Whole-algae application extraction methods and uses of algal polysaccharides

Abstract

This invention discloses a whole-algae extraction method for algal polysaccharides and its applications. The method allows for the extraction of algal polysaccharides with prebiotic activity and rich in polysaccharides from algal raw materials, while also producing algal byproducts during the extraction process. Therefore, this whole-algae extraction method can produce specialized algal polysaccharides suitable for use in health foods or skincare products, and significantly reduces extraction waste, thus achieving environmental sustainability.

Description

[Technical Field]

[0001] This invention relates to an algae extraction technology and the application of the products obtained therefrom, particularly to a whole-algae extraction method for algal polysaccharides and its uses. [Previous Technology]

[0002] Note: Seaweed, also known as macroalgae, is a type of multicellular algae that grows in the sea and is visible to the naked eye. Common types include green algae, brown algae, red algae, and blue-green algae (also known as cyanobacteria). Because seaweed contains many nutrients, such as essential amino acids, proteins, vitamins, minerals, and polysaccharides, and recent studies have shown that seaweed has various health and beauty benefits, it has been widely used in food, health products, and skincare products.

[0003] To obtain the active ingredients in seaweed, extraction techniques are currently widely used to extract the desired components from seaweed. For example, seaweed polysaccharides are separated from seaweed thallus using methanol, ethyl acetate, and n-butanol as extraction solvents. However, while conventional extraction techniques can obtain the desired components, they still have the following drawbacks:

[0004] Firstly, the overall extraction process is cumbersome, requires the use of multiple organic solvents, and cannot be carried out in a continuous extraction manner, which leads to increased production costs;

[0005] Secondly, since a large amount of extraction waste is generated after extraction, it not only increases the cost of waste disposal for manufacturers, but also makes it difficult to achieve the goal of sustainable environmental protection. [Summary of the Invention]

[0006] The main objective of this invention is to provide a whole-algae extraction method for algae polysaccharides and their uses. The algae polysaccharides extracted by the method disclosed in this invention are rich in polysaccharides and special sugars, and have activities such as anti-allergy, regulation of intestinal flora, anti-inflammation, and promotion of wound repair. Therefore, they can be used as the main active ingredients in food or health care products.

[0007] Another objective of this invention is to provide a whole-algae extraction method for algal polysaccharides and its uses. Since the algal polysaccharide extraction method disclosed in this invention can produce usable algal by-products during the extraction process, it can effectively reduce the generation of extraction waste and achieve the effect of being environmentally friendly.

[0008] Therefore, in order to achieve the above-mentioned objectives, the present invention provides a whole-algae application extraction method for algal polysaccharides. The algal polysaccharides produced by the method disclosed in the present invention are rich in polysaccharides and have prebiotic, anti-allergic, anti-inflammatory, wound-healing and anti-ultraviolet activities. Therefore, by administering an effective amount of the algal polysaccharides disclosed in the present invention or a composition containing such algal polysaccharides to an individual, it is possible to regulate the intestinal flora and effectively achieve the effects of preventing or improving inflammation-related diseases, diseases related to intestinal flora imbalance, anti-skin aging and wound-healing.

[0009] The total sugar content of the algal polysaccharide is above 80 (w / w)%, the water soluble component is above 70 (w / w)%, and the viscosity is 2200-2510 cps. Furthermore, the monosaccharide composition of the algal polysaccharide includes: 56-63% galactose, 27-33% glucose, 5-6% xylose, and 3-5% mannose.

[0010] In one embodiment of the present invention, the total sugar content of the algal polysaccharide is 90 (w / w)% or more.

[0011] Furthermore, the whole-algae extraction method for algal polysaccharides disclosed in one embodiment of the present invention comprises the following three-stage extraction procedure, which is executed sequentially and continuously. Specifically, the first-stage extraction procedure involves extracting an algal raw material with a first extraction solvent to obtain a first extraction mixture; the second-stage extraction procedure involves collecting a first precipitate from the first extraction mixture and extracting it with a second extraction solvent to obtain a second extraction mixture; and the third-stage extraction procedure involves collecting a second precipitate from the second extraction mixture and extracting it with a third extraction solvent to obtain a third extraction mixture; filtering the third extraction mixture to obtain a third filtrate, which contains an algal polysaccharide, and the algal polysaccharide is rich in polysaccharides.

[0012] The extraction solvent used in the second stage extraction process is different from the extraction solvent used in the first and third stage extraction processes; and the extraction temperature of the first stage extraction process and the second stage extraction process is lower than the extraction temperature of the third stage extraction process.

