Preparation method of high-purity xylooligosaccharide

Abstract

Disclosed is a method for preparing high-purity xylo-oligosaccharides, comprising: pretreating a hemicellulose-rich agroforestry by-product to obtain a hemicellulose substrate, adding xylanase to perform a first enzymatic hydrolysis reaction to generate a first mixed enzymatic hydrolysis liquid containing xylan, xylo-oligosaccharides, xylose and glucose; adding glucose oxidase, catalase, xylose isomerase and borate to perform a second enzymatic hydrolysis reaction and a third enzymatic hydrolysis reaction, catalytically converting glucose into sodium gluconate, catalytically converting xylose into xylulose, and in-situ forming a stable complex of borate and xylulose; after the second enzymatic hydrolysis reaction and the third enzymatic hydrolysis reaction, a second mixed enzymatic hydrolysis liquid is obtained; and after enzyme inactivation, filtration, concentration, ion exchange and drying, high-purity xylo-oligosaccharides are obtained. The application does not need fermentation to remove impurities, has no risk of bacterial contamination, can significantly improve the actual yield of xylo-oligosaccharides and the purity of the product, and the yield of xylo-oligosaccharides is greater than or equal to 60%, and the purity is greater than or equal to 99%.

Description

Technical Field

[0001] This application relates to the field of functional oligosaccharide preparation technology, specifically to a method for preparing high-purity xylooligosaccharides. Background Technology

[0002] Xylooligosaccharides (XOS) are functional oligosaccharides composed of 2 to 9 xylose units linked by β-1,4-glycosidic bonds. As a highly effective bioactive agent, they can selectively promote the growth of beneficial gut bacteria such as Bifidobacteria, and their demand is increasing in the food, feed, and pharmaceutical industries. Currently, the industrial production of xylooligosaccharides mainly uses xylan-rich biomass such as corn cobs and straw as raw materials, and obtains them through targeted enzymatic hydrolysis by xylanase.

[0003] However, traditional xylan enzymatic hydrolysis processes generally suffer from product inhibition and difficulty in controlling byproducts. Commercially available xylanase preparations often inevitably contain contaminating enzymes such as cellulase, β-xylosidase, and arabinofuranase. During actual enzymatic hydrolysis, these enzymes can cause excessive degradation of some xylan and even cellulose in the raw material, generating monosaccharides such as glucose, xylose, and arabinose. The large accumulation of these monosaccharide byproducts not only directly reduces the purity of xylooligosaccharides in the product, but more importantly, monosaccharides such as glucose and xylose have a significant feedback inhibition effect on xylanase, significantly inhibiting the main enzymatic hydrolysis reaction, leading to incomplete conversion of xylan substrates and low xylooligosaccharide yield. At the same time, high monosaccharide residues also increase the burden on subsequent purification stages such as decolorization, desalting, and chromatographic separation, further increasing production costs and making it difficult for the purity of the final product to meet the requirements of high-end applications.

[0004] In summary, existing enzymatic hydrolysis processes based on single xylanases or simple commercial enzymes generally suffer from drawbacks such as low xylooligosaccharide yield, high monosaccharide residue, and insufficient product purity, making it difficult to achieve both high conversion rate and high selectivity. Therefore, developing a novel preparation process that can effectively eliminate product inhibition, reduce the accumulation of monosaccharide byproducts, and improve the yield and purity of xylooligosaccharides has become an urgent technical problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a method for preparing high-purity xylooligosaccharides. The entire process utilizes enzymatic catalysis, with synergistic effects of multiple enzymes. The process is green and mild, requiring no fermentation for impurity removal and eliminating the risk of bacterial contamination. This significantly improves the actual yield and purity of xylooligosaccharides.

[0006] The technical solution of this application is as follows: 1. A method for preparing high-purity xylooligosaccharides, characterized by comprising the following steps: Hemicellulose enzymatic hydrolysis: Agricultural and forestry by-products rich in hemicellulose are pretreated to obtain hemicellulose substrates. Xylanase is added to the hemicellulose substrates to carry out the first enzymatic hydrolysis reaction, generating a first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose and glucose. In-situ conversion of glucose and xylose: Glucose oxidase, catalase, xylose isomerase and borate are added to the first mixed enzymatic hydrolysate to carry out a second enzymatic hydrolysis reaction and a third enzymatic hydrolysis reaction. In the second enzymatic hydrolysis reaction, the glucose is catalyzed to convert to sodium gluconate, and in the third enzymatic hydrolysis reaction, the xylose is catalyzed to convert to xylulose. The borate and xylulose form a stable complex in situ. After the second and third enzymatic hydrolysis reactions, a second mixed enzymatic hydrolysate is obtained. Post-processing and refining: The second mixed enzymatic hydrolysate is subjected to enzyme inactivation, filtration, concentration, ion exchange, and drying to obtain high-purity xylooligosaccharides.

[0007] 2. The method according to item 1, characterized in that, in the first enzymatic hydrolysis step, the hemicellulose-rich agricultural and forestry by-product is selected from any one or more of corn cobs, sugarcane bagasse, and straw.

[0008] 3. The method according to item 1 or 2, characterized in that the pretreatment is steam explosion pretreatment, dilute organic acid pretreatment or hot water extraction pretreatment; Preferably, the recovery rate of hemicellulose after pretreatment is ≥70%.

[0009] 4. The method according to any one of items 1 to 3, characterized in that, in the hemicellulose enzymatic hydrolysis step, the xylanase is a highly selective endo-β-1,4-xylanase; Preferably, the xylanase has an enzyme activity of 5000~20000 U / g; Preferably, the amount of xylanase added is 5-15 U / g of the dry basis of the hemicellulose substrate; Preferably, the temperature of the first enzymatic hydrolysis reaction is 40~50℃, and the reaction time is 12~24h; Preferably, the first enzymatic hydrolysis reaction is carried out at a pH of 4.5 to 6.

[0010] 5. The method according to any one of items 1 to 4, characterized in that, when the first enzymatic hydrolysis reaction reaches equilibrium, preferably when the yield of the xylooligosaccharide is ≥55%, glucose oxidase, catalase, xylose isomerase and borate are added to the first mixed enzymatic hydrolysate; Preferably, the mass ratio of glucose oxidase to catalase added is 1:(1~2), more preferably 1:(1.1~1.4); Preferably, the total mass of the added glucose oxidase and catalase is 0.05% to 1.5% of the dry weight of the first mixed enzymatic hydrolysate, more preferably 0.1% to 0.5%. Preferably, the activity of the glucose oxidase is 8000~10000 U / ml, and the activity of the catalase is 200,000~300,000 U / ml.

[0011] 6. The method according to any one of items 1 to 5, characterized in that the temperature of the second enzymatic hydrolysis reaction is 45 to 50°C; Preferably, the second enzymatic hydrolysis reaction is carried out at a pH of 5.0 to 7.0; Preferably, the second enzymatic hydrolysis reaction is carried out until the glucose content in the second mixed enzymatic hydrolysate is ≤0.5wt%.

[0012] 7. The method according to any one of items 1 to 6, characterized in that the xylose isomerase is a room-temperature xylose isomerase, and its applicable temperature range is 25 to 60°C; Preferably, the xylose isomerase has an enzyme activity of 10,000 to 30,000 U / g; Preferably, the amount of xylose isomerase added is 3~10 U / g of the first mixed enzymatic hydrolysate on a dry basis; Preferably, the borate is sodium borate and / or potassium borate; Preferably, the molar ratio of the borate to the xylose is (0.3~1):1.

[0013] 8. The method according to any one of items 1 to 7, characterized in that, in the in-situ conversion of glucose and xylose step, the temperature of the third enzymatic hydrolysis reaction is 45 to 50°C; Preferably, the third enzymatic hydrolysis reaction is carried out at a pH of 6.0 to 7.0; Preferably, the third enzymatic hydrolysis reaction is carried out until the xylose conversion rate is ≥95% and the xylose content in the second mixed enzymatic hydrolysate is ≤0.5wt%.

[0014] 9. The method according to any one of items 1 to 8, characterized in that the second enzymatic hydrolysis reaction and the third enzymatic hydrolysis reaction are carried out simultaneously at 45 to 50°C and pH 6.0 to 6.5.

[0015] 10. The method according to any one of items 1 to 9, characterized in that, in the post-processing purification step, the second mixed enzymatic hydrolysate is subjected to enzyme inactivation, microfiltration, ultrafiltration, nanofiltration concentration, ion exchange, and drying to obtain high-purity xylooligosaccharides; Preferably, the purity of the high-purity xylooligosaccharide is ≥99%, and the mass percentage of xylooligosaccharides with a degree of polymerization of 2 to 6 is ≥90% of the mass of the high-purity xylooligosaccharide.

