Method for simultaneously preparing low-molecular-weight algin and fucoidin

Abstract

The invention discloses a method for simultaneously preparing low-molecular-weight algin and fucoidin, and belongs to the field of food processing. The multi-dimensional swing type high-energy nano impact mill is adopted, through rapid multi-dimensional swing type movement of the tank body, irregular movement is increased, huge impact force is generated, the movement track of a grinding medium is prolonged, impact energy is improved, impact blind spots are reduced, and the crushing speed is greatly increased; and preparing algin and fucoidin with low molecular weight. Through a physical crushing technology, the fucoidin does not need to be treated after algin and polysaccharide are extracted and separated, pre-freezing is not needed, and the undaria pinnatifida sporophyll is crushed and extracted by using a multi-dimensional swing type high-energy nano impact mill, so that the method is environment-friendly, safe and efficient; and compared with known ultrasonic and infrared technologies, the method has the advantages of higher yield, simple process, wide application range and the like, and is suitable for preparing low-molecular-weight algin and fucoidin from various sources.

Description

Technical Field

[0001] This invention belongs to the field of food processing and relates to a method for simultaneously preparing low molecular weight alginate and fucoidan. Background Technology

[0002] Brown algae are a type of marine algae, including kelp, Sargassum, wakame, and Sargassum fusiforme, widely distributed in oceans worldwide, with an annual yield of hundreds of millions of tons, making them one of the richest biomass resources in the ocean. Alginate and fucoidan are important polysaccharides found in brown algae. Currently, my country is the world's largest producer of alginate, accounting for approximately 50% of the global market. In recent years, the large-scale production of fucoidan has also developed rapidly. Reports indicate that the global fucoidan market reached nearly $300 million in 2024, and my country has become one of the world's major fucoidan producers.

[0003] Alginate typically exists in the form of sodium alginate and is widely used in the food and pharmaceutical industries. However, because alginate is a highly polymerized polysaccharide, it possesses characteristics such as strong gelling properties, high viscosity, poor water solubility, and poor absorption, which limits its application in fields such as biomedicine. Low molecular weight alginate, on the other hand, is characterized by strong water solubility and easy absorption. Moreover, experimental and clinical studies have revealed that low molecular weight alginate possesses bioactivities such as antioxidant, antifreeze, antitumor, antibacterial, blood pressure regulation, lipid-lowering, blood sugar-lowering, and neuroprotective effects, showing broad development and application prospects in functional foods, drug development, green agriculture, and cosmetics manufacturing.

[0004] Currently, there are some reports on the technology of degrading sodium alginate in the existing technology. For example, Chinese patent CN106565852 A (a method for degrading sodium alginate) discloses a method for degrading sodium alginate, which places sodium alginate in an alcohol and acid solution and carries out heterogeneous degradation under acidic conditions. By controlling the reaction time and reaction temperature, sodium alginate with different molecular weights can be prepared.

[0005] For example, Chinese patent CN 113004434 A (A method for degrading sodium alginate based on bismuth tungstate photocatalyst) discloses a method for degrading sodium alginate using bismuth tungstate photocatalyst.

[0006] While some of the aforementioned technical solutions mention methods for degrading sodium alginate, acid degradation is a simple and common chemical method, but the reaction is violent, easily leading to equipment corrosion and generating a large amount of waste liquid. Furthermore, degrading sodium alginate using bismuth tungstate photocatalysts requires the preparation of specific photocatalysts. Therefore, a simple, environmentally friendly method for preparing low molecular weight alginate is needed.

[0007] Fucoidan (FUC), a core active ingredient in the spores of *Wakame seaweed*, enhances the body's disease resistance by activating immune cells such as macrophages and T lymphocytes. Furthermore, this polysaccharide can inhibit α-glucosidase activity, delaying the rise in postprandial blood glucose and assisting in the regulation of glucose and lipid metabolism, thus possessing dietary therapeutic value for people with diabetes and hyperlipidemia. However, due to its large molecular weight, fucoidan is not easily absorbed by the intestines, but its unique biological activity suggests significant application potential in food and pharmaceuticals. Studies have shown that reducing the molecular weight of fucoidan can enhance its absorption and utilization, thereby increasing its efficacy after oral administration.

