Enzymolysis method for efficiently preparing seaweed oligosaccharide from sargassum

Abstract

The invention provides an enzymolysis method for efficiently preparing seaweed oligosaccharide from sargassum, and belongs to the technical field of high-value utilization of seaweed. According to the enzymolysis method provided by the invention, an enzyme composition consisting of algin degrading enzyme, pectinase and cellulase is used for carrying out enzymolysis reaction on the sargassum powder, the enzymolysis rate of the obtained sargassum solid matter reaches 70% or above, and the proportion of high-activity oligomeric alginate oligosaccharide in the enzymatic hydrolysate reaches 80%. According to the method, the synergistic effect of three seaweed degrading enzymes in the same reaction system is realized, the enzyme dosage and cost are reduced, the technical process of sargassum enzymatic degradation is greatly simplified, and the yield of oligomeric alginate oligosaccharide is also effectively improved while the enzymolysis rate of sargassum solids is effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of high-value utilization of seaweed, and in particular relates to an enzymatic hydrolysis method for efficiently preparing seaweed oligosaccharides from Sargassum. Background Art

[0002] my country is one of the main production areas of Sargassum, and its resources are very rich. Sargassum is the main feed ingredient for the cultivation of important marine treasures such as sea cucumbers and abalone. At the same time, the seaweed polysaccharides and oligosaccharides it contains have good biological activities in anti-tumor, antibacterial, antioxidant, and immunomodulatory aspects, and can be used in agriculture, medicine, food and other industries. The resource development and application of brown algae polysaccharides and oligosaccharides have become one of the hot spots of current research. Among them, brown algae oligosaccharides refer to sugar chains composed of 2-20 monosaccharides with a polymerization degree of 2-20. They have the characteristics of low molecular weight, good water solubility, safety and non-toxicity. They have antioxidant, antibacterial, anti-inflammatory, anti-tumor, immunity enhancement, and plant growth promotion effects. They are increasingly valued in the fields of agriculture and medicine. Adding brown algae oligosaccharides to aquatic feed can significantly improve the growth and immune performance of aquatic animals; treating plants with brown algae oligosaccharides can significantly enhance their stress resistance and disease resistance. Brown algae oligosaccharides and their derivatives can be used to prevent and treat major diseases such as human cardiovascular disease, Alzheimer's disease, and anti-tumor.

[0003] Although Sargassum oligosaccharides show good application potential in agriculture, medicine, cosmetics, food and other fields, the preparation efficiency of Sargassum oligosaccharides is limited due to the structure of its cell wall. Sargassum cell wall is mainly composed of cellulose and fucoidan. Alginate is composed of mannuronic acid and guluronic acid to form a long-chain macromolecular structure, and is cross-linked with fucoidan, protein, polyphenols and other components to form a network structure. At present, three methods are mainly used to degrade Sargassum to prepare oligosaccharides. ① Physical degradation method: including radiation method, hot dissolution method, high temperature and high pressure method, ultrasonic method, etc. The final molecular weight of the physical decomposition product is about 50kDa, and oligosaccharides with smaller molecular weight cannot be obtained. Therefore, it is not often used alone for the preparation of oligosaccharides, but as an auxiliary method for other methods. ② Chemical decomposition method: mainly includes oxidative decomposition, acid degradation and alkaline degradation. The oxidative degradation reaction is relatively violent and has a certain destructive effect on the reducing group of the degraded oligosaccharide. Acid hydrolysis is a common degradation method with low production cost and mature technology. Alkaline degradation will cause changes in the structure of polysaccharides and is generally not used. Chemical decomposition method easily produces a large amount of acid and alkali waste, causing great pollution to the environment. ③Biodegradation method: Using enzyme catalysis to degrade alginate is an ideal and environmentally friendly method.

