Brown algae extract with immunity enhancing function and application thereof

Abstract

The invention provides a brown algae extract with an immunity enhancing function. The brown algae extract is extracted from brown algae fermentation liquor. Effective components in the brown algae extract account for 40-60% by mass; the effective component comprises oligosaccharide formed by a carbohydrate chain formed by connecting guluronic acid and mannuronic acid with the polymerization degree of 2-10 or heterozygous guluronic acid and mannuronic acid through a beta-1, 4 glucosidic bond; the brown algae extract contains brown algae polysaccharide accounting for 20-35% by mass of the brown algae extract. Through immune organ, immune globulin, T cells, B cells, macrophages, NK cells, immune cell active factors and complement experiments, the influence of brown algae extracts with different concentrations on specific immune and non-specific immune functions of a mouse body is studied. Results show that intake of the brown algae extract with a low dosage (4mg / mL) has good effects of delaying thymus atrophy decline of immune organs and improving immune cell activity.

Description

Technical Field

[0001] The invention belongs to the field of organic macromolecular compounds, and specifically relates to a brown algae extract and application thereof. Background Art

[0002] Brown algae extract is a biologically active organic macromolecular substance extracted from seaweed. Brown algae extract has been widely used in fertilizer, feed and other industries, and its efficacy in the field of medicine and health is also being widely studied. The production and preparation methods of brown algae extract are mainly chemical, physical and bio-enzymatic methods. Among them, chemical high temperature and high pressure acid-base extraction is the main technical form in current production, but the chemical method has a certain destructive effect on the active ingredients in seaweed, and its extraction rate is also low; the physical method mainly includes mechanical crushing and low temperature blasting. This method can well retain the natural active ingredients in seaweed, is environmentally friendly and pollution-free, but the equipment investment is high and the product purity is difficult to guarantee. Bio-enzymatic hydrolysis technology uses enzymes such as alginate lyase, cellulase, protease, pectinase, etc. to effectively degrade brown algae to obtain brown algae extract.

[0003] As a natural barrier of the body, the immune system has the functions of immune surveillance, defense and regulation, and can be divided into specific immunity and nonspecific immunity. Specific immunity is divided into cellular immunity and humoral immunity, which are respectively exerted by T lymphocytes and B lymphocytes; nonspecific immunity is mainly exerted by innate immune cells, such as monocytes and macrophages, NK cells, etc. At present, the effect of brown algae extracts obtained by fermentation on enhancing immunity has not been studied. The evaluation of the immune function of brown algae extracts can be carried out in the way of modern medicine from four aspects: cellular immunity, humoral immunity, monocyte-macrophage phagocytic ability and NK cell activity.

[0004] Many known strains can secrete enzymes to achieve enzymatic hydrolysis of brown algae. The fermentation broth of the strain contains more biologically active components; different types of strains produce different oligosaccharide structures and polymerization degrees, and some process conditions can also control the oligosaccharide content and different polymerization degrees. It is a very challenging research direction to appropriately select strains and fermentation conditions, study the role of brown algae extracts in enhancing immunity from aspects such as cellular immunity, and obtain brown algae extracts with immunity-enhancing functions. Summary of the invention

[0005] In view of the shortcomings of the prior art, the first object of the present invention is to provide a brown algae extract with the function of enhancing immunity.

[0006] The second object of the present invention is to provide the application of the brown algae extract.

[0007] The technical solution for achieving the above-mentioned purpose of the present invention is:

[0008] A brown algae extract with the function of enhancing immunity, the brown algae extract is extracted from brown algae fermentation broth; the brown algae extract contains effective ingredients accounting for 40-60% by mass; the effective ingredients include oligosaccharides formed by sugar chains formed by guluronic acid (G) with a polymerization degree of 2-10, mannuronic acid (M) or a hybrid of the two connected by β-1,4 glycosidic bonds; the brown algae extract contains brown algae polysaccharides accounting for 20-35% by mass.

[0009] The brown algae extract contains alginate oligosaccharides with a polymerization degree of 2 to 6.

