Method for preparing high-purity xylooligosaccharide through cooperation of low-concentration trifluoroacetic acid and xylanase

Abstract

The invention discloses a method for preparing high-purity xylooligosaccharide from low-concentration trifluoroacetic acid in cooperation with xylanase, and belongs to the technical field of bioactive substance conversion. The method comprises the following steps: crushing and sieving a wood fiber raw material, mixing with trifluoroacetic acid, carrying out a catalytic reaction, carrying out rotary evaporation to recover trifluoroacetic acid, adding xylanase into a residual solid-liquid mixture, carrying out an enzymatic hydrolysis reaction, carrying out solid-liquid separation, and carrying out enzyme inactivation, filtration, decoloration, concentration treatment and spray drying on enzymatic hydrolysate to obtain xylooligosaccharide powder. The used trifluoroacetic acid permeates into the raw materials, hydrolyzes ester bonds and hydrogen bonds between hemicellulose and lignin, part of low-molecular-weight xylan and lignin are dissolved, wrapped cellulose microfibers are exposed, the structure of the raw materials becomes loose and porous, the accessibility is greatly improved, trifluoroacetic acid has high volatility, and the preparation method is suitable for industrial production. The xylooligosaccharide can be recycled through distillation, so that the cost and the product purification difficulty are reduced, and the purity of the xylooligosaccharide product is improved.

Description

Technical Field

[0001] This invention belongs to the field of bioactive substance conversion technology, specifically relating to a method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase. Background Technology

[0002] Xylooligosaccharides (XOS) are functional oligosaccharides with important physiological functions and are widely used in food, beverages, and healthcare. XOS with a degree of polymerization of 2-3, in particular, exhibit significant bifidobacteria proliferation effects, require small dosages, are acid-resistant and heat-resistant, and have broad market prospects. Any biomass raw material with a xylan content of 20% or higher is an ideal raw material for producing XOS, and birch wood, due to its rich xylan content and natural acetyl groups, is a high-quality raw material for XOS production.

[0003] Traditional acid hydrolysis processes, especially those using inorganic acids such as sulfuric acid and hydrochloric acid, typically require high temperatures (usually 120°C to 180°C) and high pressures to achieve sufficient reaction rates and conversion rates. These harsh reaction conditions result in a significant energy burden and lead to serious equipment corrosion and production safety hazards. Simultaneously, high temperatures exacerbate the degradation of monosaccharides, generating numerous byproducts (such as furfural), which not only reduces the yield and purity of the target product, xylooligosaccharides, but also increases the difficulty and cost of subsequent separation and purification.

[0004] In existing xylanase hydrolysis technologies, most processes do not adequately pretreat the lignocellulose raw materials, directly affecting the subsequent enzymatic hydrolysis effect. Patent analysis shows (e.g., CN107488688A) that conventional physical crushing or mild chemical pretreatment is insufficient to fully destroy the dense structure of lignocellulose, resulting in low contact efficiency between xylanase and substrate, thus leading to low yield of xylooligosaccharides. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing high-purity xylooligosaccharides by low concentration trifluoroacetic acid synergistically with xylanase. This method can achieve efficient conversion of xylan in birch raw materials at a lower temperature, improve the yield and quality of xylooligosaccharides, and at the same time reduce energy consumption and by-product generation.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase involves pulverizing and sieving lignocellulose raw materials, mixing them with trifluoroacetic acid, carrying out a catalytic reaction, recovering trifluoroacetic acid by rotary evaporation, adding xylanase to the remaining solid-liquid mixture for enzymatic hydrolysis, separating the solid and liquid, inactivating the enzyme in the hydrolysate, filtering, decolorizing, concentrating, and spray drying to obtain xylooligosaccharide powder.

[0008] Furthermore, the wood fiber raw material is selected from one of birch, corn cob, and sugarcane bagasse.

[0009] Furthermore, the wood fiber raw material is crushed and sieved to a mesh size of 20-80.

