Chitosan oligosaccharide derivative with antibacterial property as well as preparation method and application thereof

Abstract

The invention relates to an antibacterial chitosan oligosaccharide derivative and a preparation method and application thereof.The main raw materials of the antibacterial chitosan oligosaccharide derivative all belong to green and pure natural raw materials, other synthetic raw materials belong to low-toxicity or non-toxic reagents and are cheap and easy to obtain, the reaction is simple, the reaction condition is mild, the yield is high, the reaction can be quantified, and industrial mass production is easy to achieve; and most of byproducts, such as acetic acid, triethylamine hydrochloride, pivalic acid and the like, involved in the synthesis process can be recycled, so that the preparation cost is greatly saved. According to the scheme, the chitosan oligosaccharide has a relatively strong antibacterial effect, a protective layer similar to a'sugar coating 'chitosan oligosaccharide derivative can be formed on the surface of a feed, and the derivative has antibacterial performance and is small in addition amount, so that the dosage of antibiotics in the feed can be reduced.

Description

Technical Field

[0001] The invention relates to the field of feed additives, and in particular to a chitosan oligosaccharide derivative with antibacterial properties and a preparation method and application thereof. Background Art

[0003] At present, there are many types of alternatives to antibiotics in feed, mainly including plant extracts, probiotics, enzyme preparations, antimicrobial peptides, acidifiers, etc. These alternatives have their own characteristics. In recent years, prebiotics, as a type of oligosaccharide substances that are not easily digested, such as inulin, oligoxylose, oligofructose, oligoisomaltose, resistant starch, etc., are considered to be a very potential alternative to antibiotics in the future because they can selectively promote the growth of beneficial microorganisms such as bifidobacteria and lactobacilli in the animal intestine, thereby producing a variety of beneficial effects on the host, such as promoting growth and weight gain.

[0004] Chitosan oligosaccharide, also known as chitosan oligosaccharide or low-molecular chitosan, is an oligosaccharide product with a degree of polymerization between 2 and 20 obtained by enzymatic hydrolysis or acid hydrolysis of chitosan. The molecular weight is ≤3200Da. It is a low molecular weight product with good water solubility, great functional effects and high biological activity. Chitosan oligosaccharide is the only positively charged cationic alkaline amino oligosaccharide in nature. It is non-toxic, non-pyrogenic, non-mutational, and is an animal cellulose. Chitosan oligosaccharide used as a feed additive can improve the metabolic activity of microorganisms in the intestines of animals, enhance the body's immunity and disease resistance, and has a significant effect on improving the growth of livestock and poultry.

[0005] Similar to other prebiotics, chitosan oligosaccharides themselves have a weak bactericidal effect. Under acidic or weakly acidic conditions, the free amino groups in the chitosan oligosaccharide molecules will be protonated, and the protonated ammonium can interact with the bacterial cell membrane, thereby interfering with the function of the bacterial cell membrane and inhibiting the growth of fungi and microorganisms. However, this antibacterial effect will only become significant under high-dose conditions. Therefore, when chitosan oligosaccharides are used as feed additives, they are generally only used to assist in disease resistance and prevention. Therefore, if the antibacterial properties of oligosaccharides can be improved in a targeted manner, it will be of great help to improve their overall performance as feed additives. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention proposes a chitosan oligosaccharide derivative with antibacterial properties and a preparation method and application thereof. The technical solution of the present invention is as follows: A chitosan oligosaccharide derivative with antibacterial properties, the molecular structure of which is as follows: Denoted as CC(A)GCO; where n=2~20.

[0007] A method for preparing the chitosan oligosaccharide derivative as described above comprises the following steps: Step 1) In a clean round-bottom flask, add coumarin-3-carboxylic acid and freshly distilled SOCl 2 Reflux in an oil bath for 2 h, and remove the remaining SOCl by rotary evaporation under reduced pressure. 2 , to obtain a pale yellow solid (coumarin-3-carbonyl chloride).

