阅读说明：本技术 灵芝及酵母β-葡聚糖复合多糖体组合物 (Ganoderma lucidum and yeast β -glucan composite polysaccharide composition ) 是由 金晋德 于 2019-08-08 设计创作，主要内容包括：一种灵芝及酵母β-葡聚糖复合多糖体组合物,以重量百分比计,包含3.2％至12.8％的一灵芝菌丝体及子实体萃取液、3％至12％的一酵母β-葡聚糖萃取液、2.5％至10.0％的一云芝菌丝体萃取液、3.0％至12.0％的一白木耳萃取液、2.5％至10.0％的一黑木耳萃取液、0.5％至2.0％的一猴头菇萃取液以及水。(A composite polysaccharide composition of Ganoderma lucidum and yeast β -glucan comprises, by weight, 3.2% to 12.8% of a Ganoderma lucidum mycelium and fruiting body extract, 3% to 12% of a yeast β -glucan extract, 2.5% to 10.0% of a Coriolus versicolor mycelium extract, 3.0% to 12.0% of a Tremella fuciformis extract, 2.5% to 10.0% of a Auricularia auricula extract, 0.5% to 2.0% of a Hericium erinaceus extract, and water.)

1. A ganoderma and yeast β -glucan compound polysaccharide composition is characterized by comprising, by weight, 3.2% to 12.8% of a ganoderma mycelium and fruiting body extraction liquid, 3% to 12% of a yeast β -glucan extraction liquid, 2.5% to 10% of a coriolus versicolor mycelium extraction liquid, 3% to 12% of a tremella fuciformis extraction liquid, 2.5% to 10% of a black fungus extraction liquid, 0.5% to 2% of a hericium erinaceus extraction liquid and water.

2. The ganoderma and yeast β -glucan complex polysaccharide composition of claim 1, comprising a flavoring agent, wherein said flavoring agent comprises citric acid.

3. The composition of Ganoderma lucidum and yeast β -glucan complex polysaccharide of claim 1, comprising a sweetener, wherein the sweetener comprises acesulfame potassium.

4. The ganoderma and yeast β -glucan complex polysaccharide composition of claim 1, comprising a juice concentrate, wherein said juice concentrate comprises orange juice concentrate.

5. The ganoderma and yeast β -glucan complex polysaccharide composition according to claim 1, wherein the ganoderma and yeast β -glucan complex polysaccharide composition comprises a powder, a drink or a capsule.

6. A process for preparing the composite polyose composition of ganoderma and yeast β -glucan includes such steps as proportionally mixing the fermented thallus and sporophore powder of ganoderma, the fermented thallus of yeast, the fermented thallus of coriolus versicolor, the sporophore powder of tremella, the sporophore powder of auricularia auricula and the sporophore powder of hericium erinaceus with water in the weight ratio of 10: 1-40: 1, stirring at 70-100 deg.C for 2-6 hr, press filtering to remove solid, concentrating, heating for sterilization to obtain ganoderma mycelia and sporophore extract, yeast β -glucan extract, coriolus versicolor mycelia extract, tremella extract, auricularia auricula extract and hericium erinaceus extract, and mixing all said extracts together.

7. The method for preparing the ganoderma and yeast β -glucan complex polysaccharide composition according to claim 6, wherein the step of preparing the ganoderma lucidum mycelia fermentation product, the yeast mycelia fermentation product or the coriolus versicolor mycelia fermentation product comprises inoculating a ganoderma lucidum mycelia, a yeast mycelia or a coriolus versicolor mycelia in a culture medium containing 0.5 to 5.0% by weight of a carbon source, 0.1 to 1.5% by weight of a nitrogen source, other trace substances and an acid-base number of 5.0 to 6.5, and culturing at 20 to 30 ℃ for 2 to 7 days with aeration and stirring.

8. The method for preparing the ganoderma lucidum and yeast β -glucan complex polysaccharide composition as claimed in claim 6, further comprising the step of blending 3.2 to 12.8% by weight of the ganoderma lucidum mycelium and fruiting body extract, 3 to 12% by weight of the yeast β -glucan extract, 2.5 to 10% by weight of the coriolus versicolor mycelium extract, 3 to 12% by weight of the tremella fuciformis extract, 2.5 to 10% by weight of the auricularia auricula extract, and 0.5 to 2% by weight of the hericium erinaceus extract.

9. The method for preparing the ganoderma lucidum and yeast β -glucan composite polysaccharide composition as claimed in claim 6, wherein the polysaccharide specification of the ganoderma lucidum mycelium and fruiting body extract is 5-7 g/L, the polysaccharide specification of the yeast β -glucan extract is 10-12 g/L, the polysaccharide specification of the coriolus versicolor mycelium extract is 5-7 g/L, the polysaccharide specification of the tremella fuciformis extract is 10-12 g/L, the polysaccharide specification of the auricularia auricula extract is 10-12 g/L, and the polysaccharide specification of the hericium erinaceus extract is 5-7 g/L.

