A method for preparing chitooligosaccharides

Abstract

The application discloses a preparation method of chitooligosaccharide, which comprises the following steps: mixing chitin biomass with L molecular sieve, ball-milling and grinding, adding a solvent to dissolve, separating the L molecular sieve from the solid-liquid, and drying to obtain the chitooligosaccharide; the L molecular sieve has a chemical composition of 1-5K2O*1.0Al2O3*5-10SiO2*1-5H2O and has straight pore channels; the L molecular sieve with the straight pore channels can selectively cut the glycosidic bond of chitin to prepare the chitooligosaccharide, does not need a large amount of water or other solvents to participate in the reaction, has the advantages of environmental friendliness, low cost, easy separation of the product, higher catalytic efficiency, reusability and better industrialization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomass conversion and utilization, and particularly relates to a method for preparing chitooligosaccharide from chitin biomass or chitin. BACKGROUND

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

[0003] At present, chitooligosaccharide is generally prepared by hydrolysis of chitin, mainly including chemical degradation method and enzymatic degradation method. The chemical degradation method generally uses concentrated acid hydrolysis, although the degradation efficiency is high, but it has the disadvantages of harsh reaction conditions, difficult to control, low product quality, high equipment requirements and environmental pollution, and the product is mostly below tetrasaccharide; the enzymatic degradation method is a mild method, and the enzymes used include chitinase and chitosanase, etc. Chinese patent (application number: 2011102588936) discloses a chitin-degrading strain and a method for preparing chitooligosaccharide therefrom. The method first converts chitin into colloidal chitin, then cultures the strain to produce enzymes, and then uses the chitinase produced by the strain to enzymatically degrade the prepared colloidal chitin. Chinese patent (application number: 200610080091X) discloses a method for preparing chitooligosaccharide, which uses fermentation to produce enzymes to degrade chitin colloids to prepare bioactive chitooligosaccharide. However, both of them have the problems of low enzyme activity and low oligosaccharide yield. Since chitin is insoluble in water and most solvents, chitin needs to be pretreated and the process is relatively complex, 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 the sugar chains, resulting in high crystallinity and structural compactness. In addition, the chitin in chitin biomass is also embedded with calcium carbonate, protein and the like, which affects the contact of macromolecules such as enzymes, resulting in low hydrolysis efficiency. Therefore, the efficiency of preparing chitooligosaccharide from chitin by hydrolysis still needs to be improved, and it cannot meet the production demand at present. SUMMARY

[0005] The present application aims to provide a method for preparing chitooligosaccharide, in order to solve the problem of low efficiency of preparing chitooligosaccharide from chitin by hydrolysis 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, ball milling, adding solvent to dissolve, then solid-liquid separation of the L molecular sieve, and drying to obtain chitosan oligosaccharides.

[0007] Preferably, the chitosan biomass includes 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 present invention provides an L molecular sieve having the chemical composition of xK2O·1.0Al2O3·ySiO2·zH2O and 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 pores have a pore size of 0.71 and a topological structure of LTL L-type zeolite molecular sieve.

[0013] The present invention provides a preparation method of L molecular sieve, comprising the following steps: taking a silicon source and a potassium source, mixing them and performing a first grinding, then adding an aluminum source, performing a second grinding, crystallizing at 150-200° C., and directly calcining at 400-600° C. in an air atmosphere to obtain the 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; and 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 chitosan 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 pore size just matches the size of the chitin sugar chain, which can selectively cleave the glycosidic bonds of chitin to prepare chitosan oligosaccharides; based on the solid structure characteristics of the molecular sieve and 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 being environmentally friendly, low cost, and easy to separate products; on the other hand, the present invention uses native chitin biomass directly as raw material, and utilizes the chimeric structure of chitin and calcium carbonate in the native chitin biomass to more efficiently destroy hydrogen bonds and quickly hydrolyze. The solid molecular sieve catalyst in the chitosan oligosaccharide preparation method of this patent has higher catalytic efficiency and is reusable. 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 in 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 chitosan oligosaccharides obtained by ball-milling chitin with L molecular sieve in the present invention. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

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

[0023] The invention provides a method for preparing chitosan oligosaccharides, 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 oligosaccharides, separating the molecular sieves through solid-liquid separation, and drying the solution to obtain the chitosan oligosaccharides.