[0010] The present invention further proposes that the brown algae extract is obtained by the following steps:

[0011] 1) Bacterial fermentation: Bacillus subtilis is used to activate and enrich the bacterial strain; it is inoculated into an alginate functional medium, the temperature is controlled at 28° C. to 30° C., the pH is controlled at 6.5 to 8.0, and the strain is fermented for 20 to 24 hours to obtain a bacterial fermentation liquid;

[0012] 2) Preparation of alginate oligosaccharides: Prepare a food-grade sodium alginate solution with a mass concentration of 5% to 10%, add the bacterial fermentation broth obtained in step 1), carry out biological reaction at 30° C. to 35° C. for 28 to 30 hours, remove insoluble polysaccharides by centrifugal filtration, and then filter the clear liquid obtained by centrifugation through a membrane to obtain oligosaccharides with a degree of polymerization of 2 to 6, and spray-dry the obtained solution at a temperature below 100° C.

[0013] This project studies the application of brown algae extract in the preparation of products with immune-enhancing functions, so the raw materials must be harmless and edible. Among the fermentable bacteria, Bacillus subtilis is a GRAS (Generally Recognized as Safe) strain included in the US FDA and is a food-grade microorganism, so it is used in the preparation of brown algae extract.

[0014] The culture medium for activating and enriching the bacterial strains can be the same as the formula of the alginate functional culture medium, but agar is added to make it a solid culture medium.

[0015] A preferred technical solution of the present invention is that the alginic acid functional medium contains the following components in mass ratio: sodium alginate 3-5 g / L, tryptone 3-5 g / L, glucose 10 g / L, K2 HPO4 5-8 g / L, chloramphenicol 2-10 μg / mL, magnesium sulfate 1-2 g / L, ferrous sulfate 0.1-0.2 g / L, and the pH value is 6.5-7.0. The pH value is adjusted with 0.1-2 M HCl.

[0016] Preferably, in step 1), the temperature is controlled at 28° C. to 30° C., the pH is controlled at 6.5 to 7.0, and the strain is fermented and cultured for 20 to 24 hours.

[0017] Wherein, in step 2), the mass ratio of the food-grade sodium alginate solution to the bacterial fermentation broth is 100:2-6.

[0018] More preferably, in step 2), the biological reaction is carried out at 30° C.-35° C. for 28 to 30 hours.

[0019] Wherein, in step 2), the centrifugal conditions are: 4000-10000 rpm, centrifugation for 10-20 min.

[0020] The invention discloses an application of the brown algae extract in preparing a product having the function of enhancing immunity.

[0021] Wherein, the percentage of brown algae extract in the immunity enhancing functional product is 0.4% to 8%.

[0022] The beneficial effects of the present invention are:

[0023] The maximum solubility concentration of the brown algae extract in water proposed by the present invention is 600 mg / mL, and even at this dosage, it will not cause toxic reactions to mice, indicating that the brown algae extract can be safely consumed. The intervention of brown algae extract in different dosage groups of 4 to 80 mg / mL can delay the atrophy and decline of the thymus, an immune organ of mice, and improve the activity of immune cells to varying degrees, which is beneficial to the enhancement of the body's immunity.

[0024] The present invention studies the effects of different concentrations of brown algae extract on the specific and nonspecific immune functions of mice through experiments on immune organs, immunoglobulins, T cells, B cells, macrophages, NK cells, immune cell activity factors and complement. The results show that the intake of low-dose (4 mg / mL) brown algae extract has a good effect on delaying the atrophy and decline of the immune organ thymus and improving the activity of immune cells. The experimental results show that the intake of medium-dose brown algae extract (12 mg / mL) has a good effect on delaying the atrophy and decline of the immune organ thymus, improving the activity of immune cells, and improving the body's anti-tumor and anti-infection (virus and fungus) capabilities. The intake of high-dose (36 mg / mL) brown algae extract has a good effect on delaying the atrophy and decline of the immune organ thymus, improving the activity of immune cells, and improving the body's anti-tumor and anti-infection (virus and fungus) capabilities. The intake of extremely high-dose brown algae extract (80 mg / mL) has a good effect on delaying the atrophy and decline of the immune organ thymus, improving the activity of immune cells, and improving the body's anti-tumor and anti-infection (virus and fungus) capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is the liquid chromatogram of the 2-6 sugar mixed standard.

[0026] Figure 2 It is the liquid chromatogram of the product of Example 3.

