A probiotic composition with anti-allergy and immune-boosting functions

Abstract

This invention provides a probiotic composition with anti-allergic and immune-enhancing functions. The probiotic composition includes Bifidobacterium animalis. Bifidobacterium animalum Lactobacillus reuteri Lactobacillus reuteri Akkermania Akkermansia muciniphila Clostridium butyricum Clostridium butyricum and Bacillus coagulans Bacillus coagulans This invention, through the scientific compounding of multiple probiotics, can significantly enhance anti-allergy effects while effectively boosting the body's immunity, with a gentle and stable overall effect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a probiotic composition with anti-allergic and immune-enhancing functions. Background Technology

[0002] Allergic reactions are essentially closely related to immune imbalance. When the immune system responds abnormally to external stimuli, it can easily trigger allergic symptoms in multiple parts of the body, including the skin, respiratory tract, and digestive tract. Long-term, recurrent attacks can further reduce the body's resistance and affect quality of life. Currently, interventions for allergies and immune regulation are limited in their specificity and safety with long-term use, making it difficult to simultaneously meet the needs for safety, gentleness, and long-term conditioning.

[0003] Currently, intervention methods for allergic diseases and immune dysfunction still have many limitations. Most existing probiotic products are based on single strains or combinations of a few strains. These strains differ in physiological function, tolerance characteristics, and immunomodulatory capabilities. The comprehensive improvement capacity of a single strain on allergies and immune regulation is limited, and there is still room for further optimization and synergistic enhancement. These methods cannot meet the needs for daily, long-term, gentle, and safe health maintenance. Therefore, finding novel intervention programs that are natural, safe, suitable for long-term use, and capable of regulating immune status holistically has become an important research direction in this field.

[0004] The gut microbiota ecosystem is closely related to the establishment, maintenance, and regulation of the body's immune function. A stable gut microbiota structure and intact mucosal barrier function are crucial foundations for ensuring immune homeostasis. Probiotics, as live microorganisms capable of regulating gut microbiota balance and producing beneficial physiological effects on the host, have been widely proven to participate in intestinal barrier maintenance, immune cell differentiation regulation, and inflammatory response regulation, demonstrating significant application potential in the field of immune-related health. Different types and sources of probiotics exhibit significant differences in physiological function, tolerance characteristics, colonization ability, and metabolic activity. The scope and regulatory capacity of a single strain are limited, making it difficult to meet the diverse regulatory needs under complex physiological conditions. Therefore, rational strain combinations and functional optimization are important pathways to improve the application effects of probiotics.

[0005] Therefore, developing a probiotic composition that combines anti-allergy and immune-enhancing effects, with a dosage form that is suitable for industrial application, is of great practical significance for meeting the health needs of allergy sufferers and people with weakened immunity, and for enriching functional microecological products. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention proposes a probiotic composition with anti-allergic and immune-enhancing functions. The core active ingredients of this invention are a specific blend of Bifidobacterium animalis, Lactobacillus reuteri, Akkermansia, Clostridium butyricum, and Bacillus coagulans. These are further combined with screened anti-allergic active peptides of specific sequences to form a synergistic system. Suitable protective agents are used to prepare stable dosage forms such as lyophilized powder. Through functional complementarity between strains and the synergistic effect of probiotics and peptides, a systematic regulation of the body's immune homeostasis and allergic reactions is achieved. This composition can be used to prepare functional foods or health products.

[0007] This invention provides a probiotic composition comprising Bifidobacterium animalis. Bifidobacterium animalum Lactobacillus reuteri Lactobacillus reuteri Akkermania Akkermansia muciniphila Clostridium butyricum Clostridium butyricum and Bacillus coagulans Bacillus coagulating .

[0008] The selection of the above-mentioned strains for compounding in this invention is based on the complementary functions of the strains and the synergistic effect of the immune regulatory network, achieving systematic regulation of the body's immune homeostasis and allergic response. Each strain participates in the construction of the intestinal microecology, the strengthening of the mucosal barrier, and immune signal transduction through different targets, forming a multi-dimensional intervention system. This overcomes the shortcomings of single strains having limited function and regulatory range, providing a core bioactive basis for anti-allergy and enhanced immunity. Strains such as Bifidobacterium, Lactobacillus reuteri, and Clostridium butyricum can directionally colonize the intestinal mucosa, competitively inhibiting the adhesion and colonization of harmful bacteria, and maintaining the stability of the microbiota structure. Among them, Clostridium butyricum can metabolize to produce short-chain fatty acids, promoting the expression of tight junction proteins in intestinal epithelial cells, enhancing intestinal barrier function, reducing the penetration of exogenous allergens, and reducing the probability of allergy triggering from the source. Akkermansia can target and regulate the thickness of the mucus layer, further strengthening the mucosal physical barrier and reducing susceptibility to allergens.

