A method for extracting fucoidan from undaria pinnatifida

Abstract

The application belongs to the field of polysaccharide preparation technology, and discloses a method for extracting fucoidan from Undaria pinnatifida as raw material, which comprises the following steps: adding Undaria pinnatifida dry powder into ultrapure water, pre-treating by ultrasonic method, extracting fucoidan by using multiple enzymes, secondary extracting of residue, and obtaining fucoidan crude product by processing. The application uses ultrasonic pre-treatment, multiple enzyme combination assisted extraction and secondary processing of residue to improve the extraction rate of fucoidan, and obtains fucoidan by membrane separation. The method of the application not only greatly improves the extraction rate of fucoidan from Undaria pinnatifida raw material, but also prevents excessive use of organic reagents in the extraction process from polluting polysaccharide.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide preparation technology, and relates to a method for extracting fucoidan from wakame seaweed. Background Technology

[0002] Over the past 20 years, the scale of seaweed farming and production has increased significantly, playing a vital role in fisheries worldwide. According to the 2019 China Fisheries Yearbook, my country's mariculture production of wakame seaweed reached 175,000 tons in 2018, with Liaoning province ranking first nationally with 116,000 tons. One of the most important bioactive components of wakame is fucoidan. In 2017, the U.S. Food and Drug Administration approved fucoidan extracted from wakame seaweed as a Generally Recognized As Safe (GRAS) novel food and food supplement.

[0003] Research on the bioactivity and functional applications of fucoidan has attracted widespread attention. Studies have shown that fucoidan possesses a variety of bioactivities, including excellent antioxidant, antiviral, antitumor, anti-inflammatory, and antithrombotic effects, with minimal side effects. It is a new generation of functional raw materials for the food, pharmaceutical, and cosmetic fields. The chemical composition and structure of fucoidan are highly variable. Factors such as the type of brown algae, growth season, algal part, and extraction method all influence the structure and composition of fucoidan, including sulfate content, molecular weight, and monosaccharide composition and ratio. Currently, the main extraction methods for fucoidan include water extraction, acid extraction, enzyme-assisted extraction, and ultrasound-assisted extraction. Too mild extraction conditions will result in a low extraction rate of fucoidan. However, excessively harsh extraction conditions will lead to the decomposition of fucoidan, affecting its structure and composition.

[0004] In recent years, the extraction and purification processes of fucoidan have attracted much attention. Patent CN110590970B (publication date: October 15, 2021) describes a method for preparing fucoidan and sodium alginate-rich brown algae polysaccharide and its application, but the resulting polysaccharide is not purified and has an overly complex composition. Patent CN109851687B (publication date: June 22, 2021) describes a method for separating and preparing fucoidan and alginate from kelp, but the process is cumbersome and the polysaccharide extraction rate is low. Summary of the Invention

[0005] The present invention aims to provide a method for extracting fucoidan from wakame seaweed.

[0006] The technical solution of this invention: A method for extracting fucoidan from wakame seaweed, comprising the following steps: (a) Wash and dry wakame seaweed, grind it in a pulverizer and pass it through a 60-mesh sieve to obtain wakame seaweed powder; add ultrapure water at a material-to-liquid ratio of 1:20~1:60 g / ml, stir thoroughly, and then pre-treat it with ultrasound at a frequency of 20~60 kHz and a maximum output power of 400~1200 W to obtain an aqueous solution of wakame seaweed powder. (b) Slowly add dilute hydrochloric acid solution to the aqueous solution of dried wakame powder to adjust the pH to 3-7, and add 0.5%-2.0% (by weight) of cellulase, pectinase, amylase and papain of dried wakame powder respectively; control the first extraction temperature at 30℃-70℃ and stir at 200 r / min for 2-6 hours. (c) After the extraction is completed and the system has cooled to room temperature, filter the first extract and the first residue using a centrifuge at 2000 rpm. Add ultrapure water to the first residue according to the same material-to-liquid ratio as the first extraction for a second extraction. Control the temperature of the second extraction at 30℃~70℃ and stir at 200 r / min for 2~6 hours to obtain the second extract and the second residue. Discard the second residue. Combine the extracts obtained from the two extractions and inactivate the enzymes at high temperature for 5~40 min. Adjust the pH with sodium bicarbonate. (d) Add anhydrous calcium chloride to the extract, precipitate for 8-12 hours, centrifuge and take the supernatant to remove 70% water by rotary evaporation, then separate the extract through a hollow fiber membrane to obtain fucoidan extract, add anhydrous ethanol to the fucoidan extract and let it stand to precipitate fucoidan, centrifuge and filter, and freeze dry to obtain fucoidan.

