Method for preparing alginate oligosaccharide through enzymolysis

Abstract

The invention relates to a method for preparing alginate oligosaccharide through enzymolysis, and belongs to the field of environmental protection. The method comprises the following steps: inoculating flavobacterium B2 into a culture medium, and carrying out shake culture; transferring into a fermentation culture medium according to the inoculum size of 1-3% of the volume of the transferred culture medium, carrying out shake culture and centrifugation, and collecting supernate; adding ammonium sulfate into the supernate, standing, centrifuging and collecting precipitate; dissolving with a phosphate buffer solution and dialyzing to obtain a crude enzyme solution; adding the crude enzyme liquid into purified water, redissolving, heating, carrying out membrane filtration, carrying out vacuum concentration, and freeze-drying to obtain alginate oligosaccharide; according to the alginate oligosaccharide prepared by the method, the reducing sugar generation amount is relatively high, and the extraction efficiency is relatively high; an antibacterial modified ultrafiltration membrane is selected for membrane filtration, the PMP ultrafiltration membrane is placed in a plasma reaction device, mixed gas of nitrogen, hydrogen and cysteine steam is introduced, and plasma treatment is carried out; introducing mixed gas of anacardol distillation gas and piceatannol distillation gas, and continuing plasma treatment; and washing with absolute ethyl alcohol for three times, and drying.

Description

Technical Field

[0001] The invention relates to the field of environmental protection, and in particular to a method for preparing brown algae oligosaccharides by enzymolysis. Background Art

[0002] Brown algae oligosaccharides are oligomers of alginate. They are highly soluble, easily absorbed, safe and non-toxic compounds. Studies have shown that they have antioxidant and antibacterial activities, can promote plant growth, and can treat human diseases.

[0003] Chinese patent CN117987490A: It relates to the field of oligosaccharide preparation technology, and specifically discloses a method for preparing brown algae oligosaccharides. A method for preparing brown algae oligosaccharides comprises the following steps: mixing: mixing sodium alginate with water, heating to 42-45°C and stirring for 1-1.5h to form a colorless colloid, wherein the mass ratio of sodium alginate to water is 5:90-100; enzymolysis: adding alginate lyase to the colorless colloid, and obtaining an enzymolysis solution after enzymolysis; filtering: centrifuging the enzymolysis solution, taking the centrifuge, filtering the centrifuge through a filter membrane, and freeze-drying the filtrate to obtain brown algae oligosaccharides.

[0004] Chinese patent CN118290605A: provides a brown algae oligosaccharide and a preparation method thereof and a water-soluble fertilizer, belonging to the technical field of fertilizer manufacturing. The preparation method of the brown algae oligosaccharide of the present invention comprises: crushing the brown algae, adding a composite enzymatic hydrolysis solution for enzymolysis, filtering, mixing the filtrate with an ethanol solution for alcohol precipitation, centrifuging, and precipitating crude brown algae polysaccharide; adding an acid solution to the crude brown algae polysaccharide for acid hydrolysis, adjusting the pH value to neutral, centrifuging, and decompressing and concentrating the supernatant to obtain the brown algae oligosaccharide.

[0005] Chinese patent CN118028402A: discloses a G-type brown algae oligosaccharide and its preparation method and application, which relates to the technical field of G-type brown algae oligosaccharide preparation. The present invention uses kelp as raw material when preparing G-type brown algae oligosaccharide, organically combines chemical, physical and biological enzymatic hydrolysis methods, and obtains a G-type brown algae oligosaccharide with a content of more than 90.12%.

[0006] The conversion rate of brown algae oligosaccharides prepared by the above patents and prior art is low, and the free radical scavenging rate needs to be further improved. Macromolecular impurities, such as incompletely degraded polysaccharides and proteins, are not easy to separate from the target product brown algae oligosaccharides, thereby improving the purity of the product. If the crude enzyme solution contains a high salt content (such as ammonium sulfate), the high salt concentration will have an adverse effect on the subsequent process steps. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a method for preparing brown algae oligosaccharides by enzymatic hydrolysis, and the operating steps are as follows:

[0008] S1: Inoculate Flavobacterium B2 into the culture medium and culture with shaking for 10-16h;

