Preparation method of chitosan oligosaccharide

Abstract

The preparation method comprises the following steps: mixing chitin biomass with an L molecular sieve, carrying out ball milling, adding a solvent for dissolving, carrying out solid-liquid separation on the L molecular sieve, and drying to obtain the chitosan oligosaccharide, the chemical composition of the L molecular sieve is as follows: 1 to 5 percent of K2O, 1.0 percent of Al2O3, 5 to 10 percent of SiO2 and 1 to 5 percent of H2O, and the L molecular sieve has a straight pore channel. The L molecular sieve with the straight pore channels can selectively shear glucosidic bonds of chitin to prepare the chitosan oligosaccharide, a large amount of water or other solvents are not needed to participate in the reaction, and the L molecular sieve has the advantages of being environmentally friendly, low in cost, easy in product separation, higher in catalytic efficiency, capable of being repeatedly used and more beneficial to industrialization.

Description

Technical Field

[0001] The invention belongs to the technical field of biomass conversion and utilization, and specifically relates to a method for preparing chito-oligosaccharides from chitin biomass or chitin. Background Art

[0002] Chitosan oligosaccharide is an oligosaccharide formed by connecting N-acetylglucosamine through β-1,4 glycosidic bonds, and the degree of polymerization is generally 2-7. Chitosan oligosaccharide has a variety of biological activities, including inducing plant disease resistance, promoting plant growth, and resisting stress; it also has physiological activities and functions such as enhancing immunity, anti-tumor, lowering blood pressure, blood sugar, blood lipids, and regulating the balance of beneficial intestinal microorganisms. It is widely used in the fields of biological pesticides, biological fertilizers, health foods, food additives, etc.

[0003] At present, chito-oligosaccharides are generally prepared by hydrolysis of chitin, which mainly includes chemical degradation and enzymatic degradation. Chemical degradation generally uses concentrated acid hydrolysis. Although the degradation efficiency is high, it has the disadvantages of harsh reaction conditions, difficult control, low product quality, high equipment requirements and environmental pollution, and the products are mostly below tetrasaccharides; Enzyme degradation is a mild method, and the enzymes used include chitinase and chitosanase. Chinese patent (application number: 2011102588936) discloses a chitin degradation strain and a method for preparing chito-oligosaccharides, which first converts chitin into colloidal chitin, then cultivates the strain and produces enzymes, and then uses the chitinase produced by the strain to perform enzymatic hydrolysis on the prepared colloidal chitin; Chinese patent (application number: 200610080091X) discloses a method for preparing chito-oligosaccharides, which uses fermentation enzyme production to degrade chitin colloid to prepare bioactive chito-oligosaccharides, but both have problems such as insufficient enzyme activity and low oligosaccharide yield. Since chitosan is insoluble in water and most solvents, chitosan requires preliminary treatment and the process is relatively complicated, the production cycle is long, and the cost is high.

[0004] The reason why chitin is difficult to degrade is that a large number of hydrogen bonds are formed between its sugar chains, resulting in high crystallinity and structural density; in addition, chitin in chitin biomass is also interlocked with calcium carbonate, protein, etc., which affects the contact of macromolecules such as enzymes, resulting in low hydrolysis efficiency. Therefore, the efficiency of chitin hydrolysis to prepare chitosan oligosaccharides still needs to be improved and currently cannot meet production needs. Summary of the invention

[0005] The present invention aims to provide a method for preparing chitosan oligosaccharides to solve the problem of low efficiency in preparing chitosan oligosaccharides by hydrolysis of chitin in the prior art.

[0006] To achieve the above object, the present invention provides a method for preparing chitosan oligosaccharides, comprising the following steps: mixing chitosan biomass with L molecular sieve, grinding with a ball mill, adding a solvent to dissolve, separating the L molecular sieve from the solid and liquid, and drying to obtain chitosan oligosaccharides.