Technical Field

The invention belongs to the technical field of polysaccharide compositions, and particularly relates to a ganoderma lucidum and yeast β -glucan composite polysaccharide composition.

Background

Polysaccharides existing in mushrooms have the effect of enhancing immune function, and in the case of ganoderma lucidum, β -Glucan (β -Glucan) has been widely used for a long time as a raw material of nutritional and health foods, and is very relevant to the immune function of polysaccharides.

The conventional polysaccharide health-care products have single-component products and also have compound products, and the compound products have better immunoregulation effect and can be favored by consumers. With the increasing demand of consumers for health-care food in modern society, the development of compound polysaccharide products with flavor and health-care efficacy is urgently needed.

Disclosure of Invention

In view of the above, the present invention aims to provide a ganoderma lucidum and yeast β -glucan complex polysaccharide composition, which can improve immune function.

In order to achieve the above object, the present invention provides a ganoderma lucidum and yeast β -glucan complex polysaccharide composition, which comprises, by weight, 3.2% to 12.8% of a ganoderma lucidum mycelium and fruiting body extract, 3% to 12% of a yeast β -glucan extract, 2.5% to 10% of a coriolus versicolor mycelium extract, 3% to 12% of a tremella alba extract, 2.5% to 10% of a black fungus extract, 0.5% to 2% of a hericium erinaceus extract, and water.

In order to achieve the above object, the present invention also provides a method for producing a ganoderma and yeast β -glucan complex polysaccharide composition, comprising the steps of mixing a fungus fermentation product and a fruiting body powder of ganoderma lucidum, a fungus fermentation product of yeast, a fungus fermentation product of coriolus versicolor, a fruiting body powder of tremella, a fruiting body powder of auricularia auricula and a fruiting body powder of hericium erinaceus with water at a weight ratio of 10: 1 to 40: 1, respectively, mixing the mixtures at 70 to 100 ℃ for 2 to 6 hours, performing filter pressing to remove solids, concentrating and heating to sterilize the filtrates, and obtaining a ganoderma mycelium and fruiting body extract, a yeast β -glucan extract, a coriolus versicolor mycelium extract, a tremella extract, a black fungus extract and a hericium erinaceus extract, and then blending all the extracts to obtain the ganoderma and yeast β -glucan complex polysaccharide composition.

The compound polysaccharide composition has the beneficial effects that the specific and non-specific immune functions are effectively improved through the comprehensive action of the compound polysaccharide composition.

Drawings

FIG. 1 is a graph of the weekly average body weight change of test animals in a specific immunoregulatory function assessment assay;

FIG. 2 is a graph of spleen immune cell proliferative capacity of test animals in a specific immunoregulatory function assessment assay;

FIG. 3 is a graph showing the secretion amounts of the spontaneous cytokines IL-2, TNF- α and IFN-. gamma.in test animals in the evaluation test of specific immunoregulatory function;

FIG. 4 is a graph showing the secretion of IL-2, a cytokine after OVA stimulation, in test animals for a specific immunoregulatory function assessment test;

FIG. 5 is a graph showing the secretion of TNF- α, a cytokine after OVA stimulation, in test animals for evaluation of specific immunoregulatory function;

FIG. 6 is a graph showing the secretion of IFN-. gamma.as a cytokine after OVA stimulation in test animals for evaluation of specific immunoregulatory function;

FIG. 7 is a graph showing the amount of anti-OVA IgG2a antibody produced in test animals in a test for evaluating specific immunoregulatory function;

FIG. 8 is a graph of the weekly average body weight change of test animals in a non-specific immunoregulatory function assessment assay;

FIG. 9 is a graph of spleen immune cell proliferative capacity of test animals in a non-specific immunoregulatory function assessment assay;

FIG. 10 is a graph of natural killer cell activity in test animals for non-specific immunoregulatory function assessment assay;

FIG. 11 is a graph of phagocyte activity of test animals in a nonspecific immunoregulatory function assessment assay;

FIG. 12 is a graph showing the secretion amounts of IL-2 and IFN-. gamma.which are the spontaneous cytokines of test animals in the non-specific immunoregulatory function assessment test;

FIG. 13 is a graph showing the secretion amount of cytokine IL-2 after Con A stimulation of spleen immune cells of test animals in the nonspecific immunoregulatory function assessment test;

FIG. 14 is a graph showing the secretion of IL-2, a cytokine, after stimulation with LPS, of spleen immune cells of test animals in the evaluation test of nonspecific immunoregulatory function;

FIG. 15 is a graph showing IFN-. gamma.secretion amounts of cytokines after Con A stimulation of spleen immune cells of test animals in the nonspecific immunoregulatory function assessment test;

FIG. 16 is a graph showing IFN-. gamma.secretion amounts of cytokines after LPS stimulation of spleen immune cells of test animals in the nonspecific immunoregulatory function evaluation test.