[0009] S2: transfer the culture medium to the fermentation medium at an inoculum volume of 1-3% of the transferred culture medium volume, culture with shaking for 30-36 hours, centrifuge for 5-10 minutes, and collect the supernatant;

[0010] S3: Add ammonium sulfate to the supernatant at 4°C until the saturation is 50-60%, let stand for 18-24 hours, centrifuge for 5-10 minutes, collect the precipitate, dissolve it with 15-20 mmol / L phosphate buffer and dialyze to obtain a crude enzyme solution;

[0011] S4: 10-20 parts of the crude enzyme solution are added to 60-100 parts of purified water for redissolution, and after heating treatment under ventilation conditions, membrane filtration, reduced pressure concentration, and freeze-drying are performed to obtain brown algae oligosaccharides.

[0012] Furthermore, the shaking culture temperature is 25-30°C and the rotation speed is 150-180r / min.

[0013] Furthermore, the centrifugal speed is 8000-10000r / min.

[0014] Furthermore, the heating treatment temperature is 50-60°C and the time is 30min-60min.

[0015] Furthermore, the preparation method of the culture medium is:

[0016] Add 1-5 parts of peptone, 0.1-1 parts of yeast powder and 0.001-0.01 parts of ferric chloride to 800-1000 parts of artificial seawater to dissolve, and sterilize at 100-120°C for 10-20 minutes.

[0017] Furthermore, the preparation method of the fermentation medium is:

[0018] Add 1-5 parts of sodium alginate, 0.2-2 parts of peptone and 0.1-1 parts of potassium dihydrogen phosphate into 800-1000 parts of artificial seawater to dissolve, and sterilize at 100-120°C for 10-20 minutes.

[0019] Furthermore, the membrane filtration of S4 uses an antibacterial modified ultrafiltration membrane: the effective area of ​​a single PMP ultrafiltration membrane is 50-200 cm 2 , using poly 4-methylpentene-1 polymer material; its preparation method is:

[0020] A1: Accurately weigh 4-6 portions of cleaned PMP ultrafiltration membranes and place them in a plasma reaction device. Select a carrier gas medium of nitrogen, hydrogen and cysteine ​​vapor mixed gas, with a gas flow of 25-60 ml / min, a discharge voltage of 28-72 V, and a power of 75-160 W for plasma treatment for 6-15 minutes;

[0021] A2: A mixed gas of cardanol distillation gas and piceatannol distillation gas is introduced again, with the gas flow rate of 10-20 ml / min, and the plasma treatment is continued for 20-40 minutes;

[0022] A3: Post-treatment method: wash three times with anhydrous ethanol and dry in vacuum at 60-80℃.

[0023] Furthermore, the volume ratio of nitrogen:hydrogen:cysteine ​​vapor in the plasma treatment gas A1 is 1:0.5-1:0.05-0.1.

[0024] Furthermore, in A2, the volume ratio of the gas distilled from cardanol in the plasma treatment gas: the gas distilled from piceatannol is 1:0.5-1.

[0025] Reaction mechanism

[0026] 1. Chemical grafting reaction:

[0027] During the plasma treatment process, the surface of the PMP ultrafiltration membrane is activated to generate active groups containing amino and thiol groups; cardanol and piceatannol are converted into a gaseous state through distillation and come into contact with these active groups; the hydroxyl groups in cardanol and the carbon-carbon double bonds in piceatannol can chemically react with the thiol groups on the membrane surface to form stable thioether bonds; the formation of this chemical bond ensures that cardanol and piceatannol are firmly grafted to the surface of the PMP ultrafiltration membrane.

[0028] 2. Michael addition reaction:

[0029] The amino groups on the membrane surface act as a catalyst, promoting the Michael addition reaction; the thiol group reacts with the carbon-carbon double bonds in cardanol and piceatannol to form a stable CS bond; this reaction enhances the functionality of the membrane surface and creates favorable conditions for subsequent sterilization operations.

[0030] Technical Effects

[0031] The method for preparing brown algae oligosaccharides by enzymatic hydrolysis of the present invention has the following significant effects compared with the prior art:

[0032] 1. The brown algae oligosaccharide prepared by the present invention has a high reducing sugar production amount and a high extraction efficiency;

[0033] 2. The brown algae oligosaccharide prepared by the present invention has a high free radical scavenging rate and strong antioxidant capacity;

[0034] 3. Improve sterilization efficiency: The surface of the grafted PMP ultrafiltration membrane has enhanced antibacterial properties; cardanol and piceatannol themselves have antibacterial properties, and the phenolic hydroxyl and olefin groups in their structures have inhibitory effects on microorganisms. The efficient sterilization performance reduces the risk of cross-contamination.

