A kind of sargassum for complex enzyme preparation and method for preparing fucoidan by enzymolysis sargassum
By employing a three-stage stepwise enzymatic hydrolysis process and multi-enzyme synergistic technology, the problem of low degradation efficiency of Sargassum has been solved, enabling the efficient production of high-value brown algae oligosaccharides, which are applicable to fields such as pharmaceuticals, functional foods, and cosmetics.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG ACAD OF MARINE SCI (QINGDAO NAT MARINE SCI RES CENT)
- Filing Date
- 2026-04-29
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies for degrading Sargassum have problems such as low efficiency, high cost, serious pollution, wide molecular weight distribution of products, poor uniformity, and easy destruction of active structures, making it difficult to efficiently produce high-value fucoidan oligosaccharides.
A three-stage enzymatic hydrolysis process was adopted. First, alginate lyase, pectinase and cellulase were used to target and degrade the cell wall. Then, papain was used to remove the protein barrier. Finally, xylanase and mannanase were used to deeply degrade the residual hemicellulose. The enzyme system ratio was optimized to establish a cascade catalytic reaction.
It significantly improves the extraction rate and solid conversion rate of brown algae oligosaccharides, with concentrated molecular weight distribution and high bioactivity, making it suitable for high-end fields such as pharmaceuticals, functional foods, and cosmetics, and meeting the requirements of green manufacturing.
Smart Images
Figure CN122104833A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of enzyme engineering technology, and particularly relates to a compound enzyme preparation for Sargassum and a method for preparing brown algae oligosaccharides by enzymatic hydrolysis of Sargassum. Background Technology
[0002] Sargassum ( Sargassum ) belongs to the phylum Phaeophyta ( Phaeophyta ), round-shaped class ( Cyclosporeae ), Fucoidales ( Fucales Sargassum family ( Sargassaceae ), mainly distributed in warm and temperate sea areas. Among the active ingredients contained in brown algae such as Sargassum, alginate ( Alginic acid Alginate is an important class of natural high-molecular-weight polysaccharides, but its high molecular weight, high degree of polymerization, and high viscosity make it difficult to cross physiological barriers in organisms, resulting in a significant reduction in its bioavailability and severely limiting its application value as a functional active ingredient. Therefore, in order to improve the utilization value of alginate, it is usually necessary to convert it into alginic oligosaccharides with lower molecular weight and simpler structure.
[0003] To achieve the conversion of alginate into oligosaccharides, there are currently three main degradation methods: physical degradation, chemical degradation, and biodegradation. However, physical degradation suffers from low oligosaccharide release efficiency and high energy consumption; chemical degradation generates large amounts of difficult-to-treat, highly polluting wastewater, is violent and poorly controllable, resulting in a wide molecular weight distribution and poor uniformity of the product, and easily damages the natural active structure, potentially generating toxic byproducts, thus limiting high-end applications; in biodegradation, single alginate lyases suffer from incomplete degradation and poor substrate accessibility due to substrate sequence specificity and the multi-component physical barrier of Sargassum cell walls, resulting in a solid conversion rate of less than 30%; commercial compound enzymes suffer from high costs and low yields of the target product, oligosaccharides; and multi-enzyme synergistic technology in enzymatic processes still faces significant bottlenecks.
[0004] Therefore, there is an urgent need to develop a novel compound enzyme preparation or process that can overcome the above-mentioned defects. Summary of the Invention
[0005] This invention addresses the technical problems mentioned above, such as the low degradation efficiency of Sargassum using single enzyme preparations and the significant incompatibility of existing commercial compound enzyme preparations when directly applied to Sargassum degradation. It proposes a method for preparing brown algal oligosaccharides from Sargassum using a compound enzyme preparation and enzymatic hydrolysis of Sargassum. This method can target the degradation of polysaccharide components such as cellulose and pectin in the cell wall of Sargassum, and produce high-value oligosaccharides with a degree of polymerization of 2 to 8 in a targeted and efficient manner without reducing the total solids conversion efficiency.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for preparing brown algal oligosaccharides by enzymatic hydrolysis of Sargassum fusiforme, comprising the following steps: Under the conditions of a material-to-liquid ratio of 1:39.5, pH of 6.0-7.0, and enzymatic hydrolysis temperature of 44-46℃, alginate lyase, pectinase, and cellulose were added to the reaction system, and the first enzymatic hydrolysis reaction was carried out for 11-13 hours. Adjust the pH to 5.5-6.0, add papain to the reaction system, and carry out a second enzymatic hydrolysis reaction for 6-8 hours at an enzymatic hydrolysis temperature of 44-46℃. Then add xylanase and mannanase to the reaction system, raise the temperature to 49~51℃, and carry out the third enzymatic hydrolysis reaction for 12~16h.