[0013] In another embodiment of the present invention, the extraction temperatures of the first stage extraction process and the second stage extraction process are 10-40°C, and the extraction temperature of the third stage extraction process is approximately 90-100°C.

[0014] In another embodiment of the present invention, the extraction solvent used in the first and third stage extraction processes is water, while the extraction solvent used in the second stage extraction process is a lower alcohol, such as ethanol.

[0015] Furthermore, the extraction process at each stage of the whole-algae application extraction method for algal polysaccharides disclosed in this invention produces algal byproducts that can be used industrially, thus achieving the effects of whole-algae utilization, environmental protection, and enhanced economic value. Specifically, the first-stage extraction process produces an extract containing phycobiliproteins; the second-stage extraction process produces an extract containing phytochemicals; and the third-stage extraction process produces an extract containing algal polysaccharides and an extract containing seaweed fiber.

[0016] In a further embodiment of the present invention, it is disclosed that the algal polysaccharide produced by the whole algae application extraction method disclosed in the present invention can be used to prepare compositions for regulating intestinal flora, compositions for resisting ultraviolet damage, or cytokine inhibitors.

[0017] Wherein, the composition or the inhibitor may be a food, a health care product or a nutritional supplement.

[0018] When the algal polysaccharide is used to prepare a composition for regulating intestinal flora, the composition for regulating intestinal flora can be used to regulate the growth of good and bad bacteria in the intestine, so as to balance the intestinal flora. That is, the composition for regulating intestinal flora can increase the number of bacteria classified as Bifidobacteriaceae, Lactobacillusaceae, Muribaculaceae or Bacteroidaceae in the intestine, and increase the content of short-chain fatty acids in the intestine, while also inhibiting the growth of microorganisms classified as Clostridiaceae or Enterobacteriaceae.

[0019] Wherein, when the algal polysaccharide is used to prepare a composition for resisting ultraviolet damage, the composition for resisting ultraviolet damage can be used to increase the skin barrier so as to avoid skin damage or aging caused by ultraviolet radiation.

[0020] When the algal polysaccharide is used to prepare a cytokine inhibitor, the cytokine inhibitor can be used to inhibit the concentration of IL-4, IL-6, IL-8 or histamine, thereby achieving anti-allergic and / or anti-inflammatory effects.

[0021] When the algal polysaccharide is used to prepare a wound-healing composition, the wound-healing composition can enhance the expression of MMP-9, VEGF-A or KRT16 genes and promote epithelial cell migration, thereby promoting wound healing and repair.

Implementation Method

[0022] This invention discloses a whole-algae extraction method for algal polysaccharides and its uses. The whole-algae extraction method for algal polysaccharides disclosed in this invention mainly includes a three-stage extraction process, in which different extraction solvents and conditions are used to extract algal polysaccharides rich in polysaccharides from an algal raw material, and algal by-products are produced in each stage of the extraction process. The algal polysaccharides produced by the extraction method disclosed in this invention have a variety of biological activities, such as regulating intestinal flora, resisting ultraviolet damage, anti-inflammatory, and anti-allergic. Therefore, by administering an effective amount of algal polysaccharides to an individual, it is possible to improve allergy-related diseases, inflammation or related diseases, intestinal flora imbalance-related diseases, anti-skin aging, and promote wound healing.

[0023] Specifically, the total sugar content of the algal polysaccharide disclosed in this invention is higher than 80 (w / w)%, the water-soluble component is higher than 70 (w / w)%, and the viscosity is 2200-2510 cps. The main monosaccharides of the algal polysaccharide are galactose, glucose, xylose and mannose, with contents of 56-63%, 27-33%, 5-6%, and 0.5-2%, respectively, and the contents of other sugars are about 3-5%.

[0024] The extraction solvent used in the second stage extraction process is different from the extraction solvent used in the first and third stage extraction processes. For example, the extraction solvent used in the first and third stage extraction processes is water, while the extraction solvent used in the second stage extraction process is a lower alcohol, such as ethanol with a concentration of 75-90%; or the extraction method used in the first and third stage extraction processes is solvent extraction, and the extraction solvent used is water, while the extraction method used in the second stage extraction process is supercritical extraction, and the extraction solvent used is carbon dioxide.