[0027] Figure 3 This is the effect of brown algae extract on the body weight of mice. There is no significant difference in the body weight of each group of mice.

[0028] Figure 4 The figure shows the effect of brown algae extract on the immune organ index of mice. In the figure, "*" represents significant difference between the groups (P < 0.05); "**" represents extremely significant difference between the groups (P < 0.01).

[0029] Figure 5 This is the effect of brown algae extract on mouse immunoglobulin. In the figure, "**" represents extremely significant differences between the groups, P < 0.01.

[0030] Figure 6 The effect of brown algae extract on mouse T cells.

[0031] Figure 7 This is the effect of brown algae extract on mouse B cells. In the figure, "*" represents a significant difference, P < 0.05; "**" represents an extremely significant difference, P < 0.01.

[0032] Figure 8 The effect of brown algae extract on the phagocytic index of mouse macrophages;

[0033] Fig. 9 Effects of brown algae extract on the activity of natural killer (NK) cells in mice. Note: "**" indicates extremely significant difference, P < 0.01.

[0034] Fig.10 The effect of brown algae extract on the functional factor group of mouse immune cell activity. Note: "*" represents significant difference P < 0.05; "**" represents extremely significant difference P < 0.01.

[0035] Fig.11 This is the effect of brown algae extract on the inflammatory factor group of mouse immune cell activity. In the figure, "*" represents a significant difference, P < 0.05; "**" represents an extremely significant difference, P < 0.01.

[0036] Fig.12 The figure shows the effect of brown algae extract on the complement C3 and C4 content of mice. In the figure, "*" represents a significant difference, P < 0.05; "**" represents an extremely significant difference, P < 0.01. DETAILED DESCRIPTION

[0037] The following examples are used to illustrate the present invention but should not be used to limit the scope of the present invention.

[0038] The strain used in the examples is commercially available Bacillus subtilis, with the accession number ATCC6633.

[0039] In the examples, unless otherwise specified, all means used are conventional techniques in the art; and all raw materials used are commercially available unless otherwise specified.

[0040] Example 1 Preparation of brown algae extract

[0041] 1) Bacterial fermentation: Bacillus subtilis was used as the bacterial strain, which was activated and enriched. The strain was inoculated into a functional medium of alginate, the temperature was controlled at 28°C-30°C, the pH was controlled at 6.5-8.0, and the strain was fermented for 22 hours to obtain a bacterial fermentation liquid.

[0042] 2) Preparation of brown algae extract: prepare 10% food-grade sodium alginate solution, add the bacterial fermentation broth obtained in step 1), the mass ratio of food-grade sodium alginate solution to the bacterial fermentation broth is 100:5. Perform biological reaction at 30°C-35°C for 28 hours, and remove insoluble polysaccharides by centrifugation. The centrifugation conditions are: 6000 rpm, and centrifugation for 10 minutes.

[0043] Oligosaccharides with a degree of polymerization of 2 to 6 are obtained by membrane filtration, and the obtained solution is spray-dried at 85° C. to obtain a brown algae extract, wherein the content of oligosaccharides with a degree of polymerization of 2 to 6 reaches 40.1%.

[0044] The alginic acid functional medium contains the following components in mass ratio: sodium alginate 5g / L, tryptone 4g / L, glucose 10g / L, K2 HPO4 5g / L, chloramphenicol 2μg / mL, magnesium sulfate 1g / L, ferrous sulfate 0.2g / L, and pH value is 6.5-7.0. The pH value is adjusted with 1M HCl. After preparation with distilled water, the temperature is 121°C and sterilized for 15 minutes. The medium formula used for activation and enrichment of the strain is the same, but 20g / L agar is added.

[0045] The filter membrane model is selected according to the molecular weight range. The following table shows the molecular weight of oligosaccharides with a degree of polymerization of 2-6. The yield is 50.2%.

[0046] Table 1: Molecular weight of oligosaccharides with a degree of polymerization of 2-6

[0047]

[0048]

[0049] Example 2 Preparation of brown algae extract

[0050] 1) Bacterial fermentation: Bacillus subtilis was used as the bacterial strain, which was activated and enriched. The strain was inoculated into an alginate functional medium, the temperature was controlled at 28°C-30°C, the pH was controlled at 6.5-8.0, and the strain was fermented for 24 hours to obtain a bacterial fermentation liquid. The culture medium was the same as in Example 1.

