Refining and purifying technology of high-purity alginate oligosaccharide

Abstract

The invention discloses a refining and purifying technology of high-purity alginate oligosaccharide. The average polymerization degree of the prepared alginate oligosaccharide is 2-6. The refining and purifying technology of the high-purity alginate oligosaccharide comprises the steps of enzymolysis, ceramic membrane microfiltration, ceramic membrane ultrafiltration, concentration and crystallization, step-by-step alcohol precipitation, gradient cooling and vacuum drying. According to the method disclosed by the invention, the alginate oligosaccharide after enzymolysis is subjected to fractional separation by adopting ultrafiltration and ethanol precipitation methods, so that the alginate oligosaccharide with low molecular weight can be obtained; the technology combination of membrane filtration, graded alcohol precipitation, gradient cooling and the like is adopted, industrial production of alginate oligosaccharides of different specifications is achieved, the product purity is larger than 99%, the production cost is low, and certain economic and social benefits are achieved.

Description

Technical Field

[0001] The present invention relates to the field of bioseparation engineering, and particularly to a technology for refining and purifying high-purity fucoidan oligosaccharides from enzymatic hydrolysates. Background Art

[0002] Fucoidan oligosaccharides are mainly obtained by degrading alginate in the cell walls of brown algae plants. Compared with the polysaccharide form, fucoidan oligosaccharides have higher solubility and bioavailability, and have various biological activities such as antioxidant, immunomodulatory, and anti-tumor effects. Among them, antioxidant activity is the basis for the generation of other biological activities and can slow down the cell and tissue damage caused by reactive oxygen free radicals in the body. Relevant research also shows that the excessive production and accumulation of free radicals are closely related to the occurrence and development of a series of diseases such as cancer and Parkinson's disease, and fucoidan oligosaccharides have a certain degree of improvement effect on the above diseases.

[0003] Research shows that the antioxidant activity of fucoidan oligosaccharides is not only related to their preparation methods, M / G ratios, etc., but also related to the molecular weight of the components; the components with a molecular weight lower than 1.0 kDal have strong hydroxyl radical scavenging ability, and the influence degree of the molecular weight on the antioxidant performance is greater than the M / G ratio. At present, the main methods for separating and purifying fucoidan oligosaccharides include sedimentation separation method, membrane separation method, and chromatographic separation method, etc. The sedimentation separation method has incomplete separation and low product purity, and is suitable for preliminary separation; the membrane separation method is easily affected by factors such as temperature, pressure, and mixed solution concentration, its separation effect is not easy to control, and the membrane cleaning cost is relatively high; the chromatographic separation method has a large number of solvents, low efficiency, and a large amount of sewage. The existing fucoidan oligosaccharide extraction technology has a complicated process, high cost, and low extraction purity, which is easy to cause waste. At present, fucoidan oligosaccharides have received more attention as functional factors, and it is necessary to establish an efficient, safe, and simple preparation process for low-molecular-weight and high-purity fucoidan oligosaccharides to promote its industrial process. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a technology for refining and purifying fucoidan oligosaccharides with a simple process, low production cost, low molecular weight and high purity of the product, and suitable for industrial production.

[0005] The technical solution of the present invention is as follows: A technology for refining and purifying high-purity fucoidan oligosaccharides, the specific steps are as follows: S1. Ceramic membrane microfiltration: The crude solution of fucoidan oligosaccharides obtained after enzymatic hydrolysis is microfiltered by a ceramic membrane with a pore size of 50 nm to remove small particle impurities such as Bacillus subtilis contained in the aqueous solution after enzymatic hydrolysis, and the particles with a diameter > 50 nm are intercepted and removed, and the material temperature is maintained at 60 - 70 °C. Under this process condition, the removal rate of bacterial proteins can reach more than 97%.

