A method for preparing a seaweed fertilizer rich in fucoidan

Abstract

The application relates to the field of seaweed processing, and particularly discloses a preparation method of seaweed fertilizer rich in fucoidan, which comprises the following steps: S1, washing raw materials of Ascophyllum nodosum, then crushing to obtain algae bodies; S2, performing hot water steaming on the algae bodies, and performing filtration separation to obtain supernatant and algae body precipitate; S3, concentrating the supernatant to obtain a fucoidan solution; S4, adding water and sodium carbonate to the algae body precipitate, adjusting pH, then adding an enzyme to perform enzymolysis, and finally concentrating to obtain an enzymolysis solution; and S5, uniformly mixing and stirring the fucoidan solution and the enzymolysis solution, and performing spray drying to obtain finished seaweed fertilizer; the seaweed fertilizer is rich in fucoidan.

Description

Technical Field

[0001] This application relates to the field of seaweed processing, and more specifically, to a method for preparing seaweed fertilizer rich in fucoidan. Background Technology

[0002] Fucoidan, also known as fucoidan, is a natural active polysaccharide containing sulfate groups, unique to brown algae. It is mainly found in the cell wall matrix, intercellular spaces, and secreted mucus of brown algae such as kelp, wakame, giant kelp, and fucoidan. It is widely distributed on the surface of brown algae such as kelp, wakame, bubbly seaweed, and fucoidan, and plays important roles in antibacterial, moisturizing, and radiation protection. In agricultural production, fucoidan can induce plant stress resistance, effectively alleviate abiotic stress, and inhibit the development of plant rhizobium, tomato gray mold, and tobacco mosaic virus infection, showing broad application prospects in agricultural production.

[0003] The content of fucoidan in *Leptochloa crus-galli* is relatively high. *Leptochloa crus-galli* mainly lives along the North Atlantic coast. Due to the low temperature and strong salinity, the growth environment is quite harsh. The temperature in the North Atlantic does not exceed 27°C all year round. Therefore, under the harsh growth conditions of low temperature and other harsh environments, *Leptochloa crus-galli* has a strong ability to enrich and absorb nutrients, making it rich in active ingredients such as fucoidan, alginic acid, brown algal starch, protein, fat, and cellulose.

[0004] Therefore, extracting fucoidan from *Tetracentron sinense* is an unsolved problem. Current methods for producing seaweed fertilizer from *Tetracentron sinense* generally involve washing, crushing, enzymatic hydrolysis, hot water extraction, concentration, and spray drying, without extracting fucoidan. In fact, fucoidan is often washed away during the washing process, resulting in a low fucoidan content in current seaweed fertilizers. Summary of the Invention

[0005] In order to prepare a seaweed fertilizer rich in fucoidan, this application provides a method for preparing a seaweed fertilizer rich in fucoidan.

[0006] This application provides a method for preparing seaweed fertilizer rich in fucoidan, which adopts the following technical solution:

[0007] A method for preparing seaweed fertilizer rich in fucoidan includes the following steps:

[0008] S1. Take the raw material of *Alternaria bubblyleaf*, wash it with water, and then crush it to obtain the algae;

[0009] S2. The algae are boiled in hot water and filtered to separate them, resulting in a supernatant and algae precipitate.

[0010] S3. The supernatant was concentrated to obtain fucoidan solution;

[0011] S4. After the algal precipitate is treated with water and sodium carbonate, the pH is adjusted, then enzymes are added for enzymatic hydrolysis, and finally the solution is concentrated to obtain the enzymatic hydrolysate.

[0012] S5, fucoidan solution and enzymatic hydrolysate are mixed and stirred evenly, and then spray-dried to obtain the finished seaweed fertilizer.

[0013] By adopting the above technical solution, after washing with water to remove surface impurities, mud, and other particles from *Alternaria latifolia*, it is then crushed to increase the surface area of ​​the algae. During hot water cooking, fucoidan is gradually extracted under the action of hot water, and the supernatant is fucoidan solution. The algae precipitate is then treated with sodium carbonate to release sodium alginate, thereby ensuring the enzymatic hydrolysis effect and efficiency. This results in the enzymatic hydrolysis product being rich in sodium alginate, alginate oligosaccharides, alginate polyphenols, amino acids, plant hormones, trace elements, etc. Finally, the fucoidan solution and the enzymatic hydrolysis solution are mixed and spray-dried, resulting in a finished seaweed fertilizer with a high content of fucoidan and other nutrients. This fertilizer can be used in agriculture to prevent and inhibit tobacco mosaic virus, promote growth, increase stress resistance and disease resistance, and improve the quality of fruits or crops.

