated carbon is used and passed through a 200-mesh sieve. The sieved activated carbon is added to deionized water to prepare an activated carbon solution with a concentration of 1% (w / v). The solution is activated by stirring for 30 minutes and then used. Large particulate impurities are removed by filtration before use.

[0036] Preparation Example 2: This preparation example provides a method for preparing an activated carbon solution, including the following steps: Food-grade powdered activated carbon is used and passed through a 200-mesh sieve. The sieved activated carbon is added to deionized water to prepare an activated carbon solution with a concentration of 1.5% (w / v). The solution is activated by stirring for 30 minutes and then used. Large particulate impurities are removed by filtration before use.

[0037] Preparation Example 3: This preparation example provides a method for preparing an activated carbon solution, including the following steps: Food-grade powdered activated carbon is used and passed through a 200-mesh sieve. The sieved activated carbon is added to deionized water to prepare an activated carbon solution with a concentration of 2% (w / v). The solution is activated by stirring for 30 minutes and then used. Large particulate impurities are removed by filtration before use.

[0038] Examples 1-6: Example 1: This embodiment provides a method for extracting chitin from glutathione fermentation residue. The raw material is the fermentation residue obtained from the deep fermentation of brewing yeast in the industrial fermentation production of glutathione, followed by centrifugation to extract the glutathione product. This residue is stored at 4°C for 20 hours before use. The residue (before dehydration) has a moisture content of 75%, a chitin content of 22%, and a protein content of 35%. The method includes the following steps: 1. Raw material pretreatment: Collect the glutathione fermentation residue, press and dehydrate it, and control the moisture content of the residue to 45%; then crush the dehydrated residue and pass it through an 80-mesh sieve to obtain residue powder. The mushroom residue powder was placed in a vacuum drying oven and dried at 60℃ and 0.08MPa for 3.5h to remove free moisture and volatile impurities. After cooling to room temperature, it was ready for use. The moisture content of the dried mushroom residue powder was 6.2%.

[0039] 2. Impurity removal and protein removal: Add the pretreated fungal residue powder from step 1 to a 2.0% (w / v) sodium hydroxide solution. The ratio of sodium hydroxide solution to fungal residue powder is 1:12 (g / mL). Adjust the system temperature to 55℃, stir at 200 r / min, and stir at a constant temperature for 2.5 h. After the reaction was completed, centrifugation was performed at a speed of 4500 r / min for 18 min. After centrifugation, the supernatant was clear and free of suspended solids, and the water content of the precipitate was 65%. The precipitate was collected and washed four times with deionized water. After the washing liquid was stirred evenly, the supernatant was tested with precision pH paper (accuracy 0.1). The pH value was 7.2 for two consecutive tests, and the deproteinized bacterial residue was obtained. The supernatant obtained by centrifugation is concentrated and spray-dried to prepare feed-grade protein additives.

[0040] 3. Deacetylation treatment: Add the deproteinized bacterial residue obtained in step 2 to a 12% (w / v) sodium hydroxide solution. The ratio of concentrated sodium hydroxide solution to deproteinized bacterial residue is 1:10 (g / mL). Adjust the system temperature to 85℃, stir at 160 r / min, and stir at a constant temperature for 5 hours. During the reaction, add 12% (w / v) concentrated sodium hydroxide solution every 1 hour. Before adding, preheat the alkali solution to 85℃. After adding, stir until the system temperature returns to 85℃. The amount added is 8% of the initial volume of concentrated alkali solution. After the reaction was completed, the mixture was cooled to room temperature and centrifuged (4000 r / min, 12 min). The supernatant was clear and free of suspended solids after centrifugation. The water content of the precipitate was 63%. The precipitate was collected and washed with deionized water until the washing liquid was stirred evenly. The supernatant was then taken and the pH value was measured to be 7.3 using precision pH paper (accuracy 0.1), thus obtaining the deacetylated product.

