Method for preparing peanut shell oligosaccharide by xylanase method

Abstract

The invention discloses a method for preparing peanut shell oligosaccharide by a xylanase method. The method comprises four steps of pretreatment of peanut shells, extraction of peanut shell polysaccharide, enzymolysis reaction of xylanase and determination of the peanut shell oligosaccharide. The method comprises the following steps: selecting a mildew-free peanut shell raw material, removing impurities such as soil, stones and residual peanut kernels, cleaning with clear water for multiple times until the water is clear and free of turbidity, and drying and crushing the cleaned peanut shells; the peanut shells with low utilization rate are used as raw materials and are converted into oligosaccharide with high added value through directional degradation, the problems of resource waste and environmental pollution caused by discarding of the peanut shells are solved, the comprehensive economic benefits of the peanut processing industry are improved, the xylanase method is adopted for preparation, the reaction is mild, the specificity is high, the enzymolysis efficiency is high, and the molecular weight distribution of products is uniform. The defects of pollution of a chemical method and high energy consumption of a physical method are avoided; the preparation process is simple in steps, required equipment is conventional equipment, the investment cost is low, and industrial large-scale production is easy to realize.

Description

Technical Field

[0001] This invention relates to the field of oligosaccharide preparation technology, specifically a method for preparing peanut shell oligosaccharides using xylanase. Background Technology

[0002] Peanuts, as one of my country's top-producing oilseed and cash crops, generate a large amount of peanut shells as a byproduct during processing, with an annual output reaching tens of millions of tons. However, the utilization rate of peanut shells is currently extremely low, with the vast majority being directly burned or discarded at will. This not only causes a serious waste of valuable biomass resources but also creates additional pressure on ecological and environmental governance due to the harmful gases produced by burning and the environmental pollution caused by dumping and accumulation.

[0003] Studies have found that peanut shells are rich in nutrients, with a high proportion of hemicellulose, the main component of which is xylan. Xylan can be converted into peanut shell oligosaccharides through targeted degradation. Peanut shell oligosaccharides are a class of low-molecular-weight sugar compounds formed by 2-10 monosaccharide molecules linked by glycosidic bonds. They possess a variety of excellent physiological activities, such as regulating the balance of human intestinal flora, enhancing the body's immune function, inhibiting the rise in blood sugar, and exerting antioxidant effects. They have broad application prospects in food processing, health product development, feed additives, and other fields. Developing efficient peanut shell oligosaccharide preparation technology has significant economic and social benefits.

[0004] Currently, the preparation methods for oligosaccharides are mainly classified into three categories: chemical degradation, physical degradation, and enzymatic hydrolysis. Chemical degradation is the traditional mainstream method, often using strong acid solutions such as hydrochloric acid and sulfuric acid as degrading agents, and degrading polysaccharides through heating and reflux. Physical degradation mainly includes ultrasonic-assisted degradation and microwave-assisted degradation, which use physical energy to break glycosidic bonds to reduce the molecular weight of polysaccharides. Enzymatic hydrolysis has become the preferred method due to its mild reaction conditions and strong substrate specificity. Xylanase, as a complex enzyme system capable of specifically breaking glycosidic bonds in xylan molecules, can directionally degrade xylan in peanut shells into oligosaccharides. Related technologies have been initially applied in the field of oligosaccharide preparation, but research on the xylanase method for preparing peanut shell oligosaccharides is limited.

[0005] While physical degradation methods can shorten reaction time to some extent, they have obvious limitations. On the one hand, they consume a lot of energy; for example, the energy consumption of ultrasound-assisted degradation is several times that of conventional methods, which does not conform to the industrial development trend of energy conservation and emission reduction. On the other hand, the product stability is poor, and the equipment investment required for large-scale production is huge, and the production process is difficult to control, which is not conducive to industrial promotion.