[0014] Preferably, the specific steps of the S1 water washing are as follows: first, wash with clean water 2-3 times for 5-8 minutes, then soak the raw material of *Alternaria latifolia* in calcium gluconate solution for 5-10 minutes, then filter to separate the *Alternaria latifolia*, and rinse with water for 5-10 minutes.

[0015] By adopting the above technical solution, the first step is to wash with clean water for a short period of time. This ensures that impurities and dirt on the surface of *Tetracentron sinense* are removed while minimizing the removal of fucoidan from the surface. After the initial washing, the *Tetracentron sinense* raw material is mixed with a calcium gluconate solution. Because fucoidan contains abundant carboxyl and hydroxyl groups, the hydroxyl groups in calcium gluconate and calcium ions are easily adsorbed and attached to the surface of *Tetracentron sinense*. The calcium ions and hydroxyl groups in glucose cross-link with the fucoidan, sealing the fucoidan on the surface of *Tetracentron sinense*. This ensures that the fucoidan adheres stably to the surface of *Tetracentron sinense*, making it difficult to wash or break it up, thus affecting the fucoidan content on the surface of *Tetracentron sinense*. The second water rinse further removes bacteria and impurities from the surface of *Tetracentron sinense*. During the rinsing process, the sealing and binding effect of calcium gluconate on fucoidan ensures the adhesion stability of fucoidan on the surface of *Tetracentron sinense*, and minimizes the impact of bacteria and impurities on the extraction of fucoidan, thereby ensuring that the finished seaweed fertilizer contains a high content of fucoidan.

[0016] During the hot water cooking process, the cross-linked structure and hydrogen bonds can be destroyed by the high temperature of the hot water, which facilitates the extraction of fucoidan and ensures the fucoidan content in the supernatant. During the washing process of Bufota bufota, fucoidan is not easily lost. In the subsequent extraction of fucoidan on the surface of Bufota bufota, it does not affect the separation of fucoidan to obtain the supernatant, thus ensuring that fucoidan in Bufota bufota can be extracted efficiently to prepare seaweed fertilizer.

[0017] Preferably, the temperature during the S2 hot water steaming process is 90-100℃, and the time is 2-2.5h.

[0018] By adopting the above technical solution and limiting the cooking temperature and time, it is possible to ensure that fucoidan on the surface of *Leptochloa crus-galli* is extracted efficiently, while also ensuring that the cross-linking structure and chemical bonds formed by fucoidan and calcium gluconate are destroyed, resulting in a high content of fucoidan in the supernatant and thus a high content of fucoidan in the finished seaweed fertilizer.

[0019] Preferably, before heating up the hot water for the S2 cooking process, the algae, chitosan microsphere carrier polyethylene glycol, and water are mixed and stirred evenly at a mass ratio of 1:0.1-0.2:10-15, and then heated and cooked.

[0020] By adopting the above technical solution, the algae, chitosan microspheres, polyethylene glycol, and water phase are combined. Since the chitosan microspheres are positively charged and the calcium ions on the surface of fucoidan are also positively charged, the chitosan microspheres and polyethylene glycol can be easily dispersed on the surface of *Tetracentron sinense* where there is no fucoidan. With the lubricating properties of water-soluble polyethylene glycol, the fucoidan on the surface of *Tetracentron sinense* is easily detached from the surface of *Tetracentron sinense*, ensuring that the seaweed fertilizer is rich in fucoidan.

[0021] After the supernatant and algal precipitate are separated, the chitosan microspheres precipitate together with the algal precipitate. The chitosan microspheres are evenly dispersed in the algal precipitate. With the addition of water, the hydrophilic and adsorption effects of the chitosan microspheres facilitate the adsorption of water to the interior of the algal precipitate, ensuring the rapid disintegration and dispersion of the algal precipitate. This allows the enzyme to come into uniform contact with the algal precipitate, while improving the enzyme's decomposition efficiency of substances in the algal precipitate. As a result, the content of other nutrients in the seaweed fertilizer is increased, meeting the growth needs of various crops.

[0022] Preferably, the solid content of the supernatant after concentration in S3 is 10-12%.

[0023] By adopting the above technical solution, the solid content after concentration is limited, ensuring that fucoidan can be uniformly mixed with the enzymatic hydrolysate, so that the finished seaweed fertilizer has the advantages of high fucoidan and high nutrient content, meeting the needs of agricultural use.

[0024] Preferably, the enzyme in S4 is composed of alginate lyase, cellulase, pectinase, and protease in a mass ratio of 1:1-2:1-2:0.2-0.5.

