Pretreatment method of raw materials for preparing xylooligosaccharide
By using an ultrasonic-assisted method involving amylase, protease, and a complex enzyme to process wheat bran, the problems of high energy consumption and pollution emissions in the wheat bran pretreatment process have been solved, the yield and purity of xylooligosaccharides have been improved, and clean and efficient xylooligosaccharide production has been achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MICROBIOLOGY INST OF SHAANXI
- Filing Date
- 2025-12-16
- Publication Date
- 2026-04-17
AI Technical Summary
Existing technologies for bran pretreatment suffer from high energy consumption, pollution emissions, and low efficiency, making it difficult to effectively break down the dense structure of bran, resulting in high production costs and low yields of xylooligosaccharides.
Pretreatment was performed using an ultrasound-assisted method involving amylase, protease, and a complex enzyme, including substrate preparation, removal of starch and protein, and loosening of cell wall structure by ultrasound with the complex enzyme. The synergistic effect of ferulic acid esterase and lignin-degrading enzyme was used to cleave and oxidize lignin, exposing the surface of hemicellulose and cellulose.
This method achieves clean and efficient bran pretreatment, reduces subsequent enzymatic hydrolysis costs, improves the accessibility of xylan and the yield of xylooligosaccharides, simplifies separation and purification steps, and conforms to green chemistry principles.
Smart Images
Figure CN121874285A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology and functional sugar preparation technology, specifically relating to a pretreatment method for raw materials for the preparation of xylooligosaccharides. Background Technology
[0002] Xylooligosaccharides are functional oligosaccharides with excellent prebiotic properties. They can effectively promote the proliferation of beneficial bacteria such as Bifidobacteria in the human gut and have broad application prospects in the food, health product, and pharmaceutical fields. Wheat bran, as a major by-product of grain processing, is rich in xylan and is an ideal and inexpensive raw material for producing xylooligosaccharides.
[0003] However, in the cell walls of wheat bran, xylan and cellulose microfibrils are tightly bound together by hydrogen bonds, and simultaneously cross-linked with lignin through ester and ether bonds formed by ferulic acid and other substances, forming a dense and complex lignocellulose network structure. This structure severely hinders the effective contact and degradation of the xylan backbone by endoxylanases, resulting in low yield, poor efficiency, and high cost in the direct enzymatic hydrolysis method for producing xylooligosaccharides.
[0004] To overcome this structural barrier, traditional pretreatment methods often employ physicochemical approaches such as acid treatment, alkali treatment, or steam explosion. While these methods can disrupt the structure to some extent, they have numerous drawbacks: acid and alkali treatments generate large amounts of wastewater, severely corrode equipment, and produce fermentation inhibitors such as furfural; steam explosions are energy-intensive and may lead to sugar degradation. None of these methods meet the requirements of green and sustainable modern industry.
[0005] Therefore, developing a pretreatment method that is mild, environmentally friendly, highly specific, and efficient in disrupting the dense structure of bran is of great significance for improving the yield of xylooligosaccharides, reducing production costs, and achieving cleaner production. Summary of the Invention
[0006] The purpose of this invention is to provide a pretreatment method for raw materials used in the preparation of xylooligosaccharides, comprising the following steps: Step 1: Substrate preparation; Step 2: Remove starch with amylase; Step 3: Protein removal using protease; Step 4: Use a compound enzyme and ultrasound to loosen the cell wall structure.
[0007] Furthermore, the specific process of step 1, substrate preparation, is as follows: according to the mass fraction, 1 part of crushed and sieved bran is mixed with 10 to 32 parts of boiling water and stirred evenly to obtain the pretreated substrate.
[0008] Furthermore, the specific process of step 2, starch removal with amylase, is as follows: the substrate obtained in step 1 is boiled in a water bath to gelatinize the starch for 5-15 minutes, the pH is adjusted to 6.5-7.5, and the temperature is 50-95℃; α-thermoresistant amylase preparation is added for enzymatic pretreatment, the amount of enzyme preparation added is 20-800 U / g substrate, the treatment time is 1-10 minutes, and the mixture is repeatedly rinsed with distilled water and filtered to obtain destarched bran.
[0009] Furthermore, the specific process of step 3, protein removal by protease, is as follows: according to the mass fraction, add 1 part of the destarched bran after enzyme treatment in step 2 to 6-10 parts of distilled water, stir evenly, adjust the pH to 7.0-8.0, and the temperature to 55-58℃; add plant protease enzyme preparation for enzymatic hydrolysis pretreatment, the amount of enzyme preparation added is 1%-2%, the treatment time is 8-24 hours, rinse repeatedly with distilled water, filter, and obtain deproteinized bran.