[0007] Preferably, the chitosan biomass comprises shrimp shells, crab shells, crayfish shells, and krill shells.

[0008] Preferably, the mass ratio of the chitosan biomass to the L molecular sieve is 1 to 20:1; the mass ratio of chitin in the chitosan biomass to the L molecular sieve is 1 to 10:1.

[0009] Preferably, the ball milling beads used in ball milling are one or more of zirconia ball milling beads, alumina ball milling beads, and agate ball milling beads; the diameter of the ball milling beads is 1 to 10 mm, and the mass ratio of the chitin biomass to the ball milling beads is 1:1 to 20.

[0010] Preferably, the ball milling time is 1 to 10 hours, the grinding speed is 100 to 1000 rpm; the temperature is maintained at 25 to 60°C; the solvent is water, and the amount of water added is 2 to 10 times the mass of the chitin biomass; the solid-liquid separation method includes centrifugation and filtration; the drying method includes rotary evaporation drying, spray drying, and oven drying.

[0011] The invention provides an L molecular sieve, whose chemical composition is: xK2O·1.0Al2O3·ySiO2·zH2O, and has straight pores; wherein x ranges from 1 to 5; y ranges from 5 to 10; and z ranges from 1 to 5.

[0012] Preferably, the straight pore size is 0.71, and the topological structure is LTL L-type zeolite molecular sieve.

[0013] The invention provides a method for preparing L molecular sieve, comprising the following steps: taking a silicon source and a potassium source, mixing them and grinding them for the first time, adding an aluminum source, grinding them for the second time, crystallizing at 150-200° C., and directly calcining at 400-600° C. in an air atmosphere to obtain L molecular sieve.

[0014] Preferably, the molar ratio of silicon source: potassium source: aluminum source is 5-20:1-10:0.1-2.0; the first grinding time is 10-120 minutes, and the second grinding time is 5-60 minutes; the crystallization time is 4-24 hours; the calcination time is 1-5 hours; the silicon source is one or a mixture of fumed silica, potassium silicate, and sodium silicate; the potassium source is one or a mixture of potassium hydroxide and potassium bromide; the aluminum source is one or a mixture of aluminum hydroxide, sodium aluminate, and aluminum sulfate.

[0015] The present invention provides an application of an L molecular sieve for preparing a catalyst for chito-oligosaccharide.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] Based on a large number of experiments, this patent has developed an L molecular sieve with straight pores, and the size of the pores just matches the size of the chitin sugar chain, which can selectively shear the glycosidic bonds of chitin to prepare chitosan oligosaccharides; based on the solid structure characteristics of the molecular sieve, combined with the ball milling method, this patent has developed a technology that uses L molecular sieve as a catalyst to ball mill chitin biomass or chitin with L molecular sieve, which can efficiently prepare chitosan oligosaccharides. On the one hand, this technical solution does not require a large amount of water or other solvents to participate in the reaction, and has the advantages of environmental friendliness, low cost, and easy product separation; on the other hand, the present invention uses the original ecological chitosan biomass directly as raw material, and utilizes the chimeric structure of chitin and calcium carbonate in the original ecological chitosan biomass to more efficiently destroy hydrogen bonds and quickly hydrolyze. The solid molecular sieve catalyst in the preparation method of chitosan oligosaccharides in this patent has higher catalytic efficiency and can be reused. Compared with the existing technology, it has obvious advantages and is more conducive to industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The XRD pattern of the L molecular sieve prepared by the present invention;

[0019] Figure 2 The XRD pattern of L molecular sieve prepared by the hydrothermal method in the prior art;

[0020] Figure 3 This is the mass spectrum of chito-oligosaccharides obtained by ball-milling chitin with L molecular sieve in the present invention. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0022] The instruments, reagents, materials, etc. involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0023] The invention provides a method for preparing chitosan oligosaccharide, comprising the following steps: mixing chitosan biomass or chitin with molecular sieves, adding ball milling beads, grinding with a ball mill for a certain time, adding a certain amount of water to dissolve the chitosan oligosaccharide, separating the molecular sieve from solid and liquid, and drying the solution to obtain the chitosan oligosaccharide.