[0025] By adopting the above technical solutions, cellulase and pectinase can destroy the cell wall of *Alternaria latifolia*, allowing the nutrients in *Alternaria latifolia* to be released. Combined with alginate lysis buffer and protease, sodium alginate is cleaved into alginate oligosaccharides, and large protein molecules are broken down into small protein molecules, which is more conducive to crop absorption, meets agricultural applications, and promotes the growth of grain crops, vegetables, and fruits.

[0026] Preferably, the enzymatic hydrolysis temperature is 40-50℃ and the enzymatic hydrolysis time is 12-24h.

[0027] By adopting the above technical solution and limiting the enzymatic hydrolysis temperature and time, the enzymatic hydrolysis products are rich in sodium alginate, alginate oligosaccharides, algal polyphenols, amino acids, plant hormones, trace elements, etc., so that the seaweed fertilizer contains a high content of nutrients, which can meet the nutritional needs of various crops at various stages and can be used for agricultural production to promote the growth of crops or fruits.

[0028] Preferably, the cellulase is a silk fibroin-loaded cellulase and the pectinase is a biological cellulose-loaded pectinase.

[0029] By adopting the above technical solution, the silk fibroin-loaded cellulase and the biological cellulose-loaded pectinase are combined. Both silk fibroin and biological cellulose have high porosity and good adsorption effect, which can adsorb and load cellulase and pectinase, thus facilitating uniform contact between the enzyme and the algal precipitate. Furthermore, the hydroxyl groups in silk fibroin and biological cellulose can interact with the amino and carboxyl groups in chitosan microspheres, thereby further promoting the enzyme to enter the interior of the algal precipitate and improving the decomposition efficiency and nutrient content of nutrients.

[0030] Silk fibroin is a protein, while biocellulose is a type of cellulose. Silk fibroin is decomposed by proteases, and biocellulose is decomposed by cellulases, ensuring the inherent stability of both the silk fibroin carrier cellulase and the biocellulose carrier pectinase. Utilizing the cross-linking effect of the amino and carboxyl groups in silk fibroin combined with the hydroxyl groups in biocellulose, a cross-linking network can be easily formed on the surface of the cell wall of *Alternaria latifolia*. After cellulase decomposes the cellulose at the cell wall surface, nearby pectinase further decomposes the cell wall, thereby accelerating the decomposition and destruction of the cell wall and further promoting the extraction of nutrients from *Alternaria latifolia*. This results in seaweed fertilizer with a high content of nutrients, which can be used in agriculture to prevent tobacco mosaic virus and promote the growth of crops or fruits.

[0031] Preferably, the silk fibroin carrier cellulase is prepared by the following method:

[0032] A sucrose ester solution was uniformly sprayed onto the surface of silk fibroin at a mass ratio of 1:0.2-0.3. Then, cellulase was added, with a mass ratio of silk fibroin to cellulase of 1:0.2-0.4. After air drying and dispersion, silk fibroin-loaded cellulase was obtained.

[0033] By employing the above technical solution, the adhesive effect of sucrose esters combined with the adsorption effect of silk fibroin allows the sucrose ester solution to easily adhere to the surface of silk fibroin, thereby loading cellulase. The sucrose ester solution is not degraded by cellulase. Utilizing the high specific surface area and good loading effect of silk fibroin, combined with the partial lubrication effect of sucrose esters, the silk fibroin-loaded cellulase can easily penetrate into the algae, allowing the cellulase to enter the algal precipitate and accelerate the decomposition efficiency of cellulose in the algal precipitate. Furthermore, the presence of hydroxyl groups in sucrose esters ensures hydrophilicity and cross-linking effects. Even if the cellulase eventually detaches from the silk fibroin, it can still increase the decomposition efficiency of cellulose in the algal precipitate, ensuring the nutrient content of the seaweed fertilizer. This enables the seaweed fertilizer to be used in agriculture to prevent tobacco mosaic disease.

[0034] Preferably, the bio-cellulose carrier pectinase is prepared by the following method:

[0035] Biological cellulose was dispersed in a polyethylene glycol solution at a mass ratio of 1:0.2-0.4, and then pectinase was added at a mass ratio of biological cellulose to pectinase of 1:0.3-0.5. After air drying and dispersion, biological cellulose-loaded pectinase was obtained.