[0041] 4. Decolorization and purification: Add the deacetylated product obtained in step 3 to 4% (v / v) hydrogen peroxide solution. The ratio of hydrogen peroxide solution to deacetylated product is 1:15 (g / mL). Adjust the system temperature to 35℃, stir at 130 r / min, and decolorize for 40 min. After decolorization, centrifugation was performed (3500 r / min, 12 min). The supernatant was clear and free of suspended solids after centrifugation. The water content of the precipitate was 62%. The precipitate was collected and washed three times with deionized water until the pH was neutral. The precipitate was then added to a 0.8% (v / v) dilute hydrochloric acid solution at a material-to-liquid ratio of 1:12 (g / mL). The mixture was stirred at room temperature for 18 min and then centrifuged again (3500 r / min for 12 min). After centrifugation, the supernatant was clear and free of suspended solids, and the precipitate had a water content of 60%. The precipitate was collected and washed with deionized water until the washing liquid was thoroughly stirred. The supernatant was then tested with precision pH paper (accuracy 0.1) and the pH value was found to be 7.0, yielding crude chitin (chitosan). After neutralization, the decolorized waste liquid is reserved for use in the preparation of sodium hydroxide solution in step 2.

[0042] 5. Drying and shaping: The crude chitin (chitosan) obtained in step 4 is placed in a vacuum drying oven and dried at 55°C and 0.07MPa for 2.5 hours to remove moisture; After drying, it is crushed and passed through a 110-mesh sieve to remove coarse particles, thus obtaining a high-purity chitin (chitosan) product.

[0043] Example 2: This embodiment provides a method for extracting chitin from glutathione fermentation residue. The raw material is fermentation residue obtained from deep fermentation of *E. coli* in the industrial fermentation production of glutathione. This residue is frozen at -20°C for 7 days and then thawed naturally before use. The residue (before dehydration) has a moisture content of 70%, a chitin content of 15%, and a protein content of 30%. The method includes the following steps: 1. Raw material pretreatment: The glutathione fermentation residue is pressed and dehydrated to a moisture content of 40%, then pulverized through a 60-mesh sieve to obtain residue powder; The fungal residue powder was placed in a vacuum drying oven and dried at 55℃ and 0.07MPa for 4 hours. After cooling to room temperature, the moisture content of the fungal residue powder after drying was 6.8%.

[0044] 2. Removal of impurities and protein: Add 1.5% (w / v) potassium hydroxide solution, with a material-to-liquid ratio of 1:10 (g / mL), adjust the temperature to 50℃, stir at 180 r / min, and stir at a constant temperature for 3 hours; Centrifuge (4000 r / min, 20 min). After centrifugation, the supernatant is clear and free of suspended solids. The water content of the precipitate is 68%. Collect the precipitate and wash it three times with deionized water. After stirring the washing liquid evenly, take the supernatant and test it twice with precision pH test paper (accuracy 0.1). The pH value is 7.0, and the deproteinized bacterial residue is obtained. The supernatant was concentrated and dried to prepare feed-grade protein.

[0045] 3. Deacetylation treatment: Add 10% (w / v) sodium hydroxide solution at a material-to-liquid ratio of 1:8 (g / mL), adjust the temperature to 80℃, stir at 150 r / min, and stir at a constant temperature for 6 hours. Add 10% (w / v) concentrated sodium hydroxide solution every 1 hour, preheating to 80℃ before each addition. The amount added is 5% of the initial concentrated alkali solution volume. After cooling, centrifuge (3500 r / min, 15 min). The supernatant after centrifugation is clear and free of suspended solids. The water content of the precipitate is 67%. After washing the precipitate until the washing liquid is evenly stirred, take the supernatant and test the pH with precision pH paper (accuracy 0.1) to obtain the deacetylated product.

[0046] 4. Decolorization and purification: Add 3% (v / v) hydrogen peroxide solution, with a material-to-liquid ratio of 1:12 (g / mL), at 30℃ and a stirring speed of 120 r / min, for 45 min of decolorization; Centrifuge (3000 r / min, 15 min). After centrifugation, the supernatant is clear and free of suspended solids. The water content of the precipitate is 66%. The precipitate is washed 3 times until the pH is neutral. Add 0.5% (v / v) dilute hydrochloric acid solution at a material-to-liquid ratio of 1:10 (g / mL). Stir at room temperature for 20 min. Centrifuge and wash until the washing liquid is homogeneous. Take the supernatant and test the pH with precision pH paper (accuracy 0.1) to obtain crude chitin (chitosan). The decolorization waste liquid is neutralized and then recycled for the protein removal process.

