A preparation process and application of immune-regulating Bifidobacteria

Abstract

This invention discloses a process for preparing live Bifidobacterium bacteria that regulate immunity and its application. Using *Bifidobacterium animalis*, *Bifidobacterium breve*, and *Bifidobacterium longum* as production strains, a five-in-one preparation process is employed: solid-state fermentation of oat nutrient substrate with yeast; secondary anaerobic fermentation under starvation stress and assisted feeding resuscitation; low-temperature high-speed centrifugation for lossless concentration; emulsification with a compound stress-resistant protective agent; and gradient-temperature freeze-drying. The final product has a live bacteria count ≥ 2.0 × 10⁻⁶. 11 A highly active Bifidobacterium preparation with CFU / g, water activity ≤0.22, gastric acid resistance survival rate ≥85%, and accelerated survival rate of ≥85% after 3 months at 37℃ significantly enhances the colonization ability, immunomodulatory activity, intestinal tolerance, and long-term storage stability of Bifidobacterium.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation engineering, probiotic live bacteria preparations, and immunomodulatory functional food technology, specifically to a preparation process and application of highly active, highly stable, and immunomodulatory Bifidobacterium live bacteria. Background Technology

[0002] Bifidobacteria are core beneficial probiotics in the human gut. They can enhance systemic immunity by colonizing the intestinal mucosa, activating macrophages, promoting the secretion of immune factors IL-6 / IL-10 / IFN-γ, enhancing mucosal barrier function, and regulating the balance of gut microbiota. They are currently the core probiotic raw material in the fields of functional foods and health foods.

[0003] However, the existing Bifidobacterium live bacteria preparation technology has the following key defects, resulting in low live bacteria count, poor stability, and insufficient immune efficacy: (i) Poor safety and compatibility of culture medium: Conventional culture medium contains animal-derived / inorganic components such as beef extract, peptone, and manganese sulfate, which have a strong odor and low safety. Moreover, the nutritional composition has not been optimized for the immune activity of Bifidobacterium, which is not conducive to the cultivation of highly active bacteria; (ii) Crude fermentation process: Most fermentation is a single fermentation, resulting in low bacterial density, insufficient metabolism, insufficient accumulation of immune-related active substances, and weak immune regulation activity; (iii) Post-processing damage to live bacteria: Centrifugation, homogenization, and drying processes can easily cause bacterial damage and lysis, resulting in low survival rate and poor storage stability; (iv) Lack of targeted immune enhancement process: Targeted process optimization has not been carried out for resistance to gastric acid, bile, intestinal colonization ability, and immune activation, resulting in poor intestinal tolerance and low bioavailability of the product.

[0004] In existing technologies, the viable count of Bifidobacteria prepared using traditional liquid oat culture medium and a single fermentation process is only 6.2 × 10⁻⁶. 9 CFU / g, with a gastric acid resistance survival rate of 52%, and an accelerated survival rate of 47% after 3 months at 37℃; commercially available high-end Bifidobacterium live bacteria products have a live bacteria count of only 1.0×10⁻⁶. 11 With a CFU / g concentration, a gastric acid resistance survival rate of 65%, and a macrophage phagocytic rate increase of only 18%, it cannot simultaneously meet the requirements of high viable bacterial count, high stability, and strong immune activity. Summary of the Invention

[0005] The present invention aims to overcome the above-mentioned defects of existing Bifidobacterium live bacteria preparation processes and provide an innovative, whole-chain process for preparing highly active, highly stable, and strongly immunomodulatory Bifidobacterium live bacteria, solving problems such as low live bacteria count, poor stability, weak acid and bile salt resistance, and insufficient immune efficacy in existing technologies.

[0006] This invention innovates across the entire chain, including substrate pretreatment, fermentation metabolism regulation, non-destructive cell collection, stress resistance protection, and drying stabilization, and adopts the following technical solutions:

[0007] A process for preparing live Bifidobacterium bacteria that regulate immunity includes the following steps:

[0008] (1) Strain selection: One or more compound strains of Bifidobacterium animalis, Bifidobacterium breve, and Bifidobacterium longum are selected as production strains;

[0009] (2) Preparation of yeast solid fermentation oat culture medium: After the oats are peeled and soaked, yeast is inoculated for solid fermentation. The fermentation product is crushed, water extracted and filtered, and glucose and agar are added to obtain oat culture medium.

