A method for preparing a cello-oligosaccharide

Abstract

The application discloses a preparation method of codlane oligosaccharide, and comprises the following steps: mixing codlane polysaccharide 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 codlane oligosaccharide; the L molecular sieve has a chemical composition of 1-5K2O*1.0Al2O3*5-10SiO2*1-5H2O and has straight pores; the L molecular sieve with the straight pores can selectively cut the glycosidic bond of the codlane to prepare the codlane oligosaccharide; thus, the codlane oligosaccharide can be efficiently prepared, and the L molecular sieve can be repeatedly used; the method does not need a large amount of water or other solvents to participate in the reaction, has the advantages of environmental friendliness and low cost, and has obvious advantages compared with the prior art, and is more beneficial to industrialization.

Description

Technical Field

[0001] The invention belongs to the technical field of biomass conversion and utilization, and particularly relates to a method for preparing curdlan oligosaccharides from curdlan polysaccharides. Background Art

[0002] Curdlan oligosaccharides are oligosaccharides composed of glucose linked by β-1,3-glycosidic bonds, with a degree of polymerization generally ranging from 2 to 10. Curdlan oligosaccharides exhibit a variety of biological activities, including inducing plant disease resistance, promoting plant growth, and combating stress. They also have physiological activities and functions such as enhancing immunity, lowering blood sugar and lipids, and regulating the balance of beneficial intestinal microorganisms. They are widely used in biopesticides, biofertilizers, health foods, and food additives.

[0003] Currently, Curdlan (also known as Curdlan, Curdlan gum, English name: Curdlan) is a β-1,3-glucan produced by microbial fermentation. It has a large molecular weight and is insoluble in water and common solvents. Curdlan oligosaccharides are generally prepared by hydrolyzing Curdlan polysaccharides, mainly including chemical degradation and enzymatic degradation methods. Chemical degradation generally uses concentrated acid hydrolysis. Although the degradation efficiency is high, it has disadvantages such as harsh reaction conditions, difficulty in control, low product quality, high equipment requirements, and environmental pollution. Enzymatic degradation is a mild method that uses enzymes such as glucanase and glucomananase, but both methods suffer from problems such as insufficient enzyme activity and low enzyme yield. Since Curdlan polysaccharide is insoluble in water and most solvents, Curdlan polysaccharide hydrolysis requires relatively complex pre-treatment, long production cycle, and high cost.

[0004] The reason why kodlan is difficult to degrade is that its sugar chains form a large number of hydrogen bonds, resulting in high crystallinity and a dense structure. It is insoluble in water and common solvents and difficult to hydrolyze. Ball milling is a method that can help polysaccharide molecules break some hydrogen bonds and reduce crystallinity, but it cannot degrade polysaccharide chains to produce oligosaccharides. It is often used as an auxiliary tool in conjunction with other polysaccharide hydrolysis methods. For example, ball milling is used before enzymatic hydrolysis to improve the efficiency of enzymatic hydrolysis. Despite this, the efficiency of kodlan hydrolysis to produce kodlan oligosaccharides still needs to be improved and currently cannot meet production needs. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing Curdlan oligosaccharide to solve the technical problem of low efficiency in preparing Curdlan oligosaccharide by hydrolysis of Curdlan in the prior art.

[0006] To achieve the above object, the present invention provides a method for preparing Curdlan oligosaccharide, comprising the following steps: mixing Curdlan polysaccharide with L molecular sieve, ball milling, adding solvent to dissolve, then solid-liquid separation of L molecular sieve, and drying to obtain Curdlan oligosaccharide.

[0007] Preferably, the mass ratio of the codlane polysaccharide to the L molecular sieve is 10:1 to 1:1.

[0008] Preferably, the ball milling beads used in the ball milling include 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 codlane polysaccharide to the ball milling beads is 1:1 to 1:20.

[0009] Preferably, the ball milling time is 1 to 10 hours, the grinding rotation speed is 100 to 1000 rpm, the temperature is kept at 25 to 60 DEG C, the solvent is water, the amount of water added is 2 to 10 times the mass of the codlane polysaccharide, the solid-liquid separation method includes centrifugation and filtration, and the drying method includes rotary evaporation drying, spray drying, and oven drying.

[0010] The application provides an L molecular sieve, which has a straight pore and a chemical composition of 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.

[0011] Preferably, the pore size of the straight pore is 0.71 nm, and the L-type zeolite molecular sieve has an LTL topology structure.