[0024] Among them, the molecular sieve is L molecular sieve; the chitin biomass is shrimp shell, crab shell, crayfish shell and krill shell; the mass ratio of chitin biomass to molecular sieve is: 20:1~1:1; the mass ratio of chitin to molecular sieve is: 10:1~1:1.

[0025] The ball milling beads are zirconium oxide ball milling beads, aluminum oxide ball milling beads and / or agate ball milling beads; the diameter of the ball milling beads is 1 to 10 mm; the amount of ball milling beads added is: the mass ratio of chitin biomass and / or chitin to ball milling beads is 1:1 to 1:20; the ball mill grinding time is 1 to 10 hours, preferably 1 to 4 hours; the grinding speed is 100-1000 rpm; the temperature is maintained at 25 to 60° C., preferably 30 to 40° C.; the amount of water added is 2 to 10 times the mass of chitin biomass and / or chitin; the solid-liquid separation method includes centrifugation and filtration at 5000 rpm; the drying method includes rotary evaporation drying, spray drying, and drying.

[0026] The chitin degree of chitosan oligosaccharide is 2-7.

[0027] The preparation method of L molecular sieve is characterized in that the molar ratio of silicon source: potassium source: aluminum source is 5-20:1-10:0.1-2.0; the first grinding time is 10-120 minutes, and the second grinding time is 5-60 minutes; the crystallization time is 4-24 hours; the calcination time is 1-5 hours; the silicon source is one or a mixture of fumed silica, potassium silicate, and sodium silicate; the potassium source is one or a mixture of potassium hydroxide and potassium bromide; the aluminum source is one or a mixture of aluminum hydroxide, sodium aluminate, and aluminum sulfate.

[0028] The chemical composition of L molecular sieve is: xK2O·1.0Al2O3·ySiO2·zH2O, wherein x ranges from 1 to 5; y ranges from 5 to 10; and z ranges from 1 to 5. L molecular sieve is an L-type zeolite molecular sieve with a pore size of 0.71 nm and a topological structure of LTL.

[0029] Example 1: Preparation of L molecular sieve by solvent-free method of the present invention

[0030] Solvent-free synthesis of L molecular sieve: Weigh 3.60g of fumed silica and 3.366g of potassium hydroxide as solid raw materials, pour them into a mortar and grind for 10 minutes, then add 0.468g of aluminum hydroxide and grind for another 5 minutes, add the reaction raw materials into a polytetrafluoroethylene stainless steel reactor, crystallize at 170°C for 24h, and the obtained product does not need to be filtered or washed, but is directly calcined at 550°C for 4h in an air atmosphere to obtain the final L molecular sieve. The proportion of the reaction raw materials is as follows:

[0031] 10K2O:1Al2O3:20SiO2.

[0032] X-ray diffraction analysis showed that the structure was L zeolite molecular sieve. Figure 1 The XRD pattern synthesized by this method is consistent with the typical XRD peaks of L molecular sieve.

[0033] By adding different amounts of aluminum hydroxide, L molecular sieves with different Si / Al ratios can be prepared, for example: L molecular sieves with Si / Al=2.5, L molecular sieves with Si / Al=15, and L molecular sieves with Si / Al=20.

[0034] Compared with the traditional hydrothermal method for synthesizing L molecular sieve:

[0035] According to the reference (Catalysts 2019,9,1073; doi:10.3390 / catal9121073), L molecular sieve was prepared by traditional hydrothermal method. The specific synthesis process is as follows:

[0036] Weigh 3.018g potassium hydroxide and 1.442g aluminum sulfate, pour them into a beaker filled with 18.208g deionized water, stir at room temperature for 10min to form a slightly turbid solution, namely liquid A; weigh 6.875g HS-40 silica sol, pour it into a beaker filled with 9.958g deionized water, stir at room temperature for 5min, namely liquid B; add liquid B dropwise into liquid A and stir well for 10min to obtain liquid C, which is transferred to a polytetrafluoroethylene stainless steel reactor and crystallized at 180℃ for 72h. The obtained product is washed with deionized water until neutral and dried, and then calcined at 480℃ for 4h in air atmosphere to obtain the final L molecular sieve.

