CN108588150A - Batch feeding-oxidation pre-treatment auxiliary enzymes hydrolyzing chitosan prepares chitosan oligosaccharide method - Google Patents

Abstract

Batch feeding oxidation pre-treatment auxiliary enzymes hydrolyzing chitosan prepares chitosan oligosaccharide method, is related to the preparation method of low-molecular weight chitoglycan.The batch feeding oxidative degradation of chitosan pre-processes, and obtains degradation solution；Hydrogen peroxide removes；The enzymatic hydrolysis of chitosan inhibits with side reaction；Product detaches and drying, obtains chitosan oligosaccharide.Concentration of substrate in degradation of chitosan system is improved by batch feeding mode in oxidation pre-treatment, initial hydrolysis is realized, so that chitosan is further hydrolyzed eventually by the effect of enzyme.With the highly concentrated Hydrolyze method of chitosan of early development comparatively, preprocess method has equipment simple, adaptable, it is easy to be combined with technologies such as enzyme mebrane reactor, immobilised enzymes, has larger application prospect in terms of the chitosan oligosaccharide of preparation Narrow Molecular Weight Distribution.

Description

Method for preparing chitooligosaccharides by fed-batch-oxidative pretreatment-assisted enzymatic hydrolysis of chitosan

technical field

The invention relates to a preparation method of low-molecular-weight chitosan, in particular to a method for preparing chitosan oligosaccharide by fed-batch-oxidation pretreatment assisting enzyme hydrolysis of chitosan.

Background technique

Chitosan is a linear macromolecule formed by connecting glucosamine and acetylglucosamine through β-1,4 glycosidic bonds. Its molecular weight ranges from hundreds of thousands to millions. It is the only naturally occurring positively charged polymer in nature. Sugar is insoluble in neutral and alkaline solutions, and cannot be dissolved in organic solvents. Although it is soluble in acidic aqueous solutions, its solubility is limited and its viscosity is extremely high, which seriously affects the application of chitosan. Low-molecular-weight chitosan is an incomplete hydrolysis product of chitosan, which retains the basic properties of chitosan such as adsorption, positive charge, reduction and alkalinity, and has good solubility, low viscosity, and high biological activity. The advantages, especially the potential application of chitosan oligosaccharides in antitumor drugs and improving immunity have attracted extensive attention of researchers at home and abroad. As a functional sugar, low molecular weight chitosan has broad application prospects in agriculture, medicine, functional food, materials and other fields.

The degradation methods of chitosan mainly include physical method, chemical method, enzymatic method and various combined hydrolysis methods. Physical methods such as the commonly used gamma radiation method are prone to chitosan cross-linking and disproportionation side reactions during the degradation process, affecting further degradation, and large-scale radiation equipment increases production costs. The chemical method is to use strong acid or oxidant to destroy the β-1,4 glycosidic bond to achieve hydrolysis. This method is cheap, especially the oxidative degradation process using hydrogen peroxide as the oxidant has the advantages of no residue, cleanliness, and high efficiency. However, the preparation of chitosan oligosaccharides by chemical methods also has great disadvantages. For example, the acid hydrolysis process is difficult to control, and the products are mainly products with low biological activity such as monosaccharides and disaccharides, and a large amount of acid is unfavorable to the purification of later products and waste liquid treatment; hydrogen peroxide oxidative degradation of chitosan is very important for the preparation of high molecular weight Chitosan with a low molecular weight of more than 10000 is more favorable, and further degradation has the problem of "peroxidation" of the product, that is, the structure of the product changes significantly, so it is difficult to obtain low-molecular-weight chitosan only by chemical methods. The enzymatic method is a mild method, especially the specific chitosanase can produce oligosaccharide products with almost no monosaccharides. However, the high cost of specific enzyme preparations and poor thermal stability make it difficult to apply on a large scale. In addition, the high viscosity of chitosan solution greatly hinders the collision between enzyme molecules and chitosan macromolecules, which is not conducive to the effective shearing of enzymes. Experiments have proved that when the concentration of chitosan is higher than 5% (mass volume ratio), chitosan dissolves slowly, it is difficult to form a uniform system, and the viscosity is extremely high, and the dispersibility is extremely poor. The low-concentration hydrolysis of chitosan leads to high energy consumption, high water consumption, low utilization rate of equipment, troublesome post-processing, etc. Therefore, the low concentration and high viscosity of the chitosan degradation system are still the biggest bottlenecks hindering the efficient production of low molecular weight chitosan. Chinese patent 201010172203.0 discloses a method for quickly dissolving chitosan and preparing chitosan oligosaccharides with high substrate concentration enzymatically, using a high-speed shearing disperser to assist in dissolving chitosan, and gradually adding chitosan, acetic acid and hydrolase The method is to make the final concentration of the substrate in the chitosan degradation system reach 10%-20%. However, this method requires the use of high-speed dispersing equipment, which may affect the activity of the enzyme. In addition, the continuous high-viscosity system during the degradation process is not conducive to the conversion of the enzyme molecular conformation and molecular diffusion, so this process requires high enzyme activity and dosage (20 ~ 80U /g), the production cost is higher.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies such as high viscosity, low substrate concentration and low production efficiency in the existing chitosan oligosaccharide production technology, and provide fed-batch-oxidative pretreatment auxiliary enzyme hydrolysis chitosan to prepare chitosan oligosaccharide method.

