Probiotic composition and application thereof in treatment of immune diseases

Abstract

The invention discloses a probiotic composition and application thereof in treatment of immune diseases. The probiotic composition is prepared from the following raw materials in parts by weight: 10 to 20 parts of probiotics, 5 to 15 parts of black garlic extract, 1 to 5 parts of eucheuma gelatinae polysaccharide and 0.5 to 1 part of stevioside. The probiotic composition disclosed by the invention has good acid-resistant and cholate-resistant activity, can increase the generation amount of B cells of antibody production cells of normal mice, can improve the antibody level of the normal mice, and has an enhancement effect on the humoral immune function of the normal mice.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and specifically relates to a probiotic composition and application thereof in treating immune diseases. Background Art

[0002] Immunity is the body's own defense mechanism, which plays an important role in identifying and eliminating foreign invading substances (such as viruses, bacteria, etc.) and dealing with aging, damaged or dead cells in the body. Low immunity may make the body susceptible to infection by pathogens. Therefore, protecting the health of the immune system is very important for improving immunity and preventing diseases. The immune system has evolved to maintain a symbiotic relationship between the host and microorganisms.

[0003] The microbiome is a dense and complex ecosystem that is increasingly recognized as an important component of host immune regulation. Studies have found that the intestinal flora often plays an important role in immune regulation, and the dysregulation of these interactions can lead to related diseases such as inflammation. In fact, the intestinal flora has been found to have a wide range of effects on inflammatory / autoimmune diseases. When the homeostasis of the host and its intestinal microorganisms is disrupted, the expression of inflammatory factors such as IL-0 and TNF-α is reduced, resulting in a loss of microbiome diversity, which may cause diseases such as inflammatory bowel disease. Probiotics can improve host immunity by maintaining the epithelial barrier, inhibiting the adhesion of pathogens to the intestinal surface, and regulating and properly maturing the immune system. Therefore, the use of probiotics to control the intestinal microbiome may be an effective treatment or preventive measure to regulate the composition of the intestinal flora, enhance immunity, provide many health benefits to the host, and actively regulate the composition of the intestinal microbiome and the function of the immune system.

[0004] There are more and more products developed using probiotics to treat immune diseases. Among these probiotic products, what really works on the human body is not the amount of probiotics taken in, but the number of probiotics taken in and colonized in the intestines. Traditional probiotic preparations cannot guarantee the number of live probiotics before consumption, and whether they can be colonized in the intestines after passing through gastric acid. People often cannot tell whether the probiotics are not working or the number of live bacteria is insufficient, thus misleading consumers and producing adverse effects.

[0005] Therefore, how to ensure the vitality of probiotics and their colonization efficiency in the body and fundamentally solve the problem of low immunity is an urgent problem that needs to be solved. Summary of the invention

[0006] In view of this, the present invention aims to provide a probiotic composition and its application in the treatment of immune diseases. The probiotic composition has good acid and bile salt resistance activity, can increase the production of antibody-producing cells B cells in normal mice, and can improve the antibody level of normal mice, and has an enhancing effect on the humoral immune function of normal mice.

[0007] The first object of the present invention is to provide a probiotic composition, comprising the following raw materials in parts by weight: 10 to 20 parts of probiotics, 5 to 15 parts of black garlic extract, 1 to 5 parts of Eucheuma polysaccharide and 0.5 to 1 part of stevioside.

[0008] The probiotics are at least one of Lactobacillus rhamnosus, Lactobacillus reuteri, Bifidobacterium animalis and Bifidobacterium breve.

[0009] Preferably, the Lactobacillus rhamnosus is Lactobacillus rhamnosus LGG, the Lactobacillus reuteri is Lactobacillus reuteri DSM17938, the Bifidobacterium animalis is Bifidobacterium animalis BB-12, and the Bifidobacterium breve is Bifidobacterium breve M-16V.

[0010] The preparation steps of the black garlic extract are:

[0011] (1) drying and crushing the black garlic, adding water and stirring to mix, then adding hemicellulase and cellulase to perform enzymolysis at 45-60° C. for 1-2 h, then heating to 95-100° C. for extraction for 1-2 h, filtering, collecting the first filtrate, and retaining the filter residue;

[0012] (2) adding ethanol solution to the filter residue, extracting at 65-75° C. for 1-2 h, filtering, and collecting the second filtrate;

[0013] (3) combining the first filtrate and the second filtrate, concentrating and drying to obtain a black garlic extract.