[0006] Chemical degradation methods have many insurmountable drawbacks. Not only are the reaction conditions harsh, leading to uneven molecular weight distribution of the prepared oligosaccharides and low product purity, but they also produce toxic byproducts such as furfural, making subsequent treatment difficult and causing serious environmental pollution. Existing research on the preparation of peanut shell oligosaccharides using xylanase also has significant shortcomings, mainly manifested in low enzymatic hydrolysis efficiency, generally low oligosaccharide yield, and uneven molecular weight distribution of the products, affecting product quality and application effects. These problems seriously limit the industrial application of this technology. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing peanut shell oligosaccharides using xylanase, so as to solve the problems in the background art mentioned above.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing peanut shell oligosaccharides using a xylanase method, comprising the following steps performed sequentially:

[0009] (1) Peanut shell pretreatment: Select peanut shell raw materials without mold, remove impurities such as soil, stones, and residual peanut kernels, wash with clean water multiple times until the water is clear and turbid, dry and crush the washed peanut shells, sieve to obtain peanut shell powder with uniform particle size, and seal and store for later use.

[0010] (2) Extraction of peanut shell polysaccharide: The peanut shell powder obtained in step (1) was mixed with the solvent at a ratio of 1:30 g / mL. It was first acidified and pretreated with 4.50% acetic acid, and then fully mixed with 14% NaOH solution. The mixture was reacted for 4 min under microwave-assisted conditions. After the reaction, the solid-liquid separation was achieved by cooling and vacuum filtration. The initial filtrate and washing liquid were collected. The pH of the filtrate was adjusted to neutral with concentrated hydrochloric acid. After vacuum concentration, the filtrate was purified by dialysis using a dialysis bag. Three times the volume of 95% ethanol was added to the purified solution for alcohol precipitation. After standing overnight, the solution was redissolved with distilled water to obtain peanut shell polysaccharide solution.

[0011] (3) Xylan enzymatic hydrolysis reaction: Add xylanase to the pretreatment solution obtained in step (2), control the amount of xylanase added to be 300-700U / mL, maintain the pH value of the system at 8.0, and place the system in a constant temperature shaking environment at 50℃ and a shaking rate of 200rpm for enzymatic hydrolysis reaction;

[0012] (4) Determination of oligosaccharides and preparation of finished products: The total sugar content was determined by the phenol-sulfuric acid method and the reducing sugar content was determined by the DNS method. The molecular weight of oligosaccharides was determined by high performance liquid chromatography. The concentrated solution after separation and purification was freeze-dried to obtain peanut shell oligosaccharide finished products.

[0013] As a preferred embodiment of the present invention, the washing with clean water in step (1) is performed 3 times, the drying temperature is controlled at 60-80℃, the peanut shell moisture content is dried to less than 8%, and after crushing, it is passed through an 80-100 mesh sieve to obtain peanut shell powder with uniform particle size.

[0014] As a preferred embodiment of the present invention, the power of the microwave-assisted reaction in step (2) is 300-500W, the pressure of vacuum filtration is 0.06-0.08MPa, the dialysis purification time is 24-36h, and the dialysis fluid is replaced every 8h during the process.

[0015] As a preferred embodiment of the present invention, the amount of xylanase added in step (3) is 500 U / mL, and the system is stirred once every 30 minutes during the enzymatic hydrolysis reaction for 1-2 minutes to ensure that the enzyme and substrate are in full contact.

[0016] As a preferred embodiment of the present invention, the acetic acidification pretreatment in step (2) takes 15-20 minutes, and the mixture is continuously stirred during the acidification process at a stirring rate of 100-150 rpm to ensure that the peanut shell powder and the acetic acid solution are in full contact.

[0017] As a preferred embodiment of the present invention, the isothermal oscillation environment in step (3) is an isothermal shaker, the oscillation mode of the shaker is reciprocating oscillation, and the total duration of the enzymatic hydrolysis reaction is 4-6 hours until the reducing sugar content in the system no longer increases significantly.

[0018] As a preferred embodiment of the present invention, the vacuum concentration in step (2) is carried out at a temperature of 45-50°C, and the concentration is carried out to 1 / 5-1 / 4 of the original filtrate volume. During the concentration process, the vacuum degree is controlled at 0.07-0.09 MPa.

[0019] As a preferred embodiment of the present invention, the determination conditions for high performance liquid chromatography in step (4) are as follows: the chromatographic column is a gel permeation chromatography column, the mobile phase is ultrapure water, the column temperature is 30℃, the flow rate is 0.8-1.0 mL / min, the injection volume is 20 μL, and the molecular weight of oligosaccharides is calculated by the standard curve method.