[0024] 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 to 1:1; and the mass ratio of chitin to molecular sieve is 10:1 to 1:1.

[0025] The ball milling beads are zirconia ball milling beads, alumina 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 the 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 oven drying.

[0026] The chitin degree of chitosan oligosaccharide is 2 to 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; and the aluminum source is one or a mixture of aluminum hydroxide, sodium metaaluminate, and aluminum sulfate.

[0028] The chemical composition of the 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. The 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 the solvent-free method of the present invention

[0030] Solvent-free synthesis of L molecular sieve: Weigh 3.60g of solid raw materials, fumed silica, and 3.366g of potassium hydroxide, 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 and crystallize at 170°C for 24 hours. The obtained product does not need to be filtered or washed, and is directly calcined at 550°C in an air atmosphere for 4 hours to obtain the final L molecular sieve. The ratio of the reaction raw materials is as follows:

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

[0032] X-ray diffraction analysis showed that its 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 sieve with Si / Al=2.5, L molecular sieve with Si / Al=15, and L molecular sieve 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, and stir at room temperature for 10 minutes to form a slightly turbid solution, which is liquid A; weigh 6.875g HS-40 silica sol, pour it into a beaker filled with 9.958g deionized water, and stir at room temperature for 5 minutes to form liquid B; liquid B is added dropwise to liquid A and stirred thoroughly for 10 minutes to obtain liquid C, which is transferred to a polytetrafluoroethylene stainless steel reactor and crystallized at 180°C for 72 hours. The obtained product is washed with deionized water until neutral and dried, and then calcined at 480°C in an air atmosphere for 4 hours to obtain the final L molecular sieve.

[0037] The proportions of the reaction materials are as follows:

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

[0039] X-ray diffraction analysis showed that its 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, the L molecular sieve synthesized by solvent-free method in the present invention contains 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 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 of potassium silicate and 1.19g of potassium bromide as solid raw materials, pour them into a mortar and grind for 15 minutes, then add 0.246g of sodium aluminate and grind for another 10 minutes. Add the reaction raw materials into a polytetrafluoroethylene stainless steel reactor and crystallize at 170°C for 24 hours. The obtained product does not need to be filtered or washed, and is directly calcined at 550°C in an air atmosphere for 4 hours 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 metaaluminate, 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.

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

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

[0052] In the milling jar of a high-energy planetary ball mill F-P2000, 2.0 g of chitin, different masses of solid catalysts (L molecular sieve with different Si / Al ratios, activated carbon, oxidized activated carbon, ZSM-5 molecular sieve with Si / Al = 10, Hβ molecular sieve with Si / Al = 10, and nanosilica), 10.0 g of zirconia ball milling beads (Ø = 5 mm), and 0.2 g of water were added. The mixture was milled at 500 rpm and 1000 rpm for different times. The milling chamber temperature was controlled at 40°C. After milling, 50.0 mL of water was added, and the mixture was stirred at 200 rpm for 30 minutes. The mixture was filtered, and the filtrate was rotary evaporated to dryness to obtain chitosan oligosaccharides. The chitosan oligosaccharide yield was calculated based on the mass of chitin added. The product was analyzed by electrospray ionization (ESI-MS) to determine the degree of polymerization of the 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 hydrolyzing crab shells using L molecular sieve ball milling:

[0057] In the milling jar of a high-energy planetary ball mill F-P2000, 8.0 g of dried crab shells (containing 25.3% chitin by weight), different amounts of solid catalysts (L molecular sieve with varying Si / Al ratios, activated carbon, oxidized activated carbon, ZSM-5 molecular sieve with Si / Al = 10, Hβ molecular sieve with Si / Al = 10, and nanosilica), 10.0 g of zirconia ball milling beads (Ø = 5 mm), and 0.2 g of water were added. The mixture was milled at 500 rpm and 1000 rpm for different times. The milling chamber temperature was controlled at 40°C. After milling, 50.0 mL of water was added, the mixture was stirred at 200 rpm for 30 minutes, filtered, and the filtrate was rotary evaporated to dryness to obtain chitosan oligosaccharides. The chitosan oligosaccharide yield was calculated based on the amount of chitin added. The product was analyzed by electrochemical spectroscopy (ESI-MS) to determine the degree of polymerization of the 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 sieves