[0036] By employing the above technical solution, the partial viscosity of the polyethylene glycol solution, combined with the adsorption and loading effect of biocellulose, facilitates the entry of the polyethylene glycol solution into the biocellulose, where it binds pectinase. The hydroxyl groups in the polyethylene glycol interact with those in the biocellulose, attracting and connecting with the sucrose ester hydroxyl groups on the surface of the silk fibroin-modified cellulase. This facilitates the formation of a network on the cell wall surface, accelerating cell wall decomposition. Furthermore, the flow and lubrication effect of polyethylene glycol facilitates the entry of the biocellulose-loaded pectinase into the algal precipitate, further accelerating cell wall breakdown. This ensures uniform decomposition of nutrients throughout the algal precipitate, increasing the nutrient content of the seaweed fertilizer. Therefore, seaweed fertilizer can be applied in agriculture to prevent tobacco mosaic disease.

[0037] In summary, this application has the following beneficial effects:

[0038] 1. After washing with water to remove surface impurities, mud, and other particles from *Alternaria latifolia*, the algae are crushed to increase their surface area. During hot water cooking, fucoidan is gradually extracted, and the supernatant is the fucoidan solution. The algae precipitate is treated with sodium carbonate to release sodium alginate, thus ensuring the effectiveness and efficiency of enzymatic hydrolysis. This results in enzymatic hydrolysis products rich in sodium alginate, alginate oligosaccharides, alginate polyphenols, amino acids, plant hormones, and trace elements. Finally, the fucoidan solution is mixed with the enzymatic hydrolysis solution and spray-dried to produce a finished seaweed fertilizer with a high content of fucoidan and other nutrients, which can be widely used in agricultural production.

[0039] 2. The combination of calcium gluconate, chitosan microspheres loaded with polyethylene glycol, silk fibroin loaded with cellulase, and biological cellulose loaded with pectinase, ensures that fucoidan is not removed during the washing process through the binding effect of calcium gluconate and chitosan microspheres. Then, the dispersing effect of the chitosan microspheres loaded with polyethylene glycol facilitates the uniform dispersion of algal precipitate and contact with enzymes. The silk fibroin loaded with cellulase and the biological cellulose loaded with pectinase further promote the uniform enzymatic hydrolysis of algal precipitate, and the resulting nutrients can be used in the agricultural field.

[0040] 3. The combination of silk fibroin-loaded cellulase and bio-cellulose-loaded pectinase, both of which have high porosity and good adsorption effects, can adsorb and load cellulase and pectinase, thus facilitating uniform contact between the enzyme and algal precipitate. Furthermore, the hydroxyl groups in silk fibroin and bio-cellulose facilitate interaction with the amino and carboxyl groups in chitosan microspheres, thereby further promoting the entry of enzyme into the algal precipitate and improving the decomposition efficiency and nutrient content of nutrients.

[0041] 4. The silk fibroin carrier cellulase and the biological cellulose carrier pectinase work together, and the enzymes that degrade each other are coated, so that the enzymes that degrade silk fibroin and biological cellulose are released slowly. When added to the algal precipitate, silk fibroin and biological cellulose will not be rapidly degraded, but will take a period of time. During this period, the silk fibroin and biological cellulose are ensured to adhere to the cell wall surface, promoting the decomposition of the cell wall, thus facilitating the extraction of nutrients. Detailed Implementation

[0042] The present application will be further described in detail below with reference to the embodiments.

[0043] Example of preparation of polyethylene glycol as a carrier for chitosan microspheres

[0044] Preparation Example 1: The chitosan microsphere carrier, polyethylene glycol, was prepared by the following method:

[0045] 1 kg of chitosan microspheres were placed in 10 kg of polyethylene glycol solution and ultrasonically dispersed at 20 kHz for 20 min. The chitosan microspheres were then separated by filtration, dried, and dispersed until they did not stick together or agglomerate, thus obtaining the finished product. The chitosan microspheres passed through a 150-mesh sieve, and the average porosity of the chitosan microspheres was 50%. The polyethylene glycol solution was a 5% (w / w) polyethylene glycol ethanol solution, and the polyethylene glycol was polyethylene glycol 8000 with a (w / w) ethanol content of 75%.

[0046] Example of preparation of cellulase for silk fibroin carrier

[0047] Preparation Example 2: Silk fibroin carrier cellulase was prepared using the following method:

[0048] 0.3 kg of sucrose ester solution was uniformly sprayed onto the surface of 1 kg of silk fibroin. The silk fibroin was passed through an 80-mesh sieve. The sucrose ester solution was a 1% (w / w) sucrose ester ethanol solution with a 99% (w / w) ethanol content. Then, 0.4 kg of cellulase was added. The cellulase was a dextran exoglucanase. The cellulase was passed through a 150-mesh sieve, air-dried, and dispersed until the silk fibroin proteins did not stick together or agglomerate, thus obtaining the silk fibroin-loaded cellulase.