[0025] The extraction temperatures of the first-stage extraction procedure and the second-stage extraction procedure are 10-40℃, respectively, and the temperature of the third-stage extraction procedure is higher than that of the first-stage extraction procedure and the second-stage extraction procedure. For example, the extraction temperature of the first-stage extraction procedure is 20-40℃, such as 20℃, 25℃, 30℃, 35℃, and 40℃; the extraction temperature of the second-stage extraction procedure is 10-30℃, such as 10℃, 15℃, 18℃, 20℃, 25℃, 28℃, and 30℃; and the extraction temperature of the third-stage extraction procedure is 90-100℃, such as 90℃, 95℃, 98℃, and 100℃.

[0026] Furthermore, the algal by-products produced in each stage of the extraction process of the whole algae application extraction method of the present invention can be utilized by different industries or different products, thus achieving the effects of whole algae utilization, enhancing economic value and environmental sustainability.

[0027] Specifically, the first stage extraction process produces an extract containing phycobiliproteins; the second stage extraction process produces an extract containing phytochemicals; and the third stage extraction process produces the algal polysaccharide disclosed in this invention and an algal fiber.

[0028] The crude products obtained in each stage of the extraction process are then dried to improve the convenience of transportation and storage. The drying process is a moisture removal technique commonly used by those skilled in the art, such as freeze drying, spray drying, and hot air drying.

[0029] Unless otherwise specified, the scientific terms or nouns contained in the specification and claims of this invention are interpreted based on textbooks, dictionaries or common knowledge in the technical field to which this invention pertains.

[0030] The term "algae raw material" refers to seaweed that has been washed, powdered, crushed, cut, dried or otherwise processed, such as algae powder; wherein the seaweed includes red algae, brown algae, etc.

[0031] The term "sea fungus (Sarcodia montagneana)" refers to a type of large red algae that grows in the ocean.

[0032] The term "phycobiliprotein" refers to water-soluble proteins found in algae, which are complexes composed of various proteins.

[0033] The term "phytochemicals" refers to essential elements for algal growth. They can provide algae with color and odor, and also provide algae with the ability to protect themselves, such as by scavenging free radicals.

[0034] The term "seaweed fiber" refers to the fiber obtained from seaweed.

[0035] The term "composition" refers to a mixture containing the algal polysaccharide disclosed in this invention, such as a health product, food, or nutritional supplement. For example, if the composition is a food product, it contains the algal polysaccharide disclosed in this invention and a food-acceptable component, and can be produced in different forms as needed, such as beverages, biscuits, or jellies; if the composition is a health product, its main active ingredient contains the algal polysaccharide disclosed in this invention, and a health product-acceptable carrier and / or excipient; if the composition is a nutritional supplement, its main active ingredient contains the algal polysaccharide disclosed in this invention, and a nutritional supplement-acceptable carrier and / or excipient.

[0036] The term "active ingredient" refers to an ingredient that can achieve its claimed efficacy or use. For example, if A is the active ingredient of a blood sugar lowering composition, it means that A has the activity of lowering or stabilizing blood sugar.

[0037] The term "metabolic disease" in this invention refers to metabolic diseases related to intestinal flora imbalance, such as hyperglycemia, hyperlipidemia or obesity caused by intestinal flora imbalance.

[0038] The term "gut microbiota imbalance-related diseases" refers to diseases caused by the number of bad bacteria in the gut being greater than the number of good bacteria. According to recent studies, gut microbiota imbalance-related diseases include depression, Alzheimer's disease, and immune diseases.

[0039] The numerical values ​​disclosed in this invention are interpreted as including the actual value of the value and the value plus or minus an acceptable error value in the technical field to which this invention pertains, such as 10% of the actual value of the value. For example, 20°C disclosed in this specification should be interpreted as 20±2°C.

[0040] Hereinafter, in order to illustrate the technical features of the present invention, an embodiment will be described in detail.

[0041] Please refer to Figure 1. The first embodiment of the present invention discloses a whole-algae extraction method 10 for obtaining algal polysaccharides from an algal raw material, which includes a first-stage extraction process 100, a second-stage extraction process 200, and a third-stage extraction process 300; wherein:

[0042] The first-stage extraction procedure of the 100 series includes the following steps:

[0043] Step 101: Obtain a seaweed powder; wherein the seaweed powder is purchased from the market or obtained by sequentially washing, air-drying (40°C), and crushing seaweed.

[0044] Step 102: Extract the algae powder with cold water for 30-60 minutes to obtain a first extraction mixture, wherein the weight ratio of the sea fungus algae powder to the cold water is 1:20-30; the extraction temperature is 20-40℃;

[0045] Step 103: Filter the first extraction mixture to obtain a first filtrate and a first precipitate;

[0046] Step 104: Collect the first filtrate and remove the water from the first filtrate by freeze drying technology to obtain a phycobiliprotein extract.

