A method for preparing oligosaccharides from biomass

Abstract

The present invention belongs to the field of biomass conversion and specifically relates to a method for preparing oligosaccharides from biomass. The method comprises pre-treating the biomass to obtain a sugar solution; enzymatically hydrolyzing the sugar solution to obtain an enzymatic hydrolyzate; decolorizing the enzymatic hydrolyzate; preparing copper-doped porous silica; grafting hydroxymethyl groups onto the surface of the porous silica to obtain modified silica; and purifying the decolorized enzymatic hydrolyzate using the modified silica to remove acid and xylose. The method can separate small molecular byproducts and impurities such as acid and xylose produced during the hydrolysis process, thereby reducing the cost of preparing and purifying xylooligosaccharides.

Description

Technical Field

[0001] The present invention belongs to the field of biomass conversion, and in particular relates to a method for preparing oligosaccharides from biomass. Background Art

[0002] Xylo-oligosaccharides are functional oligosaccharides composed of xylose molecules linked by β-1,4 glycosidic bonds. They are highly thermally stable and are prebiotic oligosaccharides with benefits such as regulating intestinal ecological balance, preventing cardiovascular disease, combating dental caries, and reducing the risk of colon cancer. Furthermore, xylo-oligosaccharides have a sweetness approximately 40% that of sucrose, resulting in a pure sweetness that is not absorbed by the human body. They can be used as a sugar substitute for individuals with obesity and diabetes. Xylo-oligosaccharides are primarily derived from the degradation and conversion of xylan from lignocellulosic biomass. Boiling the lignocellulosic material in aqueous solution, combined with the hydronium ions produced by water ionization, depolymerizes and deacetylates the xylan, producing xylo-oligosaccharides with a lower degree of polymerization. This method of producing xylo-oligosaccharides is environmentally friendly, but it produces numerous byproducts such as xylose monosaccharides and dehydrated substances. Furthermore, the reaction requires high temperatures, resulting in a darker color and impurities such as soluble lignin. Enzymatic hydrolysis to produce oligoxylose also produces a variety of byproducts, including xylose, enzyme proteins, acetic acid, and pigments. These biomass products themselves contain a high level of impurities, leading to a complex and complex refining process, high production costs, and a consistently high price. Decolorization with activated carbon can remove most color-forming substances, but it remains ineffective against small molecules like acetic acid and xylose. Summary of the Invention

[0003] In order to solve the problem in the prior art that by-products and impurities are difficult to separate during the hydrolysis process, resulting in high costs for the preparation and purification of xylo-oligosaccharides, the present invention mainly provides a method for preparing xylo-oligosaccharides using biomass at low cost, and the technical solution is as follows:

[0004] A method for preparing oligosaccharides from biomass comprises pretreating the biomass to obtain a sugar solution; enzymatically hydrolyzing the sugar solution to obtain an enzymatic hydrolyzate; decolorizing the enzymatic hydrolyzate; preparing copper-doped porous silica; grafting hydroxymethyl groups on the surface of the porous silica to obtain modified silica; and purifying the decolorized enzymatic hydrolyzate using the modified silica to remove acid and xylose therein.

[0005] Furthermore, the mass ratio of the dry weight of the enzymatic hydrolysate to the modified silica is 3-8:1; the modified silica and the decolorized enzymatic hydrolysate are stirred and reacted for 1-2 hours, and then ethanol is added and the reaction is continued at 40-60° C. for 1-2 hours.

[0006] Furthermore, the preparation of the porous silica includes the following steps: tetrabutyl titanate, ethyl orthosilicate and copper nitrate are fully dissolved in water, hexadecyltrimethylammonium bromide is added, and hydrochloric acid is added to the system, and the mixture is fully stirred and mixed, and then crystallized at 100-110° C. for 20-30 hours to obtain crystals; and the crystals are calcined at 500-600° C. for 4-8 hours in a nitrogen atmosphere to obtain the obtained product.