[0009] Furthermore, the animal Bifidobacterium Bifidobacterium animalum Lactobacillus reuteri Lactobacillus reuteri Akkermania Akkermansia muciniphila Clostridium butyricum Clostridium buttermilk and Bacillus coagulans Bacillus coagulansThe mass ratio is (1~3):(1~3):(0.5~3):(1~3):(2:3), preferably (1~2):(2~3):(0.5~2):(2~3):(2~2.5); more preferably (2:2:1:3:2), (1:3:2:2:2) or (2:2:0.5:3:2.5); the above ratio range can maximize the positive synergistic effect between strains, optimize the intensity of immunomodulation and the efficiency of allergy suppression, avoid functional antagonism or effect attenuation caused by imbalance of ratio, and ensure the stable and prominent efficacy of the preparation.

[0010] It should be understood that when adjusting the ratio of each strain within the above-mentioned numerical range, those skilled in the art can obtain a combination scheme with similar technical effects through conventional experiments and appropriate optimization based on the technical teachings disclosed in this invention.

[0011] Furthermore, the animal Bifidobacterium Bifidobacterium animalum for Bifidobacterium animal ATCC 25527 Bifidobacterium animalum ATCC 27672 and Bifidobacterium animal Any one of CGMCC 1.1852; The Lactobacillus reuteri Lactobacillus reuteri for Lactobacillus reuteri DSM17938 Lactobacillus reuteri ATCC PTA6475 and Lactobacillus reuteri Any one of JCM 1112; The Akkermania Akkermansia muciniphila for Akkermansia muciniphila ATCC BAA-835; The butyric acid clostridium Clostridium butyricum for Clostridium butyricum CGMCC 0313.1 Clostridium butyricum MIYAIRI 588 and Clostridium butyricum Any one of ATCC 19398; The Bacillus coagulans Bacillus coagulans for Bacillus coagulans MTCC 5856 Bacillus coagulans ATCC 7050 and Bacillus coagulans Any of the following in DSM 1.

[0012] Furthermore, the probiotic composition also includes active peptides, the amino acid sequence of which is shown in SEQ ID NO. 1. The active peptides compounded in this invention can directly inhibit mast cell degranulation and block the initiation of allergy signals; the probiotics focus on long-term immune regulation and barrier repair. The combined use of the two forms a synergistic mode of rapid relief and long-term conditioning: the active peptides rapidly reduce allergy symptoms, while the probiotics continuously repair immune homeostasis, resulting in a significant improvement in overall efficacy.

[0013] Furthermore, the mass ratio of the active peptide to the probiotic composition is (0.5~1):1000; based on the optimal concentration effect of bioactivity synergy, the active peptide can play its role in a highly efficient and low-interference manner while ensuring that the core function of the probiotic is not affected, avoiding antagonism and failure caused by excessively high or low concentrations, and maximizing synergistic efficacy.

[0014] Furthermore, the final concentration of the active peptide in the probiotic composition is 0.5~1 mg / g.

[0015] Furthermore, the total viable bacterial concentration of the strains in the probiotic composition is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g; preferably, the total viable bacteria concentration is 1×10⁻⁶. 9 CFU / mL ~5×10 10 CFU / mL.

[0016] Furthermore, the dosage form of the probiotic agent is selected from solutions, lyophilized powders, capsules, tablets, or granules; based on the principle of adapting bioavailability and stability under different application scenarios, it can meet diverse usage needs, improve the applicability, convenience, and in vivo absorption efficiency of the composition, and expand the scope of application.

[0017] Furthermore, the probiotic agent is a lyophilized powder; The lyophilized powder also includes a protectant.

[0018] Furthermore, the protective agent includes any one or a combination of at least several of the following: skim milk powder, trehalose, mannitol, gelatin, sucrose, lactose, dextran, dextrin, gum arabic, sodium alginate, polyvinylpyrrolidone, sorbitol, xylooligosaccharides, fructooligosaccharides, or xylitol. Using a composite protective agent such as skim milk powder, trehalose, and mannitol, a protective film can be formed on the surface of the bacteria during freeze-drying, reducing ice crystal damage, maintaining cell membrane integrity, and maximizing the preservation of viable bacterial activity. When formulated as a freeze-dried powder, it can reduce water activity, inhibit bacterial metabolism, improve the product's storage and transportation resistance, and ensure stable efficacy.