The present invention comprises the following steps:

1) Fed-batch-oxidative degradation pretreatment of chitosan to obtain a degradation solution;

In step 1), the method of fed-batch-oxidative degradation pretreatment of described chitosan can be: chitosan powder is dispersed in water, add acid to dissolve, then add hydrogen peroxide reaction, add acid and batchwise Chitosan until the chitosan concentration reaches 6% to 30%; the chitosan powder is dispersed in water, and the chitosan powder is dispersed in 25 to 50 times the water according to the mass volume ratio, wherein the chitosan powder is Calculated by mass, water is calculated by volume; the addition of acid can be dissolved according to 0.25～0.35mL acid/g chitosan; the mass final concentration of the hydrogen peroxide can be added and can be 0.5%～10%; the temperature of the reaction can be 40 ~70°C; the time of the pretreatment can be 10 ~ 60min; the acid can be acetic acid; the addition of acid and chitosan in batches, the amount of chitosan that can be added each time does not exceed the amount of chitosan The initial addition amount is 5-10 minutes each time, so that the final concentration of chitosan reaches 6%-30%. No blue, the amount of catalase is 0.0015 ~ 0.003g/mL.

2) hydrogen peroxide removal;

In step 2), the method for removing hydrogen peroxide can be: add catalase to the degradation solution obtained in step 1) to remove residual hydrogen peroxide, and the dosage is 0.0015-0.003g/mL pretreatment solution until the liquid cannot be used KI-starch test paper turns blue.

3) enzymatic hydrolysis and side reaction inhibition of chitosan;

In step 3), the enzymatic hydrolysis of chitosan and side reaction inhibition can be: use one of ammonia water, sodium hydroxide or potassium hydroxide solution to adjust the pH value of the liquid obtained in step 2) to 4-6 , adding chitosan hydrolase according to 1% to 20% of the mass of chitosan, and adding one of sodium bisulfite, sodium sulfite, sodium pyrosulfite, etc. during the enzymolysis process to inhibit the browning reaction, and the dosage of the inhibitor is 0.05- 0.15g/g chitosan, the reaction temperature is 40-60°C, the reaction time is 4-24h, until the required molecular weight; the ammonia water, sodium hydroxide or potassium hydroxide solution can be used with a mass concentration of 5-10M; the enzyme Including one of chitosanase, cellulase, hemicellulase and the like.

4) The product is separated and dried to obtain chitosan oligosaccharide.

In step 4), the method for separating and drying the product can be: adjust the pH value of the liquid obtained in step 3) to 7 with one of ammonia water, sodium hydroxide or potassium hydroxide solution, and use a 10-20kDa ultrafiltration membrane Remove the enzyme, and obtain chitosan oligosaccharide after drying, the chitosan oligosaccharide is a light yellow chitosan product, and the deacetylation degree of chitosan is 80% to 100%; the drying can be spray drying, freeze drying, vacuum drying One of a kind.