[0007] Furthermore, the mass ratio of the tetrabutyl titanate to the ethyl orthosilicate is 0.1-0.2:1; the mass ratio of the copper nitrate to the ethyl orthosilicate is 0.08-0.2:1; the mass ratio of the hexadecyltrimethylammonium bromide to the ethyl orthosilicate is 0.3-0.6:1; and the mass concentration of the hydrochloric acid in the system is 5-7%.

[0008] Further, the mixture is stirred at 40-60° C. for 18-24 hours.

[0009] Furthermore, the preparation of the modified silica comprises the following steps:

[0010] Expose the porous silica to light for 5-9 hours and then dry it to obtain a precursor;

[0011] Dissolve thionyl chloride in N,N-dimethylformamide to obtain a thionyl chloride solution, fully disperse the precursor in the thionyl chloride solution, and react at 70-80°C for 6-10 hours; evaporate excess thionyl chloride, wash and dry to obtain acyl chloride-modified silica;

[0012] A tetrahydrofuran solution of borane is prepared; acyl chloride-modified silica is dispersed in tetrahydrofuran, and then the tetrahydrofuran solution of borane is added at 0-5° C., followed by stirring and reacting for 10-12 hours; the obtained precipitate is washed with methanol and water respectively, and then dried to obtain modified silica.

[0013] Furthermore, the illumination intensity of the illumination is 50-100 mW / cm 2 .

[0014] Furthermore, the mass ratio of the precursor to thionyl chloride is 1:5-10; the mass ratio of the borane to the acyl chloride-modified silica is 1:1-2.

[0015] Further, the biomass is crushed to obtain crumbs with a D90 of 0.1 to 1 mm; the crumbs are ground at a speed of 150 to 300 rpm for 2 to 4 hours, with rest periods of 3 to 10 minutes after every 3 to 10 minutes, to obtain a powder; the powder is hydrothermally reacted at 165 to 180° C. for 0.5 to 2 hours to obtain a sugar solution; the xylanase is fully dispersed in a weakly acidic buffer solution, and then the sugar solution is added, and enzymatic hydrolysis is carried out at 35 to 40° C. for 6 to 10 hours, and the enzyme is removed to obtain an enzymatic hydrolyzate.

[0016] Furthermore, the mass ratio of the dry weight of the sugar solution to the enzyme is 3-5:1.

[0017] By adopting the above scheme, the method of the present invention has the following advantages:

[0018] 1. The preparation method of the present invention can control the degree of polymerization of the raw materials, reduce the excessive hydrolysis of oligoxylose, and can selectively adsorb and separate small molecular substances such as acetic acid and xylose, thereby improving the purity of the oligoxylose product. The preparation method has low energy consumption and the purification agent can be repeatedly used, which is conducive to wide promotion.

[0019] 2. The present invention utilizes the electrostatic attraction between copper oxide and carboxylic acid to adsorb byproducts represented by acetic acid without changing the adsorption structure, thereby purifying the enzymatic hydrolyzate.

[0020] 3. The present invention prepares porous silica particles doped with titanium and copper, combines doped titanium dioxide and copper oxide, and enables the porous silica to have the ability to photocatalytically generate active free radicals, so that the hydroxyl groups on the surface of the porous silica can be easily oxidized to carboxyl groups without the intervention of external reagents such as strong oxidants, which is environmentally friendly and has low production costs.

[0021] 4. The present invention performs incomplete calcination of the template, so that active carbon and part of the carbon-nitrogen complex remain on the surface of the porous silica, providing more modified matrix for the carboxylation modification of the porous silica and improving the light response ability of the carboxylation of the porous silica.

[0022] 5. The present invention utilizes the structural difference between xylose and oligoxylose. The hydroxymethyl groups grafted on the surface of porous silica, especially in the pores, are similarly soluble in xylose. The interaction between the hydroxymethyl groups and xylose attracts xylose into the pores for adsorption, thereby reducing the xylose content in the enzymatic hydrolyzate.