[0019] The present invention also provides the use of the probiotic composition in the preparation of food or health products.

[0020] The present invention also provides an anti-allergic probiotic agent, wherein the active ingredient in the probiotic agent is the probiotic composition described above.

[0021] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) This invention can significantly improve the anti-allergy effect by scientifically compounding a variety of probiotics, while effectively enhancing the body's immunity. The overall effect is mild and stable.

[0022] (2) The probiotic composition of the present invention can be made into various dosage forms such as freeze-dried powder. With the help of special protective agents, it has good stability and is easy to store and use.

[0023] (3) By combining with active peptides, this invention can exert a synergistic effect, further enhance the anti-allergy effect, and has a wider range of applications. It can be used to prepare functional foods and health products, and has good application prospects and market value. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0026] Animal Bifidobacterium ( Bifidobacterium animalum ): Bifidobacterium animal CGMCC1.1852, purchased from Shanghai Center for Preservation of Biotechnology, abbreviated as BA.

[0027] Lactobacillus reuteri ( Lactobacillus reuteri ): Lactobacillus reuteri DSM 17938, purchased from Baosai Biotechnology, abbreviated as LR.

[0028] Akkermania ( Akkermansia muciniphila ): Akkermansia muciniphila ATCC BAA-835, catalog number TS263615, purchased from Taisto Biotechnology, abbreviated as AM.

[0029] Clostridium butyricum ( Clostridium butyricum ): Clostridium butyricum ATCC 19398, purchased from Fuxiang Biotechnology, abbreviated as CB.

[0030] Bacillus coagulans ( Bacillus coagulans ): Bacillus coagulans ATCC 7050, purchased from Warner Biotech, abbreviated as BC.

[0031] Example 1: Activation and Cultivation of Probiotics This embodiment provides a method for preparing a compound probiotic composition, wherein the probiotic composition includes Bifidobacterium, Lactobacillus reuteri, Akkermansia mucinosa, Clostridium butyricum, and Bacillus coagulans. The specific steps are as follows: (1) Activation of microbial strains: Each of the above-mentioned strains was removed from its -80℃ cryopreservation tube and allowed to thaw naturally at room temperature for approximately 2–5 minutes before being inoculated into its corresponding liquid culture medium for activation culture. Specifically, *Bifidobacterium* CGMCC1.1852 and *Lactobacillus reuteri* DSM 17938 were inoculated into MRS liquid medium and cultured anaerobically at 37℃ for 24 h (purged with nitrogen, oxygen concentration 0.5%). *Ackermania mucinica* ATCC BAA-835 was inoculated into modified BHI liquid medium containing 0.2% (w / v) mucin and cultured strictly anaerobicly at 37℃ (purged with a nitrogen-carbon dioxide mixture, volume ratio 8:2) for 36 h. *Clostridium butyricum* ATCC 19398 was inoculated into Reinforced Clostridial Medium (RCM) liquid medium and cultured anaerobicly at 37℃ for 24 h; *Bacillus coagulans* ATCC 7050 was inoculated into LB liquid medium and cultured aerobically at 37℃ for 18 h. Once the culture medium becomes noticeably turbid and the bacterial cells have entered the logarithmic growth phase, it can be used for subsequent large-scale culture.

[0032] (2) Large-scale culture of strains The activated bacterial culture was transferred to 500 mL of the corresponding fresh culture medium at an inoculation rate of 2% (v / v) for expansion culture.

[0033] The culture conditions are maintained as shown in Table 1: Table 1. Culture conditions for different strains

[0034] (3) Collection and washing of bacterial cells After expansion culture, the bacterial suspensions were transferred to sterile centrifuge tubes and centrifuged at 5000 rpm for 10 min at 4°C. The supernatant (containing culture medium components and metabolic waste) was discarded, and the bacterial pellet was collected. 20 mL of sterile phosphate-buffered saline (PBS, pH 7.4) was added to the pellet, and the suspension was resuspended by vortexing. The pellet was then centrifuged again at 5000 rpm for 10 min at 4°C. This washing step was repeated twice. Finally, the bacterial suspension was resuspended in 10 mL of sterile physiological saline, and the bacterial concentration of each strain was adjusted to a uniform 1.0 × 10⁻⁶ using plate counting. 10 CFU / mL.

[0035] Example 2 Preparation of Compound Probiotics On a sterile operating table, mix the bacterial suspensions of each strain with the adjusted concentration according to the following ratio of live bacteria (based on live bacteria count).

