Preparation method and application of low molecular weight laminarin

Abstract

This invention discloses a method for preparing and applying low molecular weight kelp polysaccharide. The method includes: washing, drying, and pulverizing kelp, then inoculating it with a compound inoculum of Bacillus subtilis and Lactobacillus casei for fermentation; extracting the fermentation product with hot water, removing proteins, and precipitating with alcohol to obtain kelp polysaccharide; dissolving it in water, adding hydrochloric acid and a compound protective agent composed of borate, glycerol, and sodium alginate for acid hydrolysis; after acid hydrolysis, neutralizing, ultrafiltration, dialysis, and freeze-drying to obtain the final product. This invention improves the polysaccharide conformation through fermentation, and the compound protective agent synergistically inhibits the breakage of sulfate ester bonds during acid hydrolysis. The obtained polysaccharide has a molecular weight of 7.5 × 10⁻⁶. 3 ~8.5×10 3 Da, with a sulfate content of ≥28%, is fast-dissolving, low-viscosity, has good transdermal absorption, and excellent antioxidant activity, making it suitable for use in anti-wrinkle and moisturizing skincare products.

Description

Technical Field

[0001] This invention relates to the technical field of polysaccharide preparation methods, specifically to a method for preparing low molecular weight kelp polysaccharide and its application. Background Technology

[0002] Kelp (Laminaria japonica) is an important economic seaweed in my country, with abundant resources and huge yields, and has been widely used in the food, feed, and marine biomedicine fields. Fucoidan (also known as fucoidan sulfate), extracted from kelp, is a type of water-soluble heteropolysaccharide containing sulfate groups. Its main monosaccharide component is fucose, and it also contains galactose, mannose, xylose, glucuronic acid, etc. The sulfate groups are usually attached to the C-2 or C-4 positions of the sugar units. Extensive research has confirmed that fucoidan sulfate possesses a variety of important biological functions, including anticoagulation, antiviral, antitumor, immunomodulatory, anti-inflammatory, and antioxidant effects, making it a natural active polysaccharide with broad application prospects.

[0003] However, natural fucoidan sulfates typically have high molecular weights, often reaching millions of Daltons. This high molecular weight results in poor water solubility, high solution viscosity, and low bioavailability, making them difficult for the body to effectively absorb and utilize, thus significantly limiting their full biological activity. Studies have shown that the molecular weight of polysaccharides is one of the key factors affecting their biological activity. Appropriately reducing the molecular weight can improve the water solubility and rheological properties of polysaccharides, increase their transmembrane permeability and bioavailability, thereby enhancing their antitumor, immunomodulatory, and anti-inflammatory biological activities. Therefore, preparing low molecular weight fucoidan sulfates through degradation methods has become an important technical approach to improve their biological activity and expand their application range.

[0004] Currently, commonly used polysaccharide degradation methods mainly include physical degradation methods (such as ultrasonic degradation and microwave degradation), chemical degradation methods (such as acid hydrolysis and oxidative degradation), and enzymatic degradation methods. Among them, acid hydrolysis is one of the most classic, simplest, and lowest-cost chemical degradation methods. By controlling conditions such as acid concentration, hydrolysis temperature, and time, high molecular weight polysaccharides can be effectively degraded into low molecular weight products, facilitating industrial-scale production. However, traditional acid hydrolysis has a long-standing key technical problem: during the process of reducing the molecular weight of polysaccharides through acid hydrolysis, the sulfate ester bonds on the polysaccharide chains are also sensitive to acid and easily break, leading to the loss of a large number of sulfate groups. The sulfate group is a key active functional group for the anticoagulant, antitumor, and immunomodulatory biological functions of fucoidan sulfate esters, and its loss significantly weakens the biological activity of the product. Studies have shown that during traditional hydrochloric acid or trifluoroacetic acid hydrolysis, the sulfate groups of fucoidan are severely damaged, resulting in a significant decrease in its original biological activities, creating a contradiction of reduced molecular weight and loss of activity.

[0005] To address the aforementioned issues, researchers have attempted to prepare low-molecular-weight kelp sulfated polysaccharides using hydrogen peroxide oxidation degradation. While this method can maintain the sulfate group and total sugar content to a certain extent, its degradation rate is slow, and the adjustability of molecular weight control is limited, making it difficult to accurately obtain products within the target molecular weight range. Other studies have used a combination of ultrasonic-assisted extraction, ultrafiltration fractionation, enzymatic hydrolysis, and acid hydrolysis to prepare fucoidan sulfates of different molecular weights. However, this method involves complex processes, demanding equipment, and high production costs, hindering large-scale production. More importantly, none of these methods fundamentally solve the technical challenge of easily breaking sulfate bonds during acid hydrolysis. Furthermore, in the extraction and preparation of fucoidan sulfates, traditional hot water extraction methods have low extraction efficiency and product yield, and the crude product contains a significant amount of protein, pigments, and other impurities, increasing the difficulty of subsequent purification. Although there are reports of cellulase-assisted extraction, the enzyme type is limited, the cell wall disruption effect is limited, and the release of polysaccharides is insufficient, leaving considerable room for improvement in the extraction rate.

[0006] Therefore, developing a method for preparing low molecular weight kelp sulfated polysaccharides that can effectively protect or even increase the sulfate group content while reducing the molecular weight is of great academic research value and industrial application prospects for fully utilizing the bioactivity of brown algae polysaccharide sulfate esters and expanding their application in the fields of biomedicine and health. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing low molecular weight kelp polysaccharides. This method first improves the extraction rate and purity of kelp polysaccharides through microbial fermentation pretreatment, and then effectively protects the sulfate bonds while reducing the molecular weight of the polysaccharide by adding a specific protective agent during acid hydrolysis, thereby obtaining a low molecular weight kelp polysaccharide product with low molecular weight, high sulfate group content and excellent biological activity.

