Method for preparing chitosan oligosaccharide based on compound enzyme

Abstract

The invention belongs to the technical field of chitosan oligosaccharide preparation, and particularly relates to a method for preparing chitosan oligosaccharide based on compound enzyme. According to the method for preparing the chitosan oligosaccharide based on the compound enzyme, a multi-enzyme synergistic system is matched with the design of a specific protective agent, chitosanase and chitinase can cooperatively cut beta-1, 4 glycosidic bonds of chitosan and control molecular weight distribution of products, meanwhile, chitinase can directionally cut the beta-1, 4 bonds at the tail end of chitosan, and the molecular weight distribution of the products is controlled. The papain is used for removing protein impurities in the chitosan to realize accurate control of the polymerization degree. Besides, a specific protective agent matched with the multi-enzyme synergistic system is specifically designed, the specific protective agent and the multi-enzyme composition act together, the molecular weight distribution width can be remarkably reduced, the yield and the DP 4-8 proportion can be increased, meanwhile, physical field ultrasonic assistance is added in the enzymolysis treatment process, and the enzymolysis treatment efficiency is improved. The combination of the substrate and the enzyme is further promoted, and the reaction time is shortened.

Description

Technical Field

[0001] The present application belongs to the technical field of chitosan oligosaccharide preparation, and specifically relates to a method for preparing chitosan oligosaccharide based on a complex enzyme. Background Art

[0002] Chitosan oligosaccharides, also known as amino oligosaccharides or oligochitosans, are derived from the degradation of chitosan. They are typically chain-like, low-molecular-weight, alkaline, water-soluble oligosaccharides composed of 2-10 glucosamines linked by β-1,4 glycosidic bonds. They are the only naturally occurring positively charged polysaccharide found in nature and have unique applications in fine chemicals, biopharmaceuticals, health foods, agriculture, forestry, and animal husbandry.

[0003] Enzymatic hydrolysis is currently a commonly used method for preparing chitosan oligosaccharides. The prior art records that chitosan can be hydrolyzed by various enzymes such as papain and chitosanase to obtain chitosan oligosaccharide products with different degrees of polymerization and properties. The enzymatic hydrolysis method has the advantages of mild reaction conditions, easy product control, and environmental friendliness. However, there are still many technical problems in actual operation: for example, patent CN102492664B records the use of a complex of cellulase, lysozyme, amylase, lipase, glucosidase and papain to prepare chitosan oligosaccharides. Due to technical problems such as the optimal pH / temperature conflict between lysozyme and lipase, and the substrate overlap of amylase and glucosidase, the yield is low, and the proportion of the average molecular weight in the range of 1000KDa-5000KDa is high, and the overall relative molecular weight is large; and the preparation method of chitosan recorded in patent CN112980905B is by adding the complex enzyme in batches, and the heating rate needs to be strictly controlled during the heating process to avoid enzyme inactivation. The process is complicated, the process requirements are high, and the yield is unstable.

[0004] Therefore, it is necessary to further optimize the preparation process based on complex enzymes to develop a preparation process for chitosan oligosaccharides with high yield and small average molecular weight. Summary of the Invention

[0005] Based on this, an embodiment of the present application provides a method for preparing chitosan oligosaccharides based on a complex enzyme, which can effectively improve On the one hand, the present application provides a method for preparing chitosan oligosaccharides based on a complex enzyme, comprising: Performing a first ultrasonic treatment on the chitosan raw material to prepare a first preparatory material; The first prepared material is subjected to enzymatic hydrolysis treatment using a complex enzyme composition, and simultaneously subjected to a second ultrasonic treatment to prepare a chitosan enzymatic hydrolyzate; The chitosan enzymatic hydrolyzate is subjected to centrifugation and ultrafiltration to prepare chitosan; The complex enzyme composition comprises: 20-30 parts of chitosanase, 10-20 parts of chitinase, 5-15 parts of papain and a protective agent.