[0014] In step (1), the amount of hemicellulase and cellulase added is 0.5-1% of the mass of the black garlic powder.

[0015] In step (2), the solid-liquid ratio of the filter residue to the ethanol solution is 1:3-4 g / mL, and the concentration of the ethanol solution is 10-15% by volume.

[0016] The polysaccharide of Eucheuma truncatum is prepared by conventional process, and can be prepared by water extraction method or ultrasonic extraction method. The present invention adopts ultrasonic extraction method for preparation, and specifically, its preparation process includes the following steps:

[0017] The Eucheuma truncatum was cleaned, dried and crushed, passed through an 80-mesh sieve, added with water at a solid-liquid ratio of 1:100 g / mL and soaked for 0.5 h, extracted for 30 min under the conditions of ultrasonic power of 100 W and ultrasonic temperature of 60 ° C, filtered, collected the filtrate, concentrated the filtrate to 2 / 5 of the original volume, and then added 95% ethanol (3 times the volume of the concentrate), placed in a 4 ° C refrigerator for 24 h, filtered, collected the filtrate, and dried to obtain Eucheuma truncatum polysaccharide.

[0018] The second object of the present invention is to provide a method for preparing the above-mentioned probiotic composition, comprising the step of mixing probiotics, black garlic extract, Eucheuma polysaccharide and stevioside.

[0019] The third object of the present invention is to provide the use of the above-mentioned probiotic composition in the preparation of medicines for treating immune diseases.

[0020] The fourth object of the present invention is to provide the use of the above-mentioned probiotic composition in the preparation of health products, medicines or foods for improving immunity.

[0021] Preferably, the probiotic composition is formulated into powder, granules, tablets or capsules.

[0022] The probiotic composition of the present invention can effectively enhance the acid and bile resistance activity of probiotics by adding black garlic extract, Eucheuma polysaccharide and stevioside, so that the probiotics can be effectively colonized in the intestine, thereby fully exerting the role of immunoregulation by improving the intestinal environment. The probiotic composition can increase the production of antibody-producing cells B cells in normal mice, and can increase the volume and level of antibodies in normal mice, and has an enhancing effect on the humoral immune function of normal mice.

[0023] Among them, black garlic extract contains polysaccharides and oligosaccharides, organic acids, sulfur compounds, amino acids, melanoidins, lactic acid fermentation products, dietary fiber and other ingredients, which can promote the growth of probiotics and help improve the activity of probiotics. It also improves the body's defense against pathogens by improving the phagocytic function of macrophages in the body, improving the activity of immune organs, regulating the production of immune active substances, and having antioxidant and anti-inflammatory effects.

[0024] Eucheuma polysaccharides are mainly composed of galactose and 3,6-ether galactose, and also contain a small amount of glucose, xylose, talose and idose, etc. Polysaccharide sulfate refers to polysaccharides with sulfate groups on sugar hydroxyl groups, also known as sulfated polysaccharides. Studies have shown that Eucheuma polysaccharides show a protective effect on the immune function of mice in the case of radiation damage. Eucheuma polysaccharides can enhance the resistance of immune cells to radiation and promote the repair of radiation damage. The present invention has found that Eucheuma polysaccharides can enhance the activity of probiotics, especially when used in combination with black garlic extract and stevioside, which can effectively enhance the acid and bile resistance of probiotics.

[0025] Stevioside is a natural sweetener extracted from Stevia rebaudiana, which can stimulate the body's innate immune response, thereby inhibiting the release of proinflammatory cytokines, inhibiting the activity of the NF-κB pathway by inhibiting the degradation of nuclear factor κB inhibitor α (IκBα), and then exerting an immunomodulatory effect. The present invention found that the combination of stevioside with black garlic extract and Eucheuma polysaccharide can not only enhance the acid and bile resistance of probiotics, but also significantly enhance the immunomodulatory activity of probiotics. The obtained probiotic composition is expected to be used in the development of drugs for the treatment of immune diseases, or in the preparation of health products, medicines or foods that can improve immunity, and has broad prospects.

[0026] The probiotic composition of the present invention has good stability and is conducive to being developed into various preparations to meet the needs of different groups of people. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The effect of the probiotic composition on antibody-producing cells in normal mice. Compared with the negative control group (NC), * p<0.05, ** p<0.01, *** p<0.001.