[0023] 6. The present invention adopts grinding and hydrothermal treatment of biomass to fully dissolve the hemicellulose in the biomass, and by controlling the temperature and time, avoids the large-scale decomposition of hemicellulose into oligosaccharides, thereby controlling the polymerization degree of the enzymatic hydrolysis product and reducing the probability of oligosaccharides caused by wide distribution being excessively hydrolyzed into xylose.

[0024] 7. The present invention combines mechanical grinding with hydrothermal and enzymatic hydrolysis to improve the accessibility of enzymes and oligosaccharide-producing raw materials, improve production efficiency, and reduce the energy consumption of grinding and the temperature of hydrothermal reaction; the biomass is fully refined to generate xylan with a narrow degree of polymerization distribution, which is then hydrolyzed by a highly specific enzyme to reduce the production of by-products and increase the yield of oligoxylose. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0026] Example 1: (1) 1.5 g of tetrabutyl titanate, 10 g of ethyl orthosilicate and 1.5 g of copper nitrate were fully dissolved in water, 5 g of hexadecyltrimethylammonium bromide was added, and hydrochloric acid was added to the system so that the mass concentration of hydrochloric acid in the system was 6%; the mixture was stirred at 50° C. for 20 h, and then crystallized at 100-110° C. for 24 h to obtain crystals; the crystals were calcined at 550° C. for 6 h in a nitrogen atmosphere to obtain the product;

[0027] (2) The porous silica was heated at 80 mW / cm 2 The precursor was reacted under light of irradiation intensity of for 7 hours, and dried to obtain a precursor; 26 mL of thionyl chloride was dissolved in 10 mL of N, N-dimethylformamide to obtain a thionyl chloride solution, 3 g of the precursor was placed in the thionyl chloride solution and fully dispersed, and reacted at 75°C for 8 hours; excess thionyl chloride was evaporated, and after washing and drying, acyl chloride-modified silica was obtained;

[0028] (3) Prepare a tetrahydrofuran solution of borane by taking 2 g of borane; disperse 2.5 g of acyl chloride-modified silica in tetrahydrofuran, then add the tetrahydrofuran solution of borane at 0-5 °C, and then stir and react for 10-12 h; wash the obtained precipitate with methanol and water respectively, and then dry to obtain modified silica;

[0029] (4) The corn stalks were crushed to obtain powder with a D90 of 0.5-0.8 mm; the powder was ground at 200 rpm for 3 h, with a 5-min grinding interval and a 5-min grinding interval, to obtain a powder; the powder was hydrothermally reacted at 175 °C for 1.5 h to obtain a sugar solution; the xylanase was fully dispersed in a weakly acidic buffer solution, and then the sugar solution was added, with the dry weight of the sugar solution and the enzyme weight ratio being controlled to be 4:1; the enzyme was hydrolyzed at 38 °C for 8 h, and the enzyme solution was obtained after the enzyme was removed;

[0030] (5) After the high molecular weight substances are removed by membrane separation, the enzymatic hydrolysate is decolorized using a resin; then the modified silica is placed in the enzymatic hydrolysate, and the mass ratio of the modified silica to the dry weight of the enzymatic hydrolysate is controlled to be 1:6. The mixture is mixed and reacted at a speed of 150 r / min for 1.5 h. Ethanol 3 times the mass of the enzymatic hydrolysate is added, and the mixture is refluxed at 50°C for 1.5 h to remove the acid and xylose therein to obtain an oligosaccharide solution. The solution is filtered, and the upper filtrate is evaporated to remove the solvent to obtain oligosaccharides.