[0008] Currently, there are some technical reports on the preparation of low molecular weight fucoidan in the existing technology. For example, Chinese patent CN109055460B (A low molecular weight fucoidan and its application in the preparation of cosmetics) discloses a method for preparing low molecular weight fucoidan as follows: using Flavobacterium RC2-3mut strain with accession number CGMCC No.14855 to prepare fucoidan enzyme by fermentation; dissolving fucoidan enzyme in distilled water and mixing it with an equal volume of fucoidan solution with a mass concentration of 0.2%-2%; placing it in a water bath shaker for enzymatic hydrolysis; placing it in a boiling water bath for inactivation of enzyme; centrifuging and taking the supernatant; and using an ultrafiltration system to fractionate the supernatant into low molecular weight fucoidan of different molecular weight ranges.

[0009] For example, Chinese patent CN 117164734 A (A low molecular weight fucoidan from *Tetracentron sinense* and its preparation method and application) discloses a technical method for obtaining low molecular weight fucoidan from *Tetracentron sinense* by adding deionized water to fucoidan, stirring evenly, adding TiO-2 and H-2O-2, and irradiating with a light source to carry out a degradation reaction.

[0010] For example, Chinese patent CN 106432544 A (A method for infrared degradation of marine sulfated polysaccharides) discloses a technical solution for obtaining low-molecular-weight kelp fucoidan by treating marine sulfated polysaccharide dry powder with infrared lamps.

[0011] For example, Chinese patent CN 103087214 A (Preparation method of low molecular weight sea cucumber fucoidan sulfate) discloses a technical solution for obtaining low molecular weight sea cucumber fucoidan sulfate by breaking glycosidic bonds through ultrasonic treatment.

[0012] For example, Chinese patent CN 115490780 A (Extraction method and application of crude extract of fucoidan from Sargassum fusiforme) discloses a technical solution for preparing crude extract of fucoidan by ultra-fine pulverization of Sargassum fusiforme to obtain ultra-fine pulverized Sargassum fusiforme powder, followed by hot water extraction, ultrasonication, centrifugation, concentration of the supernatant, alcohol precipitation, and dialysis.

[0013] While some of the aforementioned technical solutions mention using physical methods to break glycosidic bonds and degrade the molecular weight of fucoidan, minimizing damage to the product structure and offering advantages over chemical methods (no side reactions, no residues, and easy molecular weight control), the physical methods used in these solutions primarily focus on processing the extracted fucoidan. They do not explore the feasibility of using other physical methods to directly process the raw material for polysaccharide degradation. Ultrasonic treatment is generally used for liquid solutions because the energy of ultrasound requires a medium for conduction, and the solvent in the solution can effectively serve as this medium. However, this leads to additional energy consumption and product loss (products tend to adhere to the container walls) during subsequent product extraction from the solution, whether using heating, precipitation centrifugation, or freeze-drying. Furthermore, ultrasonic degradation yields are low; not all fucoidan after ultrasonic treatment is degraded into low-molecular-weight polysaccharides, and some is converted into other unusable substances, resulting in a low yield of low-molecular-weight polysaccharides and significant fucoidan loss. Furthermore, all ultrasonic degradation treatments have focused on extracted and purified polysaccharides, with no evidence of in-situ degradation of polysaccharides directly within biological tissues. Direct ultrasonic treatment of solids not only yields poor degradation results but also presents challenges in large-scale industrial continuous production, including heat generation, harsh noise, and damage to equipment (mechanical fatigue). Additionally, irradiating fucoidan with infrared lamps generates high temperatures, easily causing dehydration, carbonization, or Maillard reactions, leading to darker product color, functional group destruction resulting in sulfate removal, and even the formation of harmful substances. Moreover, the raw material used is extracted and separated fucoidan, making pre-degradation treatment cumbersome. Due to the generally low extraction rates of existing polysaccharide extraction methods, this method results in significant raw material loss and a relatively low yield of low molecular weight fucoidan. Therefore, it is necessary to explore other suitable physical methods to directly treat the raw materials from which fucoidan is extracted, thereby addressing losses during polysaccharide extraction, dissolution, and reprecipitation, the shortcomings of industrial ultrasonic production, and the need to reduce the molecular weight of fucoidan.

[0014] In addition, in order to achieve efficient extraction and degradation of fucoidan, some existing technologies have proposed the concept of nano-pulverizing sea cucumbers. For example, CN101451157A (a method for preparing low molecular weight sea cucumber polysaccharide) discloses that sea cucumbers are freeze-dried, coarsely pulverized, air-jet pulverized and nano-pulverized to make nano-sea cucumber powder, and then sea cucumber polysaccharide is extracted. The molecular weight of sea cucumber polysaccharide is effectively reduced to 30kDa. However, this patent does not mention the preparation of low molecular weight fucoidan.