[0004] Using Sargassum as raw material, the bioenzymatic method is used to prepare brown algae oligosaccharides, mostly using multi-enzyme composite degradation technology supplemented by physical crushing technology, such as CN 110295209 A "A process for extracting alginate oligosaccharides by enzymatic hydrolysis of Sargassum" adopts physical crushing steps, water infiltration steps, softening steps, and multi-enzyme composite degradation steps to achieve deep degradation of Sargassum, and the yield of alginate oligosaccharides can reach 40%. The composite enzyme used in this technology includes 8 enzymes: protease, cellulase, pectinase, xylanase, mannanase, β-glucanase, neutral amylase, and alginate lyase. Although this technology improves the yield of brown algae oligosaccharides, the process steps are still relatively complicated. And the above technology only mentions the compounding ratio of various enzymes in the composite enzyme preparation, and does not mention the dosage of the enzyme preparation. The patented technology of CN 105483183 A is to degrease with ethanol; deproteinize with Sevag method / trifluorotrichloroethane method / trichloroacetic acid method; dialysis; rotary evaporation; sequentially add alginate lyase, mannanase, xylanase, and pectinase for enzymolysis; and finally add ethanol to precipitate and remove undegraded polysaccharides to obtain an oligosaccharide solution. The steps are very complicated, and the technology does not mention the final oligosaccharide polymerization degree and yield. Therefore, a more feasible method that can effectively degrade Sargassum and simultaneously improve the yield of the obtained oligomeric brown algae oligosaccharides has not been proposed in the prior art. Summary of the invention

[0005] The present invention provides an enzymatic hydrolysis method for efficiently preparing seaweed oligosaccharides from Sargassum. The method greatly simplifies the existing technical process of enzymatic degradation of Sargassum, so that the enzymatic hydrolysis rate of Sargassum solids reaches more than 70%. At the same time, the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolysis solution reaches about 80%, effectively improving the yield of brown algae oligosaccharides.

[0006] In order to achieve the above-mentioned object, the present invention provides an enzymatic hydrolysis method for efficiently preparing seaweed oligosaccharides from Sargassum, wherein an enzyme composition composed of alginate degrading enzyme, pectinase and cellulase is used to perform enzymatic hydrolysis reaction on ultrafine powder of Sargassum, and the enzymatic hydrolysis rate of the obtained Sargassum solid reaches more than 70%, and the proportion of highly active oligosaccharides of brown algae in the enzymatic hydrolysis liquid reaches 80%.

[0007] Preferably, the addition amount of alginate degrading enzyme is 300-500U / g Sargassum powder, the addition amount of pectinase is 3000-5000U / g Sargassum powder, and the addition amount of cellulase is 240-400U / g Sargassum powder.

[0008] Preferably, the addition amount of alginate degrading enzyme is 500U / g Sargassum powder, the addition amount of pectinase is 5000U / g Sargassum powder, and the addition amount of cellulase is 320U / g Sargassum powder.

[0009] Preferably, the temperature of the enzymatic hydrolysis reaction is 45-50°C, the pH is 5-6, and the enzymatic hydrolysis time is 8-18h.

[0010] Preferably, the temperature of the enzymatic hydrolysis reaction is 47° C., the pH is 5.5, and the enzymatic hydrolysis time is 8 h.

[0011] It is understandable that in the above scheme, the key of the present application is to perform deep enzymolysis reaction on Sargassum under specific conditions by using an enzyme composition composed of three enzymes to achieve the expected effect. The reason is that the cell wall and intercellular matrix of Sargassum contain multiple components. In theory, according to its chemical composition, the deep degradation of Sargassum can be achieved by using multiple enzymes to work together. However, as a biological catalyst, each enzyme has its own suitable action temperature and pH conditions. To achieve the joint action of different enzymes under the same action conditions, it is necessary to weigh the activity retention rates of multiple enzymes under different conditions and explore a condition suitable for multiple enzymes to work. The present invention has established such a process condition that can maximize the synergistic effect of three seaweed degrading enzymes in the same reaction system through a large number of experimental explorations. Compared with the prior art, some choose to use two key enzymes in consideration of the maintenance of enzyme activity, and some use step-by-step enzymolysis (the steps are relatively cumbersome), such as using enzyme A to enzymolyze for 6h under condition a, then changing the enzymolysis condition b, adding enzyme B to continue enzymolyzing for 8h, etc., which not only simplifies the cumbersome steps, but also obtains better technical effects.