[0007] In step (a), the ratio of extract to liquid is 1:30 to 1:40 g / ml, and the extract is ultrasonically treated for 20 to 45 minutes at a frequency of 30 kHz and a maximum output power of 950 W.

[0008] In step (b), at the beginning of extraction, 1.2-1.8% of cellulase and 1.2-1.8% of pectinase based on the weight of the dried wakame powder used for extraction are added respectively. After 2 hours of extraction, 1.0-1.5% of amylase and 1.0-1.5% of papain based on the weight of the dried wakame powder used for extraction are added respectively. The pH value is 4.5-5.0, the extraction temperature is 50-55℃, and the total extraction time is 4-5 hours.

[0009] In step (c), no additional cellulase, pectinase, amylase, or papain are added during the secondary extraction process. The extraction temperature is 50–55℃, the extraction time is 2–3 hours, the enzymes are inactivated at a high temperature of 80–100℃ for 10 minutes, and the pH value is 4.5–5.0.

[0010] In step (d), add 6-8% anhydrous calcium chloride equivalent to the mass of the dried wakame powder used for extraction, stir thoroughly and precipitate for 12 hours, and use hollow fiber membrane with specifications of 10-25 kDa; add anhydrous ethanol to the fucoidan extract until the ethanol volume fraction reaches more than 70%, and let it stand for precipitation for 12 hours.

[0011] The beneficial effects of this invention are as follows: By combining ultrasonic pretreatment with compound enzymatic hydrolysis and secondary extraction, the polysaccharides from *Wakame seaweed* are released to the maximum extent, thus improving the extraction rate of fucoidan. This reduces the number of extraction steps for fucoidan, minimizes component loss, and increases the yield. The invention focuses on comparing the dosage and addition time of various enzymes during the extraction process, obtaining the enzyme combination conditions that yield the highest polysaccharide extraction rate. The fucoidan extract obtained through enzymatic hydrolysis degrades macromolecules such as cellulose, starch, and protein obtained during the extraction process, simplifying subsequent polysaccharide purification operations. Attached Figure Description

[0012] Figure 1 This is the effect of cellulase dosage on fucoidan extraction rate in Example 1 of this application; Figure 2 This is the effect of pectinase dosage on fucoidan extraction rate in Example 1 of this application; Figure 3 This is the effect of amylase dosage on the extraction rate of fucoidan in Example 1 of this application; Figure 4 Example 2 of this application illustrates the effect of pH on the extraction rate of fucoidan. Figure 5 This describes the effect of extraction time on the extraction rate of fucoidan in Example 3 of this application. Figure 6 This is a molecular weight distribution diagram of fucoidan from Example 4 of this application; the red curve represents the molecular weight distribution, and the blue curve represents the cumulative percentage. Figure 7 This is a gel chromatography curve of fucoidan from Example 4 of this application; the red curve represents the integration range, and the blue curve represents the elution curve. Figure 8 This is the effect of the combined enzyme process on the extraction rate of fucoidan in Comparative Example 1 of this application. Detailed Implementation