[0016] Beneficial effects of the invention This application uses agricultural and forestry byproducts rich in hemicellulose as raw materials. After pretreatment to obtain hemicellulose substrate, xylanase, glucose oxidase, catalase, xylose isomerase, and borate are used to perform in-situ conversion of glucose and xylose in the product after xylanase enzymatic hydrolysis. This completely eliminates the dual inhibition of xylanase by monosaccharides, enabling continuous and efficient enzymatic hydrolysis. Finally, high-purity xylooligosaccharide is obtained through purification. The entire process uses enzymatic catalysis, which is green and mild, requires no fermentation for impurity removal, eliminates the risk of microbial contamination, and significantly improves the actual yield and purity of xylooligosaccharide.

[0017] This application obtains a higher purity xylooligosaccharide product by further controlling the addition ratio and amount of glucose oxidase and catalase, as well as the enzyme activity and addition amount of xylose isomerase. Furthermore, it improves the xylooligosaccharide yield to ≥60%, with xylooligosaccharides of degree of polymerization 2-6 accounting for ≥90% of the finished product, achieving a purity of ≥99% and a monosaccharide residue of ≤1%. The process described in this application has lower costs, is suitable for large-scale industrial production, and can be widely applied in food, feed, health products, and other fields. Detailed Implementation

[0018] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0019] It should be noted that the terms "comprising" or "including" used throughout the specification and claims are open-ended terms and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are intended to illustrate the general principles of this application and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0020] This application provides a method for preparing high-purity xylooligosaccharides, which includes the following steps: Hemicellulose enzymatic hydrolysis: Agricultural and forestry by-products rich in hemicellulose are pretreated to obtain hemicellulose substrates. Xylanase is added to the hemicellulose substrates to carry out the first enzymatic hydrolysis reaction, generating a first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose and glucose. In-situ conversion of glucose and xylose: Glucose oxidase, catalase, xylose isomerase, and borate are added to the first mixed enzymatic hydrolysate to carry out the second and third enzymatic hydrolysates. In the second enzymatic hydrolysate, glucose is catalyzed to be converted into sodium gluconate, and in the third enzymatic hydrolysate, xylose is catalyzed to be converted into xylulose. Furthermore, borate and xylulose form a stable complex in situ. After the second and third enzymatic hydrolysates, the second mixed enzymatic hydrolysate is obtained. Post-processing and refining: The second mixed enzymatic hydrolysate is subjected to enzyme inactivation, filtration, concentration, ion exchange, and drying to obtain high-purity xylooligosaccharides.

[0021] This application uses agricultural and forestry byproducts rich in hemicellulose as raw materials. After pretreatment to obtain hemicellulose substrate, xylanase is used for targeted enzymatic hydrolysis. Then, glucose oxidase and catalase are added to convert the glucose produced by enzymatic hydrolysis into sodium gluconate without inhibition in situ. Xylose isomerase and borate are added to continuously catalyze the xylose byproduct to xylulose. Borate and xylulose form a stable complex in situ, completely eliminating the dual inhibition of xylanase by glucose and xylose, and achieving continuous and efficient enzymatic hydrolysis. Finally, high-purity xylooligosaccharide is obtained after purification. This application discloses a method for the synergistic production of high-purity xylooligosaccharides using multiple enzymes, solving the product inhibition problem caused by the accumulation of monosaccharide byproducts such as glucose, xylose, and arabinose in the traditional xylan enzymatic hydrolysis process for xylooligosaccharide preparation. It overcomes the defects of low xylooligosaccharide yield, high monosaccharide residue, and insufficient purity in existing processes. The entire process uses enzymatic catalysis, which is green and mild, requires no fermentation for impurity removal, eliminates the risk of microbial contamination, and significantly improves the actual yield and purity of xylooligosaccharides.

[0022] In some implementations, in the first enzymatic hydrolysis step, the hemicellulose-rich agricultural and forestry by-product is selected from any one or more of corn cobs, bagasse, and straw.

[0023] In some implementations, the straw used in this application may be selected from any one or more of corn straw, wheat straw, rice straw, barley straw, oat straw, sugarcane straw, and sorghum straw.

[0024] In some implementations, the hemicellulose content in agricultural and forestry by-products rich in hemicellulose may be, for example, above 20 wt%.

[0025] In some implementations, the pretreatment is steam explosion pretreatment, dilute organic acid pretreatment, or hot water extraction pretreatment.

[0026] The "steam explosion treatment" of this application involves allowing steam to penetrate the raw material under high temperature and pressure, followed by instantaneous pressure release. This utilizes mechanical tearing and the autocatalytic hydrolysis effect of acetic acid to degrade and dissolve hemicellulose. In some embodiments, the steam explosion treatment involves: cutting straw into 2-5 cm short sections, adding water at a material-to-liquid ratio of 1:(5-10) and soaking for 10-30 minutes at room temperature; draining and then conveying it to the explosion chamber, closing the inlet and outlet valves of the explosion chamber, and continuously introducing high-temperature saturated steam; raising the pressure to 1.5-1.8 MPa and the temperature to 180-190℃ and maintaining this temperature for 150-240 seconds; rapidly opening the pressure relief valve to quickly reduce the pressure inside the chamber to atmospheric pressure; after the steam explosion, the released material is filtered, and the resulting clear liquid is the hemicellulose substrate. The "dilute organic acid pretreatment" of this application refers to using organic acids (such as acetic acid, formic acid, phosphoric acid, or oxalic acid) under heating conditions to catalyze the hydrolysis of hemicellulose, causing it to dissolve from the raw material and enter the liquid phase. In some implementation schemes, the pretreatment of dilute organic acids is as follows: straw is cut into short sections of 2-5 cm and mixed with an acid solution (acetic acid solution, formic acid solution or oxalic acid solution) with a mass fraction of 88%-90% at a solid-liquid ratio of 1:(8-10); the mixture is then placed in a high-pressure reactor and stirred at a speed of 300-500 rpm and a temperature of 155-175℃ for 40-60 minutes; after the reaction, the mixture is rapidly cooled to ≤50℃ to terminate the hydrolysis, and the clear liquid obtained by plate and frame filtration is a hemicellulose substrate.

[0027] In some implementations, "hot water extraction pretreatment" utilizes high-temperature liquid water under autocatalytic conditions (water dissociates into H+ at high temperatures). + The process involves the degradation and dissolution of hemicellulose by the shedding of acetyl groups from the raw materials to generate acetic acid. In some embodiments, the hot water extraction pretreatment is as follows: the straw is cut into 2-5 cm sections, deionized water is added at a solid-liquid ratio of 1:10, the mixture is placed in a high-pressure reactor, and the stirring is controlled at 300-500 rpm, 170-180℃, and 0.6-1.2 MPa for 40-60 min. After the reaction, cooling water is immediately passed through to cool the mixture to ≤60℃ within 10 min. The clear liquid obtained by plate and frame filtration is the hemicellulose substrate.

[0028] In some implementations, the hemicellulose substrate includes xylan, D-xylose, glucose, L-arabinose, lignin, etc. The "xylooligosaccharide" in this application, also known as xylooligosaccharide, is a linear or branched low-degree polymeric sugar composed of 2 to 9 xylose molecules linked by β-1,4-glycosidic bonds. The xylooligosaccharide content in this application can be determined with reference to Appendix B of GB / T 23528.3-2025 "Quality Requirements for Oligosaccharides Part 3: Xylooligosaccharides".

[0029] The "xylan" in this application is a linear polysaccharide composed of D-xylose linked by β-1,4-glycosidic bonds. The xylan content in this application can be determined according to GB / T 45556-2025 "Determination of Pentosan Content in Plant-Derived Products - GC-MS Method".

[0030] In this application, "hemicellulose recovery rate" refers to the percentage of hemicellulose mass in the pretreated hemicellulose substrate relative to the total hemicellulose mass in the agricultural and forestry by-products of the raw material. The hemicellulose recovery rate in this application can be determined using dilute acid hydrolysis-high performance liquid chromatography (HPLC) according to the detection standard GB / T 23528.3-2025.

[0031] In some implementation schemes, after pretreatment of hemicellulose-rich agricultural and forestry by-products, the recovery rate of hemicellulose in the hemicellulose substrate is ≥70%, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, etc.

[0032] In some embodiments, the xylanase is a highly selective endo-β-1,4-xylanase. The term "highly selective endo-β-1,4-xylanase" in this application refers to a class of xylanases that exhibit high catalytic activity towards the β-1,4-glycosidic bonds of the long-chain xylan backbone, while possessing extremely low hydrolytic activity towards the already formed short-chain xylooligosaccharides (XOS). This enzyme can prevent xylooligosaccharides from being further degraded into monosaccharides such as xylose, thereby effectively reducing the accumulation of monosaccharide byproducts. This is its core characteristic that distinguishes it from conventional endo-xylanases.

[0033] In some embodiments, the xylanase activity is 5000~20000 U / g, for example, it can be 5000 U / g, 5500 U / g, 6000 U / g, 6500 U / g, 7000 U / g, 7500 U / g, 8000 U / g, 8500 U / g, 9000 U / g, 9500 U / g, 10000 U / g, 10500 U / g, 11000 U / g, 11500 U / g, 12000 U / g, 1 2500 U / g, 13000 U / g, 13500 U / g, 14000 U / g, 14500 U / g, 15000 U / g, 15500 U / g, 16000 U / g, 16500 U / g, 17000 U / g, 17500 U / g, 18000 U / g, 18500 U / g, 19000 U / g, 19500 U / g or 20000 U / g, etc., preferably 18000~20000 U / g.