[0010] (3) Activation of strain: The strain from step (1) is anaerobic cultured at 36-38℃, with pH controlled at ≤5.0, until OD 600 ≥1, no bacteria detected under microscopic examination;

[0011] (4) Graded expansion culture: The activated strain is subjected to first-level, second-level and third-level anaerobic expansion culture to obtain high-vitality seed liquid;

[0012] (5) Primary fermentation: After inoculating the seed liquid into the culture medium, anaerobic fermentation is carried out at 36-38℃ until the total sugar is ≤0.5wt% and the pH is ≤4.5 to complete the primary fermentation;

[0013] (6) Secondary fermentation: Add glucose, stachyose and seed liquid from the same plant to the primary fermentation liquid, and anaerobic ferment at 36-38℃ until pH≥4.5 to complete the feed recovery fermentation;

[0014] (7) Low-temperature high-speed centrifugation concentration: control the centrifugation temperature at 4-10℃ and collect the bacterial cells;

[0015] (8) Emulsification: Add a compound stress protectant to the collected bacterial cells and emulsify thoroughly;

[0016] (9) Gradient temperature freeze drying: control the final product temperature ≥25℃, and the water activity of the dried product ≤0.22;

[0017] (10) Sterilely pulverize and package to obtain a live Bifidobacterium preparation.

[0018] As one specific embodiment, the preservation information of the strain described in this invention is as follows:

[0019] Strain name: Bifidobacterium animalis subsp. lactis V9, classified as Bifidobacterium animalis subsp. lactis; deposit date: April 8, 2025; depositary institution: China General Microbiological Culture Collection Center (CGMCC); deposit address: Beijing, China; accession number: CGMCC No. 34121.

[0020] Strain name: Bifidobacterium longum BB36, classified as Bifidobacterium longum; deposit date: April 8, 2025; depositary institution: China General Microbiological Culture Collection Center (CGMCC); deposit address: Beijing, China; deposit number: CGMCC No. 34123.

[0021] Strain name: Bifidobacterium breve BB-K9, classified as Bifidobacterium breve; Deposit date: April 14, 2025; Depository institution: China General Microbiological Culture Collection Center (CGMCC); Deposit address: Beijing, China; Deposit number: CGMCC No. 34184.

[0022] As a specific implementation method, in step (2), the solid-state fermentation conditions are: after inoculating yeast, solid-state fermentation is carried out at 20-30℃ for 5-10 days; the water extraction conditions are: after the fermentation product is crushed, deionized water is added at a material-to-liquid ratio of 1:50-100, kept warm at 85-90℃, stirred and filtered, and passed through a 300-mesh sieve.

[0023] As a specific implementation method, in step (4), during the anaerobic amplification process: the primary seed inoculation amount is 1% to 2%, and the culture is carried out anaerobically at 36 to 38°C for 10 to 12 hours until OD. 600 ≥1.0; secondary seed inoculation amount 5%~10%, anaerobic culture at 36~38℃ for 8~10h until OD 600 ≥1.5; 10%–15% of the seed was used as a third-grade seed inoculation, and the seed was anaerobically cultured at 36–38℃ for 6–8 hours until the OD reached its maximum. 600 ≥2.0, viable count ≥1.0×10 9 CFU / mL was used to obtain a highly viable seed culture.

[0024] In one specific implementation, in step (5), the culture medium for the primary fermentation consists of: 40-80 wt% oat culture medium, 0.1-0.2 wt% compound vitamins, 0.1-0.2 wt% urea, and the remainder is seed liquid.

[0025] As a specific implementation, in step (6), the supplemented glucose, stachyose and seed liquid of the same plant are calculated based on the total weight of the primary fermentation liquid as follows: glucose 0.5-2wt%, stachyose 0.05-0.1wt%, and seed liquid of the same plant 2-20wt%.