[0047] 5. Drying and shaping: The crude chitin (chitosan) product is dried at 50℃ and vacuum degree of 0.06MPa for 3 hours, then pulverized and passed through a 100-mesh sieve to obtain the finished product.

[0048] Example 3: This embodiment provides a method for extracting chitin from glutathione fermentation residue. The raw material is fermentation residue obtained from deep fermentation of brewing yeast in the industrial fermentation production of glutathione. This residue is stored at 4°C for 24 hours before use. The residue (before dehydration) has a moisture content of 80%, a chitin content of 25%, and a protein content of 40%. The method includes the following steps: 1. Raw material pretreatment: The glutathione fermentation residue is pressed and dehydrated to a moisture content of 50%, then pulverized through a 100-mesh sieve to obtain residue powder; The fungal residue powder was placed in a vacuum drying oven and dried at 65℃ and 0.09MPa for 3 hours. After cooling to room temperature, the moisture content of the fungal residue powder after drying was 6.0%.

[0049] 2. Removal of impurities and protein: Add 2.5% (w / v) sodium hydroxide solution, with a material-to-liquid ratio of 1:15 (g / mL), adjust the temperature to 60℃, stir at 220 r / min, and stir at a constant temperature for 3 hours; Centrifuge (5000 r / min, 15 min). After centrifugation, the supernatant is clear and free of suspended solids. The water content of the precipitate is 62%. Collect the precipitate and wash it 4 times with deionized water. After stirring the washing liquid evenly, take the supernatant and test it with precision pH paper (accuracy 0.1) twice. The pH value is 7.5, and the deproteinized bacterial residue is obtained. The supernatant was concentrated and dried to prepare feed-grade protein.

[0050] 3. Deacetylation treatment: Add 15% (w / v) sodium hydroxide solution at a material-to-liquid ratio of 1:12 (g / mL), adjust the temperature to 90℃, stir at 180 r / min, and stir at a constant temperature for 6 hours. Add 15% (w / v) concentrated sodium hydroxide solution every 1 hour, preheating to 90℃ before each addition. The amount added is 10% of the initial concentrated alkali solution volume. After cooling, centrifuge (4500 r / min, 10 min). The supernatant after centrifugation is clear and free of suspended solids. The water content of the precipitate is 60%. After washing the precipitate until the washing liquid is evenly stirred, take the supernatant and test the pH with precision pH paper (accuracy 0.1) to obtain the deacetylated product.

[0051] 4. Decolorization and purification: Add 5% (v / v) hydrogen peroxide solution, with a material-to-liquid ratio of 1:18 (g / mL), at 40℃ and stirring speed of 150 r / min, for 30 min of decolorization; Centrifuge (4000 r / min, 10 min). After centrifugation, the supernatant is clear and free of suspended solids. The water content of the precipitate is 61%. The precipitate is washed twice until the pH is neutral. Add 1.0% (v / v) dilute hydrochloric acid solution, with a material-to-liquid ratio of 1:15 (g / mL), stir at room temperature for 15 min, centrifuge and wash until the washing liquid is uniform, take the supernatant and test the pH with precision pH test paper (accuracy 0.1) to obtain crude chitin (chitosan); the decolorization waste liquid is neutralized and recycled for the deproteinization process.

[0052] 5. Drying and shaping: The crude chitin (chitosan) product is dried at 60℃ and vacuum degree of 0.08MPa for 2 hours, then pulverized and passed through a 120-mesh sieve to obtain the finished product.

[0053] Example 4: This example provides a method for extracting chitin from glutathione fermentation residue.

[0054] The only difference between this embodiment and Embodiment 1 is the type of decolorizing agent in step 4. The composition of the other raw materials and the operation process and parameters of steps 1, 2, 3 and 5 are exactly the same as those in Embodiment 1.

