Composition for improving immunity and application thereof

Abstract

The invention discloses a composition for improving immunity and application thereof. The composition comprises the following raw materials: mulberry leaf powder, ampelopsis grossedentata leaf powder, ginkgo leaf powder and ginkgo fruit powder. The preparation method of the composition for improving immunity comprises the following steps: respectively sieving mulberry leaf powder, ampelopsis grossedentata leaf powder, ginkgo leaf powder and ginkgo fruit powder, and uniformly mixing to obtain mixed powder; adding sterile deionized water into the mixed powder, and sterilizing to obtain a mixed solution; inoculating a mixed strain solution into the mixed solution, and stirring and fermenting; and after the fermentation is finished, filtering, and freeze-drying the filtrate to obtain the composition for improving immunity. The plant extract is extracted through mixed fermentation of various microorganisms, natural active substances in the plant extract are enriched, the variety of the natural active substances is enriched, and the effect of enhancing immunity is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of food health care, and in particular relates to an immunity improving composition and application thereof. Background Art

[0002] People pay more and more attention to preventive health care for non-disease populations, and enhancing immunity is the key to preventing diseases. However, some health foods with immune-enhancing effects on the market currently have certain shortcomings.

[0003] As people pay more and more attention to health, the demand for immunity-enhancing preparations is also increasing year by year. Immunity-enhancing preparations prepared by microbial fermentation will be favored by more and more consumers due to their natural, safe and effective advantages. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide an immunity enhancing composition prepared based on microbial fermentation technology and its application.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a composition for improving immunity, comprising the following raw materials;

[0007] Mulberry leaf powder, Ampelopsis macrophylla leaf powder, Ginkgo leaf powder, Ginkgo nut powder.

[0008] In a second aspect, the present invention provides a method for preparing the composition for improving immunity according to the first aspect, comprising the following steps:

[0009] S1: sifting mulberry leaf powder, Ampelopsis grossedentata leaf powder, Ginkgo leaf powder, and Ginkgo nut powder respectively, and mixing them to obtain a mixed powder;

[0010] S2: adding sterile deionized water to the mixed powder, sterilizing it, and obtaining a mixed solution;

[0011] S3: inoculate the mixed bacterial liquid into the mixed liquid, stir and ferment for 36-48h;

[0012] S4: After the fermentation is completed, filtering and freeze-drying the filtrate to obtain the immunity enhancing composition;

[0013] The mixed bacterial liquid is composed of Inonotus obliquus, Rhizopus arrhizus and sterile deionized water, and the effective viable bacteria count in the mixed bacterial liquid is 1×10 8 CFU / mL-1×10 10CFU / mL, the ratio of the viable counts of the birch inonotus to the rhizopus arrhizus is 1:0.5-0.7, the inoculation amount of the mixed bacterial liquid is 10-12v / v% of the mixed liquid, the preservation number of the birch inonotus is: CGMCCNO.20206, and the preservation number of the rhizopus arrhizus is: CGMCCNO.19912.

[0014] Preferably, the mass ratio of mulberry leaf powder, Ampelopsis grossedentata leaf powder, Ginkgo leaf powder and Ginkgo nut powder in the mixed powder is 1:5-7:2-4:1-3.

[0015] Preferably, in step S2, the mass ratio of the mixed powder to the sterile deionized water is 1:4-6.

[0016] Preferably, the temperature of the stirring fermentation in step S3 is 30±2°C.

[0017] Preferably, the ventilation volume of the stirring fermentation in step S3 is 1-3 vvm.

[0018] Preferably, the stirring speed of the stirring fermentation in step S3 is 100-300 rpm.

[0019] In a third aspect, the present invention provides a use of the immunity enhancing composition prepared by the first aspect or the second aspect in the preparation of a health product for enhancing immunity.

[0020] Preferably, the dosage form of the immunity enhancing health product is any one of tablets, hard capsules, soft capsules, pastes, granules and powders.