[0047] The second-stage extraction process 200 is performed following the first-stage extraction process 100 and includes the following steps:

[0048] Step 201: Collect the first precipitate;

[0049] Step 202: Extract the first precipitate with ethanol of 75-95% concentration for 30-60 minutes to obtain a second extraction mixture, wherein the weight ratio of the first precipitate to the ethanol is 1:20-30, and the extraction temperature is 10-30℃.

[0050] Step 203: Filter the second extraction mixture to obtain a second filtrate and a second precipitate;

[0051] Step 204: Collect the second filtrate and remove the water from the second filtrate by freeze drying technology to obtain a phytochemical extract.

[0052] The third-stage extraction process 300 is performed following the second-stage extraction process 200 and includes the following steps:

[0053] Step 301: Obtain the second precipitate;

[0054] Step 302: Extract the second precipitate with hot water for 60-120 minutes to obtain a third extraction mixture, wherein the weight ratio of the second precipitate to the hot water is 1:30-50, and the extraction temperature is approximately or equal to 100°C.

[0055] Step 303: Filter the third extraction mixture to obtain a third filtrate and a third precipitate;

[0056] Step 304: Collect the third filtrate and remove the water from the third filtrate by spray drying technology to obtain algae polysaccharide extract (brown).

[0057] Step 305: Collect the third precipitate and remove the water from the third precipitate by hot air drying technology to obtain a seaweed fiber.

[0058] Before carrying out the filtration process in each of the above stages, the extraction process can be completed by checking the flow rate, moisture content, weight and other checkpoints.

[0059] Furthermore, in another embodiment of the present invention, the whole algae application extraction method for algae polysaccharides is generally the same as that in the above embodiment. However, the difference is that the extraction technology used in the second stage extraction process is supercritical extraction technology, that is, extraction is carried out by using carbon dioxide as the extraction solvent to obtain the second extraction mixture.

[0060] Furthermore, the polysaccharide content and viscosity of different batches of algal polysaccharide extracts obtained by the above extraction method were detected, and monosaccharide composition analysis and FTIR (Fourier transform infrared spectroscopy) analysis were performed. The results are shown in Tables 1 to 3 and Figure 2. The viscosity was determined by the Brookfield viscosity method.

[0061] Table 1: Analysis of the polysaccharide monosaccharide composition of algal polysaccharides Monosaccharide composition First batch Second batch Third batch Galactose 58.61% 62.54% 56.96% glucose 29.89% 27.32% 32.15% Xylose 5.85% 5.12% 6.31% Mannose 0.93% 1.92% 0.50% Other sugars 4.70% 3.10% 4.10%

[0062] Table 2: Viscosity Analysis of Algal Polysaccharides Viscosity (cps) First batch 2430 Second batch 2200 Third batch 2510

[0063] Table 3: Polysaccharide properties analysis of algal polysaccharides Property Analysis First batch Second batch Third batch Solid weight of sample after drying 1g 0.84g 1.1g Water-soluble (water-soluble dietary fiber) 76 (w / w)% 81 (w / w)% 70 (w / w)% Total sugar / sugar content analysis 90 (w / w)% 83 (w / w)% 92 (w / w)% Free sugar analysis 0 0 3

[0064] As can be seen from the above test results, the polysaccharide content of the algal polysaccharide obtained by the extraction method disclosed in this invention is 83-92 (w / w)%, the water-soluble component is 70-81 (w / w)%, and the viscosity is 2200-2510 cps. The main monosaccharides of the algal polysaccharide are galactose, glucose, xylose and mannose, with contents of 56-63%, 27-33%, 5-6%, and 0.5-2%, respectively, and the contents of other sugars are about 3-5%.

[0065] As can be seen from the results in Figure 2, the algal polysaccharide disclosed in this invention has a strong broad signal at wavenumbers of 3600-3000, and sulfate signals at wavenumbers of 1249, 1037, and 841, indicating that the algal polysaccharide disclosed in this invention is an α-configuration sugar unit and is rich in sulfate.

[0066] In addition, the algal polysaccharide disclosed in this invention was subjected to heavy metal testing and in vitro cytotoxicity tests. The test results showed that the algal polysaccharide obtained by the extraction method disclosed in this invention met the heavy metal testing standards and did not have cytotoxicity.