[0010] Furthermore, the specific process of step 4, the ultrasonic-assisted loosening of cell wall structure by compound enzyme, is as follows: Add 1 part by weight of the deproteinized bran treated with enzymes in step 3 to 10-30 parts of distilled water, stir evenly, adjust the pH to 5.0-7.0, and the temperature to 30-45℃; add 5-20 U / g of ferulic acid esterase substrate, and simultaneously add the supernatant of Bacillus pumilus fermentation broth for ultrasonic-assisted enzymatic hydrolysis pretreatment for 4-24 hours, boil for 5-10 minutes to inactivate the enzyme, adjust the pH to neutral with sodium hydroxide, filter, rinse the solid residue 3-5 times with 75% ethanol, repeatedly rinse with distilled water, filter, dry at 40℃-60℃, and grind into powder in a mortar to obtain a partially lignin-free, loosely structured, low-impurity, and finely refined bran pretreatment material.
[0011] Furthermore, the supernatant of the fermentation broth contains a lignin-degrading enzyme system, including lignin peroxidase, manganese peroxidase and laccase. The contents of the three enzymes in the supernatant are 900-4600 U / mL, 2500-12300 U / mL and 6-30 U / mL, respectively, and the enzyme addition amount is 6-60 U / g substrate.
[0012] Furthermore, the ultrasonic frequency of the ultrasonic synergy is 40kHz and the power is 360W.
[0013] The advantages of this invention are: the pretreatment method for the raw materials for preparing xylooligosaccharides provided by this invention has the following beneficial effects: 1. Clean and efficient: The enzymatic hydrolysis process is carried out in the aqueous phase, without the need for any strong acids or alkalis, resulting in no pollution emissions and conforming to the principles of green chemistry.
[0014] 2. Good impurity removal effect: In addition to cellulose and hemicellulose, the main components in wheat bran are starch and protein. Removing starch and protein before exposing xylan can effectively reduce impurities in the system, which facilitates xylan extraction and the separation and purification of xylooligosaccharides.
[0015] 3. High Specificity and Synergistic Effect: This invention creatively combines ferulic acid esterase and lignin-degrading enzyme. Ferulic acid esterase cleaves the ferulic acid ester bonds between xylan and lignin; lignin-degrading enzyme gently oxidizes and modifies some lignin, exposing the surface of hemicellulose and cellulose, making the cell structure more porous and improving the pretreatment effect. The two work synergistically to "disintegrate" the cross-linked structure of the cell wall from different dimensions, greatly improving the accessibility of xylan and laying a solid foundation for subsequent enzymatic hydrolysis.
[0016] 4. Improved economic efficiency: This pretreatment reduces the amount of endoxylanase used, shortens the enzymatic hydrolysis time, significantly improves the yield of xylooligosaccharides, and makes the overall process more economical. Attached Figure Description
[0017] Figure 1 This is a flowchart of the pretreatment method for the raw materials used in the preparation of xylooligosaccharides.
[0018] Figure 2 It is a picture of bran that is high in starch and protein and has a tight cell wall structure without any pretreatment.
[0019] Figure 3 This is a diagram of bran with low impurities and a loose cell wall structure after ultrasonic treatment with a compound enzyme.
[0020] Figure 4 These are graphs showing the starch removal effects under different treatment conditions.
[0021] Figure 5 These are graphs showing the protein removal effects under different treatment conditions.
[0022] Figure 6 This is a graph showing the hydrothermal extraction rate of xylan under different pretreatment schemes. Detailed Implementation
[0023] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description is provided in conjunction with the specific embodiments, structural features, and effects of the present invention.
[0024] The technical solutions of the present invention will be clearly and completely described below through embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0025] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.
[0026] Any feature disclosed in this specification (including any appended claims) may be replaced by other equivalent or similar features, unless specifically stated otherwise. That is, unless specifically stated otherwise, each feature is merely one example of a series of equivalent or similar features.
[0027] Example 1
[0028] One such Figure 1 The pretreatment method for the raw materials used in the preparation of xylooligosaccharides shown includes the following steps: Step 1: Substrate preparation; Step 2: Remove starch with amylase; Step 3: Protein removal using protease; Step 4: Use a compound enzyme and ultrasound to loosen the cell wall structure.
[0029] Furthermore, the specific process of step 1, substrate preparation, is as follows: according to the mass fraction, 1 part of crushed and sieved bran is mixed with 10 to 32 parts of boiling water and stirred evenly to obtain the pretreated substrate.