[0062] In the ball mill tank of high-energy planetary ball mill F-P2000, dry crayfish shell 8.0 g (chitin mass percentage content is 22.5%) was added, different mass of solid catalyst (L zeolite with different Si / Al, activated carbon, oxidized activated carbon, ZSM-5 zeolite with Si / Al = 10, Hβ zeolite with Si / Al = 10, nano silicon dioxide), zirconium oxide ball milling beads (Ф = 5 mm) 10.0 g, 0.2 g of water, and ball milling at 500 rpm and 1000 rpm for different time; the ball milling chamber temperature was controlled at 40℃. After 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 chitooligosaccharide. The yield of chitooligosaccharide was calculated based on the mass of chitin added; the product was analyzed by ESI-MS to analyze the degree of polymerization of chitooligosaccharide.

[0063] Table 4. Preparation of chitooligosaccharide by ball milling hydrolysis of crayfish shell with different solid catalysts

[0064]

[0065]

[0066] Example 7: Life experiment of L zeolite ball milling hydrolysis of chitin to prepare chitooligosaccharide

[0067] In the ball mill tank of high-energy planetary ball mill F-P2000, chitin 2.0 g, 0.4 g of L zeolite with Si / Al = 10, zirconium oxide ball milling beads (Ф = 5 mm) 10.0 g, 0.2 g of water were added, and ball milling at 500 rpm for 2 h; the ball milling chamber temperature was controlled at 40℃. After 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 chitooligosaccharide. The yield of chitooligosaccharide was 90.3% based on the mass of chitin added; the L zeolite obtained by filtration was repeatedly used in the above process four times, and the yield of chitooligosaccharide was 90.5%, 90.1%, 90.1% and 89.5% respectively, indicating that the catalyst activity did not decrease obviously and could be reused. The product was analyzed by ESI-MS, and the degree of polymerization of chitooligosaccharide was 2-7.

[0068] As can be seen from the above examples, L zeolite with different Si / Al can efficiently hydrolyze chitin and chitin biomass to prepare chitooligosaccharide under different ball milling parameters, and the degree of polymerization is 2-7; other solid acid catalysts, including ZSM-5 zeolite, Hβ zeolite, activated carbon, oxidized activated carbon and nano silicon dioxide, can also catalyze the hydrolysis of chitin to prepare chitooligosaccharide, but the efficiency is not high, and the degree of polymerization of oligosaccharide is either too low (less than 3) or contains monosaccharide, affecting the quality of chitooligosaccharide. The ball milling catalysis of L zeolite described in this patent for preparing chitooligosaccharide from chitin and chitin biomass has significant technical advantages.

[0069] For any skilled person familiar in the art, many possible variations and modifications, or equivalent embodiments of equivalent variations, can be made to the technical solutions of the present application by using the technical contents disclosed above without departing from the scope of the technical solutions of the present application. Therefore, any simple modification, equivalent variation and modification made to the above embodiments according to the technical essence of the present application without departing from the content of the technical solutions of the present application should still belong to the scope of protection of the technical solutions of the present application.

Claims

1. A method for preparing chitosan oligosaccharide, characterized in that: The following steps are involved: Chitosan biomass is mixed with L molecular sieve, ball-milled, and then dissolved in a solvent, followed by solid-liquid separation of the L molecular sieve, and dried to obtain chitosan oligosaccharides. The chemical composition of L molecular sieve is: xK2O·1.0Al2O3·ySiO2·zH2O, with straight pores; where x ranges from 1 to 5; y ranges from 5 to 10; and z ranges from 1 to 5. The preparation method of L molecular sieve is as follows: take silicon source and potassium source, mix them and grind them for the first time, then add aluminum source and grind them for the second time, crystallize them at 150~200℃, and calcine them at 400~600℃ in air atmosphere to obtain it.

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

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

1.

4. The method for preparing chitosan oligosaccharides according to claim 1, wherein: 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 1-10 mm, and the mass ratio of the chitin biomass to the ball milling beads is 1:1-20.

5. The method for preparing chitosan oligosaccharides according to claim 1, wherein 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. The method for preparing chitosan oligosaccharides according to claim 1, wherein: The straight pores have a pore size of 0.71 nm and a topological structure of an LTL L-type zeolite molecular sieve.

7. The method for preparing chitosan oligosaccharides according to claim 1, wherein 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; and the aluminum source is one or a mixture of aluminum hydroxide, sodium aluminate, and aluminum sulfate.

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