[0026] In one specific implementation, in step (8), the composite stress protectant is composed of the following components: trehalose, skim milk powder, sodium ascorbate, fructooligosaccharides, and γ-aminobutyric acid; wherein, based on the total weight of the bacterial cells, the components are: trehalose 10-12 wt%, skim milk powder 6-8 wt%, sodium ascorbate 0.8-1.0 wt%, fructooligosaccharides 4.0-5.0 wt%, and γ-aminobutyric acid 0.3-0.5 wt%.

[0027] As a specific implementation method, in step (9), the conditions for gradient temperature freeze drying are: pre-freezing at -40℃ for 3 to 6 hours, first sublimation at -10℃ for 6 to 10 hours, and desorption drying at 25℃ for 5 to 8 hours.

[0028] As one specific embodiment, the viable count of the Bifidobacterium live bacteria preparation obtained by the present invention is ≥2.0×10⁻⁶. 11 CFU / g, water activity ≤0.22, gastric acid resistance survival rate ≥85%, bile resistance survival rate ≥80%, 37℃ accelerated survival rate for 3 months ≥85%, and cell integrity rate ≥98%.

[0029] The present invention also provides the application of the Bifidobacterium live bacteria preparation obtained by the above-mentioned Bifidobacterium live bacteria preparation process in the preparation of health foods, functional foods, solid beverages, capsules, tablets and oral liquids that regulate immunity, regulate intestinal flora, help protect gastric mucosa and have antioxidant effects.

[0030] The Bifidobacterium live bacteria preparation process described in this invention has the following advantages:

[0031] (1) Yeast solid-state fermentation of oat substrate: Through solid-state fermentation of yeast, oat macromolecules are degraded into small molecule sugars, peptides and growth factors, which completely replace animal-derived culture medium. It has the advantages of being odorless and highly safe, and can significantly enhance the activity and immune activity of Bifidobacteria. There is no such substrate option in the current technology.

[0032] (2) Starvation stress-feed recovery secondary fermentation (metabolic flow directed regulation technology): Primary fermentation induces the accumulation of immune active substances through low sugar and low pH stress, and secondary fermentation achieves high density and high activity synergy through precise feeding. Unlike conventional expansion culture process, there is no such fermentation regulation idea in the existing technology.

[0033] (3) Low temperature high speed non-destructive concentration technology: Through specific centrifugation parameters, only the bacterial cells are concentrated, and the bacterial cell integrity rate is ≥98%.

[0034] (4) Composite stress-resistant protective agent system: Multi-component compounding can simultaneously achieve freeze-drying protection, acid and bile salt resistance protection and enhanced colonization ability. A single protective agent cannot achieve this synergistic effect.

[0035] (5) Gradient temperature rise freeze drying process: segmented temperature control avoids damage to the microorganisms, and the final water activity is ≤0.22, ensuring the long-term stability of the product.

[0036] Compared with existing technologies, the live Bifidobacterium bacteria prepared by the process described in this invention have the following advantages:

[0037] (1) Significantly increased viable count: The viable count of the obtained preparation was ≥2.0×10⁻⁶. 11 The CFU / g is more than twice that of commercially available products and more than 30 times that of traditional single-fermentation processes.

[0038] (2) Excellent stability: water activity ≤0.22, gastric acid resistance survival rate ≥85%, bile resistance survival rate ≥80%, accelerated survival rate at 37℃ for 3 months ≥85%, and survival rate at 25℃ room temperature for 12 months can still reach 78%.

[0039] (3) Strong immune activity: The phagocytic rate of macrophages increased by ≥32%, and the secretion of IL-6 and NO was significantly upregulated; mouse animal experiments showed that the spleen index increased by 28%, the serum IgG content increased by 32%, the phagocytic rate of peritoneal macrophages increased by 39%, and the delayed-type hypersensitivity increased by 31%, and the immune regulation effect was significantly better than the existing technology.

[0040] (4) Strong intestinal colonization ability: intestinal adhesion rate ≥75%, which is significantly higher than the level of about 50% of existing products.

[0041] (5) High safety: The culture medium is made entirely of plant sources, with no animal-derived ingredients, no preservatives, no chemical additives, and has a mild odor, making it suitable for the development of health food products. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to specific embodiments and comparative examples, but the scope of protection of the present invention is not limited thereto.