[0028] Figure 2 The effect of the probiotic composition on serum hemolysin in normal mice. Compared with the negative control group (NC), * p<0.05, ** p<0.01, *** p<0.001. DETAILED DESCRIPTION

[0029] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0030] Stevioside was purchased from Shandong Fengtai Biotechnology Co., Ltd.

[0031] Example 1

[0032] Preparation of black garlic extract:

[0033] (1) The black garlic is dried and crushed, passed through a 60-mesh sieve, and the powder is dispersed in water at a ratio of 1:20 g / mL, and 0.5% of hemicellulase and 1% of cellulase by weight of the black garlic powder are added to perform enzymolysis at 50° C. for 1 h, and then the temperature is raised to 100° C. for extraction for 2 h, filtered, the first filtrate is collected, and the filter residue is retained.

[0034] (2) Add 10% ethanol solution by volume to the filter residue at a solid-liquid ratio of 1:3 g / mL, extract at 70° C. for 1 h, filter, and collect the second filtrate.

[0035] (3) combining the first filtrate and the second filtrate, concentrating and drying to obtain a black garlic extract.

[0036] Example 2

[0037] Preparation of Eucheuma polysaccharide:

[0038] The Eucheuma truncatum was cleaned, dried and crushed, passed through an 80-mesh sieve, added with water at a solid-liquid ratio of 1:100 g / mL, soaked for 0.5 h, extracted for 30 min under the conditions of ultrasonic power of 100 W and ultrasonic temperature of 60 ° C, filtered, collected the filtrate, concentrated the filtrate to 2 / 5 of the original volume, and then added with 95% volume fraction ethanol (3 times the volume of the concentrate), placed in a 4 ° C refrigerator for 24 h, filtered, collected the filtrate, and freeze-dried to obtain Eucheuma truncatum polysaccharide.

[0039] Example 3

[0040] A probiotic composition, characterized in that it comprises the following raw materials in parts by weight: 3 parts of Lactobacillus rhamnosus LGG, 3 parts of Lactobacillus reuteri DSM17938, 4 parts of Bifidobacterium animalis BB-12, 5 parts of black garlic extract, 1 part of Eucheuma polysaccharide and 0.5 parts of stevioside.

[0041] Preparation steps: Lactobacillus rhamnosus LGG, Lactobacillus reuteri DSM17938, and Bifidobacterium animalis BB-12 are premixed at a temperature of 20±2°C and a relative humidity of less than 40%, and then black garlic extract, Eucheuma polysaccharide, and stevioside are added and stirred to obtain a probiotic having a live bacterial content of ≥1×10 10 cfu / g.

[0042] Example 4

[0043] A probiotic composition, characterized in that it comprises the following raw materials in parts by weight: 5 parts of Lactobacillus rhamnosus LGG, 5 parts of Lactobacillus reuteri DSM17938, 5 parts of Bifidobacterium animalis BB-12, 10 parts of black garlic extract, 3 parts of Eucheuma polysaccharide and 1 part of stevioside.

[0044] Preparation steps: Lactobacillus rhamnosus LGG, Lactobacillus reuteri DSM17938, and Bifidobacterium animalis BB-12 are premixed at a temperature of 20±2°C and a relative humidity of less than 40%, and then black garlic extract, Eucheuma polysaccharide, and stevioside are added and stirred to obtain a probiotic having a live bacterial content of ≥1×10 10 cfu / g.

[0045] Example 5

[0046] A probiotic composition, characterized in that it comprises the following raw materials in parts by weight: 6 parts of Lactobacillus rhamnosus LGG, 6 parts of Lactobacillus reuteri DSM17938, 8 parts of Bifidobacterium animalis BB-12, 15 parts of black garlic extract, 5 parts of Eucheuma polysaccharide and 1 part of stevioside.

[0047] Preparation steps: Lactobacillus rhamnosus LGG, Lactobacillus reuteri DSM17938, and Bifidobacterium animalis BB-12 are premixed at a temperature of 20±2°C and a relative humidity of less than 40%, and then black garlic extract, Eucheuma polysaccharide, and stevioside are added and stirred to obtain a probiotic having a live bacterial content of ≥1×10 10 cfu / g.

[0048] Comparative Example 1

[0049] The difference between Comparative Example 1 and Example 5 is that the black garlic extract is not contained, and the other ingredients remain unchanged.

