Method for preparing chitosan from ganoderma lucidum spore wall waste residues and application of chitosan

Abstract

The invention provides a method for preparing chitosan from ganoderma lucidum spore wall waste residues and application, and belongs to the technical field of waste resource utilization. The method comprises the following steps: 1) crushing the ganoderma lucidum spore wall waste residues, performing electron beam radiation treatment, mixing the ganoderma lucidum spore wall waste residues with water, sequentially adding cellulase and papain under an ultrasonic condition to perform a step-by-step hydrolysis reaction, centrifuging after the reaction is finished, and collecting a precipitate; 2) mixing the precipitate obtained in the step 1) with a sodium hydroxide solution, adding a surfactant, and treating in an ultrasonic-microwave reactor; and adjusting the pH value to be neutral, centrifuging, and collecting the precipitate to obtain the chitosan. According to the method, low-dose electron beam radiation treatment is adopted, and cellulase and papain are subjected to step-by-step hydrolysis, so that the yield and the deacetylation degree of the chitosan can be improved, and the biocompatibility of the chitosan is improved. When the obtained chitosan is used in the barrier film, the light transmittance can be reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of waste resource utilization, and particularly relates to a method for preparing chitosan from Ganoderma lucidum spore wall waste residue and its application. Background Art

[0002] Chitosan (CS), also known as deacetylated chitin, is a biocopolymer composed of β-(1,4)-2-amino-D-glucopyranose units. Chitosan is a colorless, odorless, non-toxic white or light yellow powder, with hygroscopicity and adsorbability, as well as various biological activity functions, such as antibacterial property and biocompatibility, etc., and has wide applications in food antibacterial, packaging, additives, etc.

[0003] Currently, industrially produced chitosan is mainly obtained from the waste of seafood such as shrimp and crab shells. However, the sources of shrimp and crab shells are affected by regions and seasons, and the differences are difficult to control, with poor batch-to-batch stability. At the same time, there are problems such as different degrees of metal residues and allergenic pollutants. With the continuous expansion of the application fields of chitosan and the continuous increase in market demand, it is necessary to explore new chitosan preparation methods and raw material sources.

[0004] Ganoderma lucidum spores have important medicinal values. The cell wall of Ganoderma lucidum spores consists of a double-wall structure, with a complex and tight structure. Since the human body lacks enzymes for degrading chitin, the bioactive components inside Ganoderma lucidum spores cannot be absorbed by the human body. Generally, the active components such as Ganoderma lucidum spore oil in Ganoderma lucidum spores are extracted before consumption. After extracting the active substances from Ganoderma lucidum spores, a large amount of spore wall residue is generated. The spore wall residue is very hard and difficult to degrade. Therefore, there are few studies on efficiently and greenly preparing chitosan directly from Ganoderma lucidum spore wall waste residue.

[0005] In the prior art, generally, fine raw materials with ultra-fine pulverization (particle size reaching 100 nm) are used, and high-concentration alkali liquor (while consuming concentrated acid to adjust pH), high temperature, long-time treatment, etc. are adopted. The chitosan is greatly damaged and the degree of deacetylation is low, which limits the application range of chitosan. The extraction yield of the prior art is low, and at the same time, there are disadvantages such as long time consumption, high energy consumption, and waste liquid polluting the environment, which is not conducive to industrial production.

[0006] For example, the journal literature (Cong Wenqin, Optimization of the Separation and Extraction Process of Ganoderma lucidum Spore Chitosan, 2022) discloses a process for extracting chitosan from Ganoderma lucidum spore residues. The experimental conditions are determined through single-factor experiments and an orthogonal experiment design is carried out. Then, the optimal extraction process is determined through the orthogonal experiment, and the quality analysis of the chitosan extracted under these process conditions is carried out. The results show that the optimal conditions for chitosan extraction are a NaOH mass fraction of 40%, a temperature of 90 °C, and a water bath time of 4 h. The degree of deacetylation of the chitosan obtained under these conditions is 91.52%. However, this scheme uses a high-concentration strong alkali solution for the deacetylation reaction of chitin, and a large amount of water is required for washing to neutrality after the reaction, which has the disadvantages of high energy consumption and environmental pollution by waste liquid.