Detailed Description

In order that the invention may be more clearly described, reference will now be made in detail to the embodiments illustrated in the accompanying drawings.

The invention relates to a ganoderma and yeast β -glucan compound polysaccharide composition, which is prepared from extract liquor of various mushroom fungi, and mainly comprises a ganoderma mycelium and fruiting body extract liquor, a yeast β -glucan extract liquor, a corious versicolor mycelium extract liquor, a tremella extract liquor, a black fungus extract liquor, a hericium erinaceus extract liquor and water.

A method for preparing the composite polysaccharide composition of glossy ganoderma and yeast β -glucan

1. Method for extracting polysaccharide from thallus fermentation product

1.1 preparation of a fermentation broth of the fungus

Respectively inoculating the strains in a culture medium containing 0.5-5.0 wt% of carbon source (such as glucose or sucrose), 0.1-1.5 wt% of nitrogen source (such as yeast extract, yeast peptone or soybean peptone), other trace substances (such as trace elements and inorganic substances), and pH 5.0-6.5, and culturing at 20-30 deg.C under aeration and stirring for 2-7 days to obtain thallus fermentation product.

1.2 obtaining polysaccharide extract

Mixing the thallus fermentation product with water according to a weight ratio of 10: 1 to 40: 1, stirring the mixture at 70-100 ℃ for 2-6 hours, performing pressure filtration on the mixture, removing solids, concentrating the filtrate, and heating for sterilization to obtain polysaccharide extract.

2. Method for obtaining polysaccharides from fruiting body

2.1 preparation of a mixture of fruit bodies

Mixing the fruit body powder and water in a weight ratio of 10: 1 to 40: 1, and stirring the mixture at 70 ℃ to 100 ℃ for 2 to 6 hours.

2.2 obtaining polysaccharide extract

And respectively carrying out pressure filtration on the mixture, removing solids, concentrating the filtrate, and heating and sterilizing to obtain polysaccharide extract.

3. Measurement of polysaccharide specifications

3.1 calculation of β -Glucan content

Adding yeast β -glucan extract into buffer solution with proper volume, mixing uniformly, sequentially adding α -amylase (α -amylase), proteolytic enzyme (Protease) and Amyloglucosidase (amylogucosidase), treating, precipitating with 4 times volume of alcohol, settling and separating β -glucan from the solution, collecting precipitate, cleaning the precipitate with alcohol, drying, hydrolyzing the dried precipitate with strong acid and high temperature, neutralizing with acid and alkali, measuring glucose, and calculating β -glucan content.

3.2 detection of polysaccharide concentration

Respectively diluting ganoderma lucidum mycelium and fruiting body extract, yeast β -glucan extract, coriolus versicolor mycelium extract, tremella extract, black fungus extract and hericium erinaceus extract to proper concentrations, injecting the diluted solutions into a dialysis membrane (MW 6000-8000), dialyzing the dialyzed solutions for 48 hours with flowing water (flow rate: 0.2L/min), and determining the polysaccharide concentration of the dialyzed solutions by using a Phenol-sulfuric acid method (Phenol-sulfuric acid assay).

4. Examples of the invention

In this example, polysaccharide extracts were prepared from the strains of Ganoderma lucidum, Saccharomyces cerevisiae and Coriolus versicolor, and the fruiting bodies of Ganoderma lucidum, Tremella, Auricularia and Hericium erinaceus in the manner described above.

Name of article	In percentage by weight	Polysaccharide Specification (g/L)
			Ganoderma mycelium and fruiting body extractive solution	3.2～12.8％	5～7
Yeast β -dextran extract	3.0～12％	10～12
			Coriolus versicolor mycelium extractive solution	2.5～10％	5～7
Tremella extract	3.0～12％	10～12
			Black fungus extract	2.5～10％	10～12
Hericium erinaceus extract	0.5～2％	5～7
			Citric acid	0.13～0.52％	-
Acetylsulfanilic acid	0.035～0.14％	-
			Orange juice concentrate	3.4～13.6％	-
Water (W)	Water is supplemented to 100%	-

The ganoderma and yeast β -glucan complex polysaccharide composition of the present invention may further comprise a flavoring agent such as citric acid, a sweetener such as acesulfame potassium (acesulfame potassium), and a juice concentrate such as orange juice concentrate.