[0007] In one embodiment, in the step of adding xylanase and mannanase to the reaction system, xylanase and mannanase are added when the moisture content of the algal residue is ≤30%.
[0008] In one embodiment, the extraction rate of fucoidan oligosaccharides was 14.60-15.74%, and the solid conversion rate was 61.23-62.79%.
[0009] In one embodiment, the degree of polymerization of the fucoidan oligosaccharide is 2 to 8.
[0010] In another aspect, the present invention provides a complex enzyme preparation for Sargassum, applied to the method of preparing brown algae oligosaccharides by enzymatic hydrolysis of Sargassum. The complex enzyme preparation for Sargassum includes alginate lyase, pectinase, cellulase, papain, xylanase, and mannanase. Based on the dry weight of Sargassum, the amount of alginate lyase added is 0.2-0.5%, the amount of pectinase added is 1-1.5%, the amount of cellulase added is 1.5-2%, the amount of papain added is 0.5-1%, the amount of xylanase added is 1-2%, and the amount of mannanase added is 0.5-1.5%.
[0011] In one embodiment, based on the dry weight of Sargassum, the amount of alginate lyase added was 0.28%, pectinase added was 1.18%, cellulose added was 1.645%, papain added was 0.815%, xylanase added was 1.5%, and mannanase added was 1%.
[0012] In one embodiment, the enzyme activity ratio of alginate lyase (total enzyme activity of pectinase and cellulase):papain (total enzyme activity of xylanase and mannanase) is 1:(4.0-4.5):(5.5-6.0):(0.8-1.2).
[0013] In one embodiment, the alginate lyase was isolated from Shewanella. Shewanella sp. HD5 The original alginate lyase gene was obtained through heterologous expression in Shewanella. Shewanella sp. HD5 It was deposited on September 22, 2025, at the China Center for Type Culture Collection, Wuhan, Hubei Province, with accession number CCTCC NO: M20252078.
[0014] In one embodiment, the nucleotide sequence of the gene encoding alginate lyase is shown in SEQ ID NO: 1.
[0015] In one embodiment, the optimal pH for the alginate lyase is 6.0; the half-life at 45°C is ≥8 h; and it can be soluble in Al 3+ Activated, with relative enzyme activity ≥300%.
[0016] Compared with the prior art, the advantages and positive effects of the present invention are as follows: (1) The enzymatic hydrolysis method of the present invention solves the technical bottleneck of conflicting optimal reaction conditions when multiple enzymes are combined by controlling the reaction conditions step by step, and finally achieves an oligosaccharide extraction rate of 14.60~15.74%. Compared with the existing commercial compound enzyme preparations (9.68%), the extraction efficiency is increased by more than 60%, and a solid conversion rate of 61.23~62.79% is achieved. Sargassum is highly utilized, and the resulting oligosaccharide products have a more concentrated molecular weight distribution (DP=2~8), a clearer structure, higher biological activity and better consistency. It has a broader application prospect in high-end fields such as medicine, functional food and cosmetics. The enzymatic hydrolysis method of the present invention has mild reaction conditions, no strong acid or strong alkali is used, and no chemical pollution is generated, which meets the requirements of green manufacturing and sustainable development. This invention employs a three-step enzymatic hydrolysis process targeting the complex structure of Sargassum cell walls. The first step involves the synergistic action of alginate lyase, cellulase, and pectinase to target and degrade the three core structural components of the cell wall. The second step utilizes papain to remove protein barriers and deeply expose the substrate. The third step uses xylanase and mannanase to degrade residual hemicellulose. By overcoming the substrate limitations of single enzymes through the synergistic effect of multiple enzymes, this invention significantly improves the degradation efficiency of Sargassum and enables precise control over the composition of the products.