[0050] Comparative Example 2

[0051] The difference between Comparative Example 2 and Example 5 is that Comparative Example 2 does not contain Eucheuma polysaccharide, and the other ingredients remain unchanged.

[0052] Comparative Example 3

[0053] The difference between Comparative Example 3 and Example 5 is that Comparative Example 3 does not contain stevioside, and the other ingredients remain unchanged.

[0054] Comparative Example 4

[0055] The difference between Comparative Example 4 and Example 5 is that the black garlic extract and Eucheuma polysaccharide are not contained, and the other ingredients remain unchanged.

[0056] Comparative Example 5

[0057] The difference between Comparative Example 5 and Example 5 is that the black garlic extract and stevioside are not contained, and the other ingredients remain unchanged.

[0058] Comparative Example 6

[0059] The difference between Comparative Example 6 and Example 5 is that it does not contain Eucheuma polysaccharide and stevioside, and the other ingredients remain unchanged.

[0060] Example 6: Stability test of probiotic composition

[0061] The probiotic compositions prepared in Examples 3-5 and Comparative Examples 1-6 were subjected to simulated gastrointestinal digestion experiments, respectively, and the specific steps were as follows: 1.0 g of the product was weighed into 20 mL of simulated gastric juice (pH 3.5), and then placed in a 37°C, 90 r / min water bath for oscillation digestion for 2 h, and then 80 mL of simulated intestinal juice was added, and the oscillation digestion was continued in a 37°C, 120 r / min water bath for 2 h, simulating the gastrointestinal digestion process under anaerobic conditions. The number of viable bacteria was determined by the plate count method, and three parallel experiments were performed at the same time, and the survival rate of the probiotics was calculated. The results are shown in Table 1.

[0062] The simulated gastric fluid was prepared by adjusting the pH value of 0.1 mol / L potassium phosphate buffer to 3.5, adding pepsin (10 g / L) after sterilization, and mixing to fully dissolve the mixture to obtain simulated gastric fluid, which was preheated at 37°C before use.

[0063] Preparation of simulated intestinal fluid: adjust the pH of potassium phosphate buffer to 6.8 and sterilize, add trypsin (10 g / L) and porcine bile salt (3 g / L), mix to fully dissolve, and obtain simulated intestinal fluid. Preheat to 37°C before use.

[0064] Table 1 Tolerance of probiotic composition to simulated gastrointestinal fluid

[0065] Group Survival rate (%) Example 3 90.18 Example 4 92.52 Example 5 93.84 Comparative Example 1 79.36 Comparative Example 2 85.60 Comparative Example 3 89.07 Comparative Example 4 64.12 Comparative Example 5 71.06 Comparative Example 6 77.82

[0066] As shown in Table 1, the probiotic composition of the present invention has acid and choline resistance activity, and its survival rate remains above 90% under the action of acidic gastric juice and high bile salt osmotic pressure in the intestine, indicating that the probiotics of the present invention can well resist the digestive tract environment to achieve intestinal colonization. As shown in Comparative Examples 1-6, black garlic extract, Eucheuma polysaccharide and stevioside play an important role in improving the stability of probiotics, and the lack of any of these components will affect the stability of probiotics.

[0067] Example 7: Effect of the probiotic composition on antibody-producing cells in normal mice

[0068] 1. Reagents and drugs: sterile defibrinated sheep blood (Shanghai Puzhen Biotechnology Co., Ltd., Kramar), transfer factor oral solution (Nanjing Ruier Pharmaceutical Co., Ltd.), sheep red blood cells (SRBC) (Beijing Borsi Technology Co., Ltd.), Hank's solution (Beijing Biolab Technology Co., Ltd.); RPMI1640 culture medium (GIBCO), red blood cell lysis solution (Sigma-Aldrich).

[0069] 2. Experimental process

[0070] (1) Animal grouping and dosing: 6-week-old SPF male BALB / c mice were selected and housed in a controlled environment with a temperature of 24±2°C, a relative humidity of 45-60%, and a 12h / 12h light-dark cycle. The mice were free to eat during the entire experiment. After 1 week of adaptation, the mice were randomly divided into 11 groups (n=10 in each group), namely, a negative control group (NC), a positive control group (PC), Example 3-5 groups (T3-T5), and Comparative Example 1-6 groups (D1-D6). The animals in the negative control group were gavaged with purified water, and the gavage volume was 20mL / kg body weight. The animals in the positive control group were gavaged with transfer factor oral liquid, with a dose of 20mg / kg body weight. The animals in Example 3-5 groups were gavaged with the probiotic composition prepared in Example 3-5, with a dose of 2.0g / kg body weight. The animals in Comparative Example 1-6 groups were gavaged with the probiotic composition prepared in Comparative Example 1-6, with a dose of 2.0g / kg body weight, once a day, for 30 consecutive days. During the experiment, the survival, body shape, coat, skin, feces, muscle tension, gait, spirit, breathing and other conditions of the animals were observed once a day. The animals were weighed once a week and once before the end of the experiment.