The invention relates to a ganoderma lucidum and yeast β -glucan compound polysaccharide composition, which comprises, by weight, 3.2% to 12.8% of ganoderma lucidum mycelium and fruiting body extract, 3% to 12% of yeast β -glucan extract, 2.5% to 10% of coriolus versicolor mycelium extract, 3% to 12% of tremella fuciformis extract, 2.5% to 10% of black fungus extract, 0.5% to 2% of hericium erinaceus extract, 0.13% to 0.52% of citric acid, 0.035% to 0.14% of acesulfame potassium, 3.4% to 13.6% of orange concentrated juice and water.

To evaluate the ability of the Ganoderma lucidum and Yeast β -glucan complex polysaccharide compositions of the present invention to promote the proliferation of immune cells, the following animal tests were conducted to evaluate the specific and non-specific immunomodulatory effects, respectively.

Second, the effect evaluation of the composition of Ganoderma lucidum and yeast β -glucan complex polysaccharide of the invention

1. Specific immunomodulating efficacy

1.1 test group design and test substance addition

The animal test was performed by selecting 7-week-old female BALB/c mice, as shown in table 1, the test groups were designed with a negative control group, a low dose group, a medium dose group, a high dose group, and a normal control group, each group of test animals was 10 animals, wherein the negative control group was added with sterile water only, the low dose group was added with a test substance at a dose of 1 time the recommended dose for human body, the medium dose group was 2 times the recommended dose for human body, the high dose group was 4 times the recommended dose for human body, and the normal control group was added with sterile water only the recommended dose for human body of the ganoderma and yeast β -glucan complex polysaccharide composition of the present invention was 180mL/day, according to the method announced by the food and drug administration in 2005, the number of reduction per body weight of kg of human body was 12.3, the method was converted for each group of mice to add the dose, the ganoderma and yeast β -glucan complex polysaccharide composition of the present invention was prepared into powder by freeze-drying, after the test substances were disposed in water, the test tubes were directly added to the test animals every day, and the test solution was added with 1 kg of sterile water, i.e., once per day, and the test solution was added with 10 kg of negative control solution.

TABLE 1 test group design and test substance addition

1.2 sensitized mice

After 4 weeks of test substance addition, Ovalbumin (OVA) was used as an immunogen to sensitize the test mice, 100. mu.L of a mixture of 25. mu.g of OVA and Freund's complete adjuvant (complete Freund's adjuvant) was intraperitoneally administered, and after 2 weeks, 100. mu.L of a mixture of 25. mu.g of OVA and Freund's incomplete adjuvant (incomplete Freund's adjuvant) was intraperitoneally administered again to sensitize the test mice.

1.3 Experimental animal observations

During the test period, the activity, hair color and reaction of the test animals are not abnormal, and the conditions of depilation, abnormal clinical symptoms or death and the like are not caused. The mean body weight of each group was about 17.3 to 17.8g at the start of the test and about 20.1 to 20.7g at the end of the test. There was no significant difference in body weight between the groups of animals tested during the test period, as shown in fig. 1. In addition, the relative weight of spleen and body weight was significantly higher in the OVA-induced groups than in the normal control group, which was a phenomenon caused by OVA sensitization. However, there was no difference in the relative weight of spleen and body weight between the OVA-sensitized groups (including the negative control group, the low dose group, the medium dose group and the high dose group), as shown in table 2.

TABLE 2 mean weekly weight changes and relative weight ratio of spleen to body weight for test animals

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences. The relative weight ratio of spleen to body weight was calculated as [ weight of spleen (g)/weight of body (g) ] X100%

1.4 Immunocytocyte proliferation assay

In a 96-well plate, add quantitative 1.0X 10⁵cells/well spleen cells, OVA-stimulated cells, after 72 hours of culture, reacted (

Aqueous One Solution Cell proliferative assay), using OD₄₉₀The absorbance is measured at nm, from which the effect of the test substance on lymphocyte proliferation is evaluated. The test results are expressed as Stimulation index (S.I.) and calculatedThe method is as follows: stimulation index (S.I.) -. OD after OVA Stimulation₄₉₀nm/non-schizont stimulated OD₄₉₀nm。

As shown in table 3 and fig. 2, after spleen immune cells were stimulated by OVA, the groups sensitized by OVA included a negative control group, a low dose group, a medium dose group, and a high dose group, which all showed a significant increase (p <0.05) compared to the normal control group (i.e., no OVA sensitization), in the mice sensitized by OVA, the groups added with the test substance (including the low dose group, the medium dose group, and the high dose group) were significantly higher than the negative control group (p <0.05), which indicates that the ganoderma lucidum and yeast β -glucan complex polysaccharide composition of the present invention has the efficacy of promoting OVA-induced immune cell proliferation.