[0012] The application provides a preparation method of the L molecular sieve, which comprises the following steps: taking a silicon source and a potassium source, mixing them, performing first grinding, adding an aluminum source, performing second grinding, crystallizing at 150 to 200 DEG C, and calcining directly at 400 to 600 DEG C in an air atmosphere to obtain the L molecular sieve.

[0013] Preferably, the molar ratio of the silicon source to the potassium source to the aluminum source is 5 to 20:1 to 10:0.1 to 2.0, the first grinding time is 10 to 120 minutes, the second grinding time is 5 to 60 minutes, the crystallization time is 4 to 24 hours, the calcination time is 1 to 5 hours, the silicon source is one or a mixture of several of fumed silicon dioxide, potassium silicate, and sodium silicate, the potassium source is one or a mixture of several of potassium hydroxide and potassium bromide, and the aluminum source is one or a mixture of several of aluminum hydroxide, sodium aluminate, and aluminum sulfate.

[0014] The application also provides an application of the L molecular sieve, which is used as a catalyst for preparing codlane oligosaccharide.

[0015] Compared with the prior art, the application has the following beneficial effects: 1.

[0016] Based on a large number of experiments, the present patent develops an L molecular sieve with a straight pore, and the pore size is just matched with the size of codlane sugar chain, which can selectively cut the glycosidic bond of codlane to prepare codlane oligosaccharide; based on the solid structure characteristics of the molecular sieve, combined with the ball milling method, the present patent develops a technology of ball milling codlane polysaccharide with L molecular sieve as a catalyst, which can efficiently prepare codlane oligosaccharide and can be reused. On the one hand, the present technical scheme does not need a large amount of water or other solvents to participate in the reaction, and has the advantages of environmental friendliness and low cost; compared with the existing reports, the technology described in the present patent has obvious advantages and is more conducive to industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 XRD pattern of the L molecular sieve prepared in the present application;

[0018] Figure 2 Mass spectrum of codlane oligosaccharide obtained by ball milling codlane polysaccharide with the L molecular sieve prepared in the present application;

[0019] Figure 3 XRD pattern of the L molecular sieve prepared by the hydrothermal method in the prior art. DETAILED DESCRIPTION

[0020] The present application will be further described below in conjunction with specific examples, but the present application is not limited in any way by the examples.

[0021] In the following examples, the instruments, reagents, materials and the like involved, if not specifically stated, are conventional instruments, reagents, materials and the like in the prior art, which can be obtained through regular commercial channels. In the following examples, the experimental methods, detection methods and the like involved, if not specifically stated, are conventional experimental methods, detection methods and the like in the prior art.

[0022] The present application provides a preparation method of codlane oligosaccharide, which comprises the following steps: mixing codlane polysaccharide with L molecular sieve, adding ball milling beads, grinding with a ball mill for a certain time, dissolving codlane oligosaccharide by adding a certain amount of water, separating the molecular sieve after solid-liquid separation, and drying the solution to obtain codlane oligosaccharide.

[0023] In the present application, the mass ratio of codlane polysaccharide to molecular sieve is 10:1-1:1; 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 1-10 millimeters; the mass ratio of codlane polysaccharide to ball milling beads is 1:1-1:20; the grinding time of the ball mill is 1-10 hours, preferably 1-4 hours; the grinding speed is 100 rpm-1000 rpm; the temperature is kept at 25℃-60℃, preferably 30℃-40℃; and the amount of water added is 2-10 times the mass of codlane polysaccharide.

[0024] The solid-liquid separation method includes centrifugation at 5000 rpm and filtration; the drying method includes rotary evaporation drying, spray drying, and oven drying.

[0025] The degree of polymerization of Curdlan oligosaccharide is 2~10.

[0026] The preparation method of the L molecular sieve of the present invention comprises the following steps: taking a silicon source and a potassium source, mixing them and performing a first grinding, then adding an aluminum source and 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.

[0027] 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.

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

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

[0030] 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 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.

[0034] The L molecular sieve prepared by the present invention is ball-milled and hydrolyzed to prepare the oligomeric

[0035] In the milling jar of a high-energy planetary ball mill F-P2000, 2.0 g of Curdlan, different masses of solid catalysts (L molecular sieves 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 nano-silica), 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 Curdlan oligosaccharides. The yield of Curdlan oligosaccharides was calculated based on the mass of Curdlan added. The product was analyzed by ESI-MS to determine the degree of polymerization of Curdlan oligosaccharides.