[0007] The invention patent with the publication number CN108017724B discloses a method for preparing plant-derived chitosan. The method is as follows: (1) Deionized water is added to Ganoderma lucidum spore powder, and ultrasonic extraction is carried out in a water bath, followed by centrifugation. Ethanol is added and ultrasonic treatment is carried out, and then centrifugation is carried out again. Deionized water is added, and soaking and centrifugation are carried out, and then drying is carried out to obtain crude chitin; (2) The crude chitin is placed in an H2O2 solution, placed in a water bath, adjusted to neutral with a NaOH solution, centrifuged, an alkali solution is added, water bath treatment is carried out, adjusted to neutral with an HCl solution, and drying is carried out to obtain a crude chitosan product. This method still uses a strong alkali solution to complete the deacetylation reaction of chitin, which has the disadvantages of high energy consumption and environmental pollution by waste liquid.

[0008] The invention patent with the publication number CN119350525A discloses a method and application for the separation and utilization of all components of Ganoderma lucidum spores. The method includes: S1 preparing an emulsion and spore wall shells; S2 concentrating and alcohol-precipitating the emulsion to obtain a supernatant and a precipitate; S3 drying the supernatant and the precipitate respectively to obtain a small molecule peptide component and a polysaccharide component; S4 enzymatically hydrolyzing the spore wall shells with composite enzyme A and composite enzyme B, and performing solid-liquid separation to obtain an enzymatic hydrolysate and a residue; composite enzyme A includes cellulase and chitinase, and composite enzyme B includes papain, bromelain, and α-amylase; S5 preparing soluble dietary fiber from the enzymatic hydrolysate; S6 drying and extracting the residue to obtain Ganoderma lucidum spore oil and a residue; S7 preparing chitosan from the residue. It can be seen that in this method, composite enzyme A (including cellulase and chitinase) and composite enzyme B (papain, bromelain, and α-amylase) are used for enzymatic hydrolysis to mainly obtain soluble dietary fiber. Among them, cellulase and chitinase enzymatically hydrolyze the cellulose and chitin components in the Ganoderma lucidum spore wall shells, making the structure of the spore wall shells loose.

[0009] The invention patent with the publication number CN108623703A discloses a method for preparing crude polysaccharide from the cell wall of Ganoderma lucidum spores by irradiation, which includes the following steps: (1) irradiating the waste material of Ganoderma lucidum spore extraction, irradiating with an electron beam to a dose of 500 - 900 kGy to obtain irradiated waste material; the waste material of Ganoderma lucidum spore extraction is the cell wall of Ganoderma lucidum spores remaining after wall breaking, water extraction and degreasing of Ganoderma lucidum spores; (2) mixing the irradiated waste material with water, heating for extraction, and performing solid-liquid separation to obtain a crude polysaccharide extract; (3) drying the crude polysaccharide extract to obtain crude polysaccharide from the cell wall of Ganoderma lucidum spores. This method irradiates the waste material of Ganoderma lucidum spore extraction to destroy the cell structure, further releasing the active polysaccharides inside the cells, and can significantly increase the content of active polysaccharides in the waste material, but does not perform the purification of chitosan.

[0010] In summary, in order to prepare chitosan from the waste residue of Ganoderma lucidum spore wall more greenly and efficiently, improvement is needed. Summary of the Invention

[0011] Aiming at the problems existing in the prior art, the present invention provides a method for preparing chitosan from the waste residue of Ganoderma lucidum spore wall and its application. The present invention uses low-dose electron beam radiation treatment, cellulase and papain for stepwise hydrolysis, which can improve the yield and degree of deacetylation of chitosan, improve the biocompatibility of chitosan, and expand its application. The obtained chitosan can be used in a barrier film to reduce the light transmittance.

[0012] To achieve the above object, the technical scheme adopted by the present invention is as follows:

[0013] In the first aspect, the present invention provides a method for preparing chitosan from the waste residue of Ganoderma lucidum spore wall, which includes the following steps:

[0014] 1) After the waste residue of Ganoderma lucidum spore wall is pulverized and irradiated with an electron beam, it is mixed with water, and under ultrasonic conditions, cellulase and papain are added successively for stepwise hydrolysis reaction. After the reaction ends, centrifugation is performed to collect the precipitate;

[0015] 2) Mix the precipitate obtained in step 1) with sodium hydroxide solution, add a surfactant, and then place it in an ultrasonic-microwave reactor for treatment; adjust the pH value to neutral, perform centrifugation, and collect the precipitate to obtain chitosan.

