Fucoidan and preparation method and application thereof

Abstract

The application discloses fucoidan oligosaccharide and a preparation method and application thereof, and belongs to the technical field of substance extraction and preparation; the preparation method of the fucoidan oligosaccharide provided by the application comprises the following steps: hydrogen peroxide solution and nano-mesoporous material are added into a fucoidan polysaccharide aqueous solution, and after uniform stirring, a degradation reaction is carried out under the assistance of microwaves; after the degradation reaction is completed, catalase is added, and then filtration is carried out, and a filter cake is collected; sodium chloride solution is added into the filter cake for elution replacement, and an eluate is collected and concentrated through nanofiltration, washed, and freeze-dried to obtain the fucoidan oligosaccharide. The obtained fucoidan oligosaccharide has uniform molecular structure, relatively complete sulfate group, relatively narrow molecular weight distribution and high anti-inflammatory activity; and the preparation method provided by the application has high degradation efficiency, does not need high-cost enzymes or complex equipment, is simple to operate and easy to realize actual production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of substance extraction and preparation, and particularly relates to fucoidan oligosaccharide and a preparation method and application thereof. BACKGROUND

[0002] Fucoidan, also known as fucoidan, is a natural active polysaccharide containing sulfate groups unique to brown algae, and its structure is shown in Figure 1 The fucoidan is mainly present in the cell wall matrix, intercellular space and secreted mucus of brown algae such as kelp, wakame, giant kelp and rockweed. Fucoidan has various physiological effects, such as anti-Helicobacter pylori, anti-tumor, immune enhancement (vaccine adjuvant), anti-thrombus, blood pressure reduction, anti-oxidation and the like, and has become a research hotspot and market highlight. According to reports, the general standard molecular weight of fucoidan is 30,000-150,000 Dalton (Dalton), and the large molecular weight is not conducive to the absorption of the organism, is difficult to digest, and the chemical composition and structure of the polysaccharide extracted from different algae are also different. Due to its complexity, the biological activity thereof is weakened to some extent, and the application of fucoidan in various fields is also limited. Therefore, the related research on fucoidan oligosaccharide which is more easily absorbed and has a relatively simple structure has gradually risen.

[0003] Fucoidan oligosaccharide is a low molecular weight polymer obtained by degrading fucoidan. Compared with fucoidan, fucoidan oligosaccharide has small molecular weight, strong solubility, stronger stability, can be better absorbed by the organism, and has biological activities such as immune regulation, anti-tumor, anti-oxidation, anti-inflammatory and anti-virus. Studies have shown that in the system of human intestinal microorganisms degrading fucoidan in vitro, the content of fucoidan oligosaccharide is significantly reduced, and the monosaccharide composition of the fermentation product does not change, indicating that it is completely degraded and utilized by human intestinal microorganisms; the degradation and utilization degree of macromolecular fucoidan (20 kDa) by human intestinal microorganisms is very low, and the proportion of galactose and mannose in the product is significantly reduced; at the same time, it is found that the main short-chain fatty acids generated after the decomposition of fucoidan oligosaccharide are acetic acid, propionic acid and butyric acid, which can provide energy for the growth and reproduction of intestinal microorganisms and colon mucosa, maintain mucosal immune cells, reduce the generation of pro-inflammatory factors, repair mucosal inflammation, and are beneficial to the health of human body. However, branched-chain fatty acids (isobutyric acid, isovaleric acid) and valeric acid are also generated in the fermentation liquor of macromolecular fucoidan, which can further produce harmful substances such as ammonia, phenol and indole to the human body.