[0013] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0014] Example 1 The effect of cellulase dosage on fucoidan yield was investigated, including the following steps: Grinding: After washing and drying the Dalian wakame seaweed, grind it through a 60-mesh sieve to obtain dried wakame seaweed powder; Ultrasonic pretreatment: Weigh four 10 g portions of kelp powder and add 400 m of ultrapure water as a solvent. Ultrasonic treatment is carried out for 30 min at a frequency of 30 kHz and a maximum output power of 950 W. Enzyme-assisted method: 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% (by weight of dried wakame powder) of cellulase and 1.0% of pectinase were added to the extract as a control experiment. The pH of the extract was adjusted to 5 with dilute hydrochloric acid. After two hours of extraction, 1.5% of papain and amylase were added. The extract was then subjected to a 50°C hot water bath for 4 hours. The solid and liquid were separated by high-speed centrifugation to obtain the supernatant. The dried residue was then subjected to a second extraction. Secondary extraction: The dry residue is subjected to secondary extraction under the same conditions to separate and remove the dry residue, and a secondary extract is obtained; Precipitation: Combine the two extracts, inactivate the enzymes at high temperature for 10 minutes, adjust the pH to neutral with sodium bicarbonate, add 6% anhydrous calcium chloride by weight of the dried wakame powder, stir thoroughly and precipitate for 12 hours. Membrane separation: After centrifugation, the supernatant was evaporated to remove 70% of the water. The extract was then separated using a hollow fiber membrane. Anhydrous ethanol was added to precipitate fucoidan. After standing and allowing the precipitate to settle, the mixture was centrifuged and filtered. The precipitate was then freeze-dried in a vacuum freeze dryer to obtain fucoidan. The product was weighed and divided by the weight of the wakame powder to calculate the fucoidan yield. Figure 1 The results are shown in Table 1. As the cellulase dosage increased from 0.5% to 2.5%, the fucoidan extraction rate continued to increase and remained stable. The increased cellulase dosage disrupted the cell walls of *Wakame seaweed*, allowing more fucoidan to leach out. However, when the cellulase dosage exceeded 2.0%, the extraction rate remained essentially unchanged. This may be because the enzymatic hydrolysis of the *Wakame seaweed* cell walls reached saturation at excessively high cellulase dosages. Considering all factors, a fixed cellulase dosage of 1.5% was deemed optimal.

[0015] Table 1. Effect of cellulase dosage on fucoidan extraction rate in Example 1

[0016] Example 2 The effect of pectinase dosage on fucoidan yield includes the following steps: Only the enzyme-assisted method was adjusted; other operating procedures and processes were carried out under the same conditions as in Example 1. Enzyme-assisted method: 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% (by weight of dried wakame powder) of pectinase and 1.5% of cellulase were added to the extract, respectively. The pH of the extract was adjusted to 5 with dilute hydrochloric acid. After two hours of extraction, 1.5% of papain and amylase were added. The extract was then subjected to a hot water bath at 50°C for 4 hours. The solid and liquid were separated by high-speed centrifugation to obtain the supernatant. The dried residue was then subjected to a second extraction. The yield of fucoidan was calculated based on Example 1. Figure 2 The results are shown in Table 2. As the amount of pectinase increased from 0.5% to 2.5%, the extraction rate of fucoidan continued to increase and remained stable at a dosage of 1.0%. Considering both the extraction rate and economic benefits, the optimal amount of pectinase was fixed at 1.0%.

[0017] Table 2. Effect of pectinase dosage on fucoidan extraction rate in Example 2

[0018] Example 3 The effect of amylase dosage on fucoidan yield was investigated, including the following steps: Only the enzyme-assisted method was adjusted; all other procedures and operations were performed under the same conditions as in Example 1. Enzyme-assisted method: 1.5% cellulase and 1.0% pectinase by weight of dried wakame powder were added to the extract. The pH of the extract was adjusted to 5 with dilute hydrochloric acid. After two hours of extraction, 1.5% papain and 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% amylase were added as control experiments. The extract was carried out in a hot water bath at 50°C for 4 hours. The solid and liquid were separated by high-speed centrifugation to obtain the supernatant. The dried residue was then subjected to a second extraction. The yield of fucoidan was calculated based on Example 1. Figure 3 The results are shown in Table 3. As the amount of amylase increased from 0.5% to 2.5%, the extraction rate of fucoidan continued to increase and remained stable at a dosage of 1.5%. Considering both the extraction rate and economic benefits, the optimal amount of amylase was fixed at 1.5%.