[0016] First, the waste residue of Ganoderma lucidum spore wall is pulverized. Preferably, after the waste residue of Ganoderma lucidum spore wall is pulverized, it is sieved through a 60 - 100 mesh sieve, and then irradiated with an electron beam. The treatment intensity of electron beam irradiation is 50 - 600 kGy, which can be 50 kGy, 200 kGy, 300 kGy, 500 kGy, 600 kGy, preferably 300 - 500 kGy, and more preferably 500 kGy.

[0017] After the Ganoderma lucidum spore wall waste residue is crushed and treated by electron beam radiation, it is mixed with water, and the material-liquid ratio is preferably 1 g: 10 - 30 mL, more preferably 1 g: 20 mL.

[0018] Then, under ultrasonic conditions, cellulase and papain are added successively for stepwise hydrolysis reaction. The ultrasonic power of the ultrasonic wave is 100 - 300 W, preferably 100 - 150 W, more preferably 100 W. The stepwise hydrolysis reaction includes:

[0019] First, add cellulase with a mass fraction of 3 - 5‰ and react for 2 - 4 h. Preferably, add cellulase with a mass fraction of 3.5 - 5‰ and react for 2 - 2.5 h. More preferably, add cellulase with a mass fraction of 5‰ and react for 2 h. Then, add papain with a mass fraction of 1 - 3‰ and react for 0.5 - 2 h. Preferably, add papain with a mass fraction of 2‰ and react for 1 h.

[0020] After the reaction is completed, centrifuge and collect the precipitate.

[0021] Preferably, the precipitate is post-treated. The post-treatment is decolorization treatment. The decolorization treatment specifically includes: placing the sample and 20% H2O2 in a ratio of 1:15 (g:L) at 60°C for bleaching for 3 h, then adjusting the pH value to neutral, and collecting the precipitate.

[0022] Mix the obtained precipitate or the post-treated precipitate with sodium hydroxide solution. Preferably, mix in a ratio of 1 g: 10 - 30 mL, more preferably 1 g: 20 mL. The mass fraction of the sodium hydroxide solution is 5 - 15%, preferably 8 - 10%, more preferably 8%; then add a surfactant. The surfactant is Tween 80. The addition amount of Tween 80 is 0.01 - 0.05% volume fraction, preferably 0.01 - 0.03% volume fraction, more preferably 0.01% volume fraction; place it in an ultrasonic-microwave reactor for treatment. In the ultrasonic-microwave reactor, the ultrasonic power is 100 - 500 W, preferably 200 - 400 W, more preferably 400 W; the microwave power is 200 - 600 W, preferably 300 - 400 W, more preferably 400 W; the treatment time is 10 - 40 min, preferably 20 - 30 min, more preferably 30 min.

[0023] After the treatment is completed, adjust the pH value to neutral, centrifuge, and collect the precipitate to obtain chitosan.

[0024] In a second aspect, the present invention provides an application of the chitosan prepared by the method according to the above technical solution in the preparation of a barrier film.

[0025] Preferably, the barrier film includes but is not limited to a food wrap.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] First, the present invention uses low-dose electron beam radiation to treat Ganoderma lucidum spore wall waste residue, adds cellulase first and then papain in an ultrasonic field for stepwise enzymatic hydrolysis to obtain a pretreated sample; then places the sample in an ultrasonic-microwave system, adds a low-concentration alkali solution and a surfactant for deacetylation reaction, and the obtained chitosan has better film-forming properties and wide applications.

[0028] Second, the method of directly using Ganoderma lucidum spore wall waste residue to efficiently prepare high-quality chitosan at low temperature in the present invention significantly improves the product yield and degree of deacetylation, reduces the usage amount of concentrated alkali and concentrated acid, as well as the energy consumption in the preparation process, and is simple to operate and convenient for industrial production.

[0029] Third, the present invention realizes the efficient resource utilization of agricultural waste, significantly increases the added value, reduces environmental pollution, has important economic, environmental and social benefits, and has broad industrial application prospects.

[0030] Third, when the chitosan obtained by the present invention is used in a barrier film, the light transmittance can be reduced. Description of the Drawings

[0031] Figure 1 It is a graph of the light opacity results of CS-1 film and CS-2 film;

[0032] Figure 2 It is a graph of the light opacity results of chitosan from different sources and the antioxidant active substance gallic acid forming a film. Detailed Embodiments

[0033] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments usually follow conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0034] Cellulase was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.;

[0035] Papain was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.;

[0036] Ganoderma lucidum spore wall waste residue was provided by Zhejiang Shouxian Valley Pharmaceutical Co., Ltd. The Ganoderma lucidum spore wall waste residue used in the following embodiments and comparative examples is the same batch of waste.