[0010] Furthermore, the mass concentration of the trifluoroacetic acid is 0.5%-2%.

[0011] Furthermore, the mass-to-volume ratio of the wood fiber raw material to trifluoroacetic acid is 1:10.

[0012] Furthermore, the catalytic reaction temperature is 80~110℃ and the time is 60~120min.

[0013] Furthermore, the amount of xylanase added is 3 U / mL.

[0014] Furthermore, the enzymatic hydrolysis temperature is 50°C and the time is 8 hours.

[0015] Furthermore, the pH value of the enzymatic hydrolysis is 5.5.

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

[0017] (1) The trifluoroacetic acid used in this invention permeates into the raw material, hydrolyzes the ester bonds and hydrogen bonds between hemicellulose and lignin, dissolves some low molecular weight xylan and lignin, and exposes the encapsulated cellulose microfibers; the raw material structure becomes loose and porous, and its accessibility is greatly improved.

[0018] (2) Compared with the traditional acid hydrolysis method, the reaction temperature of the present invention is significantly reduced, the energy consumption is greatly reduced, the xylooligosaccharide product obtained has low xylose content, high purity of xylooligosaccharide, and few by-products.

[0019] (3) The trifluoroacetic acid used in this invention has strong volatility and can be recycled by distillation, which reduces costs and the difficulty of product purification, and improves the purity of xylooligosaccharide products.

[0020] (4) The combination of the two steps of “low-concentration trifluoroacetic acid pretreatment” and “xylan enzymatic hydrolysis” in this invention produces a synergistic effect of “1+1>2”, that is, the pretreatment creates an unprecedentedly efficient reaction environment for enzymatic hydrolysis, while enzymatic hydrolysis makes up for the lack of product selectivity of simple acid hydrolysis.

[0021] (5) The xylanase of the present invention achieves controlled degradation of hemicellulose by specifically recognizing and cleaving the β-1,4-glycosidic bonds of the xylan backbone. This process can further depolymerize the highly polymerized xylooligosaccharides produced by the preceding acid hydrolysis, thereby significantly increasing the total concentration of xylooligosaccharides in the system. Through precise control of the enzymatic hydrolysis conditions, this hydrolysis process exhibits selectivity for components with specific degrees of polymerization, effectively enriching the two most bioactive core components, xylobiose (X2) and xylotriose (X3), resulting in a significant increase in the proportion of X2 and X3 in the final product, greatly optimizing the functional characteristics and application value of the product. Attached Figure Description

[0022] Figure 1 This is a flowchart of the method for preparing xylooligosaccharides according to this application. Detailed Implementation

[0023] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0024] In the following examples, the corn cob raw material was sourced from Dongtai, Jiangsu Province; the birch sawdust raw material was sourced from a furniture factory in Shandong, China; and the sugarcane bagasse raw material was sourced from Hainan Province, China. Xylanase was purchased from Sigma-Aldrich, model X2753.

[0025] All calculation formulas involved in the following examples are as follows.

[0026]

[0027]

[0028]

[0029]

[0030] In the following examples, the xylooligosaccharide content was detected using high-performance anion exchange chromatography. Chromatographic conditions: The detection and analysis of xylooligosaccharides (X2-X6) were performed on a Thermo ICS-3000 high-performance anion exchange chromatography system equipped with a CarboPac™ PA200 column, using a gradient elution program of 100 mM sodium hydroxide and 1.0 M sodium acetate, with a flow rate controlled at 0.3 mL / min.

[0031] Figure 1 The process flow diagram for preparing xylooligosaccharides according to this application includes the following steps:

[0032] 1) After drying, the wood fiber raw material is crushed and sieved to 20-80 mesh. 0.5%-2% trifluoroacetic acid is added at a solid-liquid ratio of 1:10 (g / mL). The mixture is then reacted in an oil bath at 80-110℃ for 60-120 min.