[0034] In some implementations, the xylanase addition amount is 5-15 U / g hemicellulose substrate (dry basis). If the xylanase addition amount is higher than this range, the performance improvement of the high-purity xylooligosaccharide product is very limited or remains essentially unchanged, but it results in cost waste. If the xylanase addition amount is lower than this range, it will reduce the xylose conversion rate. For example, the xylanase addition amount can be 5 U / g hemicellulose substrate (dry basis), 5.5 U / g hemicellulose substrate (dry basis), 6 U / g hemicellulose substrate (dry basis), 6.5 U / g hemicellulose substrate (dry basis), 7 U / g hemicellulose substrate (dry basis), 7.5 U / g hemicellulose substrate (dry basis), 8 U / g hemicellulose substrate (dry basis), 8.5 U / g hemicellulose substrate (dry basis), 9 U / g hemicellulose substrate (dry basis), 9.5 U / g hemicellulose substrate (dry basis), 10 U / g hemicellulose substrate (dry basis), etc. Dry basis of cellulose substrate, dry basis of 10.5 U / g hemicellulose substrate, dry basis of 11 U / g hemicellulose substrate, dry basis of 11.5 U / g hemicellulose substrate, dry basis of 12 U / g hemicellulose substrate, dry basis of 12.5 U / g hemicellulose substrate, dry basis of 13 U / g hemicellulose substrate, dry basis of 13.5 U / g hemicellulose substrate, dry basis of 14 U / g hemicellulose substrate, dry basis of 14.5 U / g hemicellulose substrate, or dry basis of 15 U / g hemicellulose substrate.

[0035] In this application, "U" refers to an enzyme activity unit. At 37°C and pH 5.5, 1U is defined as the amount of enzyme required to catalyze the conversion of 1 μmol of substrate to product per minute. For xylanase, 1U is defined as the amount of enzyme required to release 1 μmol of reducing sugar (calculated as xylose) per minute from a 5 mg / mL xylan solution. For glucose oxidase, 1U is defined as the amount of enzyme required to oxidize 1.0 μmol of β-D-glucose to D-gluconic acid and hydrogen peroxide per minute at pH 5.1 and 35°C. For catalase, 1U is defined as the amount of enzyme required to catalyze the decomposition of 1.0 μmol of hydrogen peroxide per minute at pH 7.0 and 25°C. For xylose isomerase, 1U is defined as the amount of enzyme required to catalyze the decomposition of 1.0 μmol of hydrogen peroxide per minute at 70°C, pH 7.5, and Mg2+. 2+ Under certain conditions, the amount of enzyme required to catalyze the production of 1 mg of D-fructose per hour is defined as 1 U.

[0036] In this application, “U / g” refers to the enzyme activity units contained in each gram of enzyme preparation.

[0037] In this application, "U / ml" refers to the enzyme activity units contained in each milliliter of enzyme preparation.

[0038] In this application, the unit for "enzyme addition amount" is "U / g substrate dry basis". "1U / g substrate dry basis" means that the amount of enzyme added is 1U per gram of substrate dry basis. The substrates involved in this application are hemicellulose substrate and the first mixed enzymatic hydrolysate.

[0039] In this application, "dry basis" refers to the absolute dryness of the material after all moisture has been removed.

[0040] The enzyme activity of glucose oxidase in this application was determined by colorimetric method in accordance with GB / T 44828-2024 "Methods for Determination of Glucose Oxidase Activity".

[0041] The activity of catalase in this application was determined by ultraviolet spectrophotometry in accordance with QB / T 4614-2013 "Industrial Catalase Preparations".

[0042] The enzyme activity of xylose isomerase in this application was determined by spectrophotometry in accordance with GB / T 23527.4-2025 "Quality Requirements for Enzyme Preparations Part 4: Immobilized Glucose Isomerase Preparations".

[0043] In some embodiments, the temperature of the first enzymatic hydrolysis reaction is 40-50°C, for example, 40°C, 40.5°C, 41°C, 41.5°C, 42°C, 42.5°C, 43°C, 43.5°C, 44°C, 44.5°C, 45°C, 45.5°C, 46°C, 46.5°C, 47°C, 47.5°C, 48°C, 48.5°C, 49°C, 49.5°C, or 50°C, etc., and the reaction time is 12-24 hours, for example, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, etc.

[0044] In some implementations, the first enzymatic hydrolysis reaction is carried out at a pH of 4.5 to 6.0, for example, pH can be 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0.

[0045] In some embodiments, the xylanase is a highly selective endo-β-1,4-xylanase with an enzyme activity of 5000~20000 U / g and an addition amount of 5~15 U / g of hemicellulose substrate on dry basis.

[0046] In some implementations, the temperature of the first enzymatic hydrolysis reaction is 40-50°C, the reaction time is 12-24 h, and the reaction is carried out at a pH of 4.5-6.0.

[0047] In some embodiments, the xylanase is a highly selective endo-β-1,4-xylanase with an enzyme activity of 5000~20000 U / g, and the amount added is 5~15 U / g of dry hemicellulose substrate. The temperature of the first enzymatic hydrolysis reaction is 40~50℃, and the time of the first enzymatic hydrolysis reaction is 12~24h.

[0048] In some embodiments, the xylanase is a highly selective endo-β-1,4-xylanase with an enzyme activity of 5000~20000 U / g, and the amount added is 5~15 U / g of dry hemicellulose substrate. The temperature of the first enzymatic hydrolysis reaction is 40~50℃, the time of the first enzymatic hydrolysis reaction is 12~24h, and the first enzymatic hydrolysis reaction is carried out at a pH of 4.5~6.0.

[0049] In some embodiments, when the first enzymatic hydrolysis reaction reaches equilibrium, glucose oxidase, catalase, xylose isomerase, and borate are added to the first mixed enzymatic hydrolysate. In this application, "the reaction reaches equilibrium" means that the concentrations of the reactants remain constant.

[0050] In this application, "yield of xylooligosaccharides" refers to the percentage of the total mass of xylooligosaccharides in the intermediate or final product relative to the mass of hemicellulose in the raw material. The formula for calculating the xylooligosaccharide yield is: Xylooligosaccharide yield = Total xylooligosaccharides produced ÷ Total hemicellulose in the raw material × 100%. The hemicellulose content in the raw material is determined according to GB / T 23747.

[0051] In some embodiments, when the first enzymatic hydrolysis reaction proceeds to a xylooligosaccharide yield ≥55%, glucose oxidase, catalase, xylose isomerase, and borate are added to the first mixed enzymatic hydrolysate. In this embodiment, the xylooligosaccharide yield refers to the percentage of the total mass of xylooligosaccharides in the first mixed enzymatic hydrolysate relative to the mass of xylan in the hemicellulose-rich agricultural and forestry by-products.

[0052] In some embodiments, glucose oxidase and catalase are added simultaneously. In some embodiments, glucose oxidase and catalase are added first, followed by xylose isomerase and borate. In some embodiments, glucose oxidase and catalase are added first, followed by xylose isomerase and borate. In some embodiments, xylose isomerase and borate are added first, followed by glucose oxidase and catalase. In some embodiments, xylose isomerase and borate are added first, followed by glucose oxidase and catalase. In some embodiments, the second and third enzymatic hydrolysis reactions occur simultaneously, achieving synchronous completion of glucose removal and xylose conversion, without terminating the xylan enzymatic hydrolysis reaction (i.e., the first enzymatic hydrolysis reaction). In some embodiments, glucose oxidase, catalase, and xylose isomerase are added simultaneously. In some embodiments, glucose oxidase, catalase, xylose isomerase, and borate are added simultaneously.

[0053] In some embodiments, the mass ratio of glucose oxidase to catalase is 1:(1~2), for example, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc., preferably 1:(1.1~1.4). This application controls the ratio of glucose oxidase to catalase within the above range to maximize the efficiency and conversion rate of glucose to sodium gluconate. If the ratio is lower than this, the glucose conversion rate is insufficient; if the ratio is higher than this, the purity of the high-purity xylooligosaccharide product remains essentially unchanged, and catalase is wasted.

[0054] In some embodiments, the total mass of added glucose oxidase and catalase is 0.05% to 1.5% of the dry basis mass of the first mixed enzymatic hydrolysate, for example, it can be 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%, preferably 0.1% to 0.5%. Adding glucose oxidase and catalase within this range is the most economical and efficient; adding less than this amount will limit the reaction rate, while adding more will not significantly change the purity of the high-purity xylooligosaccharide product and will increase costs.

[0055] In some embodiments, the mass ratio of glucose oxidase to catalase is 1:(1~2), and the total mass of the two is 0.05%~1.5% of the dry basis mass of the first mixed enzymatic hydrolysate.

[0056] In some embodiments, the mass ratio of glucose oxidase to catalase is 1:(1.1~1.4), and the total mass of the two is 0.1%~0.5% of the dry basis mass of the first mixed enzymatic hydrolysate.