[0004] At present, the methods for preparing fucoidan oligosaccharides by degrading fucoidan at home and abroad mainly include acid degradation method, oxidation degradation method and enzyme degradation method. The acid degradation method has the disadvantages of easy production of reaction byproducts, difficult control of degradation degree, wide molecular weight distribution range of prepared fucoidan oligosaccharides and poor stability; for example, a preparation method of fucoidan oligosaccharides is disclosed in Chinese patent CN109970823A, an acid solution of fucoidan is prepared, acid hydrolysis is carried out at a certain temperature for 2-12 hours, the pH is adjusted to neutral after the degradation is completed, centrifugation and ultrafiltration are carried out to obtain degradation products of multiple molecular weight segments, and the degradation products are respectively subjected to freeze-drying to obtain freeze-dried powder, which is fucoidan oligosaccharides. The fucoidan oligosaccharides obtained by the method have a wide molecular weight distribution, and the part less than 5kDa only accounts for 20-30%, which is not conducive to exerting the efficacy of fucoidan oligosaccharides. The enzyme degradation method can utilize the specificity of enzymes to degrade polysaccharide glycosidic bonds, and has the advantages of mild conditions and less byproducts, but is currently only in the laboratory research stage, and has the disadvantages of low enzyme degradation efficiency and high cost of enzyme screening and culture; for example, Chinese patent CN114181923A provides a process for preparing fucoidan oligosaccharides catalyzed by continuous endo-1, 3-fucoidanase, which adopts an endo-1, 3-fucoidanase Fun168D to specifically cut the glycosidic bond between 2-position sulfated fucose, and the action mode is continuous endo, which can continuously cut the glycosidic bond along the polysaccharide main chain to continuously produce specific structure oligosaccharides; the purity of the product oligosaccharides without purification is higher than 70%, and the purity after one-step chromatography purification is higher than 95%, which can be applied to the large-scale preparation of fucoidan oligosaccharides with specific structure. However, the enzyme is difficult to obtain, the culture cost is high, and the enzyme degradation efficiency is not high. The most common oxidation degradation method is H2O2 / copper ion oxidation method, which utilizes the hydroxyl radicals generated by the decomposition of H2O2 to oxidize and break the glycosidic bond between polysaccharides, and has little damage to the functional groups (sulfate groups) of fucoidan; however, the method has the disadvantages of low degradation efficiency, complex operation and high operation cost due to the need of chelating resin adsorption to remove metal ions after the degradation is completed. It is reported that the fucoidan is degraded by the H2O2 / copper ion oxidation degradation method, and 19.6kDa fucoidan with a sulfate group content of 28.2% can be obtained after 4 hours of reaction; however, subsequent operations need to use sodium hydroxide to precipitate and remove copper ions, and use chelating resin to remove trace residual copper ions. The existing technologies have the following obvious defects: 1. low degradation efficiency and long reaction time; 2. wide molecular weight distribution of obtained oligosaccharides and difficult control of narrow molecular weight distribution; and 3. complex post-treatment, need to use acid and alkali reagents or metal salts, and is not environment-friendly. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provide fucoidan oligosaccharides with uniform molecular structure, relatively complete sulfate groups, narrow molecular weight distribution, high degradation efficiency, simple operation and high anti-inflammatory activity, and a preparation method and application thereof.

[0006] To achieve the above object, the technical scheme adopted by the present application is as follows: a preparation method of fucoidan oligosaccharide, the preparation method comprising the following steps:

[0007] (1) fucoidan is prepared into a fucoidan aqueous solution, then hydrogen peroxide solution and nano-mesoporous material are added into the fucoidan aqueous solution, and after uniform stirring, a degradation reaction is carried out under microwave assistance;

[0008] (2) after the degradation reaction is completed, catalase is added, then filtration is carried out, and the filter cake is collected;

[0009] (3) sodium chloride solution is added to the filter cake for elution replacement, the eluate is collected, concentrated by nanofiltration, and freeze-dried to obtain fucoidan oligosaccharide.

[0010] In the preparation method of fucoidan oligosaccharide provided by the present application, microwave-assisted hydrogen peroxide is used for degradation, and nano-mesoporous material is added at the same time, so that fucoidan oligosaccharide with uniform molecular structure, relatively complete sulfate group, narrow molecular weight distribution and high anti-inflammatory activity can be obtained, wherein the molecular weight is between 500-3000 Da, the sulfate group content is between 16.8-22.4%, which is close to the sulfate group content in the provided fucoidan substance, the molecular weight distribution index is 1.06-1.12, and in the later animal anti-inflammatory experiment, it is found that the transcription level of pro-inflammatory factors is obviously down-regulated; and the preparation method provided by the present application has high degradation efficiency, does not need high-priced enzymes or complex equipment, is simple to operate, and is easy to realize actual production.