[0037] The ratio of the reaction materials is as follows:

[0038] 10K2O:1Al2O3:20SiO2:800H2O.

[0039] X-ray diffraction analysis showed that the structure was L zeolite molecular sieve. Figure 2 The XRD pattern synthesized by this method is consistent with the typical XRD peaks of L molecular sieve.

[0040] Table 1 shows the pyridine-infrared analysis characterization results of L molecular sieves synthesized by solvent-free method and traditional hydrothermal method. Compared with L molecular sieves synthesized by traditional hydrothermal method, L molecular sieves synthesized by solvent-free method in the present invention contain higher Lewis acid / The acid ratio is 5.961, which is significantly higher than 0.86 prepared by traditional hydrothermal method.

[0041] Table 1. Pyridine-IR analysis characterization results of L molecular sieves prepared by traditional hydrothermal method and solvent-free method

[0042]

[0043] Example 2: Preparation of L molecular sieve by solvent-free method

[0044] Weigh 1.8g potassium silicate and 1.19g potassium bromide as solid raw materials, pour them into a mortar and grind for 15 minutes, then add 0.246g sodium aluminate and grind for another 10 minutes, add the reaction raw materials into a polytetrafluoroethylene stainless steel reactor, crystallize at 170°C for 24h, and the obtained product does not need to be filtered or washed, but is directly calcined at 550°C for 4h in an air atmosphere to obtain the final L molecular sieve. The ratio of the reaction raw materials is as follows:

[0045] 3.5K2O:1Al2O3:15SiO2.

[0046] By adding different amounts of sodium aluminate, L molecular sieves with different Si / Al ratios can be prepared, for example: L molecular sieves with Si / Al=2.5, L molecular sieves with Si / Al=10, and L molecular sieves with Si / Al=20.

[0047] Example 3: Preparation of L molecular sieve by solvent-free method

[0048] Weigh 1.45g sodium silicate and 1.19g potassium bromide as solid raw materials, pour them into a mortar and grind for 15 minutes, then add 4.0g aluminum sulfate and grind for another 10 minutes, add the reaction raw materials into a polytetrafluoroethylene stainless steel reactor, crystallize at 170℃ for 24h, and the obtained product does not need to be filtered or washed, but is directly calcined at 550℃ for 4h in an air atmosphere to obtain the final L molecular sieve. The proportion of the reaction raw materials is as follows:

[0049] 3.5K2O:2Al2O3:15SiO2.

[0050] By adding different amounts of aluminum sulfate, L molecular sieves with different Si / Al ratios can be prepared, for example: L molecular sieve with Si / Al=2.5, L molecular sieve with Si / Al=10, and L molecular sieve with Si / Al=20.

[0051] Example 4: Preparation of chitosan oligosaccharides by ball milling and hydrolysis of chitin using L molecular sieve:

[0052] 2.0 g of chitin, different masses of solid catalysts (L molecular sieves with different Si / Al, activated carbon, oxidized activated carbon, ZSM-5 molecular sieves with Si / Al=10, Hβ molecular sieves with Si / Al=10, nano-silica), 10.0 g of zirconium oxide ball milling beads (Ф=5 mm), and 0.2 g of water were added to the ball milling jar of the high-energy planetary ball mill F-P2000, and ball milling was performed at 500 rpm and 1000 rpm for different times; the temperature of the ball milling chamber was controlled at 40°C. After the ball milling, 50.0 mL of water was added, stirred at 200 rpm for 30 min, filtered, and the filtrate was rotary evaporated and dried to obtain chitosan oligosaccharides. The chitosan oligosaccharide yield was calculated based on the mass of chitin added; the product was analyzed by ESI-MS to analyze the degree of polymerization of chitosan oligosaccharides.