[0055] The specific step 4 in this embodiment is as follows: 4. Decolorization and purification: The deacetylated product obtained in step 3 was added to the 1% (w / v) activated carbon solution prepared in Preparation Example 1. The ratio of activated carbon solution to deacetylated product was 1:15 (g / mL). The system temperature was adjusted to 35°C, the stirring speed was 130 r / min, and the mixture was stirred at a constant temperature for 40 min for decolorization. After decolorization, centrifugation was performed (3500 r / min, 12 min). The supernatant was clear and free of suspended solids after centrifugation. The water content of the precipitate was 62%. The precipitate was collected and washed three times with deionized water until the pH was neutral. The precipitate was then added to a 0.8% (v / v) dilute hydrochloric acid solution at a material-to-liquid ratio of 1:12 (g / mL). The mixture was stirred at room temperature for 18 min and then centrifuged again (3500 r / min for 12 min). After centrifugation, the supernatant was clear and free of suspended solids, and the precipitate had a water content of 60%. The precipitate was collected and washed with deionized water until the washing liquid was thoroughly stirred. The supernatant was then tested with precision pH paper (accuracy 0.1) and the pH value was found to be 7.0, yielding crude chitin (chitosan). After neutralization, the decolorized waste liquid is reserved for use in the preparation of sodium hydroxide solution in step 2.

[0056] Example 5: This example provides a method for extracting chitin from glutathione fermentation residue.

[0057] The only difference between this embodiment and Embodiment 1 is the type of decolorizing agent in step 4. The composition of the other raw materials and the operation process and parameters of steps 1, 2, 3 and 5 are exactly the same as those in Embodiment 1.

[0058] The specific step 4 in this embodiment is as follows: 4. Decolorization and purification: The deacetylated product obtained in step 3 was added to the activated carbon solution with a concentration of 1.5% (w / v) prepared in Preparation Example 2. The ratio of activated carbon solution to deacetylated product was 1:15 (g / mL). The system temperature was adjusted to 35°C, the stirring speed was 130 r / min, and the mixture was stirred at a constant temperature for 40 min for decolorization. After decolorization, centrifugation was performed (3500 r / min, 12 min). The supernatant was clear and free of suspended solids after centrifugation. The water content of the precipitate was 62%. The precipitate was collected and washed three times with deionized water until the pH was neutral. The precipitate was then added to a 0.8% (v / v) dilute hydrochloric acid solution at a material-to-liquid ratio of 1:12 (g / mL). The mixture was stirred at room temperature for 18 min and then centrifuged again (3500 r / min for 12 min). After centrifugation, the supernatant was clear and free of suspended solids, and the precipitate had a water content of 60%. The precipitate was collected and washed with deionized water until the washing liquid was thoroughly stirred. The supernatant was then tested with precision pH paper (accuracy 0.1) and the pH value was found to be 7.0, yielding crude chitin (chitosan). After neutralization, the decolorized waste liquid is reserved for use in the preparation of sodium hydroxide solution in step 2.

[0059] Example 6: This example provides a method for extracting chitin from glutathione fermentation residue.

[0060] The only difference between this embodiment and Embodiment 1 is the type of decolorizing agent in step 4. The composition of the other raw materials and the operation process and parameters of steps 1, 2, 3 and 5 are exactly the same as those in Embodiment 1.

[0061] The specific step 4 in this embodiment is as follows: 4. Decolorization and purification: The deacetylated product obtained in step 3 was added to the activated carbon solution with a concentration of 2.0% (w / v) prepared in Preparation Example 3. The ratio of activated carbon solution to deacetylated product was 1:15 (g / mL). The system temperature was adjusted to 35°C, the stirring speed was 130 r / min, and the mixture was stirred at a constant temperature for 40 min for decolorization. After decolorization, centrifugation was performed (3500 r / min, 12 min). The supernatant was clear and free of suspended solids after centrifugation. The water content of the precipitate was 62%. The precipitate was collected and washed three times with deionized water until the pH was neutral. The precipitate was then added to a 0.8% (v / v) dilute hydrochloric acid solution at a material-to-liquid ratio of 1:12 (g / mL). The mixture was stirred at room temperature for 18 min and then centrifuged again (3500 r / min for 12 min). After centrifugation, the supernatant was clear and free of suspended solids, and the precipitate had a water content of 60%. The precipitate was collected and washed with deionized water until the washing liquid was thoroughly stirred. The supernatant was then tested with precision pH paper (accuracy 0.1) and the pH value was found to be 7.0, yielding crude chitin (chitosan). After neutralization, the decolorized waste liquid is reserved for use in the preparation of sodium hydroxide solution in step 2.

