Seven-probiotic composition for improving immunity and application thereof

Abstract

The invention relates to a composition of seven probiotics for improving immunity and application of the composition. The bacterial strains in the probiotic composition comprise a combination of a streptococcus thermophilus CT-02 bacterial strain, a bifidobacterium animalis subsp. Lactis C-2 bacterial strain, a bifidobacterium longum subsp. Infantis C-3 bacterial strain, a lactobacillus delbrueckii subsp. Bulgaricus CB-01 bacterial strain, a lactobacillus acidophilus CL-01 bacterial strain, a lactobacillus paracasei CL-02 bacterial strain and a lactobacillus rhamnosus CL-03 bacterial strain. Potential interaction exists among the seven strains, the seven strains can be mutually matched and synergistically interacted when being combined, and the seven strains have excellent effects in the aspects of improving immunity, relieving inflammatory response, improving the health degree of the intestinal environment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a seven-probiotic composition for enhancing immunity and its application. Background Art

[0002] As the core defense system for the human body to resist pathogen invasion, the functional integrity of the immune system depends on the dynamic synergistic effects of immune organs, immune cells and active substances. Immunity is an important defense mechanism for the human body to resist pathogen invasion and maintain its own health.

[0003] As the largest immune organ in the human body, about 10 14 microorganisms colonized in the intestine form a bidirectional regulatory network with the host immune system. The intestinal flora can not only directly activate the innate immune response by metabolizing active substances such as short-chain fatty acids (SCFAs), but also regulate the differentiation of T lymphocyte subsets and cytokine secretion through the gut-immune axis. However, external interfering factors such as the intake of a high-fat diet are likely to cause intestinal flora disorders, thereby weakening the immune barrier function and forming a vicious cycle of "flora imbalance-immune suppression".

[0004] Probiotics have become a research hotspot due to their inherent immunomodulatory properties. Specific probiotics (such as Lactobacillus rhamnosus GG strain) can activate dendritic cells through the TLR4 / MyD88 signaling pathway and promote the secretion of Th1-type cytokine IFN-γ; Bifidobacterium can up-regulate the expression of intestinal epithelial tight junction proteins, thereby enhancing the physical barrier function. In the prior art, mostly single strains or simple combinations of two to three strains are used, and their action mechanisms are limited to the regulation of a single immune index (such as enhanced IgA secretion). Due to the high heterogeneity of the human intestinal microbial environment (including more than 1000 kinds of flora competing for ecological niches), it is difficult for a single strain to break through the colonization resistance to achieve stable proliferation, and simple compounding is prone to cause metabolic competition between strains, resulting in insufficient coverage of immune regulation nodes. Therefore, how to develop a composite probiotic composition product designed based on the multi-strain metabolic interaction mechanism, which can achieve coordinated synergistic effects for multiple immune links such as maintaining the intestinal barrier, activating immune cells, resisting oxidative stress or regulating inflammatory responses, has become an important path to break through the existing technical barriers. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a seven-probiotic composition for enhancing immunity and its application.

[0006] To achieve the purpose of this invention, the following technical solutions are adopted by the present invention:

[0007] In a first aspect, the present invention provides a probiotic composition of seven strains for enhancing immunity. The strains in the probiotic composition include a combination of Streptococcus thermophilus CT-02 strain with the preservation number of GDMCC No: 64474, Bifidobacterium animalis subsp. lactis C-2 strain with the preservation number of GDMCC No: 65455, Bifidobacterium longum subsp. infantis C-3 strain with the preservation number of GDMCC No: 64541, Lactobacillus delbrueckii subsp. bulgaricus CB-01 strain with the preservation number of GDMCC No: 64475, Lactobacillus acidophilus CL-01 strain with the preservation number of GDMCC No: 64731, Lacticaseibacillus paracasei CL-02 strain with the preservation number of GDMCC No: 64476, and Lacticaseibacillus rhamnosus CL-03 strain with the preservation number of GDMCC No: 65725.