[0020] As a preferred embodiment of the present invention, the prepared peanut shell oligosaccharide has an average degree of polymerization of 2.93 and a molecular weight range of 421-4321 Da, wherein when the amount of xylanase added is 500 U / mL, the molecular weight of the product is 1292 Da.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. This invention uses peanut shells, a byproduct with huge output and extremely low utilization rate, as raw materials and transforms them into high-value-added peanut shell oligosaccharide products through targeted degradation technology. This not only effectively solves the problem of resource waste and environmental pollution caused by discarding peanut shells, but also significantly improves the overall economic benefits of the peanut processing industry, and has the dual significance of resource conservation and environmental protection.

[0023] 2. This invention uses xylanase to prepare oligosaccharides. The reaction conditions are mild, the enzymatic hydrolysis temperature is only 50℃, and the pH value of the system is stable at 8.0. No harsh reaction conditions are required, which effectively avoids the environmental pollution problems caused by strong acids and alkalis in chemical methods, and also overcomes the drawback of excessive energy consumption in physical methods. In addition, xylanase has extremely high specificity for xylan, high enzymatic hydrolysis efficiency, and can directionally degrade xylan to generate oligosaccharides. The molecular weight distribution of the products is uniform (421-4321 Da), and the average degree of polymerization is 2.93.

[0024] 3. The preparation process in this invention has only four core steps, which are simple and clear, and easy to operate and master. At the same time, the equipment required for the process are all conventional equipment in the fields of agricultural product deep processing and food processing, without the need for special high-end equipment. The equipment investment cost is low and it is easy to achieve industrial-scale production. Attached Figure Description

[0025] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] In the description of this invention, it should be understood that the terms "length," "width," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, in the description of this invention, "first," "second," "third," and "fourth" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0028] In the description of this invention, it should also be noted that, unless otherwise explicitly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or a connection through an intermediate medium; and they can refer to the internal communication between two components. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances. "Multiple" means two or more, and unless otherwise explicitly limited, all such meanings fall within the scope of protection of this invention.

[0029] Example 1

[0030] 1. Peanut shell pretreatment: Select peanut shells free of mold, remove impurities such as soil, stones, and residual peanut kernels, and wash them three times with clean water until the water is clear and free of turbidity. Place the washed peanut shells in a 70℃ oven to dry until the moisture content is below 8%, then crush them and pass them through a 90-mesh sieve to obtain peanut shell powder with uniform particle size. Seal and store for later use.

[0031] 2. Extraction of peanut shell polysaccharides: The peanut shell powder and solvent were mixed evenly at a material-to-liquid ratio of 1:30 g / mL. First, 4.50% acetic acid was added, and the mixture was pretreated by acidification at 120 rpm for 18 min. Then, 14% NaOH solution was added and thoroughly mixed. The mixture was placed under 400W microwave power for 4 min to assist the reaction. After the reaction, the temperature was lowered, and solid-liquid separation was achieved by vacuum filtration at 0.07 MPa. The initial filtrate and washings were collected. The pH of the filtrate was adjusted to neutral with concentrated hydrochloric acid, and concentrated under reduced pressure at 48℃ and 0.08 MPa to 1 / 4 of the original filtrate volume. The solution was then purified by dialysis using a dialysis bag for 30 h, with the dialysis fluid replaced every 8 h. Three volumes of 95% ethanol were added to the purified solution for alcohol precipitation. After standing overnight, the solution was reconstituted with distilled water to obtain a peanut shell polysaccharide solution.

[0032] 3. Xylan enzymatic hydrolysis: Xylanase was added to the peanut shell polysaccharide pretreatment solution, with the addition amount controlled at 500 U / mL, and the pH of the system was maintained stable at 8.0. The system was placed in a reciprocating constant temperature shaker at 50℃ and a shaking speed of 200 rpm for enzymatic hydrolysis. The total reaction time was 5 hours. During the reaction, the system was stirred for 1.5 minutes every 30 minutes to ensure sufficient contact between the enzyme and the substrate.