[0008] To achieve the above technical solution, the present invention adopts the following technical solution: A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash fresh or dried kelp to remove mud and impurities, dry it at 40-60℃ to constant weight, and pulverize it through a 40-80 mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water according to the material-liquid ratio, adjust the pH to 6.5, sterilize, and then inoculate with compound microbial agent. Ferment for 1-2 days at a temperature of 25-35℃ and a humidity of 70-80%, and obtain the fermentation product after inactivation. S3, extract kelp polysaccharides from the fermentation product obtained in step S2; S4, dissolve the kelp polysaccharide obtained in step S3 in water to obtain a kelp polysaccharide solution, add hydrochloric acid and a composite protective agent to it and then perform acid hydrolysis treatment; S5. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 6.8-7.2 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to obtain the supernatant. The supernatant is purified by ultrafiltration membrane. The retentate is further dialyzed. The dialysate is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0009] Preferably, in step S2, the ratio of kelp powder to distilled water is 1g:20mL.

[0010] Preferably, the compound microbial agent is composed of Bacillus subtilis and Lactobacillus casei in a mass ratio of 2:1; the inoculation amount of the compound microbial agent is 0.5% to 1.5% of the mass of dried kelp powder.

[0011] Preferably, the specific method for extracting kelp polysaccharides in step S3 is as follows: add 3 times the amount of distilled water to the kelp fermentation product, heat and extract 2-3 times at 90-95℃, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to a final concentration of 70%, let stand at 4℃ for alcohol precipitation for 12-16 h, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharides.

[0012] Preferably, the mass concentration of the kelp polysaccharide solution in step S4 is 5-8%.

[0013] Preferably, in step S4, the composite protective agent is composed of borate, glycerol, and sodium alginate in a mass ratio of 2:1:1; the amount of the composite protective agent added is 0.5~1.5% of the mass of kelp polysaccharide.

[0014] Preferably, the borate is one or more of sodium borate, potassium borate, ammonium borate, and sodium tetraborate.

[0015] Preferably, in step S4, the final concentration of hydrochloric acid is 0.3~0.5 mol / L, the acidolysis temperature is 80~120℃, and the hydrolysis time is 1~1.5h.

[0016] Preferably, the ultrafiltration membrane in step S5 has a molecular weight cutoff of 5 kDa; the dialysis bag has a molecular weight cutoff of 500-1000 Da; and the dialysis time is 48-72 hours.

[0017] This invention also provides an application of the low molecular weight kelp polysaccharide prepared by the above method in the preparation of anti-wrinkle and moisturizing skin care products.

[0018] Compared with the prior art, the beneficial effects of the present invention are: (1) In this invention, a compound bacterial agent of Bacillus subtilis and Lactobacillus casei is used to co-ferment kelp powder. The weak organic acids (such as lactic acid) accumulated in the fermentation broth and the extracellular polymeric substances secreted by the bacteria can gently break the hydrogen bond crosslinking between the residual cellulose and polysaccharides in the kelp cell wall. This can not only improve the extraction rate of kelp polysaccharides, but also change the aggregated structure of the extracted kelp polysaccharides from a tight bundle to a relatively loose extended and compliant conformation. The loose conformation makes the glycosidic bonds more exposed and more accessible during acid hydrolysis. This invention only requires a lower acid concentration and a shorter time to achieve efficient degradation, avoiding the excessive impact of high-intensity acid hydrolysis on the sulfate groups. At the same time, the polysaccharide chains obtained by this invention provide more spatial sites and hydroxyl binding sites, so that the subsequently added composite protective agent can be more evenly wrapped around the sugar chains to form a dense protective layer.

[0019] (2) In this invention, a composite protective agent composed of borate, glycerol, and sodium alginate is added during the acid hydrolysis process. Borate can form reversible borate ester bonds with the hydroxyl groups on the polysaccharide chains, providing scaffold protection for the sugar chain conformation and mitigating excessive acid attack on the glycosidic bonds. Glycerol, as a hydrogen bond donor, can reduce the dielectric constant of the reaction system, weakening the electrophilic attack of protons on the sulfate ester bonds. Sodium alginate forms a physical cross-linking network with the sulfate esters of brown algae polysaccharides through intermolecular interactions, further shielding the hydrated protons around the sulfate groups. The synergistic effect of these three agents ensures that acid hydrolysis effectively reduces the molecular weight while significantly inhibiting the hydrolytic breakage of sulfate ester bonds, resulting in a significantly higher sulfate group content in the final product compared to products from traditional acid hydrolysis methods. This ensures that the low molecular weight product still maintains excellent biological activity.

[0020] (3) The low molecular weight kelp polysaccharide prepared by this invention can rapidly dissolve into a clear and transparent highly fluid solution within minutes at room temperature. Under the same solid content, its solution viscosity is significantly reduced compared to before degradation, and there is no flocculation or turbidity. This rapid dissolution, low viscosity, and high clarity characteristic makes it easy to be compounded in various skin care lotion systems as an efficacy additive. At the same time, the increased sulfate group content and decreased molecular weight significantly improve the transdermal absorption rate of the polysaccharide. In vitro activity tests show that its antioxidant, anti-inflammatory, and fibroblast proliferation-promoting activities are all superior to those of undegraded high molecular weight kelp polysaccharide, and it can be directly used as an efficacy additive in anti-wrinkle and moisturizing skin care products, with broad application prospects. The entire preparation method has a clear process route, mild operating conditions, and safe and inexpensive reagents. No special equipment is required, it is easy to scale up production, and the cost is controllable, resulting in good economic and social benefits. Attached Figure Description

[0021] Figure 1 The following are the 1H NMR spectra of the low molecular weight kelp polysaccharides prepared in Example 3 and Comparative Example 1 of this invention; Figure 2The following are the Fourier transform infrared (FT-IR) spectra of the low molecular weight kelp polysaccharides prepared in Example 3 and Comparative Example 1 of this invention. Figure 3 XPS images of the low molecular weight kelp polysaccharides prepared in Example 3 and Comparative Example 1 of this invention; Figure 4 The graphs show the DPPH free radical scavenging rate of the low molecular weight kelp polysaccharides prepared in Example 3 and Comparative Examples 1-7 of this invention as a function of concentration. Figure 5 The graphs show the superoxide radical scavenging rate of the low molecular weight kelp polysaccharides prepared in Example 3 and Comparative Examples 1-7 of this invention as a function of concentration. Figure 6 The graphs show the scavenging rate of low molecular weight kelp polysaccharides prepared in Example 3 and Comparative Examples 1-7 of this invention as a function of concentration. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to specific embodiments, but is not limited thereto. The Bacillus subtilis used in this invention was purchased from Shanxi Luyin Biotechnology Co., Ltd., with a specification of 100 cfu / g; Lactobacillus casei was purchased from Shandong Jiacheng Biotechnology Co., Ltd., with a specification of 10 billion 100 cfu / g.