[0017] (2) The Sargassum compound enzyme preparation of the present invention establishes a cascade catalytic reaction by optimizing the enzyme system ratio, which can target and degrade polysaccharide components such as cellulose and pectin in the cell wall of Sargassum, eliminate steric hindrance, generate more diverse functional oligosaccharide fragments, improve the oligosaccharide extraction rate and solid enzymatic hydrolysis rate, and obtain specific degree of polymerization active oligosaccharides, providing high-quality raw materials for the development of functional foods and drug carriers. Attached Figure Description
[0018] Figure 1The image shown is an SDS-PAGE electrophoresis diagram of the alginate lyase provided in Example 1 of this invention. M represents the protein marker, 1 represents the recombinant strain before induction, 2 represents the crude enzyme solution after induction, 3 represents the protein purification flow-through solution, and 4 represents the purified product. Figure 2 This is an HPLC analysis chromatogram of the enzymatic hydrolysis product in Example 1 of the present invention; Figure 3 This is an HPLC analysis chromatogram of the enzymatic hydrolysis product in Example 2 of the present invention; Figure 4 This is an HPLC analysis chromatogram of the enzymatic hydrolysis product in Example 3 of the present invention; Figure 5 This is an HPLC analysis chromatogram of the enzymatic hydrolysis product in Example 4 of the present invention. Detailed Implementation
[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0020] This invention provides a compound enzyme preparation for Sargassum and a method for preparing brown algae oligosaccharides by enzymatic hydrolysis of Sargassum, thereby improving the extraction rate and solid enzymatic hydrolysis rate of Sargassum oligosaccharides. This invention first screens Shewanella bacteria from kelp. Shewanella sp. HD5 The alginate lyase gene of Sargassum fusiforme was identified, and a recombinant expression vector was constructed and transformed into Pichia pastoris GS115 for heterologous expression. The yield of soluble protein was increased by optimizing induction conditions, and high-purity recombinant alginate lyase was obtained using affinity chromatography. This recombinant enzyme was then combined with pectinase, cellulase, papain, xylanase, and mannanase in a specific ratio to obtain a complex enzyme preparation. Optimized enzymatic hydrolysis conditions further improved the degradation efficiency of alginate and the yield of target oligosaccharides. This invention provides a three-stage stepwise enzymatic hydrolysis process for extracting alginate oligosaccharides from Sargassum fusiforme. This process can target and degrade polysaccharide components such as cellulose and pectin in the cell wall of Sargassum fusiforme, producing high-value oligosaccharides in a directed and efficient manner without reducing the total solids conversion efficiency. The degree of polymerization (DP) of the enzymatic hydrolysis product, alginate oligosaccharide, is between 2 and 8.
[0021] The present invention provides a method for preparing brown algal oligosaccharides from Sargassum using a specific compound enzyme preparation, comprising the following steps: Under the initial reaction conditions of a material-to-liquid ratio of 1:39.5, a pH of 6.0-7.0, and an enzymatic hydrolysis temperature of 44-46℃, 0.2-0.5% alginate lyase, 1-1.5% pectinase, and 1.5-2% cellulase were simultaneously added to the Sargassum substrate, and the enzymatic hydrolysis reaction was carried out for 11-13 hours. Adjust the pH to 5.5-6.0, add 0.5-1% papain to the reaction system, and continue enzymatic hydrolysis for 6-8 hours; Add 1-2% xylanase and 0.5-1.5% mannanase to the reaction system, adjust the reaction temperature to 49-51℃, and continue the enzymatic hydrolysis reaction for 12-16 hours.