[0012] Preferably, the highly active oligosaccharide is an oligosaccharide with a molecular weight of less than 2000 Da.

[0013] Preferably, the highly active brown algae oligosaccharide is mainly composed of brown algae oligosaccharide with a polymerization degree of 2-saccharide, 3-saccharide and 4-saccharide.

[0014] As a preference, the method comprises the following steps:

[0015] The dried Sargassum superfine powder crushed to 300 mesh, alginate lyase, pectinase and cellulase are weighed and put into a centrifuge tube for mixing, a buffer solution is added, mixed, and put into a metal bath at a preset temperature for heat preservation and enzymolysis;

[0016] During the enzymatic hydrolysis process, the enzymatic hydrolysis system is shaken every 20-40 minutes to allow the Sargassum powder to be fully enzymatically hydrolyzed. After the enzymatic hydrolysis is completed, the metal bath temperature is immediately adjusted to 90-100°C for 10-20 minutes to inactivate the enzyme in the reaction system, and then the system is taken out and naturally cooled to room temperature;

[0017] The enzymatically hydrolyzed Sargassum solution was centrifuged at 4000-5000×g for 15-20 minutes and the separation was repeated twice.

[0018] Preferably, the mass ratio of the added Sargassum powder to the buffer solution is 1:10.

[0019] Preferably, the preset temperature of the metal bath is 40-60°C.

[0020] Compared with the prior art, the advantages and positive effects of the present invention are:

[0021] 1. The enzymatic hydrolysis method provided by the present invention targets the structural characteristics of the cell wall of Sargassum, selects three lyases (algin degrading enzyme, cellulase, and pectinase) to hydrolyze Sargassum, and uses the response surface methodology to optimize the enzymatic hydrolysis compound ratio and enzymatic hydrolysis process conditions, thereby achieving deep enzymatic hydrolysis of Sargassum and obtaining oligosaccharides with a degree of polymerization mainly consisting of 2-sugar, 3-sugar, and 4-sugar.

[0022] 2. The enzymatic hydrolysis method established by the present invention has undergone two rounds of optimization processes, one of which is the optimization of the compound ratio of the enzyme, so that the solid enzymatic hydrolysis rate reaches about 60%, and the other is the further optimization of the enzymatic hydrolysis conditions, so that the solid enzymatic hydrolysis rate is increased to about 70%. Through two rounds of optimization, the synergistic effect of the three seaweed degradation enzymes in the same reaction system is achieved, the enzyme dosage and cost are reduced, and the technical process of enzymatic degradation of Sargassum is greatly simplified. While the enzymatic hydrolysis rate of Sargassum solids reaches more than 70%, the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate reaches about 80%, which effectively increases the yield of brown algae oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a high pressure liquid phase analysis spectrum of the Sargassum enzymatic hydrolysate provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1 Optimization of the ratio of multiple enzymes for enzymatic hydrolysis of Sargassum by response surface methodology

[0026] 1.1 Experimental materials and enzymatic hydrolysis steps:

[0027] Alginate lyase 10000U / g; Pectinase 100000U / g; Cellulase 8000U / g; Sargassum powder 300 mesh; Enzyme buffer: citric acid-disodium hydrogen phosphate buffer