[0015] For example, CN 119586736 A (A green processing technology for sea cucumber powder with directional enrichment to improve polysaccharide release rate) and CN 119655407A (A green deodorization method applicable to ultrafine sea cucumber powder) disclose a technical solution of placing coarse sea cucumber powder in the extraction vessel of a supercritical fluid extractor to deodorize the sea cucumber powder, and then pulverizing it with an ultrafine pulverizer to obtain sea cucumber powder. However, none of these technical solutions mention the method for preparing low molecular weight fucoidan. Summary of the Invention

[0016] To address the aforementioned problems, this invention provides a method for simultaneously preparing low molecular weight alginate and fucoidan. By employing a multidimensional oscillating high-energy nano-impact mill, the rapid multidimensional oscillating motion of the mill body increases the irregular motion, generating a huge impact force, extending the movement trajectory of the grinding media, increasing the impact energy, reducing impact blind spots, and significantly improving the pulverization efficiency. This method can directly break down alginate and fucoidan molecules in algae, thereby achieving the simultaneous preparation of low molecular weight alginate and low molecular weight fucoidan.

[0017] This invention provides a method for simultaneously preparing low molecular weight alginate and fucoidan, comprising the following steps:

[0018] S1. Dry wakame spore leaves are pulverized into coarse wakame spore leaf powder of less than 30 mesh;

[0019] S2. The coarse powder of wakame spore leaves obtained in step S1 is pulverized into ultrafine powder to obtain powder. The ultrafine powder pulverization is carried out on a multi-dimensional oscillating high-energy nano-impact mill with a rotation speed of 200-500 rpm and a pulverization time of 8-12 hours.

[0020] S3. Take the ultrafine powder of wakame spore leaves obtained in S2, add disodium hydrogen phosphate-citric acid buffer solution (pH 5.0), then add the compound enzyme, and after enzymatic hydrolysis at 50℃ for 4 hours, heat to 100℃ to inactivate the enzyme and maintain for 15 minutes.

[0021] S4. After the temperature of the extract has dropped to room temperature, centrifuge the sample solution to remove impurities. The centrifugation conditions are: 4000 rpm, 15 min, 4℃ (the same below) to obtain the supernatant.

[0022] S5. While stirring, add an excess of anhydrous CaCl2 to the supernatant above, and centrifuge to obtain low molecular weight alginate and supernatant.

[0023] S6. Add hexadecyltrimethylammonium bromide (CTAB) to the supernatant above, stir well, let stand for 12 h, and collect the precipitate by centrifugation. Dissolve the precipitate in 3M CaCl2 solution, then add 2-4 times the amount of anhydrous ethanol, let stand at 4℃ for 12 h, and centrifuge to obtain the precipitate.

[0024] S7. After the above precipitate has dried, it is redissolved in deionized water and placed in a 0.5-3.5 kDa dialysis bag. Dialysis with tap water for 48 hours, followed by dialysis with deionized water for 72 hours. The dialysis solution is concentrated using a rotary evaporator and freeze-dried to obtain low molecular weight fucoidan (UPF).

[0025] In one embodiment of the present invention, the mass ratio of the ultrafine powder of *Wagtail spores* to the disodium hydrogen phosphate-citric acid buffer solution in S3 is 1:30, and the complex enzyme is 0.5% of the mass of the ultrafine powder of *Wagtail spores*.

[0026] In one embodiment of the present invention, the complex enzyme in S3 is obtained by mixing cellulase, pectinase and papain in a mass ratio of 7:1:1.

[0027] The present invention also provides low molecular weight alginate and low molecular weight fucoidan prepared by the above-described method.

[0028] In one embodiment of the present invention, the low molecular weight alginate has a molecular weight of 57.6-314.7 kDa, and the low molecular weight fucoidan has a molecular weight of 35.7-49.6 kDa.

[0029] The present invention also provides the application of any of the above-described methods in the preparation of low molecular weight alginate and fucoidan from various sources.

[0030] In one embodiment of the present invention, the source is kelp, wakame, or various brown algae.

[0031] The present invention also provides the application of the aforementioned low molecular weight alginate and low molecular weight fucoidan in food and health products.

[0032] The beneficial effects of this invention are:

[0033] 1. This invention employs a multi-dimensional oscillating high-energy nano-impact mill. Through the rapid multi-dimensional oscillating motion of the tank, the irregular motion generates a huge impact force, extends the movement trajectory of the grinding media, increases the impact energy, reduces impact blind spots, and significantly improves the pulverization efficiency. It can directly break down alginate and fucoidan molecules in algae, thereby achieving the preparation of low molecular weight alginate and low molecular weight fucoidan.