[0033] 2) After the reaction, trifluoroacetic acid is recovered by rotary evaporation, and the remaining solid-liquid mixture is added to 0.5 times its volume of distilled water;

[0034] 3) Add xylanase to the solid-liquid mixture and hydrolyze it at 50°C for 8 hours to enrich xylobiose and xylotriose;

[0035] 4) Solid-liquid separation: The enzymatic hydrolysate is inactivated by enzyme, filtered, decolorized, concentrated, and spray-dried to obtain xylooligosaccharide powder.

[0036] Example 1

[0037] A method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase includes the following steps:

[0038] (1) Grind the dried corn cob and sieve it to 40 mesh. Add 1wt% trifluoroacetic acid at a solid-liquid ratio of 1:10 (g / mL) and react in an oil bath at 100℃ for 80 min.

[0039] (2) After the reaction, trifluoroacetic acid was recovered by rotary evaporation, and the remaining solid-liquid mixture was added to 0.5 times the volume of distilled water.

[0040] (3) Adjust the pH of the solid-liquid mixture to 5.5, add 3 U / mL xylanase, and hydrolyze it at 50℃ and 150 rpm for 8 hours to enrich xylobiose and xylotriose.

[0041] (4) After enzymatic hydrolysis, the hydrolysate was kept at 90℃ for 15 min to inactivate the enzyme, cooled, and then filtered. Activated carbon was added to the filtrate at 2% of the dry matter mass, and the solution was decolorized at 60℃ for 30 min and then filtered. After decolorization, the solution was concentrated by rotary evaporation to a solid content of approximately 30%. Finally, it was spray-dried (inlet air 180℃, outlet air 85-90℃, feed 5 mL / min) to obtain white xylooligosaccharide powder.

[0042] (5) After preparing a 10 g / L mother liquor from a trace amount of xylooligosaccharide powder, the xylooligosaccharide content was determined by high performance anion exchange chromatography and high performance liquid chromatography. The results are shown in Table 1.

[0043] Table 1 Yield of Enzyme Hydrolysate Components

[0044]

[0045] As shown in Table 1, the purification process is highly efficient and the product has high purity: after purification through the "inactivation, filtration, decolorization, and drying" system, white xylooligosaccharide powder was obtained, with a xylooligosaccharide content exceeding 70%. This data indicates that the purification steps effectively remove impurities such as pigments, proving the effectiveness of the purification process and yielding xylooligosaccharide products that meet high-quality application requirements.

[0046] The enrichment effect of the target components is significant: among the total xylooligosaccharides, the most bioactive core components, xylobiose (X2) and xylotriose (X3), achieved yields of 36.3% and 19.4%, respectively, accounting for more than 79.0% of the total xylooligosaccharides. This proves that the method can effectively achieve targeted enrichment of high-value-added components (X2 and X3) through condition control (pH 5.5, 50℃, 8h enzymatic hydrolysis), and the purity of xylooligosaccharides reached 77.3%, with clear product functionality and high commercial value.

[0047] Example 2

[0048] A method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase includes the following steps:

[0049] (1) Grind the dried corn cob and sieve it to 40 mesh. Add trifluoroacetic acid solutions with concentrations of 0.5wt%, 1.0wt%, 1.5wt%, and 2.0wt% at a solid-liquid ratio of 1:10 (g / mL) and react in an oil bath at 100℃ for 80 min.

[0050] (2) After the reaction, trifluoroacetic acid was recovered by rotary evaporation, and the remaining solid-liquid mixture was added to 0.5 times the volume of distilled water.

[0051] (3) Adjust the pH of each solid-liquid mixture to 5.5, add 3 U / mL xylanase, and hydrolyze by shaking at 50℃ and 150 rpm for 8 h.

[0052] (4) After the enzymatic hydrolysis was completed, samples of the enzymatic hydrolysate from each experimental group were directly taken for component analysis. The yield data of xylooligosaccharides are shown in Table 2.