[0053] Table 2. Preparation of chitosan oligosaccharides by ball-milling hydrolysis of chitin using different solid catalysts

[0054]

[0055]

[0056] Example 5: Preparation of chitosan oligosaccharides by ball milling and hydrolyzing crab shells with L molecular sieve:

[0057] Add 8.0g of dried crab shell (of which the mass percentage of chitin is 25.3%), solid catalysts of different masses (L molecular sieves with different Si / Al, activated carbon, oxidized activated carbon, ZSM-5 molecular sieves with Si / Al=10, Hβ molecular sieves with Si / Al=10, nano-silica), 10.0g of zirconium oxide ball milling beads (Ф=5mm), and 0.2g of water into the ball milling jar of the high-energy planetary ball mill F-P2000, and mill for different times at 500rpm and 1000rpm; the temperature of the ball milling chamber is controlled at 40°C. After the ball milling, add 50.0mL of water, stir at 200rpm for 30min, filter, and rotary evaporate the filtrate to dry to obtain chitosan oligosaccharides. The yield of chitosan oligosaccharides is calculated based on the mass of chitin added; the product is analyzed by ESI-MS to analyze the degree of polymerization of chitosan oligosaccharides.

[0058] Table 3. Preparation of chitosan oligosaccharides by ball milling and hydrolysis of crab shells using different solid catalysts

[0059]

[0060]

[0061] Example 6: Preparation of chitosan oligosaccharides by ball milling and hydrolysis of crayfish shells using L molecular sieve

[0062] Add 8.0g of dried crayfish shells (of which the mass percentage of chitin is 22.5%), solid catalysts of different masses (L molecular sieves with different Si / Al, activated carbon, oxidized activated carbon, ZSM-5 molecular sieves with Si / Al=10, Hβ molecular sieves with Si / Al=10, nano-silica), 10.0g of zirconium oxide ball milling beads (Ф=5mm), and 0.2g of water into the ball milling jar of the high-energy planetary ball mill F-P2000, and mill for different times at 500rpm and 1000rpm; the temperature of the ball milling chamber is controlled at 40°C. After the ball milling, add 50.0mL of water, stir at 200rpm for 30min, filter, and rotary evaporate the filtrate to dry to obtain chitosan oligosaccharides. The chitosan oligosaccharide yield is calculated based on the mass of chitin added; the product is analyzed by ESI-MS to analyze the polymerization degree of chitosan oligosaccharides.

[0063] Table 4. Preparation of chitosan oligosaccharides by ball-milling hydrolysis of crayfish shells using different solid catalysts

[0064]

[0065]

[0066] Example 7: Lifespan experiment of preparing chitosan oligosaccharides by ball milling and hydrolyzing chitin with L molecular sieve

[0067] Add 2.0g chitin, 0.4g L molecular sieve with Si / Al of 10, 10.0g zirconium oxide ball milling beads (Ф=5mm), and 0.2g water to the ball milling jar of high-energy planetary ball mill F-P2000, and ball mill for 2h at 500rpm; the temperature of the ball milling chamber is controlled at 40℃. After the ball milling, add 50.0mL water, stir at 200rpm for 30min, filter, and rotary evaporate the filtrate to obtain chitosan oligosaccharides. The chitosan oligosaccharide yield is 90.3% based on the mass of chitin added; the L molecular sieve obtained by filtration is repeatedly used in the above process four times, and the chitosan oligosaccharide yields are 90.5%, 90.1%, 90.1% and 89.5% respectively, indicating that the catalyst activity has not decreased significantly and can be reused. The product is analyzed by ESI-MS, and the polymerization degree of chitosan oligosaccharides is 2-7.