[0021] Compared with the prior art, the present invention has the following beneficial effects: in the composition provided by the present invention, mulberry leaf powder, Agave sphenanthera leaf powder, ginkgo leaf powder and ginkgo fruit powder are rich in flavonoids, and fermented by Inonotus obliquus and Rhizopus arrhizus, the content and bioavailability of bioactive components such as polysaccharides, flavonoids and amino acids in the raw materials are significantly improved; the polysaccharides produced by fermentation can activate immune cells and promote their proliferation and differentiation; the antioxidant and anti-inflammatory effects of flavonoids help maintain the stability of the immune system; the amino acids and small molecule peptides produced during the fermentation process provide necessary nutritional support for the synthesis and repair of immune cells; in addition, the fermentation products indirectly enhance the intestinal barrier function by regulating the balance of intestinal flora, inhibiting the growth of harmful bacteria and promoting the proliferation of beneficial bacteria, thereby further enhancing the body's immunity. Specific implementation methods

[0022] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples, wherein the terms used in the examples are for describing specific embodiments and do not constitute limitations on the scope of protection of the present invention.

[0023] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0024] Some raw materials and their sources are as follows:

[0025] Mulberry leaf powder: purchased from Shaanxi Huaze Biotechnology Co., Ltd.;

[0026] Ampelopsis grossedentata leaf powder: purchased from Lanzhou Waterless Biotechnology Co., Ltd.;

[0027] Ginkgo leaf powder: purchased from Shaanxi Huaze Biotechnology Co., Ltd.;

[0028] Ginkgo fruit powder: purchased from Shaanxi Junhe Biotechnology Co., Ltd.;

[0029] Inonotus obliquus: from China General Microbiological Culture Collection Center, collection number: CGMCCNO.20206.

[0030] Rhizopus arrhizus: from China General Microbiological Culture Collection Center, collection number: CGMCCNO.19912.

[0031] Other raw materials and reagents were purchased commercially.

[0032] Preparation of composition for improving immunity

[0033] Composition 1:

[0034] S1: sieve mulberry leaf powder, ampelopsis serrulata leaf powder, ginkgo leaf powder and ginkgo nut powder through an 80-mesh sieve respectively, and mix them to obtain a mixed powder, wherein the mass ratio of mulberry leaf powder, ampelopsis serrulata leaf powder, ginkgo leaf powder and ginkgo nut powder in the mixed powder is 1:6:3:2;

[0035] S2: adding sterile deionized water to the mixed powder, and sterilizing by ultra-high temperature to obtain a mixed solution, wherein the mass ratio of the mixed powder to the sterile deionized water is 1:5;

[0036] S3: inoculating the mixed bacterial liquid into the mixed liquid, stirring and fermenting for 42 hours, wherein the temperature of the stirring fermentation is 30° C., the ventilation volume of the stirring fermentation is 2 vvm, and the stirring speed of the stirring fermentation is 200 rpm;

[0037] S4: After the fermentation is completed, filtering and freeze-drying the filtrate to obtain the immunity enhancing composition;

[0038] The mixed bacterial liquid is composed of Inonotus obliquus, Rhizopus arrhizus and sterile deionized water, and the effective viable bacteria count in the mixed bacterial liquid is 1×10 9CFU / mL, the ratio of the number of live bacteria of Inonotus obliquus to Rhizopus arrhizus is 1:0.6; the inoculation amount of the mixed bacterial liquid is 11v / v% of the mixed liquid, the preservation number of the Inonotus obliquus is: CGMCCNO.20206, and the preservation number of Rhizopus arrhizus is: CGMCC NO.19912.

[0039] Composition 2:

[0040] S1: sieve mulberry leaf powder, ampelopsis serrulata leaf powder, ginkgo leaf powder and ginkgo nut powder through an 80-mesh sieve respectively, and mix them to obtain a mixed powder, wherein the mass ratio of mulberry leaf powder, ampelopsis serrulata leaf powder, ginkgo leaf powder and ginkgo nut powder in the mixed powder is 1:5:4:1;

[0041] S2: adding sterile deionized water to the mixed powder, and sterilizing by ultra-high temperature to obtain a mixed solution, wherein the mass ratio of the mixed powder to the sterile deionized water is 1:4;

[0042] S3: inoculating the mixed bacterial liquid into the mixed liquid, stirring and fermenting for 48 hours, wherein the temperature of the stirring fermentation is 28° C., the ventilation volume of the stirring fermentation is 3 vvm, and the stirring speed of the stirring fermentation is 100 rpm;