[0037] In the following embodiments and comparative examples, the calculation formula for the chitosan yield is: chitosan yield (%) = chitosan mass / initial Ganoderma lucidum spore wall waste residue mass.

[0038] Example 1

[0039] The method for preparing chitosan from Ganoderma lucidum spore wall waste residue is specifically as follows:

[0040] 1) Weigh 10 g of Ganoderma lucidum spore wall waste residue, pass it through a 60-mesh sieve, and then place it in a linear accelerator device for electron beam radiation treatment. The electron beam radiation dose is 600 kGy to obtain the waste residue after radiation treatment. Mix the waste residue after radiation treatment with water at a ratio of 1 g:20 mL evenly, and then place it in an ultrasonic reaction tank. Under the ultrasonic condition with an ultrasonic power of 300 W, add cellulase with a mass fraction of 3‰ to hydrolyze for 3 h and papain with a mass fraction of 2‰ to hydrolyze for 1 h in sequence. After the stepwise hydrolysis reaction, centrifuge at a speed of 10,000 rpm for 10 min, collect the precipitate and perform decolorization treatment. The specific decolorization treatment is as follows: Place the sample and 20% H2O2 at a ratio of 1:15 (g:L) at 60 °C for bleaching for 3 h, and then adjust the pH value to neutral to obtain the sample after decolorization treatment.

[0041] 2) Mix the sample after decolorization treatment with sodium hydroxide solution at a ratio of 1 g:20 mL. The mass fraction of the NaOH solution is 15%, and add Tween 80 with an addition amount of 0.02% volume fraction. Then place it in an ultrasonic-microwave reactor for treatment. The ultrasonic power is 500 W, the microwave power is 400 W, and the reaction time is 30 min. After the reaction, adjust the pH value of the reaction system to neutral, centrifuge at a speed of 10,000 rpm for 10 min, collect the precipitate, and obtain 2.59 g of chitosan sample. The chitosan yield is 25.88%.

[0042] Example 2

[0043] The method for preparing chitosan from Ganoderma lucidum spore wall waste residue is specifically as follows:

[0044] 1) Weigh 10 g of Ganoderma lucidum spore wall waste residue, pass it through an 80-mesh sieve, and then place it in a linear accelerator device for electron beam radiation treatment. The electron beam radiation dose is 500 kGy to obtain the waste residue after radiation treatment. Mix the waste residue after radiation treatment with water at a ratio of 1 g:20 mL evenly, and then place it in an ultrasonic reaction tank. Under the ultrasonic condition with an ultrasonic power of 200 W, add cellulase with a mass fraction of 5‰ to hydrolyze for 2 h and papain with a mass fraction of 2‰ to hydrolyze for 1 h in sequence. After the stepwise hydrolysis reaction, centrifuge at a speed of 10,000 rpm for 10 min. The specific decolorization treatment is as follows: Place the sample and 20% H2O2 at a ratio of 1:15 (g:L) at 60 °C for bleaching for 3 h, and then adjust the pH value to neutral to obtain the sample after decolorization treatment.

[0045] 2) Mix the decolorized sample and sodium hydroxide solution evenly at a ratio of 1 g: 20 mL. The mass fraction of the NaOH solution is 8%, and Tween 80 is added with an addition amount of 0.01% by volume fraction. Then place it in an ultrasonic-microwave reactor for treatment. The ultrasonic power is 200 W, the microwave power is 600 W, and the reaction time is 10 min. After the reaction, adjust the pH value of the reaction system to neutral, centrifuge at a speed of 10,000 rpm for 10 min, collect the precipitate, and obtain 2.63 g of chitosan sample. Calculate the chitosan yield to be 26.31%.