[0057] In some implementations, the activity of glucose oxidase is 8000-10000 U / ml, for example, 8000 U / ml, 8200 U / ml, 8400 U / ml, 8600 U / ml, 8800 U / ml, 9000 U / ml, 9200 U / ml, 9400 U / ml, 9600 U / ml, 9800 U / ml, or 10000 U / ml, etc., and the activity of catalase is 200,000-300,000 U / ml, for example, 200,000 U / ml, 20.5 10,000 U / ml, 210,000 U / ml, 215,000 U / ml, 220,000 U / ml, 225,000 U / ml, 230,000 U / ml, 235,000 U / ml, 240,000 U / ml, 245,000 U / ml, 250,000 U / ml, 25 .50,000 U / ml, 260,000 U / ml, 265,000 U / ml, 270,000 U / ml, 275,000 U / ml, 280,000 U / ml, 285,000 U / ml, 290,000 U / ml, 295,000 U / ml or 300,000 U / ml, etc.

[0058] In some embodiments, the mass ratio of glucose oxidase to catalase is 1:(1~2), and the total mass of the two is 0.05%~1.5% of the dry basis mass of the first mixed enzymatic hydrolysate. The enzyme activity of glucose oxidase is 8000~10000 U / ml, and the enzyme activity of catalase is 200,000~300,000 U / ml.

[0059] In some embodiments, the mass ratio of glucose oxidase to catalase is 1:(1~2), and the total mass of the two is 0.05%~1.5% of the dry basis mass of the first mixed enzymatic hydrolysate. The enzyme activity of glucose oxidase is 8000~10000 U / ml, and the enzyme activity of catalase is 200,000~300,000 U / ml.

[0060] In some embodiments, the mass ratio of glucose oxidase to catalase is 1:(1.1~1.4), and the total mass of the two is 0.1%~0.5% of the dry basis mass of the first mixed enzymatic hydrolysate. The enzyme activity of glucose oxidase is 8000~10000 U / ml, and the enzyme activity of catalase is 200,000~300,000 U / ml.

[0061] In some embodiments, the mass ratio of glucose oxidase to catalase is 1:(1.1~1.4), and the total mass of the two is 0.1%~0.5% of the dry basis mass of the first mixed enzymatic hydrolysate. The enzyme activity of glucose oxidase is 8000~10000 U / ml, and the enzyme activity of catalase is 200,000~300,000 U / ml.

[0062] In some embodiments, the second enzymatic hydrolysis reaction is carried out at a temperature of 45-50°C, for example, 45°C, 45.5°C, 46°C, 46.5°C, 47°C, 47.5°C, 48°C, 48.5°C, 49°C, 49.5°C, or 50°C. In some embodiments, the second enzymatic hydrolysis reaction is carried out at a pH of 5.0-7.0, for example, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. In some embodiments, the second enzymatic hydrolysis reaction is carried out at a temperature of 45-50°C and at a pH of 5.0-7.0.

[0063] In some embodiments, the second enzymatic hydrolysis reaction is carried out until the glucose content in the second mixed enzymatic hydrolysate is ≤0.5wt%, for example, it can be 0.05wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, or 0.5wt%, etc. The glucose content in this application is determined using the HPLC-RID method in accordance with GB 5009.8-2023 "National Food Safety Standard - Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Food".

[0064] In some embodiments, the xylose isomerase is a room-temperature xylose isomerase with an applicable temperature range of 25–60°C. The "applicable temperature range of room-temperature xylose isomerase" in this application refers to: with the optimal enzyme activity at 50°C as 100% as a baseline, the relative enzyme activity is 50%–58% below 25°C, with lower activity at lower temperatures; above 60°C, the relative enzyme activity is 75%–85%, with lower activity at higher temperatures.

[0065] In some implementations, the activity of xylose isomerase is 10,000 to 30,000 U / g, for example, it can be 10,000 U / g, 11,000 U / g, 12,000 U / g, 13,000 U / g, 14,000 U / g, 15,000 U / g, 16,000 U / g, 17,000 U / g, 18,000 U / g, 19,000 U / g, 20,000 U / g, 21,000 U / g, 22,000 U / g, 23,000 U / g, 24,000 U / g, 25,000 U / g, 26,000 U / g, 27,000 U / g, 28,000 U / g, 29,000 U / g, or 30,000 U / g, etc.

[0066] In some embodiments, the amount of xylose isomerase added is 3-10 U / g of the first mixed enzymatic hydrolysate, for example, it can be 3 U / g, 3.5 U / g, 4 U / g, 4.5 U / g, 5 U / g, 5.5 U / g, 6 U / g, 6.5 U / g, 7 U / g, 7.5 U / g, 8 U / g, 8.5 U / g, 9 U / g, 9.5 U / g, or 10 U / g, preferably 8-10 U / g. By controlling the amount of xylose isomerase added within the above range, both efficiency and conversion rate can be considered. If the amount of xylose isomerase added is lower than this range, the conversion rate and efficiency will be low. If the amount of xylose isomerase added is higher than this range, the conversion rate will not be improved, resulting in waste of raw materials.

[0067] In some embodiments, the borate is sodium borate and / or potassium borate.

[0068] In some embodiments, the molar ratio of borate to xylose in the first mixed enzymatic hydrolysate is (0.3~1):1, for example, it can be 0.3:1, 0.35:1, 0.4:1, 0.45:1, 0.5:1, 0.55:1, 0.6:1, 0.65:1, 0.7:1, 0.75:1, 0.8:1, 0.85:1, 0.9:1, 0.95:1 or 1:1, etc.

[0069] In some implementations, the temperature of the third enzymatic hydrolysis reaction is 45~50℃, for example, it can be 45℃, 45.5℃, 46℃, 46.5℃, 47℃, 47.5℃, 48℃, 48.5℃, 49℃, 49.5℃ or 50℃, etc.

[0070] In some implementations, the third enzymatic hydrolysis reaction is carried out at a pH of 6.0 to 7.0, such as 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0.

[0071] In some implementations, the third enzymatic hydrolysis reaction is carried out at a temperature of 45-50°C and at a pH of 6.0-7.0.

[0072] In some embodiments, the conversion rate of xylose to xylose in the third enzymatic hydrolysis reaction is ≥95%, for example, it can be 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%, and the xylose content in the second mixed enzymatic hydrolysate is ≤0.5wt%, for example, it can be 0.5wt%, 0.45wt%, 0.4wt%, 0.35wt%, 0.3wt%, 0.25wt%, 0.2wt%, 0.15wt%, 0.1wt%, or 0.05wt%, etc. The "xylose conversion rate" in this application refers to the percentage of the mass of xylose converted to xylulose relative to the total mass of xylose in the reaction system at the beginning of the third enzymatic hydrolysis reaction. The mass of xylose converted to xylulose is equal to the total mass of xylose in the reaction system at the beginning of the third enzymatic hydrolysis reaction (i.e., the mass of xylose in the first mixed enzymatic hydrolysate) minus the mass of residual xylose in the second mixed enzymatic hydrolysate after the third enzymatic hydrolysis reaction. The xylose content in this application was determined using the amino column HPLC differential method in accordance with GB / T 23528.3-2025 "Quality Requirements for Oligosaccharides Part 3: Xylooligosaccharides".

[0073] In some implementations, the second and third enzymatic hydrolysis reactions are carried out simultaneously at 45–50°C and pH 6.0–6.5.

[0074] In some implementations, enzyme inactivation is performed in the post-processing purification step by incubating at 85-95°C for 10-15 minutes, for example at 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C; the incubation time can be 10 minutes, 10.5 minutes, 11 minutes, 11.5 minutes, 12 minutes, 12.5 minutes, 13 minutes, 13.5 minutes, 14 minutes, 14.5 minutes, or 15 minutes, etc.

[0075] In some embodiments, the second mixed enzymatic hydrolysate is subjected to enzyme inactivation, microfiltration, ultrafiltration, nanofiltration concentration, ion exchange, and drying to obtain high-purity xylooligosaccharides.

[0076] This application does not limit the microfiltration method, as long as it can remove suspended solids and coarse lignin. In some embodiments, microfiltration is carried out using a ceramic membrane with a pore size of 0.2 μm at a temperature of 40–55 °C and a transmembrane pressure of 0.2–0.4 MPa.

[0077] This application does not limit the method of ultrafiltration, as long as it can remove macromolecular impurities such as residual enzymes, proteins, and polysaccharide polymers. In some embodiments, ultrafiltration is carried out using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da at a temperature of 40~50°C and a transmembrane pressure of 0.15~0.3 MPa.

[0078] This application does not limit the method of nanofiltration concentration, as long as it can retain xylooligosaccharides with a degree of polymerization ≥2, and allow the passage of monosaccharides, salts, or sodium gluconate. In some embodiments, nanofiltration concentration is carried out using a nanofiltration membrane with a molecular weight cutoff of 200 Da at a temperature of 30~45°C and a transmembrane pressure of 2.0~3.5 MPa.