[0033] 4. Oligosaccharide Determination and Finished Product Preparation: The total sugar content was determined by the phenol-sulfuric acid method, and the reducing sugar content was determined by the DNS method. The molecular weight of oligosaccharides was determined by high-performance liquid chromatography (HPLC) (chromatographic conditions: gel permeation column, mobile phase: ultrapure water, column temperature: 30℃, flow rate: 0.9 mL / min, injection volume: 20 μL, molecular weight calculated using the standard curve method). The concentrated solution after separation and purification was freeze-dried to obtain the peanut shell oligosaccharide product.

[0034] Beneficial Effects: This embodiment uses the xylanase addition amount (500 U / mL) recommended in the claims, combined with reciprocating isothermal oscillation and periodic stirring, which significantly improves the contact efficiency between the enzyme and the substrate, resulting in a more complete enzymatic hydrolysis reaction. The final peanut shell oligosaccharide has an average degree of polymerization of 2.93 and a molecular weight of 1292 Da, which is within the excellent range of 421-4321 Da. Meanwhile, key process parameters such as microwave power, reduced pressure concentration parameters, and dialysis time are all selected from the median values ​​within the range defined in the claims, ensuring strong process stability and effectively reducing the risk of parameter fluctuations during production. Furthermore, precise control of drying temperature and sieve mesh during peanut shell pretreatment improves raw material utilization and reduces the difficulty of subsequent extraction, making it suitable for large-scale production applications.

[0035] Example 2

[0036] 1. Peanut shell pretreatment: Select peanut shells free of mold, remove impurities, and wash them three times with clean water until the water is clear. Place the washed peanut shells in a 65℃ oven to dry until the moisture content is below 8%, then crush them and pass them through an 80-mesh sieve to collect peanut shell powder with uniform particle size. Seal and store for later use.

[0037] 2. Extraction of peanut shell polysaccharides: Peanut shell powder and solvent were mixed at a material-to-liquid ratio of 1:30 g / mL. 4.50% acetic acid was added, and the mixture was pretreated with acidification at 100 rpm for 1 hour. Then, 14% NaOH solution was added and mixed thoroughly. The mixture was then reacted under 350 W microwave power for 4 minutes. After the reaction, the mixture was cooled and filtered under reduced pressure at 0.06 MPa. The pH of the filtrate was adjusted to neutral, and then concentrated to 1 / 5 of the original filtrate volume at 45℃ and 0.07 MPa vacuum. The filtrate was purified by dialysis for 24 hours, with the external solution replaced every 8 hours. Three volumes of 95% ethanol were added for precipitation. After standing overnight, the mixture was centrifuged and reconstituted with distilled water to obtain the peanut shell polysaccharide solution.

[0038] 3. Xylan enzymatic hydrolysis: Add xylanase to the pretreatment solution at a rate of 350 U / mL, maintain the pH of the system at 8.0, and place it in a reciprocating constant temperature shaker at 50℃ and 200 rpm for 4.5 h of enzymatic hydrolysis, stirring for 1 min every 30 min to ensure uniform enzymatic hydrolysis.

[0039] 4. Oligosaccharide determination and product preparation: The total sugar and reducing sugar content were determined by the phenol-sulfuric acid method and the DNS method, respectively. The molecular weight was determined by high performance liquid chromatography (HPLC) (chromatographic conditions: gel permeation column, ultrapure water as mobile phase, column temperature 30℃, flow rate 0.8 mL / min, injection volume 20 μL). The purified concentrate was freeze-dried to obtain the peanut shell oligosaccharide product.

[0040] Beneficial Effects: This embodiment selects a relatively low xylanase dosage (350 U / mL), effectively reducing the cost of enzyme preparations while ensuring enzymatic hydrolysis, resulting in better economic benefits. The process parameters are selected near the lower limit of the range defined in the claims, such as microwave power 350 W and dialysis time 24 h, which reduces energy consumption and production cycle, improving production efficiency. The final prepared peanut shell oligosaccharides have a molecular weight range of 512-4186 Da, which meets the optimal range defined in the claims, and the product has high purity and stable total sugar content. Simultaneously, the use of a lower drying temperature and a coarser sieve during pretreatment reduces equipment energy consumption and pulverization difficulty, making it suitable for small- to medium-sized production applications, balancing product quality and production economy.

