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CN-122012646-A - Method for preparing peanut chitosan oligosaccharide by xylanase method

CN122012646ACN 122012646 ACN122012646 ACN 122012646ACN-122012646-A

Abstract

The invention discloses a method for preparing peanut shell oligosaccharide by a xylanase method, which comprises four steps of peanut shell pretreatment, extraction of peanut shell polysaccharide, enzymolysis reaction of xylan and determination of peanut shell oligosaccharide, wherein mildew-free peanut shell raw materials are selected, impurities such as soil, stones and residual peanut kernels are removed, the peanut shells are repeatedly washed by clear water until the water quality is clear and free from turbidity, and the washed peanut shells are dried and crushed. The invention takes low-utilization peanut shells as raw materials, converts the low-utilization peanut shells into high-added-value oligosaccharides through directional degradation, solves the problems of resource waste and environmental pollution caused by discarding the peanut shells, improves the comprehensive economic benefit of the peanut processing industry, adopts a xylanase method for preparation, has mild reaction, strong specificity, high enzymolysis efficiency and uniform molecular weight distribution of products, avoids the defects of high energy consumption of chemical pollution and physical method, simplifies the preparation process steps, adopts conventional equipment, has low input cost, and is easy for industrialized large-scale production.

Inventors

  • WU QIONG
  • LI ZHENTAO
  • LI SHUANG
  • Du Junxiao
  • DAI YONGGANG

Assignees

  • 长春大学
  • 吉林省农业科学院(中国农业科技东北创新中心)

Dates

Publication Date
20260512
Application Date
20260206

Claims (9)

  1. 1. The method for preparing the peanut chitosan oligosaccharide by using the xylanase method is characterized by comprising the following steps of: (1) Pretreating peanut shells, namely selecting a mildew-free peanut shell raw material, removing impurities such as soil, stones and residual peanut kernels, cleaning the peanut shells with clear water for many times until the water quality is clear and free of turbidity, drying and crushing the cleaned peanut shells, sieving to obtain peanut shell powder with uniform particle size, and sealing and preserving the peanut shell powder for later use; (2) Uniformly mixing peanut shell powder obtained in the step (1) with a solvent according to a feed-liquid ratio of 1:30g/mL, performing acidification pretreatment by using acetic acid with a concentration of 4.50%, fully mixing with a NaOH solution with a mass concentration of 14%, reacting for 4min under a microwave-assisted condition, cooling and decompressing and filtering after the reaction is finished to realize solid-liquid separation, collecting initial filtrate and washing liquid, regulating the pH value of the filtrate to be neutral by using concentrated hydrochloric acid, dialyzing and purifying by using a dialysis bag after decompressing and concentrating, adding 95% ethanol with a volume of 3 times into the purifying liquid for alcohol precipitation, standing overnight, centrifuging to obtain precipitate, and re-dissolving by using distilled water to obtain a peanut shell polysaccharide solution; (3) Adding xylanase into the pretreatment liquid obtained in the step (2), controlling the adding amount of xylanase to be 300-700U/mL, maintaining the pH value of the system to be 8.0, and placing the system in a constant-temperature oscillation environment with the temperature of 50 ℃ and the oscillation rate of 200 rpm for enzymolysis; (4) And (3) determining the total sugar content by using a phenol sulfuric acid method, determining the reducing sugar content by using a DNS method, determining the molecular weight of the oligosaccharide by using a high performance liquid chromatography, and performing freeze drying treatment on the peanut chitosan oligosaccharide solution to obtain the peanut chitosan oligosaccharide finished product.
  2. 2. The method according to claim 1, wherein the number of times of cleaning with clean water in the step (1) is 3, the temperature of the drying treatment is controlled to be 60-80 ℃, the drying is carried out until the water content of the peanut shells is lower than 8%, and the peanut shells are crushed and then pass through a 80-100-mesh sieve, so that peanut shell powder with uniform particle size is obtained.
  3. 3. The method according to claim 1, wherein the power of the microwave-assisted reaction in step (2) is 300-500W, the pressure of the reduced pressure suction filtration is 0.06-0.08MPa, the dialysis purification time is 24-36h, and the dialysis external fluid is replaced every 8h.
  4. 4. The method of claim 1, wherein the xylanase in step (3) is added in an amount of 500U/mL, and the system is stirred every 30: 30min during the enzymolysis reaction for 1-2min to ensure sufficient contact between the enzyme and the substrate.
  5. 5. The method of claim 1, wherein the acetic acid acidification pretreatment in step (2) is carried out for 60 minutes with continuous stirring during acidification at a stirring rate of 100 to 150 rpm to ensure adequate contact of the peanut shell powder with the acetic acid solution.
  6. 6. The method according to claim 1, wherein the constant temperature oscillation environment in the step (3) is a constant temperature shaking table, the shaking mode of the shaking table is reciprocating oscillation, and the total duration of the enzymolysis reaction is 4-6h until the content of the reducing sugar in the system is not increased significantly any more.
  7. 7. The method according to claim 1, wherein the reduced pressure concentration in step (2) is carried out at a temperature of 45-50 ℃ and concentration is carried out to 1/5-1/4 of the volume of the original filtrate, and the vacuum degree is controlled to be 0.07-0.09MPa during concentration.
  8. 8. The method according to claim 1, wherein the high performance liquid chromatography in the step (4) is performed under the conditions that the chromatographic column is a gel permeation chromatographic column, the mobile phase is ultrapure water, the column temperature is 30 ℃, the flow rate is 0.8-1.0mL/min, the sample injection amount is 20 mu L, and the molecular weight of the oligosaccharide is calculated by a standard curve method.
  9. 9. The method according to any one of claims 1 to 9, wherein the peanut chitosan prepared has an average degree of polymerization of 2.93 and a molecular weight in the range of 421 to 4321Da, wherein the molecular weight of the product is 1292Da when the xylanase is added in an amount of 500U/mL.