[0023] Example 1 A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash fresh or dried kelp to remove mud and impurities, dry it at 40-60℃ to constant weight, and pulverize it through a 40-80 mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water at a ratio of 1g:20mL, adjust the pH to 6.5, sterilize, and then inoculate with a compound microbial agent. The inoculation amount of the compound microbial agent is 0.5% of the kelp powder mass. Ferment for 2 days at a temperature of 25℃ and a humidity of 70%, and obtain the fermentation product after inactivation. The compound microbial agent is composed of Bacillus subtilis and Lactobacillus casei in a mass ratio of 2:1.

[0024] S3, extract kelp polysaccharides from the fermentation product obtained in step S2; S4. Dissolve the kelp polysaccharide obtained in step S3 in water to obtain a 5% kelp polysaccharide solution. Add hydrochloric acid and a composite protectant to the solution and then perform acid hydrolysis. The final concentration of hydrochloric acid is 0.3 mol / L, the acid hydrolysis temperature is 100℃, and the acid hydrolysis time is 1.5 h. The amount of composite protectant added is 0.5% of the mass of kelp polysaccharide. The composite protectant is composed of sodium borate, glycerol, and sodium alginate in a mass ratio of 2:1:1.

[0025] S5. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 6.8 using saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to obtain the supernatant. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The retentate is then further dialyzed. The molecular weight cutoff of the dialysis bag is 500~1000 Da, and the dialysis time is 48~72 hours. The dialysis solution is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0026] The specific method for extracting kelp polysaccharides in step S3 is as follows: Add 3 times the amount of distilled water to the kelp fermentation product, heat and extract 2-3 times at 90℃, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for 12 hours for alcohol precipitation, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharides.

[0027] Example 2 A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash fresh or dried kelp to remove mud and impurities, dry it at 40-60℃ to constant weight, and pulverize it through a 40-80 mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water at a ratio of 1g:20mL, adjust the pH to 6.5, sterilize, and then inoculate with a compound microbial agent. The inoculation amount of the compound microbial agent is 1.5% of the mass of the kelp powder. Ferment for 1 day at a temperature of 35℃ and a humidity of 80%, and obtain the fermentation product after inactivation. The compound microbial agent is composed of Bacillus subtilis and Lactobacillus casei in a mass ratio of 2:1.

[0028] S3, extract kelp polysaccharides from the fermentation product obtained in step S2; S4. Dissolve the kelp polysaccharide obtained in step S3 in water to obtain a kelp polysaccharide solution with a mass concentration of 8%. Add hydrochloric acid and a composite protective agent to the solution and then perform acid hydrolysis treatment. The final concentration of hydrochloric acid is 0.5 mol / L, the acid hydrolysis temperature is 120℃, and the acid hydrolysis time is 1h. The amount of composite protective agent added is 1.5% of the mass of kelp polysaccharide. The composite protective agent is composed of potassium borate, glycerol, and sodium alginate in a mass ratio of 2:1:1.

[0029] S5. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 7.2 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to obtain the supernatant. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The retentate is then further dialyzed. The molecular weight cutoff of the dialysis bag is 500~1000 Da, and the dialysis time is 72 hours. The dialysis solution is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0030] The specific method for extracting kelp polysaccharides in step S3 is as follows: Add 3 times the amount of distilled water to the kelp fermentation product, heat and extract 2-3 times at 95℃, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for 16 h for alcohol precipitation, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharides.

[0031] Example 3 A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash fresh or dried kelp to remove mud and impurities, dry it at 40-60℃ to constant weight, and pulverize it through a 40-80 mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water at a ratio of 1g:20mL, adjust the pH to 6.5, sterilize, and then inoculate with a compound microbial agent. The inoculation amount of the compound microbial agent is 1.0% of the mass of the kelp powder. Ferment for 2 days at a temperature of 30℃ and a humidity of 75%, and obtain the fermentation product after inactivation. The compound microbial agent is composed of Bacillus subtilis and Lactobacillus casei in a mass ratio of 2:1.

[0032] S3, extract kelp polysaccharides from the fermentation product obtained in step S2; S4. Dissolve the kelp polysaccharide obtained in step S3 in water to obtain a 6% kelp polysaccharide solution. Add hydrochloric acid and a composite protectant to the solution and then perform acid hydrolysis. The final concentration of hydrochloric acid is 0.4 mol / L, the acid hydrolysis temperature is 110℃, and the acid hydrolysis time is 1.2 h. The amount of composite protectant added is 1.0% of the mass of kelp polysaccharide. The composite protectant is composed of ammonium borate, glycerol, and sodium alginate in a mass ratio of 2:1:1.

[0033] S5. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 7.0 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to obtain the supernatant. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The retentate is then further dialyzed. The molecular weight cutoff of the dialysis bag is 500~1000 Da, and the dialysis time is 60 hours. The dialysis solution is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0034] The specific method for extracting kelp polysaccharides in step S3 is as follows: Add 3 times the amount of distilled water to the kelp fermentation product, heat and extract 2-3 times at 95℃, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for 14 h for alcohol precipitation, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharides.