[0046] Example 3

[0047] The method for preparing chitosan from Ganoderma lucidum spore wall waste residue specifically comprises the following steps:

[0048] 1) Weigh 10 g of Ganoderma lucidum spore wall waste residue, pass it through a 100-mesh sieve, and then place it in a linear accelerator device for electron beam radiation treatment with a radiation dose of 50 kGy to obtain the radiation-treated waste residue. Mix the radiation-treated waste residue and water evenly at a ratio of 1 g: 20 mL, and then place it in an ultrasonic reaction tank. Under the ultrasonic condition with an ultrasonic power of 100 W, add cellulase with a mass fraction of 4‰ to hydrolyze for 3 h and papain with a mass fraction of 2‰ to hydrolyze for 1 h in sequence. After the stepwise hydrolysis reaction, centrifuge at a speed of 10,000 rpm for 10 min, collect the precipitate and perform decolorization treatment. The specific decolorization treatment is as follows: Place the sample and 20% H2O2 at a ratio of 1:15 (g:L) at 60 °C for bleaching for 3 h, and then adjust the pH value to neutral to obtain the decolorized sample.

[0049] 2) Mix the decolorized sample and sodium hydroxide solution evenly at a ratio of 1 g: 20 mL. The mass fraction of the NaOH solution is 13%, and Tween 80 is added with an addition amount of 0.03% by volume fraction. Then place it in an ultrasonic-microwave reactor for treatment. The ultrasonic power is 500 W, the microwave power is 200 W, and the reaction time is 30 min. After the reaction, adjust the pH value of the reaction system to neutral, centrifuge at a speed of 10,000 rpm for 10 min, collect the precipitate, and obtain 2.71 g of chitosan sample. The chitosan yield is 27.07%.

[0050] Example 4

[0051] The method for preparing chitosan from Ganoderma lucidum spore wall waste residue specifically comprises the following steps:

[0052] 1) Weigh 10 g of Ganoderma lucidum spore wall waste residue, pass it through a 80-mesh sieve, and then place it in a linear accelerator device for electron beam radiation treatment with an electron beam radiation dose of 200 kGy to obtain the waste residue after radiation treatment. Mix the waste residue after radiation treatment with water at a ratio of 1 g:20 mL evenly, and then place it in an ultrasonic reaction cell. Under the ultrasonic condition with an ultrasonic power of 200 W, add cellulase with a mass fraction of 4‰ to hydrolyze for 4 h and papain with a mass fraction of 2‰ to hydrolyze for 1 h in sequence. After the stepwise hydrolysis reaction is completed, centrifuge at a rotation speed of 10,000 rpm for 10 min, collect the precipitate and perform decolorization treatment. The specific decolorization treatment is as follows: Place the sample and 20% H2O2 at a ratio of 1:15 (g:L) at 60 °C for bleaching for 3 h, and then adjust the pH value to neutral to obtain the sample after decolorization treatment.

[0053] 2) Mix the sample after decolorization treatment with sodium hydroxide solution at a ratio of 1 g:20 mL evenly. The mass fraction of the NaOH solution is 5%, and add Tween 80 with an addition amount of 0.05% volume fraction. Then place it in an ultrasonic-microwave reactor for treatment with an ultrasonic power of 100 W, a microwave power of 400 W, and a reaction time of 40 min. After the reaction is completed, adjust the pH value of the reaction system to neutral, centrifuge at a rotation speed of 10,000 rpm for 10 min, collect the precipitate, and obtain 2.69 g of chitosan sample with a chitosan yield of 26.93%.

[0054] Example 5

[0055] The method for preparing chitosan from Ganoderma lucidum spore wall waste residue specifically comprises the following steps:

[0056] 1) Weigh 10 g of Ganoderma lucidum spore wall waste residue, pass it through a 80-mesh sieve, and then place it in a linear accelerator device for electron beam radiation treatment with an electron beam radiation dose of 300 kGy to obtain the waste residue after radiation treatment. Mix the waste residue after radiation treatment with water at a ratio of 1 g:20 mL evenly, and then place it in an ultrasonic reaction cell. Under the ultrasonic condition with an ultrasonic power of 150 W, add cellulase with a mass fraction of 3.5‰ to hydrolyze for 2.5 h and papain with a mass fraction of 2‰ to hydrolyze for 1 h in sequence. After the stepwise hydrolysis reaction is completed, centrifuge at a rotation speed of 10,000 rpm for 10 min, collect the precipitate and perform decolorization treatment. The specific decolorization treatment is as follows: Place the sample and 20% H2O2 at a ratio of 1:15 (g:L) at 60 °C for bleaching for 3 h, and then adjust the pH value to neutral to obtain the sample after decolorization treatment.