[0036] Preparation Example 1: Strains ratio (based on viable count) BA:LR:AM:CB:BC = 2:2:1:3:2. After mixing, gently invert the centrifuge tube 10 times to ensure even distribution of the strains. The overall viable concentration of the resulting composite bacterial solution was 1.0 × 10⁻⁶. 10 CFU / mL. Add a sterile preservative solution to the above-mentioned compound bacterial solution to make the final system have the following mass percentages: skim milk powder 8%, trehalose 5%, mannitol 3%, and gelatin 2%. After mixing evenly, let stand for 10 min to obtain the bacterial suspension before lyophilization.

[0037] The bacterial suspension was dispensed into 10 mL lyophilization bottles, each containing 5 mL of liquid. After dispensing, the bottles were quickly capped (leaving a small gap to allow moisture to evaporate during pre-freezing). The bottles were then placed in a -40°C freezer for pre-freezing for 8 hours. The pre-frozen samples were then transferred to a freeze dryer and lyophilized under the following conditions: vacuum: 20 Pa, cold trap temperature: -50°C, drying time: 30 hours. After lyophilization, a pale yellow powdery compound probiotic preparation was obtained.

[0038] Preparation Example 2: Strains ratio (based on viable count) BA:LR:AM:CB:BC = 1:3:2:2:2. Each strain was activated and cultured according to the process described in Example 1 above. After collection and washing, the bacterial concentration was adjusted to 1.0 × 10⁻⁶. 10 CFU / mL, mixed under aseptic conditions in the above proportions, with the same protective agent added.

[0039] Preparation Example 3: Strain strain ratio (based on viable cell count): BA:LR:AM:CB:BC = 2:2:0.5:3:2.5. Each strain was activated and cultured according to the process described in Example 1 above. After collection and washing, the cell concentration was adjusted to 1.0 × 10⁻⁶. 10CFU / mL, mixed under aseptic conditions in the above proportions, with the same protective agent added.

[0040] Preparation Example 4: The preparation ratio was the same as in Preparation Example 1. After collection and washing, the bacterial cell concentration was adjusted to 1.0 × 10⁻⁶. 9 CFU / mL, mixed under aseptic conditions in the above proportions, with the same protective agent added.

[0041] Preparation Example 5: The preparation ratio was the same as in Preparation Example 1. After collection and washing, the bacterial cell concentration was adjusted to 5.0 × 10⁻⁶. 10 CFU / mL, mixed under aseptic conditions in the above proportions, with the same protective agent added.

[0042] Comparative examples: Preparations containing only BA (Comparative Example 1), LR (Comparative Example 2), AM (Comparative Example 3), CB (Comparative Example 4), or BC (Comparative Example 5) were prepared separately, and the bacterial cell concentration was adjusted to 1.0 × 10⁻⁶. 10 The single-strain formulation was obtained by using the same process as in Example 1, including activation, expansion culture, collection and washing, addition of the same protective agent, and pre-freezing and drying.

[0043] Example 3: In vitro anti-allergy function verification experiment (mast cell degranulation inhibition experiment) The compound probiotic preparations of Examples 1-5 and the single-strain preparations of Comparative Examples 1-5 were selected (all were lyophilized powders prepared above, reconstituted with sterile physiological saline to the corresponding concentration before use to ensure that the viable bacteria concentration after reconstitution was consistent with that of the preparation examples and comparative examples); positive control drugs: sodium cromoglycate (an anti-allergy positive control drug) and transfer factor oral solution (an immune-enhancing positive control drug) were used as experimental samples. The RBL-2H3 mast cell degranulation model was used, and the anti-allergy activity of each preparation was evaluated by detecting the β-hexosinosinase release rate. The specific steps are as follows: 1. Cell Culture: RBL-2H3 mast cells were seeded into 96-well cell culture plates at a density of 1 × 10⁶ cells per well. 5 Add 100 μL of DMEM medium containing 10% fetal bovine serum and 1% penicillin to each well and incubate at 37°C in a 5% CO2 incubator for 24 h until the cells adhere stably.

[0044] 2. Experimental Groups: A blank control group, a positive control group, preparation example experimental groups (preparation examples 1-5), and a comparative example experimental group (comparative examples 1-5) were set up, with 6 replicate wells in each group. The specific groupings are as follows: Blank control group (only cells and culture medium were added, without samples, antibodies, or allergens); Positive control group (sodium cromoglycate solution was added to a final concentration of 100 μg / mL); Preparation example experimental groups (reconstituted bacterial suspensions from each preparation example were added, with the final concentration corresponding to the viable cell concentration of each preparation example, added at 100 μL / well); Comparative example experimental groups (reconstituted single-strain bacterial suspensions from each comparative example were added to a final concentration of 1.0 × 10⁻⁶). 10 CFU / mL, 100μL / well).

