Preparation method of curdlan oligosaccharide

Abstract

The invention discloses a preparation method of curdlan oligosaccharide, which comprises the following steps: mixing curdlan with an L molecular sieve, performing ball milling, adding a solvent for dissolving, performing solid-liquid separation on the L molecular sieve, and drying to obtain the curdlan 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. According to the L molecular sieve with the straight pore channels, glucosidic bonds of curdlan can be selectively sheared to prepare curdlan oligosaccharides; therefore, efficient preparation of curdlan oligosaccharide is realized, and the curdlan oligosaccharide can be repeatedly used; the method does not need a large amount of water or other solvents to participate in the reaction, and has the advantages of environmental friendliness, low cost and the like; compared with existing reports, the method has obvious advantages 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 oligosaccharide is an oligosaccharide formed by glucose connected by β-1,3 glycosidic bonds, and the degree of polymerization is generally 2-10. Curdlan 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, lowering blood sugar and 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, Curdlan (also known as Curdlan, Curdlan gum, English name: Curdlan) is a β-1,3-glucan prepared by microbial fermentation. It has a large molecular weight and is insoluble in water and general solvents. Curdlan oligosaccharides are generally prepared by hydrolyzing Curdlan polysaccharides, mainly including chemical degradation and enzyme degradation. The chemical degradation method 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. The enzyme degradation method is a mild method. The enzymes used include glucanase and glucomananase, but they all have problems such as insufficient enzyme activity and low enzyme yield. Since Curdlan polysaccharides are insoluble in water and most solvents, Curdlan polysaccharides require relatively complex pre-treatment before hydrolysis, long production cycle and high cost.

[0004] The reason why kodlan 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. It is insoluble in water and general solvents and difficult to hydrolyze. Ball milling is a method that can assist polysaccharide molecules to open some hydrogen bonds and reduce crystallinity, but it cannot degrade polysaccharide chains to prepare oligosaccharides. It is mostly used as an auxiliary tool for crops, used in conjunction with other polysaccharide hydrolysis methods, such as ball milling before enzymatic hydrolysis to improve the efficiency of enzyme hydrolysis. Despite this, the efficiency of hydrolysis of kodlan to prepare kodlan oligosaccharides still needs to be improved and currently cannot meet production needs. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing Curdlan oligosaccharide, so as to solve the technical problem that the efficiency of preparing Curdlan oligosaccharide by hydrolysis of Curdlan is low 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, grinding by ball milling, adding solvent to dissolve, separating the L molecular sieve from the solid and liquid, and drying to obtain Curdlan oligosaccharide.

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

[0008] Preferably, 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; and the mass ratio of the curdlan 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 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.

[0010] 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.

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

[0012] 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.

[0013] 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.

[0014] An application of L molecular sieve, characterized in that it is used as a catalyst for preparing kodlan oligosaccharides.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 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 kohlrabi sugar chain, which can selectively cut the glycosidic bond of kohlrabi to prepare kohlrabi 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 kohlrabi polysaccharides with L molecular sieves, which can efficiently prepare kohlrabi oligosaccharides and can be reused. 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 and low cost; compared with existing reports, the technology described in this patent has obvious advantages and is more conducive to industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The XRD pattern of the L molecular sieve prepared by the present invention;

[0018] Figure 2 The mass spectrum of Curdlan oligosaccharide obtained by ball-milling Curdlan polysaccharide with L molecular sieve prepared in the present invention;

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

[0020] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0021] 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.

[0022] The invention provides a method for preparing Curdlan oligosaccharide, comprising the following steps: mixing Curdlan polysaccharide with L molecular sieve, adding ball milling beads, grinding with a ball mill for a certain time, adding a certain amount of water to dissolve Curdlan oligosaccharide, separating the molecular sieve from solid and liquid, and drying the solution to obtain Curdlan oligosaccharide.

[0023] The mass ratio of Curdlan polysaccharide to molecular sieve is 10:1 to 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 to 10 mm; the mass ratio of Curdlan polysaccharide 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 rpm-1000 rpm; the temperature is maintained at 25°C to 60°C, preferably 30°C to 40°C; the amount of water added is 2-10 times the mass of Curdlan 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 to 10.

[0026] The preparation method of L molecular sieve of the present invention comprises the following steps: taking silicon source and potassium source, grinding them for the first time after mixing, adding aluminum source, grinding them for the second time, crystallizing at 150-200° C., and calcining them directly at 400-600° C. in air atmosphere to obtain L molecular sieve.

[0027] Among them, 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 the L molecular sieve of the present invention is: xK2O·1.0Al2O3·ySiO2·zH2O, with straight channels; wherein x ranges from 1 to 5; y ranges from 5 to 10; z ranges from 1 to 5; the pore size of the straight channels is 0.71 nm, and the topological structure is LTL, 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, 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:

[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.

[0034] Preparation of Curdlan oligosaccharide by ball milling and hydrolyzing Curdlan with L molecular sieve prepared by the present invention

[0035] Add 2.0g of Curdlan, 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 Curdlan oligosaccharide. The yield of Curdlan oligosaccharide is calculated based on the mass of Curdlan added; the product is analyzed by ESI-MS to analyze the degree of polymerization of Curdlan oligosaccharide.

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

[0037]

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

[0039] Add 2.0g of Curdlan polysaccharide, 0.4g of L molecular sieve with Si / Al of 10, 10.0g of zirconium oxide ball milling beads (Ф=5mm), and 0.2g of water into 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 of water, stir at 200rpm for 30min, filter, and rotary evaporate the filtrate to obtain Curdlan oligosaccharide. The yield of Curdlan oligosaccharide is 95.3% calculated based on the mass of Curdlan polysaccharide added; the L molecular sieve obtained by filtration is repeatedly used in the above process four times, and the yields of Curdlan oligosaccharide are 94.7%, 94.3%, 94.1% and 93.6% 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 Curdlan oligosaccharide is 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), the L molecular sieve was prepared by the traditional hydrothermal method. The specific synthesis process is as follows:

[0043] 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°C for 72h. The obtained product is washed with deionized water until neutral and dried, and then calcined at 480°C for 4h in an air atmosphere to obtain the final L molecular sieve.

[0044] The ratio of the reaction materials is as follows:

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

[0046] X-ray diffraction analysis showed that the 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 4 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.

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

[0049]

[0050] It can be seen from the above examples that L molecular sieves with different Si / Al can efficiently hydrolyze Curdlan polysaccharide to prepare Curdlan oligosaccharide under different ball milling parameters, and the degree of polymerization is 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 prepare Curdlan oligosaccharide, but the efficiency is not high, and the degree of polymerization of oligosaccharide is either low or contains monosaccharide, which affects the quality of Curdlan oligosaccharide. The preparation of Curdlan oligosaccharide by ball milling of Curdlan polysaccharide catalyzed by L molecular sieve described in this patent has significant technical advantages.

[0051] 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 kodlan oligosaccharide, characterized in that: The method comprises the following steps: mixing Curdlan polysaccharide 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 Curdlan oligosaccharide.

2. The method for preparing Curdlan oligosaccharide according to claim 1, characterized in that: 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, characterized in that: 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 kodran polysaccharide to the ball milling beads is 1:1 to 1:

20.

4. The method for preparing Curdlan oligosaccharide 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.

5. 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.

6. The L molecular sieve according to claim 5, 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.

7. A method for preparing L molecular sieve, 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.

8. The method for preparing L molecular sieve according to claim 7, 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.

9. An application of L molecular sieve, characterized in that: Catalyst for the preparation of kudlan oligosaccharides.

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