[0011] Specifically, the microwave is a high-frequency electromagnetic wave, when it passes through the medium, the polar molecules in the medium oscillate at high frequency in the electromagnetic wave generated by the microwave, so that the medium is degraded by breaking the intramolecular or intermolecular chemical bond, thereby greatly shortening the reaction time. The mesoporous material has the characteristics of extremely high specific surface area, regular and ordered pore structure, narrow pore size distribution, and continuous adjustable pore size, so that it can complete the adsorption and separation process of macromolecules which is difficult for microporous zeolite mesoporous material; the nano-mesoporous material has smaller particle size, so more active centers can be exposed, and it has larger specific surface area and higher intracrystalline diffusion rate; when the molecule is small to a certain extent, its kinetic diameter is smaller than the pore size of the nano-mesoporous material, the molecule can enter the inner pore of the nano-mesoporous material and be adsorbed, and at the same time, due to the space steric hindrance effect and chemical inertness effect in the pore, the reaction activity and reaction ability of the molecule will be affected. That is, as a whole, under the action of the microwave, the macromolecular fucoidan is uniformly dispersed in water, and the hydrogen peroxide plays its oxidizing role, so that the fucoidan is gradually degraded into oligosaccharide; at the same time, the addition of the nano-mesoporous material can make the degraded oligosaccharide enter the pore of the nano-mesoporous material, and due to the space steric hindrance and chemical inertness in the pore, the further degradation of the oligosaccharide is prevented, so that the fucoidan oligosaccharide with relatively uniform molecular structure and low relative molecular weight is obtained; in addition, due to the mild oxidation effect of hydrogen peroxide, the organic sulfate group with strong binding ability is difficult to be destroyed, so that the relatively complete organic sulfate group can be retained.

[0012] As a preferred embodiment of the preparation method of the present application, the mass ratio of the fucoidan, the hydrogen peroxide solution and the nano-mesoporous material is fucoidan: hydrogen peroxide solution: nano-mesoporous material = 100: (0.1-2): (0.1-0.5).

[0013] The inventors have found that when the mass ratio of the hydrogen peroxide solution and the nano-mesoporous material to the fucoidan is within the above range, on the one hand, the fucoidan oligosaccharide with the target molecular weight range can be obtained, so that a higher anti-inflammatory activity can be achieved, and on the other hand, a higher preparation yield can be ensured.

[0014] As a preferred embodiment of the preparation method of the present application, the mass concentration of the hydrogen peroxide is 30%.

[0015] As a preferred embodiment of the preparation method of the present application, the nano-mesoporous material is monodisperse nano-mesoporous carbon microspheres, and the pore size of the monodisperse nano-mesoporous carbon microspheres is 3.5-5 nm.

[0016] The inventors have found that, in the process of degrading fucoidan, when the fucoidan is degraded to a molecular weight below 3000 Da, the molecular particle size is reduced to below 4 nm. In combination with the molecular weight range of the present application, the monodisperse nano-mesoporous carbon microspheres with a pore size of 3.5-5 nm are preferred, so that the oligosaccharides with a molecular weight below 3000 Da can enter the pores of the monodisperse mesoporous carbon nanomicrospheres, preventing further degradation of the oligosaccharides, thereby obtaining fucoidan oligosaccharides with a molecular weight distribution of 500-3000 Da.

[0017] As a preferred embodiment of the preparation method of the present application, in the microwave-assisted degradation reaction, the output power of the microwave is 10-2500 w, the working frequency of the microwave is 2400-2500 MHz, and the degradation reaction time is 0.5-12 h.

[0018] Preferably, in the microwave-assisted degradation reaction, the output power of the microwave is 100-500 w, the working frequency of the microwave is 2450 MHz, and the degradation reaction time is 1-2 h.

[0019] The inventors have found that, under the above microwave output power and degradation reaction time, the fucoidan in the present application can be obtained with a higher yield.

[0020] As a preferred embodiment of the preparation method of the present application, the mass concentration of the fucoidan aqueous solution is 0.1-10%.