[0006] The present application provides a method for preparing chitosan oligosaccharides based on a composite enzyme through the design of a multi-enzyme synergistic system in combination with a specific protective agent, wherein chitosanase and chitinase can synergistically cut the β-1,4 glycosidic bonds of chitosan to control the molecular weight distribution of the product. At the same time, chitinase can also directionally cut the terminal β-1,4 bonds of chitosan, and papain is used to remove protein impurities in chitosan to achieve precise control of the degree of polymerization. However, since there may be certain problems of unstable enzyme activity after combining the above-mentioned enzymes, the present application specifically designs a specific protective agent that is compatible with the above-mentioned multi-enzyme synergistic system. The specific protective agent and the multi-enzyme composition act together to significantly reduce the molecular weight distribution width, improve the yield and DP 4-8 ratio, and at the same time, physical field ultrasonic assistance is added during the enzymatic hydrolysis process to further promote the binding of substrate and enzyme, shorten the reaction time, and the protective agent can also effectively ensure the activity of the enzyme, enable the enzyme to be recovered and reused, and save production costs.

[0007] In one embodiment, the protective agent includes one or more of 0.25-0.35 parts of maltodextrin, 0.04-0.06 parts of poloxamer, 0.15-0.25 parts of glycerol, and 0.01-0.03 parts of ascorbic acid.

[0008] The protective agent of the present application is prepared through a specific selection ratio, wherein maltodextrin stabilizes the enzyme structure through hydrogen bonds, and poloxamer is used to reduce the interfacial tension of the system, enhance the binding efficiency of the enzyme and substrate, reduce ineffective adsorption, and avoid ion interference caused by traditional protective agents (such as CaCl2).

[0009] In one embodiment, the complex enzyme composition comprises: 25 parts of chitosanase, 15 parts of chitinase, 10 parts of papain, 0.30 parts of maltodextrin and poloxamer, 0.2 parts of glycerol and 0.02 parts of ascorbic acid.

[0010] In one embodiment, the frequency of the first ultrasonic treatment is 30 kHz-40 kHz, and the duration is 8 min-12 min.

[0011] In one embodiment, the parameters of the second ultrasonic treatment include: power of 180W-220W, treatment time of 4min-6min every 25min-35min, repeated 2-3 times.

[0012] The first ultrasound of the present application is used to destroy the crystalline structure of the raw material, and the second ultrasound further enhances the substrate permeability through intermittent ultrasound, promotes the binding of the substrate and the enzyme, and shortens the reaction time.

[0013] In one embodiment, the deacetylation degree of the chitosan raw material is ≥85%.

[0014] In one embodiment, the enzymatic treatment conditions include a temperature of 48° C. to 52° C. and a pH of 5.5 to 6.0. This temperature and pH range are suitable for the multi-enzyme composition synergistic reaction system of the present application, at which the enzyme activity is high and the reaction is more thorough.

[0015] In one embodiment, the complex enzyme composition further comprises the steps of embedding with magnetic nanoparticles and recovering after enzymatic hydrolysis.

[0016] In one embodiment, the magnetic nanoparticles include Fe3O4.

[0017] Due to the addition of the specific protective agent of the present application, after the enzymatic hydrolysis reaction is completed, the composite enzyme embedded in magnetic nanoparticles (Fe3O4) is used to separate the immobilized enzyme from the reaction solution using magnetic separation technology (such as magnet, adsorption), thereby realizing enzyme recovery and significantly improving the reuse rate of the enzyme.

[0018] In one embodiment, the molecular weight of the fragment isolated by ultrafiltration is less than 10 kDa.

[0019] On the other hand, the present application provides a chitosan oligosaccharide, which is prepared by the above-mentioned preparation method.