[0067] As can be seen from the composition and description of the above steps, the whole-algae application extraction method for algal polysaccharides disclosed in this invention can safely and quickly separate galactose-rich algal polysaccharides and other algal by-products from algal raw materials through a continuous extraction technology. This not only significantly reduces the extraction waste generated during the extraction process, but also obtains high-purity and highly biosafety algal polysaccharides through a specific multi-stage extraction procedure, thereby achieving the effect of increasing the economic value of algae and reducing production costs.

[0068] In the second embodiment of the present invention, the steps are generally the same as those in the first embodiment described above. However, the difference is that in step 305, the third precipitate is bleached before drying to obtain seaweed fiber with a uniform color, thereby improving the possibility of subsequent applications.

[0069] Furthermore, in order to verify that the algal polysaccharide system disclosed in this invention has complex biological activity, several examples will be given below and described in detail with reference to the figures.

[0070] The sea fungus polysaccharides used in the following examples were prepared in accordance with the contents disclosed in the first embodiment above.

[0071] Example 1: Intestinal Microbiota Analysis

[0072] A plurality of 6-week-old BALB / c mice were randomly divided into 3 groups and treated for 14 days according to the following conditions:

[0073] Group 1: Feed with RO water daily.

[0074] Group 2: Orally administer 20mg / kg / day of sea fungus polysaccharide.

[0075] Group 3: 40mg / kg / day of sea fungus polysaccharide administered orally daily.

[0076] Feces from mice in each group were collected on day 1 and day 14 of the experiment for next-generation sequencing (NGS) analysis, and the content of short-chain fatty acids (SCFA) in the feces was detected by gas chromatography-mass spectrometry (GC-MS). The results are shown in Figures 3 to 12.

[0077] The next-generation sequencing (NGS) analysis was performed by extracting fecal DNA from the fecal sample using a commercial kit (QIAamp Fast DNA Stool Mini Kit, Germany) and then performing sequencing analysis.

[0078] The analysis of short-chain fatty acid (SCFA) content involved taking 30 mg of fecal sample, adding deionized water (300 μL), homogenizing and centrifuging; taking 150 μL of supernatant and acidifying it with 50 μL of 50% sulfuric acid; adding 10 μL of 2-ethylbutyric acid and 400 μL of diethyl ether, and mixing evenly; centrifuging at 9000 rpm and 4℃ for 10 minutes, taking the ether layer, adding anhydrous sodium sulfate for dehydration, and performing GC / MS analysis; and detecting the gaseous ionic short-chain fatty acids in the sample according to the mass-to-charge ratio: acetic acid, propionic acid, and butyric acid.

[0079] As shown in Figures 3 and 4, the proliferation rate of beneficial bacteria in the intestines of mice in groups 2 and 3 was significantly higher than that in group 1. For example, the Bifidobacteriaceae flora, including Bifidobacteria, and the Lactobacillusceae flora, including Lactobacillus acidophilus (A bacteria), were significantly higher. The proliferation rate of beneficial bacteria in the intestines of mice in group 3 was higher than that in group 2, indicating that high-dose phytohexidine gluconate (Hydrogen Proteus) was beneficial. Auricularia auricula polysaccharide has a better effect on increasing the proliferation rate of beneficial bacteria. As shown in Figures 5 and 6, the proliferation rates of intestinal symbiotic bacteria, such as Muribacteraceae and Bacteroidaceae, in the second and third groups of mice were significantly higher than those in the first group of mice. In particular, the proliferation rate of intestinal symbiotic bacteria in the third group of mice was higher than that in the second group of mice, indicating that high doses of Auricularia auricula polysaccharide have a better effect on increasing the number of intestinal symbiotic bacteria.

[0080] As shown in Figures 7 and 8, the relative abundance of harmful bacteria in the intestines of mice in groups 2 and 3 was lower than that in group 1. For example, Clostridium difficile, which includes Clostridium difficile, and Enterobacteriaceae, which includes Escherichia coli. Among them, the number of pathogenic bacteria in the intestines of mice in group 3 was significantly lower than that in group 2, indicating that the higher the dose of seaweed polysaccharide, the better the inhibitory effect on pathogenic bacteria in the intestines.

[0081] Furthermore, as shown in Figure 9, the relative abundance of Bacteroidetes in the intestines of mice in groups 2 and 3 was significantly increased, further confirming that the sea fungus polysaccharide disclosed in this invention can promote the proliferation of beneficial bacteria in the intestines. Based on the fact that Bacteroidetes are related to the decomposition and fermentation of polysaccharides and the production of metabolites such as propionate, and can increase the production of acetate and propionate, the results in Figure 9 also show that the sea fungus polysaccharide disclosed in this invention has activities such as improving individual intestinal metabolic health, anti-intestinal inflammation, and regulating metabolism.