[0068] It can be seen from the above examples that L molecular sieves with different Si / Al can efficiently hydrolyze chitin and chitosan biomass to prepare chitosan oligosaccharides under different ball milling parameters, and the degree of polymerization is 2-7; other solid acid catalysts, including ZSM-5 molecular sieve, Hβ molecular sieve, activated carbon, oxidized activated carbon and nano-silica, can also catalyze the hydrolysis of chitin to prepare chitosan oligosaccharides, but the efficiency is not high, and the degree of polymerization of oligosaccharides is either low (less than 3) or contains monosaccharides, which affects the quality of chitosan oligosaccharides. The L molecular sieve ball milling catalyzed chitin and chitosan bio-preparation of chitosan oligosaccharides described in this patent has significant technical advantages.

[0069] For any technician familiar with the art, without departing from the scope of the technical solution of the present invention, the technical content disclosed above can be used to make many possible changes and modifications to the technical solution of the present invention, or modified into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention should still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing chitosan oligosaccharides, characterized in that: The following steps are involved: The chitosan biomass is mixed with L molecular sieve, ball-milled, and then dissolved in a solvent, and then the L molecular sieve is separated from the solid and liquid, and dried to obtain chitosan oligosaccharides.

2. The method for preparing chitosan oligosaccharides according to claim 1, characterized in that: The chitosan biomass includes shrimp shells, crab shells, crayfish shells, and krill shells.

3. The method for preparing chitosan oligosaccharides according to claim 1, characterized in that: The mass ratio of the chitosan biomass to the L molecular sieve is 1 to 20:1; the mass ratio of chitin in the chitosan biomass to the L molecular sieve is 1 to 10:

1.

4. The method for preparing chitosan oligosaccharides according to claim 1, characterized in that: The ball milling beads used in ball milling are one or more of zirconium oxide ball milling beads, aluminum oxide ball milling beads and agate ball milling beads; the diameter of the ball milling beads is 1 to 10 mm, and the mass ratio of the chitin biomass to the ball milling beads is 1:1 to 20.

5. The method for preparing chito-oligosaccharides according to claim 1, characterized in that: The ball milling time is 1 to 10 hours, the grinding speed is 100 to 1000 rpm; the temperature is maintained at 25 to 60° C.; the solvent is water, and the amount of water added is 2 to 10 times the mass of the chitin biomass; the solid-liquid separation method includes centrifugation and filtration; the drying method includes rotary evaporation drying, spray drying, and oven drying.

6. An L molecular sieve, characterized in that Its chemical composition is: xK2O·1.0Al2O3·ySiO2·zH2O, with straight channels; wherein x ranges from 1 to 5; y ranges from 5 to 10; and z ranges from 1 to 5.

7. The L molecular sieve according to claim 6, characterized in that: The straight pores have a pore size of 0.71 nm and a topological structure of an LTL L-type zeolite molecular sieve.

8. A method for preparing the L molecular sieve according to any one of claims 6-7, characterized in that: The following steps are involved: Take a silicon source and a potassium source, mix them and grind them for the first time, then add an aluminum source and grind them for the second time, crystallize at 150-200° C., and calcine at 400-600° C. in an air atmosphere to obtain the product.

9. The method for preparing L molecular sieve according to claim 8, characterized in that: The molar ratio of silicon source: potassium source: aluminum source is 5-20:1-10:0.1-2.0; the first grinding time is 10-120 minutes, and the second grinding time is 5-60 minutes; the crystallization time is 4-24 hours; the calcination time is 1-5 hours; the silicon source is one or a mixture of fumed silica, potassium silicate, and sodium silicate; the potassium source is one or a mixture of potassium hydroxide and potassium bromide; the aluminum source is one or a mixture of aluminum hydroxide, sodium aluminate, and aluminum sulfate.

10. An application of the L molecular sieve according to any one of claims 6 to 9, characterized in that: Catalyst for the preparation of chitosan oligosaccharides.

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