[0053] Table 2. Yields of enzymatic hydrolysis products after treatment with different concentrations of trifluoroacetic acid

[0054]

[0055] Table 2 shows that trifluoroacetic acid concentration plays a crucial regulatory role in the pretreatment efficiency of corn cobs and the distribution of enzymatic hydrolysis products. Experimental results indicate that when the trifluoroacetic acid concentration increased from 0.5% to 1.5%, the total yield of xylooligosaccharides (X2-X6) significantly increased from 29.7% to 35.0%; however, further increasing the concentration to 2.0% resulted in a decrease in the total yield to 27.3%, indicating that excessively high acid concentrations lead to excessive degradation or the formation of inhibitors, resulting in decreased efficiency. Furthermore, under different concentration conditions, xylobiose (X2) and xylotriose (X3) maintained a high proportion in the total xylooligosaccharides, mainly due to the specific hydrolytic action of xylanase on the xylan chains, effectively achieving the targeted enrichment of these two highly bioactive components.

[0056] Example 3

[0057] A method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase includes the following steps:

[0058] (1) Grind the dried birch wood chips and sieve them to 40 mesh. Add a 2.0wt% trifluoroacetic acid solution at a solid-liquid ratio of 1:10 (g / mL) and react them in oil baths at 80℃, 90℃, 100℃ and 110℃ for 70 min respectively.

[0059] (2) After the reaction, trifluoroacetic acid was recovered by rotary evaporation, and the remaining solid-liquid mixture was added to 0.5 times the volume of distilled water.

[0060] (3) Adjust the pH of each solid-liquid mixture to 5.5, add 3 U / mL xylanase, and hydrolyze by shaking at 50℃ and 150 rpm for 8 h.

[0061] (4) After the enzymatic hydrolysis was completed, samples of the enzymatic hydrolysate from each experimental group were directly taken for component analysis. The yield data of xylooligosaccharides are shown in Table 3.

[0062] Table 3. Effect of different catalytic reaction temperatures on the yield of pretreatment and enzymatic hydrolysis products.

[0063]

[0064] As shown in Table 3, under the conditions of 2.0% trifluoroacetic acid and a reaction time of 70 min, 90℃ is the optimal temperature for birch sawdust pretreatment. This temperature can effectively enrich xylobiose and xylotriose while achieving a high conversion rate, indicating that increasing the temperature can promote the formation of xylooligosaccharides. However, excessively high temperatures (≥100℃) will lead to a significant decrease in product yield and quality, indicating that high temperatures cause excessive degradation of xylan into monosaccharides or other byproducts. Therefore, in actual production, the temperature needs to be precisely controlled within a suitable range.

[0065] Example 4

[0066] A method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase includes the following steps:

[0067] (1) Grind the dried sugarcane bagasse and sieve it to 40 mesh. Add a 1.5wt% trifluoroacetic acid solution at a solid-liquid ratio of 1:10 (g / mL) and react in an oil bath at 100℃ for 60 min, 80 min, 100 min and 120 min respectively.

[0068] (2) After the reaction, trifluoroacetic acid was recovered by rotary evaporation, and the remaining solid-liquid mixture was added to 0.5 times the volume of distilled water.

[0069] (3) Adjust the pH of each solid-liquid mixture to 5.5, add 3 U / mL xylanase, and hydrolyze by shaking at 50℃ and 150 rpm for 8 h.

[0070] (4) After the enzymatic hydrolysis was completed, samples of the enzymatic hydrolysate from each experimental group were directly taken for component analysis. The yield data of xylooligosaccharides are shown in Table 4.