[0008] The present invention has developed a brand-new probiotic compounding method and a brand-new strategy for enhancing immunity, that is, compounding and using in combination seven strains, namely Streptococcus thermophilus CT-02 strain, Bifidobacterium animalis subsp. lactis C-2 strain, Bifidobacterium longum subsp. infantis C-3 strain, Lactobacillus delbrueckii subsp. bulgaricus CB-01 strain, Lactobacillus acidophilus CL-01 strain, Lacticaseibacillus paracasei CL-02 strain, and Lacticaseibacillus rhamnosus CL-03 strain. It is found that there are potential interactions among the seven strains, which can cooperate with each other and synergistically enhance the effect. When the amount of bacteria used is the same, compared with the strain intervention method lacking any one strain, the compounding of the seven strains significantly improves the effect in enhancing immunity.

[0009] Preferably, the viable count ratio of the CT-02 strain, C-2 strain, C-3 strain, CB-01 strain, CL-01 strain, CL-02 strain, and CL-03 strain is (1 - 10):(1 - 10):(0.1 - 5):(1 - 10):(5 - 15):(1 - 10):(0.1 - 5).

[0010] Among them, the above "1 - 10" can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0011] The above "0.1 - 5" can be, for example, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0012] The above "5 - 15" can be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. Any other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0013] Based on the potential interaction relationships among the seven strains, the present invention also discovers that when the seven strains are compounded and used in the above - mentioned specific viable - bacteria number ratio, the efficacy in improving the immunity level is more remarkable.

[0014] Preferably, the total viable - bacteria concentration of the strains in the probiotic composition is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g, such as 1×10 8 CFU / mL (CFU / g), 2×10 8 CFU / mL (CFU / g), 5×10 8 CFU / mL (CFU / g), 8×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), etc. Any other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0015] In a second aspect, the present invention provides a probiotic agent for improving immunity, and the probiotic agent includes the probiotic composition described in the first aspect.

[0016] Preferably, the dosage form of the probiotic agent is selected from solution, freeze - dried powder, capsule, tablet or granule.

[0017] Preferably, the probiotic agent further includes a protective agent.

[0018] Preferably, the protective agent includes any one or a combination of at least two of skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, arabic gum, sodium alginate, polyvinylpyrrolidone, sorbitol, xylo - oligosaccharide, fructo - oligosaccharide or xylitol.

[0019] Preferably, the dosage form of the probiotic agent is a solution, which is prepared by the following method:

[0020] The strains are respectively inoculated into a culture medium and sequentially activated and fermented to obtain a fermentation broth; the fermentation broth is centrifuged respectively and resuspended with a solvent to obtain a bacterial suspension of the corresponding strain, and the bacterial suspensions are mixed according to the viable count ratio to obtain the probiotic agent.

[0021] Alternatively, the dosage form of the probiotic agent is a freeze-dried powder, which is prepared by the following method:

[0022] The strains are respectively inoculated into a culture medium and sequentially activated and fermented to obtain a fermentation broth; the fermentation broth is centrifuged respectively, mixed with a cryoprotectant and then freeze-dried to obtain the bacterial powder of the corresponding strain, and the bacterial powders are mixed according to the viable count ratio to obtain the probiotic agent.

[0023] Preferably, the culture medium includes MRS culture medium or M17 culture medium.

[0024] Preferably, the MRS culture medium includes, by concentration: peptone 8 - 12 g / L, beef extract 8 - 12 g / L, glucose 15 - 25 g / L, sodium acetate 1 - 3 g / L, yeast powder 3 - 7 g / L, diammonium hydrogen citrate 1 - 3 g / L, K2PO4·3H2O 2 - 3 g / L, MgSO4·7H2O 0.05 - 0.2 g / L, MnSO4 0.01 - 0.1 g / L, Tween 80 0.5 - 2 mL / L, cysteine hydrochloride 0.1 - 1 g / L.

[0025] Preferably, the M17 culture medium includes, by concentration: soy peptone 3 - 7 g / L, yeast extract 3 - 7 g / L, casein peptone 3 - 7 g / L, ascorbic acid 0.1 - 1 g / L, beef extract 1 - 10 g / L, β-glycerophosphate disodium 15 - 25 g / L.

[0026] In the third aspect, the present invention provides an application of the probiotic composition as described in the first aspect or the probiotic agent as described in the second aspect in the preparation of food or health products.