[0021] As a preferred embodiment of the preparation method of the present application, the molecular weight of the fucoidan is 20-1000 kDa, and the fucoidan is derived from any one of the following: kelp, laminaria, undaria pinnatifida, sargassum fusiforme, sargassum thunbergii, sargassum siliquosum, and sea cucumber.

[0022] As a preferred embodiment of the preparation method of the present application, in step (2), the catalase is added to inactivate the uncompletely consumed hydrogen peroxide in the system.

[0023] As a preferred embodiment of the preparation method of the present application, in step (2), the filtration is divided into two stages. The first stage is plate-and-frame filtration, and the filtrate is collected for the second stage filtration. The second stage filtration is performed using a 0.44 μm microporous filter.

[0024] The two-stage filtration can effectively retain the monodisperse nano-mesoporous carbon microspheres and discard the supernatant containing incompletely degraded macromolecular substances. The discarded supernatant can be further collected, dried, and then subjected to degradation reaction again.

[0025] As a preferred embodiment of the preparation method of the present application, the mass concentration of the sodium chloride solution is 10-30%.

[0026] Preferably, the sodium chloride solution has a mass concentration of 20%.

[0027] As a preferred embodiment of the preparation method of the present application, the temperature of the elution displacement is 50-70℃.

[0028] The sodium chloride solution with the above mass concentration is added, and the elution displacement is carried out at the above temperature, which can sufficiently displace the fucoidan oligosaccharides adsorbed in the pores of the monodisperse nanometer mesoporous carbon microspheres, thereby ensuring the preparation yield.

[0029] In addition, the present application also provides a fucoidan oligosaccharide prepared by the preparation method of the present application.

[0030] As a preferred embodiment of the fucoidan oligosaccharide of the present application, the molecular weight of the fucoidan oligosaccharide is 500-3000 Da, and the molecular weight distribution coefficient is 1.06-1.12.

[0031] The fucoidan oligosaccharide in the above molecular weight range and the molecular weight distribution coefficient range provided by the present application has excellent anti-inflammatory effect.

[0032] In addition, the present application also provides the use of the fucoidan oligosaccharide in the preparation of a composition with anti-inflammatory activity.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] In the preparation method of the fucoidan oligosaccharide provided by the present application, microwave-assisted hydrogen peroxide is used for degradation, and nanometer mesoporous material is added at the same time, so that the fucoidan oligosaccharide with uniform molecular structure, relatively complete sulfate group, narrow molecular weight distribution and high anti-inflammatory activity can be obtained, wherein the molecular weight is between 500-3000 Da, the sulfate group content is between 16.8-22.4%, which is close to the sulfate group content in the provided fucoidan polysaccharide, the molecular weight distribution index is 1.06-1.12, and in the later animal anti-inflammatory experiment, it is found that it can significantly down-regulate the transcription level of pro-inflammatory factors; and the degradation efficiency of the preparation method provided by the present application is high, without the need for high-priced enzymes or complex equipment, the operation is simple, and the actual production is easy to realize. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is a structural schematic diagram of fucoidan polysaccharide;

[0036] Figure 2 It is a process flow diagram of the preparation method of the present application. DETAILED DESCRIPTION

[0037] For the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific embodiments.

[0038] The reagents, methods and apparatus employed in the present application are conventional in the art unless otherwise specified.

[0039] Monodisperse nanometer mesoporous carbon microspheres: Beijing Zhongke Keyou Technology Co., Ltd.