[0034] 2. This invention uses algae as raw material for ultrafine pulverization, which can simultaneously prepare low molecular weight alginate and low molecular weight fucoidan, making it highly efficient and convenient. Other reported polysaccharide degradation methods, such as ultrasound, infrared spectroscopy, and photocatalysis, target alginate or fucoidan, not the algae themselves. To obtain low molecular weight alginate and low molecular weight fucoidan, the alginate and fucoidan need to be processed separately.

[0035] 3. This invention uses algae as raw material for ultrafine pulverization, which significantly reduces the viscosity of the extract. Typically, the production of alginate requires a large amount of water for dilution to ensure smooth filtration of the extract. However, in this invention, the reduced viscosity of the alginate significantly reduces water usage during filtration, lowering the extraction cost of alginate and fucoidan and making the process more environmentally friendly. Attached Figure Description

[0036] Figure 1 A biological microscope image of the coarse powder of *Undaria pinnatifida* sporophytes in Comparative Example 1.

[0037] Figure 2 Biological microscope image of Undaria pinnatifida spore powder in Comparative Example 2.

[0038] Figure 3 A biological microscope image of the Undaria pinnatifida spore powder after 2 hours of ultrafine grinding in Comparative Example 3.

[0039] Figure 4 A biological microscope image of the Undaria pinnatifida spore powder after 4 hours of ultrafine grinding in Comparative Example 4.

[0040] Figure 5 A biological microscope image of Undaria pinnatifida spore powder after 8 hours of ultrafine grinding in Example 1.

[0041] Figure 6 A biological microscope image of the Undaria pinnatifida spore powder after 12 hours of ultrafine grinding in Example 2.

[0042] Figure 7 Particle size distribution of coarse powder from *Undaria pinnatifida* spore leaves in Comparative Example 1.

[0043] Figure 8 Particle size distribution of Undaria pinnatifida spore powder in Comparative Example 2.

[0044] Figure 9 Particle size distribution of Undaria pinnatifida spore powder after 2 hours of ultrafine grinding in Comparative Example 3.

[0045] Figure 10 Particle size distribution of Undaria pinnatifida spore powder after 4 hours of ultrafine grinding in Comparative Example 4.

[0046] Figure 11 Particle size distribution of Undaria pinnatifida spore powder after 8 hours of ultrafine grinding in Example 1.

[0047] Figure 12 Particle size distribution diagram of Undaria pinnatifida spore powder after 12 hours of ultrafine grinding in Example 2

[0048] Figure 13 Molecular weight distribution of brown alginate from *Undaria pinnatifida* sporophytes in Comparative Example 1 and Examples 1 and 2.

[0049] Figure 14 Molecular weight distribution of fucoidan from *Undaria pinnatifida* sporophytes in Comparative Examples 1 and 2.

[0050] Figure 15 Molecular weight distribution of fucoidan from *Undaria pinnatifida* sporophytes in Examples 1 and 2 and Comparative Examples 1, 3, and 4.

[0051] Figure 16 Infrared spectra of fucoidan from *Undaria pinnatifida* sporophytes of different molecular weights in Examples 1, 2, and Comparative Examples 1, 3, and 4. Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0053] Example 1

[0054] (1) After pulverizing the dried wakame spore leaves, pass them through a 30-mesh sieve to obtain coarse wakame spore leaf powder. Then, the coarse wakame spore leaf powder is pulverized using a multi-dimensional oscillating high-energy nano-impact mill at a speed of 380 rpm for 8 hours to obtain wakame spore leaf powder.

[0055] (2) Take the ultrafine powder of wakame spore leaves after grinding for 8 hours and add disodium hydrogen phosphate-citric acid buffer solution (pH 5.0). Then add the compound enzyme (in a ratio of cellulase, pectinase and papain of 7:1:1), stir at 50℃ for 4 hours, and then heat to 100℃ and maintain for 15 minutes; wherein, the mass ratio of wakame spore leaf ultrafine powder to disodium hydrogen phosphate-citric acid buffer solution is 1:30, and the compound enzyme is 0.5% of the mass of wakame spore leaf ultrafine powder.

[0056] (3) After the temperature of the extract solution drops to room temperature, centrifuge the sample solution to remove impurities and obtain the supernatant. Add excess anhydrous CaCl2 to the supernatant while stirring, and centrifuge to obtain low molecular weight alginate and supernatant; the centrifugation conditions mentioned above are: 4000 rpm, 15 min, 4℃.