[0071] Table 4. Effect of different reaction times on the yield of enzymatic hydrolysis products

[0072]

[0073] Table 4 shows that the pretreatment reaction time has a significant impact on the conversion efficiency of sugarcane bagasse. Within a reaction time of 60 to 100 minutes, the total yield of xylooligosaccharides increased from 24.6% to 37.9%, with xylobiose and xylotriose consistently accounting for over 75%, indicating that pretreatment during this period effectively improved the overall yield of the target product. When the reaction time was extended to 120 minutes, the total yield plummeted to 22.3%, and the combined proportion of xylobiose and xylotriose decreased simultaneously, indicating that excessive acid hydrolysis had triggered deep degradation of high-value-added components. Therefore, selecting an appropriate reaction time is crucial for balancing conversion efficiency and preventing excessive degradation.

[0074] Example 5

[0075] A method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase includes the following steps:

[0076] (1) Grind the dried corn cob, birch sawdust and sugarcane bagasse into powder and sieve them to 40 mesh. Add a 1.5wt% trifluoroacetic acid solution at a solid-liquid ratio of 1:10 (g / mL) and react in an oil bath at 100℃ for 80 min.

[0077] (2) After the reaction, trifluoroacetic acid was recovered by rotary evaporation, and the remaining solid-liquid mixture was added to 0.5 times the volume of distilled water.

[0078] (3) Adjust the pH of each solid-liquid mixture to 5.5, add 3 U / mL xylanase, and hydrolyze by shaking at 50℃ and 150 rpm for 8 h.

[0079] (4) After enzymatic hydrolysis, samples of the enzymatic hydrolysate from each experimental group were directly taken for component analysis. The yield data of xylooligosaccharides are shown in Table 5.

[0080] Table 5. Yields of enzymatic hydrolysates after pretreatment of different raw materials

[0081]

[0082] Table 5 shows that this method exhibits good applicability to three different lignocellulose raw materials: corn cob, birch sawdust, and bagasse. Under the same pretreatment (1.5% trifluoroacetic acid, 100℃, 80 minutes) and enzymatic hydrolysis conditions, the total yield of xylooligosaccharides from all three raw materials reached over 32%, with corn cob yielding the highest (35.0%), indicating that its xylan is more easily hydrolyzed by mild acid and efficient enzymatic hydrolysis. Furthermore, xylobiose and xylotriose predominated in the enzymatic hydrolysis products of each raw material, and the final xylooligosaccharide purity ranged from 65.4% to 73.2%. This demonstrates that the "low-concentration trifluoroacetic acid pretreatment synergistic xylan enzymatic hydrolysis" process route has broad applicability to various raw materials.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase, characterized in that, The lignocellulose raw material is crushed and sieved, mixed with trifluoroacetic acid, and subjected to a catalytic reaction. The trifluoroacetic acid is recovered by rotary evaporation. The remaining solid-liquid mixture is added to xylanase for enzymatic hydrolysis. The solid and liquid are separated, and the hydrolysate is subjected to enzyme inactivation, filtration, decolorization, concentration, and spray drying to obtain xylooligosaccharide powder.

2. The method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase according to claim 1, characterized in that: The wood fiber raw material is selected from one of birch, corn cob, and sugarcane bagasse.

3. The method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase according to claim 1, characterized in that: The wood fiber raw material is crushed and sieved to a mesh size of 20-80.

4. The method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase according to claim 1, characterized in that: The mass concentration of the trifluoroacetic acid is 0.5%-2%.

5. The method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase according to claim 1, characterized in that: The mass-to-volume ratio of the wood fiber raw material to trifluoroacetic acid is 1:

10.

6. The method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase according to claim 1, characterized in that: The catalytic reaction temperature is 80~110℃ and the time is 60~120min.

7. The method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase according to claim 1, characterized in that: The amount of xylanase added was 3 U / mL.

8. The method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase according to claim 1, characterized in that: The enzymatic hydrolysis temperature was 50℃ and the time was 8 hours.

9. The method for preparing high-purity xylooligosaccharides using low-concentration trifluoroacetic acid in conjunction with xylanase according to claim 1, characterized in that: The pH value of the enzymatic hydrolysis is 5.5.

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