[0031] Example 2: The difference from Example 1 is that:

[0032] (1) Dissolve 1.5 g of tetrabutyl titanate, 10 g of ethyl orthosilicate, and 0.8 g of copper nitrate in water, add 5 g of hexadecyltrimethylammonium bromide, and add hydrochloric acid to the system so that the mass concentration of hydrochloric acid in the system is 6%; stir and mix at 50°C for 20 h, then crystallize at 105°C for 24 h to obtain crystals; calcine the crystals at 550°C in a nitrogen atmosphere for 6 h to obtain.

[0033] Example 3: The difference from Example 1 is that:

[0034] (1) Dissolve 1.5 g of tetrabutyl titanate, 10 g of ethyl orthosilicate, and 2 g of copper nitrate in water, add 5 g of hexadecyltrimethylammonium bromide, and add hydrochloric acid to the system so that the mass concentration of hydrochloric acid in the system is 6%; stir and mix at 50°C for 20 h, then crystallize at 105°C for 24 h to obtain crystals; calcine the crystals at 550°C in a nitrogen atmosphere for 6 h to obtain.

[0035] Example 4: The difference from Example 1 is that:

[0036] (1) Dissolve 1.5 g of tetrabutyl titanate, 10 g of ethyl orthosilicate and 1.5 g of copper nitrate in water, add 3 g of hexadecyltrimethylammonium bromide, and add hydrochloric acid to the system so that the mass concentration of hydrochloric acid in the system is 6%; stir and mix at 50°C for 20 h, then crystallize at 100-110°C for 24 h to obtain crystals; calcine the crystals at 550°C in a nitrogen atmosphere for 6 h to obtain.

[0037] Example 5: The difference from Example 1 is that:

[0038] (2) The porous silica was heated at 80 mW / cm 2 The precursor was obtained by light illumination for 5 hours at a light intensity of , and then dried. 26 mL of thionyl chloride was dissolved in 10 mL of N, N-dimethylformamide to obtain a thionyl chloride solution. 3 g of the precursor was placed in the thionyl chloride solution and fully dispersed. The product was reacted at 75° C. for 8 hours. Excess thionyl chloride was evaporated, and the product was washed and dried to obtain acyl chloride-modified silica.

[0039] Example 6: The difference from Example 1 is that:

[0040] (2) The porous silica was heated at 80 mW / cm 2 The precursor was obtained by light illumination for 9 hours at a light intensity of , and then dried; 26 mL of thionyl chloride was dissolved in 10 mL of N, N-dimethylformamide to obtain a thionyl chloride solution, 3 g of the precursor was placed in the thionyl chloride solution and fully dispersed, and the reaction was carried out at 75° C. for 8 hours; excess thionyl chloride was evaporated, and the product was washed and dried to obtain acyl chloride-modified silica.

[0041] Example 7: The difference from Example 1 is that:

[0042] (4) The corn stalks were crushed to obtain crumbs with a D90 of 0.5-0.8 mm; the crumbs were ground at 200 rpm for 3 h, with a 5-min grinding interval and a 5-min rest period, to obtain a powder; the powder was hydrothermally reacted at 180 °C for 1.5 h to obtain a sugar solution; the xylanase was fully dispersed in a weakly acidic buffer solution, and then the sugar solution was added to control the mass ratio of the sugar solution to the enzyme to be 4:1; the enzyme was hydrolyzed at 38 °C for 8 h, and the enzyme solution was obtained after the enzyme was removed.

[0043] Example 8: The difference from Example 1 is that:

[0044] (4) The corn stalks were crushed to obtain crumbs with a D90 of 0.5-0.8 mm. The crumbs were ground at 200 rpm for 3 h, with a 5-min grinding interval and a 5-min rest period, to obtain a powder. The powder was hydrothermally reacted at 175 °C for 2 h to obtain a sugar solution. The xylanase was fully dispersed in a weakly acidic buffer solution, and then the sugar solution was added to control the mass ratio of the sugar solution to the enzyme to be 4:1. The enzyme was hydrolyzed at 38 °C for 8 h, and the enzyme solution was obtained after the enzyme was removed.