[0020] Since the above preparation method is adopted in this embodiment, the chitosan oligosaccharide obtained has a lower molecular weight distribution width and a higher DP 4-8 ratio, and has high application value in the fields of biomedicine, health food, etc. DETAILED DESCRIPTION

[0021] Below in conjunction with embodiment and example, the application is described in further detail.Should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description hereinafter, a large amount of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0023] the term Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings: The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0024] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0025] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0026] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0027] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0028] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0029] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0030] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional values ​​within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoints of the numerical range, as well as every integer between the two numerical endpoints. In this document, this is equivalent to directly listing each integer. For example, "t is an integer selected from 1 to 10" means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges may be combined. In other words, unless otherwise specified, ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0031] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0032] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0033] All documents mentioned in this application are cited as references in this application, just as each document is cited as a reference individually. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0034] On the one hand, the present application provides a method for preparing chitosan oligosaccharides based on a complex enzyme, comprising: Performing a first ultrasonic treatment on the chitosan raw material to prepare a first preparatory material; The first prepared material is subjected to enzymatic hydrolysis treatment using a complex enzyme composition, and simultaneously subjected to a second ultrasonic treatment to prepare a chitosan enzymatic hydrolyzate; The chitosan enzymatic hydrolyzate is subjected to centrifugation and ultrafiltration to prepare chitosan; The complex enzyme composition comprises: 20-30 parts of chitosanase, 10-20 parts of chitinase, 5-15 parts of papain and a protective agent.

[0035] The present application provides a method for preparing chitosan oligosaccharides based on a composite enzyme through the design of a multi-enzyme synergistic system in combination with a specific protective agent, wherein chitosanase and chitinase can synergistically cut the β-1,4 glycosidic bonds of chitosan to control the molecular weight distribution of the product. At the same time, chitinase can also directionally cut the terminal β-1,4 bonds of chitosan, and papain is used to remove protein impurities in chitosan to achieve precise control of the degree of polymerization. However, since there may be certain problems of unstable enzyme activity after combining the above-mentioned enzymes, the present application specifically designs a specific protective agent that is compatible with the above-mentioned multi-enzyme synergistic system. The specific protective agent and the multi-enzyme composition act together to significantly reduce the molecular weight distribution width, improve the yield and DP 4-8 ratio, and at the same time, add physical field ultrasonic assistance during the enzymatic hydrolysis process to further promote the binding of substrate and enzyme and shorten the reaction time.

[0036] In one embodiment, the protective agent includes one or more of 0.25-0.35 parts of maltodextrin, 0.04-0.06 parts of poloxamer, 0.15-0.25 parts of glycerol, and 0.01-0.03 parts of ascorbic acid.

[0037] For example, the number of parts of chitosan is 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, 26 parts, 27 parts, 28 parts, 29 parts or 30 parts and any numerical value in between; for example, the number of parts of chitinase is 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts or 20 parts and any numerical value in between.

[0038] For example, the amount of maltodextrin is 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34 or 0.35, and any values ​​therebetween; and the amount of poloxamer is 0.04, 0.05 or 0.06.

[0039] For example, the amount of glycerol is 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25, and any values ​​therebetween.

[0040] For example, the amount of ascorbic acid is 0.01 part, 0.02 part or 0.03 part and any value therebetween.

[0041] The protective agent of the present application is prepared through a specific selection ratio, wherein maltodextrin stabilizes the enzyme structure through hydrogen bonds, and poloxamer is used to reduce the interfacial tension of the system, enhance the binding efficiency of the enzyme and substrate, reduce ineffective adsorption, and avoid ion interference caused by traditional protective agents (such as CaCl2).

[0042] In one embodiment, the complex enzyme composition comprises: 25 parts of chitosanase, 15 parts of chitinase, 10 parts of papain, 0.30 parts of maltodextrin, 0.05 parts of poloxamer, 0.2 parts of glycerol and 0.02 parts of ascorbic acid.

[0043] In one embodiment, the first ultrasonic treatment has a frequency of 30 kHz to 40 kHz and a duration of 8 min to 12 min, for example, a frequency of 30 kHz, 31 kHz, 32 kHz, 33 kHz, 34 kHz, 35 kHz, 36 kHz, 37 kHz, 38 kHz, 39 kHz, or 40 kHz, and any values ​​therebetween; and for example, a duration of 8 min, 9 min, 10 min, 11 min, or 12 min, and any values ​​therebetween.