[0049] Preparation Example 3: The difference between this preparation example and Preparation Example 2 is that:

[0050] 0.2 kg of sucrose ester solution was uniformly sprayed onto the surface of 1 kg of silk fibroin. The silk fibroin was passed through an 80-mesh sieve. The sucrose ester solution was a 1% sucrose ester ethanol solution with a 99% ethanol content. Then, 0.2 kg of cellulase was added. The cellulase was passed through a 150-mesh sieve and then air-dried and dispersed until the silk fibroin did not stick together or agglomerate. This yielded the silk fibroin-loaded cellulase.

[0051] Example of preparation of pectinase from biological cellulose carrier

[0052] The pectinase in the following raw materials was purchased from Guangzhou Huayu Biotechnology Co., Ltd., with an enzyme activity of 30,000 and in powder form; the other raw materials are all commercially available.

[0053] Preparation Example 4: Pectinase from biological cellulose carriers was prepared using the following method:

[0054] 1 kg of bio-cellulose was dispersed in 0.4 kg of polyethylene glycol solution. The bio-cellulose was passed through an 80-mesh sieve. The polyethylene glycol solution was a 5% (w / w) polyethylene glycol ethanol solution with a 75% (w / w) ethanol content. Then, 0.5 kg of pectinase was added. The pectinase was passed through a 150-mesh sieve. After air drying, the bio-cellulose was dispersed to prevent it from sticking together and agglomerating, thus obtaining bio-cellulose-loaded pectinase.

[0055] Preparation Example 5: The difference between this preparation example and Example 4 is that:

[0056] 1 kg of bio-cellulose was dispersed in 0.2 kg of polyethylene glycol solution. The bio-cellulose was passed through an 80-mesh sieve. The polyethylene glycol solution was a 5% (w / w) polyethylene glycol ethanol solution with a 75% (w / w) ethanol content. Then, 0.3 kg of pectinase was added. The pectinase was passed through a 150-mesh sieve. After air drying, the bio-cellulose was dispersed to prevent it from sticking together and agglomerating, thus obtaining bio-cellulose-loaded pectinase.

[0057] Example

[0058] The alginate lyase in the following raw materials was purchased from Shanghai Huashangxiang Biotechnology Co., Ltd., and was in powder form; the other raw materials were all commercially available.

[0059] Example 1: A method for preparing seaweed fertilizer rich in fucoidan:

[0060] S1. Take 100kg of *Alternaria buergeriana* raw material, wash it twice with water for 5 minutes, and then crush it to obtain algae. The average particle size of the algae is 1cm.

[0061] S2. Add 15 times the amount of water to the algae, add citric acid to adjust the pH to 3.5, then heat to 100℃ and cook in hot water for 2 hours. After cooking, filter and separate the algae, pass through a 200-mesh sieve to obtain the supernatant and algae precipitate.

[0062] S3. The supernatant is concentrated to a solid content of 10% to obtain fucoidan solution;

[0063] S4. Add water and sodium carbonate to the algal precipitate and stir. The ratio of algal precipitate, water and sodium carbonate is 1:8:0.03. Then adjust the pH to 7.0 with potassium dihydrogen phosphate. Add enzyme for enzymatic hydrolysis. The amount of enzyme is 1 / 20 of the algal precipitate. The hydrolysis temperature is 45℃ and the hydrolysis time is 20h. The enzymes are alginate lyase, cellulase, pectinase and protease in a mass ratio of 1:2:2:0.5. The protease is papain. Finally, concentrate to a solid content of 10% to obtain the enzymatic hydrolysate.

[0064] S5, fucoidan solution and enzymatic hydrolysate are mixed and stirred evenly, and then spray-dried to obtain the finished seaweed fertilizer, which is then passed through a 60-mesh sieve.

[0065] Example 2: The difference between this example and Example 1 is that:

[0066] S1. Take 100kg of *Leptochloa crus-galli* raw material and wash it 3 times with water for 8 minutes. Then soak the *Leptochloa crus-galli* raw material in calcium gluconate solution for 10 minutes. The calcium gluconate solution is a 1% (w / w) calcium gluconate aqueous solution. Then filter to separate the *Leptochloa crus-galli*, rinse with water for 10 minutes, and then crush to obtain algae. The average particle size of the algae is 1cm.