[0008] Step 2) The product of step 1) is dissolved in DMF, glycine and sodium carbonate are dissolved in deionized water, cooled to 0°C, and the above DMF solution is added dropwise, and the reaction is stirred for 2-4 hours to terminate; acidification is performed with concentrated hydrochloric acid to pH = 1-2, and then extraction and washing are performed with ethyl acetate, followed by drying with anhydrous sodium sulfate for 4-6 hours; the desiccant is removed by filtration, and the filtrate is subjected to rotary evaporation to obtain a white solid product, coumarin-3-formylglycine; Step 3) dissolving acetylglycine and the product of step 2) in DMF in a certain ratio to obtain a DMF mixed solution, adding triethylamine (TEA), cooling to 0°C, adding an equivalent amount of pivaloyl chloride solution dropwise, gradually heating to room temperature and reacting for 2h, filtering to remove the white precipitate, and obtaining a mixed anhydride solution; Step 4) Weigh appropriate amounts of chitosan oligosaccharide and sodium carbonate and dissolve them in a certain amount of deionized water, add the mixed anhydride solution obtained in step 3) dropwise, react for 3-4 hours, filter the reaction solution, precipitate and filter with n-propanol, concentrate the centrifuged liquid to 1 / 4, precipitate the filtrate with solvent A, centrifuge and vacuum dry to obtain the chitosan oligosaccharide derivative (CC(A)GCO).

[0009] Preferably, the molar ratio of the raw materials in step 1) is: coumarin-3-carboxylic acid: thionyl chloride = 1:5-20.

[0010] Preferably, the molar ratio of the raw materials in step 2) is: glycine: sodium carbonate: coumarin-3-carbonyl chloride = 1: 1.1-1.3: 1.1-1.4.

[0011] Preferably, the molar ratio of the raw materials in step 3) is: acetylglycine: coumarin-3-formylglycine = 1:1-3.

[0012] Preferably, the molar ratio of the raw materials in step 3) is: DMF mixed solution: triethylamine: pivaloyl chloride = 1: 1.0-1.05: 1.0-1.05.

[0013] Preferably, the molar ratio of the raw materials in step 4) is: mixed anhydride solution: sodium carbonate: chitosan oligosaccharide = 1: 1.0-1.05: 0.9-1.05.

[0014] Preferably, the solvent A used in step 4) is selected from tetrahydrofuran, ethyl acetate or propyl acetate.

[0015] The crude product obtained by the above process can also be used to obtain fine products according to the needs of different users by the following method: dissolve the crude chitosan oligosaccharide derivative in an appropriate amount of water, add n-propanol for secondary precipitation, centrifuge, and vacuum dry to obtain a product with higher purity.

[0016] A use of the chitosan oligosaccharide derivative as described above as a feed additive.

[0017] Additives with their own antibacterial effects can significantly extend the storage time of feed, allowing enterprises to mass-produce finished feed during the low-price period of agricultural and sideline products, reducing the probability of losses caused by microbial contamination. In terms of the method of adding additives, most of them adopt the step-by-step mixing method, that is, first select a small amount of feed and additives for premixing, then add a certain amount of feed to mix, and finally add all the feed to mix thoroughly. This is mainly because the amount of additives added is very small, and the concentration must be controlled uniformly and appropriately. The graded mixing method can adsorb a thin layer of additives on the surface of most feed solids (this layer may adsorb feed containing additives with finer particle size). Therefore, the key to the antiseptic effect of this additive is to form a protective layer with antibacterial effect on the surface of the feed solid. In view of this, if chitosan oligosaccharide derivatives with strong antibacterial effect are sprayed on the surface of the feed solid, because the molecular weight of the chitosan oligosaccharide derivatives is large, it is difficult to penetrate into the interior of the feed solid, and the chitosan oligosaccharide derivatives are easy to form a film, so this film can wrap the solid surface of the feed well to form a protective layer with antibacterial effect. In addition, because the concentration of solid matter is much higher than that in solution, its antibacterial property is much greater than that in liquid solution state.

[0018] Coumarin-3-carboxylic acid is a natural substance found in the seeds of coumarin beans, which has multiple biological activities. Because of the structure of benzo α-pyrone in its molecular structure, it has obvious biological activity and has pharmacological effects on the human body, such as anticoagulation, antibacterial, anti-inflammatory, antioxidant, hypoglycemic, anti-osteoporosis, anti-hypertension, and anti-cancer. Coumarin compounds and their derivatives are regarded as potential substitutes for antibiotics because of their low toxicity, high efficiency, and easy degradation. In recent years, coumarin-3-carboxylic acid has become one of the research hotspots due to its significant antibacterial activity. Combining coumarin compounds with chitosan oligosaccharides, taking the advantages of both, a new type of antibiotic-free substitute is synthesized, which uses the good film-forming properties of chitosan oligosaccharides to adhere to the surface of feed solids, and relies on the antibacterial properties of coumarin-3-carboxylic acid itself to protect the feed from microbial contamination. Moreover, since this additive only forms a protective layer similar to "sugar coating", the dosage is very small, and it has a broad application prospect.