[0082] As shown in Figures 10 to 12, on day 14 of the experiment, the concentrations of acetic acid, propionic acid, and butyric acid in the intestines of mice in groups 2 and 3 were all higher than those in group 1, with group 3 showing the highest concentrations of these substances. These results indicate that the *Auricularia auricula-judae* polysaccharide disclosed in this invention has the activity of increasing the production of short-chain fatty acids in the intestines.

[0083] The results disclosed in this example confirm that the algal polysaccharide disclosed in this invention has the activity of promoting the proliferation of beneficial bacteria in the intestine, increasing the metabolic activity of intestinal symbiotic bacteria, inhibiting the growth of pathogenic bacteria in the intestine, reducing the number of pathogenic bacteria, and increasing the production of short-chain fatty acids in the intestine. That is, by administering an effective amount of algal polysaccharide or a composition containing algal polysaccharide to an individual, it is possible to maintain the balance of intestinal flora, promote the repair of the intestinal barrier, and resist inflammation, thereby achieving the effects of improving or preventing metabolic diseases or diseases related to intestinal flora imbalance, and maintaining individual metabolic function.

[0084] Example 2: Allergy Test

[0085] P815 mast cells were seeded at a concentration of 1x106 cells / ml in 24-well culture dishes and cultured at 37°C and 5% CO2 for 24 hours. Then, different concentrations (0, 125, 250, 500 μg / ml) of Auricularia auricula polysaccharide were added and co-cultured with P815 mast cells for 1 hour. 10 μg / ml of compound 48 / 80 was added and cultured for 6 hours. The culture medium was collected, and the concentrations of IL-4 and histamine in the culture medium were detected by ELISA. The results are shown in Figures 13 and 14. Among them, P815 cells without the addition of Auricularia auricula polysaccharide and without induction were used as the control group; P815 cell lines without the addition of Auricularia auricula polysaccharide but with induction were used as the induction group.

[0086] As can be seen from the results in Figures 13 and 14, the concentrations of IL-4 and histamine in the cell groups with added seaweed polysaccharide were significantly lower than those in the induced group. Specifically, the concentrations of IL-4 and histamine in the cell groups with added 500 μg / ml seaweed polysaccharide were 43% and 33% lower than those in the induced group, respectively.

[0087] The results show that the seaweed polysaccharide disclosed in this invention can reduce the production of IL-4 and histamine by mast cells due to degranulation, thereby reducing allergic reactions. In other words, by administering an effective amount of the seaweed polysaccharide disclosed in this invention to an individual, it is possible to effectively improve or prevent the occurrence of allergic diseases.

[0088] Example 3: Anti-inflammatory test

[0089] RAW264.7 cells were seeded in 96-well culture dishes at a concentration of 2x10⁵ cells / ml. Different concentrations (0, 125, 250, 500 μg / ml) of *Auricularia auricula-judae* polysaccharide were added, followed by LPS (1 μg / ml), and cultured for 24 hours. After culture, the culture medium was collected, and the concentrations of IL-6 and IL-8 in the culture medium were detected by ELISA. The results are shown in Figures 15 and 16. Cells without *Auricularia auricula-judae* polysaccharide and without LPS induction were used as the control group; cell lines without *Auricularia auricula-judae* polysaccharide but induced by LPS were used as the LPS group.

[0090] As shown in Figures 15 and 16, the concentrations of IL-6 and IL-8 in the cell groups with added seaweed polysaccharide were significantly lower than those in the LPS group. Specifically, the concentrations of IL-6 and IL-8 in the cell groups with added 500 μg / ml seaweed polysaccharide were 50% and 40% lower than those in the LPS group, respectively.

[0091] Since research indicates that Malassezia, which causes dandruff, upregulates the expression of the inflammatory factor IL-8 and has chemotaxis towards neutrophils and irritating fatty acids, and the results of Figures 15 and 16 show that the seaweed polysaccharide system disclosed in this invention has the activity of reducing inflammatory factors, it can be inferred from the results of this example that the algae polysaccharide system disclosed in this invention can be used to treat or improve inflammation or its related conditions, and can improve dandruff or soothe the scalp by reducing the production of inflammatory factors.