[0067] S2. Add 15 times the amount of water to the algae and add 0.2 kg of polyethylene glycol, the chitosan microsphere carrier prepared in Example 1. After mixing evenly, add citric acid to adjust the pH to 3.5, and then heat to 100°C for hot water cooking for 2 hours. After cooking, filter and separate the algae, and pass it through a 200-mesh sieve to obtain the supernatant and algae precipitate.

[0068] S3. The supernatant was concentrated to a solid content of 12% to obtain fucoidan solution;

[0069] S4. Water and sodium carbonate were added to the algal precipitate and stirred. The ratio of algal precipitate, water, and sodium carbonate was 1:8:0.03. The pH was then adjusted to 7.0 using potassium dihydrogen phosphate. Enzymes were added for enzymatic hydrolysis. The amount of enzyme used was 1 / 20 of the algal precipitate. The hydrolysis temperature was 50℃, and the hydrolysis time was 12 hours. The enzymes used were alginate lyase, cellulase, pectinase, and protease in a mass ratio of 1:2:2:0.5. The cellulase used was the cellulase prepared in Preparation Example 2, and the pectinase used was the pectinase from the biological cellulose carrier prepared in Preparation Example 4. Finally, the solution was concentrated to a solid content of 12% to obtain the enzymatic hydrolysate.

[0070] S5, fucoidan solution and enzymatic hydrolysate are mixed and stirred evenly, and then spray-dried to obtain the finished seaweed fertilizer, which is then passed through a 60-mesh sieve.

[0071] Example 3: The difference between this example and Example 1 is that:

[0072] S1. Take 100kg of *Leptochloa crus-galli* raw material and wash it twice with water for 5 minutes. Then, soak the *Leptochloa crus-galli* raw material in a calcium gluconate solution for 5 minutes. The calcium gluconate solution is a 1% (w / w) calcium gluconate aqueous solution. Then filter to separate the *Leptochloa crus-galli*, rinse with water for 5 minutes, and then crush to obtain algae. The average particle size of the algae is 1cm.

[0073] S2. Add 10 times the amount of water to the algae and add 0.1 kg of polyethylene glycol, the chitosan microsphere carrier prepared in Example 1. After mixing evenly, add citric acid to adjust the pH to 3.5, and then heat to 90°C for hot water cooking for 2.5 h. After cooking, filter and separate the algae, pass through a 200-mesh sieve to obtain the supernatant and algae precipitate.

[0074] S3. The supernatant is concentrated to a solid content of 10% to obtain fucoidan solution;

[0075] S4. Water and sodium carbonate were added to the algal precipitate and stirred. The ratio of algal precipitate, water, and sodium carbonate was 1:8:0.03. The pH was then adjusted to 7.0 using potassium dihydrogen phosphate. Enzymes were added for enzymatic hydrolysis. The amount of enzyme used was 1 / 20 of the algal precipitate. The hydrolysis temperature was 40℃, and the hydrolysis time was 24 hours. The enzymes used were alginate lyase, cellulase, pectinase, and protease in a mass ratio of 1:1:1:0.2. The cellulase used was the cellulase prepared in Preparation Example 3, and the pectinase used was the pectinase from the biological cellulose carrier prepared in Preparation Example 5. Finally, the solution was concentrated to a solid content of 10% to obtain the enzymatic hydrolysate.

[0076] S5, fucoidan solution and enzymatic hydrolysate are mixed and stirred evenly, and then spray-dried to obtain the finished seaweed fertilizer, which is then passed through a 60-mesh sieve.

[0077] Example 4: The difference between this example and Example 1 is that:

[0078] In S1, calcium gluconate is replaced with an equal mass of glucose.

[0079] Example 5: The difference between this example and Example 1 is that:

[0080] In S2, the polyethylene glycol carrier for the chitosan microspheres is replaced with chitosan microspheres of equal mass.

[0081] Example 6: The difference between this example and Example 1 is that:

[0082] In S2, the chitosan microsphere carrier polyethylene glycol is replaced with polyethylene glycol of equal mass.

[0083] Example 7: The difference between this example and Example 1 is that:

[0084] In the preparation of silk fibroin-loaded cellulase, an equal mass of sucrose solution was used to replace the sucrose ester solution.

[0085] Example 8: The difference between this example and Example 1 is that:

[0086] In the preparation of pectinase using biological cellulose carriers, the polyethylene glycol solution was replaced with an equal mass of carboxymethyl cellulose solution. The carboxymethyl cellulose solution was a 1% (w / w) aqueous solution of carboxymethyl cellulose with a molecular weight of 200,000.