[0062] Comparative example: The difference compared to Example 1 is as follows: This comparative example uses a traditional extraction process, replacing the raw material with fresh shrimp and crab shells (after cleaning, removing impurities, and drying), and adding a step of "soaking in 5% (w / v) hydrochloric acid solution at room temperature for 2 hours to remove calcium carbonate" before deproteinization; Meanwhile, the removal of impurities and proteins uses a 3% (w / v) sodium hydroxide solution. No stepwise addition of alkali solution is performed during the deacetylation process. The decolorization and purification uses conventional activated carbon decolorization, and there is no waste liquid recovery and recycling step.

[0063] The other routine operations, such as centrifugation, washing to neutral, and vacuum drying and shaping, are the same.

[0064] Test Example 1-3: Test Example 1: Extraction Rate and Purity Tracking Test of Each Processing Unit This test case mainly focuses on the pretreatment, deproteinization, deacetylation, decolorization and purification and drying processes in Example 1, and tracks and monitors the evolution of the extraction rate and purity of chitin (chitosan) in the target material.

[0065] Sampling points were set at the end of each processing step in Example 1 to collect solid materials from six nodes: the original fermentation residue, the pretreated powder, the deproteinized residue, the deacetylated product, the crude chitin, and the final dried product.

[0066] The obtained wet sample was placed in a vacuum freeze dryer to achieve constant weight, in order to eliminate systematic errors caused by moisture fluctuations in the calculation of mass percentage, and to accurately record the total dry weight at each stage.

[0067] A fixed amount of dried powder from each stage was taken, and the absolute mass of chitin / chitosan in the sample was determined by elemental analysis (by measuring the total nitrogen content and deducting the amount of free protein residue).

[0068] The target extraction rate is calculated based on the ratio of the absolute mass measured at each node to the initial theoretical chitin content in the bacterial residue. At the same time, the stage purity is determined by the ratio of this absolute mass to the total dry matter weight at the current sampling point.

[0069] Table 1. Chitin component purification tracking data at each process node

[0070] Summarize: Based on the data in Table 1 and the changes in material balance during the processing, the extraction pattern of chitin components in glutathione fermentation residue was derived. The original glutathione fermentation residue contained culture medium residue and unused microbial cells, with an initial purity of 18.6% for the target product. After pressing, dehydration, and vacuum drying, free water was removed, cell wall structures shrank and broke down, and the components in the dry material were initially concentrated, increasing the chitin purity to 20.3%.

[0071] After alkaline deproteinization treatment, as shown at each process node in Test Example 1 of this invention... Figure 2 As shown, the purity increased from 20.3% to 68.5%. Using an alkaline solution with a concentration of 1.5%-2.5% can cleave protein peptide bonds, generating soluble amino acids which are discharged with the supernatant. Furthermore, the low concentration condition avoids the damage of strong alkali to the polysaccharide backbone. This is further demonstrated by the process steps corresponding to each step in Test Example 1 of this invention. Figure 1 It can be seen that the deproteinization process increased the material enrichment and extraction rate to 65.3%.

[0072] The deacetylation process, using high-temperature, concentrated alkaline conditions, induces the amide bond cleavage of the groups, leading to depolymerization within the polymer. This process removes residual organic impurities, increasing the product purity to 85.7%. Subsequently, hydrogen peroxide and dilute hydrochloric acid are added for decolorization and purification, removing pigment groups and calcium and magnesium inorganic salt impurities. The final product purity reaches 92.3%, with a total chitin extraction rate of 80.2%. Test results demonstrate that the stepwise extraction process can progressively remove impurities from the fermentation residue, achieving chitin extraction and purification.

[0073] Test Example 2: Comparative Test of Physicochemical Properties of Finished Products Weigh 5.00g of each of the dried chitin (chitosan) products prepared in Examples 1 to 6 and Comparative Example 1, and place them in a silica gel desiccator to equilibrate to room temperature for later use.