[0051] 2) Preparation of brown algae extract: Prepare 10% food-grade sodium alginate solution, add the bacterial fermentation broth obtained in step 1), the mass ratio of food-grade sodium alginate solution to the bacterial fermentation broth is 100:5. Perform biological reaction at 30℃-35℃ for 26h, remove insoluble polysaccharides by centrifugation, and the centrifugation conditions are: 6000 rpm, centrifugation for 10min. Oligosaccharides with a degree of polymerization of 2-6 are obtained by membrane filtration, and the obtained solution is spray-dried at 85℃ to obtain brown algae extract, wherein the content of oligosaccharides with a degree of polymerization of 2-6 reaches 40.1%.

[0052] The yield was 48.7%.

[0053] Example 3 Preparation of brown algae extract

[0054] 1) Bacterial fermentation: Bacillus subtilis was used as the bacterial strain, which was activated and enriched. The strain was inoculated into an alginate functional medium, the temperature was controlled at 28°C-30°C, the pH was controlled at 6.8, and the strain was fermented for 24 hours to obtain a bacterial fermentation liquid. The culture medium was the same as in Example 1.

[0055] 2) Preparation of brown algae extract: prepare 10% food-grade sodium alginate solution, add the bacterial fermentation broth obtained in step 1), the mass ratio of the food-grade sodium alginate solution to the bacterial fermentation broth is 100:5. Perform bioreaction at 30°C to 35°C for 28 hours, remove insoluble polysaccharides by centrifugation, obtain oligosaccharides with a degree of polymerization of 2-6 by membrane filtration, spray-dry the obtained solution at 85°C to obtain a brown algae extract, wherein the content of oligosaccharides with a degree of polymerization of 2 to 6 is 42.8%.

[0056] The yield was 51.8%. The liquid chromatography test results of the standard and the product are shown in Figure 1 and Figure 2 .

[0057] Test example

[0058] The experiment used the brown algae extract prepared in Example 3 and prepared it into solutions of different concentrations with purified water.

[0059] Mouse toxicology experiment: The brown algae extract was designed to be administered intragastrically at doses of 150, 300, and 600 mg / mL, and the mice were observed for death multiple times within 24 hours. The results showed that no mice died after intragastric administration of different doses of brown algae extract, indicating that no dose of brown algae extract would cause toxic reactions in mice.

[0060] The maximum solubility concentration of brown algae extract is 600 mg / mL, and even at this dose it does not cause toxic reactions in mice, indicating that brown algae extract can be consumed safely.

[0061] Phase I animal experiment: 40 male adult (8 weeks) mice were randomly divided into 4 groups according to body weight, with 10 mice in each group. Group 1 was the control group, and groups 2-4 were intervention groups. They were given low, medium, and high doses (4, 12, and 36 mg / mL) of brown algae extract by gavage for 45 consecutive days, once a day. Mouse serum was extracted, and the levels of immunoglobulins IgA, IgD, IgE, IgG, and IgM in the serum were determined by Elisa method.

[0062] The second phase of animal experiments: 40 male adult mice (8 weeks old) were randomly divided into 5 groups according to body weight, with 8 mice in each group. Group 1 was the control group (the control in the figure represents the control group, that is, the group without gavage of brown algae extract), and groups 2-5 were intervention groups, which were gavaged with four doses of brown algae extract (4, 12, 36, and 80 mg / mL), including low, medium, high, and extremely high doses, for 45 consecutive days, once a day. The mouse immune organs were extracted, and the body weight, thymus, spleen, macrophage and NK cell function, B cell and T cell subsets, immune cell activity, and complement content of mice in different groups were measured.

[0063] Effects of brown algae extract on body weight and immune organs in mice: Figure 3 As can be seen from Table 2, under the intervention of different doses (4, 12, 36, 80 mg / mL) of brown algae extract, the body weight of mice in each group had no significant difference from that of mice in the blank control group, indicating that the intake of brown algae extract at various doses in this experiment had no effect on the normal growth of mice.