[0045] 3. Cell sensitization and sample interaction: Anti-DNP IgE solution was added to each well (except for the blank control group) to a final concentration of 0.5 μg / mL, and incubated in a 37℃, 5% CO2 incubator for 12 h to induce cell sensitization. After sensitization, the corresponding sample solution was added to each experimental group and the positive control group, and an equal volume of sterile physiological saline was added to the blank control group. The cells were incubated at 37℃ for 1 h to allow the sample to fully interact with the cells.

[0046] 4. Degranulation induction and detection: DNP-BSA allergen solution was added to each well (except the blank control group) to a final concentration of 100 ng / mL, and incubated at 37°C for 30 min to induce mast cell degranulation. After the reaction, 50 μL of supernatant from each well was transferred to a new 96-well plate, and 50 μL of 1 mmol / L PNPG substrate solution was added to each well. The plate was incubated at 37°C for 60 min, and then 150 μL of 0.1 mol / L sodium carbonate solution was added to terminate the reaction. The absorbance (OD value) of each well was measured at 405 nm using a microplate reader.

[0047] 5. Calculate the inhibition rate: The inhibition rate of β-hexosinosinase release was calculated using the formula: Inhibition rate (%) = [(OD value of positive control group - OD value of sample group) / (OD value of positive control group - OD value of blank control group)] × 100.

[0048] The higher the inhibition rate, the stronger the anti-allergic activity of the preparation; the experiment was repeated 3 times, the average value was taken, and the inhibition rate data of each experimental group were recorded.

[0049] The results are shown in Table 2: Table 2 Statistical results of β-hexosamine sidase release inhibition rate

[0050] This indicates a comparison with the model control group. P <0.05; This indicates a comparison with the model control group. P <0.01. △ This indicates a comparison with Comparative Example 4. P <0.05; △△ This indicates a comparison with Comparative Example 4. P <0.01.

[0051] As shown in Table 2, the anti-allergic effect of the single-strain control group was poor, and the inhibition rate of each preparation was significantly higher than that of the corresponding single-strain control group (P<0.05 or P<0.01), indicating that the present invention significantly improved the anti-allergic effect of the bacterial agent by combining various strains. Among them, preparation 5 had the highest inhibition rate, which was slightly higher than that of the positive control group.

[0052] Example 4: In vivo anti-allergy and immune-enhancing experiments SPF-grade BALB / c mice (6-8 weeks old, weighing 18-22g, half male and half female) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Healthy mice were screened before the experiment, excluding those with abnormal weight, lethargy, messy fur, or respiratory or intestinal diseases to ensure baseline consistency. All mice were housed in an SPF-grade animal facility under strictly controlled conditions: temperature 22-25℃, relative humidity 50%-60%, and a circadian rhythm of 12h light / 12h darkness (light intensity 150-200 lx). Sterile transparent plastic cages were used, with 5 mice per cage (males and females separated). Cages, bedding (sterile corn cobs), waterers, and feeders were all sterilized by autoclaving at 121℃ and 101 kPa for 30 minutes and then cooled before use.

[0053] Healthy mice were randomly divided into four groups (n=10 per group, half male and half female) after acclimatization for 3 days: a blank control group, a model control group, a positive control group, experimental groups of prepared cases (prepared cases 1-5), and experimental groups of comparative cases (comparative cases 1-5). Each group continued acclimatization for 1 day to ensure the mice adapted to their assigned environment. Except for the blank control group, allergic rhinitis models were established in all other groups. The specific modeling steps were as follows: On days 1, 7, and 14, mice were intraperitoneally injected with a sensitizing solution (containing 10 μg ovalbumin + 2 mg aluminum hydroxide adjuvant, diluted to 0.2 mL / mouse with sterile saline). The mice's abdomens were gently massaged to ensure even diffusion of the sensitizing solution. Starting on day 21, ovalbumin solution (concentration 100 μg / mL) was instilled into the nasal cavity daily, 10 μL per nostril. After instillation, the nostrils were gently pinched for 10-15 seconds to ensure the solution entered the nasal cavity. This instillation was continued for 7 days to induce allergic rhinitis attacks. Drug treatment was administered concurrently during the modeling period.