[0022] This invention optimizes a three-step enzymatic hydrolysis process, resolving the conflict of optimal conditions among multiple enzymes. This allows each enzyme to function at its best, resulting in a synergistic effect. The three-step process involves two main steps: the first step utilizes the synergistic effect of the three enzymes to break down the main structure of the Sargassum cell wall, creating favorable conditions for subsequent hydrolysis; the second step removes the protein barrier and deeply exposes the substrate, fully utilizing the protease activity of papain to hydrolyze protein components intertwined with polysaccharides in the cell wall and intracellular storage proteins, further removing structural barriers and deeply exposing encapsulated soluble polysaccharides and recalcitrant components, thus improving the overall accessibility of the substrate. Through the first two enzymatic hydrolysis steps, the yield of brown algae oligosaccharides can reach 11.21%, and the solid conversion rate can reach 39.7%. The third enzymatic hydrolysis can achieve deep degradation and transformation of the hemicellulose components remaining from the previous enzymatic hydrolysis and optimize the final product. It can also refine the algal residue remaining after the previous enzymatic hydrolysis, specifically degrade the remaining hemicellulose components such as hydrolysates and mannans, thereby maximizing the extraction efficiency of brown algae oligosaccharides and maximizing the utilization of biomass. The final extraction rate of brown algae oligosaccharides can reach 15.74%, and the solid enzymatic hydrolysis rate can reach 62.79%, which is better than traditional commercial compound enzyme preparations. This shows that the enzymatic hydrolysis method of this invention achieves efficient and thorough degradation and transformation of Sargassum biomass.
[0023] This invention also provides a specific compound enzyme preparation for Sargassum, applied to the method of enzymatically hydrolyzing Sargassum to prepare brown alginic oligosaccharides. The Sargassum compound enzyme preparation includes alginate lyase, pectinase, cellulase, papain, xylanase, and mannanase. Based on the dry weight of Sargassum, the alginate lyase content is 0.2-0.5%, preferably 0.28%; the pectinase content is 1-1.5%, preferably 1.18%; the cellulase content is 1.5-2%, preferably 1.645%; the papain content is 0.5-1%, preferably 0.815%; the xylanase content is 1-2%, preferably 1.5%; and the mannanase content is 0.5-1.5%, preferably 1%.
[0024] To more clearly and in detail introduce the complex enzyme preparation for Sargassum and the method for preparing brown algae oligosaccharides by enzymatic hydrolysis of Sargassum provided in the embodiments of the present invention, the following description will be based on specific embodiments.
[0025] Example 1: Recombinant alginate lyase gene sequence, recombinant expression, and recombinase preparation. 1. Strains and Gene Sources The original alginate lyase gene was screened from Shewanella bacteria derived from kelp. Shewanella sp. HD5 The strain is deposited at the China Center for Type Culture Collection, Wuhan, Hubei Province, with accession number CCTCC NO: 20252078. Shewanella Shewanella sp. HD5 The samples were collected in April 2024 in Yantai City, Shandong Province, China. The amino acid sequence of the original alginate lyase is shown in SEQ ID NO: 2. To improve the heterologous expression efficiency of the original alginate lyase gene in the Pichia pastoris GS115 system, its full-length nucleotide sequence was codon-optimized to make the optimized gene sequence more suitable for the Pichia pastoris GS115 expression host, laying the foundation for efficient heterologous expression.
[0026] 2. Construction of recombinant expression vectors The expression vector used was pPIC9K, with EcoRI / NotI restriction sites, and a 6*His tag was introduced downstream of the target protein. The amino acid sequence of alginate lyase after removing the signal peptide is shown in SEQ ID NO: 3. Based on this sequence, a codon-optimized nucleotide sequence was designed and synthesized, as shown in SEQ ID NO: 4. This optimized sequence was inserted into the expression vector pPIC9K via EcoRI and NotI restriction sites, and a 6*His tag was added to the C-terminus of the target protein to construct a recombinant plasmid capable of inducible expression.
[0027] 3. Construction of recombinant engineered bacteria Pichia pastoris GS115 culture, cultured to mid-log phase, was incubated on ice for 30 min, centrifuged at 4000×g for 5 min at 4℃, and the supernatant was discarded. The cells were gently resuspended in an equal volume of pre-cooled sterile ddH2O, and this process was repeated once. The cells were then resuspended in 1 mol / L sorbitol, centrifuged at 4000×g for 5 min at 4℃, and the supernatant was discarded. The cells were then gently resuspended in 1 mL of pre-cooled 1 mol / L sorbitol to obtain Pichia pastoris GS115 competent cells. 80 μL of competent cells were taken, and 5 μg of linearized recombinant plasmid was added. After mixing, the cells were incubated on ice for 5 min for electroporation. The electroporated solution was then plated on SD-HIS screening plates to screen for recombinant engineered bacteria.