[0019] The chitosan oligosaccharide molecular structure of the present invention is rich in highly polar hydroxyl and amino groups, and has strong hydrophilicity. Because the feed surface is not all hydrophilic, in most cases, the surface hydrophilic region and the hydrophobic region are mixed. However, if the feed surface has good adhesion, it must have a certain hydrophobicity at the same time to form an amphiphilic structure. Since the amino group is more active than the hydroxyl group and is evenly distributed on both sides of the glucose structural unit with a large interval, a derivatization is carried out on the amino group of chitosan oligosaccharide, that is, a certain amount of hydrophobic groups are introduced through the amino group. However, this inevitably causes the antibacterial ability of chitosan oligosaccharide to be lost, and thus the hydrophobic groups introduced on the amino group must have sufficient antibacterial ability (preferably having multiple physiological functions at the same time). Coumarin-3-carboxylic acid can meet the requirements.

[0020] In terms of molecular design, the present invention does not directly couple coumarin-3-carboxylic acid to chitosan oligosaccharides, but connects them through certain amino acids as bridges. First, the chain length of the hydrophobic end of the amphiphilic molecule cannot be too short. Generally, a carbon chain length of more than 8 is required to have sufficient hydrophobicity, and a benzene ring is equivalent to 6 to 8 carbon atoms (empirical value), so the carbon chain needs to be extended; second, the methylene group on the pyran ring of chitosan oligosaccharides also has a certain hydrophobicity. In order to prevent the extended hydrophobic part on the amino group from shrinking to the pyran ring, the derivative part on the amino group needs to have a certain rigidity. When using amino acids as bridges, a small segment of a rigid peptide chain structure can be formed to avoid hydrophobic contraction; third, because the exocyclic carbonyl group in the molecular structure of coumarin-3-carboxylic acid can form a conjugated system with the connected carbon-carbon double bond and benzene ring, the entire structure is The chitosan oligosaccharide molecule itself is rigid, so when such a rigid side chain is introduced to the amino group, the molecular structure is very regular and easier to disperse (favorable to form a more regular membrane); on the other hand, this hydrophobic region is easy to bind to the hydrophobic region on the surface of the microbial cell membrane. Due to the role of the amino acid bridge (equivalent to extending the length of 2 carbon chains), the benzopyran ring can be inserted into the interior of the hydrophobic protein (mainly the enzyme on the surface of the cell membrane), which not only affects the transmission of external nutrients, but also directly affects the activity of the enzyme; especially after the lactone bond is hydrolyzed, the phenolic hydroxyl structure has a strong physiological activity, which has a great influence on the activity of the enzyme. Therefore, this chitosan oligosaccharide derivative will have a certain bactericidal or antibacterial effect.

[0021] In the present invention, glycine is selected as the amino acid used. This is because when the mixed anhydride is prepared by the pivaloyl chloride method, the amino group can theoretically attack any carbonyl carbon atom in the anhydride. From the molecular structure of the anhydride, due to the steric hindrance and electron-donating effect of the tert-butyl group, the reaction activity of the nucleophilic reaction on the carbonyl carbon atom of the anhydride connected to the tert-butyl group is low, so the amino group mainly attacks the other carbonyl carbon atom of the anhydride (i.e., the carbonyl carbon atom at the end of the amino acid) and the carbonyl carbon atom on the target carboxylic acid side.

[0022] Compared with other amino acids, glycine is selected as the supporting amino acid in this case to reduce the influence of effective coupling caused by the steric hindrance caused by the side chain residues. Since the anhydride has high activity, the coupling efficiency is also high.

[0023] Another object of the present invention is to provide the application of the CC(A)GCO in protecting feed. Taking the protection of soybean feed as an example, a CC(A)GCO solution of 0.1%-1% by mass is prepared with sterile water, and evenly sprayed on the surface of the soybean feed, mixed well, dried at 50°C, and packaged to obtain the finished feed.