[0079] This application does not limit the method of ion exchange, as long as it can desalinate and deeply remove sodium ions and borate ions. In some embodiments, ion exchange is carried out using a strongly acidic styrene cation exchange resin and a strongly basic styrene anion exchange resin at a feed flow rate of 4~6 BV / h, an outlet conductivity ≤10μs / cm, and a pH of 5.0~6.0.

[0080] This application does not limit the drying method, as long as it can remove moisture. For example, spray drying can be used. In some embodiments, spray drying can be carried out at a feed temperature of 40~50℃, an air inlet temperature of 180~200℃, and an atomization pressure of 2.0~3.0MPa.

[0081] In some embodiments, the high-purity xylooligosaccharides obtained in this application have a purity ≥99%, and the mass percentage of xylooligosaccharides with a degree of polymerization of 2-6 accounts for ≥90% of the mass of the high-purity xylooligosaccharides. The purity of the xylooligosaccharides can be, for example, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%, and the mass percentage of xylooligosaccharides with a degree of polymerization of 2-6 can be, for example, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%, etc. The xylooligosaccharides with a degree of polymerization of 2-6 in this application can be determined according to Appendix A of GB / T 23528.3-2025 "Quality Requirements for Oligosaccharides Part 3: Xylooligosaccharides". X2 xylobiose, X3 xylotriose, X4 xylotetraose, X5 xylopentose, and X6 xylohexaose can be quantified separately, and XOS can be obtained by directly summing them. 2-6 .

[0082] Example The materials and test methods used in the embodiments of this application are described in a general and / or specific manner. In the following embodiments, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0083] The monosaccharide content in this application refers to the total content of glucose, xylose, and arabinose. Specifically, the glucose content was determined using HPLC-RID method according to GB 5009.8-2023 "National Food Safety Standard - Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Food". The xylose and arabinose contents were determined using amino column HPLC differential method according to GB / T 23528.3-2025 "Quality Requirements for Oligosaccharides Part 3: Xylooligosaccharides".

[0084] The content of xylooligosaccharides in the embodiments of this application was determined in accordance with Appendix B of GB / T 23528.3-2025 "Quality Requirements for Oligosaccharides Part 3: Xylooligosaccharides".

[0085] The xylan content in the embodiments of this application was determined in accordance with GB / T 45556-2025 "Determination of pentosan content in plant-derived products - GC-MS method".

[0086] In this embodiment, the xylose conversion rate = (mass of xylose in the first mixed enzymatic hydrolysate - mass of xylose remaining in the second mixed enzymatic hydrolysate after the third enzymatic hydrolysis) / total mass of xylose in the reaction system at the beginning of the third enzymatic hydrolysis * 100%.

[0087] The content of xylooligosaccharides with a degree of polymerization of 2 to 6 in the embodiments of this application was determined by Appendix A of GB / T 23528.3-2025 "Quality Requirements for Oligosaccharides Part 3: Xylooligosaccharides".

[0088] Example 1 (1) Hemicellulose enzymatic hydrolysis: 100 kg of corn cob (hemicellulose content of 32%) was used as raw material. After treatment with 1000 kg of formic acid with a mass fraction of 88%, 31.04 kg of hemicellulose substrate was obtained (of which xylan 86%, xylose 6%, glucose 2%, arabinose 0.2%, and lignin impurities 5.8%). After pretreatment, the recovery rate of hemicellulose was 88.5%. Highly selective endo-β-1,4-xylanase (Shandong Longket Enzyme Preparation Co., Ltd.) with an enzyme activity of 20000 U / g was added to the hemicellulose substrate at a dosage of 5 U / g of dry hemicellulose substrate. The first enzymatic hydrolysis reaction was carried out at a reaction temperature of 50℃, a reaction system pH of 6.0, and a reaction time of 12 h to generate a first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose, and glucose. (2) In-situ conversion of glucose and xylose: When the first enzymatic hydrolysis reaction reaches equilibrium, glucose oxidase with an activity of 10,000 U / ml, catalase with an activity of 300,000 U / ml, and room-temperature xylose isomerase (Shandong Longket Enzyme Preparation Co., Ltd.) and sodium borate with an activity of 10,000 U / g are simultaneously added to the above first mixed enzymatic hydrolysate to carry out the second and third enzymatic hydrolysis reactions. The temperature of the second and third enzymatic hydrolysis reactions is 50℃, and the pH of the system is 6.0; among which, glucose oxidase and catalase... The mass ratio of hydrogenase to hydrogenase is 1:1.1, and the total amount of both added is 0.1% of the dry weight of the first mixed enzymatic hydrolysate. A second enzymatic hydrolysis reaction is carried out until the glucose content in the system is 0.1 wt%. The amount of room-temperature xylose isomerase added is 10 U / g of the dry weight of the first mixed enzymatic hydrolysate. The molar ratio of sodium borate to xylose in the first mixed enzymatic hydrolysate is 0.3:1. A third enzymatic hydrolysis reaction is carried out until the xylose conversion rate is 95% and the xylose residue is 0.3 wt%. After the second and third enzymatic hydrolysis reactions, the second mixed enzymatic hydrolysate is obtained. (3) Post-processing and refining: The second mixed enzymatic hydrolysate is kept at 95℃ for 10 min to inactivate the enzyme, and then successively passes through ceramic membrane microfiltration to remove slag (ceramic membrane pore size 0.2μm, temperature 50℃, transmembrane pressure 0.3MPa), ultrafiltration to remove macromolecular impurities (ultrafiltration membrane molecular weight cutoff 3000Da, temperature 45℃, transmembrane pressure 0.2MPa), nanofiltration concentration (nanofiltration membrane molecular weight cutoff 200Da, temperature 40℃, transmembrane pressure 3.0MPa), ion exchange desalination (using strong acid styrene cation resin (Zhejiang Zhengguang Industrial D001 type) and strong acid styrene cation resin (Zhejiang Zhengguang Industrial D201 type), feed flow rate 5BV / h, output conductivity ≤10μs / cm), decolorization (activated carbon addition 1‰, temperature 75℃), and spray drying (feed temperature 45℃, air inlet temperature 200℃, atomization pressure 3.0MPa) to obtain high-purity xylooligosaccharide product.

[0089] In the finished xylooligosaccharide product, the xylooligosaccharide yield is 61.2%, the purity of the high-purity xylooligosaccharide product is 99.1%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 92.1%, and the monosaccharide content is 0.6 wt%.

[0090] Example 2 (1) Hemicellulose enzymatic hydrolysis: 100 kg of sugarcane bagasse (hemicellulose content of 28%) was used as raw material. After steam explosion pretreatment, 24.64 kg of hemicellulose substrate (xylan 81.6%, xylose 10%, glucose 2.1%, arabinose 0.1%, lignin impurities 6.2%) was obtained. After pretreatment, the recovery rate of hemicellulose was 78.4%. Highly selective endo-β-1,4-xylanase with an enzyme activity of 5000 U / g was added to the hemicellulose substrate. The amount added was 15 U / g of dry hemicellulose substrate. The first enzymatic hydrolysis reaction was carried out at a reaction temperature of 45℃, a reaction system pH of 4.5, and a reaction time of 24 h. The first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose and glucose was generated. (2) In-situ conversion of glucose and xylose: When the first enzymatic hydrolysis reaction reaches equilibrium, glucose oxidase with an activity of 8000 U / ml, catalase with an activity of 200,000 U / ml, xylose isomerase with an activity of 30000 U / g and potassium borate are added simultaneously to the first mixed enzymatic hydrolysate to carry out the second and third enzymatic hydrolysis reactions. The temperature of the second and third enzymatic hydrolysis reactions is 45℃, and the pH of the system is 6.5; wherein, the mass ratio of glucose oxidase to catalase is... The ratio of potassium borate to xylose in the first mixed enzymatic hydrolysate is 1:1.4, and the total amount of both added is 0.5% of the dry weight of the first mixed enzymatic hydrolysate. A second enzymatic hydrolysis is performed until the glucose content in the system is 0.4 wt%. The amount of room-temperature xylose isomerase added is 3 U / g of the dry weight of the first mixed enzymatic hydrolysate. The molar ratio of potassium borate to xylose in the first mixed enzymatic hydrolysate is 1:1. A third enzymatic hydrolysis is performed until the xylose conversion rate is 99% and the xylose residue is 0.1 wt%. After the second and third enzymatic hydrolysis reactions, the second mixed enzymatic hydrolysate is obtained. (3) Post-processing and refining: The second mixed enzymatic hydrolysate is kept at 85℃ for 15 min to inactivate the enzyme, and then successively passes through ceramic membrane microfiltration to remove slag (ceramic membrane pore size 0.2μm, temperature 50℃, transmembrane pressure 0.3MPa), ultrafiltration to remove macromolecular impurities (ultrafiltration membrane molecular weight cutoff 3000Da, temperature 45℃, transmembrane pressure 0.2MPa), nanofiltration concentration (nanofiltration membrane molecular weight cutoff 200Da, temperature 40℃, transmembrane pressure 3.0MPa), ion exchange desalination (using strong acid styrene cation exchange resin (Zhejiang Zhengguang Industrial D001 type) and strong acid styrene cation exchange resin (Zhejiang Zhengguang Industrial D201 type), feed flow rate 5BV / h, discharge conductivity ≤10μs / cm), decolorization (activated carbon addition 1‰, temperature 75℃), and spray drying (feed temperature 45℃, air inlet temperature 200℃, atomization pressure 3.0MPa) to obtain high-purity xylooligosaccharide product.