[0035] 4. Reduce operational difficulty: The modified PMP ultrafiltration membrane has better mechanical strength and chemical stability; the service life of the membrane is improved, thereby reducing long-term operating costs; due to the improved antibacterial properties of the membrane, the use of additional chemical disinfectants can be reduced or avoided, simplifying the operating process. DETAILED DESCRIPTION

[0036] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following is a detailed description in combination with embodiments and comparative examples:

[0037] 1. Determination of reducing sugar production: 3,5-dinitrosalicylic acid (DNS) method was used to determine the reducing sugar production;

[0038] 2. Determination of free radical scavenging rate: Prepare 0.04g / LDPPH solution with anhydrous ethanol; take 2mL brown algae oligosaccharide and polysaccharide solution, add an equal volume of DPPH solution, mix well and place at room temperature for 30min, centrifuge at 5000r / min for 10min, take the supernatant and measure the absorbance at 517nm; use vitamin C (VC) as a positive control; calculate the free radical scavenging rate using the following formula:

[0039]

[0040] A0—absorbance value of the mixture of anhydrous ethanol and DPPH solution;

[0041] A1—absorbance value of the mixture of sample solution and DPPH solution;

[0042] A2—Absorbance value of the mixture of sample solution and anhydrous ethanol.

[0043] 3. Colony count: The brown algae oligosaccharides prepared in the examples and comparative examples were diluted to a concentration of about 106 CFU / mL, and the diluted samples were spread on LB plates to count the colonies.

[0044] Example 1

[0045] A method for preparing brown algae oligosaccharides by enzymatic hydrolysis, the operating steps of which are:

[0046] S1: Inoculate Flavobacterium B2 into the culture medium and culture with shaking for 10 h;

[0047] S2: transfer the culture medium to the fermentation medium at an inoculum volume of 1% of the transferred medium volume, culture with shaking for 30 h, centrifuge for 5 min, and collect the supernatant;

[0048] S3: Add ammonium sulfate to the supernatant at 4°C to 50% saturation, let stand for 18 hours, centrifuge for 5 minutes, collect the precipitate, dissolve it in 15mmol / L phosphate buffer and dialyze to obtain a crude enzyme solution;

[0049] S4: 10 g of the crude enzyme solution was added to 60 g of purified water for re-dissolution, and the mixture was heated under ventilation conditions, membrane filtered, concentrated under reduced pressure, and freeze-dried to obtain brown algae oligosaccharides.

[0050] The shaking culture temperature is 25° C. and the rotation speed is 150 r / min.

[0051] The centrifugal speed is 8000r / min.

[0052] The heating treatment temperature is 50° C. and the time is 30 minutes.

[0053] The preparation method of the culture medium is:

[0054] Add 1 g of peptone, 0.1 g of yeast powder and 0.001 g of ferric chloride into 800 g of artificial seawater, and sterilize at 100°C for 10 min.

[0055] The preparation method of the fermentation medium is as follows:

[0056] 1 g of sodium alginate, 0.2 g of peptone, and 0.1 g of potassium dihydrogen phosphate were added to 800 g of artificial seawater to dissolve, and sterilized at 100°C for 10 min.

[0057] The membrane filtration of S4 uses an antibacterial modified ultrafiltration membrane: the effective area of ​​a single PMP ultrafiltration membrane is 50 cm 2 , using poly 4-methylpentene-1 polymer material; its preparation method is:

[0058] A1: Accurately weigh 4g of cleaned PMP ultrafiltration membrane and place it in a plasma reaction device. Select the carrier gas medium as a mixed gas of nitrogen, hydrogen and cysteine ​​vapor, the gas flow rate is 25ml / min, the discharge voltage is 28V, and the power is 75W for plasma treatment for 6 minutes;

[0059] A2: A mixed gas of cardanol distillation gas and piceatannol distillation gas was introduced again, with the gas flow rate of 10 ml / min, and the plasma treatment was continued for 20 minutes;

[0060] A3: Post-treatment method: wash three times with anhydrous ethanol and dry in vacuum at 60℃.