[0024] The advantages of the present invention are: (1) From the perspective of molecular design, the advantages of coumarin-3-carboxylic acid and chitosan oligosaccharide were combined to prepare chitosan oligosaccharide derivatives with good antibacterial properties and film-forming properties; (2) Different acyl groups are introduced into the amino group of glycine, and then the acylated glycine is coupled to the amino group of the chitosan oligosaccharide molecule. The ratio of the two acyl groups is controlled. On the one hand, the water solubility of the derivative is improved by acetylglycine, and on the other hand, the antibacterial property of the coumarin-3-formyl group is used to impart antibacterial properties to the derivative. (3) All the main raw materials are green and pure natural raw materials, and other synthetic raw materials are low-toxic or non-toxic reagents, cheap and easy to obtain, simple reaction, mild reaction conditions, high yield, quantitative reaction, and easy to achieve industrial mass production. Most of the by-products involved in the synthesis process (such as acetic acid, triethylamine hydrochloride, pivalic acid, etc.) can be recycled, thereby greatly saving the preparation cost.

[0025] The beneficial effects of the present invention are as follows: 1. It can achieve the strong antibacterial effect of chitosan oligosaccharide.

[0026] 2. It can form a protective layer similar to the "sugar-coated" chitosan oligosaccharide derivatives on the surface of feed.

[0027] 3. Since the derivative itself has antibacterial properties, a small amount of addition can reduce the amount of antibiotics used in feed.

[0028] 4. The present invention adopts the pivaloyl chloride method to couple carboxylic acid and amino group, that is, carboxylic acid reacts with pivaloyl chloride to generate corresponding carboxylic acid-pivalic anhydride, which is then coupled with amino group. This method has high yield and low production cost and is very suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the H NMR spectrum of coumarin-3-formylglycine prepared in Example 1.

[0030] Figure 2 This is the infrared spectrum of CC(A)GCO prepared in Example 1.

[0031] Figure 3 This is the UV spectrum of CC(A)GCO prepared in Example 1.

[0032] Figure 4 The antibacterial effect comparison chart of the prepared CC(A)GCO at the concentration of 20 mg / mL; A: Staphylococcus aureus; B: Escherichia coli.

[0033] Figure 5 This is a comparison chart of soybeans treated with CC(A)GCO (A: untreated; B: CC(A)GCO treated).

[0034] Figure 6 A comparison of the surface appearance of a single soybean before and after treatment (A: untreated; B: CC(A)GCO treated). DETAILED DESCRIPTION

[0035] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] Embodiment 1: The molecular reaction formula for synthesizing chitosan oligosaccharide derivatives is as follows: Among them, n=2~20.

[0038] Step 1) In a clean round-bottom flask, add 2 g of coumarin-3-carboxylic acid, 15 mL of freshly distilled SOCl 2 Reflux in an oil bath for 2 h, and remove the remaining SOCl by rotary evaporation under reduced pressure. 2 , to obtain 2.1 g of a light yellow solid.

[0039] Step 2) Dissolve 150 mg of glycine and 212 mg of sodium carbonate in an appropriate amount of deionized water, cool to 0°C, add 10 mL of DMF solution containing 430 mg of coumarin-3-carbonyl chloride dropwise, and stir for 2 to 4 hours to complete the reaction. Acidify with concentrated hydrochloric acid to pH = 1 to 2, then extract with 5 mL of ethyl acetate three times, wash, add anhydrous sodium sulfate and dry for 4 to 6 hours; filter to remove the desiccant, and rotary evaporate the filtrate to obtain a white solid product, coumarin-3-carbonylglycine; Characterized by NMR, H spectrum (see attached Figure 1 )The results are as follows: 1H NMR (400 MHz, DMSO-d6) δ 13.06(s, 1H),9.17 – 8.89 (m, 1H), 8.74 (s, 1H), 7.96 (dd, J = 42.1, 7.0 Hz, 1H),7.75 (dt, J = 14.2, 7.1 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.43 (p, J = 8.1,7.6 Hz, 2H), 4.06 (d, J = 5.5 Hz, 1H). Step 3) Dissolve 61 mg of acetylglycine and 295 mg of the product of step 2) in 8 ml of DMF, add 287 μL of triethylamine, cool to 0°C, add 254 μL of pivaloyl chloride solution dropwise, gradually warm to room temperature and react for 2 h, filter to remove the white precipitate, and obtain a mixed anhydride solution; Step 4) Weigh 322 mg of chitosan oligosaccharide and 212 mg of sodium carbonate and dissolve them in a certain amount of deionized water, add the mixed anhydride solution obtained in step 3) dropwise, react for 3-4 hours, filter the reaction solution, precipitate and filter with n-propanol, concentrate the centrifuged liquid to 1 / 4, precipitate the filtrate with ethyl acetate after filtration, centrifuge and vacuum dry to obtain the chitosan oligosaccharide derivative.