[0027] In the fourth aspect, the present invention provides an application of the probiotic composition as described in the first aspect or the probiotic agent as described in the second aspect in the preparation of a preparation for preventing or alleviating liver injury caused by inflammatory factors, spleen injury caused by inflammatory factors or inflammatory bowel disease.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention has developed a brand-new probiotic compounding method and a brand-new strategy for enhancing immunity, that is, a combination of seven strains, namely Streptococcus thermophilus strain CT-02, Bifidobacterium animalis subsp. lactis strain C-2, Bifidobacterium longum subsp. infantis strain C-3, Lactobacillus delbrueckii subsp. bulgaricus strain CB-01, Lactobacillus acidophilus strain CL-01, Lactobacillus paracasei strain CL-02, and Lactobacillus rhamnosus strain CL-03. It is found that there are potential interactions among the seven strains, which can cooperate with each other and synergistically enhance the effect. When the amount of bacteria used is the same, compared with the strain intervention method lacking any one strain, the compounding of the seven strains significantly improves the effect in enhancing immunity.

[0030] The CT-02 strain involved in the present invention is classified and named as Streptoccus thermophilus. The preservation unit is the Guangdong Provincial Microbial Culture Collection Center, the preservation number is GDMCC No: 64474, the preservation date is March 29, 2024, and the preservation address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0031] The C-2 strain involved in the present invention is classified and named as Bifidobacterium animalis subsp. lactis. The preservation unit is the Guangdong Provincial Microbial Culture Collection Center, the preservation number is GDMCC No: 65455, the preservation date is November 8, 2024, and the preservation address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0032] The C-3 strain involved in the present invention is classified and named as Bifidobacterium longum subsp. infantis. The preservation unit is the Guangdong Provincial Microbial Culture Collection Center, the preservation number is GDMCC No: 64541, the preservation date is April 22, 2024, and the preservation address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0033] The CB-01 strain involved in the present invention is classified and named as Lactobacillus delbrueckii subsp. bulgaricus. The preservation unit is the Guangdong Provincial Microbial Culture Collection Center, the preservation number is GDMCC No: 64475, the preservation date is March 29, 2024, and the preservation address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0034] The taxonomic name of the CL-01 strain involved in the present invention is Lactobacillus acidophilus. The preservation unit is the Guangdong Provincial Culture Collection Center of Microorganisms. The preservation number is GDMCC No: 64731. The preservation date is June 7, 2024. The preservation address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou;

[0035] The taxonomic name of the CL-02 strain involved in the present invention is Lacticaseibacillus paracasei. The preservation unit is the Guangdong Provincial Culture Collection Center of Microorganisms. The preservation number is GDMCC No: 64476. The preservation date is March 29, 2024. The preservation address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou;

[0036] The taxonomic name of the CL-03 strain involved in the present invention is Lacticaseibacillus rhamnosus. The preservation unit is the Guangdong Provincial Culture Collection Center of Microorganisms. The preservation number is GDMCC No: 65725. The preservation date is February 21, 2025. The preservation address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou. Description of the Drawings

[0037] Figure 1 It is a statistical result chart of the weight changes of mice in each group before and after modeling;

[0038] Figure 2 It is a statistical result chart of the content levels of TNF-α, IL-6, IFN-γ and IL-10 factors in the sera of mice in each group;

[0039] Figure 3 It is a statistical result chart of the IgA and IgG levels in the sera of mice in each group;

[0040] Figure 4 It is a histological observation and analysis result chart of the liver sections of mice in groups S1, S10 and S11;

[0041] Figure 5 It is a statistical result chart of the alanine aminotransferase levels in the sera of mice in each group;

[0042] Figure 6 It is a histological observation and analysis result chart of the spleen sections of mice in groups S1, S10 and S11;

[0043] Figure 7 It is a statistical result chart of the ratio of the content levels of reduced glutathione and oxidized glutathione in the spleen tissues of mice in each group;

[0044] Figure 8It is a figure showing the results of histopathological observation and analysis of intestinal sections of mice in groups S1, S10, and S11;

[0045] Figure 9 It is a figure showing the statistical results of the contents of TNF-α, IL-6, INF-γ, and IL-10 factors in the intestinal tissues of mice in each group;

[0046] Figure 10 It is a figure showing the statistical results of the IgA level in the fecal samples of mice in each group. Detailed implementation manners