[0064] Table 2 Effects of brown algae extract on body weight, thymus index and spleen index of mice

[0065]

[0066] The thymus and spleen are the most important immune organs in the body. Changes in the immune organ index can directly reflect the stimulating effect of the test drug on the immune system. Therefore, the thymus index and spleen index are important indicators for mouse immune function testing. Figure 4As shown in (a) and Table 2, the thymus index of mice gradually increased with the increase in the concentration of brown algae extract, and reached the highest value in the extremely high dose group (80 mg / mL). Compared with the blank control group mice, the thymus index of mice in the low dose group (4 mg / mL) and the medium dose group (12 mg / mL) significantly increased by 19.3% and 15.8%, respectively (P<0.05), while the thymus index of mice in the high dose group (36 mg / mL) and the extremely high dose group (80 mg / mL) increased significantly by 50.7% and 74.6% (P<0.01), which were 1.5 and 1.7 times that of the blank control group, respectively. However, there was no significant change in the spleen index of mice after intervention with different doses of brown algae extract ( Figure 4 (b)). After taking brown algae extract, the weight of the thymus of mice increased significantly, indicating that brown algae extract can significantly promote the regeneration and development of immune cells in mice, thereby achieving the effect of enhancing the immunity of mice.

[0067] Effect of brown algae extract on immunoglobulin in mice: Serum immunoglobulins mainly include IgA, IgG, IgM, IgD, and IgE. This study used Elisa method to measure the immunoglobulin content in serum and evaluated the humoral immune regulation effect of brown algae extract intake on mice at the molecular level. The results are as follows Figure 5 As shown. Figure 5 (c) shows that compared with the blank control group, the brown algae extracts in different dose groups can increase the level of immunoglobulin IgE in mouse serum after intragastric administration, especially after intragastric administration of the high-dose group of brown algae extract (36 mg / mL), the content of IgE in mouse serum increased significantly by 11.3% (P < 0.01), while the levels of immunoglobulins IgA, IgD, IgG and IgM in mouse serum after intervention with different doses of brown algae extracts did not change significantly. The above experimental results show that brown algae extracts can enhance the immunity of mice by increasing the level of immunoglobulin IgE in mouse serum.

[0068] Table 3 Effects of brown algae extract on mouse immunoglobulin

[0069]

[0070] Effects of brown algae extract on mouse T cells and B cells: Cellular immunity is mainly mediated by T lymphocytes. The proliferation capacity of T lymphocytes determines the number of effector T cells and the intensity of cellular immune response. T lymphocytes are the most numerous and most important functional cells in the body's immune cells. T lymphocytes have two different functional subsets - CD4 + and CD8 + Among them, CD4 + It is a helper T cell, which plays a major auxiliary and inductive role in the body's immune process, and CD8 +It has an inhibitory effect, thus maintaining CD4 + / CD8 + The balance of CD4 is the key to the normal immune system. + and CD8 + T cells were tested and the results were as follows Figure 6 Compared with the blank control group, the CD4 + / CD8 + The results were significantly higher than those in the blank control group (P < 0.01), and increased by 17.7% and 30.2% respectively, indicating that the intake of brown algae extract has a good promoting effect on inducing T lymphocyte proliferation in mice. In addition, the increase of T lymphocytes can also assist B cells involved in humoral immunity to differentiate into plasma cells to produce antibodies and secrete related factors.

[0071] Table 4 Effects of brown algae extract on mouse T cells and B cells

[0072]

[0073] Note: "**" indicates extremely significant difference, P < 0.01.

[0074] like Figure 7 As shown, compared with the blank control group, the B cell percentages of mice in the low-dose group (4 mg / mL) and the medium-dose group (12 mg / mL) were significantly increased (P < 0.01), which were increased by 64.6% and 47.3% respectively compared with the blank control group, indicating that these two doses of brown algae extracts have the effect of improving the proliferation ability of mouse B cells.

[0075] In summary, when the brown algae extract concentration was 12 mg / mL, the proliferation and differentiation of mouse T lymphocytes and B lymphocytes were simultaneously promoted. Therefore, the effect of brown algae extract intake on mouse specific immunity is mainly reflected in the ability to increase the proliferation and differentiation of T lymphocytes and B lymphocytes, thereby increasing the secretion of plasma cells, increasing serum antibody concentrations, and regulating the balance of T lymphocyte subsets.