[0040] According to elemental analysis, the content of each element is: C56.1%, N7.9%, O36%, which is basically consistent with the theoretical results (C56.0%, N8.2%, O35.6%).

[0041] The infrared spectrum of CC(A)GCO prepared by this method is shown in the attached figure. Figure 2 shown.

[0042] In the attached Figure 2 Middle 3407cm -1 The broad peaks near the 2935 cm-1 are mainly the stretching vibration peaks of OH on the hydroxyl group and NH on the amide group of glucosamine. -1 ~2831 cm -1 Nearby is the stretching vibration peak of the CH bond; 1631 cm -1 ~1591 cm -1 The absorption at 1351 cm is mainly the conjugation of the stretching vibration peak of the amide bond and the characteristic absorption peak of the benzene ring skeleton vibration. -1 The peaks nearby are mainly the in-plane bending vibration peaks of the CH bonds.

[0043] The UV spectra of CC(A)GCO and raw chitosan oligosaccharide (CO) prepared by this method are shown in the attached figure. Figure 3As shown in the figure, the raw chitosan oligosaccharide has almost no absorption peak in the wavelength range of 200 nm – 400 nm, and after the reaction, it has obvious absorption in the range of 248nm-398nm, and the absorption peak is located at 298nm. The peak shape is similar to the ultraviolet spectrum of coumarin-3-carboxylic acid in the literature, indicating the presence of coumarin-3-formyl in the product.

[0044] Attached Figure 4 The picture shows the inhibitory effect of the prepared CC(A)GCO on the culture of Escherichia coli and Staphylococcus aureus at a concentration of 20 mg / mL (Oxford cup method). Figure 4 It can be seen that CC(A)GCO forms obvious inhibition zones or rings (both with diameters greater than 7 mm) for both bacteria at the action concentration, and the inhibition zone for Staphylococcus aureus is larger than that for Escherichia coli, indicating that CC(A)GCO has significant antibacterial effects on both bacteria. The reason for the formation of the inhibition zone for Staphylococcus aureus may be that the CC(A)GCO sample solution is added to the Oxford cup, and CC(A)GCO produces a diffusion effect on the culture medium. Since the derivatives are composed of oligomers of different molecular weights, oligomers with small molecular weight diffuse faster, while those with large molecular weight diffuse relatively slowly. Oligomers with small molecular weight bind to bacteria in large numbers and have good antibacterial effects, while oligomers with large molecular weight bind to bacteria less and have poor antibacterial effects. After the water in the Oxford cup diffuses, the residual oligomers with a high concentration that have not diffused remain, so the Oxford cup also produces an antibacterial effect.

[0045] Embodiment 2: Weigh 0.2g CC(A)GCO, dissolve it in 40mL sterile water, and transfer it to a 50mL spray bottle; take about 5g of commercially available soybeans, place them in a petri dish, spray CC(A)GCO solution while shaking, mix well, put them in a 50℃ oven to dry for 90min, put them into a packaging bag after cooling, and store them at room temperature. No mold was found after 12 months.

[0046] Attached Figure 5 The following is a comparison of soybeans treated with CC(A)GCO (A: untreated; B: treated with CC(A)GCO). Figure 5 It can be seen that the color of soybeans becomes significantly darker and more uniform after being treated with CC(A)GCO, indicating that the additive has a good adhesion effect.

[0047] Attached Figure 6 The following is a comparison of the surface appearance of a single soybean before and after treatment (A: untreated; B: CC(A)GCO treated). Figure 6 It can be seen that the outer layer of soybeans becomes bright and has good continuity after being treated with CC(A)GCO, indicating that the additive forms a continuous film on the surface of soybeans.

[0048] Embodiment 3: In order to confirm that CC(A)GCO was encapsulated on the surface of soybean, the following method was used.