[0019] Table 3. Effect of amylase dosage on fucoidan extraction rate in Example 3

[0020] Example 4 The effect of pH on the yield of fucoidan includes the following steps: Grinding: After washing and drying the Dalian wakame seaweed, grind it through a 60-mesh sieve to obtain dried wakame seaweed powder; Ultrasonic pretreatment: Weigh four 10 g portions of kelp powder and add 400 m of ultrapure water as a solvent. Ultrasonic treatment is carried out for 30 min at a frequency of 30 kHz and a maximum output power of 950 W. Enzyme-assisted method: 1.5% cellulase and 1.0% pectinase by weight of dried wakame powder were added to the extract, respectively. The pH of the extract was adjusted to 2, 3, 4, 5 and 6 with dilute hydrochloric acid as a control experiment. After two hours of extraction, 1.5% papain and amylase were added. The extract was then extracted for 4 hours in a hot water bath at 50℃. The solid and liquid were separated by high-speed centrifugation to obtain the supernatant. The dried residue was then subjected to a second extraction. Secondary extraction: The dry residue is subjected to secondary extraction under the same conditions to separate and remove the dry residue, and a secondary extract is obtained; Precipitation: Combine the two extracts, inactivate the enzymes at high temperature for 10 minutes, adjust the pH to neutral with sodium bicarbonate, add 6% anhydrous calcium chloride by weight of the dried wakame powder, stir thoroughly and precipitate for 12 hours. Membrane separation: After centrifugation, the supernatant was rotary evaporated to remove 70% of the water. The extract was then separated using a hollow fiber membrane. Anhydrous ethanol was added to precipitate fucoidan. After standing and allowing the precipitate to settle, the mixture was centrifuged and filtered. The precipitate was then freeze-dried in a vacuum freeze dryer to obtain fucoidan. The resulting product was weighed and divided by the weight of the dried wakame seaweed powder to calculate the fucoidan yield. Figure 4 The results are shown in Table 4. The extraction rate of fucoidan reached its highest at pH 5. This indicates that this pH condition may be most favorable for cellulase activity, thus achieving the best polysaccharide extraction effect. As the pH value further decreased, the extraction rate gradually declined. This may be because excessively low pH inhibits the activity of the combined enzymes, thereby reducing the extraction efficiency.

[0021] Table 4. Effect of pH on fucoidan extraction rate in Example 4

[0022] Example 5 The effect of extraction time on the yield of fucoidan includes the following steps: Grinding: After washing and drying the Dalian wakame seaweed, grind it through a 60-mesh sieve to obtain dried wakame seaweed powder; Ultrasonic pretreatment: Weigh four 10 g portions of kelp powder and add 400 m of ultrapure water as a solvent. Ultrasonic treatment is carried out for 30 min at a frequency of 30 kHz and a maximum output power of 950 W. Enzyme-assisted method: 1.5% cellulase and 1.0% pectinase by weight of dried wakame powder were added to the extract. The pH of the extract was adjusted to 5 with dilute hydrochloric acid. After two hours of extraction, 1.5% papain and amylase were added. The extraction was carried out for 2, 3, 4, 5 and 6 hours in a hot water bath at 50℃ as a control experiment. The solid and liquid were separated by high-speed centrifugation to obtain the supernatant. The dried residue was then subjected to a second extraction. Secondary extraction: The dry residue is subjected to secondary extraction under the same conditions to separate and remove the dry residue, and a secondary extract is obtained; Precipitation: Combine the two extracts, inactivate the enzymes at high temperature for 10 minutes, adjust the pH to neutral with sodium bicarbonate, add 6% anhydrous calcium chloride by weight of the dried wakame powder, stir thoroughly and precipitate for 12 hours. Membrane separation: After centrifugation, the supernatant was rotary evaporated to remove 70% of the water. The extract was then separated using a hollow fiber membrane. Anhydrous ethanol was added to precipitate fucoidan. After standing and allowing the precipitate to settle, the mixture was centrifuged and filtered. The precipitate was then freeze-dried in a vacuum freeze dryer to obtain fucoidan. The resulting product was weighed and divided by the weight of the dried wakame seaweed powder to calculate the fucoidan yield. Figure 5 The results are shown in Table 5. The extraction rate increased as the extraction time increased from 2 hours to 4 hours, but remained relatively stable between 4 and 6 hours. Considering all factors, an extraction time of 4 hours is optimal.