[0030] Furthermore, the specific process of step 2, starch removal with amylase, is as follows: the substrate obtained in step 1 is boiled in a water bath to gelatinize the starch for 5-15 minutes, the pH is adjusted to 6.5-7.5, and the temperature is 50-95℃; α-thermoresistant amylase preparation is added for enzymatic pretreatment, the amount of enzyme preparation added is 20-800 U / g substrate, the treatment time is 1-10 minutes, and the mixture is repeatedly rinsed with distilled water and filtered to obtain destarched bran. Figure 4 The images show the starch removal effects under different treatment conditions. Group 1 consists of untreated wheat bran; Group 2 consists of wheat bran treated with 80 U / g substrate at 80℃ for 5 min; Group 3 consists of wheat bran treated with 70 U / g substrate at 80℃ for 10 min; Group 4 consists of wheat bran treated with 80 U / g substrate at 80℃ for 10 min; and Group 5 consists of wheat bran treated with 70 U / g substrate at 60℃ for 10 min. All enzymatic hydrolysis was performed at the original pH range of 6.5–7.0. The starch removal rates for groups 2–5 were 79.96%, 90.41%, 96.66%, and 77.87%, respectively.
[0031] Furthermore, the specific process of step 3, protein removal by protease, is as follows: according to the mass fraction, add 1 part of the destarched bran after enzyme treatment in step 2 to 6-10 parts of distilled water, stir evenly, adjust the pH to 7.0-8.0, and the temperature to 55-58℃; add plant protease enzyme preparation for enzymatic hydrolysis pretreatment, the amount of enzyme preparation added is 1%-2%, the treatment time is 8-24 hours, rinse repeatedly with distilled water, filter, and obtain deproteinized bran. Figure 5 The images show the protein removal effects under different treatment conditions. Group 1 consisted of untreated bran; Group 2 consisted of 1% protease hydrolysed at 55°C for 4 hours; Group 3 consisted of 1% protease hydrolysed at 55°C for 12 hours; Group 4 consisted of 1% protease hydrolysed at 55°C for 16 hours; and Group 5 consisted of 1% protease hydrolysed at 55°C for 8 hours. The enzymatic hydrolysis pH for all groups was 7.5. The protein removal rates for groups 2-5 were 62.83%, 69.96%, 74.03%, and 67.92%, respectively.
[0032] Furthermore, the specific process of step 4, the ultrasonic-assisted loosening of cell wall structure by compound enzyme, is as follows: Add 1 part by weight of the deproteinized bran treated with enzymes in step 3 to 10-30 parts of distilled water, stir evenly, adjust the pH to 5.0-7.0, and the temperature to 30-45℃; add 5-20 U / g of ferulic acid esterase substrate, and simultaneously add the supernatant of Bacillus pumilus fermentation broth for ultrasonic-assisted enzymatic hydrolysis pretreatment for 4-24 hours, boil for 5-10 minutes to inactivate the enzyme, adjust the pH to neutral with sodium hydroxide, filter, rinse the solid residue 3-5 times with 75% ethanol, repeatedly rinse with distilled water, filter, dry at 40℃-60℃, and grind into powder in a mortar to obtain a partially lignin-free, loosely structured, low-impurity, and finely refined bran pretreatment material. Figure 6 This is a graph showing the hydrothermal extraction rates of xylan under different pretreatment schemes. Group 1 represents xylan obtained by first treating with amylase, then ultrasonically treating with lignin-degrading enzyme, followed by high-temperature hydrothermal extraction; Group 2 represents xylan obtained by first treating with amylase, then with protease, then ultrasonically grinding with ferulic acid esterase combined with lignin-degrading enzyme, followed by high-temperature hydrothermal extraction; Group 3 represents xylan obtained by first treating with amylase, then with protease, then ultrasonically grinding with lignin-degrading enzyme, followed by high-temperature hydrothermal extraction; Group 4 represents xylan obtained by directly high-temperature hydrothermal extraction after protease treatment alone; Group 5 represents xylan obtained by directly high-temperature hydrothermal extraction after amylase treatment alone; and Group 6 represents xylan obtained by direct hydrothermal extraction without any enzyme treatment. The xylan extraction rates for groups 1-6 are 69.54%, 85.3%, 78.66%, 9.98%, 10.01%, and 8.74%, respectively.