[0036] Table 1. Preparation of Curdlan oligosaccharides by ball-milling hydrolysis of Curdlan using different solid catalysts

[0037] .

[0038] Lifespan experiment of preparing Curdlan oligosaccharide by ball-milling and hydrolyzing Curdlan polysaccharide with L molecular sieve prepared by the present invention

[0039] In the milling jar of a high-energy planetary ball mill F-P2000, 2.0 g of curdlan polysaccharide, 0.4 g of L molecular sieve with a Si / Al ratio of 10, 10.0 g of zirconia ball milling beads (Ø = 5 mm), and 0.2 g of water were added and milled at 500 rpm for 2 h. 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 min. The mixture was filtered, and the filtrate was rotary evaporated to dryness to obtain curdlan oligosaccharide. The yield of curdlan oligosaccharide, calculated based on the mass of curdlan polysaccharide added, was 95.3%. The L molecular sieve obtained by filtration was reused four times in the same process, with yields of 94.7%, 94.3%, 94.1%, and 93.6%, respectively. This indicates that the catalyst activity did not decrease significantly and the product can be reused. ESI-MS analysis of the product revealed a degree of polymerization (DP) of 2-10.

[0040] Comparative Example:

[0041] Compared with the traditional hydrothermal method for synthesizing L molecular sieve:

[0042] 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:

[0043] Weigh 3.018 g of potassium hydroxide and 1.442 g of aluminum sulfate, pour them into a beaker filled with 18.208 g of deionized water, and stir at room temperature for 10 minutes to form a slightly turbid solution, which is liquid A; weigh 6.875 g of HS-40 silica sol, pour it into a beaker filled with 9.958 g of 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 air atmosphere for 4 hours to obtain the final L molecular sieve.

[0044] The proportions of the reaction materials are as follows:

[0045] 10K2O:1Al2O3:20SiO2:800H2O.

[0046] X-ray diffraction analysis showed that its structure was L zeolite molecular sieve. Figure 3 The XRD pattern synthesized by this method is consistent with the typical XRD peaks of L molecular sieve.

[0047] Table 2 shows the pyridine-infrared analysis characterization results of L molecular sieves synthesized by the solvent-free method and the traditional hydrothermal method. Compared with the L molecular sieve synthesized by the traditional hydrothermal method, the L molecular sieve synthesized by the solvent-free method in the present invention contains a higher Lewis acid / Brønsted acid ratio of 5.961, which is significantly higher than the 0.86 prepared by the traditional hydrothermal method.

[0048] Table 2. Pyridine-IR characterization results of L molecular sieves prepared by traditional hydrothermal method and solvent-free method

[0049] .

[0050] As can be seen from the above examples, L molecular sieves with different Si / Al ratios can efficiently hydrolyze Curdlan polysaccharide to produce Curdlan oligosaccharides under different ball milling parameters, with a degree of polymerization of 2-10; 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 Curdlan to produce Curdlan oligosaccharides, but the efficiency is not high, and the degree of polymerization of the oligosaccharides is either low or contains monosaccharides, which affects the quality of Curdlan oligosaccharides. The L molecular sieve ball milling catalyzed Curdlan polysaccharide to produce Curdlan oligosaccharides described in this patent has significant technical advantages.

[0051] Anyone skilled in the art will be able to utilize the above-disclosed technical content to make many possible changes and modifications to the technical solution of the present invention, or to modify it into equivalent embodiments with equivalent changes, without departing from the scope of the technical solution of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention that do not depart from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing Curdlan oligosaccharide, characterized in that: The method comprises the following steps: mixing Curdlan polysaccharide with L molecular sieve, grinding with ball mill, adding solvent to dissolve, separating the L molecular sieve from the solid and liquid, and drying to obtain Curdlan oligosaccharide; 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 Curdlan oligosaccharide according to claim 1, wherein The mass ratio of the Curdlan polysaccharide to the L molecular sieve is 10:1 to 1:

1.

3. The method for preparing Curdlan oligosaccharide according to claim 1, wherein The ball milling beads used in ball milling include 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; the mass ratio of the curdlan polysaccharide to the ball milling beads is 1:1 to 1:

20.

4. The method for preparing Curdlan oligosaccharide 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 curdlan polysaccharide; the solid-liquid separation method includes centrifugation and filtration; the drying method includes rotary evaporation drying, spray drying, and oven drying.

5. The method for preparing Curdlan oligosaccharide 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.

6. The method for preparing Curdlan oligosaccharide 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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