[0057] (4) Add hexadecyltrimethylammonium bromide (CTAB) to the supernatant above, stir well, let stand for 12 h, and collect the precipitate by centrifugation. Dissolve the precipitate in 3M CaCl2 solution, then add anhydrous ethanol, let stand at 4℃ for 12 h, and centrifuge to obtain the precipitate.

[0058] (5) After the above precipitate is air-dried, it is redissolved in deionized water, placed in a 3.5 kDa dialysis bag, and dialyzed with tap water for 48 h, followed by dialyzed with deionized water for 72 h. The dialyzed solution is concentrated by rotary evaporator and freeze-dried to obtain low molecular weight Wrigley's sporophyll fucoidan.

[0059] Example 2

[0060] (1) After pulverizing the dried wakame spore leaves, pass them through a 30-mesh sieve to obtain coarse wakame spore leaf powder. Then, the coarse wakame spore leaf powder is pulverized using a multi-dimensional oscillating high-energy nano-impact mill at a speed of 380 rpm for 12 hours to obtain wakame spore leaf powder.

[0061] (2) Take the ultrafine powder of wakame spore leaves after grinding for 12 hours and add disodium hydrogen phosphate-citric acid buffer solution (pH 5.0). Then add the compound enzyme (in a ratio of cellulase, pectinase and papain of 7:1:1), stir at 50℃ for 4 hours, and then heat to 100℃ and maintain for 15 minutes; wherein, the mass ratio of wakame spore leaf ultrafine powder to disodium hydrogen phosphate-citric acid buffer solution is 1:30, and the compound enzyme is 0.5% of the mass of wakame spore leaf ultrafine powder.

[0062] (3) After the temperature of the extract solution drops to room temperature, centrifuge the sample solution to remove impurities and obtain the supernatant. Add excess anhydrous CaCl2 to the supernatant while stirring, and centrifuge to obtain low molecular weight alginate and supernatant; the centrifugation conditions mentioned above are: 4000 rpm, 15 min, 4℃.

[0063] (4) Add hexadecyltrimethylammonium bromide (CTAB) to the supernatant above, stir well, let stand for 12 h, and collect the precipitate by centrifugation. Dissolve the precipitate in 3M CaCl2 solution, then add anhydrous ethanol, let stand at 4℃ for 12 h, and centrifuge to obtain the precipitate.

[0064] (5) After the above precipitate is air-dried, it is redissolved in deionized water, placed in a 3.5 kDa dialysis bag, and dialyzed with tap water for 48 h, followed by dialyzed with deionized water for 72 h. The dialyzed solution is concentrated by rotary evaporator and freeze-dried to obtain low molecular weight Wrigley's sporophyll fucoidan.

[0065] Comparative Example 1

[0066] (1) After the dried wakame spore leaves are crushed, they are passed through a 30-mesh sieve to obtain coarse wakame spore leaf powder.

[0067] (2) Take the coarse powder of wakame spore leaves and add disodium hydrogen phosphate-citric acid buffer solution (pH 5.0). Then add the compound enzyme (in a ratio of cellulase, pectinase and papain of 7:1:1), stir at 50°C for 4 hours, and then heat to 100°C and maintain for 15 minutes; wherein, the mass ratio of wakame spore leaf ultrafine powder to disodium hydrogen phosphate-citric acid buffer solution is 1:30, and the compound enzyme is 0.5% of the mass of wakame spore leaf ultrafine powder.

[0068] (3) After the temperature of the extract solution drops to room temperature, centrifuge the sample solution to remove impurities and obtain the supernatant. Add excess anhydrous CaCl2 to the supernatant while stirring, and centrifuge to obtain alginate and supernatant; the centrifugation conditions mentioned above are: 4000 rpm, 15 min, 4℃.

[0069] (4) Add hexadecyltrimethylammonium bromide (CTAB) to the supernatant above, stir well, let stand for 12 h, and collect the precipitate by centrifugation. Dissolve the precipitate in 3M CaCl2 solution, then add anhydrous ethanol, let stand at 4℃ for 12 h, and centrifuge to obtain the precipitate.

[0070] (5) After the above precipitate is air-dried, it is redissolved in deionized water, placed in a 3.5 kDa dialysis bag, and dialyzed with tap water for 48 h, followed by dialyzed with deionized water for 72 h. The dialyzed solution is concentrated by rotary evaporator and freeze-dried to obtain Wrigley's spore leaf fucoidan.

[0071] Biological microscopy revealed that the coarse powder particles were large in size, with large lumps and large gaps between them.