[0006] S2. Ceramic membrane ultrafiltration: Using a ceramic membrane with a pore size of 2 nm to intercept pigments and other macromolecules with a molecular weight > 3000 Da in the solution after microfiltration, a yellow clear liquid can be obtained; S3. Concentration: Vacuum concentrate the ultrafiltrate to a concentration of 30% - 35%. By controlling the temperature and concentration ratio, the appearance and purity of the final product can be ensured; S4. Stepwise alcohol precipitation and gradient cooling: Add an appropriate multiple of 90 - 99.5% ethanol to the concentrated solution. Heat and stir the mixture until it is completely dissolved. Adopt the gradient cooling crystallization technology and repeat the above steps to precipitate fucoidan oligosaccharide crystals with different molecular weights and a purity > 99%; S5. Low - temperature vacuum drying: Filter, rinse, and dry the crystal mixture to obtain a fucoidan oligosaccharide powder product.

[0007] As a preferred solution, the preparation method of the above - mentioned fucoidan oligosaccharide stock solution is as follows: S1. Slant culture: Inoculate Bacillus subtilis onto a slant medium under sterile conditions and incubate it in an inverted position. The culture temperature is 28°C and the culture time is 48 h.

[0008] S2. Seed expansion culture: Pick colonies from the slant medium in the above step, dilute with water to obtain a cell solution, inoculate the cell solution onto a seed medium for expansion culture. The inoculation amount is 10% (V / V). The expansion culture temperature is 28°C, the shaker speed is 120 r / min, and the culture time is 12 h; S3. Fermentation culture: Inoculate the expanded cell solution into a fermentation broth medium. The inoculation amount is 10% (V / V). The expansion culture temperature is 30°C, the shaker speed is 150 r / min, and the culture time is 36 h; S4. Strain amplification: Inoculate the fermentation broth after fermentation culture in the above step into the fermentation medium of a first - stage amplified fermentation tank. The inoculation amount is 10%. Control the tank pressure to 0.05 MPa and culture at 28°C for 48 hours. The stirring speed is 400 rpm and the air volume is 10 L / min; When the first - stage amplified culture tank reaches the process index, transfer it to the second - stage amplified culture tank for continuous culture. Stop the culture when the second - stage culture tank reaches the process index, sample and detect the stock solution index. The production workshop inputs raw materials, auxiliary materials, and water into the first - stage and second - stage amplified culture tanks according to the requirements of the process document, and a fucoidan lyase aqueous solution is obtained after fermentation.

[0009] S5. Enzymatic hydrolysis: Using high - shear emulsification technology, slowly add sodium alginate into the fucoidan lyase aqueous solution with an enzyme content of 500 - 1000 U / mL. The feeding amount can reach 35% to obtain a fucoidan oligosaccharide stock solution.

[0010] The present invention provides a refined purification technology for fucoidan oligosaccharides. It has the following beneficial effects: 1. Use ceramic membranes instead of organic membranes, which are simple to maintain, low cost and have a wide range of applications. The existing technology uses organic membrane filtration, which is difficult to clean, has a large amount of sewage, a short lifespan, is not resistant to high temperature, acid and alkali, and increases production costs.

[0011] 2. The enzymatically hydrolyzed brown algae oligosaccharides were fractionated by ultrafiltration and ethanol precipitation, and four components with weight-average molecular weights of 0.84, 1.40, 2.25 and 34.56 kDal were obtained, with yields of 50.82%, 5.15%, 11.48% and 12.00%, respectively. Each component has a certain reducing ability and can effectively scavenge DPPH and hydroxyl free radicals. Among them, the antioxidant activity of low molecular weight component A is the most significant, and its ability to scavenge hydroxyl free radicals is similar to that of vitamin C.

[0012] 3. Use the method of step-by-step alcohol precipitation and gradient cooling to obtain brown algae oligosaccharides with low molecular weight. By controlling the amount of ethanol added and the temperature, brown algae oligosaccharides of different specifications can be produced.

[0013] 4. The product yield and product quality are improved through the combination of new extraction processes. The extraction yield of brown algae oligosaccharides reaches 95% and the product purity reaches 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0015] Figure 1 TLC spectrum of four components of brown algae oligosaccharide Figure 2 HPGPC chromatogram of brown algae oligosaccharide (fraction A and fraction B) Figure 3 HPGPC chromatogram of brown algae oligosaccharides (fraction C and fraction D) Figure 4 Mass spectrometry scanning of brown algae oligosaccharide products Figure 1 Figure 5 Mass spectrometry scanning of brown algae oligosaccharide products Figure 2 DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0017] For the instruments, reagents, and materials involved in the following examples, unless otherwise specified, they are all conventional instruments, reagents, and materials existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, they are all conventional experimental methods and detection methods existing in the prior art.