[0047] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0048] The information of the bacterial strains involved below is as follows:

[0049] ① The classification and naming of the CT-02 strain involved below is Streptococcus thermophilus, and the preservation number is GDMCC No: 64474;

[0050] ② The classification and naming of the C-2 strain involved below is Bifidobacterium animalis subsp. lactis, and the preservation number is GDMCC No: 65455;

[0051] ③ The classification and naming of the C-3 strain involved below is Bifidobacterium longum subsp. infantis, and the preservation number is GDMCC No: 64541;

[0052] ④ The classification and naming of the CB-01 strain involved below is Lactobacillus delbrueckii subsp. bulgaricus, and the preservation number is GDMCC No: 64475;

[0053] ⑤ The classification and naming of the CL-01 strain involved below is Lactobacillus acidophilus, and the preservation number is GDMCC No: 64731;

[0054] ⑥ The classification and naming of the CL-02 strain involved below is Lacticaseibacillus paracasei, and the preservation number is GDMCC No: 64476;

[0055] ⑦ The taxonomic naming of the CL-03 strain involved below is Lacticaseibacillus rhamnosus, and the deposit number is GDMCC No: 65725.

[0056] The culture medium and its formula involved below are as follows:

[0057] The mice involved below are C57BL / 6J male mice (6 weeks old), sourced from Beijing Sparf Bioscience Co., Ltd.

[0058] The preparation methods of the bacterial suspension and bacterial powder involved below are as follows: After activating the strain, it is inoculated into the culture medium for cultivation respectively to obtain the culture solution; the culture solution is centrifuged, and the bacterial cells are resuspended to obtain the bacterial suspension, or further, a cryoprotectant is added for freeze-drying to prepare the freeze-dried bacterial powder product.

[0059] The histogram is made using GraphPad 8.0 statistical software, and the values are expressed as mean ± SEM.

[0060] Example 1

[0061] This example explores the improvement ability of the compound probiotic on various indexes of inflammatory mice

[0062] (1) Test animals: 6-week-old C57BL / 6J male mice, housed under specific pathogen-free conditions in the laboratory, with a daily light cycle of 12 h, a temperature of 22 ± 2 °C, and a relative humidity of 55% ± 10%

[0063] (2) Animal grouping and modeling: The random grouping of the mice after one week of adaptive feeding is shown in Table 1 as follows:

[0064] Table 1

[0065]

[0066] Among them, there are 6 mice in each group in the above table, and the total viable bacteria count in each of the S1 - S9 groups is 1×10 9 CFU / mL.

[0067] (3) Intervention method: During the test, the mice in each group were allowed to eat and drink freely.

[0068] From the 8th day to the 35th day of the test, the S1 - S9 groups were intragastrically administered the corresponding compound probiotic bacterial suspension twice a day, 250 μL each time, and the S10 and S11 groups were intragastrically administered an equal amount of sterile normal saline twice a day. On the 36th day, except for the S11 group, the remaining groups were intraperitoneally injected with 5 mg / Kg b.w. lipopolysaccharide (LPS, Escherichia coli O55:B5). After modeling, the mice were anesthetized and sacrificed 24 h later, and the samples were collected.

[0069] (4) Index testing

[0070] ① Mouse body weight analysis

[0071] Before and after modeling, the body weights of mice in each group were counted respectively, and the difference in body weight changes of mice on the 36th day (before modeling) and the 37th day (after modeling) of the experiment was calculated. The results are as Figure 1 shown. At the same time, the fecal shape and health status of mice were observed, and the results are shown in Table 2.

[0072] Table 2

[0073]

[0074] It can be seen from the statistical results in the figure that after the experiment, the body weights of mice in the S10 (model group) group decreased significantly, and there were soft stools, sparse stools, disordered back hair, curled up and less active, listless and slow to respond, etc., proving that the modeling was successful. After intervention with probiotics, the state of body weight loss in mice in each probiotic intervention group was alleviated to varying degrees. Among them, by comparing the S1 group and the S3-S9 groups, it can be seen that when any one of the seven strains CT-02, C-2, C-3, CB-01, CL-01, CL-02, and CL-03 was missing in the compound probiotics used, the effect was worse than that of the S1 group. It can be seen that the present invention uses the above seven specific probiotics for compound combination, which can synergistically increase the effect of improving the level of body weight loss in inflammatory mice.