[0076] Effects of brown algae extract on mouse macrophages and NK cells: Changes in phagocytic rate and phagocytic index of mouse macrophages after intervention with different concentrations of brown algae extract Figure 8 As shown, compared with the blank control group, the phagocytic index of mice in the medium-dose group (12 mg / mL) increased significantly by 14.9% (P<0.01), indicating that the brown algae extract in the medium-dose group (12 mg / mL) can improve the phagocytic ability of mouse macrophages and enhance the nonspecific immune response function of mice.

[0077] Table 5 Effects of brown algae extract on the phagocytic index of mouse macrophages and natural killer (NK) cell activity

[0078]

[0079] Natural killer (NK) cells are innate immune effector cells that can perform immune clearance and immune surveillance on the body. Their killing range is much greater than that of T cells. They can also play an immune function in the absence of allergens, killing most infected cells and protecting the body from infection. Fig. 9 As shown, compared with the blank control group, under the action of brown algae extract in the medium dose group (12 mg / mL) and the high dose group (36 mg / mL), the NK cell activity of mice was significantly increased by 23.9% and 25.4% respectively (P<0.01), indicating that the brown algae extract at this dose can enhance the killing activity of NK cells in the nonspecific immunity of mice. The higher the activity of NK cells, the stronger the immune regulation function. The above results show that brown algae extract can improve the immune ability of mice by enhancing the activity of NK cells.

[0080] Effects of brown algae extract on immune cell active factors in mice

[0081] (1) Immune cell activity functional factor group: The content of immune cell activity functional factor in mice after intervention with different concentrations of brown algae extracts Fig.10 As shown in a, b, and c, compared with the blank control group, the IL-2 and IL-4 concentrations of mice in different dose groups gradually increased. Under the action of brown algae extract in the high dose group (36 mg / mL) and the extremely high dose group (80 mg / mL), the IL-2 concentrations of mice were significantly increased by 16.1% and 40.8% respectively compared with the blank control group (P<0.01); under the action of brown algae extract in the medium dose group (12 mg / mL), the IL-4 concentration of mice was significantly increased by 10.5% compared with the blank control group (P<0.05), and under the action of brown algae extract in the high dose group (36 mg / mL) and the extremely high dose group (80 mg / mL), the IL-4 concentration of mice was significantly increased by 26.9% and 40.6% respectively compared with the blank control group (P<0.01).

[0082] The levels of interferon-γ (IFN-γ) in mice after intervention with different concentrations of brown algae extracts Fig.10As shown in (d), compared with the blank control group, the IFN-γ content of mice in the high-dose group (36 mg / mL) was significantly increased by 27.0% (P<0.05), while the IFN-γ content of mice in the very high-dose group (80 mg / mL) was extremely significantly increased by 47.0% (P<0.01). Tumor necrosis factor (TNF-α) is a direct manifestation of inflammation in the body. The TNF-α content of mice after intervention with different concentrations of brown algae extract is shown in Figure 2. Fig.10 As shown in (e). Compared with the blank control group, the TNF-α content of mice in the high-dose group (36 mg / mL) was extremely significantly reduced by 15.0% (P<0.01), indicating that the intake of this dose of brown algae extract can significantly reduce the expression of TNF-α in mice, thereby having a certain immunomodulatory effect on the body.

[0083] Table 6 Effects of brown algae extract on the functional factors of mouse immune cell activity

[0084]

[0085] (2) Immune cell active inflammatory factor group: Interleukin-6 (IL-6) is an interleukin that mediates inflammation and has the function of promoting B cell differentiation into plasma cell factors; Interleukin-10 (IL-10) is a cytokine with pleiotropic effects in immune regulation and inflammation, which has the function of enhancing B cell proliferation and differentiation; Interleukin-12 (IL-12) has the function of stimulating T cell growth. Fig.11 (b) shows that compared with the blank control group, the IL-10 concentration of mice in the low-dose group (4 mg / mL) increased significantly by 8.8% (P<0.05), while the IL-10 concentration of mice in the high-dose group (36 mg / mL) and the very high-dose group (80 mg / mL) increased significantly by 17.5% and 30.6% respectively (P<0.01). Under the action of different doses of brown algae extract, the IL-12 concentration of mice increased with the increase of dose, and the IL-12 concentration of mice in the high-dose group (36 mg / mL) and the very high-dose group (80 mg / mL) increased significantly by 61.7% and 126.7% respectively (P<0.01) compared with the blank control group. Fig.11 c).