[0061] The volume ratio of nitrogen: hydrogen: cysteine ​​vapor in the plasma treatment gas A1 is 1:0.5:0.05.

[0062] The volume ratio of the plasma-treated gas used for the distillation of cardanol to the gas used for the distillation of piceatannol in A2 is 1:0.5.

[0063] Example 2

[0064] A method for preparing brown algae oligosaccharides by enzymatic hydrolysis, the operating steps of which are:

[0065] S1: Inoculate Flavobacterium B2 into the culture medium and culture with shaking for 12 h;

[0066] S2: transfer the culture medium to the fermentation medium at an inoculum volume of 2% of the transferred medium volume, culture with shaking for 32 h, centrifuge for 6 min, and collect the supernatant;

[0067] S3: Add ammonium sulfate to the supernatant at 4°C to 55% saturation, let stand for 20 hours, centrifuge for 6 minutes, collect the precipitate, dissolve it in 16mmol / L phosphate buffer and dialyze it to obtain a crude enzyme solution;

[0068] S4: 13 g of the crude enzyme solution was added to 70 g of purified water for re-dissolution, and the mixture was heated under ventilation conditions, membrane filtered, concentrated under reduced pressure, and freeze-dried to obtain brown algae oligosaccharides.

[0069] The shaking culture temperature is 26° C. and the rotation speed is 160 r / min.

[0070] The centrifugal speed is 8500r / min.

[0071] The heating treatment temperature is 55° C. and the time is 40 minutes.

[0072] The preparation method of the culture medium is:

[0073] 2 g of peptone, 0.5 g of yeast powder and 0.005 g of ferric chloride were added to 850 g of artificial seawater and dissolved, and sterilized at 105°C for 15 min.

[0074] The preparation method of the fermentation medium is as follows:

[0075] 2 g of sodium alginate, 1 g of peptone, and 0.5 g of potassium dihydrogen phosphate were added to 850 g of artificial seawater to dissolve, and sterilized at 105° C. for 15 min.

[0076] The membrane filtration of S4 uses an antibacterial modified ultrafiltration membrane: the effective area of ​​a single PMP ultrafiltration membrane is 100 cm 2 , using poly 4-methylpentene-1 polymer material; its preparation method is:

[0077] A1: Accurately weigh 5g of the cleaned PMP ultrafiltration membrane and place it in a plasma reaction device. Select the carrier gas medium as a mixed gas of nitrogen, hydrogen and cysteine ​​vapor, the gas flow rate is 30ml / min, the discharge voltage is 36V, and the power is 100W for plasma treatment for 9 minutes;

[0078] A2: A mixed gas of cardanol distillation gas and piceatannol distillation gas was introduced again, with the gas flow rate of 13 ml / min, and the plasma treatment was continued for 25 minutes;

[0079] A3: Post-treatment method: wash with anhydrous ethanol three times and dry in vacuum at 65℃.

[0080] The volume ratio of nitrogen: hydrogen: cysteine ​​vapor in the plasma treatment gas A1 is 1:0.6:0.06.

[0081] The volume ratio of the plasma-treated gas used for the distillation of cardanol to the gas used for the distillation of piceatannol in A2 is 1:0.6.

[0082] Example 3

[0083] A method for preparing brown algae oligosaccharides by enzymatic hydrolysis, the operating steps of which are:

[0084] S1: Inoculate Flavobacterium B2 into the culture medium and culture with shaking for 14 h;

[0085] S2: transfer the culture medium to the fermentation medium at an inoculum volume of 2% of the transferred medium volume, culture with shaking for 34 h, centrifuge for 8 min, and collect the supernatant;

[0086] S3: Add ammonium sulfate to the supernatant at 4°C to 55% saturation, let stand for 22 hours, centrifuge for 8 minutes, collect the precipitate, dissolve it in 18 mmol / L phosphate buffer and dialyze to obtain a crude enzyme solution;

[0087] S4: 18 g of the crude enzyme solution was added to 90 g of purified water for re-dissolution, and the mixture was heated under ventilation conditions, membrane filtered, concentrated under reduced pressure, and freeze-dried to obtain brown algae oligosaccharides.

[0088] The shaking culture temperature is 28° C. and the rotation speed is 170 r / min.