[0045] Comparative Example 1: The difference from Example 1 is:

[0046] (5) After membrane separation to remove high molecular weight substances, the enzymatic hydrolysate is decolorized using resin to obtain oligosaccharides.

[0047] Comparative Example 2: The difference from Example 1 is:

[0048] (5) After the high molecular weight substances are removed by membrane separation, the enzymatic hydrolysate is decolorized using a resin; then the modified silica is placed in the enzymatic hydrolysate, and the mass ratio of the modified silica to the dry weight of the enzymatic hydrolysate is controlled to be 1:6. The mixture is mixed and reacted at a speed of 150 r / min for 1.5 h. Ethanol 0.5 times the mass of the enzymatic hydrolysate is added, and the mixture is refluxed at 50°C for 1.5 h to remove the acid and xylose therein to obtain an oligosaccharide solution. The solution is filtered, and the upper filtrate is evaporated to remove the solvent to obtain oligosaccharides.

[0049] Example sample test:

[0050] The contents of xylobiose, xylotriose, xylotetraose, xylose, and acetic acid in the oligosaccharides were determined by high performance liquid chromatography. The yields of oligosaccharides (xylobiose, xylotriose, and xylotetraose) and xylose were calculated based on the solid content of the enzymatically hydrolyzed sugar solution. The results are as follows:

[0051]

[0052] Comparative Example 1 does not adopt modified silica of the present invention to purify oligosaccharides. In the product obtained, the xylose yield is high and the acetic acid content is also high, indicating that the modified silica of the present invention can significantly reduce the xylose and acetic acid in the product and improve the purity of oligosaccharides. The copper content added in Example 2 and Example 3 is different. The acetic acid content of Example 3 decreases significantly, indicating that copper oxide is beneficial to the adsorption separation of acetic acid in porous silica. However, the acetic acid content in Example 3 is not significantly changed compared with Example 1, and the xylose yield 1 increases slightly, indicating that the adsorption capacity is close to the upper limit, and further increasing the copper oxide content will affect the amount of sites generating hydroxymethyl. The template added in Example 4 is less, and the xylose yield and acetic acid content both increase, wherein the xylose yield increases significantly, indicating that the content of the template has a great influence on the adsorption performance of porous silica, which not only affects the adsorption pore size, but also affects the removal of xylose and acetic acid, and also affects the amount of sites generating hydroxymethyl.

[0053] When carrying out silica modification, the light application time of Example 5 is less than that of Example 6, and the xylose yield of Example 5 is significantly higher than that of Example 6, while the oligosaccharide yield decreases, indicating that a longer light application time can increase the generation rate of hydroxymethyl, thereby improving the adsorption separation performance of xylose. Comparing Example 6 and Example 1, the xylose content of Example 6 still increases to some extent, indicating that a long light application time is also not conducive to increasing the generation rate of hydroxymethyl, and a long time may cause the occurrence of more side reactions. When carrying out purification treatment, the hydrothermal reaction temperature of Example 7 is higher, and the hydrothermal time of Example 8 is longer, and the xylose yields of the two examples increase, indicating that a higher temperature and a longer time are easy to cause the obvious hydrolysis of xylan in the hydrothermal stage, affecting the yield of xylooligosaccharides.

[0054] During purification, the product was first treated in an aqueous solvent environment, which facilitated the adsorption of acetic acid. Ethanol was then added, leveraging the affinity of hydroxymethyl groups for ethanol and the difference in solubility between xylo-oligosaccharides and xylose in ethanol to cause more xylose to accumulate within the modified silica and be adsorbed by the modified silica. The more ethanol, the more pronounced the adsorption effect, resulting in a significant increase in the xylose yield in Comparative Example 2, where too little ethanol was added.