[0091] In the finished xylooligosaccharide product, the xylooligosaccharide yield is 65.5%, the purity of the high-purity xylooligosaccharide product is 99.3%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 93.7%, and the monosaccharide content is 0.6 wt%.

[0092] Example 3 (1) Hemicellulose enzymatic hydrolysis: 100 kg of wheat straw (hemicellulose content of 26%) was used as raw material. After hot water extraction pretreatment, 21.32 kg of hemicellulose substrate (77.9% xylan, 10% xylose, 3% glucose, 0.1% arabinose, and 9% lignin impurities) was obtained. After pretreatment, the recovery rate of hemicellulose was 70.5%. Highly selective endo-β-1,4-xylanase with an enzyme activity of 10000 U / g was added to the substrate system. The amount added was 10 U / g of dry hemicellulose substrate. The first enzymatic hydrolysis reaction was carried out at a reaction temperature of 47℃, a reaction system pH of 5.5, and a reaction time of 18 h. The first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose, and glucose was generated. (2) In-situ conversion of glucose and xylose: When the first enzymatic hydrolysis reaction reaches equilibrium, glucose oxidase with an activity of 9000 U / ml, catalase with an activity of 250,000 U / ml, and xylose isomerase with an activity of 20000 U / g at room temperature, along with sodium borate and potassium borate, are simultaneously added to the first mixed enzymatic hydrolysate to carry out the second and third enzymatic hydrolysis reactions. The temperature of the second and third enzymatic hydrolysis reactions is 47℃, and the pH of the system is 6.2. Among them, the mass ratio of glucose oxidase to catalase is 1:1.3, and the total amount of both added is... The first mixed enzymatic hydrolysate was 0.3% of its dry weight. A second enzymatic hydrolysis was performed until the glucose content in the system was 0.2 wt%. The amount of xylose isomerase added at room temperature was 5 U / g of the dry weight of the first mixed enzymatic hydrolysate. The molar ratio of potassium borate to sodium borate was 1:1, and the ratio of the total moles of potassium borate and sodium borate to the moles of xylose in the first mixed enzymatic hydrolysate was 0.5:1. A third enzymatic hydrolysis was performed until the xylose conversion rate in the system was 98% and the xylose residue was 0.2 wt%. After the second and third enzymatic hydrolysis reactions, the second mixed enzymatic hydrolysate was obtained. (3) Post-processing and refining: The second mixed enzymatic hydrolysate is kept at 90℃ for 13 min to inactivate the enzyme, and then successively passes through ceramic membrane microfiltration to remove slag (ceramic membrane pore size 0.2μm, temperature 50℃, transmembrane pressure 0.3MPa), ultrafiltration to remove macromolecular impurities (ultrafiltration membrane molecular weight cutoff 3000Da, temperature 45℃, transmembrane pressure 0.2MPa), nanofiltration concentration (nanofiltration membrane molecular weight cutoff 200Da, temperature 40℃, transmembrane pressure 3.0MPa), ion exchange desalination (using strong acid styrene cation resin (Zhejiang Zhengguang Industrial D001 type) and strong acid styrene cation resin (Zhejiang Zhengguang Industrial D201 type), feed flow rate 5BV / h, discharge conductivity ≤10μs / cm), decolorization (activated carbon addition 1‰, temperature 75℃), and spray drying (feed temperature 45℃, air inlet temperature 200℃, atomization pressure 3.0MPa) to obtain high-purity xylooligosaccharide product.

[0093] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 64.2%, the purity of the high-purity xylooligosaccharide finished product is 99.5%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 92.3%, and the monosaccharide content is 0.5 wt%.

[0094] Example 4 The difference between this embodiment and embodiment 2 is that the mass ratio of glucose oxidase to catalase added in step (2) is 1:1.1.

[0095] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 62.4%, the purity of the high-purity xylooligosaccharide finished product is 99.2%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 93.3%, and the monosaccharide content is 0.4 wt%.

[0096] Example 5 The difference between this embodiment and embodiment 2 is that the mass ratio of glucose oxidase to catalase added in step (2) is 1:0.8.

[0097] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 58.7%, the purity of the high-purity xylooligosaccharide finished product is 84.2%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 87.5%, and the monosaccharide content is 1.3 wt%.

[0098] Example 6 The difference between this embodiment and embodiment 2 is that the mass ratio of glucose oxidase to catalase added in step (2) is 1:2.

[0099] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 63.5%, the purity of the high-purity xylooligosaccharide finished product is 99.4%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 94.3%, and the monosaccharide content is 0.3 wt%.

[0100] Example 7 The difference between this embodiment and embodiment 2 is that the total amount of glucose oxidase and catalase added in step (2) is 0.1% of the dry basis mass of the first mixed enzyme hydrolysate.

[0101] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 64.0%, the purity of the high-purity xylooligosaccharide finished product is 99.3%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 95.1%, and the monosaccharide content is 0.2 wt%.

[0102] Example 8 The difference between this embodiment and embodiment 2 is that the total amount of glucose oxidase and catalase added in step (2) is 0.05% of the dry basis mass of the first mixed enzyme hydrolysate.

[0103] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 59.6%, the purity of the high-purity xylooligosaccharide finished product is 83.5%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 85.2%, and the monosaccharide content is 1.5 wt%.

[0104] Example 9 The difference between this embodiment and embodiment 2 is that the total amount of glucose oxidase and catalase added in step (2) is 1.5% of the dry basis mass of the first mixed enzyme hydrolysate.

[0105] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 64.4%, the purity of the high-purity xylooligosaccharide finished product is 99.5%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 93.0%, and the monosaccharide content is 0.2 wt%.

[0106] Example 10 The difference between this embodiment and embodiment 2 is that the amount of room temperature xylose isomerase added in step (2) is 10 U / g of the dry basis of the first mixed enzyme hydrolysate.

[0107] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 63.1%, the purity of the high-purity xylooligosaccharide finished product is 99.3%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 92.7%, and the monosaccharide content is 0.4 wt%.

[0108] Example 11 The difference between this embodiment and embodiment 2 is that the amount of room temperature xylose isomerase added in step (2) is 2 U / g of the dry basis of the first mixed enzyme hydrolysate.

[0109] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 59.3%, the purity of the high-purity xylooligosaccharide finished product is 99.1%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 90.2%, and the monosaccharide content is 0.8 wt%.

[0110] Example 12 The difference between this embodiment and embodiment 2 is that the amount of room temperature xylose isomerase added in step (2) is 12 U / g of the dry basis of the first mixed enzyme hydrolysate.

[0111] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 65.5%, the purity of the high-purity xylooligosaccharide finished product is 99.3%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 94.2%, and the monosaccharide content is 0.4 wt%.

[0112] Example 13 The difference between this embodiment and embodiment 2 is that the enzyme activity of the room temperature xylose isomerase in step (2) is 10000 U / g.

[0113] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 64.1%, the purity of the high-purity xylooligosaccharide finished product is 99.2%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 92.8%, and the monosaccharide content is 0.3 wt%.

[0114] Example 14 The difference between this embodiment and Example 2 is that the amount of highly selective endo-β-1,4-xylanase added is 4 U / g dry basis of hemicellulose substrate.

[0115] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 59.3%, the purity of the high-purity xylooligosaccharide finished product is 99.1%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 91.1%, and the monosaccharide content is 0.7 wt%.

[0116] Example 15 The difference between this embodiment and Example 2 is that the amount of highly selective endo-β-1,4-xylanase added is 17 U / g dry basis of hemicellulose substrate.

[0117] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 65.2%, the purity of the high-purity xylooligosaccharide finished product is 99.5%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 92.1%, and the monosaccharide content is 0.3 wt%.

[0118] Example 16 The difference between this embodiment and Example 1 is that the highly selective endo-β-1,4-xylanase is replaced with a broad-spectrum xylanase (Shandong Longket Enzyme Preparation Co., Ltd.).

[0119] In the high-purity xylooligosaccharide finished product, the xylooligosaccharide yield is 59.3%, the purity of the high-purity xylooligosaccharide finished product is 82.7%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 83.6%, and the monosaccharide content is 1.8 wt%.