[0043] 1. The experimental method for determining gastric acid tolerance as described in this invention:

[0044] An artificial gastric fluid simulation method was used. An artificial gastric fluid (pH 2.0, containing 3 g / L pepsin) was prepared. A 1% inoculum of live Bifidobacterium preparation was added to the artificial gastric fluid, mixed thoroughly, and treated anaerobically at 37°C for 2 hours. The bacterial cultures before and after 2 hours of treatment were serially diluted, plated on MRS agar plates, and anaerobically incubated at 37°C for 48 hours. The viable bacterial count was then performed. The gastric acid resistance survival rate was calculated using the following formula:

[0045] Gastric acid resistance survival rate (%) = number of viable bacteria after treatment / initial number of viable bacteria × 100%.

[0046] 2. The experimental method for determining bile salt tolerance as described in this invention:

[0047] The 0.3% ox bile salt in vitro simulation method was used. Live Bifidobacterium preparations were inoculated into PBS buffer (pH 7.2) containing 0.3% ox bile salt and anaerobically treated at 37°C for 2 h. The bacterial cultures before and after treatment were serially diluted, plated on MRS plates, and anaerobically incubated for 48 h. The bacterial counts were then performed, and the bile salt tolerance survival rate was calculated using the following formula:

[0048] Bile salt tolerance survival rate (%) = number of viable bacteria after treatment / initial number of viable bacteria × 100%.

[0049] 3. Experimental method for determining intestinal adhesion ability as described in this invention:

[0050] An in vitro adhesion model using Caco-2 cells was employed. Bifidobacteria were co-cultured with Caco-2 cells for 2 hours. Unadhered cells were removed by washing with PBS, and the cells were lysed, diluted, and plated for counting. The number of adherent colonies was used to represent intestinal adhesion ability.

[0051] 4. The method for determining the immunomodulatory activity of macrophages described in this invention is as follows:

[0052] I. Experimental methods for macrophage phagocytosis rate:

[0053] Mouse peritoneal macrophages at 1×10 6 Cells were seeded per well in 24-well plates and incubated at 37°C with 5% CO2 for 2 hours to allow adherence. Live Bifidobacterium preparation (MOI=10:1) was added and co-cultured for 24 hours, followed by incubation with neutral red indicator or fluorescently labeled E. coli for 1 hour. The supernatant was discarded, cells were washed with PBS, lysed, and the absorbance was measured or the number of phagocytic positive cells was counted under a microscope.

[0054] Control group: No Bifidobacterium preparation was added; all other procedures were the same.

[0055] Calculation formula:

[0056] Macrophage phagocytosis rate (%) = (number of macrophages phagocytosing bacteria / total number of macrophages) × 100%.

[0057] Phagocytosis rate improvement rate (%) = (experimental group - control group) / control group × 100%.

[0058] II. Experimental methods for NO release:

[0059] Collect the cell supernatant after co-culturing macrophages with the Bifidobacterium preparation in the following examples for 24 hours, and detect the NO content using the Griess kit (with sodium nitrite NaNO2 as the standard). Measure the absorbance at 540 nm and calculate the NO concentration based on the standard curve.

[0060] Blank group: Only an equal volume of complete culture medium was added, without inoculating macrophages or adding bifidobacteria preparations. The remaining incubation, sample addition, color development, and detection steps were exactly the same as those of the experimental group. It was used to subtract the background of the culture medium and reagents.

[0061] Calculation formula:

[0062] NO release enhancement rate (%) = (Experimental group - Control group) / Control group × 100%

[0063] III. Calculation method for IL-6 secretion level (ELISA method):

[0064] Cell supernatant was collected after 24 hours of co-culturing macrophages with Bifidobacterium and measured using a mouse IL-6 ELISA kit. Following the kit instructions, the standard was serially diluted and added to the ELISA plate, followed by the supernatant sample. The plate was incubated at 37°C. After washing, enzyme-labeled secondary antibody was added and incubated at 37°C in the dark. After washing, chromogenic buffer was added and the plate was incubated in the dark. The reaction was terminated by adding stop solution. The absorbance (OD) of each well was measured at 450 nm using an ELISA reader. 450 (The standard concentration is plotted on the x-axis, and the OD value is plotted on the y-axis.) 450 Plot a standard curve on the ordinate, based on the sample OD. 450 Substitute the values ​​into the standard curve to calculate the IL-6 content.