[0018] Preparation of slant medium: Sodium alginate 10.0 - 15.0 g / L, ammonium sulfate 3.0 - 8.0 g / L, magnesium sulfate 0.5 - 3.0 g / L, dipotassium hydrogen phosphate 1.0 - 4.0 g / L, ferrous sulfate 0.01 - 0.1 g / L, and agar 15.0 - 20.0 g / L. The sterilization temperature of the slant medium is 115 °C, and the sterilization time is 15 min.

[0019] Preparation of seed medium: NaCl 20 g / L, sodium alginate 10 g / L, MgSO4 3 g / L, peptone 10 g / L, Na2HPO4 1 g / L, adjust the pH to 7.2 - 7.4 with 1 mol / L NaOH solution, and prepare with distilled water. The sterilization temperature of the seed medium is 115 °C, and the sterilization time is 15 min.

[0020] Preparation of fermentation medium: Sodium alginate 20 - 40.0 g / L, yeast extract 4 - 8.0 g / L, peptone 8 - 15.0 g / L, NaCl 2 - 6.0 g / L. The temperature for fermentation culture is 37 °C, the rotation speed is 160 r / min, and the fermentation culture time is 42 h.

[0021] Example 1: A method for preparing alginate oligosaccharide solution using Bacillus subtilis, the specific steps are as follows: A. Slant culture: Inoculate Bacillus subtilis onto the slant medium under sterile conditions and perform inverted culture. The culture temperature is 28 °C, and the culture time is 48 h; B. Seed expansion culture: Pick colonies from the slant medium in the above step, dilute with water to obtain a cell solution, inoculate the cell solution onto the seed medium for expansion culture, the inoculation amount is 10% (V / V), the expansion culture temperature is 28 °C, the shaker rotation speed is 120 r / min, and the culture time is 12 h; C. Fermentation culture: Inoculate the expanded culture broth into the fermentation broth medium, the inoculation amount is 10% (V / V), the expansion culture temperature is 30 °C, the shaker rotation speed is 150 r / min, and the culture time is 36 h; D. Strain amplification: The fermented broth after fermentation culture in the above steps is inoculated into the fermentation medium of the first-stage amplification fermenter, with an inoculation amount of 10%. The tank pressure is controlled at 0.05 MPa, and it is cultured at 28 °C for 48 hours, with a stirring speed of 400 rpm and an air volume of 10 L / min. When the first-stage amplification culture tank reaches the process index, it is transferred to the second-stage amplification culture tank for continued culture. When the second-stage culture tank reaches the process index, the culture is stopped, and the original liquid index is sampled and detected. The production workshop inputs raw and auxiliary materials and water into the first-stage and second-stage amplification culture tanks according to the requirements of the process document, and an alginate lyase aqueous solution is obtained after fermentation.

[0022] F. Enzymolysis: Using high-shear emulsification technology, sodium alginate is slowly added to the alginate lyase aqueous solution with an enzyme content of 500 - 1000 U / mL, and the feeding amount can reach 35% to obtain an alginate oligosaccharide crude liquid.

[0023] A refined purification technology for high-purity alginate oligosaccharides, the specific steps are as follows: A. Ceramic membrane microfiltration: Use a 50 nm ceramic membrane for microfiltration to remove small particle impurities such as Bacillus subtilis in the aqueous solution after enzymolysis, intercept and remove particles with a diameter greater than 50 nm, and keep the material temperature at 60 °C. Under this process condition, the removal rate of bacterial protein can reach 97%; B. Ceramic membrane ultrafiltration: Use a 2 nm ceramic membrane to remove pigments and other macromolecules with a molecular weight greater than 3000 Dal in the solution after microfiltration, and a yellow clear liquid can be obtained; C. Concentration: Vacuum concentrate the ultrafiltrate to a concentration of 30% - 35%, and control the temperature and concentration ratio to ensure the appearance and purity of the final product; D. Stepwise alcohol precipitation and gradient cooling: Add an appropriate multiple of 90 - 99.5% ethanol to the concentrated solution, heat and stir the mixture until it is completely dissolved, adopt gradient cooling crystallization technology, and repeat the above steps to precipitate alginate oligosaccharide crystals with different molecular weights and a purity greater than 99%; E. Low-temperature vacuum drying: Filter, wash, and dry the crystal mixture to obtain an alginate oligosaccharide powder product.