[0072] Comparative Example 2

[0073] (1) After the dried wakame spore leaves were crushed, they were passed through a 30-mesh sieve to obtain coarse wakame spore leaf powder. Then, the coarse wakame spore leaf powder was crushed using a Retsch high-energy ball mill at a speed of 800 rpm for 2 min, with a 1 min interval, for a total crushing time of 12 h to obtain wakame spore leaf powder.

[0074] (2) Take wakame spore leaf powder and add disodium hydrogen phosphate-citric acid buffer solution (pH 5.0). Then add a compound enzyme (with cellulase, pectinase and papain in a ratio of 7:1:1), stir at 50°C for 4 hours, then heat to 100°C and maintain for 15 minutes; wherein, the mass ratio of wakame spore leaf ultrafine powder to disodium hydrogen phosphate-citric acid buffer solution is 1:30, and the compound enzyme is 0.5% of the mass of wakame spore leaf ultrafine powder.

[0075] (3) After the temperature of the extract solution drops to room temperature, centrifuge the sample solution to remove impurities and obtain the supernatant. Add excess anhydrous CaCl2 to the supernatant while stirring, and centrifuge to obtain alginate and supernatant; the centrifugation conditions mentioned above are: 4000 rpm, 15 min, 4℃.

[0076] (4) Add hexadecyltrimethylammonium bromide (CTAB) to the supernatant above, stir well, let stand for 12 h, and collect the precipitate by centrifugation. Dissolve the precipitate in 3M CaCl2 solution, then add anhydrous ethanol, let stand at 4℃ for 12 h, and centrifuge to obtain the precipitate.

[0077] (5) After the above precipitate is air-dried, it is redissolved in deionized water, placed in a 3.5 kDa dialysis bag, and dialyzed with tap water for 48 h, followed by dialyzed with deionized water for 72 h. The dialyzed solution is concentrated by rotary evaporator and freeze-dried to obtain Wrigley's spore leaf fucoidan.

[0078] Comparative Example 3

[0079] (1) After pulverizing the dried wakame spore leaves, pass them through a 30-mesh sieve to obtain coarse wakame spore leaf powder. Then, the coarse wakame spore leaf powder is pulverized using a multi-dimensional oscillating high-energy nano-impact mill at a speed of 380 rpm for 2 hours to obtain wakame spore leaf powder.

[0080] (2) Take the ultrafine powder of wakame spore leaves after grinding for 2 hours and add disodium hydrogen phosphate-citric acid buffer solution (pH 5.0). Then add the compound enzyme (in a ratio of cellulase, pectinase and papain of 7:1:1), stir at 50℃ for 4 hours, and then heat to 100℃ and maintain for 15 minutes; wherein, the mass ratio of wakame spore leaf ultrafine powder to disodium hydrogen phosphate-citric acid buffer solution is 1:30, and the compound enzyme is 0.5% of the mass of wakame spore leaf ultrafine powder.

[0081] (3) After the temperature of the extract solution drops to room temperature, centrifuge the sample solution to remove impurities and obtain the supernatant. Add excess anhydrous CaCl2 to the supernatant while stirring, and centrifuge to obtain low molecular weight alginate and supernatant; the centrifugation conditions mentioned above are: 4000 rpm, 15 min, 4℃.

[0082] (4) Add hexadecyltrimethylammonium bromide (CTAB) to the supernatant above, stir well, let stand for 12 h, and collect the precipitate by centrifugation. Dissolve the precipitate in 3M CaCl2 solution, then add anhydrous ethanol, let stand at 4℃ for 12 h, and centrifuge to obtain the precipitate.

[0083] (5) After the above precipitate is air-dried, it is redissolved in deionized water, placed in a 3.5 kDa dialysis bag, and dialyzed with tap water for 48 h, followed by dialyzed with deionized water for 72 h. The dialyzed solution is concentrated by rotary evaporator and freeze-dried to obtain low molecular weight Wrigley's sporophyll fucoidan.

[0084] Comparative Example 4

[0085] (1) After pulverizing the dried wakame spore leaves, pass them through a 30-mesh sieve to obtain coarse wakame spore leaf powder. Then, the coarse wakame spore leaf powder is pulverized using a multi-dimensional oscillating high-energy nano-impact mill at a speed of 380 rpm for 4 hours to obtain wakame spore leaf powder.