[0086] Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory chemokine that widely regulates the functions of various immune cells, such as macrophages. Fig.11As shown in (d), compared with the blank control group, the MCP-1 concentration of mice in the high-dose group (36 mg / mL) was significantly increased by 23.6% (P<0.01). Therefore, the intake of brown algae extract can increase the concentration of inflammatory factors in the immune cell activity of mice, thereby improving the immunity of mice to a certain extent.

[0087] Table 7 Effects of brown algae extract on inflammatory factors in mouse immune cell activity

[0088]

[0089] In summary, under the action of different concentrations of brown algae extract, especially in the high-dose group (36 mg / mL), the levels of interleukins IL-2, IL-4 and IL-10, interferon IFN-γ and pro-inflammatory chemokine MCP-1 in mice increased significantly, while the expression of tumor necrosis factor TNF-α decreased, indicating that brown algae extract can increase the production of antibodies in mice, reduce the occurrence of inflammation in the body, and have a certain immunoregulatory effect on mice.

[0090] Effects of brown algae extract on complement in mice: Complement is an indispensable part of the body's immunity. Complement components play an important role in innate and adaptive immunity. Its main functions are phagocytosis, lysis of pathogen cells, dissolution of immune complexes and inflammatory response. Whether it is the classical activation pathway or the alternative activation pathway, C3 activation is required to promote the subsequent chain reaction of complement components. The level of C3 corresponds to the content of total complement. Therefore, the C3 measurement can reflect the level of total serum complement, and its level is an important indicator for measuring humoral immunity. C4 is an important component of the classical complement activation pathway. Therefore, the content of complement C3 and C4 can reflect the strength of the body's immunity. Fig.12 As shown in the results, except for the low-dose (4 mg / mL) group, the complement C3 content of mice in each dose group was significantly higher than that of the control group, among which the medium-dose group (12 mg / mL) was significantly increased by 8.6% (P<0.05) compared with the control group, while the high-dose group (36 mg / mL) and the extremely high-dose group (80 mg / mL) were significantly increased by 25.4% and 55.7% (P<0.01) compared with the control group, respectively. For the complement C4 content, compared with the blank control group, the extremely high-dose group (80 mg / mL) mice were significantly increased by 10.1% (P<0.05) compared with the control group, indicating that the brown algae extract at this dose can increase the complement C4 content of mice, so that the activation pathway proceeds smoothly, thereby improving the immunity of mice. C3 is the core component of complement, with a higher concentration in serum, while C4 has a lower content in serum, so the change of complement C3 is more significant than that of C4.

[0091] Table 8 Effects of brown algae extract on complement C3 and C4 content in mice

[0092]

[0093] The above experimental results show that low-dose (4 mg / mL) brown algae extract can significantly increase the thymus index of mouse immune organs (19.3%) and the proliferation ability of mouse B lymphocytes (64.6%), and significantly increase the content of interleukin IL-10, an immune cell activity factor in mice (8.8%); indicating that low-dose brown algae extract intake has a good effect on delaying the atrophy and decline of the immune organ thymus and improving the activity of immune cells.

[0094] Medium dose (12 mg / mL) of brown algae extract: can significantly increase the thymus index of mice immune organ (15.8%), extremely significantly increase the proliferation capacity of mice T lymphocytes and B lymphocytes (17.7% and 47.3%), macrophage phagocytosis (14.9%), NK cell activity (23.9%), and significantly increase the content of mouse immune cell activity factor interleukin IL-4 (10.5%) and complement C3 (8.6%); indicating that the intake of medium dose of brown algae extract has a good effect on delaying the atrophy and decline of the immune organ thymus, improving the activity of immune cells, and improving the body's anti-tumor and anti-infection (virus and fungus) capabilities.