[0049] Weigh 10 portions of soybeans (about 2 g) accurately in culture dishes of the same specifications, spray deionized water into one of the culture dishes under shaking, mix the yellow, and spray 0.5% CC(A)GCO aqueous solution into the remaining culture dishes, and place them in a 5050℃ oven to dry for 90 minutes. After cooling, weigh them and calculate the unit mass of CC(A)GCO attached to the soybean surface. The results are shown in Table 1.

[0050] Table 1 Unit mass of CC(A)GCO attached to the surface of soybean after spray treatment As shown in Table 1, the average mass of the 10 samples is 20.03591. The average weight loss of the water sample after 5 sprays was 0.40298g after drying, and the average weight loss of the CC(A)GCO aqueous solution sample after 5 sprays was 0.142g. The average residue on the surface of the soybean sample was 0.2609, and the unit sample residue = 0.2609 / 20.03591 = 0.013g / g. That is, the weight gain of each gram of soybean surface was 13 mg, which confirmed that CC(A)GCO remained on the soybean surface after spraying.

[0051] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A chitosan oligosaccharide derivative with antibacterial properties, characterized in that: Its molecular structure is as follows: Among them, n=2~20.

2. A method for preparing a chitosan oligosaccharide derivative as claimed in claim 1, characterized in that: The following steps are involved: Step 1) In a clean round-bottom flask, add coumarin-3-carboxylic acid and SOCl2, reflux in an oil bath for 2 h, and remove the remaining SOCl2 by rotary evaporation under reduced pressure to obtain a light yellow solid, i.e., coumarin-3-carbonyl chloride; Step 2) Dissolve glycine and sodium carbonate in deionized water, cool to 0°C, add coumarin-3-carbonyl chloride DMF solution dropwise, stir for 2-4 h to complete the reaction; acidify with concentrated hydrochloric acid to pH = 1-2, then extract with ethyl acetate, wash, add anhydrous sodium sulfate and dry for 4-6 h; filter to remove the desiccant, and rotary evaporate the filtrate to obtain a white solid product, coumarin-3-carbonylglycine; Step 3) dissolving acetylglycine and the product of step 1) in DMF in a certain ratio to obtain a DMF mixed solution, adding triethylamine, cooling to 0°C, adding an equivalent amount of pivaloyl chloride solution dropwise, gradually heating to room temperature and reacting for 2h, filtering to remove the white precipitate, and obtaining a mixed anhydride solution; Step 4) Weigh appropriate amounts of chitosan oligosaccharide and sodium carbonate and dissolve them in a certain amount of deionized water, add the mixed anhydride solution obtained in step 3) dropwise, react for 3-4 hours, filter the reaction solution, precipitate and filter with n-propanol, concentrate the centrifuged liquid to 1 / 4, precipitate the filtrate with solvent A, centrifuge and vacuum dry to obtain the chitosan oligosaccharide derivative.

3. The method for preparing the chitosan oligosaccharide derivative according to claim 2, characterized in that: The molar ratio of the raw materials in step 2) is: glycine: sodium carbonate: coumarin-3-carbonyl chloride = 1: 1.1-1.3: 1.1-1.

4.

4. The method for preparing the chitosan oligosaccharide derivative according to claim 2, characterized in that: The molar ratio of the raw materials in step 3) is: acetylglycine: coumarin-3-formylglycine = 1:1-3.

5. The method for preparing the chitosan oligosaccharide derivative according to claim 2, characterized in that: The molar ratio of the raw materials in step 3) is: DMF mixed solution: triethylamine: pivaloyl chloride = 1: 1.0-1.05: 1.0-1.

05.

6. The method for preparing the chitosan oligosaccharide derivative according to claim 2, characterized in that: The molar ratio of the raw materials in step 4) is: mixed anhydride solution: sodium carbonate: chitosan oligosaccharide = 1: 1.0-1.05: 0.9-1.

05.

7. The method for preparing the chitosan oligosaccharide derivative according to claim 2, characterized in that: The molar ratio of the raw materials in step 1) is: coumarin-3-carboxylic acid: SOCl2=1:5-20.

8. The method for preparing the chitosan oligosaccharide derivative according to claim 2, characterized in that: The solvent A used in step 4) is selected from tetrahydrofuran, ethyl acetate or propyl acetate.

9. Use of the chitosan oligosaccharide derivative as claimed in claim 1 as a feed additive.

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