[0087] Comparative Example

[0088] Comparative Example 1: The difference between this comparative example and Example 1 is that:

[0089] S2. Algae are mixed with water and sodium carbonate, and stirred. The ratio of algae precipitate, water, and sodium carbonate is 1:8:0.03. Then, the pH is adjusted to 7.0 with potassium dihydrogen phosphate. Enzymes are added for enzymatic hydrolysis. The amount of enzyme used is 1 / 20 of the algae precipitate. The hydrolysis temperature is 45℃ and the hydrolysis time is 20h. The enzymes are alginate lyase, cellulase, pectinase, and protease in a mass ratio of 1:2:2:0.5. Finally, the mixture is concentrated to a solid content of 10%, and then spray-dried to obtain the finished seaweed fertilizer, which is passed through a 60-mesh sieve.

[0090] Performance testing

[0091] 1. Detection of fucoidan content

[0092] Fucoidan solutions were prepared using the methods described in Examples 1-8 and Comparative Example 1, respectively. The content of fucoidan in the fucoidan solutions was detected and the data were recorded, referring to SC / T3404-2012.

[0093] 2. Nutrient testing

[0094] Seaweed fertilizers were prepared using the methods described in Examples 1-8 and Comparative Example 1, respectively. The alginic acid content was tested according to NY / T3174-2017, and the data were recorded.

[0095] 3. Inoculation solution for tobacco mosaic virus inhibition detection: The virus source was the common TMV strain preserved by the Plant Disease Integrated Prevention and Control Research Laboratory of the Institute of Plant and Environmental Protection, Beijing Academy of Agricultural and Forestry Sciences (using common tobacco as the reproductive host, preserved in living form). The midrib of the leaves of common tobacco infected with the common TMV strain was removed, and the leaves were mixed with 0.01mol / L PBS at pH 8.0 at a ratio of 1g / 20mL. 0.015g of carborundum was added to every 20mL of PBS, and the leaves were thoroughly ground to obtain the inoculation solution.

[0096] Test solution: The seaweed fertilizer was prepared using the preparation methods of Examples 1-8 and Comparative Example 1, respectively. The seaweed fertilizer was diluted 10 times with water and sprayed on the whole plant of Nicotiana scabra at the three to four leaf stage, with three leaves per plant, to obtain the test sample.

[0097] Inoculation: The sample to be tested was inoculated by friction with the inoculation solution. After inoculation, it was immediately rinsed with water. The culture conditions were 28°C under light for 12 hours and 18°C ​​under no light for 12 hours, alternating for 120 hours.

[0098] Calculation of necrotic spot inhibition rate: Record the number of necrotic spots on the leaves of *Tobacco Trifolium* var. *necrotic* 120 hours after inoculation. The formula for calculating the necrotic spot inhibition rate is: X(%) = (CK - Y) / CK × 100%

[0099] In Formula I, X represents the necrotic spot inhibition rate; CK represents the average number of necrotic spots on the leaves of the water control (unit: spots); and Y represents the average number of necrotic spots on the leaves after induction treatment with *Schizophyllum commune* polysaccharide extract (unit: spots).

[0100] Table 1 Performance Test Table (In the table, " / " indicates that the corresponding embodiment or comparative example did not test this item, so there is no data)

[0101]

[0102] As can be seen from Examples 1 and 2-3 and Table 1, this application has a high content of fucoidan and alginic acid, and can inhibit tobacco mosaic virus. At the same time, the extraction efficiency of fucoidan and nutrients can be further improved by combining calcium gluconate, chitosan microsphere carrier polyethylene glycol, and biological cellulose carrier pectinase.

[0103] Combining Examples 2 and 4-8 with Table 1, it can be seen that in Example 4S1, when the same mass of glucose was used to replace calcium gluconate, the content of fucoidan in Example 4 was lower than that in Example 2, and the inhibition rate was also lower. This indicates that calcium gluconate is more likely to bind with fucoidan, improving the adhesion stability of fucoidan on the surface of *Tetracentron sinense*, and making it less likely to be washed away by water. This allows the surface of *Tetracentron sinense* to retain a high content of fucoidan while minimizing the presence of impurities, bacteria, and other substances, thus ensuring the efficiency of subsequent nutrient extraction.