[0041] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for preparing peanut shell oligosaccharides via xylanase, characterized in that, The following steps are performed sequentially: (1) Peanut shell pretreatment: Select peanut shell raw materials without mold, remove impurities such as soil, stones, and residual peanut kernels, wash with clean water multiple times until the water is clear and turbid, dry and crush the washed peanut shells, sieve to obtain peanut shell powder with uniform particle size, and seal and store for later use. (2) Extraction of peanut shell polysaccharide: The peanut shell powder obtained in step (1) was mixed with the solvent at a ratio of 1:30 g / mL. It was first acidified and pretreated with 4.50% acetic acid, and then fully mixed with 14% NaOH solution. The mixture was reacted for 4 min under microwave-assisted conditions. After the reaction, the mixture was cooled and filtered under reduced pressure to achieve solid-liquid separation. The initial filtrate and washing liquid were collected. The pH of the filtrate was adjusted to neutral with concentrated hydrochloric acid. After concentration under reduced pressure, the filtrate was purified by dialysis using a dialysis bag. Three times the volume of 95% ethanol was added to the purified solution for alcohol precipitation. After standing overnight, the precipitate was centrifuged and reconstituted with distilled water to obtain peanut shell polysaccharide solution. (3) Xylan enzymatic hydrolysis reaction: Add xylanase to the pretreatment solution obtained in step (2), control the amount of xylanase added to be 300-700 U / mL, maintain the pH value of the system at 8.0, and place the system in a constant temperature shaking environment at 50℃ and a shaking rate of 200 rpm for enzymatic hydrolysis reaction; (4) Determination of oligosaccharides and preparation of finished products: The total sugar content was determined by the phenol-sulfuric acid method and the reducing sugar content was determined by the DNS method. The molecular weight of oligosaccharides was determined by high performance liquid chromatography. The peanut shell oligosaccharide solution was freeze-dried to obtain the peanut shell oligosaccharide finished product.

2. The method according to claim 1, characterized in that, The washing with clean water in step (1) is performed 3 times. The drying temperature is controlled at 60-80℃. The peanut shells are dried until the moisture content is less than 8%. After crushing, they are passed through an 80-100 mesh sieve to obtain peanut shell powder with uniform particle size.

3. The method according to claim 1, characterized in that, The power of the microwave-assisted reaction in step (2) is 300-500W, the pressure of vacuum filtration is 0.06-0.08MPa, the dialysis purification time is 24-36h, and the dialysis fluid is replaced every 8h.

4. The method according to claim 1, characterized in that, The amount of xylanase added in step (3) is 500 U / mL. During the enzymatic hydrolysis reaction, the system is stirred once every 30 min for 1-2 min to ensure that the enzyme and substrate are in full contact.

5. The method according to claim 1, characterized in that, The acetic acidification pretreatment in step (2) takes 60 minutes. During the acidification process, the mixture is continuously stirred at a speed of 100-150 rpm to ensure that the peanut shell powder and the acetic acid solution are in full contact.

6. The method according to claim 1, characterized in that, The isothermal oscillation environment described in step (3) is an isothermal shaker, the oscillation mode of the shaker is reciprocating oscillation, and the total duration of the enzymatic hydrolysis reaction is 4-6 hours until the reducing sugar content in the system no longer increases significantly.

7. The method according to claim 1, characterized in that, The vacuum concentration in step (2) is carried out at a temperature of 45-50℃, and the concentration is carried out to 1 / 5-1 / 4 of the original filtrate volume. During the concentration process, the vacuum degree is controlled at 0.07-0.09MPa.

8. The method according to claim 1, characterized in that, The determination conditions for high performance liquid chromatography in step (4) are as follows: the chromatographic column is a gel permeation chromatography column, the mobile phase is ultrapure water, the column temperature is 30℃, the flow rate is 0.8-1.0 mL / min, the injection volume is 20 μL, and the molecular weight of oligosaccharides is calculated by the standard curve method.

9. The method according to any one of claims 1-9, characterized in that, The prepared peanut shell oligosaccharides had an average degree of polymerization of 2.93 and a molecular weight range of 421-4321 Da. When the amount of xylanase added was 500 U / mL, the molecular weight of the product was 1292 Da.

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