[0071] (2) Preparation of complement: Select 5 SPF male Hartley guinea pigs, collect blood from the abdominal aorta, place at room temperature for about 40 minutes, centrifuge at 3000 rpm for 15 minutes, and take the upper serum. Add 1 mL of packed SRBC (sheep red blood cells) to 5 mL of guinea pig serum, place in a refrigerator at 4°C for 30 minutes, shake frequently, centrifuge, take the supernatant, divide into aliquots, and store at -70°C. Dilute with SA buffer at a ratio of 1:8 when used.

[0072] (3) Slide coating: Apply a thin layer of 0.5% agarose on a clean slide and allow to dry naturally before long-term storage.

[0073] (4) Immunization of animals: On the 26th day after administration, defibrinated sheep blood was collected and washed three times with sodium chloride injection. Each time, the blood was centrifuged (2000 r / min) for 10 min. The packed SRBC (sheep red blood cells) was prepared into a 2% (v / v) cell suspension with sodium chloride injection and 0.2 mL / mouse was injected intraperitoneally into each group of mice.

[0074] (5) Preparation of spleen cell suspension: 24 h after the last administration, the spleen of each group of animals was aseptically removed and placed in a plate containing an appropriate amount of sterile Hank's solution. The spleen was gently ground and filtered through a 200-mesh sieve. The suspension was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. Red blood cell lysis buffer was added at a ratio of 1:1 (v:v). After the red blood cells were lysed, the suspension was centrifuged at 1000 rpm for 10 min, and the supernatant was discarded. The cells were suspended in 3 mL of complete culture medium and the number of live cells was counted by trypan blue staining. The cell concentration was adjusted to 5 × 10 6 Pieces / mL.

[0075] (6) Detection of antibody-producing cells: Heat and dissolve the surface culture medium (1 g agarose plus double distilled water to 100 mL), place in a 45-50°C water bath to keep warm, mix with an equal amount of Hank's solution at pH 7.2-7.4 and 2 times the concentration, dispense into small test tubes, 0.5 mL per tube, add 50 μL of 10% SRBC (v / v, prepared with SA buffer) and 25 μL of spleen cell suspension to the tube, mix quickly, and pour onto a glass slide that has been brushed with a thin layer of agarose. After the agar solidifies, place the glass slide horizontally on a slide rack and incubate in a carbon dioxide incubator for 1.5 hours. Then, add complement diluted with SA buffer (1:8) into the groove of the slide rack. After continuing to incubate for 1.5 hours, count the number of hemolytic plaques. Number of hemolytic plaques = number of plaques / total spleen cells. Results are shown in Figure 1 .

[0076] Depend on Figure 1 It can be seen that the number of hemolytic plaques in Example 3-5 groups was significantly higher than that in the negative control group, with significant differences (**p<0.01, ***p<0.001). The effect of the probiotic composition of Example 3-5 on increasing the number of hemolytic plaques was better than that of the positive control group and the comparative example 1-6 groups. The results show that the probiotic composition of the present invention can increase the production of antibody-producing cells B cells in normal mice.

[0077] Example 8: Effect of probiotic composition on serum hemolysin in normal mice

[0078] 1. The selection and grouping of animals refer to the above-mentioned Example 7.

[0079] 2. Experimental process

[0080] (1) Immunization of animals: On the 26th day after administration, the animals were intraperitoneally injected with 2% (v / v) packed SRBC suspension, 0.2 mL / animal.

[0081] (2) Sampling: On the 31st day after administration (i.e., the day after the last oral gavage), the eyeballs of the animals were removed and blood was collected into centrifuge tubes. The tubes were left for about 1 hour to separate the coagulated blood from the tube wall and allow the serum to fully separate out. The tubes were centrifuged at 2000 rpm for 10 min to collect the serum.