[0043] S4: After the fermentation is completed, filtering and freeze-drying the filtrate to obtain the immunity enhancing composition;

[0044] The mixed bacterial liquid is composed of Inonotus obliquus, Rhizopus arrhizus and sterile deionized water, and the effective viable bacteria count in the mixed bacterial liquid is 1×10 10 CFU / mL, the ratio of the number of live bacteria of Inonotus obliquus to Rhizopus arrhizus is 1:0.5; the inoculation amount of the mixed bacterial liquid is 10v / v% of the mixed liquid, the preservation number of the Inonotus obliquus is: CGMCCNO.20206, and the preservation number of Rhizopus arrhizus is: CGMCC NO.19912.

[0045] Composition 3:

[0046] S1: sieve mulberry leaf powder, ampelopsis serrulata leaf powder, ginkgo leaf powder and ginkgo nut powder through an 80-mesh sieve respectively, and mix them to obtain a mixed powder, wherein the mass ratio of mulberry leaf powder, ampelopsis serrulata leaf powder, ginkgo leaf powder and ginkgo nut powder in the mixed powder is 1:7:2:3;

[0047] S2: adding sterile deionized water to the mixed powder, and sterilizing by ultra-high temperature to obtain a mixed solution, wherein the mass ratio of the mixed powder to the sterile deionized water is 1:6;

[0048] S3: inoculating the mixed bacterial liquid into the mixed liquid, stirring and fermenting for 36 hours, wherein the temperature of the stirring fermentation is 32° C., the ventilation volume of the stirring fermentation is 1 vvm, and the stirring speed of the stirring fermentation is 300 rpm;

[0049] S4: After the fermentation is completed, filtering and freeze-drying the filtrate to obtain the immunity enhancing composition;

[0050] The mixed bacterial liquid is composed of Inonotus obliquus, Rhizopus arrhizus and sterile deionized water, and the effective viable bacteria count in the mixed bacterial liquid is 1×10 8 CFU / mL, the ratio of the number of live bacteria of Inonotus obliquus to Rhizopus arrhizus is 1:0.7; the inoculation amount of the mixed bacterial liquid is 12v / v% of the mixed liquid, the preservation number of the Inonotus obliquus is: CGMCCNO.20206, and the preservation number of Rhizopus arrhizus is: CGMCC NO.19912.

[0051] Composition ①: Different from composition 1, Inonotus obliquus CGMCC NO.20206 is used to replace Rhizopus arrhizus CGMCC NO.19912, and the remaining steps and parameters are the same as composition 1.

[0052] Composition ②: Different from composition 1, Rhizopus arrhizus CGMCC NO.19912 is used to replace Inonotus obliquus CGMCC NO.20206, and the remaining steps and parameters are the same as composition 1.

[0053] Composition ③: Different from composition 1, Inonotus obliquus CGMCC NO.14549 is used to replace Inonotus obliquus CGMCC NO.20206, and the remaining steps and parameters are the same as composition 1.

[0054] Composition ④: Different from composition 1, Rhizopus arrhizus GDMCC NO.64209 is used to replace Rhizopus arrhizus CGMCC NO.19912, and the remaining steps and parameters are the same as composition 1.

[0055] Composition ⑤: Different from composition 1, Rhizopus oryzae ACCC30416 is used to replace Rhizopus arrhizus CGMCCNO.19912, and the remaining steps and parameters are the same as composition 1.

[0056] Composition ⑥: Different from composition 1, fermentation medium is used to replace the mixed liquid. The raw materials and method for configuring the fermentation medium are: glucose 10 g / L, maltose 5 g / L, potato starch 40 g / L, Nucel 845MG 15 g / L, peptone 10 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, the pH of the fermentation medium is 5.5, and the remaining steps are the same as composition 1.

[0057] Composition ⑦: Different from composition 1, mulberry leaf powder is missing, and the mass ratio of the other raw materials is 6:3:2 for Ampelopsis grossedentata leaf powder, Ginkgo leaf powder and Ginkgo nut powder. The other steps and parameters are the same as composition 1.

[0058] Composition ⑧: Different from composition 1, it lacks Ampelopsis grossedentata leaf powder, and the mass ratio of the other raw materials is mulberry leaf powder, ginkgo leaf powder, and ginkgo nut powder is 1:3:2. The other steps and parameters are the same as composition 1.