[0104] In Example 5S2, the same mass of chitosan microspheres replaced the polyethylene glycol carrier for the chitosan microspheres. In Example 6S2, the same mass of polyethylene glycol replaced the polyethylene glycol carrier for the chitosan microspheres. Compared to Example 2, the fucoidan content and alginic acid content in Examples 5 and 6 were lower than in Example 2, and the inhibition rate was lower than in Example 2. This indicates that the positive charge of the chitosan microspheres and the positive charge of the calcium ions on the surface of the fucoidan repel each other, making it easier for the polyethylene glycol carrier to disperse on the surface of *Tetracentron sinense* where there is no fucoidan. With the lubrication of the water-soluble polyethylene glycol, the fucoidan on the surface of *Tetracentron sinense* is easily detached, ensuring that the seaweed fertilizer is rich in fucoidan. The chitosan microspheres can disperse in the algal precipitate. Utilizing the hydrophilic and adsorption effects of the chitosan microspheres, the enzymes are attracted into the interior of the algal precipitate and make uniform contact with the precipitate, improving the extraction rate of nutrients in the algal precipitate and meeting the application requirements of the seaweed fertilizer.

[0105] In Example 7, during the preparation of cellulase using silk fibroin as a carrier, the sucrose ester solution was replaced with an equal mass of sucrose solution. In Example 8, during the preparation of pectinase using biological cellulose as a carrier, the polyethylene glycol solution was replaced with an equal mass of carboxymethyl cellulose solution. Compared to Example 2, the alginic acid content in Examples 7 and 8 was lower than that in Example 2, and the inhibition rate was lower than that in Example 2. This indicates that both sucrose solution and carboxymethyl cellulose solution have high viscosity, which can easily affect the contact between cellulase and pectinase and the substances inside the algal precipitate, thereby affecting the extraction efficiency of nutrients and thus affecting the inhibition rate.

[0106] Combining Example 1 and Comparative Example 1 with Table 1, it can be seen that the fucoidan content in Comparative Example 1 is lower than that in Example 1; this indicates that a higher content of fucoidan can be obtained through separation and extraction, and it is not easily lost, thus preserving the fucoidan content in the seaweed fertilizer.

[0107] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a seaweed fertilizer enriched in fucoidan, characterized in that, It comprises the following steps: S1, the bubble leaf alga raw material is first washed with clean water for 2-3 times, the washing time is 5-8 min, then the bubble leaf alga raw material is soaked in calcium gluconate solution for 5-10 min, then the bubble leaf alga is separated by filtration, and the water flow is washed for 5-10 min, then the alga is crushed to obtain the alga body; S2, before the alga body is heated and boiled, the alga body, chitosan microsphere carrier polyethylene glycol and water are mixed and stirred uniformly according to the mass ratio of 1:0.1-0.2:10-15, then the heated boiling is carried out, and the supernatant and alga body precipitate are obtained by filtration; S3, the supernatant is concentrated to obtain fucoidan liquid; S4, after the alga body precipitate is treated with water and sodium carbonate, the pH is adjusted, then the enzyme is added for enzymolysis, the enzyme is composed of alginate lyase, cellulase, pectinase and protease according to the mass ratio of 1:1-2:1-2:0.2-0.5, and finally the enzyme solution is obtained by concentration; S5, the fucoidan liquid and the enzyme solution are mixed and stirred uniformly, and the finished product of seaweed fertilizer is obtained by spray drying.

2. A process for the preparation of a fucoidin-rich seaweed fertilizer according to claim 1, characterized by, The temperature in the S2 heated boiling process is 90-100℃, and the time is 2-2.5h.

3. A process for the preparation of a fucoidin-rich seaweed fertilizer according to claim 1, characterized by, The solid content of the supernatant after concentration in S3 is 10-12%.

4. A process for the preparation of a fucoidin-rich seaweed fertilizer according to claim 1, characterized by, The temperature of the enzyme solution is 40-50℃, and the enzyme solution time is 12-24h.

5. The process for the preparation of a fucoidin-rich seaweed fertilizer according to claim 1, characterized by, The cellulase is a silk fibroin carrier cellulase, and the pectinase is a biological cellulose carrier pectinase.

6. A process for the preparation of a fucoidin-rich seaweed fertilizer according to claim 5, characterized by, The silk fibroin carrier cellulase is prepared by the following method: The silk fibroin is uniformly sprayed with sucrose ester solution according to the mass ratio of 1:0.2-0.3, then the cellulase is added, the mass ratio of silk fibroin to cellulase is 1:0.2-0.4, and the silk fibroin carrier cellulase is prepared by air drying and dispersion.

7. A process for the preparation of a fucoidin-rich seaweed fertilizer according to claim 5, characterized by, The biological cellulose carrier pectinase is prepared by the following method: The biological cellulose is dispersed in polyethylene glycol solution according to the mass ratio of 1:0.2-0.4, then the pectinase is added, the mass ratio of biological cellulose to pectinase is 1:0.3-0.5, and the biological cellulose carrier pectinase is prepared by air drying and dispersion.

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