[0044] In one embodiment, the parameters of the second ultrasonic treatment include: a power of 180W-220W, a treatment duration of 4-6 minutes every 25-35 minutes, repeated 2-3 times. For example, the ultrasonic treatment power is 180W, 185W, 190W, 195W, 200W, 205W, 210W, 215W or 220W, and any value therebetween; and a treatment duration of 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes or 35 minutes, and any value therebetween, for 4 minutes, 5 minutes or 6 minutes.

[0045] The first ultrasound of the present application is used to destroy the crystalline structure of the raw material, and the second ultrasound further enhances the substrate permeability through intermittent ultrasound, promotes the binding of the substrate and the enzyme, and shortens the reaction time.

[0046] In one embodiment, the deacetylation degree of the chitosan raw material is ≥85%.

[0047] In one embodiment, the conditions for the enzymatic treatment include a temperature of 48°C-52°C and a pH of 5.5-6.0. This temperature and pH range is suitable for the multi-enzyme composition synergistic reaction system of the present application. At this temperature, the enzyme activity is high and the reaction is more thorough. For example, the temperature is 48°C, 49°C, 50°C, 51°C or 52°C, and any values ​​therebetween; for example, the pH is 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0, and any values ​​therebetween.

[0048] In one embodiment, the complex enzyme composition further comprises a step of embedding with magnetic nanoparticles.

[0049] In one embodiment, the magnetic nanoparticles include Fe3O4.

[0050] Due to the addition of the specific protective agent of the present application, after the enzymatic hydrolysis reaction is completed, the composite enzyme embedded in magnetic nanoparticles (Fe3O4) is used to separate the immobilized enzyme from the reaction solution using magnetic separation technology (such as magnet, adsorption), thereby realizing enzyme recovery and significantly improving the reuse rate of the enzyme.

[0051] In one embodiment, the molecular weight of the fragment isolated by ultrafiltration is less than 10 kDa.

[0052] On the other hand, the present application provides a chitosan oligosaccharide, which is prepared by the above-mentioned preparation method.

[0053] Since the above preparation method is adopted in this embodiment, the chitosan oligosaccharide obtained has a lower molecular weight distribution width and a higher DP 4-8 ratio, and has high application value in the fields of biomedicine, health food, etc.

[0054] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0055] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0056] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application. Example

[0057] This embodiment provides a method for preparing chitosan oligosaccharides based on a complex enzyme, comprising the following steps: 1. Raw material pretreatment Chitosan raw material: chitosan powder with a deacetylation degree ≥ 85% (purchased from Shanghai Ciyu Chemical Technology Co., Ltd., with a deacetylation degree of 88% after testing) was selected.

[0058] First ultrasonic treatment: Chitosan powder was dispersed in 0.1 M acetate buffer (pH 5.0) at a solid-liquid ratio of 1:20 (w / v). Ultrasonic treatment was performed at a frequency of 35 kHz for 10 minutes (power 100 W) to fully swell the chitosan and obtain a uniform first pre-mix.

[0059] 2. Preparation of complex enzyme composition Enzyme ratio: weigh 25 parts of chitosanase, 15 parts of chitinase, and 10 parts of papain by weight and mix well.

[0060] Addition of protective agent: Add 0.30 parts of maltodextrin, 0.05 parts of poloxamer, 0.2 parts of glycerol and 0.02 parts of ascorbic acid and dissolve in pH 6.0 phosphate buffer to a final enzyme solution concentration of 20 mg / mL.

[0061] Magnetic nanoparticle embedding: The complex enzyme solution was mixed with Fe3O4 magnetic nanoparticles (particle size 50 nm) at a mass ratio of 1:1, stirred at room temperature for 2 hours, and the immobilized complex enzyme was obtained after magnetic separation for later use.