[0089] The centrifugal speed is 9500r / min.

[0090] The heating treatment temperature is 55° C. and the time is 50 minutes.

[0091] The preparation method of the culture medium is:

[0092] 4 g of peptone, 0.8 g of yeast powder and 0.008 g of ferric chloride were added to 950 g of artificial seawater and dissolved, and sterilized at 115°C for 15 min.

[0093] The preparation method of the fermentation medium is as follows:

[0094] 4 g of sodium alginate, 1.5 g of peptone, and 0.8 g of potassium dihydrogen phosphate were added to 950 g of artificial seawater to dissolve, and sterilized at 115° C. for 15 min.

[0095] The membrane filtration of S4 uses an antibacterial modified ultrafiltration membrane: the effective area of ​​a single PMP ultrafiltration membrane is 150cm 2 , using poly 4-methylpentene-1 polymer material; its preparation method is:

[0096] A1: Accurately weigh 5g of cleaned PMP ultrafiltration membrane and place it in a plasma reaction device. Select the carrier gas medium as a mixed gas of nitrogen, hydrogen and cysteine ​​vapor, the gas flow rate is 50ml / min, the discharge voltage is 56V, and the power is 130W. Perform plasma treatment for 13 minutes;

[0097] A2: A mixed gas of cardanol distillation gas and piceatannol distillation gas was introduced again, with the gas flow rate of 18 ml / min, and the plasma treatment was continued for 35 minutes;

[0098] A3: Post-treatment method: wash three times with anhydrous ethanol and dry in vacuum at 75℃.

[0099] The volume ratio of nitrogen: hydrogen: cysteine ​​vapor in the plasma treatment gas A1 is 1:0.8:0.08.

[0100] The volume ratio of the plasma-treated gas used for the distillation of cardanol to the gas used for the distillation of piceatannol in A2 is 1:0.8.

[0101] Example 4

[0102] A method for preparing brown algae oligosaccharides by enzymatic hydrolysis, the operating steps of which are:

[0103] S1: Inoculate Flavobacterium B2 into the culture medium and culture with shaking for 16 h;

[0104] S2: transfer the culture medium to the fermentation medium at an inoculum volume of 3% of the transferred medium volume, culture with shaking for 36 h, centrifuge for 10 min, and collect the supernatant;

[0105] S3: Add ammonium sulfate to the supernatant at 4°C to 60% saturation, let stand for 24 hours, centrifuge for 10 minutes, collect the precipitate, dissolve it in 20mmol / L phosphate buffer and dialyze to obtain a crude enzyme solution;

[0106] S4: 20 g of the crude enzyme solution was added to 100 g of purified water for re-dissolution, and the mixture was heated under ventilation conditions, membrane filtered, concentrated under reduced pressure, and freeze-dried to obtain brown algae oligosaccharides.

[0107] The shaking culture temperature is 30°C and the rotation speed is 180r / min.

[0108] The centrifugal speed is 10000r / min.

[0109] The heating treatment temperature is 60° C. and the time is 60 minutes.

[0110] The preparation method of the culture medium is:

[0111] Add 5 g of peptone, 1 g of yeast powder and 0.01 g of ferric chloride into 1000 g of artificial seawater and sterilize at 120°C for 20 min.

[0112] The preparation method of the fermentation medium is as follows:

[0113] Add 5 g of sodium alginate, 2 g of peptone and 1 g of potassium dihydrogen phosphate into 1000 g of artificial seawater to dissolve, and sterilize at 120°C for 20 min.

[0114] The membrane filtration of S4 uses an antibacterial modified ultrafiltration membrane: the effective area of ​​a single PMP ultrafiltration membrane is 200 cm 2 , using poly 4-methylpentene-1 polymer material; its preparation method is:

[0115] A1: Accurately weigh 6g of cleaned PMP ultrafiltration membrane and place it in a plasma reaction device. Select a carrier gas medium of nitrogen, hydrogen and cysteine ​​vapor mixed gas, with a gas flow of 60ml / min, a discharge voltage of 72V, and a power of 160W for plasma treatment for 15 minutes;

[0116] A2: A mixed gas of cardanol distillation gas and piceatannol distillation gas was introduced again, with the gas flow rate of 20 ml / min, and the plasma treatment was continued for 40 minutes;

[0117] A3: Post-treatment method: wash three times with anhydrous ethanol and dry in vacuum at 80℃.