[0075] ② Analysis of serum cytokine levels in mice

[0076] After the experiment, the sera of mice in each group were taken and centrifuged at 2000×g for 15 min at 4°C. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-γ (IFN-γ), immunoglobulin A (IgA), and immunoglobulin G (IgG) in the sera were measured with reference to the ELISA kit instructions. The results are respectively as Figure 2 and Figure 3 shown.

[0077] Lipopolysaccharide derived from Gram-negative bacteria can directly activate the innate immune cells of mammals to produce a large number of inflammatory mediators, and the produced inflammatory mediators can directly or indirectly damage host cells or tissues. It can be seen from the figure that compared with the S11 (blank group), the levels of pro-inflammatory factors IL-6, TNF-α, and IFN-γ in the sera of mice in the S10 (model group) were significantly increased, and the level of anti-inflammatory factor IL-10 was significantly decreased. At the same time, the release of pro-inflammatory factors IL-6 and TNF-α further stimulated B cells to produce more IgA and IgG. It can be seen that after lipopolysaccharide injection, immune cells can be activated, resulting in the production and release of a large number of inflammatory factors in mice, while inhibiting the expression of anti-inflammatory factors.

[0078] After the intervention of probiotics, the levels of pro-inflammatory factors in mice in each probiotic intervention group were reduced to varying degrees, and at the same time, the levels of anti-inflammatory factors were significantly increased. Among them, by comparing the S1 group with the S3-S9 groups, it can be seen that when any one of the seven strains CT-02, C-2, C-3, CB-01, CL-01, CL-02, and CL-03 is missing from the compound probiotics used, the effect is worse than that of the S1 group. It can be seen that the use of the above seven specific probiotics in combination in the present invention can synergistically enhance the effect, prevent the stimulation of the mouse immune system by LPS, reduce the release of inflammatory factors, inhibit excessive inflammation, and thus further reduce the over-activation of B cells and the production of immunoglobulins IgA and IgG.

[0079] ③ Analysis of liver injury level

[0080] a. After the experiment, partial liver tissues of mice in the S1 group, S10 group, and S11 group were taken for H&E staining sections, and the section analysis results are as Figure 4 shown.

[0081] As can be seen from the figure, in S11 (blank group), mouse hepatocytes are arranged in a lamellar structure centered on the central vein, and several bile ducts can be seen between liver lobules; the cytoplasm of hepatocytes is rich, and the cell nucleus is located in the center of the cell, and no obvious histopathological changes are seen; in the liver of S10 (model group) mice, mild multifocal hepatocyte swelling and degeneration can be seen, manifested as hepatocyte enlargement, transparent cytoplasm, and narrowing of the hepatic sinusoid space; in the S1 group, no obvious histopathological changes were seen in the liver of mice after probiotic intervention. It can be seen that the probiotic composition provided by the present invention can effectively relieve the symptoms related to liver injury caused by the inflammatory reaction induced by lipopolysaccharide.

[0082] b. Serum of mice in each group was taken, and the level of alanine aminotransferase (ALT) was measured using an ELISA kit, and the statistical results are as Figure 5 shown.

[0083] Intestinal-derived or exogenously administered LPS can translocate to the liver and further impair liver function. Among the biomarkers of liver injury, alanine aminotransferase (ALT) is an important indicator reflecting hepatocyte injury. When hepatocytes are damaged, ALT will be released from the cells into the blood, leading to an increase in the ALT level in the blood. As shown in the figure, intraperitoneal injection of LPS induced hepatocyte damage in mice. The ALT level in the serum of S10 (model group) mice increased significantly, and the histological section results showed a large amount of fat accumulation and infiltration of inflammatory cells in the mouse liver, indicating that the inflammatory model mice were successfully constructed. After intervention with probiotics, the degree of liver injury in mice in each probiotic intervention group was alleviated, indicating that the combination of seven strains, namely CT-02, C-2, C-3, CB-01, CL-01, CL-02, and CL-03, could have a synergistic effect and effectively alleviate liver injury caused by LPS.

[0084] ④ Splenic injury level

[0085] a. After the experiment, partial spleen tissues of mice in groups S1, S10, and S11 were taken for H&E staining sections, and the section analysis results are as Figure 6 shown.