[0028] 4. Induced expression of recombinase The recombinant engineered bacteria were inoculated into BMGY liquid medium and cultured overnight at 30°C and 250 rpm with shaking. The overnight culture was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were retained. The bacterial cells were resuspended in BMMY liquid medium and the OD of BMMY liquid medium was adjusted to 1.0. 1% methanol was added and expression was induced at 30°C and 250 rpm. 1% methanol was added every 24 h for a total of 3 times.
[0029] 5. Protein Sample Preparation After the induction expression was completed, the fermentation broth was centrifuged at 5000 r / min for 5 min, the precipitate was discarded, and the supernatant was retained as the crude enzyme solution for protein purification.
[0030] 6. Ni-NTA affinity chromatography purification The crude enzyme solution was filtered through a 0.22 μm filter membrane to remove insoluble impurities and ensure column patency. Affinity chromatography purification was performed using a 1 mL HisTrap pre-packed Ni-NTA column. Before loading, the column was fully equilibrated with binding buffer (20 mM Tris, 0.5 M NaCl, 0 mM imidazole, pH 8.0). The filtered crude enzyme solution was loaded onto the equilibrated column. After loading, the column was washed with 3-5 column volumes of binding buffer to remove unbound contaminants. Elution was performed using a stepwise imidazole concentration gradient, sequentially eluting with 20 mL each of elution buffers containing 50 mM, 100 mM, and 500 mM imidazole (20 mM Tris, 0.5 M NaCl, pH 8.0), and the eluted fractions at each stage were collected.
[0031] 7. Purity and enzyme activity identification The eluted fractions were analyzed by SDS-PAGE electrophoresis to identify the purification effect and purity of the target protein. The results are attached. Figure 1 As shown. By Figure 1 As can be seen, the recombinase purification product was identified by SDS-PAGE electrophoresis. The results showed that a single clear protein band was visible in the 48kDa~65kDa range. This band was band 4, and its position was basically consistent with the theoretical molecular weight of UMI-01, 58.1kDa. Meanwhile, no obvious protein band was seen in the protein flow-through solution corresponding to band 3, indicating that the target protein was fully adsorbed with the purification medium and the purification process was relatively complete.
[0032] The specific enzyme activity of the recombinant engineered bacteria constructed in this embodiment reached 88.3 U / mL after induced fermentation, as determined by the DNS method, indicating that the recombinant system has extremely high expression efficiency. After one-step purification by Ni-NTA affinity chromatography, the specific activity of the recombinant alginate lyase obtained was as high as 562.42 U / mg.
[0033] The DNS method includes: (1) plotting protein standard curves. The protein standard curve was generated using the BCA Protein Assay Kit (Enhanced Version). The BCA working solution was prepared fresh for each use. Using a 2 mg / mL protein standard as the stock solution, the solution was diluted with Elution-Buffer to prepare a series of protein standard solutions (using bovine serum albumin BSA as the protein standard) at concentrations of 0 mg / mL, 0.04 mg / mL, 0.20 mg / mL, 0.40 mg / mL, 0.60 mg / mL, 1.00 mg / mL, and 1.20 mg / mL. The reaction system for the BSA standard curve is shown in Table 1.
[0034] Table 1. BSA Standard Curve Reaction System
[0035] According to the reaction system in Table 1, shake and mix for 30 seconds, cover and incubate at 37℃ for 30 minutes. Then, measure the absorbance at 562 nm using a microplate reader. Three parallel experiments were set up for each treatment, and the average value was taken. Plot the standard BSA concentration on the x-axis and the absolute absorbance at 562 nm (absolute OD). 562 =Experimental Group OD 562-average - Blank group OD 562-average Plot a standard curve with y as the ordinate.
[0036] (2) Determination and calculation of enzyme protein content Add 25µL of enzyme solution sample and 200µL of BCA working solution to each well, follow the procedure in step (1), cool to room temperature, and measure the absorbance at 562nm. Use Elution-Buffer as a blank control. Repeat each experimental group three times.
[0037] Enzyme protein content = protein concentration × enzyme solution dilution factor × enzyme solution volume In the formula, the enzyme protein content is in mg; the protein concentration is calculated from the standard curve and is in mg / mL; and the enzyme solution volume is in mL.
[0038] (2) Determination of specific enzyme activity of alginate lyase Under the above experimental conditions, the ratio of the measured alginate lyase activity to the enzyme protein content is the specific enzyme activity (U·mg) of the alginate lyase. -1 The specific calculation formula is as follows: Enzyme activity = (Enzyme activity × Enzyme solution volume) / Enzyme protein content In the formula, enzyme activity is expressed in U·mg. -1 Enzyme solution volume, in mL; enzyme protein content, in mg.