[0065] Blank group: No Bifidobacterium preparation was added; the remaining treatment and testing steps were exactly the same.

[0066] Formula for calculating IL-6 improvement rate:

[0067] IL-6 increase rate (%) = (IL-6 content in experimental group - IL-6 content in blank group) / IL-6 content in blank group × 100%

[0068] Example 1: Preparation of live animal Bifidobacterium preparation

[0069] Step 1: Preparation of oat yeast solid culture medium: Peel oats and soak them in deionized water at 95℃ for 4 hours until soft; cool to room temperature and inoculate with yeast, then ferment at 25℃ for 7 days; after crushing, add deionized water at a ratio of 1:50, keep warm at 85℃ and stir for 2 hours, and filter through 300 mesh; add glucose and agar to the filtrate and mix well to obtain oat yeast composite culture medium.

[0070] Step 2, Strain Activation: Bifidobacterium animalis subsp. lactis V9 was anaerobically cultured at 37℃ until pH 4.8, OD... 600 =1.2, no bacteria were found under microscopic examination.

[0071] Step 3: MRS liquid culture medium (10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 20 g / L glucose, 5 g / L sodium acetate, 2 g / L diammonium citrate, 1 g / L Tween 80, 0.2 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O, pH 6.2–6.5); inoculate at 1% (v / v) and anaerobic incubate at 37°C for 12 h. OD 600 ≥1.0, pH 4.5~5.0, no bacteria detected under microscopy.

[0072] Secondary seed culture medium: oat liquid medium (80% yeast-fermented oat aqueous extract, 2% glucose, 0.1% stachyose, 1% peptone, 0.5% yeast extract, pH 6.0–6.3); inoculated at 5% (v / v), anaerobic culture at 37℃ for 10 h, OD 600 ≥1.5, pH 4.4~4.8.

[0073] Tertiary seed culture medium: oat yeast fermentation medium (90% oat yeast solid fermentation broth, 1.5% glucose, 0.15% compound vitamins, 0.15% urea, pH 5.8~6.2); inoculated at 10% (v / v) inoculum, anaerobic culture at 37℃ for 8 h, OD 600 ≥2.0, viable count ≥1.0×10 9 CFU / mL yields a high-vitamin seed solution.

[0074] Step 4, primary fermentation: The culture medium consists of 60wt% oat yeast culture medium, 0.15wt% compound vitamins, 0.15wt% urea, and the remainder is seed liquid; anaerobic fermentation at 37℃ for 22h, total sugar 0.42wt%, pH 4.2.

[0075] Step 5, Secondary fermentation: Add 1.0wt% glucose, 0.08wt% stachyose, and 10wt% seed liquid from the same plant (based on the total weight of the primary fermentation liquid), and anaerobic ferment at 37℃ for 7 hours until the pH rises to 4.8.

[0076] Step 6, Centrifugation Concentration: Centrifuge at 14000 rpm and 8℃ for 25 min using a tubular centrifuge, and collect the bacterial cells.

[0077] Step 7, Emulsification: Add a compound stress protectant (trehalose 10wt%, skim milk powder 6wt%, sodium ascorbate 0.8wt%, fructooligosaccharide 4wt%, γ-aminobutyric acid 0.3wt%, based on the total weight of the bacterial cells) and emulsify thoroughly.

[0078] Step 8, Gradient freeze drying: Pre-freeze at -40℃ for 4 hours, sublimate once at -10℃ for 8 hours, desorption dry at 25℃ for 6 hours, with a final water activity of 0.20.

[0079] Step 9: Aseptically pulverize and package to obtain live Bifidobacterium animalis powder.

[0080] Results data: Viable bacteria count 2.3 × 10⁻⁶ 11 CFU / g, water activity 0.20, gastric acid resistance survival rate (pH2.0, 2h) 88%, bile resistance survival rate (0.3% bile salts, 2h) 83%, 3-month accelerated survival rate at 37℃ 87%, macrophage phagocytosis rate increased by 33%, relative expression of IL-6 increased by 30%, NO release increased by 28%, and intestinal adhesion rate 78%.