[0092] Example 4: UV Resistance Test

[0093] HaCaT cells were seeded in culture dishes at a concentration of 5 x 10⁴ cells / ml, and different concentrations (0, 125, 250, 500 μg / ml) of *Auricularia auricula-judae* polysaccharide were added for culture. After removing the culture medium from each cell group, they were irradiated with UVB (100 mJ / cm², 360 seconds). After irradiation, fluorescence detection and intracellular ROS (reactive oxygen species) assays were performed on each cell group. The results are shown in Figures 17 and 18. Cells without *Auricularia auricula-judae* polysaccharide and without UVB irradiation were used as the control group; cell lines without *Auricularia auricula-judae* polysaccharide but with UVB irradiation were used as the UVB group.

[0094] As shown in Figures 17 and 18, the fluorescence region of each cell group with added seaweed polysaccharide is significantly smaller than that of the UVB group, especially the cell group with added seaweed polysaccharide at a dose of 500 μg / ml. Specifically, the fluorescence region of the cell group with added seaweed polysaccharide at 500 μg / ml decreased by 77% compared with the UVB group.

[0095] The results of this example confirm that the algal polysaccharide disclosed in this invention has the activity of reducing ROS generated by UVB irradiation, thereby enhancing the skin barrier and achieving the effects of protecting the skin from ultraviolet damage and anti-skin aging.

[0096] Example 5: Wound Healing Experiment

[0097] HaCaT cells were seeded in 6-well culture dishes at a concentration of 2x10⁵ cells / ml. After the cells attached to the monolayer, a wound was drawn on the cell layer. After removing the detached cells with phosphate buffer, the culture medium was replaced with different concentrations (0, 125, 250, 500 μg / ml) of Auricularia auricula polysaccharide. After 24 hours of wounding, 10 μL of CCK-8 reagent was added and cultured for 2 hours. The absorbance was then measured at 450 nm using an ELISA reader. The relative cell proliferation rate of each experimental group was calculated with the control group (0% Auricularia auricula polysaccharide culture medium) as 100%. The results are shown in Figure 19.

[0098] In addition, 24 hours after the scratch, HaCaT cells from each group were collected, and the expression levels of MMP-9, VEGF-A and KRT16 genes in each group were detected. The results are shown in Figures 20 to 22.

[0099] As shown in Figures 19 to 22, the culture medium containing *Auricularia auricula-judae* polysaccharide significantly promotes HaCaT cell proliferation, accelerates cell migration during repair, and enhances the expression levels of wound healing-related genes: MMP-9, VEGF-A, and KRT16. These results demonstrate that the *Auricularia auricula-judae* polysaccharide system disclosed in this invention possesses epithelial cell repair activation activity, thereby promoting wound healing and accelerating wound repair. [Simplified Explanation of the Diagram]

[0100] Figure 1 is a flowchart of the whole-algae application extraction method of the algal polysaccharide disclosed in the first embodiment of the present invention. Figure 2 is the result of FTIR (Fourier Transform Infrared Spectroscopy) analysis of the first batch of algal polysaccharides disclosed in the present invention. Figure 3 is an analysis of the relative abundance of Bifidobacteriaceae in the intestines of mice in each group. Figure 4 is an analysis of the relative abundance of Lactobacillusceae in the intestines of mice in each group. Figure 5 is an analysis of the relative abundance of Muribaculaceae in the intestines of mice in each group. Figure 6 is an analysis of the relative abundance of Bacteroidaceae in the intestines of mice in each group. Figure 7 is an analysis of the relative abundance of Clostridiumceae in the intestines of mice in each group. Figure 8 is an analysis of the relative abundance of Enterobacteriaceae in the intestines of mice in each group. Figure 9 is an analysis of the distribution of intestinal flora in mice in each group. Figure 10 shows the concentration of acetate in each group of mice before and after the experiment. Figure 11 shows the concentration of propionate in each group of mice before and after the experiment. Figure 12 shows the concentration of butyric acid in each group of mice before and after the experiment. Figure 13 shows the IL-4 concentration in P815 cells treated with different concentrations of *Auricularia auricula-judae* polysaccharide. Figure 14 shows the histamine concentration in P815 cells treated with different concentrations of *Auricularia auricula-judae* polysaccharide. Figure 15 shows the IL-6 concentration in RAW264.7 cells treated with different concentrations of *Auricularia auricula-judae* polysaccharide. Figure 16 shows the IL-8 concentration in RAW264.7 cells treated with different concentrations of *Auricularia auricula-judae* polysaccharide. Figure 17 shows the fluorescence response of HaCaT cells treated with different concentrations of *Auricularia auricula-judae* polysaccharide. Figure 18 shows the results of analyzing the fluorescence region of HaCaT cells treated with different concentrations of *Auricularia auricula-judae* polysaccharide. Figure 19 shows the cell proliferation rate of HaCaT cells treated with different concentrations of seaweed polysaccharide. Figure 20 shows the expression level of VEGF-A in HaCaT cells treated with different concentrations of seaweed polysaccharide. Figure 21 shows the expression level of MMP-9 in HaCaT cells treated with different concentrations of seaweed polysaccharide. Figure 22 shows the expression level of KRT-16 in HaCaT cells treated with different concentrations of seaweed polysaccharide.