[0039] This embodiment achieves efficient soluble heterologous expression of alginate lyase in Pichia pastoris GS115 through codon optimization, dual-tag design, low-temperature induction, and one-step Ni-NTA affinity chromatography. The preparation process is simple, efficient, and reproducible, making it suitable for large-scale production.
[0040] Figure 2 The HPLC chromatogram of the enzymatic hydrolysis products obtained in Example 1 (DP2~8 accounting for ≥65%) is shown below. Figure 2 It can be seen that the degree of polymerization of the enzymatic hydrolysis product is 2~8, which is consistent with the target product.
[0041] Example 2 The process for extracting brown algal oligosaccharides from Sargassum fusiforme in this embodiment includes the following steps: S1. Raw material pretreatment: Take dried Sargassum, crush it and pass it through an 80-mesh sieve to obtain Sargassum powder, and store it in a sealed container in a 4℃ refrigerator for later use. S2, Enzymatic hydrolysis step: S21. Accurately weigh 1.0g of the above Sargassum powder (on dry weight), place it in a 250mL Erlenmeyer flask, add 40mL of ultrapure water, adjust the pH to 7.0, and then add alginate lyase, pectinase and cellulase. The amount of alginate lyase added is 0.28% of the mass of Sargassum powder, the amount of pectinase added is 1.18% of the mass of Sargassum powder, and the amount of cellulase added is 1.65% of the mass of Sargassum powder. Then place the Erlenmeyer flask in a 45℃ constant temperature water bath shaker and shake at 150rpm for 12 hours. S22. After the reaction in step S21 is completed, adjust the pH to 5.5, add 0.82% papain by mass to the reaction system, and continue to react with shaking at 45℃ and 150 rpm for 12 hours. After sampling and determination, the oligosaccharide yield is 10.38% and the solid conversion rate is 39.7%. S23. After the reaction in step S22 is completed, xylanase and mannanase are added to the reaction system. The amount of xylanase added is 1.5% of the mass of Sargassum powder, and the amount of mannanase added is 1.5% of the mass of Sargassum powder. The system temperature is adjusted to 50℃, and the reaction is shaken at 150 rpm for 12 hours. S3. Termination of Reaction and Determination of Results: After the reaction in step S23 is completed, the reaction solution is heated in a boiling water bath for 10 minutes to terminate the enzyme reaction. After cooling, it is centrifuged at 4000 rpm for 15 minutes. The supernatant is collected, and the oligosaccharide extraction rate is determined to be 15.74%. The HPLC analysis chromatogram of the enzymatic hydrolysis products is attached. Figure 3 As shown, the degree of polymerization (DP) of the enzymatic hydrolysis product, alginate oligosaccharide, is between 2 and 6; after drying, the solid enzymatic hydrolysis rate (solid conversion rate) was calculated to be 62.79%.
[0042] Example 3 The process for extracting brown algal oligosaccharides from Sargassum fusiforme in this embodiment includes the following steps: S1. Raw material pretreatment: Take dried Sargassum, crush it and pass it through an 80-mesh sieve to obtain Sargassum powder, and store it in a sealed container in a 4℃ refrigerator for later use. S2, Enzymatic hydrolysis step: S21. Accurately weigh 1.0g of the above Sargassum powder (on a dry weight basis), place it in a 250mL Erlenmeyer flask, add 40mL of ultrapure water, and then add alginate lyase, pectinase and cellulase. The amount of alginate lyase added is 0.38% of the mass of Sargassum powder, the amount of pectinase added is 1.26% of the mass of Sargassum powder, and the amount of cellulase added is 1.79% of the mass of Sargassum powder. Adjust the pH of the reaction system to 5.5, and then place the Erlenmeyer flask in a 45℃ constant temperature water bath shaker and shake at 150rpm for 12 hours. S22. After the reaction in step S21 is completed, 0.920% papain by mass is added directly to the reaction system. After shaking for 12 hours at 45℃ and 150 rpm, samples are taken for analysis. The oligosaccharide yield is 9.46% and the solid conversion rate is 38.57%. S23. After the reaction in step S22 is completed, xylanase and mannanase are added to the reaction system. The amount of xylanase added is 1.5% of the mass of Sargassum powder, and the amount of mannanase added is 1% of the mass of Sargassum powder. The system temperature is adjusted to 40℃, and the reaction is carried out by shaking at 150 rpm for 18 hours. S3. Termination of Reaction and Result Determination: After the reaction in step S23 is completed, the reaction solution is heated in a boiling water bath for 10 minutes to terminate the enzyme reaction. After cooling, it is centrifuged at 4000 rpm for 15 minutes. The supernatant is collected, and the oligosaccharide extraction rate is determined to be 14.60%. The HPLC analysis chromatogram of the enzymatic hydrolysis product is attached. Figure 4 As shown, the degree of polymerization (DP) of the enzymatic hydrolysis product, alginate oligosaccharide, is between 2 and 7; after drying, the solid enzymatic hydrolysis rate (solid conversion rate) was calculated to be 61.23%.