[0028] Weigh 0.5g of dried Sargassum superfine powder crushed to 300 mesh, alginate lyase, pectinase, and cellulase, put them into a 10mL centrifuge tube and mix them well. Add 5mL of buffer solution, mix well, and put them into a metal bath with a pre-set temperature for enzymolysis. During the enzymolysis process, the enzymolysis system should be shaken every 30 minutes to fully enzymolyze the seaweed powder and fully leached the reducing sugar. After the enzymolysis is completed, the metal bath temperature is immediately adjusted to 100℃ for 10 minutes to inactivate the enzyme in the reaction system, and then taken out and naturally cooled to room temperature. The enzymolyzed Sargassum enzymolysis solution was centrifuged at 4000×g for 15 minutes and separated twice. The DNS colorimetric method was used to determine the reducing sugar content in the supernatant enzymolysis solution to determine the enzymolysis efficiency.

[0029] 1.2 Response surface experimental design and results analysis

[0030] The Box-Behnken design (BBD) design was adopted with the reducing sugar content of the supernatant in the system after enzymatic hydrolysis as the response value. This optimization experiment designed a set of 17 BBD experiments, including 12 factorial experiments and 5 central point repeated experiments. There were 3 factors (i.e., the amount of alginate lyase added, the amount of pectinase added, and the amount of cellulase added). During the experiment, pH = 7, seaweed powder / buffer 1:10, enzymatic hydrolysis time 8h, and enzymatic hydrolysis temperature 50℃. Table 1 shows the BBD response surface experimental scheme designed for the enzymatic hydrolysis of Sargassum powder based on the three factors of alginate lyase addition, pectinase addition, and cellulase addition, and the measurement results of the reducing sugar content of the supernatant after enzymatic hydrolysis.

[0031] Table 1 Multi-enzyme complex ratio BBD response surface experimental scheme and results

[0032]

[0033] The response surface analysis method was used to analyze the above experimental results. It can be seen that the model is significant and the lack of fit term is not significant. Therefore, the model is established, and the experimental plan and experimental results are reliable (Table 2). From the numerical values, it can be seen that "Prob>F"<0.05 means that the factors have a significant impact, so the amount of alginate lyase added has a significant effect on the enzymatic efficiency, the amount of pectinase added has an extremely significant effect on the enzymatic efficiency, and the effect of cellulase is small. The factors that affect the enzymatic efficiency are pectinase addition> alginate lyase addition> cellulase addition. In addition, CV = 6.70%, which proves that the experimental credibility and accuracy are high; R-Squared = 0.7534, with high correlation.

[0034] Table 2 Response surface design table results analysis of three factors and three levels (BBD)

[0035]

[0036] The optimal conditions obtained by optimization according to the model are: alginate lyase addition amount A = 4.98%, pectinase addition amount B = 5.00%, cellulase addition amount C = 4.30%, the predicted maximum reducing sugar content is 22.52 mg / mL, and Desirability = 1.00.

[0037] 1.3 Experimental verification of the optimal compounding ratio

[0038] Using 0.5 g of Sargassum powder and 5 mL of pH 7 buffer solution, enzymatic hydrolysis was performed at 50 ° C for 8 h. The enzymatic hydrolysis was repeated 3 times with the above-mentioned optimal compound ratio (algin lyase addition amount = 4.98%, pectinase addition amount = 5.00%, cellulase addition amount = 4.30%). The average reducing sugar content was 23.37 mg / mL, slightly higher than the predicted value. Therefore, the conditions of the compound ratio of multiple feed enzymes optimized by response surface analysis are feasible.

[0039] Example 2 Optimization of Sargassum enzymatic hydrolysis process conditions by response surface methodology

[0040] 2.1 Experimental plan and result analysis of optimization of enzymatic hydrolysis conditions by response surface methodology

[0041] The BBD design with the reducing sugar content of the supernatant in the system after enzymatic hydrolysis as the response value was adopted. A total of 17 BBD experiments were designed for the optimization experiment, including 12 factorial experiments and 5 central point repeated experiments, with a total of 3 factors (enzymatic hydrolysis temperature, enzymatic hydrolysis time, pH). The results of the response surface experiment of the composite ratio of Sargassum lyase were applied in the experiment, that is, the addition amount of alginate lyase = 4.98%, the addition amount of pectinase = 5.00%, and the addition amount of cellulase = 4.30%. The experimental design and results are shown in Table 3.