[0057] 2) Mix the decolorized sample with sodium hydroxide solution at a ratio of 1 g:20 mL. The mass fraction of the NaOH solution is 10%, and Tween 80 is added with an addition amount of 0.03% by volume fraction. Then place it in an ultrasonic-microwave reactor for treatment. The ultrasonic power is 200 W, the microwave power is 300 W, and the reaction time is 20 min. After the reaction, adjust the pH value of the reaction system to neutral, centrifuge at a speed of 10000 rpm / min for 10 min, collect the precipitate, and obtain 2.80 g of chitosan sample. The chitosan yield is 28.04%.

[0058] Example 6

[0059] The method for preparing chitosan from Ganoderma lucidum spore wall waste residue is specifically as follows:

[0060] 1) Weigh 10 g of Ganoderma lucidum spore wall waste residue, pass it through a 70-mesh sieve, and then place it in a linear accelerator device for electron beam radiation treatment with an electron beam radiation dose of 500 kGy to obtain the radiation-treated waste residue. Mix the radiation-treated waste residue with water at a ratio of 1 g:20 mL evenly, and then place it in an ultrasonic reaction tank. Under the ultrasonic condition with an ultrasonic power of 100 W, add cellulase with a mass fraction of 5‰ for hydrolysis for 2 h and papain with a mass fraction of 2‰ for hydrolysis for 1 h in sequence. After the stepwise hydrolysis reaction, centrifuge at a speed of 10000 rpm / min for 10 min, collect the precipitate and perform decolorization treatment. The specific decolorization treatment is as follows: Place the sample and 20% H2O2 at a ratio of 1:15 (g:L) at 60 °C for bleaching for 3 h, and then adjust the pH value to neutral to obtain the decolorized sample.

[0061] 2) Further, mix the decolorized sample with sodium hydroxide solution at a ratio of 1 g:20 mL evenly. The mass fraction of the NaOH solution is 8%, and Tween 80 is added with an addition amount of 0.01% by volume fraction. Then place it in an ultrasonic-microwave reactor for treatment. The ultrasonic power is 400 W, the microwave power is 400 W, and the reaction time is 30 min. After the reaction, adjust the pH value of the reaction system to neutral, centrifuge at a speed of 10000 rpm / min for 10 min, collect the precipitate, and obtain 2.93 g of chitosan sample. The chitosan yield is 29.31%.

[0062] Comparative Example 1

[0063] The method for preparing chitosan from Ganoderma lucidum spore wall waste residue is specifically as follows:

[0064] Weigh 10 g of Ganoderma lucidum spore wall waste residue, pass it through a 100-mesh sieve, mix it evenly with a strong alkali solution of 10% NaOH by mass fraction, carry out deproteinization treatment, centrifuge at a speed of 10,000 rpm for 10 min, discard the supernatant, and adjust the pH of the system to neutral; further carry out decolorization treatment. The specific decolorization treatment is as follows: place the sample and 20% H2O2 in a ratio of 1:15 (g:L) at 60 °C for bleaching for 3 h, then adjust the pH value to neutral, and then dry the decolorized sample to obtain a chitin sample; subject the obtained chitin sample to a deacetylation reaction under the conditions of 40% NaOH by mass fraction, a temperature of 90 °C, and a water bath time of 4 h; after the reaction, adjust the pH of the sample to neutral, centrifuge at a speed of 10,000 rpm for 10 min, discard the precipitate, and obtain 1.22 g of a chitosan sample, with a product yield of 12.23%.

[0065] Comparative Example 1 is the traditional preparation method of chitosan. This process requires a high-concentration alkali solution at high temperature for a long time, and at the same time, it requires a large amount of high-concentration acid to adjust the pH of the system.

[0066] Compared with Comparative Example 1, Examples 1-6 adopt a green preparation method. The average yield of chitosan reaches 27.26%, which is 122% higher than that of chitosan in Comparative Example 1. The mass fraction of the alkali solution is reduced by more than 67%, the energy consumption in the preparation process is reduced, the high-efficiency green manufacturing of chitosan at room temperature is realized, and the defects of the traditional preparation method of chitosan - the method of using a high-concentration alkali solution at high temperature for a long time are effectively improved, having the advantages of high efficiency, greenness and environmental protection.

[0067] Comparative Example 2

[0068] The difference from Example 2 is that the electron beam radiation dose is 30 kGy, and the others are the same as in Example 2, and chitosan is obtained. The chitosan yield is 17.32%.