[0023] Table 5. Effect of extraction time on the extraction rate of fucoidan in Example 5

[0024] Example 6 The determination of molecular weight, elemental composition, and sulfate content of fucoidan includes the following steps: Grinding: After washing and drying the Dalian wakame seaweed, grind it through a 60-mesh sieve to obtain dried wakame seaweed powder; Ultrasonic pretreatment: Weigh four 10 g portions of kelp powder and add 40 times their weight of ultrapure water as a solvent. Treat them for 30 min at a frequency of 30 kHz and a maximum output power of 950 W. Enzyme-assisted method: Add 1.5% cellulase and 1.0% pectinase equivalent to the weight of wakame to the extract, adjust the pH of the extract to 5 with dilute hydrochloric acid, add 1.5% papain and amylase after one hour of extraction, extract for 4 hours in a hot water bath at 50°C, centrifuge at high speed to separate solid and liquid, remove dry residue to obtain supernatant. Secondary extraction: The dry residue is subjected to secondary extraction under the same conditions to separate and remove the dry residue, and a secondary extract is obtained; Precipitation: Combine the extracts obtained from the two extractions, inactivate the enzymes at high temperature for 10 minutes, adjust the pH to neutral with sodium bicarbonate, add 6% anhydrous calcium chloride relative to the mass of the wakame seaweed sample, stir thoroughly and precipitate for 12 hours. Membrane separation: After centrifugation, the supernatant was removed by rotary evaporation to remove 70% of the water. The extract was then separated by a hollow fiber membrane. Anhydrous ethanol was added to precipitate fucoidan. After standing to precipitate, the mixture was centrifuged and filtered. The mixture was then placed in a vacuum freeze dryer for freeze drying to obtain crude fucoidan. Molecular weight determination: The crude fucoidan was added to 4 mL of mobile phase sodium nitrate aqueous solution, dissolved at room temperature for 10 minutes, filtered through a 0.45 μm filter membrane, and tested on an instrument. Chromatographic column: Waters Ultrahydrogel™ 120, TM250, TM500 water-soluble gel columns, 7.8 × 300 mm, three columns in tandem. Mobile phase: 0.1M sodium nitrate aqueous solution Flow rate: 1 mL / min Column temperature: 40℃ Accurately weigh 0.5 g (accurate to 0.0001 g) of fucoidan FUC-1 and FUC-2 respectively, place them in a hydrolysis tube, add 15 mL of 1 mol / L hydrochloric acid solution, seal the tube, hydrolyze at 105℃ for 4 h, cool to room temperature, filter using rapid quantitative filter paper, collect the filtrate in a 250 mL Erlenmeyer flask, wash the hydrolysis tube and quantitative filter paper with a small amount of water, and collect all the filtrate in the Erlenmeyer flask.

[0025] Boil the solution in the conical flask for 1 minute, add 20 mL of 10% barium chloride solution dropwise while hot, boil for another 3-6 minutes, and age in a 70℃ constant temperature water bath for 2 hours. Filter the solution using a glass frit filter funnel that has been dried to constant weight, wash the precipitate with water, and dry it in a 105℃ constant temperature drying oven using a glass frit funnel until constant weight. Calculate the sulfate content to be 6.39%.

[0026] Table 4. Elemental composition and sulfate group content of fucoidan in Example 4

[0027] Comparative Example 1 The effect of combined enzyme processes on the yield of fucoidan includes the following steps: Grinding: After washing and drying the Dalian wakame seaweed, grind it through a 60-mesh sieve to obtain dried wakame seaweed powder; Ultrasonic pretreatment: Weigh four 10 g portions of kelp powder and add 400 m of ultrapure water as a solvent. Ultrasonic treatment is carried out for 30 min at a frequency of 30 kHz and a maximum output power of 950 W. Enzyme-assisted method: 1.5% cellulase, 1.0% pectinase, 1.5% papain and amylase by weight of dried wakame powder were added to the extract. The pH of the extract was adjusted to 5 with dilute hydrochloric acid. The extract was extracted for 5 hours in a hot water bath at 50°C. The solid and liquid were separated by high-speed centrifugation to obtain the supernatant. The dry residue was then subjected to a second extraction. Secondary extraction: The dry residue is subjected to secondary extraction under the same conditions to separate and remove the dry residue, and a secondary extract is obtained; Precipitation: Combine the two extracts, inactivate the enzymes at high temperature for 10 minutes, adjust the pH to neutral with sodium bicarbonate, add 6% anhydrous calcium chloride by weight of the dried wakame powder, stir thoroughly and precipitate for 12 hours. Membrane separation: After centrifugation, the supernatant was evaporated to remove 70% of the water. The extract was then separated using a hollow fiber membrane. Anhydrous ethanol was added to precipitate fucoidan. After standing and settling, the precipitate was centrifuged and filtered. The product was then freeze-dried in a vacuum freeze dryer to obtain fucoidan. The resulting product was weighed and divided by the weight of the dried wakame powder to calculate the fucoidan yield. This yield was compared with the optimal extraction rate in Example 1. Figure 8 The result is as follows Figure 8 As shown, the polysaccharide extraction rate of the optimized Example 1 was 20.73%, which was higher than that of Comparative Example 1 (18.01%). Therefore, the optimized enzymatic hydrolysis process significantly improved the extraction rate.