[0035] Comparative Example 1 A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash the dried kelp to remove mud and impurities, dry it at 50°C to constant weight, and pulverize it through a 60-mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water at a ratio of 1g:20mL, heat and extract 3 times at 95℃, adding 3 times the amount of distilled water each time, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for 14 h for alcohol precipitation, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharide. S3, dissolve the kelp polysaccharide obtained in step S2 in distilled water to prepare a kelp polysaccharide solution with a mass concentration of 6%, add hydrochloric acid to a final concentration of 0.4 mol / L, and acid hydrolyze at 110℃ for 1.2 h without adding any protective agent during the acid hydrolysis process; S4. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 7.0 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to separate the supernatant. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The retentate is further dialyzed for 60 hours using a dialysis bag with a molecular weight cutoff of 500~1000 Da. The dialysate is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0036] This comparative example is basically the same as Example 3, except that: the microbial fermentation step in S2 is omitted, and the polysaccharide is extracted directly using the ordinary hydrothermal method, and no protective agent is used in the acid hydrolysis.

[0037] Comparative Example 2 A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash the dried kelp to remove mud and impurities, dry it at 50°C to constant weight, and pulverize it through a 60-mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water at a ratio of 1g:20mL, adjust the pH to 6.5, sterilize, and then inoculate with a compound microbial agent. The inoculation amount of the compound microbial agent is 1.0% of the mass of the kelp powder. The compound microbial agent is composed of Bacillus subtilis and Lactobacillus casei in a mass ratio of 2:1. Ferment for 2 days at a temperature of 30℃ and a humidity of 75%, and obtain the fermentation product after inactivation. S3. Extract kelp polysaccharide from the fermentation product obtained in step S2: Add 3 times the amount of distilled water to the kelp fermentation product, heat and extract 3 times at 95℃, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for alcohol precipitation for 14 h, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharide; S4. Dissolve the kelp polysaccharide obtained in step S3 in distilled water to prepare a kelp polysaccharide solution with a mass concentration of 6%. Add hydrochloric acid to the final concentration of 0.4 mol / L and acid hydrolyze at 110℃ for 1.2 h. No protective agent is added during the acid hydrolysis process. S5. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 7.0 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to separate the supernatant. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The retentate is further dialyzed for 60 hours using a dialysis bag with a molecular weight cutoff of 500~1000 Da. The dialysate is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0038] This comparative example is basically the same as Example 3, except that no protective agent is used in step S4 acid hydrolysis.

[0039] Comparative Example 3 A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash the dried kelp to remove mud and impurities, dry it at 50°C to constant weight, and pulverize it through a 60-mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water at a ratio of 1g:20mL, heat and extract 3 times at 95℃, adding 3 times the amount of distilled water each time, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for 14 h for alcohol precipitation, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharide. S3, dissolve the kelp polysaccharide obtained in step S2 in distilled water to prepare a 6% (w / w) kelp polysaccharide solution, add hydrochloric acid and a composite protective agent, and then perform acid hydrolysis treatment; the final concentration of hydrochloric acid is 0.4 mol / L, the acid hydrolysis temperature is 110℃, and the acid hydrolysis time is 1.2 h; the amount of composite protective agent added is 1.0% of the mass of kelp polysaccharide; the composite protective agent is composed of ammonium borate, glycerol, and sodium alginate in a mass ratio of 2:1:1.

[0040] S4. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 7.0 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to separate the supernatant. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The retentate is further dialyzed for 60 hours using a dialysis bag with a molecular weight cutoff of 500~1000 Da. The dialysate is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0041] This comparative example is basically the same as Example 3, except that there is no microbial fermentation process in step S2, and conventional hydrothermal extraction is used directly.

[0042] Comparative Example 4 A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash the dried kelp to remove mud and impurities, dry it at 50°C to constant weight, and pulverize it through a 60-mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water at a ratio of 1g:20mL, adjust the pH to 6.5, sterilize, and then inoculate with a compound microbial agent. The inoculation amount of the compound microbial agent is 1.0% of the mass of the kelp powder. The compound microbial agent is composed of Bacillus subtilis and Lactobacillus casei in a mass ratio of 2:1. Ferment for 2 days at a temperature of 30℃ and a humidity of 75%, and obtain the fermentation product after inactivation. S3. Extract kelp polysaccharide from the fermentation product obtained in step S2: Add 3 times the amount of distilled water to the kelp fermentation product, heat and extract 3 times at 95℃, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for alcohol precipitation for 14 h, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharide; S4. Dissolve the kelp polysaccharide obtained in step S3 in distilled water to prepare a kelp polysaccharide solution with a mass concentration of 6%. Add hydrochloric acid to a final concentration of 0.4 mol / L, and add ammonium borate at a mass of 1.0% of the kelp polysaccharide as a single protective agent. Acid hydrolysis is carried out at 110℃ for 1.2 h. S5. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 7.0 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to separate the supernatant. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The retentate is further dialyzed for 60 hours using a dialysis bag with a molecular weight cutoff of 500~1000 Da. The dialysate is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0043] This comparative example is basically the same as Example 3, except that only ammonium borate is added as a single protective agent during the acid hydrolysis process.

[0044] Comparative Example 5 A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash the dried kelp to remove mud and impurities, dry it at 50°C to constant weight, and pulverize it through a 60-mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water at a ratio of 1g:20mL, adjust the pH to 6.5, sterilize, and then inoculate with a compound microbial agent. The inoculation amount of the compound microbial agent is 1.0% of the mass of the kelp powder. The compound microbial agent is composed of Bacillus subtilis and Lactobacillus casei in a mass ratio of 2:1. Ferment for 2 days at a temperature of 30℃ and a humidity of 75%, and obtain the fermentation product after inactivation. S3. Extract kelp polysaccharide from the fermentation product obtained in step S2: Add 3 times the amount of distilled water to the kelp fermentation product, heat and extract 3 times at 95℃, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for alcohol precipitation for 14 h, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharide; S4, dissolve the kelp polysaccharide obtained in step S3 in distilled water to prepare a kelp polysaccharide solution with a mass concentration of 6%, add hydrochloric acid to a final concentration of 0.4 mol / L, and add a composite protective agent of 1.0% by mass of kelp polysaccharide, wherein the composite protective agent is composed of ammonium borate and glycerol in a mass ratio of 2:1 (excluding sodium alginate), and acid hydrolyze at 110℃ for 1.2 h; S5. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 7.0 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to separate the supernatant. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The retentate is further dialyzed for 60 hours using a dialysis bag with a molecular weight cutoff of 500~1000 Da. The dialysate is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0045] This comparative example is basically the same as Example 3, except that the composite protective agent in step S4 lacks the sodium alginate component and consists only of ammonium borate and glycerol.