[0059] Composition ⑨: Different from composition 1, ginkgo leaf powder is missing, and the mass ratio of the other raw materials is mulberry leaf powder, ampelopsis leaf powder, and ginkgo nut powder is 1:6:2. The other steps and parameters are the same as composition 1.

[0060] Composition ⑩: Different from composition 1, it lacks ginkgo nut powder, and the mass ratio of the other raw materials is mulberry leaf powder, Ampelopsis grossedentata leaf powder, and ginkgo leaf powder is 1:6:3. The other steps and parameters are the same as composition 1.

[0061] Immune efficacy validation test:

[0062] 400 healthy clean-grade ICR mice were selected, female, weighing 18-22g. The mice were adaptively fed for one week before the experiment, and given sterilized mouse food and sterilized water, with free access to food and water. 280 mice were randomly selected and divided into 14 groups, with 20 mice in each group;

[0063] Normal control group: fed with normal mouse chow and water.

[0064] Experimental group 1: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by composition 1 at a dose of 5 mg / g (body weight) per day.

[0065] Experimental Group 2: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by Composition 2 at a dose of 5 mg / g (body weight) per day.

[0066] Experimental group 3: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by composition 3 at a dose of 5 mg / g (body weight) per day.

[0067] Experimental group ①: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by composition ① at a dose of 5 mg / g (body weight) per day.

[0068] Experimental Group ②: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by Composition ② at a dose of 5 mg / g (body weight) per day.

[0069] Experimental group ③: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by composition ③ at a dose of 5 mg / g (body weight) per day.

[0070] Experimental group ④: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by composition ④ at a dose of 5 mg / g (body weight) per day.

[0071] Experimental group ⑤: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by composition ⑤ at a dose of 5 mg / g (body weight) per day.

[0072] Experimental Group ⑥: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by Composition ⑥ at a dose of 5 mg / g (body weight) per day.

[0073] Experimental Group ⑦: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by Composition ⑦ at a dose of 5 mg / g (body weight) per day.

[0074] Experimental Group ⑧: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by Composition ⑧ at a dose of 5 mg / g (body weight) per day.

[0075] Experimental group 9: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by composition 9 at a dose of 5 mg / g (body weight) per day.

[0076] Experimental group ⑩: The mice were fed with normal mouse food and water, and were fed with the immunity enhancing composition prepared by composition ⑩ at a dose of 5 mg / g (body weight) per day.

[0077] Immune index determination experiment: Dinitrofluorobenzene (DNFB)-induced delayed-type hypersensitivity (DTH) test in mice; NK cell activity determination (lactate dehydrogenase (LDH) assay); The above two test methods are based on the test methods recorded in the Functional Test and Evaluation Methods for Health Foods (2023 Edition).

[0078] The data were processed using statistical software, and the results were expressed as mean ± standard deviation.

[0079] Table 1 Results Statistics

[0080]

[0081]

[0082] Note: “*” indicates p<0.05 compared with the normal control group.

[0083] According to the results in Table 1, compared with the normal control group, the difference in left and right ear mass and NK cell activity of mice in test groups 1-3 of the present invention were significantly improved, which verified that the composition prepared by the present invention has a good effect of enhancing immunity.

[0084] The results of the comparative test group 1 and the test groups ①-⑤ show that the present invention uses birch inonotus CGMCCNO.20206 and rhizopus arrhizus CGMCC NO.19912 for mixed fermentation, which has a better effect. This may be because in the mixed fermentation process, birch inonotus provides a carbon source for rhizopus arrhizus by decomposing cellulose, and rhizopus arrhizus can decompose the protein in the culture medium into amino acids, oligosaccharides, monosaccharides and other active substances through the protease and amylase secreted by it to assist the growth of birch inonotus, and the secondary metabolites secreted by the two have antibacterial, anti-inflammatory and antioxidant biological activities. The two cooperate and coexist, and jointly enrich the active substances in the immunity-enhancing composition, which has a significant synergistic effect. The flavonoids, polyphenols and organic acids contained in mulberry leaf powder, ginkgo leaf powder and ginkgo fruit powder can significantly regulate the microbial environment and stimulate birch inonotus and rhizopus arrhizus to secrete antioxidant factors or disease-resistant factors.