[0062] 3. Enzymatic hydrolysis and second ultrasonic assistance Enzymatic hydrolysis conditions: The first prepared material was transferred to a constant temperature water bath reactor, and the complex enzyme composition (enzyme to substrate mass ratio 1:15) was added, and the temperature was adjusted to 50°C.

[0063] Second ultrasonic treatment: pulsed ultrasonic assisted treatment was used with a power of 200 W, ultrasonic treatment for 5 minutes every 30 minutes, repeated 3 times, and the total reaction time was 6 hours.

[0064] 4. Centrifugation and ultrafiltration purification Centrifugation: After the reaction is completed, the enzymatic hydrolyzate is centrifuged at 8000 rpm for 15 minutes and the supernatant is collected.

[0065] Ultrafiltration treatment: Ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa (Millipore Pellicon® 2) at a pressure of 0.2 MPa, and the permeate (chitosan oligosaccharide component with a molecular weight <10 kDa) was collected.

[0066] 5. Concentration and drying Concentration: The ultrafiltrate was concentrated under reduced pressure at 50°C to 1 / 5 of the original volume. Example

[0067] This example provides a method for preparing chitosan oligosaccharides based on a composite enzyme, which is basically the same as that of Example 1, except that the enzyme ratio in Example 2 is as follows: 20 parts of chitosanase, 10 parts of chitinase, and 5 parts of papain are weighed by weight and mixed evenly.

[0068] Addition of protective agent: Add 0.25 parts of maltodextrin, 0.04 parts of poloxamer, 0.15 parts of glycerol and 0.01 parts of ascorbic acid, and dissolve in pH 6.0 phosphate buffer to a final enzyme solution concentration of 20 mg / mL. Example

[0069] This example provides a method for preparing chitosan oligosaccharides based on a composite enzyme, which is basically the same as that of Example 1, except that the enzyme ratio in Example 3 is as follows: 30 parts of chitosanase, 20 parts of chitinase, and 15 parts of papain are weighed by weight and mixed evenly.

[0070] Addition of protective agent: Add 0.35 parts of maltodextrin, 0.06 parts of poloxamer, 0.25 parts of glycerol, and 0.03 parts of ascorbic acid, and dissolve in pH 6.0 phosphate buffer to a final enzyme solution concentration of 20 mg / mL. Example

[0071] This example provides a method for preparing chitosan oligosaccharides based on a complex enzyme, which is basically the same as that of Example 1, except that the frequency of the first ultrasonic treatment is 30 kHz and the time is 8 minutes. Example

[0072] This example provides a method for preparing chitosan oligosaccharides based on a complex enzyme, which is basically the same as that of Example 1, except that the frequency of the first ultrasonic treatment is 40 kHz and the time is 12 min. Example

[0073] This example provides a method for preparing chitosan oligosaccharides based on a complex enzyme, which is basically the same as that of Example 1, except that the power of the ultrasonic treatment is 180 W, the treatment time is 4 minutes every 25 minutes, and it is repeated twice. Example

[0074] This example provides a method for preparing chitosan oligosaccharides based on a complex enzyme, which is basically the same as Example 1, except that the power of the second ultrasonic treatment is 220 W, the treatment time is 6 minutes every 35 minutes, and it is repeated twice. Example

[0075] This example provides a method for preparing chitosan oligosaccharide based on a complex enzyme, which is basically the same as Example 1, except that 0.30 parts of CaCl2 and 0.05 parts of Tween 80 are used as protective agents. Example

[0076] This example provides a method for preparing chitosan oligosaccharides based on a complex enzyme, which is basically the same as that of Example 1, except that 0.30 parts of maltodextrin and 0.05 parts of poloxamer are used as protective agents. Example

[0077] This embodiment provides a method for preparing chitosan oligosaccharides based on a complex enzyme, which is basically the same as that of Example 1, except that the second ultrasonic treatment adopts a continuous treatment with a power of 180W-220W. Example

[0078] This example provides a method for preparing chitosan oligosaccharides based on a complex enzyme, which is basically the same as that of Example 1, except that the frequency of the first ultrasonic treatment is 20 kHz and the time is 8 minutes. Example

[0079] This example provides a method for preparing chitosan oligosaccharides based on a complex enzyme, which is basically the same as that of Example 1, except that the degree of deacetylation of the chitosan raw material is 75%.