[0074] The total nitrogen content of each sample was determined using a Kjeldahl nitrogen analyzer. The chitin purity of each sample was calculated by subtracting the nitrogen content from the total nitrogen content and the nitrogen content converted from residual protein.

[0075] Sample test slices were prepared using the KBr pellet method and placed in the optical path of a Fourier transform infrared spectrometer at 4000-400 cm⁻¹. -1 Infrared spectra were obtained by scanning within the wavenumber range, and the amide I band (1655 cm⁻¹) was recorded. -1 ) and hydroxyl / amino stretching vibration band (3450cm) -1 The absorbance of the product was measured, and the degree of deacetylation of the product was calculated using the peak absorption ratio.

[0076] Take 2.00g of the finished product sample and place it in a weighing bottle with a known mass. Place it in a drying oven at 105℃ and heat it to constant weight. Record the mass difference before and after heating and calculate the final moisture content of the sample.

[0077] After determining the moisture content, the dried sample was transferred into a porcelain crucible, placed in a muffle furnace, and calcined at 550℃ for 6 hours until constant weight was achieved. The ash content was calculated based on the mass of the remaining inorganic residue.

[0078] Prepare a 1% (v / v) aqueous solution of acetic acid, dissolve each sample in the solution to make a 1% (w / v) test solution, and measure the dynamic viscosity of the solution using a rotational viscometer under constant temperature water bath conditions of 20℃.

[0079] Table 2. Test data of the physical and chemical properties of the finished product

[0080] Summarize: According to the data in Table 2, there are differences in the physicochemical properties of the products between the Example Group and Comparative Example 1. The traditional process uses shrimp and crab shells as the extraction raw material. The shells contain embedded calcium carbonate structures. The process involves soaking the chitin in hydrochloric acid solution to remove inorganic calcium carbonate. This acidic environment causes the breakage of β-1,4-glycosidic bonds on the main chain of the chitin molecule.

[0081] In Comparative Example 1, the product viscosity decreased to 215 mPa·s, and the ash content reached 1.24%. Acid treatment caused a decrease in polymer molecular weight, and calcium salt residue in the system was not completely removed during the washing step. This method uses glutathione fermentation residue, and the biological matrix lacks calcium carbonate, eliminating the need for a strong acid decalcification process. The product viscosities of Examples 1 to 6 ranged from 386 mPa·s to 425 mPa·s, the macromolecular chain structure remained intact, and the ash content was in the range of 0.32% to 0.45%.

[0082] The degree of deacetylation reflects the degree of amide bond breakage in the molecular structure. This is relevant to the chitin (chitosan) products prepared in Example 1 and Comparative Example 1 of this invention. Figure 3 As can be seen in the figure, the solid black line represents the spectral curve of the product of Example 1, and the dark gray dashed line represents the spectral curve of the product of Comparative Example 1. The 1655 cm⁻¹ in the spectrum... -1 (Amide I band) and 1590cm -1 The difference in transmittance peaks at the (amino band) directly reflects the difference in the degree of deacetylation between the two process products.

[0083] The difference lies in the dense keratinized tissue of the shrimp and crab shell raw materials, which causes the hydroxide ion concentration in the system to decrease as the reaction proceeds after a single addition of alkali solution. The fermentation residue exhibits powdery physical properties. In the example, the alkali solution was added at regular intervals during the deacetylation process to maintain the reactant concentration and ensure the alkaline environment required for deacetylation.

[0084] The degree of deacetylation of the products in the examples ranged from 85.1% to 88.6%, while that in Comparative Example 1 was 76.5%. Regardless of whether hydrogen peroxide or activated carbon was used as the decolorizing agent, the purity in all examples remained above 89.7%. Protein and pigment impurities during the extraction process were effectively removed and discharged into the waste liquid during the stepwise treatment. Experimental data demonstrate that the chitosan extraction process from fermentation waste possesses operational feasibility and parameter stability in terms of performance index control.