[0046] Comparative Example 6 A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash the dried kelp to remove mud and impurities, dry it at 50°C to constant weight, and pulverize it through a 60-mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water at a ratio of 1g:20mL, adjust the pH to 6.5, sterilize, and then inoculate with a compound microbial agent. The inoculation amount of the compound microbial agent is 1.0% of the mass of the kelp powder. The compound microbial agent is composed of Bacillus subtilis and Lactobacillus casei in a mass ratio of 2:1. Ferment for 2 days at a temperature of 30℃ and a humidity of 75%, and obtain the fermentation product after inactivation. S3. Extract kelp polysaccharide from the fermentation product obtained in step S2: Add 3 times the amount of distilled water to the kelp fermentation product, heat and extract 3 times at 95℃, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for alcohol precipitation for 14 h, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharide; S4. Dissolve the kelp polysaccharide obtained in step S3 in distilled water to prepare a kelp polysaccharide solution with a mass concentration of 6%. Add hydrochloric acid to a final concentration of 0.4 mol / L, and add a composite protective agent of 1.0% by mass of kelp polysaccharide. The composite protective agent is composed of ammonium borate and sodium alginate in a mass ratio of 2:1 (excluding glycerol). Acid hydrolysis is carried out at 110℃ for 1.2 h. S5. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 7.0 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to separate the supernatant. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The retentate is further dialyzed for 60 hours using a dialysis bag with a molecular weight cutoff of 500~1000 Da. The dialysate is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0047] This comparative example is basically the same as Example 3, except that the composite protectant in step S4 lacks the glycerol component and consists only of ammonium borate and sodium alginate.

[0048] Comparative Example 7 A method for preparing low molecular weight kelp polysaccharide includes the following steps: S1. Wash the dried kelp to remove mud and impurities, dry it at 50°C to constant weight, and pulverize it through a 60-mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water at a ratio of 1g:20mL, adjust the pH to 6.5, sterilize, and then inoculate with a compound microbial agent. The inoculation amount of the compound microbial agent is 1.0% of the mass of the kelp powder. The compound microbial agent is composed of Bacillus subtilis and Lactobacillus casei in a mass ratio of 2:1. Ferment for 2 days at a temperature of 30℃ and a humidity of 75%, and obtain the fermentation product after inactivation. S3. Extract kelp polysaccharide from the fermentation product obtained in step S2: Add 3 times the amount of distilled water to the kelp fermentation product, heat and extract 3 times at 95℃, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevag method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for alcohol precipitation for 14 h, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharide; S4, dissolve the kelp polysaccharide obtained in step S3 in distilled water to prepare a kelp polysaccharide solution with a mass concentration of 6%, add hydrochloric acid to a final concentration of 0.4 mol / L, and add a composite protective agent of 1.0% by mass of kelp polysaccharide, wherein the composite protective agent is composed of glycerol and sodium alginate in a mass ratio of 1:1 (excluding borate), and acid hydrolyze at 110℃ for 1.2 h; S5. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 7.0 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to separate the supernatant. The supernatant is purified by ultrafiltration through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The retentate is further dialyzed for 60 hours using a dialysis bag with a molecular weight cutoff of 500~1000 Da. The dialysate is freeze-dried to obtain low molecular weight kelp polysaccharide.

[0049] This comparative example is basically the same as Example 3, except that the composite protective agent in step S4 lacks the borate component and consists only of glycerol and sodium alginate.

[0050] Characterization test cases The low molecular weight kelp polysaccharide freeze-dried products obtained in Examples 1-3 and Comparative Examples 1-7, as well as the undegraded kelp polysaccharide raw material (fermented kelp polysaccharide obtained in step S3 of Example 3 without acid hydrolysis), were subjected to the following performance tests.

[0051] 1. Determination of molecular weight The weight-average molecular weight (Mw) of each sample was determined by high-performance gel permeation chromatography (HPGPC). The chromatographic conditions were as follows: a TSK-GEL G4000PWXL column (7.8 mm × 300 mm) tandem with a TSK-GEL G2500PWXL column (7.8 mm × 300 mm); a mobile phase of 0.1 mol / L NaNO3 solution; a flow rate of 0.6 mL / min; a column temperature of 35℃; an injection volume of 20 μL; and detection using a refractive index detector (RID). Standard curves were constructed using a series of dextran standards with different molecular weights (Mw of 180, 2700, 9750, 36800, 135350, and 1000000 Da), and the molecular weight of the samples was calculated based on the retention time. The results are shown in Table 1.

[0052] Table 1 Test Results As shown in Table 1, the weight-average molecular weight of the low molecular weight kelp polysaccharides in all examples and comparative examples decreased to 7.5 × 10⁻⁶. 3 ~8.5×10 3 Within the Da range, they are at the same molecular weight level. Among them, the polysaccharide yields of Examples 1-3 and Comparative Examples 2 and 4-7, which underwent microbial fermentation pretreatment, were significantly higher than those of Comparative Examples 1 and 3, which were not fermented, indicating that the combined fermentation of Bacillus subtilis and Lactobacillus casei can effectively disrupt the cell walls of kelp and improve the polysaccharide extraction rate.