[0082] (3) Agglutination reaction determination: dilute the serum in multiples with sodium chloride injection, place the serum of different dilutions in a micro-hemagglutination test plate, 100 μL / well, add 100 μL of 0.5% (v / v) SRBC suspension, mix well, place in a moistened plate, cover, incubate at 37°C for 3 h, observe the degree of hemagglutination, and calculate the antibody level according to the following formula. The serum should be fully mixed when diluted, and no agglutination should occur at the last dilution.

[0083] Antibody level = (S1+2S2+3S3…nSn)

[0084] Wherein 1, 2, 3...n represent the index of dilution; S represents the level of agglutination: Level 0: All red blood cells sink and concentrate at the bottom of the well to form dense dots, with clear liquid around them; Level I: Most of the red blood cells sink at the bottom of the well to form dots, with a small amount of agglutinated red blood cells around them; Level II: The agglutinated red blood cells form a thin layer at the bottom of the well, with a distant red dot clearly visible in the center; Level III: The agglutinated red blood cells are evenly spread at the bottom of the well to form a thin layer, with a small red dot faintly visible in the center; Level IV: The agglutinated red blood cells are evenly spread at the bottom of the well to form a thin layer, and the clots are sometimes rolled up.

[0085] Results Figure 2 .

[0086] Depend on Figure 2 It can be seen that the total antibody volume of Example 3-5 groups is significantly higher than that of the negative control group, with significant differences (*p<0.05, **p<0.01), and the effect of increasing the antibody volume is better than that of the positive control group and the comparative example 1-6 groups. The results show that the probiotic composition of the present invention can increase the production of serum hemolysin in mice.

[0087] Humoral immunity is an immune response mediated by antibodies, immune molecules secreted by B cells. This study detected the effects of the probiotic composition on antibody-producing cells and serum hemolysin. The results showed that the probiotic composition can promote B cells to produce antibodies, and the effect is better than that of the positive control group. The probiotic composition can also increase the amount of serum hemolysin in normal mice. This result shows that the probiotic composition of the present invention has the function of significantly improving the body's humoral immunity.

[0088] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A probiotic composition, characterized in that: The preparation method comprises the following raw materials in parts by weight: 10 to 20 parts of probiotics, 5 to 15 parts of black garlic extract, 1 to 5 parts of Eucheuma polysaccharide and 0.5 to 1 part of stevioside.

2. The probiotic composition according to claim 1, characterized in that The probiotics are at least one of Lactobacillus rhamnosus, Lactobacillus reuteri, Bifidobacterium animalis and Bifidobacterium breve.

3. The probiotic composition according to claim 2, characterized in that The rhamnosus lactobacillus is rhamnosus lactobacillus LGG, the reuteri lactobacillus is reuteri lactobacillus DSM17938, the animal bifidobacterium is animal bifidobacterium BB-12, and the breve bifidobacterium is breve bifidobacterium M-16V.

4. The probiotic composition according to claim 1, characterized in that The preparation steps of the black garlic extract are: (1) drying and crushing the black garlic, adding water and stirring to mix, then adding hemicellulase and cellulase to perform enzymolysis at 45-60° C. for 1-2 h, then heating to 95-100° C. for extraction for 1-2 h, filtering, collecting the first filtrate, and retaining the filter residue; (2) adding ethanol solution to the filter residue, extracting at 65-75° C. for 1-2 h, filtering, and collecting the second filtrate; (3) combining the first filtrate and the second filtrate, concentrating and drying to obtain a black garlic extract.

5. The probiotic composition according to claim 4, characterized in that In step (1), the amount of hemicellulase and cellulase added is 0.5-1% of the mass of the black garlic powder.

6. The probiotic composition according to claim 4, characterized in that In step (2), the solid-liquid ratio of the filter residue to the ethanol solution is 1:3-4 g / mL, and the concentration of the ethanol solution is 10-15% by volume.

7. The method for preparing the probiotic composition according to any one of claims 1 to 6, characterized in that: The method comprises the steps of mixing probiotics, black garlic extract, Eucheuma polysaccharide and stevioside.

8. Use of the probiotic composition according to any one of claims 1 to 6 in the preparation of a medicament for treating immune diseases.

9. Use of the probiotic composition according to any one of claims 1 to 6 in the preparation of health products, medicines or foods for improving immunity.

10. The use according to claim 9, characterized in that: The probiotic composition is prepared into powder, granules, tablets or capsules.

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