[0043] Example 4 The process for extracting brown algal oligosaccharides from Sargassum fusiforme in this embodiment includes the following steps: S1. Raw material pretreatment: Take dried Sargassum, crush it and pass it through an 80-mesh sieve to obtain Sargassum powder, and store it in a sealed container in a 4℃ refrigerator for later use. S2, Enzymatic hydrolysis step: S21. Accurately weigh 1.0g of the above Sargassum powder (on dry weight), place it in a 250mL Erlenmeyer flask, add 40mL of ultrapure water, and then add alginate lyase, pectinase and cellulase. The amount of alginate lyase added is 0.180% of the mass of Sargassum powder, the amount of pectinase added is 1.50% of the mass of Sargassum powder, and the amount of cellulase added is 1.885% of the mass of Sargassum powder. Then place the Erlenmeyer flask in a 45℃ constant temperature water bath shaker and shake at 150rpm for 12 hours. S22. After the reaction in step S21 is completed, 0.714% papain by mass is added directly to the reaction system. After shaking for 12 hours at 45℃ and 150 rpm, samples are taken for analysis. The oligosaccharide yield is 11.21%, and the solid conversion rate is 38.89%. S23. After the reaction in step S22 is completed, xylanase and mannanase are added to the reaction system. The amount of xylanase added is 1.5% of the mass of Sargassum powder, and the amount of mannanase added is 0.5% of the mass of Sargassum powder. The system temperature is adjusted to 50℃, and the reaction is shaken at 150 rpm for 15 hours. S3. Termination of Reaction and Determination of Results: After the reaction in step S23 is completed, the reaction solution is heated in a boiling water bath for 10 minutes to terminate the enzyme reaction. After cooling, it is centrifuged at 4000 rpm for 15 minutes. The supernatant is collected, and the oligosaccharide extraction rate is determined to be 15.13%. The HPLC analysis chromatogram of the enzymatic hydrolysis products is attached. Figure 5 As shown, the degree of polymerization (DP) of the enzymatic hydrolysis product, alginate oligosaccharide, is between 2 and 6; after drying, the solid enzymatic hydrolysis rate (solid conversion rate) was calculated to be 61.84%.
[0044] Comparative Example 1 In this comparative example, 6.43% of a commercial compound enzyme preparation was added to 1.0g of Sargassum powder for single-stage enzymatic hydrolysis. The hydrolysis conditions were: material-to-liquid ratio 1:39.5, pH adjusted to 6.8, temperature 45℃, and hydrolysis time 12h. The extraction rate of brown algae oligosaccharides was 9.68%, and the solid enzymatic hydrolysis rate was 42.57%.