[0085] Test Example 3: Comprehensive Production Efficiency and Environmental Impact Assessment Test A unified single-batch feeding standard was set, and the actual dry weight of fermentation residue and shrimp and crab shell raw materials in Examples 1 to 6 and Comparative Example 1 was recorded respectively. At the end of the process, the absolute mass of the dried chitin product was weighed, and the total chitin extraction rate of each batch was calculated to evaluate the material conversion efficiency.

[0086] Collect and statistically analyze the electricity consumption, process water consumption, and actual dosage of chemical reagents such as sodium hydroxide, hydrogen peroxide, activated carbon, and hydrochloric acid during the operation of each process system. Combine this with the market procurement cost of the raw materials or the discounted price of waste disposal to calculate the raw material cost and operating cost per kilogram of chitin produced.

[0087] The protein-rich supernatant separated by centrifugation in the impurity removal and deproteinization process of Examples 1 to 6 was collected. The pH value of the waste liquid system was adjusted to the isoelectric point by acid precipitation to cause protein flocculation and precipitation. After centrifugation and drying, feed-grade protein additive was obtained. The protein purity of the by-product was determined by Kjeldahl nitrogen determination method.

[0088] Monitor the volume of waste liquid generated in each decolorization and deacetylation washing step, determine the usable volume of the waste liquid after acid-base neutralization treatment and re-adjustment to the specified concentration and reintegration into the original process flow, calculate the waste liquid recycling rate, and record the waste liquid recovery data according to the conventional direct discharge process in Comparative Example 1.

[0089] Table 3. Comprehensive Production Efficiency and Environmental Evaluation Test Data

[0090] Summarize: Based on the data in Table 3, the performance of fermentation residue as a substrate in terms of economic accounting and environmental protection was evaluated. Traditional extraction processes use shrimp and crab shells as raw materials, which are subject to procurement and transportation costs due to seasonal and regional variations; the raw material cost for Comparative Example 1 was 18.5 yuan / kg. Glutathione fermentation residue is derived from industrial solid waste; the raw material cost for Examples 1 to 6 ranged from 0.42 to 0.48 yuan / kg, with the main expenditures being material transportation and storage costs.

[0091] Since shrimp and crab shells contain calcium carbonate, traditional processes require the use of hydrochloric acid for decalcification and subsequent water washing and neutralization, increasing reagent and water consumption. Fermentation residue does not contain calcium carbonate, eliminating the need for acid washing and decalcification in this process. Alkali solution is added periodically during deacetylation, reducing the operating cost of this example to 12.3 to 14.1 yuan / kg.

[0092] Regarding the treatment of waste liquid and by-products, the acidic and alkaline waste liquid generated in Comparative Example 1 contains a mixture of calcium salts and free amino acids and is directly discharged as wastewater; the deproteinized supernatant generated in this scheme recovers protein components through isoelectric point precipitation technology to obtain feed-grade protein additives with a purity of 65.1% to 68.4%.

[0093] The waste liquid generated during the decolorization and washing processes is adjusted in concentration before being recycled into the system, with a recycling rate consistently between 71.9% and 75.2%. Test results show that, regardless of whether a hydrogen peroxide system or an activated carbon system is used, extracting chitin from fermentation residue can reduce material consumption and achieve protein component recovery and green recycling of waste liquid.

Claims

1. A method for extracting chitin from glutathione fermentation residue, characterized in that, Includes the following operations: The glutathione fermentation residue is pressed and dehydrated, then crushed and sieved to obtain residue powder. The residue powder is then dried under vacuum drying conditions to remove moisture and impurities, and cooled for later use. Alkali solution was added to the dried fungal residue powder, the temperature was adjusted and the mixture was stirred at a constant temperature to carry out the reaction. After the reaction was completed, centrifugation was performed to obtain the supernatant and the first precipitate. The first precipitate was collected and washed with deionized water until neutral to obtain deproteinized fungal residue. Concentrated sodium hydroxide solution was added to the deproteinized bacterial residue, the temperature was raised and the mixture was stirred at a constant temperature. During the reaction, the concentrated sodium hydroxide solution was added periodically to maintain the alkalinity of the reaction system. After the reaction was completed, the mixture was cooled, centrifuged and collected to obtain the second precipitate. The second precipitate was washed with deionized water until neutral to obtain the deacetylated product. A decolorizing agent was added to the deacetylated product, and the mixture was stirred at a constant temperature for decolorization. After decolorization, the product was centrifuged to obtain a decolorized waste liquid and a third precipitate. The third precipitate was washed until neutral. Then, dilute hydrochloric acid solution was added to the washed third precipitate and stirred. The product was centrifuged again to collect a fourth precipitate and washed until neutral to obtain crude chitin. The crude chitin was dried and dehydrated under vacuum drying conditions, then pulverized and sieved to obtain the finished chitin product.