[0055] Those skilled in the art can make various other corresponding changes and deformations based on the technical solutions and concepts described above, and all of these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing oligosaccharides from biomass, characterized in that: The biomass is pretreated to obtain a sugar solution; the sugar solution is enzymatically hydrolyzed to obtain an enzymatic hydrolyzate; the enzymatic hydrolyzate is decolorized; a copper-doped porous silica is prepared; a hydroxymethyl group is grafted onto the surface of the porous silica to obtain a modified silica; and the decolorized enzymatic hydrolyzate is purified using the modified silica to remove acid and xylose therefrom. The preparation of the modified silicon dioxide comprises the following steps: Tetrabutyl titanate, ethyl orthosilicate, and copper nitrate are fully dissolved in water, cetyltrimethylammonium bromide is added, and hydrochloric acid is added to the system, and the mixture is fully stirred and mixed, followed by crystallization at 100-110°C for 20-30 hours to obtain crystals; the crystals are calcined at 500-600°C in a nitrogen atmosphere for 4-8 hours to obtain porous silica; the porous silica is subjected to light-induced reaction for 5-9 hours, and then dried to obtain a precursor; Dissolve thionyl chloride in N,N-dimethylformamide to obtain a thionyl chloride solution, fully disperse the precursor in the thionyl chloride solution, and react at 70-80°C for 6-10 hours; evaporate excess thionyl chloride, wash and dry to obtain acyl chloride-modified silica; A tetrahydrofuran solution of borane is prepared; acyl chloride-modified silica is dispersed in tetrahydrofuran, and then the tetrahydrofuran solution of borane is added at 0-5° C., followed by stirring and reacting for 10-12 hours; the obtained precipitate is washed with methanol and water respectively, and then dried to obtain modified silica.

2. The method for preparing oligosaccharides from biomass according to claim 1, wherein The mass ratio of the dry weight of the enzymatic hydrolysate to the modified silicon dioxide is 3-8:1; the modified silicon dioxide and the decolorized enzymatic hydrolysate are stirred and reacted for 1-2 hours, and then ethanol is added and the reaction is continued at 40-60° C. for 1-2 hours.

3. The method for preparing oligosaccharides from biomass according to claim 1, wherein The mass ratio of the tetrabutyl titanate to the ethyl orthosilicate is 0.1-0.2:1; the mass ratio of the copper nitrate to the ethyl orthosilicate is 0.08-0.2:1; the mass ratio of the hexadecyltrimethylammonium bromide to the ethyl orthosilicate is 0.3-0.6:1; and the mass concentration of the hydrochloric acid in the system is 5-7%.

4. The method for preparing oligosaccharides from biomass according to claim 1, wherein Stir and mix at 40-60°C for 18-24 hours.

5. The method for preparing oligosaccharides from biomass according to claim 1, wherein The illumination intensity of the illumination is 50-100 mW / cm 2 .

6. The method for preparing oligosaccharides from biomass according to claim 1, wherein The mass ratio of the precursor to thionyl chloride is 1:5-10; the mass ratio of the borane to acyl chloride-modified silica is 1:1-2.

7. The method for preparing oligosaccharides from biomass according to claim 1, wherein The biomass is crushed to obtain crumbs with a D90 of 0.1 to 1 mm; the crumbs are ground at a speed of 150 to 300 rpm for 2 to 4 hours with a rest period of 3 to 10 minutes after every 3 to 10 minutes to obtain a powder; the powder is hydrothermally reacted at 165 to 180° C. for 0.5 to 2 hours to obtain a sugar solution; xylanase is fully dispersed in a weakly acidic buffer solution, and then the sugar solution is added, and enzymatic hydrolysis is carried out at 35 to 40° C. for 6 to 10 hours. After removing the enzyme, an enzymatic hydrolyzate is obtained.

8. The method for preparing oligosaccharides from biomass according to claim 7, characterized in that The mass ratio of the dry weight of the sugar solution to the enzyme is 3-5:1.

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