[0120] Comparative Example 1 The difference between this comparative example and Example 1 is that the in-situ conversion of glucose and xylose in step (2) is replaced by a fermentation step to remove monosaccharides. The specific preparation method is as follows: (1) Hemicellulose enzymatic hydrolysis: 100 kg of corn cob (hemicellulose content of 32%) was used as raw material. After treatment with 1000 kg of formic acid with a mass fraction of 88%, 31.04 kg of hemicellulose substrate (xylan 86%, xylose 6%, glucose 2%, arabinose 0.2%, lignin impurities 5.8%) was obtained. After pretreatment, the recovery rate of hemicellulose was 88.5%. Highly selective endo-β-1,4-xylanase (Shandong Longkete Enzyme Preparation Co., Ltd.) with an enzyme activity of 20000 U / g was added to the hemicellulose substrate. The amount added was 5 U / g of dry hemicellulose substrate. The first enzymatic hydrolysis reaction was carried out at a reaction temperature of 50℃, a reaction system pH of 6.0, and a reaction time of 12 h to generate a first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose and glucose. (2) Fermentation to remove monosaccharides: When the first enzymatic hydrolysis reaction reaches equilibrium, the temperature of the first mixed enzymatic hydrolysate is adjusted to 30℃ and pH=4.0. Pichia pastoris (accession number: CICC1960) and Candida parapsilosis (accession number: CICC1257) are inoculated into it, with each inoculation amount being 1% (v / v). After fermentation for 16h, 1% (v / v) of Pseudomonas leucocephala (accession number: CICC1019) is inoculated and fermented for 20h. When the glucose concentration is 0.5wt%, fermentation is stopped to obtain the fermentation broth. (3) Post-processing purification: The obtained fermentation broth was kept at 95℃ for 10 min to inactivate enzymes, and then passed through ceramic membrane microfiltration to remove residue (ceramic membrane pore size 0.2μm, temperature 50℃, transmembrane pressure 0.3MPa), ultrafiltration to remove macromolecular impurities (ultrafiltration membrane molecular weight cutoff 3000Da, temperature 45℃, transmembrane pressure 0.2MPa), nanofiltration concentration (nanofiltration membrane molecular weight cutoff 200Da, temperature 40℃, transmembrane pressure 3.0MPa), ion exchange desalination (using strong acid styrene cation exchange resin (Zhejiang Zhengguang Industrial D001 type) and strong acid styrene cation exchange resin (Zhejiang Zhengguang Industrial D201 type)), feed flow rate 5BV / h, discharge conductivity ≤10μs / cm, decolorization (activated carbon addition 1‰, temperature 75℃), and spray drying (feed temperature 45℃, air inlet temperature 200℃, atomization pressure 3.0MPa) to obtain high-purity xylooligosaccharide product.

[0121] In the xylooligosaccharide finished product, the xylooligosaccharide yield was 44.4%, the purity of the obtained xylooligosaccharide finished product was 83.1%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 was 78.1%, and the monosaccharide content was 2.7 wt%.

[0122] Comparative Example 2 The difference between this comparative example and Example 1 is that it does not include the step of in-situ conversion of glucose and xylose. The specific preparation method is as follows: (1) Hemicellulose enzymatic hydrolysis: 100 kg of corn cob (hemicellulose content of 32%) was used as raw material. After treatment with 1000 kg of formic acid with a mass fraction of 88%, 31.04 kg of hemicellulose substrate (xylan 86%, xylose 6%, glucose 2%, arabinose 0.2%, lignin impurities 5.8%) was obtained. After pretreatment, the recovery rate of hemicellulose was 88.5%. Highly selective endo-β-1,4-xylanase (Shandong Longkete Enzyme Preparation Co., Ltd.) with an enzyme activity of 20000 U / g was added to the hemicellulose substrate. The amount added was 5 U / g of dry hemicellulose substrate. The first enzymatic hydrolysis reaction was carried out at a reaction temperature of 50℃, a reaction system pH of 6.0, and a reaction time of 12 h to generate a first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose and glucose. (2) Post-processing purification: When the first enzymatic hydrolysis reaction reaches equilibrium, the first mixed enzymatic hydrolysate is incubated at 95℃ for 10 min to inactivate the enzyme, and then sequentially passed through ceramic membrane microfiltration for residue removal (ceramic membrane pore size 0.2μm, temperature 50℃, transmembrane pressure 0.3MPa), ultrafiltration to remove macromolecular impurities (ultrafiltration membrane molecular weight cutoff 3000Da, temperature 45℃, transmembrane pressure 0.2MPa), and nanofiltration for concentration (nanofiltration membrane molecular weight cutoff 200Da, temperature 40℃, transmembrane pressure 0.2MPa). The process involves three steps: ion exchange desalination (using strong acid styrene cation exchange resin (Zhejiang Zhengguang Industrial D001 type) and strong acid styrene cation exchange resin (Zhejiang Zhengguang Industrial D201 type), feed flow rate 5 BV / h, output conductivity ≤10 μs / cm), decolorization (activated carbon addition 1‰, temperature 75℃), and spray drying (feed temperature 45℃, air inlet temperature 200℃, atomization pressure 3.0 MPa) to obtain high-purity xylooligosaccharide product.

[0123] In the xylooligosaccharide finished product, the xylooligosaccharide yield was 29.3%, the purity of the obtained xylooligosaccharide finished product was 78.2%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 was 75.3%, and the monosaccharide content was 8.2 wt%.

[0124] Comparative Example 3 The difference between this comparative example and Example 1 is that sodium borate is not added in the in-situ conversion of glucose and xylose. The specific preparation method is as follows: (1) Hemicellulose enzymatic hydrolysis: 100 kg of corn cob (hemicellulose content of 32%) was used as raw material. After treatment with 1000 kg of formic acid with a mass fraction of 88%, 31.04 kg of hemicellulose substrate was obtained (of which xylan 86%, xylose 6%, glucose 2%, arabinose 0.2%, and lignin impurities 5.8%). After pretreatment, the recovery rate of hemicellulose was 88.5%. Highly selective endo-β-1,4-xylanase (Shandong Longket Enzyme Preparation Co., Ltd.) with an enzyme activity of 20000 U / g was added to the hemicellulose substrate at a dosage of 5 U / g of dry hemicellulose substrate. The first enzymatic hydrolysis reaction was carried out at a reaction temperature of 50℃, a reaction system pH of 6.0, and a reaction time of 12 h to generate a first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose, and glucose. (2) In-situ conversion of glucose and xylose: When the first enzymatic hydrolysis reaction reaches equilibrium, glucose oxidase with an activity of 10,000 U / ml, catalase with an activity of 300,000 U / ml, and room-temperature xylose isomerase (Shandong Longket Enzyme Preparation Co., Ltd.) with an activity of 10,000 U / g are added to the first mixed enzymatic hydrolysate to carry out the second and third enzymatic hydrolysis reactions. The temperature of the second and third enzymatic hydrolysis reactions is 50℃, and the pH of the system is 6.0. Among them, the mass ratio of glucose oxidase to catalase is 1:1.1, and the total amount of the two added is 0.1% of the dry basis mass of the first mixed enzymatic hydrolysate. The second enzymatic hydrolysis reaction is carried out until the glucose content in the system is 0.1 wt%. The amount of room-temperature xylose isomerase added is 10 U / g of the dry basis of the first mixed enzymatic hydrolysate. The third enzymatic hydrolysis reaction is carried out until the xylose conversion rate in the system is 70% and the xylose residue is 1.8 wt%. After the second and third enzymatic hydrolysis reactions, the second mixed enzymatic hydrolysate is obtained. (3) Post-processing and refining: The second mixed enzymatic hydrolysate is kept at 95℃ for 10 min to inactivate the enzyme, and then successively passes through ceramic membrane microfiltration to remove slag (ceramic membrane pore size 0.2μm, temperature 50℃, transmembrane pressure 0.3MPa), ultrafiltration to remove macromolecular impurities (ultrafiltration membrane molecular weight cutoff 3000Da, temperature 45℃, transmembrane pressure 0.2MPa), nanofiltration concentration (nanofiltration membrane molecular weight cutoff 200Da, temperature 40℃, transmembrane pressure 3.0MPa), ion exchange desalination (using strong acid styrene cation resin (Zhejiang Zhengguang Industrial D001 type) and strong acid styrene cation resin (Zhejiang Zhengguang Industrial D201 type), feed flow rate 5BV / h, output conductivity ≤10μs / cm), decolorization (activated carbon addition 1‰, temperature 75℃), and spray drying (feed temperature 45℃, air inlet temperature 200℃, atomization pressure 3.0MPa) to obtain high-purity xylooligosaccharide product.

[0125] In the finished xylooligosaccharide product, the xylooligosaccharide yield is 60.2%, the purity of the high-purity xylooligosaccharide product is 81.5%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 82.6%, and the monosaccharide content is 2.1 wt%.