TABLE 3 spleen Immunocytoproliferative Capacity

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were scored by One-way ANOVA

And analyzing, and performing post-hoc verification by using Duncan to analyze differences among groups. Stimulation index (S.I.) ═ spleen cells were treated with OVA

Absorbance after stimulation/absorbance without stimulation of spleen cells with schizont.

1.5 cytokine analysis

In a 24-well plate, the quantitative amount is 0.5 to 2X 10⁶cells/well spleen cells, 25. mu.g/mL OVA, at 37 ℃ with 5% CO₂Culturing for 48 to 72 hours, collecting the cell culture fluid, centrifuging (300 Xg, 4 ℃, 10 minutes), collecting the supernatant, measuring the contents of cytokines including Interleukin-2 (Interleukin-2, IL-2), Interleukin-4 (Interleukin-4, IL-4), Interleukin-5 (Interleukin-5, IL-5), Interleukin-10 (Interleukin-10, IL-10), Interferon- γ (Interferon γ, IFN- γ), etc. with ELISA Cytokine assay kit, Tumor necrosis factor (Tumor necrosis factor- α - α) was analyzed after OVA stimulation for 48 hours, thereby evaluating the test substance for secretion of cytokines from lymphocytesThe influence of (c).

1.5.1IL-2 secretion

As shown in Table 4 and FIG. 3, the spontaneous IL-2 secretion of each group was not significantly different (p >0.05) in the absence of OVA stimulation, whereas the OVA-sensitized groups (including negative control group, low dose group, medium dose group, and high dose group) were significantly higher than the normal control group (no OVA sensitization) in the presence of OVA induction, showing that the OVA sensitization pattern was successful, and IL-2 was significantly increased (p <0.05) in the OVA-sensitized groups compared to the negative control group after OVA stimulation (as shown in FIG. 4).

TABLE 4 content of cytokines secreted

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences.

1.5.2IL-4 secretion

Referring again to Table 4, there was no significant difference in the amount of spontaneous IL-4 secretion in each group without OVA stimulation (p > 0.05). Under the induction of OVA, the OVA sensitization of each group (comprising a negative control group, a low dose group, a medium dose group and a high dose group) is obviously higher than that of a normal control group (without OVA sensitization), and the OVA sensitization mode is successful. After OVA induction, the IL-4 secretion amount has no obvious difference (p is more than 0.05) among the animals sensitized by OVA, no matter the negative control group, the low dose group, the medium dose group and the high dose group.

1.5.3IL-5 secretion

Referring again to Table 4, there was no significant difference in the amount of spontaneous IL-5 secretion in each group without OVA stimulation (p > 0.05). Under the induction of OVA, the OVA sensitization of each group (comprising a negative control group, a low dose group, a medium dose group and a high dose group) is obviously higher than that of a normal control group (without OVA sensitization), and the OVA sensitization mode is successful. After OVA induction, the IL-5 secretion amount has no obvious difference (p is more than 0.05) among the animals sensitized by OVA, no matter the negative control group, the low dose group, the medium dose group and the high dose group.

1.5.4IL-10 secretion

Referring again to Table 4, there was no significant difference in the amount of spontaneous IL-10 secretion in each group without OVA stimulation (p > 0.05). Under the induction of OVA, the OVA sensitization of each group (comprising a negative control group, a low dose group, a medium dose group and a high dose group) is obviously higher than that of a normal control group (without OVA sensitization), and the OVA sensitization mode is successful. After OVA induction, the IL-10 secretion amount has no obvious difference (p is more than 0.05) among the animals sensitized by OVA, no matter the negative control group, the low dose group, the medium dose group and the high dose group.

1.5.5TNF- α secretion

Referring again to table 4 and fig. 3, there was no significant difference in the amount of spontaneous TNF- α secretion in each group without OVA stimulation (p > 0.05). whereas the OVA-sensitized groups (including the negative control group, the low dose group, the medium dose group, and the high dose group) were significantly higher than the normal control group (without OVA sensitization) showing successful OVA sensitization pattern, after OVA induction, the amount of TNF- α secretion tended to increase with increasing dose in OVA-sensitized animals compared to the negative control group, and reached a significant difference in the high dose group (p <0.05), as shown in fig. 5.