Claims

1. A method for extracting fucoidan from wakame seaweed, characterized in that, The steps are as follows: (a) Wash and dry wakame seaweed, grind it in a pulverizer and pass it through a 60-mesh sieve to obtain wakame seaweed powder; add ultrapure water at a material-to-liquid ratio of 1:20~1:60 g / ml, stir thoroughly, and then pre-treat it with ultrasound at a frequency of 20~60 kHz and a maximum output power of 400~1200 W to obtain an aqueous solution of wakame seaweed powder. (b) Slowly add dilute hydrochloric acid solution to the aqueous solution of dried wakame powder to adjust the pH to 3-7, and add 0.5%-2.0% (by weight) of cellulase, pectinase, amylase and papain of dried wakame powder respectively; control the first extraction temperature at 30℃-70℃ and stir at 200 r / min for 2-6 hours. (c) After the extraction is completed and the system has cooled to room temperature, filter the mixture at 2000 rpm to obtain the first extract and the first residue. Add ultrapure water to the first residue according to the same ratio as the first extraction for a second extraction. Control the temperature of the second extraction at 30℃~70℃ and stir at 200 r / min for 2~6 hours to obtain the second extract and the second residue. Discard the second residue. Combine the extracts obtained from the two extractions and inactivate the enzymes at high temperature for 5~40 min. Adjust the pH with sodium bicarbonate. (d) Add anhydrous calcium chloride to the extract, precipitate for 8-12 hours, centrifuge and take the supernatant to remove 70% water by rotary evaporation, then separate the extract through a hollow fiber membrane to obtain fucoidan extract, add anhydrous ethanol to the fucoidan extract and let it stand to precipitate fucoidan, centrifuge and filter, and freeze dry to obtain fucoidan.

2. The method for extracting fucoidan from wakame seaweed according to claim 1, characterized in that, In step (a), the liquid-to-solid ratio is 1:30~1:40 g / ml, and the mixture is ultrasonically treated for 20~45 min at a frequency of 30 kHz and a maximum output power of 950 W.

3. The method for extracting fucoidan from wakame seaweed according to claim 1, characterized in that, In step (b), at the beginning of the first extraction, 1.2-1.8% of cellulase and 1.2-1.8% of pectinase based on the weight of the dried wakame powder used for extraction were added respectively. After 2 hours of extraction, 1.0-1.5% of amylase and 1.0-1.5% of papain based on the weight of the dried wakame powder used for extraction were added respectively. The pH value was 4.5-5.0, the extraction temperature was 50-55℃, and the total extraction time was 4-5 hours.

4. The method for extracting fucoidan from wakame seaweed according to claim 1, characterized in that, In step (c), no additional cellulase, pectinase, amylase, or papain are added during the secondary extraction process. The extraction temperature is 50–55°C, the second extraction time is 2–3 hours, the enzymes are inactivated at a high temperature of 80–100°C for 10 minutes, and the pH value is 4.5–5.

0.

5. The method for extracting fucoidan from wakame seaweed according to claim 1, characterized in that, In step (d), add 6-8% anhydrous calcium chloride equivalent to the mass of the dried wakame powder used for extraction, stir thoroughly and precipitate for 12 hours, and use hollow fiber membrane with specifications of 10-25 kDa; add anhydrous ethanol to the fucoidan extract until the ethanol volume fraction reaches more than 70%, and let it stand for precipitation for 12 hours.

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