[0040] Example 1

[0041] The present application provides a fucoidan oligosaccharide, and a preparation process of the fucoidan oligosaccharide is shown in the figure. Figure 2 Specifically, the preparation method comprises the following steps:

[0042] (1) 100 g of fucoidan with a molecular weight of 150-280 kDa from kelp is weighed, 2000 g of deionized water is added, and stirring is performed for 60 min to completely dissolve the fucoidan, so as to prepare a fucoidan aqueous solution with a concentration of 5%; 1 g of hydrogen peroxide solution with a mass concentration of 30% and 0.5 g of monodisperse nanometer mesoporous carbon microspheres (pore size of 3.5-5 nm) are added, stirring is performed at room temperature for 30 min, after uniform stirring, the solution is transferred to an MCR-3 microwave reactor, the microwave output power is set to 250 W, and the working frequency is set to 2450 W, and degradation is performed for 1 h;

[0043] (2) After the degradation reaction is completed, cooling is performed to room temperature, 1% of a catalase preparation is added, and stirring reaction is performed for 30 min; hydrogen peroxide test paper is used for testing, until no residual hydrogen peroxide is present in the solution; then, plate and frame filtration is performed, the filtrate is collected, and then a microporous filter with a pore size of 0.44 μm is used for filtration, and the filter cake is collected;

[0044] (3) A sodium chloride solution with a mass concentration of 20% is added to the filter cake, the temperature is kept at 58-62 ℃, HPLC is used for monitoring the elution replacement of the fucoidan oligosaccharide, until no fucoidan oligosaccharide solution appears, which is the elution end point; the eluate is subjected to nanofiltration through a GE nanofiltration membrane with a molecular weight cut-off of 300 Da, and an appropriate amount of deionized water is used for washing; silver nitrate titration is used for monitoring the removal of chloride ions, until no chloride ions are present in the filtrate; nanofiltration is continuously performed until the solid content of the concentrated solution is 35%; the concentrated solution is discharged, and freeze-drying is performed, to obtain the fucoidan oligosaccharide.

[0045] Example 2

[0046] The present application provides a fucoidan oligosaccharide, and a preparation method of the fucoidan oligosaccharide comprises the following steps:

[0047] (1) take 50g of fucoidan from Undaria pinnatifida with molecular weight of 520-750kDa, add 5000g of deionized water, stir for 60min to make the fucoidan completely dissolved, and configure into a fucoidan aqueous solution with a concentration of 1%; add 0.5g of hydrogen peroxide solution with a mass concentration of 30% and 0.3g of monodisperse nanometer mesoporous carbon microspheres (pore size of 3.5-5nm), stir for 30min at room temperature, after uniform stirring, transfer the solution to a MCR-3 microwave reactor, set the microwave output power to 250W, and work frequency to 2450W for 1h of degradation;

[0048] (2) after the degradation reaction is completed, cool to room temperature, add 1% catalase preparation, stir for 30min, test with hydrogen peroxide test paper until there is no residual hydrogen peroxide in the solution; then first plate and frame filter, collect the filtrate, and then filter with a microporous filter of 0.44μm to collect the filter cake;

[0049] (3) add sodium chloride solution with a mass concentration of 30% to the filter cake, keep the temperature at 58-62℃, monitor the elution replacement of fucoidan oligosaccharide with HPLC until no oligosaccharide solution appears, which is the elution end point; elute the solution through a GE nanofiltration membrane of 300Da, and wash with an appropriate amount of deionized water, monitor the removal of chloride ions with silver nitrate titration method until there is no chloride ion in the filtrate, continue to concentrate by nanofiltration until the solid content of the concentrated solution is 25%, discharge the concentrated solution, and freeze-dry to obtain fucoidan oligosaccharide.

[0050] Example 3

[0051] The embodiment of the present application provides a fucoidan oligosaccharide, and a preparation method of the fucoidan oligosaccharide comprises the following steps:

[0052] (1) take 50g of fucoidan from Undaria pinnatifida with molecular weight of 520-750kDa, add 5000g of deionized water, stir for 60min to make the fucoidan completely dissolved, and configure into a fucoidan aqueous solution with a concentration of 1%; add 0.5g of hydrogen peroxide solution with a mass concentration of 30% and 0.3g of monodisperse nanometer mesoporous carbon microspheres (pore size of 3.5-5nm), stir for 30min at room temperature, after uniform stirring, transfer the solution to a MCR-3 microwave reactor, set the microwave output power to 250W, and work frequency to 2450W for 1h of degradation;

[0053] (2) after the degradation reaction is completed, cool to room temperature, add 1% catalase preparation, stir for 30min, test with hydrogen peroxide test paper until there is no residual hydrogen peroxide in the solution; then first plate and frame filter, collect the filtrate, and then filter with a microporous filter of 0.44μm to collect the filter cake;