1.5.6 IFN-gamma secretion

Referring to table 4 and fig. 3 again, there was no significant difference in the spontaneous IFN- γ secretion of each group in the absence of OVA stimulation (p >0.05), whereas the OVA-sensitized groups (including the negative control group, the low dose group, the medium dose group, and the high dose group) were significantly higher than the normal control group (no OVA sensitization) under OVA induction, showing that the OVA sensitization pattern was successful, the groups to which the test substance was added had significantly increased IFN- γ secretion (p <0.05) after OVA induction, as shown in fig. 6, which shows that the ganoderma and yeast β -glucan complex polysaccharide composition of the present invention had the efficacy of promoting OVA-induced IFN- γ secretion.

1.6 test of antibody content in blood

After centrifugation of whole blood, sera were harvested and stored at-80 ℃ for further analysis of OVA peptidesConcentrations of the heterologous antibody anti-OVA IgG2a, the OVA-specific antibody anti-OVA IgG1, and the OVA-specific antibody anti-OVA IgE. OVA was applied at 4 ℃ for 24 hours, washed, and then serum samples were added to the dish (triplicate) and allowed to stand at 37 ℃ for 1 hour. After washing with Phosphate Buffered Saline (PBST) containing Tween 20, it was reacted with a secondary antibody labeled with Peroxidase (HRP) and colored with SureBlueReserve TMB Microwell Peroxidase Substrate. By detecting OD₄₅₀nm to quantify antibodies in serum. The results for OVA-specific antibodies are presented as ELISA Units (EU) and are calculated as follows: ELISA Unit (EU) ═ A_sample–A_blank)/(A_positive-A_blank)。

As shown in Table 5, due to the OVA sensitization, each group sensitized by OVA comprises a negative control group, a low dose group, a medium dose group and a high dose group, and anti-OVA IgG2a, anti-OVA IgG1 and anti-OVA IgE antibodies in serum are all higher than those in a normal control group (no OVA sensitization) (p is less than 0.05), so that the OVA sensitization mode is successful, wherein the anti-OVA IgG2a in the low dose group, the medium dose group and the high dose group is remarkably increased (p is less than 0.05) compared with that in the negative control group, as shown in figure 7, the result shows that the ganoderma lucidum and yeast β -glucan complex polysaccharide composition provided by the invention can promote the generation of the anti-OVA IgG2a antibodies in OVA sensitized mice.

TABLE 5 OVA-specific serum antibody production

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences.

1.7 lymphocyte surface marker assay

Taking the quantitative value as 5X 10⁵Spleen cells of cells/well were immunofluorescent stained with fluorescent labeled antibodies, respectively, and analyzed by flow cytometry for T cells (CD 3)⁺/CD45⁺) B cell (CD 19)⁺/CD45⁺) T4 cell (CD 4)⁺/CD3⁺) T8 cell (CD 8)⁺/CD3⁺) And NK cells (PanNK)⁺/CD45⁺) The proportion of lymphocytes was adjusted to observe the change in lymphocyte subpopulation. As shown in table 6, the spleen immune cell population of the test animals included T cells, B cells, T4 cells, T8 cells, and NK cells, and was not different among the test groups.

TABLE 6 spleen immunocyte surface antigen marker assay

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences.

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences.

In summary, as shown in table 7, the ganoderma lucidum and yeast β -glucan complex polysaccharide composition of the present invention has the effects of promoting OVA-induced immune cell proliferation, increasing the content of serum anti-OVA IgG2a antibody, and increasing the secretion of IL-2, IFN- γ and TNF- α by immune cells under OVA stimulation, thereby improving specific immune function.

TABLE 7 summary of specific immunomodulatory test results

-: indicates no significant difference from the negative control group; +: with OVA stimulation

p <0.05 ↓: shows a significant difference in the increase from the negative control group

p <0.05 ↓: shows a significant difference in reduction from the negative control group

2. Non-specific immunomodulatory efficacy

2.1 test group design and test substance addition

The animal test was performed by selecting 7-week-old female BALB/c mice, as shown in Table 8, wherein the test groups were designed with a negative control group, a low dose group, a medium dose group, and a high dose group, and the number of test animals in each group was 10, wherein the negative control group was added with sterile water only, the test substance addition dose in the low dose group was 1-fold of the recommended human dose, the medium dose group was 2-fold of the recommended human dose, and the high dose group was 4-fold of the recommended human dose.

TABLE 8 test set design and test substance dosing

After 6 weeks of test substance addition, the mice were sacrificed, and blood, spleen cells and peritoneal phagocytes were collected for immune function evaluation analysis, including determination of immune cell proliferation, phagocyte activity, natural killer cell activity, immunocytohormone secretion, immune cell type, serum antibody concentration, etc., thereby evaluating the efficacy of nonspecific immunomodulation of the ganoderma and yeast β -glucan complex polysaccharide composition of the present invention.