[0054] Drug administration began simultaneously on day 1 of modeling, once daily for 14 consecutive days, via gavage (1 mL gavage syringe to avoid damaging the mouse esophagus). Specific dosages were as follows: the blank control group and model control group received an equal volume of sterile saline (0.2 mL / mouse); the positive control group received sodium cromoglycate solution via gavage at a dose of 10 mg / kg body weight (gavage volume calculated based on mouse body weight); experimental groups 1-5 received the corresponding prepared bacterial suspension via gavage, with the gavage dose adjusted according to the live bacteria concentration of each prepared case to ensure that the daily live bacteria intake of each mouse matched the formulation concentration, with a gavage volume of 0.2 mL / mouse; experimental groups 1-5 received the corresponding single-strain bacterial suspension via gavage, with a gavage dose of 1.0 × 10⁻⁶. 10 CFU / animal, gavage volume 0.2 mL / animal; administration time is fixed at 9:00-10:00 AM daily, and fasting for 1 hour after administration to avoid food affecting the absorption of the preparation.

[0055] After administration, the mice were fasted but allowed to drink water for 12 hours. After the observation of allergic symptoms, blood was collected from the orbital cavity of the mice (using a sterile capillary tube, gently pressing the inner side of the mouse's orbital cavity, and collecting blood into a sterile centrifuge tube, collecting 0.5~1mL of blood from each mouse).

[0056] Test method: (1) The allergy symptoms of mice were scored using a blinded method (two experimenters scored independently and the average was taken). Only the symptoms of allergic rhinitis (sneezing, nose scratching, runny nose) in mice during the experiment were quantified. The total score was 0-3 points. The specific scoring criteria are as follows: 0 points: No allergic symptoms, normal mouse behavior, no sneezing, no nose-scratching, no runny nose; 1 point: Mild allergy symptoms, occasional sneezing (1-2 times / 5 minutes), slight nose scratching (1-2 times / 5 minutes), no runny nose; 2 points: Moderate allergy symptoms, frequent sneezing (3-5 times / 5 minutes), frequent nose scratching (3-5 times / 5 minutes), and a small amount of runny nose (only the tip of the nose is moist, without dripping). 3 points: Severe allergy symptoms, including violent sneezing (≥6 times / 5 minutes), persistent nose scratching (≥6 times / 5 minutes), and copious runny nose (dripping from the tip of the nose, even wetting the hair around the mouth and nose).

[0057] (2) Serum immune markers (IgE, IgA, IFN-γ, IL-4) test method: After blood was collected from the orbital cavity, the blood sample was placed at 4℃ for 2 hours, then centrifuged at 4℃ and 3000rpm for 15 minutes. The upper serum layer was collected and stored at -80℃ for later use. When testing, the mouse IgE, IgA, IFN-γ, IL-4 test kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.) was strictly followed. The enzyme-linked immunosorbent assay (ELISA) was used for testing. The data are presented in the form of mean ± standard deviation.

[0058] The experimental data are shown in Table 3: Table 3

[0059] Table 3 shows that the allergy symptom scores of mice in each preparation were significantly lower than those in the model control group and the control group, with preparation 5 showing the lowest score and the best anti-allergic effect. Serum IgE and IL-4 levels in each preparation were significantly lower than those in the model control group and the control group, while IgA and IFN-γ levels were significantly higher, demonstrating the dual efficacy of the compound preparation in both anti-allergy and immune enhancement. Preparations 2 and 5 showed higher serum IgA and IFN-γ levels, consistent with their design focusing on anti-inflammation and high concentrations. The in vivo efficacy of all preparations was close to or better than that of the positive control group, demonstrating their potential for clinical application.

[0060] Example 5 Preparation of Lyophilized Peptide Powder In this embodiment, soy protein isolate (SPI) is selected as the peptide source, and the specific steps are as follows: Weigh 100 g of soy protein isolate and add 1000 mL of deionized water to prepare a 10% (w / v) protein solution. Adjust the pH of the solution to 2.0 using 0.1 mol / L hydrochloric acid and pre-equilibrate the solution in a 37°C water bath for 10 min. Then, add pepsin to the protein solution at 3% (w / w) of the protein mass and stir at 200 rpm for 2 h at 37°C to allow for initial hydrolysis of the protein.