[0080] Comparative Example 1 The complex enzyme composition of this comparative example uses a traditional complex enzyme: 20 parts of lysozyme, 10 parts of cellulase (10%) without papain, and the rest is the same as in Example 1.

[0081] Comparative Example 2 Comparative Example 2 is a variation of Example 1, in which papain is not added to the complex enzyme composition. The rest is the same as Example 1.

[0082] Comparative Example 3 Comparative Example 3 is a variation of Example 1, in which no protective agent is added to the complex enzyme composition. The rest is the same as Example 1.

[0083] Comparative Example 4 Comparative Example 4 is a variation of Example 1, in which the ratio of the complex enzyme composition is 10-20 parts of chitosanase, 20-30 parts of chitinase, 5-15 parts of papain and a protective agent, and the rest is the same as Example 1.

[0084] Comparative Example 5 Comparative Example 5 is a variation of Example 1, in which the second ultrasonic treatment is not performed during the enzymatic hydrolysis treatment, and the rest is the same as Example 1.

[0085] Verification results: The following are the test methods and corresponding technical details of the key data in the examples, covering the detection of chitosan oligosaccharide yield, molecular weight distribution and enzyme activity retention rate: 1. Chitosan oligosaccharide yield 1. Determination method: Weighing method: After the reaction is completed, collect the purified chitosan oligosaccharide powder, weigh the mass after drying, and compare it with the mass of the initial chitosan raw material to calculate the yield. Reference standard: GB / T 35818-2018.

[0086] Yield (%) = mass of chitosan oligosaccharide / mass of chitosan raw material × 100% Yield (%) = mass of chitosan raw material / mass of chitosan oligosaccharide × 100% High-performance liquid chromatography (HPLC): The content of reducing sugars (such as glucosamine) in the enzymatic hydrolysate is quantitatively analyzed by HPLC, and the yield is calculated based on the standard curve.

[0087] 2. Molecular weight distribution (DP 4-8 ratio & PDI) 1. Test method: SEC-MALS (size exclusion chromatography coupled with multi-angle light scattering), reference standard ISO 21314:2019.

[0088] The specific steps are as follows: Chromatographic conditions: chromatographic column: TSKgel G3000PWXL (7.8 mm × 30 cm, Tosoh Bioscience), mobile phase: 0.1 M ammonium acetate buffer (pH 4.5), flow rate: 0.5 mL / min, column temperature: 35°C.

[0089] Detection system: MALS detector (Wyatt Technology, DAWN HELEOS II), differential refractive index detector (RI).

[0090] Data Analysis: The weight-average molecular weight (Mw), number-average molecular weight (Mn), and PDI (Mw / Mn) were calculated using ASTRA 7.1 software.

[0091] 3. Enzyme activity retention rate 1. Determination method: Spectrophotometric method (using chitosanase as an example): Substrate: Dissolve chitosan in acetate buffer (pH 5.0) to prepare a 0.5% solution. Reaction: Mix the enzyme solution with the substrate and incubate at 50°C for 10 minutes. Immediately terminate the reaction in a boiling water bath. Detection: Add DNS reagent for color development. Measure absorbance at 540 nm. Calculate the amount of reducing sugar released based on a standard glucose curve.

[0092] Enzyme activity (U / mL) = reducing sugar amount (μg) reaction time (min) × enzyme solution volume (mL) Enzyme activity (U / mL) = reaction time (min) × enzyme solution volume (mL) reducing sugar amount (μg).