2.2 Experimental animal observations

During the test period, the activity, hair color and reaction of the test animal are not abnormal, and the conditions of depilation, abnormal clinical symptoms or death and the like are not caused. The mean body weight of each group was about 17.7 to 17.9g at the start of the test and about 19.8 to 20.6g at the end of the test. The body weight of the animals in each group increased steadily during the test period, and there was no significant difference in the body weight of the animals in each group (p >0.05), as shown in fig. 8. Furthermore, the relative weight of spleen to body weight was not significantly different between groups, as shown in table 9.

TABLE 9 mean weekly weight changes and relative weight ratio of spleen to body weight for test animals

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences. The relative weight ratio of spleen to body weight was calculated as [ weight of spleen (g)/weight of body (g) ] X100%

2.3 Immunocytocyte proliferation assay

The quantitative determination is 2.0X 10⁵Spleen cells of cells/wells stimulated the growth of T cells and B cells with the cytomerozoins Concanavalin A (Con A) and Lipopolysaccharide (LPS), respectively, and after culturing for 72 hours, reacted (CellTiter)

Aqueous One Solution Cell promotion Assay) using OD₄₉₀The absorbance is measured at nm, from which the effect of the test substance on lymphocyte proliferation is evaluated. The test results are expressed as a stimulation index (S.I.) and are calculated as follows: stimulation index (S.I) ═ OD₄₉₀nm post-schizont stimulation/OD₄₉₀no schizont stimulation at nm.

As shown in Table 10 and FIG. 9, spleen immunocytes stimulated by Con A showed a significant increase (p <0.05) in the low dose group, the medium dose group and the high dose group compared to the negative control group, and spleen immunocytes stimulated by LPS showed that only the high dose group was significantly higher than the negative control group.

TABLE 10 splenic immune cell proliferative capacity

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences.

Stimulation index (S.I.) -absorbance of spleen cells after Con A or LPS Stimulation/absorbance of spleen cells without merozoin Stimulation.

2.4 Natural killer cell Activity assay

YAC-1 cell line (i.e. mouse lymphoma cell) was used as a target for natural killer cells, YAC-1 cells were labeled with fluorescence using PKH67kit, and then quantitative spleen cells (containing natural killer cells) and YAC-1 cells labeled with fluorescence were combined in a ratio of 10: 1 and 25: 1 at 37 ℃ for 4 hours, and finally 50. mu.L of Propidium Iodide (PI) solution (0.1mg/mL) was added, followed by analysis using a flow cytometer, thereby analyzing the poisoning ability of natural killer cells.

As shown in table 11 and fig. 10, the ratio of natural killer cells to mouse lymphoma YAC-1 cells (E/T ratio) was 10: 1 and 25: 1, and the low dose group, the medium dose group, and the high dose group were significantly increased (p <0.05) compared to the negative control group, which indicates that the ganoderma lucidum and yeast β -glucan complex polysaccharide composition of the present invention contributes to enhancement of natural killer cell activity.

TABLE 11 Natural killer cell Activity

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences.

E (Effect) is a natural killer cell (NK cell); t (target) is YAC-1 cell, E/T ratio is the ratio of natural killer cell to YAC-1 cell.

2.5 phagocyte Activity assay

The phagocytic cells were phagocytized by co-culturing the quantitative enterophagocytic cells of the test animals and the fluorescence labeled e.coli at an infectious dose (m.o.i.) of 12.5, 25 and 50 at 37 ℃ for 2 hours. After the reaction, the amount of the phagocytic cells with fluorescence is detected by a flow cytometer, thereby evaluating the phagocytic activity of the phagocytic cells.

As shown in table 12 and fig. 11, phagocytic activity was significantly increased in the middle-dose group and the high-dose group compared to the negative control group (p <0.05) at an m.o.i. of 12.5, and significantly increased in the low-dose group, the middle-dose group, and the high-dose group compared to the negative control group (p <0.05) at an m.o.i. of 25 and 50 ratios, which indicates that the composition of ganoderma lucidum and yeast β -glucan complex polysaccharide of the present invention is helpful for increasing phagocytic activity.

TABLE 12 phagocytic Activity

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences.

The phagocytic capacity of the cells is the percentage of phagocytic cells with fluorescent e.

2.6 cytokine assay

Taking the quantitative ratio of 0.5 to 1 × 10⁶cells/wells were incubated at 37 ℃ with Con A and LPS, respectively, and the cell culture broth was harvested after 72 hours of stimulation with Con A or LPS, centrifuged (300 Xg, 4 ℃, 10 minutes) and the supernatant was collected and assayed for the cytokines IL-2, IL-4, IL-5, IL-10 and IFN-. gamma.using the ELISA Cytokine assay kit, and TNF- α was analyzed after 48 hours of stimulation with Con A or LPS, to evaluate the effect of the test substances on secretion of cytokines by lymphocytes.