[0024] Example 2 The preparation method of the alginate oligosaccharide crude liquid in Example 2 is exactly the same as that in Example 1.

[0025] A. Ceramic membrane microfiltration: Use a 50 nm ceramic membrane for microfiltration to remove small particle impurities such as Bacillus subtilis in the aqueous solution after enzymolysis, intercept and remove particles with a diameter greater than 50 nm, and keep the material temperature at 65 °C. Under this process condition, the removal rate of bacterial protein can reach 97%; B. Ceramic membrane ultrafiltration: Use a 2 nm ceramic membrane to remove pigments and other macromolecules with a molecular weight greater than 3000 Dal in the solution after microfiltration, and a yellow clear liquid can be obtained; C. Concentration: Vacuum concentrate the ultrafiltrate to a concentration of 30%-35%. By controlling the temperature and concentration ratio, the appearance and purity of the final product can be ensured. D. Stepwise alcohol precipitation and gradient cooling: Add ethanol with a proper multiple of 90-99.5% to the concentrated solution, heat and stir the mixture until it is completely dissolved, adopt the gradient cooling crystallization technology, and repeat the above steps to precipitate fucoidan oligosaccharide crystals with different molecular weights and a purity > 99%. E. Low-temperature vacuum drying: Filter, wash, and dry the crystal mixture to obtain the fucoidan oligosaccharide powder product. Example 3

[0026] This experimental case is used to illustrate the effect of fractionation of enzymatically hydrolyzed fucoidan oligosaccharides by ultrafiltration and ethanol precipitation methods.

[0027] As Figure 1 , component A is mainly composed of fucoidan oligosaccharides with a polymerization degree of 2-7, and its average molecular mass is speculated to be less than 1.50 kDal; components B, C, and D have larger molecular masses, and only a small amount of fucoidan oligosaccharides with a polymerization degree of 2-7. Further, the HPGPC method is used to detect the molecular mass HPGPC spectra of each component. Multiple oligomeric sugar fragments with different molecular mass distributions are contained in all 4 samples ( Figure 2 ). Component A mainly contains a component with a weight-average molecular mass of 0.48 kDal; component B mainly contains a component with a weight-average molecular mass of 5.03 kDal; component C mainly contains a component with a weight-average molecular mass of 3.68 kDal; component D mainly contains two components with weight-average molecular masses of 51.15 kDal and 68.23 kDal.

[0028]

[0029] Example 4 This experimental case is used to illustrate the in vitro antioxidant ability of fucoidan oligosaccharides (1) Scavenging ability of fucoidan oligosaccharides on DPPH DPPH is a stable free radical and can be used to measure the antioxidant ability of substances. The results show that all 4 components obtained by enzymatic hydrolysis have certain DPPH scavenging activities, and the scavenging effects increase with the increase of concentration. Among them, component A has a strong DPPH scavenging ability.

[0030]

[0031] Scavenging ability of fucoidan oligosaccharides on hydroxyl radicals Sodium alginate and all 4 components have good scavenging abilities on hydroxyl radicals. Research shows that oligosaccharides may play a hydroxyl radical scavenging activity by causing the movement of anomeric hydrogen in the internal monosaccharide groups through the hydrogen electron transfer mechanism.

[0032]

[0033] (3) Reducing ability of algal oligosaccharides The four fucoidan oligosaccharide components obtained by enzymatic hydrolysis have good scavenging effects on DPPH and hydroxyl radicals, and they have certain reducing power. Among them, component A with a relatively small average molecular weight has the highest antioxidant activity.