[0053] 2. Determination of sulfate content The sulfate content in the samples was determined using the barium chloride-gelatin turbidimetric method. 10.0 mg of the polysaccharide sample was accurately weighed and added to 1.0 mL of 1 mol / L hydrochloric acid solution. The mixture was hydrolyzed at 105 °C for 4 hours. After drying the hydrolysate under nitrogen, it was diluted to 10.0 mL with ultrapure water. An appropriate amount of the hydrolysate was added to the barium chloride-gelatin solution, mixed well, and the absorbance was measured at 360 nm. A standard curve was plotted using potassium sulfate as a standard (concentration range 10–100 μg / mL), and the mass percentage of sulfate (expressed as SO42-) was calculated. 2- (Calculation). The sulfate retention rate is calculated using the following formula: Sulfate group retention rate (%) = (Sulfate group content of polysaccharide after acid hydrolysis / Sulfate group content of polysaccharide before acid hydrolysis) × 100% The sulfate content of polysaccharides before acid hydrolysis was determined based on the undegraded kelp polysaccharide raw material (fermented kelp polysaccharide obtained in step S3 of Example 3 without acid hydrolysis).

[0054] Table 2 Test Results As shown in Table 2, the sulfate group content of Examples 1-3 of the present invention is 28.4%~29.1%, which is significantly higher than that of Comparative Examples 1 and 2 without protective agent treatment, and also significantly higher than that of Comparative Example 4 with only a single borate added and Comparative Examples 5-7 without any protective agent component. Given that the molecular weights of all examples and comparative examples are at the same level (see Table 1), the difference in sulfate group content directly demonstrates the protective effect of the composite protective agent of the present invention on the sulfate ester bond.

[0055] Specifically, comparing Example 3 and Comparative Example 3, both included a complete ternary composite protectant. However, Example 3, after microbial fermentation pretreatment, showed an 8.2 percentage point increase in sulfate group content, indicating that fermentation transformed the polysaccharide chains from a tightly bundled structure to a loosely extended conformation, providing more binding sites for the protectant and thus enhancing its protective effect. Comparing Example 3 with Comparative Examples 5-7, the absence of any one of the components—borate, glycerol, or sodium alginate—in the composite protectant resulted in a 5.2-6.6 percentage point decrease in sulfate group content, confirming the synergistic protective effect of all three components. These results demonstrate that the present invention, through a synergistic approach of microbial fermentation pretreatment and a ternary composite protectant, effectively reduces molecular weight while maximizing the retention of sulfate groups.

[0056] 3. Nuclear magnetic resonance and infrared spectroscopy (FT-IR) determination Polysaccharide samples obtained in Example 3 and Comparative Example 1 were used as test samples. Nuclear magnetic resonance (NMR) spectra were performed at 25°C with 4000 scans on a Bruker AVANCE NMR spectrometer (500 MHz, Bruker, Switzerland). Approximately 2.0 mg of the polysaccharide samples obtained in Example 3 and Comparative Example 1 were mixed with 200.0 mg of dry KBr powder, ground evenly, and pressed into transparent thin films with a diameter of 13 mm. Fourier transform infrared (FTIR) spectra were obtained from 4000 to 400 cm⁻¹. -1 Scan within range, resolution 4 cm -1 The scan was performed 32 times, with background subtraction using a blank KBr slide as the background. Results are shown below. Figures 1-2 .

[0057] In the 1H NMR spectrum ( Figure 1The polysaccharide 1H NMR structures obtained in Example 3 and Comparative Example 1 showed no significant differences. The signal at approximately 1.20 ppm was attributed to H-6 of α-L-fucopyranose, and the signal at 1.88 ppm was attributed to the methyl proton of N-acetylgalactosamine. The signal at 3.68 ppm was attributed to H-4 of α-L-fucopyranose or H-2 of α-D-galactopyranose, the signal at 3.91 ppm was attributed to H-2 of α-D-glucuronic acid, the signal at 5.34 ppm was attributed to H-1 of α-L-fucopyranose, and the signal at 4.14 ppm was designated as H-2 of β-D-mannopyranose or H-4 of α-L-fucopyranose.

[0058] In the infrared spectrum ( Figure 2 The polysaccharide structures obtained in Example 3 and Comparative Example 1 showed no significant changes, except for the sulfate groups. At 3390 cm⁻¹ -1 At this location, a relatively large absorption peak is related to the stretching vibration of OH, which is consistent with the basic structure of polysaccharides reported in the literature. (2330 cm⁻¹) -1 The nearby absorption peaks are related to the stretching vibrations of triple bonds and cumulative double bonds in polysaccharides. (1625 cm⁻¹) -1 The nearby absorption peak is a characteristic peak of C=O. (1419 cm⁻¹) -1 The absorption peak is mainly caused by the stretching vibrations of C=O and COO- in the uronic acid in the polysaccharide, indicating that the polysaccharides extracted by both methods contain uronic acid. However, the peak is more pronounced in Example 3, suggesting that fermentation and acid hydrolysis may have produced more uronic acid. This finding can also significantly enhance the antioxidant activity of the polysaccharide. 1330 cm⁻¹ -1 The nearby absorption peaks indicate the presence of sulfate in the polysaccharide, likely due to the asymmetric stretching vibrations of S=O and COS. The significant enhancement of this peak in Example 3 suggests that enzymatic and acidic hydrolysis exposed more sulfate groups. (855 cm⁻¹) -1 The weaker absorption peak nearby may be related to the CH bending of β-galactose residues, 943 cm⁻¹ -1 The absorption peak at that point may be related to the stretching vibration of galactose-3,6-hydrate COC. Therefore, the polysaccharides obtained in Example 3 and Comparative Example 1 are acidic pyranose polysaccharides with α and β configurations, rich in fucose sulfate.

[0059] 4. X-ray photoelectron spectroscopy (XPS) measurement To further verify the retention of sulfate groups at the elemental chemical state level, XPS S2p high-resolution spectra were analyzed on the samples from Example 3 and Comparative Example 1. Polysaccharide samples obtained from Example 3 and Comparative Example 1 were pressed into smooth thin slices and analyzed using an X-ray photoelectron spectroscopy (ESCALAB 250Xi, Thermo Scientific). The excitation source was monochromatic Al Kα rays (hν = 1486.6 eV), with a beam diameter of 500 μm and a power of 150 W, and charge correction was performed using a C 1s binding energy of 284.8 eV. High-resolution spectra of the S 2p orbitals (binding energy range 164–174 eV) were acquired, and peak fitting was performed using Thermo Avantage software. The sulfate group (S) was calculated based on the peak area. 6+ The relative content of ).