[0042] Table 3 BBD response surface experiment scheme and results of Sargassum enzymatic digestion conditions

[0043]

[0044] The above results were analyzed, and the results are shown in Table 4. The model is significant, and the lack of fit term is not significant. Therefore, the model is established, and the experimental plan and experimental results are reliable. From the numerical values, it can be seen that "Prob>F"<0.05 means that the factor has a significant effect, so the effect of enzymatic hydrolysis time on enzymatic hydrolysis efficiency is close to significant, pH has a significant effect on enzymatic hydrolysis efficiency, and temperature has a small effect. The factors that affect the enzymatic hydrolysis efficiency are pH>enzymatic hydrolysis time>enzymatic hydrolysis temperature. In addition, there is a strong interaction between AC, that is, there is a certain interaction between temperature and buffer solution pH. CV = 5.17%, which proves that the experimental credibility and precision are high; R-Squared = 0.9045, with high correlation. However, the interaction between AB and BC factors is weak and not significant.

[0045] Table 4 Response surface design table results analysis of three factors and three levels of Sargassum enzymatic hydrolysis conditions (BBD)

[0046]

[0047]

[0048] The optimal conditions obtained by optimization according to the model are: A (temperature) = 46.67°C, B (enzymatic hydrolysis time) = 8h, C (pH) = 5.5, the predicted maximum reducing sugar content is 25.29 mg / mL, and Desirability = 0.93.

[0049] 2.2 Verification of the optimal enzymatic hydrolysis conditions for Sargassum

[0050] The verification experiment of enzymatic hydrolysis of Sargassum powder was carried out with the best enzymatic hydrolysis conditions and the best enzyme compound ratio scheme obtained close to the best scheme. Similarly, 0.5g of Sargassum superfine powder was used, under the conditions of alginate lyase addition = 5.0%, pectinase addition = 5.0%, and cellulase addition = 4.0%, and the above-mentioned optimal conditions, i.e., enzymatic hydrolysis temperature = 47°C, enzymatic hydrolysis solution pH = 5.5, and enzymatic hydrolysis time = 8h, for enzymatic hydrolysis of Sargassum powder, which was repeated 3 times. The average reducing sugar content obtained in the enzymatic hydrolysis solution relative to the central point extraction rate was 25.473mg / mL, which shows that the enzymatic digestion conditions optimized by response surface analysis are feasible.

[0051] Example 3: Solid enzymatic hydrolysis rate of Sargassum powder

[0052] Solid enzymatic hydrolysis rate, this indicator is generally expressed as seaweed degradation rate (solid enzymatic hydrolysis rate), which is the solid content of the enzymatic hydrolysis part. The specific operation is to first take 0.5g of dry Sargassum powder and put it into an oven at 80℃ to dry to constant weight, and weigh it. Then wash and centrifuge the precipitate after centrifugation of the enzymatic hydrolysis sample tube twice, put it into an oven at 80℃ to dry to constant weight, and weigh it. Solid enzymatic hydrolysis rate (%) = (weight of original Sargassum powder after drying - weight of enzymatic hydrolysis precipitate after drying) / weight of original Sargassum powder after drying × 100.

[0053] Using the above optimized compound enzyme ratio and enzymatic hydrolysis reaction conditions, three enzymatic hydrolysis experiments were carried out on Sargassum powder, and the average enzymatic hydrolysis rate of Sargassum solids was 71.4%.