[0034]

[0035] Example 5 Mass spectrometry scanning chart of the molecular weight of fucoidan oligosaccharides The fucoidan oligosaccharides were subjected to Q1 scanning using an Osight 210 tandem mass spectrometer + LX50 liquid chromatograph for molecular weight identification. The scanning parameters and analysis results are as Figure 4 shown. It can be seen that the main products are fucoidan oligosaccharides with a molecular weight of 300 - 500, among which the fucoidan oligosaccharide with a molecular weight of 419 has the highest proportion. ( Figure 4 、 Figure 5 ) The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Refining and purification technology of high-purity fucoidan oligosaccharides, the specific steps are as follows: S1. Ceramic membrane microfiltration: The crude fucoidan oligosaccharide solution obtained after enzymatic hydrolysis is microfiltered using a ceramic membrane with a pore size of 50 nm to remove small particle impurities such as Bacillus subtilis in the aqueous solution after enzymatic hydrolysis, and particles with a diameter > 50 nm are intercepted and removed. The material temperature is maintained at 60 - 70 °C. Under these process conditions, the removal rate of bacterial protein can reach over 97%.

2. S2. Ceramic membrane ultrafiltration: Using a ceramic membrane with a pore size of 2 nm to intercept pigments and other macromolecules with a molecular weight > 3000 Da in the solution after microfiltration, a yellow clear liquid can be obtained; S3. Concentration: The ultrafiltrate is concentrated in vacuo to a concentration of 30% - 35%. By controlling the temperature and concentration ratio, the appearance and purity of the final product are ensured; S4. Stepwise alcohol precipitation and gradient cooling: An appropriate multiple of 90 - 99.5% ethanol is added to the concentrated solution, and the mixture is heated and stirred until completely dissolved. The gradient cooling crystallization technology is adopted, and the above steps are repeated to precipitate fucoidan oligosaccharide crystals with different molecular weights and a purity > 99%; S5. Low-temperature vacuum drying: The crystal mixture is filtered, rinsed, and dried to obtain a fucoidan oligosaccharide powder product.

3. The refined purification technology according to claim 1, characterized in that, The preparation method of the above-mentioned crude fucoidan oligosaccharide solution, the specific steps are as follows: S1. Slant culture: Bacillus subtilis is inoculated onto a slant medium under sterile conditions for inverted culture. The culture temperature is 28 °C, and the culture time is 48 h.

4. S2. Seed expansion culture: Colonies are picked from the slant medium in the above step, diluted with water to obtain a bacterial solution, and the bacterial solution is inoculated onto a seed medium for expansion culture. The inoculation amount is 10% (V / V). The expansion culture temperature is 28 °C, the shaker speed is 120 r / min, and the culture time is 12 h.

5. S3. Fermentation culture: The bacterial solution after expansion culture is inoculated into a fermentation broth medium. The inoculation amount is 10% (V / V). The expansion culture temperature is 30 °C, the shaker speed is 150 r / min, and the culture time is 36 h; S4. Strain amplification: The fermentation broth after fermentation culture in the above step is inoculated into the fermentation medium of a first-stage amplification fermentation tank. The inoculation amount is 10%. The tank pressure is controlled at 0.05 MPa, and it is cultured at 28 °C for 48 hours. The stirring speed is 400 rpm, and the air volume is 10 L / min; When the first-stage amplification culture tank reaches the process index, it is transferred to the second-stage amplification culture tank for continuous culture. When the second-stage culture tank reaches the process index, the culture is stopped, and the original solution index is sampled and detected. The production workshop inputs raw materials, auxiliary materials, and water into the first-stage and second-stage amplification culture tanks according to the requirements of the process document, and a fucoidan lyase aqueous solution is obtained after fermentation.

6. S5. Enzymatic hydrolysis: Using high-shear emulsification technology, sodium alginate is slowly added to the fucoidan lyase aqueous solution with an enzyme content of 500 - 1000 U / mL, and the feeding amount can reach 35% to obtain a crude fucoidan oligosaccharide solution.

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