[0086] As shown in the figure, in the spleen tissue of S11 (blank group) mice, dark blue lymphoid tissue was visible to form white pulp, which was distributed between dark red red pulp, and no obvious histopathological changes were observed; while in the spleen of S10 (model group) mice, mild necrosis of lymphocytes in the germinal center of white pulp and slight atrophy of white pulp were visible, specifically manifested as a decrease in lymphocytes in the periarteriolar lymphatic sheath; while in group S1 treated with probiotics, only mild necrosis of lymphocytes in the germinal center of white pulp was observed in the spleen tissue of mice, indicating that the seven probiotic compositions provided by the present invention can effectively alleviate the symptoms related to splenic injury induced by an increase in the inflammatory level.

[0087] b. Another part of the spleen tissues of mice in each group was homogenized, and the contents of reduced glutathione (GSH) and oxidized glutathione (GSSG) were measured with reference to the reagents and instructions, and the GSH / GSSG ratio was calculated. The results are as Figure 7 shown.

[0088] LPS can stimulate macrophages to secrete pro-inflammatory factors such as TNF-α, IL-6, and INF-γ. These pro-inflammatory factors can lead to the production and increase of reactive oxygen species by damaging mitochondrial integrity and function, exacerbate GSH consumption, and cause a decrease in the GSH / GSSG ratio. As shown in the figure, the GSH / GSSG ratio in the S10 (model group) samples was significantly lower than that in the S11 (blank group). Moreover, the results of section staining showed that a large number of immune cells (such as monocytes, lymphocytes, and neutrophils) were recruited to the spleen, resulting in splenomegaly due to the infiltration and proliferation of immune cells, and pathological changes such as inflammatory cell infiltration, congestion, hemorrhage, and necrosis occurred. After intervention with probiotics, the oxidative stress state of the spleen in mice in each probiotic intervention group was alleviated, indicating that the combination of seven strains, namely CT-02, C-2, C-3, CB-01, CL-01, CL-02, and CL-03, can work synergistically to effectively improve the oxidative stress state of the spleen tissue caused by LPS.

[0089] ⑤ Intestinal barrier function analysis

[0090] a. After the experiment, partial intestinal tissues of mice in the S1 group, S10 group, and S11 group were taken for PAS staining sections, and the results of section analysis are as Figure 8 shown.

[0091] As can be seen from the figure, in the colon mucosa of mice in the S11 (blank group), fold structures can be seen, containing relatively abundant intestinal glands. PAS staining shows that goblet cells are blue-violet, and no obvious histopathological changes are observed. Compared with the S11 (blank group), the colon mucosa of mice in the S10 (model group) showed mild to moderate atrophy, specifically manifested as shortened mucosa and intestinal gland heights, reduced number of goblet cells, and dilated intestinal lumen. In the S1 group treated with probiotics, only mild to slight atrophy symptoms were observed in the colon mucosa of mice, indicating that the composition composed of seven probiotics provided by the present invention can effectively relieve related intestinal injury symptoms.

[0092] b. Another part of the intestinal tissues of mice in each group was homogenized, and the levels of TNF-α, IL-6, INF-γ, and IL-10 factors were measured with reference to the reagents and instructions. The results are as Figure 9 shown.

[0093] As can be seen from the figure, after injecting lipopolysaccharide into the abdominal cavity of mice, the levels of pro-inflammatory factors TNF-α, IL-6, and INF-γ in the colon tissues of mice in the S10 (model group) were significantly higher than the levels of related factors in the S11 (blank group) samples, and the level of the anti-inflammatory factor IL-10 was significantly decreased. After intervention with probiotics, the damaged state of the intestinal barrier in mice in each probiotic intervention group was alleviated, indicating that the seven strains CT-02, C-2, C-3, CB-01, CL-01, CL-02, and CL-03 can work synergistically to effectively improve the damaged state of the barrier caused by LPS.

[0094] ⑥ Analysis of the levels of related immunoglobulins in intestinal contents

[0095] After the experiment, mouse feces were collected, and the levels of secretory immunoglobulin A (IgA) in the fecal samples of each group of mice were measured according to the instructions of the ELISA kit. The results are as Figure 10 shown.