[0126] Comparative Example 4 The difference between this comparative example and Example 1 is that room-temperature xylose isomerase and sodium borate are not added in step (2) of the in-situ conversion of glucose and xylose. The specific preparation method is as follows: (1) Hemicellulose enzymatic hydrolysis: 100 kg of corn cob (hemicellulose content of 32%) was used as raw material. After treatment with 1000 kg of formic acid with a mass fraction of 88%, 31.04 kg of hemicellulose substrate was obtained (of which xylan 86%, xylose 6%, glucose 2%, arabinose 0.2%, and lignin impurities 5.8%). After pretreatment, the recovery rate of hemicellulose was 88.5%. Highly selective endo-β-1,4-xylanase (Shandong Longket Enzyme Preparation Co., Ltd.) with an enzyme activity of 20000 U / g was added to the hemicellulose substrate at a dosage of 5 U / g of dry hemicellulose substrate. The first enzymatic hydrolysis reaction was carried out at a reaction temperature of 50℃, a reaction system pH of 6.0, and a reaction time of 12 h to generate a first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose, and glucose. (2) In-situ conversion of glucose: When the first enzymatic hydrolysis reaction reaches equilibrium, glucose oxidase with an activity of 10,000 U / ml and catalase with an activity of 300,000 U / ml are added to the first mixed enzymatic hydrolysate to carry out the second enzymatic hydrolysis reaction. The temperature of the second enzymatic hydrolysis reaction is 50℃ and the pH of the system is 6.0. The mass ratio of glucose oxidase to catalase is 1:1.1, and the total amount of both added is 0.1% of the dry basis mass of the first mixed enzymatic hydrolysate. The second enzymatic hydrolysis reaction is carried out until the glucose content in the system is 0.1 wt%. After the second enzymatic hydrolysis reaction, the second mixed enzymatic hydrolysate is obtained. (3) Post-processing and refining: The second mixed enzymatic hydrolysate is kept at 95℃ for 10 min to inactivate the enzyme, and then successively passes through ceramic membrane microfiltration to remove slag (ceramic membrane pore size 0.2μm, temperature 50℃, transmembrane pressure 0.3MPa), ultrafiltration to remove macromolecular impurities (ultrafiltration membrane molecular weight cutoff 3000Da, temperature 45℃, transmembrane pressure 0.2MPa), nanofiltration concentration (nanofiltration membrane molecular weight cutoff 200Da, temperature 40℃, transmembrane pressure 3.0MPa), ion exchange desalination (using strong acid styrene cation resin (Zhejiang Zhengguang Industrial D001 type) and strong acid styrene cation resin (Zhejiang Zhengguang Industrial D201 type), feed flow rate 5BV / h, output conductivity ≤10μs / cm), decolorization (activated carbon addition 1‰, temperature 75℃), and spray drying (feed temperature 45℃, air inlet temperature 200℃, atomization pressure 3.0MPa) to obtain high-purity xylooligosaccharide product.

[0127] In the finished xylooligosaccharide product, the xylooligosaccharide yield is 51.4%, the purity of the high-purity xylooligosaccharide product is 72.5%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 77.3%, and the monosaccharide content is 6.3 wt%.

[0128] Comparative Example 5 The difference between this comparative example and Example 1 is that glucose oxidase and catalase are not added in step (2) of the in-situ conversion of glucose and xylose. The specific preparation method is as follows: (1) Hemicellulose enzymatic hydrolysis: 100 kg of corn cob (hemicellulose content of 32%) was used as raw material. After treatment with 1000 kg of formic acid with a mass fraction of 88%, 31.04 kg of hemicellulose substrate was obtained (of which xylan 86%, xylose 6%, glucose 2%, arabinose 0.2%, and lignin impurities 5.8%). After pretreatment, the recovery rate of hemicellulose was 88.5%. Highly selective endo-β-1,4-xylanase (Shandong Longket Enzyme Preparation Co., Ltd.) with an enzyme activity of 20000 U / g was added to the hemicellulose substrate at a dosage of 5 U / g of dry hemicellulose substrate. The first enzymatic hydrolysis reaction was carried out at a reaction temperature of 50℃, a reaction system pH of 6.0, and a reaction time of 12 h to generate a first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose, and glucose. (2) In-situ conversion of xylose: When the first enzymatic hydrolysis reaction reaches equilibrium, room-temperature xylose isomerase (Shandong Longket Enzyme Preparation Co., Ltd.) with an enzyme activity of 10000 U / g and sodium borate are added to the first mixed enzymatic hydrolysate to carry out the second enzymatic hydrolysis reaction. The temperature of the second enzymatic hydrolysis reaction is 50℃ and the pH of the system is 6.0. The amount of room-temperature xylose isomerase added is 10 U / g of the dry basis of the first mixed enzymatic hydrolysate. The molar ratio of sodium borate to xylose in the first mixed enzymatic hydrolysate is 0.3:1. The second enzymatic hydrolysis reaction is carried out until the xylose conversion rate in the system is 95% and the xylose residue is 0.3wt%. After the second enzymatic hydrolysis reaction, the second mixed enzymatic hydrolysate is obtained. (3) Post-processing and refining: The second mixed enzymatic hydrolysate is kept at 95℃ for 10 min to inactivate the enzyme, and then successively passes through ceramic membrane microfiltration to remove slag (ceramic membrane pore size 0.2μm, temperature 50℃, transmembrane pressure 0.3MPa), ultrafiltration to remove macromolecular impurities (ultrafiltration membrane molecular weight cutoff 3000Da, temperature 45℃, transmembrane pressure 0.2MPa), nanofiltration concentration (nanofiltration membrane molecular weight cutoff 200Da, temperature 40℃, transmembrane pressure 3.0MPa), ion exchange desalination (using strong acid styrene cation resin (Zhejiang Zhengguang Industrial D001 type) and strong acid styrene cation resin (Zhejiang Zhengguang Industrial D201 type), feed flow rate 5BV / h, output conductivity ≤10μs / cm), decolorization (activated carbon addition 1‰, temperature 75℃), and spray drying (feed temperature 45℃, air inlet temperature 200℃, atomization pressure 3.0MPa) to obtain high-purity xylooligosaccharide product.

[0129] In the finished xylooligosaccharide product, the xylooligosaccharide yield is 53.1%, the purity of the high-purity xylooligosaccharide product is 74.4%, the proportion of xylooligosaccharides with a degree of polymerization of 2 to 6 is 78.8%, and the monosaccharide content is 2.5 wt%.

[0130] Some parameters of the above embodiments are listed in Table 1 below: Table 1

[0131] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A method for preparing high-purity xylooligosaccharides, characterized in that, Includes the following steps: Hemicellulose enzymatic hydrolysis: Agricultural and forestry by-products rich in hemicellulose are pretreated to obtain hemicellulose substrates. Xylanase is added to the hemicellulose substrates to carry out the first enzymatic hydrolysis reaction, generating a first mixed enzymatic hydrolysate containing xylan, xylooligosaccharides, xylose and glucose. In-situ conversion of glucose and xylose: Glucose oxidase, catalase, xylose isomerase and borate are added to the first mixed enzymatic hydrolysate to carry out a second enzymatic hydrolysis reaction and a third enzymatic hydrolysis reaction. In the second enzymatic hydrolysis reaction, the glucose is catalyzed to convert to sodium gluconate, and in the third enzymatic hydrolysis reaction, the xylose is catalyzed to convert to xylulose. The borate and xylulose form a stable complex in situ. After the second and third enzymatic hydrolysis reactions, a second mixed enzymatic hydrolysate is obtained. Post-processing and refining: The second mixed enzymatic hydrolysate is subjected to enzyme inactivation, filtration, concentration, ion exchange, and drying to obtain high-purity xylooligosaccharides.

2. The method according to claim 1, characterized in that, In the first enzymatic hydrolysis step, the hemicellulose-rich agricultural and forestry by-product is selected from any one or more of corn cobs, sugarcane bagasse, and straw.

3. The method according to claim 1, characterized in that, The pretreatment is steam explosion pretreatment, dilute organic acid pretreatment, or hot water extraction pretreatment.

4. The method according to claim 1, characterized in that, In the hemicellulose enzymatic hydrolysis step, the xylanase is a highly selective endo-β-1,4-xylanase.

5. The method according to claim 4, characterized in that, The xylanase activity is 5000~20000 U / g.

6. The method according to claim 4, characterized in that, The amount of xylanase added is 5~15 U / g of the dry basis of the hemicellulose substrate.

7. The method according to claim 1, characterized in that, When the first enzymatic hydrolysis reaction reaches equilibrium, glucose oxidase, catalase, xylose isomerase and borate are added to the first mixed enzymatic hydrolysate.

8. The method according to claim 1, characterized in that, The mass ratio of the added glucose oxidase to catalase is 1:(1~2).

9. The method according to claim 1, characterized in that, The total mass of the added glucose oxidase and catalase is 0.05% to 1.5% of the dry weight of the first mixed enzymatic hydrolysate.

10. The method according to claim 1, characterized in that, The xylose isomerase is a room-temperature xylose isomerase, with an applicable temperature range of 25~60℃.

11. The method according to claim 10, characterized in that, The activity of the xylose isomerase is 10,000~30,000 U / g.

12. The method according to claim 10, characterized in that, The amount of xylose isomerase added is 3~10 U / g of the dry basis of the first mixed enzymatic hydrolysate.

13. The method according to any one of claims 1 to 12, characterized in that, In the post-processing purification step, the second mixed enzymatic hydrolysate is subjected to enzyme inactivation, microfiltration, ultrafiltration, nanofiltration concentration, ion exchange, and drying to obtain high-purity xylooligosaccharides.

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