[0085] From the results of the comparative test group ⑥ and the normal control group, it can be seen that the fermentation culture of Inonotus obliquus CGMCCNO.20206 and Rhizopus arrhizus CGMCC NO.19912 using conventional culture medium, the obtained fermentation product also has a certain effect of enhancing immunity, and the results of the comparative test group 1 and the test group ⑥ show that the fermentation substrate has an effect on the performance of the composition. When the mixed solution containing mulberry leaf powder, Ampelopsis serrulata leaf powder, Ginkgo leaf powder, and Ginkgo fruit powder in the present invention is used to ferment and culture Inonotus obliquus CGMCC NO.20206 and Rhizopus arrhizus CGMCC NO.19912, the obtained composition has a better immunity enhancement effect.

[0086] By comparing the results of test group 1 with test groups ⑦ to ⑩, it can be seen that when the mixed solution lacks any one of mulberry leaf powder, Ampelopsis grossedentata leaf powder, Ginkgo leaf powder, and Ginkgo nut powder, the immunity enhancing effect of the obtained composition becomes worse. It can be seen that there is a significant synergistic effect between mulberry leaf powder, Ampelopsis grossedentata leaf powder, Ginkgo leaf powder, and Ginkgo nut powder in the present invention.

[0087] Toxicology Test

[0088] Healthy clean-grade ICR mice, female, weighing 18-22 g, were selected and divided into a control group and a test group. The control group was intragastrically administered with 50 mg / g of composition 1-3, ①-⑩ every day, and the test group was intragastrically administered with an equal amount of physiological saline every day. The mortality results of each group were measured 24 hours after administration.

[0089] The results showed that all the patients in both the control group and the experimental group survived and were in good condition.

[0090] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present application.

Claims

1. A composition for improving immunity, characterized in that: The composition comprises the following raw materials: mulberry leaf powder, ampelopsis serrulata leaf powder, ginkgo leaf powder, and ginkgo nut powder; The preparation method of the composition comprises the following steps: S1: sifting mulberry leaf powder, Ampelopsis grossedentata leaf powder, Ginkgo leaf powder, and Ginkgo nut powder respectively, and mixing them to obtain a mixed powder; S2: adding sterile deionized water to the mixed powder, sterilizing it, and obtaining a mixed solution; S3: inoculate the mixed bacterial liquid into the mixed liquid, stir and ferment for 36-48h; S4: After the fermentation is completed, filtering and freeze-drying the filtrate to obtain the immunity enhancing composition; The mixed bacterial liquid is composed of Inonotus obliquus, Rhizopus arrhizus and sterile deionized water, and the effective viable bacteria count in the mixed bacterial liquid is 1×10 8 CFU / mL-1×10 10 CFU / mL, the ratio of the viable counts of the birch inonotus to the rhizopus arrhizus is 1:0.5-0.7, the inoculation amount of the mixed bacterial liquid is 10-12v / v% of the mixed liquid, the preservation number of the birch inonotus is: CGMCCNO.20206, and the preservation number of the rhizopus arrhizus is: CGMCCNO.19912.

2. The composition for improving immunity according to claim 1, characterized in that: The mass ratio of mulberry leaf powder, Ampelopsis grossedentata leaf powder, ginkgo leaf powder and ginkgo nut powder in the mixed powder is 1:5-7:2-4:1-3.

3. The composition for improving immunity according to claim 1, characterized in that: The mass ratio of the mixed powder to the sterile deionized water in step S2 is 1:4-6.

4. The composition for improving immunity according to claim 1, characterized in that: The temperature of stirring and fermenting in step S3 is 30±2°C.

5. The composition for improving immunity according to claim 1, characterized in that: The ventilation volume of the stirring fermentation in step S3 is 1-3 vvm.

6. The composition for improving immunity according to claim 1, characterized in that: The stirring speed of the stirring fermentation in step S3 is 100-300 rpm.

7. Use of the immunity enhancing composition according to any one of claims 1 to 6 in the preparation of a health product for enhancing immunity.

8. The use according to claim 7, characterized in that: The dosage form of the immunity enhancing health product is any one of tablets, hard capsules, soft capsules, ointments, granules and powders.

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