[0061] After pepsin hydrolysis, the pH of the system was adjusted to 7.5 using 1 mol / L sodium hydroxide solution. Then, trypsin was added to the system at 2% (w / w) of the protein mass. The reaction was continued at 37°C with stirring at 200 rpm for 2 h to further hydrolyze the protein into a polypeptide mixture. After hydrolysis, the reaction system was heated to 95°C and held for 10 min to terminate the enzyme reaction. Subsequently, the mixture was centrifuged at 10,000 rpm for 15 min at 4°C, and the supernatant was collected to obtain the crude peptide hydrolysate.

[0062] The above crude peptide hydrolysate was sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 5 kDa, 3 kDa, and 1 kDa to fractionate multiple peptide fractions with molecular weights greater than 5 kDa, 3–5 kDa, 1–3 kDa, and less than 1 kDa. Each fraction was pre-frozen at -80°C for 12 h and then freeze-dried for 48 h to obtain the corresponding lyophilized peptide powders for later use.

[0063] Example 6 Screening of anti-allergic active peptides The anti-allergic activity of different molecular weight components in Example 5 was screened.

[0064] The anti-allergic activity was evaluated using a mast cell degranulation model. Specifically, RBL-2H3 cells were seeded in 96-well cell culture plates at a cell density of 1 × 10⁶ cells per well. 5 Cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C and 5% CO2 for 24 h. After the cells were stably adhered, anti-DNP IgE solution was added to each well to a final concentration of 0.5 μg / mL, and the cells were incubated at 37°C for 12 h to induce cell sensitization.

[0065] After cell sensitization, peptide solutions of different molecular weights were added to each well to a final concentration of 100 μg / mL, and incubated at 37°C for 1 h. Then, DNP-BSA allergen solution was added to each well to a final concentration of 100 ng / mL, and incubation continued at 37°C for 30 min to induce cell degranulation. After the reaction, 50 μL of the supernatant from each well was taken, and 1 mmol / L of p-nitrophenyl-N-acetyl-β-D-glucosinolate substrate solution was added. The reaction was incubated at 37°C for 60 min, and then 150 μL of 0.1 mol / L sodium carbonate solution was added to terminate the reaction. The absorbance was measured at 405 nm using a microplate reader.

[0066] The inhibition rate of β-hexosinosinase release was calculated using the following formula: Inhibition rate (%) = [(OD value of control group - OD value of sample group) / OD value of control group] × 100%.

[0067] When a peptide component exhibits an inhibition rate greater than or equal to 50% at a concentration of 100 μg / mL, it is considered to have anti-allergic activity and is further purified as a candidate active peptide component.

[0068] The active peptide components obtained from screening were further purified using reversed-phase high-performance liquid chromatography (RP-HPLC).

[0069] A C18 reversed-phase column (4.6 mm × 250 mm, 5 μm) was used. Mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. A linear gradient elution was used, with the proportion of mobile phase B increased from 5% to 60% over 30 min. The flow rate was set at 1.0 mL / min, and the detection wavelength was 214 nm. The fractions corresponding to each chromatographic peak were collected, and the mast cell degranulation experiment described above was performed. The single chromatographic peak fraction with the highest inhibition rate was selected as the target active peptide fraction and named AP-1.

[0070] Subsequently, the amino acid sequence of the AP-1 fraction was identified. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used for analysis. The mass spectrometer employed an electrospray ionization (ESI) source in positive ion mode, with a scan mass range of m / z 100–2000. De novo sequence analysis was performed on the acquired mass spectrometry data using PEAKS Studio software to obtain the amino acid sequence of the target active peptide. The amino acid sequence of peptide AP-1 was identified as follows: Lys-Leu-Pro-Tyr-Gly-Arg-Val-Leu-Ser-His (KLPYRGVLSH, as shown in SEQ ID NO.1).

[0071] Example 7 Preparation of probiotic-peptide complex formulation 1. Raw material preparation: Preparation Example 5 (live bacteria concentration 5.0 × 10⁻⁶) was selected. 10 (CFU / mL); the selected anti-allergic active peptide AP-1 with a purity ≥98% was commissioned to Nanjing Genscript Biotech Co., Ltd. for customized synthesis and purification using solid-phase peptide synthesis method.

[0072] 2. Compounding Ratio: The peptide AP-1 was compounded with the compound probiotic preparation of Preparation Example 5 at a mass ratio of 1:1000 (i.e., 1 mg of anti-allergic active peptide AP-1 was added to every 1000 mg of the compound probiotic preparation of Preparation Example 5), ensuring a final peptide concentration of 100 μg / mL (consistent with the peptide screening experiment concentration), and maintaining a probiotic live bacteria concentration of 5.0 × 10⁻⁶. 10 CFU / mL.