[0081] Example 2: Preparation of live Bifidobacterium breve preparation

[0082] The process is the same as in Example 1, except that the production strain is replaced with Bifidobacterium breve BB-K9.

[0083] Results data: Viable bacteria count 2.1 × 10⁻⁶ 11 CFU / g, water activity 0.21, gastric acid resistance survival rate 86%, bile resistance survival rate 81%, 37℃ accelerated survival rate for 3 months 86%, macrophage phagocytosis rate increased by 32%, and intestinal adhesion rate 76%.

[0084] Example 3: Preparation of live Bifidobacterium longum preparation

[0085] The process is the same as in Example 1, except that the production strain is replaced with Bifidobacterium longum BB36.

[0086] Results data: viable bacteria count 2.2 × 10⁻⁶ 11 CFU / g, water activity 0.20, gastric acid resistance survival rate 87%, bile resistance survival rate 82%, 37℃ accelerated survival rate for 3 months 86%, macrophage phagocytosis rate increased by 34%, and intestinal adhesion rate 77%.

[0087] Example 4: Preparation of a live bacteria preparation of compound Bifidobacterium (Bifidobacterium breve BB-K9 and Bifidobacterium longum BB36 in a 1:1 ratio)

[0088] The process is the same as in Example 1, using a compound strain of Bifidobacterium short and Bifidobacterium long in a 1:1 ratio for fermentation.

[0089] Results data: Viable bacteria count 2.7 × 10⁻⁶ 11 CFU / g, water activity 0.19, gastric acid resistance survival rate 90%, bile resistance survival rate 85%, 37℃ accelerated survival rate for 3 months 89%, macrophage phagocytosis rate increased by 38%, IL-6 increased by 35%, NO increased by 33%, and intestinal adhesion rate 82%.

[0090] Example 5: Compound Bifidobacterium (Bifidobacterium breve BB-K9, Bifidobacterium longum BB36, and Bifidobacterium animalis subsp. lactis V9 in a 1:1:1 ratio)

[0091] The preparation process is the same as in Example 1, using a compound strain of Bifidobacterium breve BB-K9, Bifidobacterium longum BB36, and Bifidobacterium animalis subsp. lactis V9 in a 1:1:1 ratio for fermentation.

[0092] Results data: Viable bacteria count: 2.9 × 10⁻⁶ 11 CFU / g, water activity: 0.19, gastric acid resistance survival rate: 91%, bile salt resistance survival rate: 86%, intestinal adhesion rate: 84%, macrophage phagocytosis rate increased by 40%; IL-6 increased by 37%; NO release increased by 35%; 37℃ accelerated survival rate for 3 months: 90%.

[0093] Example 6: Comprehensive Verification of Immune Function

[0094] The strain preparation from Example 1 was selected for mouse animal experiments. Experimental method:

[0095] Experimental animals: SPF-grade ICR mice, 18-22g, half male and half female, randomly divided into groups of 10 mice each.

[0096] Rearing environment: Temperature 22±2℃, humidity 50±5%, 12h light / 12h darkness, free access to food and water.

[0097] Control group setup:

[0098] Normal control group: 0.2 mL of 0.9% sterile saline was administered by gavage daily for 28 consecutive days.

[0099] Experimental group: 0.2 mL of sample solution was administered by gavage daily (dose 1×10⁻⁶). 9 CFU / each), for 28 consecutive days.

[0100] Spleen index, serum IgG content, peritoneal macrophage phagocytosis rate, and delayed-type hypersensitivity (DTH) were measured 24 hours after the last administration.

[0101] Detection indicators: spleen index, serum IgG, peritoneal macrophage phagocytosis rate, delayed-type hypersensitivity (DTH). The experimental group was given the Bifidobacterium live bacteria preparation of Example 1 by gavage for 28 consecutive days. 24 hours after the last administration, the following were measured: (1) spleen index; (2) serum IgG content (ELISA method); (3) peritoneal macrophage phagocytosis rate; (4) delayed-type hypersensitivity (DTH) (DNFB-induced).

[0102] The results showed that the spleen index increased by 26%, serum IgG increased by 30%, peritoneal macrophage phagocytosis rate increased by 34%, and delayed-type hypersensitivity increased by 29%.