[0054] Example 4 Determination of Oligosaccharide Composition in Sargassum Hydrolyzate

[0055] GPC gel chromatography was used to determine the molecular weight distribution of polysaccharides in the hydrolyzate. The results showed that the polysaccharides with a molecular weight of less than 1000Da accounted for 69.57% of the hydrolyzate, and the polysaccharides with a molecular weight between 1000-2000Da accounted for 12.06%. It can be seen that more than 80% of the polysaccharides (less than 2000Da) have been deeply hydrolyzed into low-polymer oligosaccharides with higher biological activity.

[0056] Table 5 Sugar molecular weight distribution of Sargassum hydrolysate

[0057]

[0058] The oligosaccharide composition in the enzymatic hydrolysate was determined by high pressure liquid chromatography (HPLC). The oligosaccharide sample was prepared into a 10 mg / mL solution, and a SuperdexTM Peptide column G10 / 300 column was used. 0.1 M ammonium bicarbonate solution was used as the mobile phase, the flow rate was 0.4 mL / min, and the detection was performed using an ultraviolet detector (detection wavelength 235 nm). The results are shown in FIG. Figure 1 As shown, after comparison with the HPLC spectrum of brown algae oligosaccharide standard, it can be seen that the peak at 37min is disaccharide, the absorption peak at 39min is trisaccharide, and the absorption peak at 42min is tetrasaccharide. It can be seen that the main components of the enzymatic hydrolysate are oligosaccharides such as disaccharides, trisaccharides, and tetrasaccharides.

[0059] Comparative Example 1

[0060] The experimental materials and enzymatic hydrolysis steps were the same as those in Example 1, except that no alginate lyase was added to the combined enzymes. In this manner, the enzymatic hydrolysis rate of the obtained Sargassum solids was 27.90%, and the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate was 21.20%.

[0061] Comparative Example 2

[0062] The experimental materials and enzymatic hydrolysis steps were the same as those in Example 1, except that pectinase was not added to the combined enzymes. In this manner, the enzymatic hydrolysis rate of the obtained Sargassum solids was 41.8%, and the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate was 34.50%.

[0063] Comparative Example 3

[0064] The experimental materials and enzymatic hydrolysis steps were the same as those in Example 1, except that no cellulase was added to the combined enzymes. In this manner, the enzymatic hydrolysis rate of the obtained Sargassum solids was 36.90%, and the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate was 52.23%.

[0065] Comparative Example 4

[0066] The experimental materials and enzymatic hydrolysis steps are the same as those in Example 2, except that the pH value during enzymatic hydrolysis is less than 5, taking 4.5 as an example. In this way, the enzymatic hydrolysis rate of the obtained Sargassum solids is 43.56%, and the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate is 34.40%.

[0067] Comparative Example 5

[0068] The experimental materials and enzymatic hydrolysis steps are the same as those in Example 2, except that the pH during enzymatic hydrolysis is greater than 6, taking 6.5 as an example. In this way, the enzymatic hydrolysis rate of the obtained Sargassum solids is 36.30%, and the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate is 43.20%.

[0069] Comparative Example 6

[0070] The experimental materials and enzymatic hydrolysis steps are the same as those in Example 2, except that the enzymatic hydrolysis time is less than 8 hours, 7.5 hours is taken as an example. In this way, the enzymatic hydrolysis rate of the obtained Sargassum solids is 65.70%, and the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate is 69.20%.

[0071] Comparative Example 7

[0072] The experimental materials and enzymatic hydrolysis steps are the same as those in Example 2, except that the enzymatic hydrolysis time is greater than 18 hours, taking 18.5 hours as an example. In this way, the enzymatic hydrolysis rate of the obtained Sargassum solids is 71.35%, and the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate is 80.11%.

[0073] Comparative Example 8

[0074] The experimental materials and enzymatic hydrolysis steps are the same as those in Example 1, except that the amount of pectinase added in the combined enzyme is less than 3000 U / g, taking 2900 U / g as an example. In this way, the enzymatic hydrolysis rate of the obtained Sargassum solids is 48.04%, and the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate is 52.33%.