The present invention increases the substrate concentration in the chitosan degradation system by feeding batches in the oxidation pretreatment process, realizes preliminary hydrolysis, and finally further hydrolyzes the chitosan through the action of enzymes. Compared with the high-concentration chitosan hydrolysis method developed in the early stage, the pretreatment method involved in the present invention has simple equipment, strong adaptability, and is easy to combine with technologies such as enzyme membrane reactors and immobilized enzymes. The distribution of chitosan oligosaccharides has great application prospects. The content of the present invention is of great significance to the high-efficiency and green production of chitosan oligosaccharides.

The present invention has the following advantages:

1) The fed-batch oxidation pretreatment method has a good effect of reducing viscosity and increasing concentration, and the method has strong adaptability, and is easy to be coupled with enzyme membrane reactor and enzyme immobilization technology. Great application prospects;

2) The method uses less chemical reagents, less pollution, high product safety, and conforms to the concept of green chemical production;

3) The reaction conditions of the entire degradation system are mild, the reaction equipment is simple, and it is easy to expand production.

Description of drawings

Fig. 1 is the product molecular weight distribution situation of chitosan obtained in the embodiment of the present invention after oxidation-enzymatic degradation. In Figure 1, (a) is 6% chitosan; (b) is 9% chitosan.

Detailed ways

The following embodiments will describe the specific implementation manners of the present invention in detail in conjunction with the accompanying drawings.

Example 1

3g of chitosan was uniformly dispersed in 96mL of water, 1mL of glacial acetic acid was added, stirred and dissolved at room temperature, then 4mL of 30% hydrogen peroxide was added, stirred and reacted at 60°C for 5min, and 1mL of glacial acetic acid and 3g of chitosan were added quickly to control the total amount of pretreatment. For 1 hour, add 0.2 g of catalase to remove hydrogen peroxide, adjust the pH value with concentrated ammonia water, add 5 g of cellulase, and react in a shaking table at 55 °C for 12 hours, adjust the pH value of the obtained degradation solution to 7 with ammonia water, and use a 10 kDa super The filter membrane removes the enzyme, and the filtrate is vacuum freeze-dried to obtain light yellow chitosan oligosaccharide products, wherein the yield of chitosan oligosaccharides that can be dissolved in 75% ethanol reaches 90%. The product was identified by TLC, as shown in Figure 1(a), S is the standard product, and 1-4 are the same samples repeated 4 times. The seven points of the standard from top to bottom are GlcN: glucosamine; (GlcN) ₂ : chitobiose; (GlcN) ₃ : chitotriose; (GlcN) ₄ : chitotetraose; (GlcN) ₅ : chitopentose sugar; (GlcN) ₆ : Chitohexaose; (GlcN) ₇ : Chitoheptaose.

Example 2

According to embodiment 1, add 1mL glacial acetic acid and 3g chitosan respectively at the 5th and 15min of pretreatment, control pretreatment total time 0.5h, add 0.2g catalase to remove hydrogen peroxide, adjust pH value with concentrated ammonia water, Add 5g of cellulase and react on a shaker at 55°C for 6 hours. The product can be dissolved under the condition of pH 7-14. Continue to react for 12 hours. Use ammonia water to adjust the pH value of the obtained degradation solution to 7, and use a 10kDa ultrafiltration membrane to remove the enzyme. , the filtrate was vacuum freeze-dried to obtain light yellow chitosan oligosaccharide products, wherein the yield of chitosan oligosaccharides that could be dissolved in 75% ethanol reached 70%. The product was identified by thin-layer chromatography, as shown in Figure 1(b). S is the standard; 5-8 are the same samples repeated 4 times. The seven points of the standard from top to bottom are GlcN: glucosamine; (GlcN) ₂ : chitobiose; (GlcN) ₃ : chitotriose; (GlcN) ₄ : chitotetraose; (GlcN) ₅ : chitopentose sugar; (GlcN) ₆ : Chitohexaose; (GlcN) ₇ : Chitoheptaose.

Example 3

According to Example 2, 1.2 g of NaHSO ₃ was added during enzymatic hydrolysis for 12 hours to obtain almost pure white chitosan oligosaccharides, wherein the yield of chitosan oligosaccharides soluble in 75% ethanol reached 48%.