[0060] The results are as follows Figure 3 As shown. Figure 3 In both Example 3 and Comparative Example 1, two characteristic peaks are observed in the S2p orbital, which are respectively attributed to S2p. 3 / 2 and S 2p 1 / 2 The difference is that in Example 3, compared to Comparative Example 1, S 2p 3 / 2 and S 2p 1 / 2 There was a slight migration, S 2p 3 / 2 Migrating from 168.8 eV to 168.7 eV, S 2p 1 / 2 The peak value migrated from 170.2 eV to 169.9 eV, but both clearly indicate that sulfur exists in the form of sulfate ester bonds, rather than other forms of sulfur (inorganic sulfates or thioethers). Comparing the peak areas of the two examples, it can be seen that the S2p characteristic peak area of ​​Example 3 is much larger than that of Comparative Example 1. Quantitative fitting calculations show that the relative content of sulfate groups in Example 3 is 11.2% (atomic percentage), while that in Comparative Example 1 is only 4.5%.

[0061] Application test cases 1. Transdermal absorption rate determination In vitro transdermal experiments were conducted using the Franz diffusion cell method. Isolated porcine skin was fixed in a diffusion cell with the stratum corneum facing the supply cell. 1 mL of a 2% aqueous solution of the polysaccharides obtained in Examples 1-3 and Comparative Examples 1-7 was added to each cell. PBS buffer (pH 7.4) was added to the receiving cell, and the mixture was stirred in a 37°C water bath. Samples were taken from the receiving cell at different time points (1, 2, 4, 6, 8, 12, and 24 h). The polysaccharide content in the receiving solution was determined using the phenol-sulfuric acid method, and the cumulative transdermal absorption rate was calculated.

[0062] Table 3 Results of transdermal absorption rate test As shown in Table 3, the cumulative transdermal absorption rate of each embodiment over 24 hours was significantly higher than that of the comparative examples, and far higher than that of the undegraded polysaccharide molecular weight raw materials. This indicates that the low molecular weight kelp polysaccharide prepared by this invention, due to its moderately reduced molecular weight and loose and flexible molecular conformation, is more likely to penetrate the stratum corneum of the skin; at the same time, the complete retention of sulfate groups also enhances the hydrophilicity of the polysaccharide molecules and their affinity with skin tissue.

[0063] With the molecular weights of the examples and comparative examples at the same level, the differences in transdermal absorption rates were mainly attributed to the different sulfate group content and molecular conformations. Comparative Example 1 showed the lowest transdermal absorption rate, while Example 2 showed the highest. The transdermal absorption rate of Comparative Example 3 was 64.1% lower than that of Example 3, further indicating that the fermentation-induced loose conformation not only facilitates sulfate group retention but also directly improves the transdermal properties of the polysaccharide.

[0064] 2. DPPH free radical scavenging activity test Take 2 mL of sample solutions of polysaccharides at different concentrations (0.1-10 mg / mL) obtained in Example 3 and Comparative Examples 1-7, respectively, add 2 mL of 0.2 mmol / L DPPH anhydrous ethanol solution, mix well, and react at 25°C in the dark for 30 minutes. Measure the absorbance A1 at 517 nm. A0 represents the reaction solution containing 2 mL of DPPH and 3 mL of ethanol. A2 represents 1 mL and 4 mL of 95% ethanol. Ascorbic acid was used as a positive control. Calculate the DPPH free radical scavenging rate. Results are shown below. Figure 4 As shown.

[0065] DPPH free radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100% like Figure 4 As shown, the DPPH radical scavenging rates of each example were significantly better than those of the comparative examples, and exhibited a good concentration-dependent effect. At a concentration of 1.0 mg / mL, the DPPH scavenging rates of Examples 1-3 were 63.4%–66.8%, while those of Comparative Example 1 were only 38.2%, Comparative Example 2 was 40.5%, and Comparative Example 3 was 45.6%. Even compared with Comparative Example 3, which had a complete protective agent added, Example 3 (65.2%) was still 19.6 percentage points higher, indicating that fermentation pretreatment not only increased the sulfate content but also exposed more active sites by changing the polysaccharide conformation, thereby enhancing antioxidant activity.

[0066] It is worth noting that, compared with Comparative Example 2, at a concentration of 1.0 mg / mL, the DPPH scavenging rate of Example 3 was much higher than that of Comparative Example 2, with a difference of 24.7 percentage points, further confirming that the sulfate group content is the key factor determining the antioxidant activity of kelp polysaccharides.

[0067] 3. Superoxide radical scavenging activity test The superoxide radical scavenging activity of the low molecular weight kelp polysaccharide prepared in Example 3 and the low molecular weight kelp polysaccharides prepared in Comparative Examples 1-7 was investigated using the NADH-PMS-NBT method. Specifically, samples of different concentrations (0.01-0.5 mg / mL) were prepared using Tris-HCl buffer (pH 8.0). For each sample, 0.1 mL was added to 1 mL of 557 μM NADH (dissolved in 16 mM Tris-HCl, pH 8.0), 1 mL of 45 μM PMS (dissolved in 16 mM Tris-HCl, pH 8.0), and 1 mL of 108 μM NBT (dissolved in 16 mM Tris-HCl, pH 8.0). The reaction was allowed to proceed at room temperature for 5 min, and the absorbance at 560 nm was measured as A1; A0 contained all reagents but no sample; A2 contained both sample and reagents, but Tris-HCl was used instead of NBT, with ascorbic acid as a positive control. Results are shown below. Figure 5 As shown.

[0068] Superoxide radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100% like Figure 5 As shown, the superoxide radical scavenging rates of each embodiment reached 42.3%–45.8% at a concentration of 0.1 mg / mL, significantly higher than Comparative Example 1 (20.5%) and Comparative Example 2 (22.3%). At a concentration of 0.5 mg / mL, the scavenging rate of Example 2 was as high as 76.8%, close to the positive control ascorbic acid (86.3%), while that of Comparative Example 1 was only 38.4%. The ranking of superoxide radical scavenging activities was basically consistent with the ranking of sulfate content. This further verifies the decisive role of sulfate content in free radical scavenging activity.