2.6.1IL-2 secretion

As shown in Table 13 and FIG. 12, there was no significant difference in spontaneous IL-2 secretion performance (no addition of cytokinin) between the test groups and the negative control group, the amount of IL-2 in each test group was significantly increased (p <0.05) compared to the negative control group after stimulation with Con A by spleen immune cells, as shown in FIG. 13, while the amount of IL-2 secretion in the high dose group was significantly higher than that in the negative control group (p <0.05) by spleen immune cells under LPS stimulation, as shown in FIG. 14.

TABLE 13 content of cytokines secreted

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences.

2.6.2IL-4 secretion

Referring again to Table 13, there was no significant difference between the experimental groups compared to the negative control group in the expression of spontaneous IL-4 secretion (no addition of cytokinin). Under the stimulation of Con A and LPS, the IL-4 secretion situation of each test group is not obviously different compared with that of a negative control group (p > 0.05).

2.6.3IL-5 secretion amount

Referring again to Table 13, there was no significant difference between the experimental groups compared to the negative control group in the expression of spontaneous IL-5 secretion (no addition of cytokinin). Under the stimulation of Con A and LPS, the IL-5 secretion situation of each test group is not obviously different compared with that of a negative control group (p > 0.05).

2.6.4IL-10 secretion

Referring again to Table 13, there was no significant difference between the experimental groups compared to the negative control group in the expression of spontaneous IL-10 secretion (no addition of cytokinin). Under the stimulation of Con A and LPS, the IL-10 secretion of each test group is not obviously different from that of the negative control group (p > 0.05).

2.6.5TNF- α secretion

Referring again to Table 13, there was no significant difference between the test groups and the negative control group in the expression of spontaneous TNF- α secretion (no addition of cytokinin). after Con A and LPS stimulation, the amount of TNF- α in each test group tended to increase with increasing dose, but was not significantly different from the negative control group (p > 0.05).

2.6.6 IFN-. gamma.secretion

Referring again to Table 13 and FIG. 12, there was no significant difference between the test groups and the negative control group in the expression of spontaneous IFN-. gamma.secretion (no addition of cytokinin). After Con A stimulation, IFN- γ levels were significantly higher in each test group than in the negative control group (p <0.05), as shown in FIG. 15; in addition, although each test group tended to increase under LPS stimulation, no significant difference was achieved compared to the negative control group (p >0.05), as shown in fig. 16.

2.7 testing of antibody content in blood

After centrifugation (2200 Xg, 15 min), serum was collected and antibody concentrations in serum were determined using Mouse IgM, IgE, IgA and IgGELISA quantification Set. As shown in Table 14, there was no significant difference in serum antibodies among IgM, IgE, IgA and IgG among the test groups (p > 0.05).

TABLE 14 serum antibody production

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences.

2.8 lymphocyte surface marker assay

Taking the quantitative value as 5X 10⁵Spleen cells of cells/well were immunofluorescent stained with fluorescent labeled antibodies, respectively, and analyzed by flow cytometry for T cells (CD 3)⁺/CD45⁺) B cell (CD 19)⁺/CD45⁺) T4 cell (CD 4)⁺/CD3⁺) T8 cell (CD 8)⁺/CD3⁺) And NK cells (PanNK)⁺/CD45⁺) The proportion of lymphocytes was adjusted to observe the change in lymphocyte subpopulation. As shown in table 15, the spleen immune cell population of the test animals included T cells, B cells, T4 cells, T8 cells, and NK cells, and were not different among the test groups.

TABLE 15 spleen immunocyte surface antigen marker assay

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA,

and Duncan is adopted for post-hoc verification, and differences among groups are analyzed.

Results are expressed as mean ± standard deviation, 10 animals tested per group. Data were analyzed by One-way ANOVA and post hoc assays were performed using Duncan to analyze inter-group differences.

As shown in table 16, the ganoderma lucidum and yeast β -glucan complex polysaccharide composition of the present invention has the effects of promoting the proliferation of immune cells, increasing the activities of phagocytes and natural killer cells, increasing the secretion of cytokines IL-2 and IFN- γ under the stimulation of cytokinin, and increasing the non-specific immune function.

TABLE 16 summary of results of nonspecific immunomodulation assays

-: shows no significant difference from the negative control group

p <0.05 ↓: shows a significant difference in the increase from the negative control group

p <0.05 ↓: shows a significant difference in reduction from the negative control group

As described above, the Ganoderma lucidum and yeast β -glucan complex polysaccharide composition of the present invention can improve specific and non-specific immune function after administration.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.