Claims (10)

1. Fed-batch-oxidative pretreatment assisted enzyme hydrolysis chitosan prepares chitosan oligosaccharide method, it is characterized in that comprising the following steps: 1) Fed-batch-oxidative degradation pretreatment of chitosan to obtain a degradation solution; 2) hydrogen peroxide removal; 3) enzymatic hydrolysis and side reaction inhibition of chitosan; 4) The product is separated and dried to obtain chitosan oligosaccharide. 2. fed-batch-oxidative pretreatment auxiliary enzyme hydrolysis chitosan as claimed in claim 1 prepares chitosan oligosaccharide method, it is characterized in that in step 1) in, the fed-batch-oxidative degradation of described chitosan The pretreatment method is as follows: disperse chitosan powder in water, add acid to dissolve, then add hydrogen peroxide to react, add acid and chitosan in batches until the concentration of chitosan reaches 6%-30%. 3. fed batches as claimed in claim 2-oxidative pretreatment auxiliary enzyme hydrolysis chitosan prepares chitosan oligosaccharide method, it is characterized in that described chitosan powder is dispersed in water, by mass volume ratio chitosan The powder is dispersed in 25-50 times of water, wherein the chitosan powder is calculated by mass, and the water is calculated by volume. 4. fed batches as claimed in claim 2-oxidative pretreatment auxiliary enzyme hydrolysis chitosan prepares chitosan oligosaccharide method, it is characterized in that described adding acid dissolution is according to 0.25～0.35mL acid/g chitosan; The hydrogen peroxide is added with a final mass concentration of 0.5%-10%; the reaction temperature is 40-70° C.; the pretreatment time is 10-60 minutes. 5. fed batches as claimed in claim 2-oxidative pretreatment auxiliary enzyme hydrolysis chitosan prepares chitosan oligosaccharide method, it is characterized in that described acid adopts acetic acid; Described adding acid and chitosan in batches, is The amount of chitosan added each time does not exceed the initial amount of chitosan added, and the time interval between each feeding is 5 to 10 minutes, so that the final concentration of chitosan reaches 6% to 30%; Remove the hydrogen peroxide in the system with catalase until the starch-potassium iodide test paper does not turn blue, and the dosage of catalase is 0.0015-0.003g/mL. 6. fed batches as claimed in claim 1-oxidative pretreatment auxiliary enzyme hydrolysis chitosan prepares chitosan oligosaccharide method, it is characterized in that in step 2) in, the method that described hydrogen peroxide removes is: in step 1) Add catalase to the obtained degradation liquid to remove residual hydrogen peroxide, and the dosage is 0.0015-0.003 g/mL pretreatment liquid until the liquid cannot turn KI-starch test paper blue. 7. fed batches as claimed in claim 1-oxidative pretreatment auxiliary enzyme hydrolysis chitosan prepares chitosan oligosaccharide method, it is characterized in that in step 3) in, the enzymatic hydrolysis of described chitosan and side reaction inhibition The method is: use one of ammonia water, sodium hydroxide or potassium hydroxide solution to adjust the pH value of the liquid obtained in step 2) to 4 to 6, add chitosan hydrolase according to 1% to 20% of the mass of chitosan, and the enzyme The solution process suppresses the browning reaction by adding one of sodium bisulfite, sodium sulfite and sodium metabisulfite. The dosage of the inhibitor is 0.05～0.15g/g chitosan, the reaction temperature is 40～60°C, and the reaction time is 4～24h. to the desired molecular weight. 8. fed batches as claimed in claim 7-oxidative pretreatment auxiliary enzyme hydrolysis chitosan prepares chitosan oligosaccharide method, it is characterized in that described ammoniacal liquor, sodium hydroxide or potassium hydroxide solution adopt mass concentration to be 5～10M ; The enzyme comprises one of chitosanase, cellulase and hemicellulase. 9. fed batches as claimed in claim 1-oxidative pretreatment auxiliary enzyme hydrolysis chitosan prepares chitosan oligosaccharide method, it is characterized in that in step 4) in, the method for described product separation and drying is: use ammoniacal liquor, One of sodium hydroxide or potassium hydroxide solution is used to adjust the pH value of the liquid obtained in step 3) to 7, and the enzyme is removed by using a 10-20 kDa ultrafiltration membrane, and the chitosan oligosaccharide is obtained after drying. 10. fed batches as claimed in claim 9-oxidative pretreatment auxiliary enzyme hydrolysis chitosan prepares chitosan oligosaccharide method, it is characterized in that described drying adopts the one in spray drying, freeze drying, vacuum drying.