[0118] The volume ratio of nitrogen: hydrogen: cysteine ​​vapor in the plasma treatment gas A1 is 1:1:0.1.

[0119] The volume ratio of the plasma-treated gas used for the distillation of cardanol to the gas used for the distillation of piceatannol in A2 is 1:1.

[0120] Comparative Example 1

[0121] The PMP ultrafiltration membrane was not modified, and the other properties were the same as in Example 1.

[0122] Comparative Example 2

[0123] No cardanol distillation gas was added, and the rest was the same as in Example 1.

[0124] Comparative Example 3

[0125] The other steps were the same as in Example 1 except that the piceatannol distillation gas was not added.

[0126]

[0127] Through the data analysis of the above examples and comparative examples, the brown algae oligosaccharide prepared by the present invention has high reducing sugar production and free radical scavenging rate and has strong antioxidant capacity; the antibacterial modified ultrafiltration membrane prepared by the present invention has enhanced antibacterial properties.

[0128] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing brown algae oligosaccharides by enzymatic hydrolysis, characterized in that: The steps are: S1: Inoculate Flavobacterium B2 into the culture medium and culture with shaking for 10-16h; S2: transfer the culture medium to the fermentation medium at an inoculum volume of 1-3% of the transferred culture medium volume, culture with shaking for 30-36 hours, centrifuge for 5-10 minutes, and collect the supernatant; S3: Add ammonium sulfate to the supernatant at 4°C until the saturation is 50-60%, let stand for 18-24 hours, centrifuge for 5-10 minutes, collect the precipitate, dissolve it with 15-20 mmol / L phosphate buffer and dialyze to obtain a crude enzyme solution; S4: 10-20 parts of the crude enzyme solution are added to 60-100 parts of purified water for redissolution, and after heating treatment under ventilation conditions, membrane filtration, reduced pressure concentration, and freeze-drying are performed to obtain brown algae oligosaccharides.

2. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis according to claim 1, characterized in that: The shaking culture temperature is 25-30°C and the rotation speed is 150-180r / min.

3. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis according to claim 1, characterized in that: The centrifugal speed is 8000-10000r / min.

4. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis according to claim 1, characterized in that: The heating treatment temperature is 50-60°C and the time is 30min-60min.

5. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis according to claim 1, characterized in that: The preparation method of the culture medium is: Add 1-5 parts of peptone, 0.1-1 parts of yeast powder and 0.001-0.01 parts of ferric chloride to 800-1000 parts of artificial seawater to dissolve, and sterilize at 100-120°C for 10-20 minutes.

6. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis according to claim 1, characterized in that: The preparation method of the fermentation medium is as follows: Add 1-5 parts of sodium alginate, 0.2-2 parts of peptone and 0.1-1 parts of potassium dihydrogen phosphate into 800-1000 parts of artificial seawater to dissolve, and sterilize at 100-120°C for 10-20 minutes.

7. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis according to claim 1, characterized in that: The membrane filtration of S4 uses an antibacterial modified ultrafiltration membrane: the effective area of ​​a single PMP ultrafiltration membrane is 50-200cm 2 , using poly 4-methylpentene-1 polymer material; its preparation method is: A1: Accurately weigh 4-6 portions of cleaned PMP ultrafiltration membranes and place them in a plasma reaction device. Select a carrier gas medium of nitrogen, hydrogen and cysteine ​​vapor mixed gas, with a gas flow of 25-60 ml / min, a discharge voltage of 28-72 V, and a power of 75-160 W for plasma treatment for 6-15 minutes; A2: A mixed gas of cardanol distillation gas and piceatannol distillation gas is introduced again, with the gas flow rate of 10-20 ml / min, and the plasma treatment is continued for 20-40 minutes; A3: Post-treatment method: wash three times with anhydrous ethanol and dry in vacuum at 60-80℃.

8. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis according to claim 7, characterized in that: The volume ratio of nitrogen: hydrogen: cysteine ​​vapor in the plasma treatment gas A1 is 1:0.5-1:0.05-0.

1.

9. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis according to claim 7, characterized in that: The volume ratio of the plasma-treated gas used for the distillation of cardanol to the gas used for the distillation of piceatannol in A2 is 1:0.5-1.

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