[0033] The pretreatment method for the raw materials of this xylooligosaccharide preparation involves adding lignin-degrading enzymes and ferulic acid esterase during the ultrasonic stage. This process degrades some lignin, breaking the chemical bonds between hemicellulose and lignin. Simultaneously, ultrasonication loosens the cell structure, maximizing xylan dissolution during high-pressure extraction. This significantly improves both the extraction rate and subsequent enzymatic hydrolysis rate, while also resulting in a low overall impurity content. This pretreatment method first employs high-temperature, high-pressure hydrothermal extraction followed by enzymatic hydrolysis. This avoids the enzyme inactivation issues associated with direct high-pressure enzymatic hydrolysis in existing technologies, and also avoids the problems of complex hydrolysis products and difficulty in separation and purification resulting from direct ultrasonic addition of mixed enzymes such as xylanase. The step-by-step pretreatment of the raw materials simplifies subsequent separation and purification steps by removing impurities at each step. Figure 2 The image shown is of untreated bran, high in starch and protein, with a tight cell wall structure. Figure 3 The diagram shows bran with low impurities and loose cell wall structure after ultrasonic treatment with compound enzymes.
[0034] Furthermore, the supernatant of the fermentation broth contains a lignin-degrading enzyme system, including lignin peroxidase, manganese peroxidase and laccase. The contents of the three enzymes in the supernatant are 900-4600 U / mL, 2500-12300 U / mL and 6-30 U / mL, respectively, and the enzyme addition amount is 6-60 U / g substrate.
[0035] Furthermore, the ultrasonic frequency of the ultrasonic synergy is 40kHz and the power is 360W.
[0036] The pretreatment method for the raw materials for preparing xylooligosaccharides provided herein has the following advantages: 1. Clean and efficient: The enzymatic hydrolysis process is carried out in the aqueous phase, without the need for any strong acids or alkalis, resulting in no pollution emissions and conforming to the principles of green chemistry.
[0037] 2. Good impurity removal effect: In addition to cellulose and hemicellulose, the main components in wheat bran are starch and protein. Removing starch and protein before exposing xylan can effectively reduce impurities in the system, which facilitates xylan extraction and the separation and purification of xylooligosaccharides.
[0038] 3. High Specificity and Synergistic Effect: This invention creatively combines ferulic acid esterase and lignin-degrading enzyme. Ferulic acid esterase cleaves the ferulic acid ester bonds between xylan and lignin; lignin-degrading enzyme gently oxidizes and modifies some lignin, exposing the surface of hemicellulose and cellulose, making the cell structure more porous and improving the pretreatment effect. The two work synergistically to "disintegrate" the cross-linked structure of the cell wall from different dimensions, greatly improving the accessibility of xylan and laying a solid foundation for subsequent enzymatic hydrolysis.
[0039] 4. Improved economic efficiency: This pretreatment reduces the amount of endoxylanase used, shortens the enzymatic hydrolysis time, significantly improves the yield of xylooligosaccharides, and makes the overall process more economical.
[0040] Example 2
[0041] Take 25g of pulverized and sieved wheat bran, add 500mL of boiling water, stir well, and heat in a boiling water bath for 15min to gelatinize the starch. Add 2000U of α-thermoresistant amylase, react at 80℃ for 10min, rinse 5 times with distilled water, filter, and discard the filtrate. Add 200mL of distilled water, adjust the pH to 7.5 with 5% NaOH, add 2.5g of plant protease preparation, and hydrolyze at 55℃ for 16h. Rinse the enzymatic hydrolysate 3 times with distilled water, filter, and discard the filtrate. Add 500mL of pH 5.0 phosphate buffer (0.05Mcitrate-phosphate buffer), stir well, and heat at 45℃. Add 375U of ferulic acid esterase, and simultaneously add the supernatant of Bacillus fermentation broth containing 750U, 750U, and 1250U of lignin peroxidase, manganese peroxidase, and laccase, respectively. Sonicate at 40kHz and 360W for 16h. Boil for 8 minutes to inactivate the enzyme, rinse 3 times, adjust the pH to neutral with 5% sodium hydroxide, and filter. The solid residue is rinsed 3 times with 75% ethanol, then rinsed 5 times with distilled water, filtered, dried at 50℃ for 48 hours, and ground into powder in a mortar to obtain a partially lignin-free, loosely structured, low-impurity, and refined bran pretreated material, which is used for subsequent high-temperature and high-pressure hydrothermal extraction of xylan and enzymatic hydrolysis to prepare xylooligosaccharides.
[0042] Example 3
[0043] Compared with Example 2, no compound enzyme pretreatment was performed. Instead, wheat bran was directly added to water and subjected to high temperature and high pressure hydrothermal extraction of xylan, which was then used for enzymatic hydrolysis to prepare xylooligosaccharides.