[0086] (2) Take the ultrafine powder of wakame spore leaves after grinding for 4 hours and add disodium hydrogen phosphate-citric acid buffer solution (pH 5.0). Then add the compound enzyme (in a ratio of cellulase, pectinase and papain of 7:1:1), stir at 50℃ for 4 hours, and then heat to 100℃ and maintain for 15 minutes; wherein, the mass ratio of wakame spore leaf ultrafine powder to disodium hydrogen phosphate-citric acid buffer solution is 1:30, and the compound enzyme is 0.5% of the mass of wakame spore leaf ultrafine powder.

[0087] (3) After the temperature of the extract solution drops to room temperature, centrifuge the sample solution to remove impurities and obtain the supernatant. Add excess anhydrous CaCl2 to the supernatant while stirring, and centrifuge to obtain alginate and supernatant; the centrifugation conditions mentioned above are: 4000 rpm, 15 min, 4℃.

[0088] (4) Add hexadecyltrimethylammonium bromide (CTAB) to the supernatant above, stir well, let stand for 12 h, and collect the precipitate by centrifugation. Dissolve the precipitate in 3M CaCl2 solution, then add anhydrous ethanol, let stand at 4℃ for 12 h, and centrifuge to obtain the precipitate.

[0089] (5) After the above precipitate is air-dried, it is redissolved in deionized water, placed in a 3.5 kDa dialysis bag, and dialyzed with tap water for 48 h, followed by dialyzed with deionized water for 72 h. The dialyzed solution is concentrated by rotary evaporator and freeze-dried to obtain Wrigley's spore leaf fucoidan.

[0090] Table 1. Particle size of *Wagtail* spore powder obtained from Comparative Examples 1-4 and Examples 1-2

[0091]

[0092]

[0093] Test Example 1

[0094] A sample of alginate from *Watanabe spore leaves* was dissolved in sodium carbonate solution, centrifuged, and the supernatant was filtered through a 0.22 μm microporous membrane before injection to determine the molecular weight of the alginate. The detection conditions were as follows: Waters 2414 HPLC equipped with a PDA detector and an RI detector; TSK G4000PWXL column (250 × 4.6 mm, 5 μm); column temperature 30℃; injection volume 10 μL; mobile phase 100 mM ammonium acetate; flow rate 0.4 mL / min; standards were 25, 40, 150, 250, and 500 kDa dextran standards.

[0095] Table 2 shows the molecular weight of alginate obtained in Comparative Example 1 and Examples 1 and 2.

[0096] Molecular weight / kDa Comparative Example 1 1488.1 Example 1 314.7 Example 2 57.6

[0097] Test Example 2

[0098] Fucoidan samples from *Wakame spore leaves* were dissolved in deionized water, filtered through a 0.22 μm microporous membrane, and then injected to determine the molecular weight of the fucoidan. The detection conditions were as follows: Waters 2414 HPLC equipped with a PDA detector and an RI detector; TSKG4000PWXL column (250 × 4.6 mm, 5 μm); column temperature 30℃; injection volume 10 μL; mobile phase 100 mM ammonium acetate; flow rate 0.4 mL / min; standards were 25, 40, 150, 250, and 500 kDa dextran standards.

[0099] Table 3 shows the molecular weights of fucoidan obtained in Comparative Examples 1-4 and Examples 1 and 2.

[0100] Molecular weight / kDa Comparative Example 1 943.4 Comparative Example 2 924.1 Comparative Example 3 799.4 Comparative Example 4 691.5 Example 1 49.6 Example 2 35.7

[0101] The molecular weight of alginate in *Watasetra spore powder* after different pulverization times was measured, such as... Figure 13 As shown, the peaks in the alginate prepared from the coarse powder of Undaria pinnatifida spores without ultrafine grinding (Comparative Example 1) and the alginate prepared from the Undaria pinnatifida spore powder after ultrafine grinding for 8 hours (Example 1) showed a significant shift to the later stage, indicating that the alginate in the processed Undaria pinnatifida spore powder was degraded, resulting in a significant reduction in the molecular weight of the alginate.

[0102] The molecular weight of fucoidan in *Watasetra spore powder* after different pulverization times was measured, such as... Figure 14 and Figure 15As shown, the peak values ​​of fucoidan obtained from Undaria pinnatifida spore powder after 2 hours of ultrafine grinding (Comparative Example 3) and fucoidan obtained from Undaria pinnatifida spore powder after 12 hours of high-energy ball milling (Comparative Example 2) did not shift significantly, and the molecular weight reduction was not significant. However, the peak values ​​of fucoidan obtained from Undaria pinnatifida spore powder without ultrafine grinding (Comparative Example 1) and fucoidan obtained from Undaria pinnatifida spore powder after 8 hours of ultrafine grinding (Example 1) showed a significant shift to the later stage, indicating that the fucoidan in the treated Undaria pinnatifida spore powder was degraded, resulting in a significant reduction in the molecular weight of fucoidan.