[0096] IgA is the main antibody produced by the intestinal mucosa. By observing the content levels of related factors in fecal samples, the intestinal health and inflammation levels of the host can be reflected to a certain extent. As can be seen from the figure, after injecting lipopolysaccharide into the abdominal cavity of mice, the level of IgA antibody in the intestines of S10 (model group) mice was significantly higher than that of the related factor levels in the S11 (blank group) samples. After intervention with probiotics, the intestinal inflammation symptoms of each probiotic intervention group of mice were alleviated, indicating that the seven strains of CT-02, C-2, C-3, CB-01, CL-01, CL-02, and CL-03 can synergistically enhance the effect and effectively improve the intestinal inflammatory symptoms caused by LPS, with excellent effects.

[0097] The applicant declares that the technical solution of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0098] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0099] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. In order to avoid unnecessary repetition, the present invention will not describe various possible combination methods separately.

Claims

1. A composition of seven probiotics for enhancing immunity, characterized in that, The strains in the probiotic composition include Streptococcus thermophilus with the preservation number GDMCC No: 64474 Streptoccus thermophilus Strain CT-02, Bifidobacterium animalis subsp. lactis with the preservation number GDMCC No: 65455 Bifidobacterium animalis subsp. lactis Strain C-2, Bifidobacterium longum subsp. infantis with the preservation number GDMCC No: 64541 Bifidobacterium longum subsp. infantis Strain C-3, Lactobacillus delbrueckii subsp. bulgaricus with the preservation number GDMCC No: 64475 Lactobacillus delbrueckii subsp. bulgaricus Strain CB-01, Lactobacillus acidophilus with the preservation number GDMCC No:64731 Lactobacillus acidophilus Strain CL-01, Lactobacillus paracasei with the preservation number GDMCC No:64476 Lacticaseibacillus paracasei Strain CL-02 and Lactobacillus rhamnosus with the preservation number GDMCCNo: 65725 Lacticaseibacillus rhamnosus The combination of strain CL-03.

2. The probiotic composition according to claim 1, wherein The viable cell count ratio of the CT-02 strain, C-2 strain, C-3 strain, CB-01 strain, CL-01 strain, CL-02 strain and CL-03 strain is (1-10):(1-10):(0.1-5):(1-10):(5-15):(1-10):(0.1-5).

3. The probiotic composition according to claim 1, characterized in that, The total viable cell concentration of the strains in the probiotic composition is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

4. A probiotic agent for enhancing immunity, characterized in that, The probiotic agent includes the probiotic composition as described in claim 1.

5. The probiotic agent according to claim 4, wherein The dosage form of the probiotic agent is selected from solution, freeze-dried powder, capsule, tablet or granule.

6. The probiotic agent according to claim 4, wherein The probiotic agent further includes a protective agent.

7. The probiotic agent according to claim 6, characterized in that, The protective agent includes any one or a combination of at least two of skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, gum arabic, sodium alginate, polyvinylpyrrolidone, sorbitol, xylo-oligosaccharide, fructo-oligosaccharide or xylitol.

8. The probiotic agent according to claim 5, characterized in that, The dosage form of the probiotic agent is solution, which is prepared by the following method: The strains are respectively inoculated into a culture medium and sequentially activated and fermented to obtain a fermentation broth; the fermentation broth is centrifuged respectively and resuspended with a solvent to obtain the corresponding strain suspensions, and the suspensions are mixed according to the viable cell count ratio to obtain the probiotic agent; Alternatively, the dosage form of the probiotic agent is freeze-dried powder, which is prepared by the following method: The strains are respectively inoculated into a culture medium and sequentially activated and fermented to obtain a fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and then freeze-dried to obtain the bacterial powder of the corresponding strain, and the bacterial powders are mixed according to the viable cell count ratio to obtain the probiotic agent.

9. Use of a probiotic composition according to any one of claims 1-3 or a probiotic agent according to any one of claims 4-8 in the preparation of a food or health product.

10. Use of a probiotic composition according to any one of claims 1-3 or a probiotic agent according to any one of claims 4-8 in the preparation of a preparation for preventing or alleviating liver injury caused by inflammatory factors, spleen injury caused by inflammatory factors or inflammatory bowel disease.

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