[0095] High dose (36 mg / mL) of brown algae extract can significantly increase the thymus index (50.7%) and immunoglobulin IgE content (11.3%) of mouse immune organs, significantly increase NK cell activity (25.4%), significantly increase mouse immune cell activity factors interleukin IL-2 (16.1%), IL-4 (26.9%), IL-10 (17.5%), IL-12 (61.7%), interferon IFN-γ (27.0%), pro-inflammatory chemokine MCP-1 content (23.6%) and complement C3 content (25.4%), and significantly reduce tumor necrosis factor TNF-α content (15.0%); indicating that high doses of brown algae extract intake have a good effect on delaying the atrophy and decline of the immune organ thymus, improving the activity of immune cells, and improving the body's anti-tumor and anti-infection (virus and fungus) capabilities.

[0096] Extremely high doses (80 mg / mL) of brown algae extract can significantly increase the thymus index of the mouse immune organ (74.6%), significantly increase the proliferation ability of mouse T lymphocytes (30.2%), and significantly increase the immune cell activity factors of mice interleukin IL-2 (40.8%), IL-4 (40.6%), IL-10 (30.6%), IL-12 (126.7%), interferon IFN-γ (47.0%), complement C3 content (55.7%) and C4 content (10.1%); indicating that the intake of extremely high doses of brown algae extract has a good effect on delaying the atrophy and decline of the immune organ thymus, improving the activity of immune cells, and improving the body's anti-tumor and anti-infection (virus and fungus) capabilities.

[0097] Although the present invention has been described in detail above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A brown algae extract having the function of enhancing immunity, characterized in that: The brown algae extract is extracted from brown algae fermentation broth; the effective ingredients in the brown algae extract account for 40-60% by mass; the effective ingredients include oligosaccharides formed by sugar chains formed by guluronic acid, mannuronic acid or a hybrid of the two with a polymerization degree of 2-10 connected by β-1,4 glycosidic bonds; the brown algae extract contains brown algae polysaccharides accounting for 20-35% by mass.

2. The brown algae extract according to claim 1, characterized in that The effective components of the brown algae extract contain alginate oligosaccharides with a polymerization degree of 2-6.

3. The brown algae extract according to claim 1, characterized in that The brown algae extract is obtained by the following steps: 1) Bacterial fermentation: using a strain of Bacillus subtilis, activating and enriching the strain; inoculating it in an alginate functional medium, controlling the temperature at 27° C. to 30° C., controlling the pH at 6.5 to 8.0, fermenting the strain for 20 to 24 hours, and obtaining a bacterial fermentation liquid; 2) Preparation of alginate oligosaccharides: Prepare a food-grade sodium alginate solution with a mass concentration of 5% to 10%, add the bacterial fermentation broth obtained in step 1), carry out biological reaction at 30° C. to 35° C. for 26 to 30 hours, remove insoluble polysaccharides by centrifugation, filter the clear liquid obtained by centrifugation through a membrane to obtain oligosaccharides with a degree of polymerization of 2 to 6, and spray dry the obtained solution at below 100° C.

4. The brown algae extract according to claim 3, characterized in that The alginic acid functional culture medium contains the following components in mass proportions: 3-5 g / L sodium alginate, 3-5 g / L tryptone, 10 g / L glucose, 5-8 g / L K2 HPO4, 2-10 μg / mL chloramphenicol, 1-2 g / L magnesium sulfate, 0.1-0.2 g / L ferrous sulfate, and a pH value of 6.5-7.

0.

5. The brown algae extract according to claim 3, characterized in that In step 1), the temperature is controlled at 28° C. to 30° C., the pH is controlled at 6.5 to 7.0, and the strain is fermented and cultured for 20 to 24 hours.

6. The brown algae extract according to claim 3, characterized in that In step 2), the mass ratio of the food-grade sodium alginate solution to the bacterial fermentation broth is 100:2-6.

7. The brown algae extract according to claim 3, characterized in that In step 2), the biological reaction is carried out at a temperature of 30°C to 35°C for 28 to 30 hours.

8. The brown algae extract according to claim 3, characterized in that In step 2), the centrifugal conditions are: 4000-10000 rpm, and centrifugation for 10-20 min.

9. Use of the brown algae extract according to any one of claims 1 to 8 in the preparation of a functional product for enhancing immunity.

10. The use according to claim 9, characterized in that: The percentage of brown algae extract in the immunity enhancing functional product is 0.4% to 8%.

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