[0103] Low molecular weight alginate and fucoidan from Undaria pinnatifida spore powder were prepared using physical pulverization technology.

[0104] Test Example 3

[0105] A sample of fucoidan from the sporophytes of *Watanabe* was mixed with KBr and pressed into a disc. The infrared spectrum was measured at room temperature using a PerkinElmer Fourier transform infrared spectrometer.

[0106] pass Figure 16 Infrared spectra of fucoidan from *Undaria pinnatifida* spores at different molecular weights show that reducing the molecular weight of fucoidan did not alter its functional groups, indicating that physical pulverization methods can break glycosidic bonds to degrade fucoidan and prepare low molecular weight fucoidan.

[0107] Test Example 4

[0108] Take the enzymatic hydrolysate of wakame spore leaf powder and measure the apparent viscosity of the seaweed powder hydrolysate at room temperature using a Brookfield digital viscometer.

[0109] Table 4. Viscosities of seaweed powder enzymatic hydrolysates obtained in Comparative Examples 1-4 and Examples 1 and 2

[0110] Viscosity / cP Comparative Example 1 734 Comparative Example 2 713 Comparative Example 3 544 Comparative Example 4 371 Example 1 155 Example 2 142

[0111] The apparent viscosity of the enzymatic hydrolysate of *Watana spore* leaf powder after different pulverization times was measured. The apparent viscosity of the enzymatic hydrolysate of *Watana spore* leaf powder was significantly reduced after ultra-fine pulverization, making the extract easier to filter and greatly reducing the use of water in the filtration process.

[0112] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for simultaneously preparing low molecular weight alginate and fucoidan, characterized in that, Includes the following steps: S1. Dry wakame spore leaves are pulverized into coarse powder of wakame spore leaves with a mesh size of less than 30 mesh. The coarse powder is then pulverized into ultrafine powder on a multidimensional oscillating high-energy nano-impact mill to obtain wakame spore leaf ultrafine powder. S2. The obtained wakame spore leaf ultrafine powder was added to disodium hydrogen phosphate-citric acid buffer solution, and then a compound enzyme was added for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was inactivated by heating, followed by cooling and centrifugation, and the supernatant was retained. S3. Add excess anhydrous CaCl2 to the supernatant and centrifuge to obtain low molecular weight alginate and supernatant. S4. Add hexadecyltrimethylammonium bromide to the supernatant obtained in S3, centrifuge to collect the precipitate, dissolve the precipitate in CaCl2 solution, then add anhydrous ethanol, let stand, centrifuge to obtain the precipitate, and finally obtain low molecular weight fucoidan by dialysis, concentration, and freeze drying.

2. The method according to claim 1, characterized in that, The multidimensional oscillating high-energy nano-impact mill in S1 has a rotation speed of 200-500 rpm and a pulverizing time of 8-12 h.

3. The method according to claim 1, characterized in that, The complex enzyme in S2 is obtained by mixing cellulase, pectinase and papain in a mass ratio of 7:1:

1.

4. The method according to claim 1, characterized in that, In S2, the mass ratio of the ultrafine powder of *Wagtail spores* leaves to the disodium hydrogen phosphate-citric acid buffer solution is 1:30, and the compound enzyme is 0.5% of the mass of the ultrafine powder of *Wagtail spores* leaves.

5. The method according to claim 1, characterized in that, The pH of the disodium hydrogen phosphate-citric acid buffer solution in S2 is 5.0; after enzymatic hydrolysis at 50℃ for 4 hours, the enzyme is inactivated by heating to 100℃ and held for 15 minutes.

6. The low molecular weight alginate and low molecular weight fucoidan prepared by any of the methods described in claims 1-5.

7. The low molecular weight alginate and low molecular weight fucoidan according to claim 6, characterized in that, The low molecular weight alginate has a molecular weight of 57.6-314.7 kDa, and the low molecular weight fucoidan has a molecular weight of 35.7-49.6 kDa.

8. The application of the low molecular weight alginate and low molecular weight fucoidan as described in claim 6 in food and health products.

9. The use of the method according to any one of claims 1-5 in the preparation of low molecular weight alginate and low molecular weight fucoidan from various sources.

10. The application according to claim 9, characterized in that, The sources mentioned are kelp, wakame, and various brown algae.

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