[0054] (3) Add 10% sodium chloride solution to the filter cake, keep the temperature at 58-62℃, monitor the elution replacement of fucoidan oligosaccharide by HPLC until no oligosaccharide solution appears, which is the elution end point; the eluate is filtered through 300Da GE nanofiltration membrane, and washed with appropriate amount of deionized water, and the removal of chloride ions is monitored by silver nitrate titration method until there is no chloride ion in the filtrate, and then the nanofiltration is continued to concentrate to a solid content of 30% in the concentrated solution, the concentrated solution is discharged, and freeze-drying is performed to obtain fucoidan oligosaccharide.

[0055] Comparative Example 1

[0056] The present application provides a kind of fucoidan oligosaccharide, and the preparation method of the fucoidan oligosaccharide includes the following steps:

[0057] (1) take 100g fucoidan with molecular weight of 150-280kDa from kelp, add 2000g deionized water, stir for 60min to make fucoidan completely dissolved, and prepare a 5% fucoidan aqueous solution; 1g of 30% hydrogen peroxide solution is added, stirred at room temperature for 30min, and after stirring uniformly, the solution is transferred to MCR-3 microwave reactor, set the microwave output power to 100W, and the working frequency is 2450W degradation 2h;

[0058] (2) after the degradation reaction is completed, cool to room temperature, add 1% catalase preparation, stir for 30min, test with hydrogen peroxide test paper until there is no residual hydrogen peroxide in the solution, then filter, collect the filtrate, pass the filtrate through a 600Da GE ultrafiltration membrane, and concentrate the permeate under reduced pressure to a solid content of 35% in the concentrated solution. The concentrated solution is discharged, and freeze-drying is performed to obtain fucoidan oligosaccharide.

[0059] Comparative Example 2

[0060] The present application provides a kind of fucoidan oligosaccharide, and the preparation method of the fucoidan oligosaccharide is only different from that of example 1 in that the mass ratio of added hydrogen peroxide to fucoidan is fucoidan: hydrogen peroxide = 100:4.

[0061] Comparative Example 3

[0062] The present application provides a kind of fucoidan oligosaccharide, and the preparation method of the fucoidan oligosaccharide is only different from that of example 1 in that the mass ratio of added nanometer mesoporous material to fucoidan is fucoidan: nanometer mesoporous material = 100:1.

[0063] Comparative Example 4

[0064] The present application provides a kind of fucoidan oligosaccharide, and the preparation method of the fucoidan oligosaccharide is only different from that of example 1 in that the pore size of the added nanometer mesoporous material is 6.5-8nm.

[0065] Comparative Example 5

[0066] The present comparative example provides a fucoidan, and the only difference between the preparation method of the fucoidan and Example 1 is that the temperature of elution displacement is 90℃.

[0067] Effect Example 1

[0068] This effect example tests the yield, number average molecular weight, molecular weight distribution coefficient, sulfate group content and sulfate group content retention rate of the fucoidan prepared in Examples 1-3 and Comparative Examples 1-5;

[0069] Among them, the yield of fucoidan is the mass ratio of fucoidan prepared in Examples 1-3 and Comparative Examples 1-5 to raw material fucoidan;

[0070] The test method of the number average molecular weight and the molecular weight distribution coefficient of the fucoidan is that the fucoidan prepared in Examples 1-3 and Comparative Examples 1-6 is dissolved with a mobile phase (0.1 mol / L sodium acetate-acetic acid aqueous solution), and a molecular weight narrow distribution polyethylene glycol standard sample (molecular weight is 600, 1470, 7100, 12600, 23000) provided by Waters Company is used as a standard curve, the regression coefficient is 0.9996, and the number average molecular weight and the molecular weight distribution coefficient of the fucoidan are obtained by gel chromatography (GPC) detection analysis;

[0071] The sulfate group content of the fucoidan is determined by barium sulfate turbidimetry, and the calculation method of the sulfate group content retention rate is: sulfate group content retention rate=(sulfate group content of fucoidan / sulfate group content in corresponding fucoidan)×100%;

[0072] The results obtained are shown in Table 1;

[0073] Table 1

[0074]

[0075] As can be seen from Table 1, the yield of the fucoidan obtained by using the technical scheme of the present application is higher, which is more than 70.2%, the molecular weight distribution is narrower, the molecular weight distribution coefficient is between 1.06-1.12, and the sulfate group content retention rate is higher, which is more than 87.50%.