[0093] Calculation of activity retention rate: Retention rate (%) = enzyme activity after reaction × initial enzyme activity × 100% Retention rate (%) = initial enzyme activity × 100%

[0094] The test results are shown in Table 1 below: Table 1 As shown in the table, Comparative Example 1, which employed a traditional protective agent, exhibited significant decreases in yield, dispersion, and DP4-8 percentage compared to Example 1. Comparative Example 2, which omitted papain, exhibited reduced yield and dispersion compared to Example 1, demonstrating that papain is essential for the specific cleavage of chitosan's β-1,4-glycosidic bonds. Comparative Example 3, which also employed an enzyme complex without a protective agent, exhibited reduced interfacial tension and low enzyme-substrate binding efficiency, resulting in similarly reduced performance. Comparative Example 4, which employed an opposite ratio of chitosanase to chitinase and a reduced endoenzyme system, exhibited incomplete cleavage of chitosan's β-1,4-glycosidic bonds. While the yield decreased slightly compared to Example 1, the molecular weight distribution and DP4-8 percentage decreased. Comparative Example 5, which omitted ultrasonic treatment, exhibited high enzyme activity retention but poor substrate permeability, and all other results were lower than those of Example 1.

[0095] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for preparing chitosan oligosaccharides based on complex enzymes, characterized in that: include: performing a first ultrasonic treatment on the chitosan raw material to prepare a first preparatory material; The first prepared material is subjected to enzymatic hydrolysis treatment using a complex enzyme composition, and simultaneously subjected to a second ultrasonic treatment to prepare a chitosan enzymatic hydrolyzate; The chitosan enzymatic hydrolyzate is subjected to centrifugation and ultrafiltration to prepare chitosan; The complex enzyme composition comprises: 20-30 parts of chitosanase, 10-20 parts of chitinase, 5-15 parts of papain and a protective agent.

2. The method according to claim 1, characterized in that The protective agent includes one or more of 0.25-0.35 parts of maltodextrin, 0.04-0.06 parts of poloxamer, 0.15-0.25 parts of glycerol and 0.01-0.03 parts of ascorbic acid.

3. The method according to claim 1, characterized in that The complex enzyme composition comprises: 25 parts of chitosanase, 15 parts of chitinase, 10 parts of papain, 0.30 parts of maltodextrin, 0.05 parts of poloxamer, 0.2 parts of glycerol and 0.02 parts of ascorbic acid.

4. The method for preparing chitosan oligosaccharides based on complex enzyme according to claim 1, characterized in that: Parameters of the first ultrasonic treatment include: a frequency of 30 kHz to 40 kHz, and a time of 8 min to 12 min.

5. The method for preparing chitosan oligosaccharides based on complex enzyme according to claim 1, characterized in that: The parameters of the second ultrasonic treatment include: power of 180 W to 220 W, treatment time of 4 min to 6 min every 25 min to 35 min, and repeated 2 to 3 times.

6. The method for preparing chitosan oligosaccharide based on complex enzyme according to claim 1, characterized in that: The deacetylation degree of the chitosan raw material is ≥85%.

7. The method for preparing chitosan oligosaccharides based on complex enzyme according to claim 1, characterized in that: The conditions for the enzymatic hydrolysis treatment include: a temperature of 48° C. to 52° C. and a pH of 5.5 to 6.

0.

8. The method for preparing chitosan oligosaccharide based on complex enzyme according to claim 1, characterized in that: The complex enzyme composition further comprises the steps of embedding with magnetic nanoparticles and recovering after enzymatic hydrolysis; The magnetic nanoparticles include Fe3O4.

9. The method for preparing chitosan oligosaccharide based on complex enzyme according to claim 1, characterized in that: The ultrafiltration treatment dissociates molecular weights less than 10 kDa.

10. A chitosan oligosaccharide, characterized in that The chitosan oligosaccharide is prepared by the method for preparing chitosan oligosaccharide based on complex enzyme according to any one of claims 1 to 9.