[0044] Effect verification The products obtained in Examples 2 and 3, with untreated bran containing 25.3% starch and 19.64% protein, and bran without starch containing 1.7% starch and bran without protein containing 4.8% protein, were subjected to hydrothermal extraction at 121°C for 20 min at a ratio of 9% for both types of bran. The xylan extraction rate was measured. Under the same conditions (pH 8.0, 55°C), the same amount of endoxylanase was added for enzymatic hydrolysis for 12 hours. The yield of the final xylooligosaccharides (XOS, calculated as xylobiose to xylohexaose) was determined.
[0045] Results: The xylan extraction rate in Example 2 reached 85% (based on a xylan content of 21.7% in the raw material), and the XOS yield from enzymatic hydrolysis reached 68% (based on the xylan content in the raw material), while the xylan extraction rate in Example 3 was only 10%, and the XOS yield was only about 6.5%. This indicates that the compound enzyme pretreatment method of the present invention can significantly improve the yield and purity of xylooligosaccharides.
[0046] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.
Claims
1. A pretreatment method for raw materials used in the preparation of xylooligosaccharides, characterized in that, Includes the following steps: Step 1: Substrate preparation; Step 2: Remove starch with amylase; Step 3: Protein removal using protease; Step 4: Use a complex enzyme and ultrasound to loosen the cell wall structure.
2. The pretreatment method for the raw materials for preparing xylooligosaccharides as described in claim 1, characterized in that: The specific process of step 1, substrate preparation, is as follows: according to the mass fraction, 1 part of crushed and sieved bran is mixed with 10 to 32 parts of boiling water and stirred evenly to obtain the pretreated substrate.
3. The pretreatment method for the raw materials for preparing xylooligosaccharides as described in claim 1, characterized in that: The specific process of step 2, starch removal with amylase, is as follows: the substrate obtained in step 1 is boiled in a water bath to gelatinize the starch for 5-15 minutes, the pH is adjusted to 6.5-7.5, and the temperature is 50-95℃; α-thermo-resistant amylase preparation is added for enzymatic pretreatment, the amount of enzyme preparation added is 20-800 U / g substrate, the treatment time is 1-10 minutes, and the mixture is repeatedly rinsed with distilled water and filtered to obtain destarched bran.
4. The pretreatment method for the raw materials for preparing xylooligosaccharides as described in claim 1, characterized in that: The specific process of step 3, protein removal with protease, is as follows: according to the mass ratio, add 1 part of the destarched bran after enzyme treatment in step 2 to 6-10 parts of distilled water, stir evenly, adjust the pH to 7.0-8.0, and the temperature to 55-58℃; add plant protease enzyme preparation for enzymatic hydrolysis pretreatment, the amount of enzyme preparation added is 1%-2%, the treatment time is 8-24 hours, rinse repeatedly with distilled water, filter, and obtain deproteinized bran.
5. The pretreatment method for the raw materials for preparing xylooligosaccharides as described in claim 1, characterized in that: The specific process of step 4, the ultrasonic-assisted loosening of cell wall structure by compound enzyme, is as follows: Add 1 part by weight of the deproteinized bran treated with enzymes in step 3 to 10-30 parts by weight of distilled water, stir evenly, adjust the pH to 5.0-7.0, and the temperature to 30-45℃; add 5-20 U / g of ferulic acid esterase substrate, and simultaneously add the supernatant of Bacillus pumilus fermentation broth for ultrasonic-assisted enzymatic pretreatment for 4-24 hours. Boil for 5-10 minutes to inactivate the enzyme, adjust the pH to neutral with sodium hydroxide, filter, rinse the solid residue 3-5 times with 75% ethanol, repeatedly rinse with distilled water, filter, dry at 40℃-60℃, and grind into powder in a mortar to obtain a partially lignin-free, loosely structured, low-impurity, and refined bran pretreated material.
6. The pretreatment method for the raw materials for preparing xylooligosaccharides as described in claim 5, characterized in that: The supernatant of the fermentation broth contains a lignin-degrading enzyme system, including lignin peroxidase, manganese peroxidase and laccase. The contents of the three enzymes in the supernatant are 900-4600 U / mL, 2500-12300 U / mL and 6-30 U / mL, respectively, and the enzyme addition amount is 6-60 U / g substrate.
7. The pretreatment method for the raw materials for preparing xylooligosaccharides as described in claim 5, characterized in that: The ultrasonic frequency of the ultrasonic synergy is 40kHz and the power is 360W.