[0073] 3. Preparation process: The compound probiotic preparation (freeze-dried bacterial powder) of Preparation Example 5 was reconstituted with sterile physiological saline to the specified concentration, and then a preset amount of anti-allergic active polypeptide AP-1 was added. The mixture was placed in a constant temperature shaker at 4℃ and shaken at 100 rpm for 30 min to fully mix the polypeptide and probiotics, thus obtaining compound preparation 1.

[0074] Example 8: Efficacy Verification of Probiotic Compound Preparation To verify the anti-allergic efficacy of compound preparation 1 in Example 7, control group 1 (probiotics only, no peptides) and control group 2 (AP-1 peptide only, concentration 100 μg / mL) were set up simultaneously. The remaining experimental conditions, group settings, and detection methods were consistent with the aforementioned in vitro experiments. The data are as follows: Table 4. In vitro anti-allergy function verification

[0075] Table 4 shows that the β-hexosinosinase release inhibition rate of compound preparation 1 (83.7±1.9) was significantly higher than that of control group 1, peptide control group 2 and positive control group in preparation example 5 (P<0.01), indicating that probiotics and anti-allergic active peptides have a synergistic anti-allergic effect, and the anti-allergic activity is significantly improved after compounding.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. The probiotic composition of the present invention can be used in the preparation of pharmaceuticals as well as in the preparation of foods or health products with anti-allergic and immune-enhancing functions. Given the microbial characteristics of probiotics, their application in food and health products is known in the prior art, but the specific compositions of the present invention can still achieve the aforementioned beneficial effects in food / health products.

[0077] sequence list SEQ ID No.1 KLPYRGVLSH.

Claims

1. A probiotic composition, characterized in that, The probiotic composition includes Bifidobacterium animalis. Bifidobacterium animalis Lactobacillus reuteri Lactobacillus reuteri Akkermania Akkermansia muciniphila Clostridium butyricum Clostridium butyricum and Bacillus coagulans Bacillus coagulans .

2. The probiotic composition according to claim 1, characterized in that, Bifidobacterium animalis Bifidobacterium animalis Lactobacillus reuteri Lactobacillus reuteri Akkermania Akkermansia muciniphila Clostridium butyricum Clostridium butyricum and Bacillus coagulans Bacillus coagulans The mass ratio is (1~3):(1~3):(0.5~3):(1~3):(2:3).

3. The probiotic composition according to claim 1, characterized in that, Bifidobacterium animalis Bifidobacterium animalis for Bifidobacterium animalis ATCC 25527 Bifidobacterium animalis ATCC 27672 and Bifidobacterium animalis Any one of CGMCC 1.1852; The Lactobacillus reuteri Lactobacillus reuteri for Lactobacillus reuteri DSM 17938 Lactobacillus reuteri ATCC PTA6475 and Lactobacillus reuteri Any one of JCM 1112; The Akkermania Akkermansia muciniphila for Akkermansia muciniphila ATCC BAA-835; The Clostridium butyricum Clostridium butyricum for Clostridium butyricum CGMCC 0313.1 Clostridium butyricum MIYAIRI 588 and Clostridium butyricum Any one of ATCC 19398; The Bacillus coagulans Bacillus coagulans for Bacillus coagulans MTCC 5856 Bacillus coagulans ATCC 7050 and Bacillus coagulans Any of the following in DSM 1.

4. The probiotic composition according to claim 1, characterized in that, The probiotic composition further includes an active peptide, the amino acid sequence of which is shown in SEQ ID NO.

1.

5. The probiotic composition according to claim 4, characterized in that, The final concentration of the active peptide in the probiotic composition is 0.5~1 mg / g.

6. The probiotic composition according to claim 1, characterized in that, The total viable bacterial concentration of the strains in the probiotic composition is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 9 CFU / g.

7. The probiotic composition according to any one of claims 1 to 6, characterized in that, The dosage form of the probiotic agent is selected from solutions, lyophilized powders, capsules, tablets, or granules.

8. The probiotic composition according to claim 7, characterized in that, The probiotic agent is a freeze-dried powder; The lyophilized powder also includes a protectant.

9. The probiotic composition according to claim 8, characterized in that, The protective agent includes any one or a combination of at least one of the following: skim milk powder, trehalose, mannitol, gelatin, sucrose, lactose, dextran, dextrin, gum arabic, sodium alginate, polyvinylpyrrolidone, sorbitol, xylooligosaccharides, fructooligosaccharides, or xylitol.

10. The use of the probiotic composition according to any one of claims 1 to 9 in the preparation of food or health products.

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