[0075] Comparative Example 9

[0076] The experimental materials and enzymatic hydrolysis steps are the same as those in Example 1, except that the amount of pectinase added in the combined enzyme is greater than 5000 U / g, taking 5100 U / g as an example. In this way, the enzymatic hydrolysis rate of the obtained Sargassum solids is 59.75%, and the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate is 73.20%.

[0077] Comparative Example 10

[0078] The experimental materials and enzymatic hydrolysis steps are the same as those in Example 1, except that no additional inactivation process is performed at the end of the enzymatic hydrolysis. In this manner, the enzymatic hydrolysis rate of the obtained Sargassum solids is 61.05%, and the proportion of highly active brown algae oligosaccharides in the enzymatic hydrolyzate is 72.50%.

Claims

1. An enzymatic hydrolysis method for efficiently preparing seaweed oligosaccharides from Sargassum, characterized in that: The ultrafine powder of Sargassum is subjected to enzymatic hydrolysis reaction using an enzyme composition composed of alginate degrading enzyme, pectinase and cellulase. The enzymatic hydrolysis rate of the obtained Sargassum solids reaches more than 70%, and the proportion of highly active oligosaccharides of brown algae in the enzymatic hydrolysis liquid reaches 80%.

2. The enzymatic hydrolysis method according to claim 1, characterized in that The addition amount of alginate degrading enzyme is 300-500U / g Sargassum powder, the addition amount of pectinase is 3000-5000U / g Sargassum powder, and the addition amount of cellulase is 240-400U / g Sargassum powder.

3. The enzymatic hydrolysis method according to claim 2, characterized in that The addition amount of alginate degrading enzyme is 500U / g Sargassum powder, the addition amount of pectinase is 5000U / g Sargassum powder, and the addition amount of cellulase is 320U / g Sargassum powder.

4. The enzymatic hydrolysis method according to claim 1, characterized in that The temperature of the enzymatic hydrolysis reaction is 45-50°C, the pH is 5-6, and the enzymatic hydrolysis time is 8-18h.

5. The enzymatic hydrolysis method according to claim 4, characterized in that The temperature of the enzymatic hydrolysis reaction was 47°C, the pH was 5.5, and the enzymatic hydrolysis time was 8 h.

6. The enzymatic hydrolysis method according to claim 1, characterized in that Highly active oligosaccharides are oligosaccharides with a molecular weight of <2000Da.

7. The enzymatic hydrolysis method according to claim 1, characterized in that Highly active brown algae oligosaccharides are mainly composed of brown algae oligosaccharides with a degree of polymerization of 2-saccharides, 3-saccharides, and 4-saccharides.

8. The enzymatic hydrolysis method according to any one of claims 1 to 7, characterized in that: The following steps are involved: The dried Sargassum superfine powder crushed to 300 mesh, alginate lyase, pectinase and cellulase are weighed and put into a centrifuge tube for mixing, a buffer solution is added, mixed, and put into a metal bath at a preset temperature for heat preservation and enzymolysis; During the enzymatic hydrolysis process, the enzymatic hydrolysis system is shaken every 20-40 minutes to allow the Sargassum powder to be fully enzymatically hydrolyzed. After the enzymatic hydrolysis is completed, the metal bath temperature is immediately adjusted to 90-100°C for 10-20 minutes to inactivate the enzyme in the reaction system, and then the system is taken out and naturally cooled to room temperature; The enzymatically hydrolyzed Sargassum solution was centrifuged at 4000-5000×g for 15-20 minutes and the separation was repeated twice.

9. The enzymatic hydrolysis method according to claim 8, characterized in that The mass ratio of the added Sargassum powder to the buffer solution is 1:

10.

10. The enzymatic hydrolysis method according to claim 8, characterized in that: The preset temperature of the metal bath is 40-60°C.

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