Description

Method for preparing peanut chitosan oligosaccharide by xylanase method Technical Field The invention relates to the technical field of oligosaccharide preparation, in particular to a method for preparing peanut chitosan oligosaccharide by a xylanase method. Background Peanut is used as oil crop and economic crop with stable and forefront yield in China, a large amount of peanut shell byproducts are generated in the processing process, and the annual yield can reach tens of millions of tons. However, the current peanut shells are extremely low in utilization rate, most of the peanut shells are directly burnt or discarded at will, so that not only is serious waste of precious biomass resources caused, but also the problems of harmful gas generation due to burning, accumulation and environmental pollution caused by discarding and the like are caused, and extra pressure is brought to ecological environment treatment. Researches show that the peanut shell contains rich nutritional ingredients, wherein hemicellulose content is relatively high, the main ingredient of the hemicellulose is xylan, and the xylan can be used for preparing peanut shell oligosaccharide through directional degradation. The peanut chitosan is a low molecular weight saccharide compound formed by connecting 2-10 monosaccharide molecules through glycosidic bonds, has various excellent physiological activities, for example, can regulate the balance of intestinal flora of a human body, strengthen the immunity of the organism, inhibit the rise of blood sugar, play an antioxidant role and the like, has wide application prospect in various fields such as food processing, health-care product research and development, feed additives and the like, and has important economic value and social benefit in developing a high-efficiency peanut chitosan preparation technology. At present, the preparation methods of the oligosaccharides are mainly divided into three categories of chemical degradation method, physical degradation method and biological enzymolysis method. The chemical degradation method is a traditional mainstream preparation method, a strong acid solution such as hydrochloric acid and sulfuric acid is mostly adopted as a degradation agent, polysaccharide is degraded in a heating reflux mode, the physical degradation method mainly comprises modes such as ultrasonic-assisted degradation and microwave-assisted degradation, the method breaks glycosidic bonds through physical energy to reduce the molecular weight of the polysaccharide, the biological enzymolysis method takes advantages of mild reaction conditions, strong substrate specificity and the like as a preferential direction, xylanase is taken as a compound enzyme system capable of specifically breaking glycosidic bonds in xylan molecules, xylan in peanut shells can be directionally degraded into oligosaccharide, and the related technology is primarily applied in the field of oligosaccharide preparation, but the research on the xylanase preparation of peanut shell oligosaccharide is less. Although the physical degradation method can shorten the reaction time to a certain extent, the method has obvious limitations, on one hand, the energy consumption is extremely high, for example, the energy consumption of ultrasonic-assisted degradation is several times of that of the conventional method, the method does not accord with the industrial development trend of energy conservation and consumption reduction, on the other hand, the stability of the product is poor, the equipment investment required by large-scale production is huge, the production process is difficult to control, and the method is not beneficial to industrialized popularization. The chemical degradation method has a plurality of defects which are difficult to overcome, the reaction condition is severe, the prepared oligosaccharide is easy to cause uneven molecular weight distribution, the purity of the product is lower, toxic byproducts such as furfural and the like are also produced, the subsequent treatment difficulty is high, serious environmental pollution is easy to cause, the research results related to the preparation of the peanut chitosan by the existing xylanase method are obviously insufficient, the main manifestation is low enzymolysis efficiency, the oligosaccharide yield is generally low, the molecular weight distribution of the product is uneven, the quality and the application effect of the product are influenced, and the problems severely limit the industrialized application process of the technology. Disclosure of Invention The invention aims to provide a method for preparing peanut chitosan oligosaccharide by using a xylanase method, which aims to solve the problems in the background technology. In order to achieve the aim, the invention provides the following technical scheme that the method for preparing the peanut chitosan oligosaccharide by using the xylanase method comprises the following steps in sequence