[0069] Comparative Example 3

[0070] The difference from Example 2 is that cellulase and papain are added together for hydrolysis for 1 h, and the others are the same as in Example 2, and chitosan is obtained. The chitosan yield is 14.86%.

[0071] Comparative Example 4

[0072] The difference from Example 2 is that the mass fraction of cellulase is 2% and the mass fraction of papain is 0.5%, and the others are the same as in Example 2, and chitosan is obtained. The chitosan yield is 16.16%.

[0073] Comparative Example 5

[0074] The difference from Example 2 is that the enzymatic hydrolysis time of cellulase is 1 h and that of papain is 0.5 h, and the others are the same as in Example 2. Chitosan is obtained, and the chitosan yield is 15.98%.

[0075] Test Example 1

[0076] Characterize the characteristics of the chitosan samples prepared in Examples 1-6 and Comparative Examples 1-5.

[0077] Detection method for the degree of deacetylation of chitosan: Determined by ultraviolet spectroscopy. Using 0.001 mol / L HCl as the solvent, prepare standard solutions of N-acetylglucosamine with different concentrations. Using 0.001 mol / L HCl as the reference solution, measure the absorbance of the solution at 199 nm, and establish a working curve of solution concentration vs. absorbance. Weigh 10-20 mg of the sample, use 0.001 mol / L HCl as the solvent, make up the volume to 100 mL, use 0.001 mol / L HCl as the reference solution, measure the absorbance at 199 nm, substitute it into the working curve, obtain the concentration of acetyl groups in the sample, and the degree of deacetylation (%) = 100% - (concentration of acetyl groups in the sample / sample concentration) * 100%.

[0078] The results of the degree of deacetylation of chitosan in Examples 1-6 and Comparative Examples 1-5 are shown in Table 1.

[0079] The results show that taking Comparative Example 1 and Example 1 as examples, it is found that the degrees of deacetylation of Comparative Example 1 and Example 1 are 80.2% and 92.4% respectively, and Example 1 shows better biocompatibility.

[0080] Table 1 Degree of deacetylation of chitosan in examples and comparative examples

[0081]

[0082]

[0083] Test Example 2

[0084] Analyze the film-forming properties of the chitosan prepared in Example 1 and Comparative Example 1. From two aspects of only using chitosan to form a film and the composite film of chitosan and antioxidant (gallic acid, GA), focus on analyzing the barrier properties of the chitosan film.

[0085] Use the chitosan of Comparative Example 1 to prepare a film. The film-forming steps are as follows: Accurately weigh the chitosan sample prepared in Comparative Example 1, dissolve it in 2% acetic acid solution so that the chitosan content is 2%, then stir magnetically at 55 °C and 800 r / min for 3 h to obtain a 2% chitosan acetic acid solution. Use the casting method to form a thin film solution in a disposable plastic petri dish, place it horizontally in an oven at 60 °C and dry for 3 h. The prepared film is placed in a desiccator for storage and standby. Named CS-1 film.

[0086] The chitosan prepared in Example 1 was used to form a film. The film-forming steps were as follows: The chitosan sample prepared in Example 1 was accurately weighed and dissolved in a 2% acetic acid solution so that the chitosan content was 2%. Then, it was magnetically stirred at 55 °C and 800 r / min for 3 h to obtain a 2% chitosan acetic acid solution. A thin film solution was formed in a disposable plastic Petri dish by the casting method and placed horizontally in an oven at 60 °C for drying for 3 h. The prepared film was placed in a desiccator for storage and named CS-2 film.

[0087] The chitosan prepared in Comparative Example 1 was used to form a film. The film-forming steps were as follows: 500 mg of gallic acid was accurately weighed and dissolved in 95% ethanol and fixed in a 25 mL volumetric flask to prepare a gallic acid solution with a concentration of 20 mg / mL. The chitosan acetic acid solution prepared in Comparative Example 1 was mixed with the gallic acid solution so that the concentration of gallic acid reached 4%. A thin film solution was formed in a disposable plastic Petri dish by the casting method and placed horizontally in an oven at 60 °C for drying for 3 h. The prepared film was placed in a desiccator for storage and named GA-CS-1 film.