2. The method for extracting chitin from glutathione fermentation residue according to claim 1, characterized in that, When pressing and dehydrating the glutathione fermentation residue, the moisture content of the material should be controlled to be 40% to 50%. The sieving standard for crushing and sieving is 60 mesh to 100 mesh; The bacterial residue powder is dried under vacuum conditions at a temperature of 55°C to 65°C, a vacuum degree of 0.07 MPa to 0.09 MPa, and a drying time of 3 to 4 hours.

3. The method for extracting chitin from glutathione fermentation residue according to claim 1, characterized in that, The alkaline solution added to the dried fungal residue powder is a sodium hydroxide solution or a potassium hydroxide solution, and the mass-volume concentration of the alkaline solution is 1.5% to 2.5%. The ratio of the alkaline solution to the bacterial residue powder is from 1g:10mL to 1g:15mL. The reaction was carried out under constant temperature stirring at a temperature of 50℃ to 60℃, a stirring speed of 180r / min to 220r / min, and a reaction time of 2.5h to 3h.

4. The method for extracting chitin from glutathione fermentation residue according to claim 1, characterized in that, The concentrated sodium hydroxide solution added to the deproteinized bacterial residue has a mass-volume concentration of 10% to 15%. The ratio of the concentrated sodium hydroxide solution to the deproteinized bacterial residue is from 1g:8mL to 1g:12mL. The temperature for heating and stirring is 80℃ to 90℃, the stirring speed is 150r / min to 180r / min, and the total reaction time is 5h to 6h.

5. The method for extracting chitin from glutathione fermentation residue according to claim 4, characterized in that, The concentrated sodium hydroxide solution is replenished at a frequency of once every 1 hour. Before replenishment, the concentrated sodium hydroxide solution is preheated to the same temperature as the current reaction temperature. The amount of each replenishment is 5% to 10% of the initial total volume of the concentrated sodium hydroxide solution.

6. The method for extracting chitin from glutathione fermentation residue according to claim 1, characterized in that, The added decolorizing agent is an activated carbon solution with a mass-volume concentration of 1% to 2%, or a hydrogen peroxide solution with a volume concentration of 3% to 5%.

7. The method for extracting chitin from glutathione fermentation residue according to claim 6, characterized in that, When the decolorizing agent is an activated carbon solution with a mass-volume concentration of 1% to 2%, the activated carbon solution is prepared in the following manner: Food-grade powdered activated carbon is passed through a 200-mesh sieve. The sieved food-grade powdered activated carbon is added to deionized water to prepare a solution of the required concentration. The solution is stirred and activated for 30 minutes. Before use, it is filtered to remove large particulate impurities.

8. The method for extracting chitin from glutathione fermentation residue according to claim 1, characterized in that, The volume concentration of the dilute hydrochloric acid solution added to the washed third precipitate is 0.5% to 1.0%, the ratio of the dilute hydrochloric acid solution to the washed third precipitate is 1g:10mL to 1g:15mL, and the stirring time at room temperature is 15min to 20min.

9. The method for extracting chitin from glutathione fermentation residue according to claim 1, characterized in that, The supernatant was concentrated and spray-dried to prepare a feed-grade protein additive. The decolorization waste liquid is treated with acid and alkali neutralization and then returned to be used to prepare the alkali solution and recycled.

10. The method for extracting chitin from glutathione fermentation residue according to claim 1, characterized in that, When the crude chitin is dried and dehydrated under vacuum drying conditions, the temperature is 50°C to 60°C, the vacuum degree is 0.06MPa to 0.08MPa, and the drying time is 2h to 3h. The sieving standard for crushing and sieving is 100 to 120 mesh.

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