Claims

1. A method for whole-algae extraction of algal polysaccharides, comprising the following steps: a first-stage extraction process: extracting an algal raw material with a first extraction solvent to obtain a first extraction mixture; a second-stage extraction process: collecting a first precipitate from the first extraction mixture and extracting it with a second extraction solvent to obtain a second extraction mixture; a third-stage extraction process: collecting a second precipitate from the second extraction mixture and extracting it with a third extraction solvent to obtain a third extraction mixture; filtering the third extraction mixture to obtain a third filtrate containing an algal polysaccharide; wherein: The extraction solvent used in the second stage extraction process is different from the extraction solvent used in the first and third stage extraction processes; the extraction temperatures of the first and second stage extraction processes are 10-40℃, and the extraction temperature of the third stage extraction process is higher than that of the first and second stage extraction processes; the total sugar content of the algal polysaccharide is 80% (w / w) or more, the water-soluble component is 70% (w / w) or more, the viscosity is 2200-2510 cps, and the monosaccharide composition of the algal polysaccharide includes: 56-63% galactose, 27-33% glucose, 5-6% xylose and 3-5% mannose.

2. The whole-algae extraction method for algal polysaccharides as described in claim 1, wherein, The first-stage extraction procedure includes the following steps: Step 101: Obtain the algal raw material; Step 102: Extract the algal raw material with the first extraction solvent at an extraction temperature of 20-40°C to obtain the first extraction mixture, wherein the first extraction solvent is water; Step 103: Filter the first extraction mixture to obtain a first filtrate and a first precipitate, wherein the first precipitate contains a phycobiliprotein.

3. The whole-algae extraction method for algal polysaccharides as described in claim 1, wherein, The second-stage extraction procedure includes the following steps: Step 201: Collect the first precipitate; Step 202: Extract the first precipitate with the second extraction solvent at an extraction temperature of 10-30°C to obtain the second extraction mixture, wherein the second extraction solvent is ethanol with a concentration of 75-95%; Step 203: Filter the second extraction mixture to obtain a second filtrate and the second precipitate, wherein the second precipitate contains phytochemicals.

4. The whole-algae extraction method for algal polysaccharides as described in claim 1, wherein, The third-stage extraction procedure includes the following steps: Step 301: Obtain the second precipitate; Step 302: Extract the second precipitate with the third extraction solvent at an extraction temperature of 90-100°C to obtain the third extraction mixture, wherein the third extraction solvent is water; Step 303: Filter the third extraction mixture to obtain the third filtrate and a third precipitate, wherein the third precipitate contains seaweed fiber.

5. The use of an algal polysaccharide in preparing a composition for regulating intestinal flora, wherein, The algal polysaccharide is prepared by whole-algae extraction as described in claims 1 to 4.

6. The use as described in claim 5, wherein, This composition for regulating gut microbiota can be used to increase the number of bacteria in the gut classified as Bifidobacteriaceae, Lactobacillusceae, Muribaculaceae, or Bacteroidaceae.

7. The use as described in claim 5, wherein, This composition for regulating the gut microbiota can be used to inhibit the growth of microorganisms classified as Clostridiaceae or Enterobacteriaceae.

8. The use as described in claim 5, wherein, This composition for regulating gut microbiota can be used to increase the content of short-chain fatty acids in the gut.

9. Use of a type of algal polysaccharide in the preparation of a composition for resisting ultraviolet damage, wherein, The algal polysaccharide is prepared by whole-algae extraction as described in claims 1 to 4.

10. The use of a type of algal polysaccharide in the preparation of a wound-healing composition, wherein: The algal polysaccharide is prepared by whole-algae extraction as described in claims 1 to 4.

11. The use as described in claim 10, wherein, This wound-healing composition can be used to enhance the expression of MMP-9, VEGF-A, or KRT16 genes.

12. The use of a type of algal polysaccharide in the preparation of cytokine inhibitors, wherein: The algal polysaccharide is prepared by extraction of whole algae as described in claims 1 to 4; the cytokine inhibitor is used to inhibit the concentration of IL-4, IL-6, IL-8 or histamine.

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