[0088] The chitosan prepared in Example 1 was used to form a film. The film-forming steps were as follows: 500 mg of gallic acid was accurately weighed and dissolved in 95% ethanol and fixed in a 25 mL volumetric flask to prepare a gallic acid solution with a concentration of 20 mg / mL. The chitosan acetic acid solution prepared in Example 1 was mixed with the gallic acid solution so that the concentration of gallic acid reached 4%. A thin film solution was formed in a disposable plastic Petri dish by the casting method and placed horizontally in an oven at 60 °C for drying for 3 h. The prepared film was placed in a desiccator for storage and named GA-CS-2 film.

[0089] The opacity is usually used to describe the ability of a material to transmit or block light, which is an important index for characterizing film materials and an important index for preventing the oxidation of packaged products.

[0090] Opacity detection method: The film was cut into a rectangle of 10×30 mm and fixed on the light-transmitting side of a quartz cuvette. Zero adjustment was made with a blank cuvette as a reference, and the chitosan film was measured at a wavelength range of 600 nm.

[0091] The opacity results of the samples CS-1 film and CS-2 film obtained by only forming a film with chitosan are as Figure 1 shown, Figure 1 The results show that for the opacity of the films formed with chitosans from different sources, the opacity of the CS-2 film is higher than that of the CS-1 film, indicating that the chitosan prepared in Example 1 has better barrier ability.

[0092] Due to problems such as poor mechanical properties, low water resistance ability, and weak antioxidant activity of pure chitosan films, it is impossible to directly prepare food preservative films that meet the application standards. Therefore, the light-blocking performance of composite films prepared by adding an antioxidant (gallic acid, GA) to chitosan was studied. As a food packaging film, the light transmittance of the composite film is an important evaluation index. It can be measured by opacity. The light-blocking ability and freshness preservation ability of the composite film increase with the increase of the opacity value.

[0093] The light transmittance results of GA-CS-1 film and GA-CS-2 film are as Figure 2 shown Figure 2 Figure of the opacity results of chitosan from different sources and the film formed by the antioxidant gallic acid. The results show that the chitosan film prepared by the present invention has good barrier properties, significantly better than the film formed by pure chitosan. It can be seen that gallic acid effectively fills the grid gaps of the chitosan film, making the structure of the GA-CS composite film more dense, achieving a good light isolation effect, and thus resulting in an increase in the opacity of the composite film.

[0094] The GA-CS-2 film is significantly higher than the GA-CS-1 film, which indicates that the chitosan prepared in Example 1 has good film-forming performance and light-blocking ability.

[0095] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Simple modifications or equivalent replacements made by those of ordinary skill in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing chitosan from Ganoderma lucidum spore wall waste residue, characterized in that, It includes the following steps: 1) After the Ganoderma lucidum spore wall waste residue is crushed, sieved, and treated by electron beam irradiation, it is mixed with water, and under ultrasonic conditions, cellulase and papain are sequentially added for stepwise hydrolysis reaction. After the reaction is completed, centrifugation is carried out to collect the precipitate; 2) The precipitate obtained in step 1) is mixed with sodium hydroxide solution, and after adding a surfactant, it is placed in an ultrasonic-microwave reactor for treatment; the pH value is adjusted to neutral, centrifuged, and the precipitate is collected to obtain chitosan.

2. The method according to claim 1, wherein In step 1), the stepwise hydrolysis reaction includes: First, cellulase with a mass fraction of 3-5‰ is added and reacted for 2-4 h, and then papain with a mass fraction of 1-3‰ is added and reacted for 0.5-2 h.

3. The method according to claim 2, characterized in that The mass fraction of the papain is 2‰, and the reaction time of the papain is 1 h.

4. The method according to claim 1, wherein In step 1), the treatment intensity of the electron beam irradiation is 50-600 kGy.

5. The method according to claim 4, characterized in that In step 1), the treatment intensity of the electron beam irradiation is 300-500 kGy.

6. The method according to claim 1, wherein In step 1), the mesh number of the sieved Ganoderma lucidum spore wall waste residue is 60-100 meshes.

7. The method according to claim 1, wherein In step 1), the ultrasonic power of the ultrasonic wave is 100-300 W.

8. The method according to claim 1, wherein In step 2), the mass fraction of the sodium hydroxide solution is 5-15%; the surfactant is Tween 80, and the addition amount of the Tween 80 is 0.01-0.05% volume fraction.

9. The method according to claim 1, wherein In the ultrasonic-microwave reactor, the ultrasonic power is 100-500 W, the microwave power is 200-600 W, and the treatment time is 10-40 min.

10. The application of the chitosan prepared by the method according to any one of claims 1-9 in the preparation of a barrier film.

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