[0076] The fucoidin oligosaccharide is prepared by using a conventional fucoidin degradation method. The fucoidin is configured into a fucoidin solution with a concentration of 0.6% by using 0.25M H2SO4, and the fucoidin aqueous solution is placed in a 70℃ water bath for 7.5h with a shaking speed of 120r / min. NaOH powder is added to the fucoidin solution to neutralize the pH of the fucoidin solution to terminate the reaction. The final solution is centrifuged at a speed of 5000r / min for 10min, and the supernatant is taken and subjected to ultrafiltration to divide the fucoidin degradation solution into fucoidin oligosaccharide powders with molecular weights of less than 5kDa, 5-10kDa, 10-30kDa, 30-50kDa, 50-100kDa and more than 100kDa, respectively. The total yield of the fucoidin oligosaccharide is 88%, and the proportions of the fucoidin oligosaccharide with different molecular weights are 24.4% for less than 5kDa, 49.7% for 5-10kDa, 16.1% for 10-30kDa, 6.6% for 30-50kDa, 1.9% for 50-100kDa and 1.3% for more than 100kDa. The preparation method of the present application can significantly improve the degradation yield of the fucoidin oligosaccharide with a specific molecular weight range compared with the conventional degradation method.

[0077] As can be seen from Example 1 and Comparative Example 1, when no nano-mesoporous material is added, the molecular weight distribution of the obtained fucoidin oligosaccharide is obviously widened, and the molecular weight distribution coefficient is 149.07% of that in Example 1. As can be seen from Example 1 and Comparative Example 2, when the amount of hydrogen peroxide added is too much, the number average molecular weight of the obtained fucoidin oligosaccharide shows a downward trend, and the yield and the retention rate of the sulfate group content also significantly decrease, and the molecular weight distribution coefficient also shows an increasing trend. Compared with Example 1, the molecular weight distribution coefficient increases by 96.30%, the yield decreases by 41.52%, and the retention rate of the sulfate group content decreases by 26.19%. As can be seen from Example 1 and Comparative Example 3, when more nano-mesoporous material is added, the retention rate of the sulfate group content has a slight upward trend compared with Example 1, but the yield decreases significantly by 10.76%, and the molecular weight distribution coefficient also shows a certain increasing trend, which increases by 14.58%. As can be seen from Example 1 and Comparative Example 4, when the pore size of the added nano-mesoporous material is larger, the number average molecular weight of the obtained product obviously increases from 1027Da to 4627Da. As can be seen from Example 1 and Comparative Example 5, changing the displacement temperature also affects the yield, the number average molecular weight, the molecular weight distribution and the retention rate of the sulfate group content of the product.