[0045] As shown above, compared with Comparative Example 1 which used a commercial compound enzyme preparation for single-stage hydrolysis, Examples 2-4, using the compound enzyme preparation of the present invention, achieved a maximum increase of 62.6% in the extraction rate of brown algae oligosaccharides through a three-stage stepwise enzymatic hydrolysis process, indicating that the compound enzyme preparation of the present invention has a synergistic effect. Examples 2-4, through the synergistic effect of multiple enzymes, can overcome the substrate limitations of single enzymes, significantly improve the degradation efficiency of Sargassum fusiforme, and achieve precise control over the composition of the products. By optimizing the enzyme system ratio to establish a cascade catalytic reaction, the three-stage stepwise enzymatic hydrolysis process is not a simple superposition of steps. The first stage mainly breaks down the main structure of the cell wall, the second stage enzymatically hydrolyzes the protein barrier, and the third stage deeply converts the hemicellulose component, improving the substrate conversion rate, targeting the degradation of polysaccharide components such as cellulose and pectin in the cell wall of Sargassum fusiforme, eliminating steric hindrance, and producing more structurally diverse functional oligosaccharide fragments, further improving the extraction rate and solid enzymatic hydrolysis rate of brown algae oligosaccharides. The obtained specific degree of polymerization active oligosaccharides provide high-quality raw materials for the development of functional foods and drug carriers.
[0046] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, evolutions, or improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.
Claims
1. A method for preparing brown alginic oligosaccharides by enzymatic hydrolysis of Sargassum, characterized in that, Includes the following steps: Under the conditions of a material-to-liquid ratio of 1:39.5, pH of 6.0-7.0, and enzymatic hydrolysis temperature of 44-46℃, alginate lyase, pectinase, and cellulose were added to the reaction system, and the first enzymatic hydrolysis reaction was carried out for 11-13 hours. Adjust the pH to 5.5-6.0, add papain to the reaction system, and carry out a second enzymatic hydrolysis reaction for 6-8 hours at an enzymatic hydrolysis temperature of 44-46℃. Then add xylanase and mannanase to the reaction system, raise the temperature to 49~51℃, and carry out the third enzymatic hydrolysis reaction for 12~16h.
2. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis of Sargassum according to claim 1, characterized in that, In the step of adding xylanase and mannanase to the reaction system, the xylanase and mannanase are added when the moisture content of the algal residue is ≤30%.
3. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis of Sargassum according to claim 1, characterized in that, The extraction rate of the fucoidan oligosaccharide was 14.60-15.74%, and the solid conversion rate was 61.23-62.79%.
4. The method for preparing brown algae oligosaccharides by enzymatic hydrolysis of Sargassum according to claim 1, characterized in that, The degree of polymerization of the brown algae oligosaccharide is 2 to 8.
5. A compound enzyme preparation for Sargassum, applied to the method for preparing brown algae oligosaccharides by enzymatic hydrolysis of Sargassum according to any one of claims 1-4, characterized in that, The Sargassum compound enzyme preparation includes alginate lyase, pectinase, cellulase, papain, xylanase, and mannanase. Based on the dry weight of Sargassum, the amount of alginate lyase added is 0.2-0.5%, the amount of pectinase added is 1-1.5%, the amount of cellulase added is 1.5-2%, the amount of papain added is 0.5-1%, the amount of xylanase added is 1-2%, and the amount of mannanase added is 0.5-1.5%.
6. The compound enzyme preparation for Sargassum according to claim 5, characterized in that, Based on the dry weight of Sargassum, the amount of alginate lyase added is 0.28%, the amount of pectinase added is 1.18%, the amount of cellulose added is 1.65%, the amount of papain added is 0.82%, the amount of xylanase added is 1.5%, and the amount of mannanase added is 1%.
7. The compound enzyme preparation for Sargassum according to claim 5, characterized in that, Based on enzyme activity, the ratio of alginate lyase (total enzyme activity of pectinase and cellulase) to papain (total enzyme activity of xylanase and mannanase) is 1:(4.0-4.5):(5.5-6.0):(0.8-1.2).
8. The Sargassum compound enzyme preparation according to claim 5, characterized in that, The alginate lyase was isolated from Shewanella. Shewanella sp. HD5 The original alginate lyase gene of *Shewanella* was obtained through heterologous expression. Shewanella sp. HD5 It was deposited on September 22, 2025, at the China Center for Type Culture Collection, Wuhan, Hubei Province, with accession number CCTCC NO: M20252078.
9. The compound enzyme preparation for Sargassum according to claim 8, characterized in that, The nucleotide sequence of the gene encoding the alginate lyase is shown in SEQ ID NO:
1.
10. The compound enzyme preparation for Sargassum according to claim 8, characterized in that, The optimal pH for the alginate lyase is 6.0; the half-life at 45°C is ≥8 hours; it can be lysed by Al 3+ Activated, with relative enzyme activity ≥300%.