[0069] 4. Hydroxyl radical scavenging activity test Polysaccharide samples of different concentrations (0.1-10.0 mg / mL) were prepared. 2 mL of each sample was added to 2 mL of 5 mmol / L FeSO4 and 2 mL of 5 mmol / L salicylic acid ethanol solution; then 2 mL of 5 mmol / L H2O2 was added. The samples were incubated at 37℃ for 30 min, cooled to room temperature, and the absorbance was measured at 510 nm (A1). Ascorbic acid was used as a positive control. A0 represents reagents (no sample); A2 represents the absence of hydroxyl radicals. Results are shown below. Figure 6 .

[0070] Hydroxyl radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100% like Figure 6As shown, the hydroxyl radical scavenging rates of each example reached 55.6%–59.2% at a concentration of 1.0 mg / mL, significantly higher than Comparative Example 1 (30.5%) and Comparative Example 2 (32.8%). At a concentration of 10.0 mg / mL, the scavenging rate of Example 2 was as high as 88.9%, which was quite close to that of the positive control ascorbic acid (97.8%). Consistent with the patterns of DPPH and superoxide radical scavenging activity, the hydroxyl radical scavenging activity also showed a high positive correlation with the sulfate group content.

[0071] Examples 1-3 of this invention exhibited significantly superior scavenging activity compared to the comparative examples in three antioxidant evaluation systems: DPPH radical, superoxide radical, and hydroxyl radical. The ranking of the three free radical scavenging activities was highly consistent with the ranking of sulfate group content, indicating that higher sulfate group content correlated with stronger antioxidant activity. This confirms that sulfate groups are the key active functional groups for the antioxidant function of kelp polysaccharides. These results demonstrate that the low molecular weight kelp polysaccharides prepared by this invention possess low molecular weight, high sulfate group content, excellent transdermal absorption, and significant antioxidant activity. They can be directly used as functional additives in the preparation of anti-wrinkle and moisturizing skincare products, showing promising application prospects.

[0072] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A method for preparing low molecular weight kelp polysaccharide, characterized in that, Includes the following steps: S1. Wash fresh or dried kelp to remove mud and impurities, dry it at 40-60℃ to constant weight, and pulverize it through a 40-80 mesh sieve to obtain dried kelp powder. S2, add kelp powder to distilled water according to the material-liquid ratio, adjust the pH to 6.5, sterilize, and then inoculate with compound microbial agent. Ferment for 1-2 days at a temperature of 25-35℃ and a humidity of 70-80%, and obtain the fermentation product after inactivation. S3, extract kelp polysaccharides from the fermentation product obtained in step S2; S4, dissolve the kelp polysaccharide obtained in step S3 in water to obtain a kelp polysaccharide solution, add hydrochloric acid and a composite protective agent to it and then perform acid hydrolysis treatment; S5. After the acid hydrolysis reaction is completed, the pH of the system is immediately adjusted to 6.8-7.2 with saturated sodium hydroxide solution to terminate the acid hydrolysis reaction. The solution is centrifuged to obtain the supernatant. The supernatant is purified by ultrafiltration membrane. The retentate is further dialyzed. The dialysate is freeze-dried to obtain low molecular weight kelp polysaccharide.

2. The method for preparing low molecular weight kelp polysaccharide according to claim 1, characterized in that, In step S2, the ratio of dried kelp powder to distilled water is 1g:20mL.

3. The method for preparing low molecular weight kelp polysaccharide according to claim 1, characterized in that, The compound microbial agent is composed of Bacillus subtilis and Lactobacillus casei in a mass ratio of 2:1; the inoculation amount of the compound microbial agent is 0.5% to 1.5% of the mass of dried kelp powder.

4. The method for preparing low molecular weight kelp polysaccharide according to claim 1, characterized in that, The specific method for extracting kelp polysaccharides in step S3 is as follows: Add 3 times the amount of distilled water to the kelp fermentation product, heat and extract 2-3 times at 90-95℃, combine the extracts, centrifuge to separate the supernatant, concentrate the supernatant under reduced pressure to 1 / 4 of the original volume, remove protein 5 times using the Sevage method, add pre-cooled 95% ethanol to the concentrated solution after protein removal to the final concentration of 70%, let stand at 4℃ for 12-16 h for alcohol precipitation, centrifuge to collect the precipitate, wash with anhydrous ethanol and acetone in sequence, and vacuum dry to obtain kelp polysaccharides.

5. The method for preparing low molecular weight kelp polysaccharide according to claim 1, characterized in that, The mass concentration of the kelp polysaccharide solution in step S4 is 5-8%.

6. The method for preparing low molecular weight kelp polysaccharide according to claim 1, characterized in that, In step S4, the composite protective agent is composed of borate, glycerol, and sodium alginate in a mass ratio of 2:1:1; the amount of the composite protective agent added is 0.5~1.5% of the mass of kelp polysaccharide.

7. The method for preparing low molecular weight kelp polysaccharide according to claim 6, characterized in that, The borate is one or more of sodium borate, potassium borate, ammonium borate, and sodium tetraborate.

8. The method for preparing low molecular weight kelp polysaccharide according to claim 1, characterized in that, In step S4, the final concentration of hydrochloric acid is 0.3~0.5 mol / L, the acidolysis temperature is 100~120℃, and the hydrolysis time is 1~1.5h.

9. The method for preparing low molecular weight kelp polysaccharide according to claim 1, characterized in that, The ultrafiltration membrane in step S5 has a molecular weight cutoff of 5 kDa; the dialysis bag has a molecular weight cutoff of 500-1000 Da, and the dialysis time is 48-72 hours.

10. The application of the low molecular weight kelp polysaccharide prepared by any one of claims 1-9 in the preparation of anti-wrinkle and moisturizing skin care products.

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