[0078] Effect Example 2

[0079] The anti-inflammatory activity of the fucoidan oligosaccharides prepared in Examples 1-3 and Comparative Examples 1-5 was tested; to evaluate the anti-inflammatory effect of the fucoidan oligosaccharides, an acute inflammatory mouse animal experiment was performed, 42 eight-week-old BALB / c mice were randomly divided into 7 groups, 6 mice in each group, corresponding to a normal treatment group, a lipopolysaccharide stimulation group, an Example 3 fucoidan oligosaccharide treatment group, an Example 3 fucoidan oligosaccharide and lipopolysaccharide stimulation combination group, a Comparative Example 1 fucoidan oligosaccharide treatment group, a Comparative Example 2 fucoidan oligosaccharide treatment group, and a Comparative Example 4 fucoidan oligosaccharide treatment group. The mice were raised in an environment with constant temperature and humidity, and after a one-week adaptation period, the mice were injected intraperitoneally with lipopolysaccharide, Example 3 fucoidan oligosaccharide, Example 3 fucoidan oligosaccharide and lipopolysaccharide, Comparative Example 1 fucoidan oligosaccharide, Comparative Example 2 fucoidan oligosaccharide, and Comparative Example 4 fucoidan oligosaccharide at a dose of 2 mg / Kg of mouse body weight, respectively. After 24 hours, tissue samples were obtained by dissection; and the contents of tumor necrosis factor alpha (TNFα), chemokine (MCP-1), and interleukin 8 (IL-8) were detected. The specific detection method was as follows: enzyme-linked immunosorbent assay (ELISA) was used, the tissue samples were washed with buffer to remove surface residues, weighed, cut into small pieces, and added to the dilution buffer, then homogenized with a glass homogenizer, further extracted by ultrasonic disruption, and centrifuged at 5000 x g for 5 minutes to collect the supernatant. The sample and positive control were added to the wells of the ELISA kit, and the antibody mixture was added, incubated on a plate shaker for 1 hour, washed the wells, added TMB substrate for color development, incubated in the dark at room temperature for 15 minutes, added STOP solution, gently shaken for a few seconds, and the absorbance at 450 nm was read by spectrophotometry to obtain the corresponding tumor necrosis factor alpha (TNFα), chemokine (MCP-1), and interleukin 8 (IL-8) content data. The data obtained is shown in Table 2.

[0080] Table 2

[0081]

[0082] As can be seen from Table 2, the mice injected with lipopolysaccharide have a large number of inflammatory reactions activated in a short time within 24 hours due to the innate immune recognition of the mice to the bacterial surface lipopolysaccharide, and the transcription levels of lung proinflammatory factors such as tumor necrosis factor alpha (TNFα), chemokine (MCP-1), and interleukin 8 (IL-8) are obviously increased; the transcription levels of the proinflammatory factors of the mice pretreated with the fucoidan oligosaccharide prepared in Example 3 of the application are obviously decreased; from the results of the fucoidan oligosaccharide treatment groups of Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 4, the decrease in the transcription levels of the proinflammatory factors in the fucoidan oligosaccharide treatment groups of Comparative Examples 1, 2, or 4 is not as obvious as that in the fucoidan oligosaccharide treatment group of Example 3, indicating that both the molecular weight distribution coefficient and the average number average molecular weight can affect the anti-inflammatory activity of the fucoidan oligosaccharide.

[0083] Finally, it should be noted that the above examples are used to illustrate the technical solutions of the application and not to limit the protection scope of the application. Although the application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the application can be modified or replaced equivalently without departing from the essence and scope of the technical solutions of the application.

Claims

1. A method for producing fucoid oligosaccharides, characterized by, The preparation method comprises the following steps: (1) fucoidan is prepared into a fucoidan aqueous solution, then hydrogen peroxide solution and nano-mesoporous material are added into the fucoidan aqueous solution, and after uniform stirring, a degradation reaction is carried out under microwave assistance; (2) after the degradation reaction is completed, catalase is added, then filtration is carried out, and a filter cake is collected; (3) sodium chloride solution is added to the filter cake for elution replacement, and the eluate is collected, concentrated by nanofiltration, washed, and freeze-dried to obtain fucoidan oligosaccharide; The mass ratio of the fucoidan, the hydrogen peroxide solution and the nano-mesoporous material is fucoidan: hydrogen peroxide solution: nano-mesoporous material = 100: (0.1-2): (0.1-0.5); The nano-mesoporous material is monodisperse nano-mesoporous carbon microspheres, and the pore size of the monodisperse nano-mesoporous carbon microspheres is 3.5-5 nm; The temperature of the elution replacement is 50-70 DEG C.

2. The production method according to claim 1, characterized by, In the microwave-assisted degradation reaction, the output power of the microwave is 10-2500 W, the working frequency of the microwave is 2400-2500 MHz, and the time of the degradation reaction is 0.5-12 h.

3. The preparation method according to claim 1, characterized in that, The mass concentration of the fucoidan aqueous solution is 0.1-10%.

4. The production method according to claim 1, characterized by, The mass concentration of the sodium chloride solution is 10-30%.

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