[0103] Example 7

[0104] The strain preparation from Example 2 was selected for mouse animal experiments. Experimental method:

[0105] Experimental animals: SPF-grade ICR mice, 18-22g, half male and half female, randomly divided into groups of 10 mice each.

[0106] Rearing environment: Temperature 22±2℃, humidity 50±5%, 12h light / 12h darkness, free access to food and water.

[0107] Control group setup:

[0108] Normal control group: 0.2 mL of 0.9% sterile saline was administered by gavage daily for 28 consecutive days.

[0109] Experimental group: 0.2 mL of sample solution was administered by gavage daily (dose 1×10⁻⁶). 9 CFU / each), for 28 consecutive days.

[0110] Spleen index, serum IgG content, peritoneal macrophage phagocytosis rate, and delayed-type hypersensitivity (DTH) were measured 24 hours after the last administration.

[0111] Detection indicators: spleen index, serum IgG, peritoneal macrophage phagocytosis rate, delayed-type hypersensitivity (DTH). The experimental group was given the Bifidobacterium live bacteria preparation of Example 1 by gavage for 28 consecutive days. 24 hours after the last administration, the following were measured: (1) spleen index; (2) serum IgG content (ELISA method); (3) peritoneal macrophage phagocytosis rate; (4) delayed-type hypersensitivity (DTH) (DNFB-induced).

[0112] The results showed that the spleen index increased by 25%, serum IgG increased by 29%, peritoneal macrophage phagocytosis rate increased by 33%, and delayed-type hypersensitivity increased by 28%.

[0113] Example 8

[0114] The strain preparation from Example 3 was selected for mouse animal experiments. Experimental method:

[0115] Experimental animals: SPF-grade ICR mice, 18-22g, half male and half female, randomly divided into groups of 10 mice each.

[0116] Rearing environment: Temperature 22±2℃, humidity 50±5%, 12h light / 12h darkness, free access to food and water.

[0117] Control group setup:

[0118] Normal control group: 0.2 mL of 0.9% sterile saline was administered by gavage daily for 28 consecutive days.

[0119] Experimental group: 0.2 mL of sample solution was administered by gavage daily (dose 1×10⁻⁶). 9 CFU / each), for 28 consecutive days.

[0120] Spleen index, serum IgG content, peritoneal macrophage phagocytosis rate, and delayed-type hypersensitivity (DTH) were measured 24 hours after the last administration.

[0121] Detection indicators: spleen index, serum IgG, peritoneal macrophage phagocytosis rate, delayed-type hypersensitivity (DTH). The experimental group was given the Bifidobacterium live bacteria preparation of Example 1 by gavage for 28 consecutive days. 24 hours after the last administration, the following were measured: (1) spleen index; (2) serum IgG content (ELISA method); (3) peritoneal macrophage phagocytosis rate; (4) delayed-type hypersensitivity (DTH) (DNFB-induced).

[0122] The results showed that the spleen index increased by 27%, serum IgG increased by 31%, peritoneal macrophage phagocytosis rate increased by 35%, and delayed-type hypersensitivity increased by 30%.

[0123] Example 9

[0124] The strain preparation from Example 4 was selected for mouse animal experiments. Experimental method:

[0125] Experimental animals: SPF-grade ICR mice, 18–22 g, half male and half female, randomly divided into groups of 10 mice each.

[0126] Rearing environment: Temperature 22±2℃, humidity 50±5%, 12h light / 12h darkness, free access to food and water.

[0127] Control group setup:

[0128] Normal control group: 0.2 mL of 0.9% sterile saline was administered by gavage daily for 28 consecutive days.

[0129] Experimental group: 0.2 mL of sample solution was administered daily (dose 1×10⁻⁶). 9 CFU / each), for 28 consecutive days.

[0130] Spleen index, serum IgG content, peritoneal macrophage phagocytosis rate, and delayed-type hypersensitivity (DTH) were measured 24 hours after the last administration.

[0131] Detection indicators: spleen index, serum IgG, peritoneal macrophage phagocytosis rate, delayed-type hypersensitivity (DTH). The experimental group was given the Bifidobacterium live bacteria preparation of Example 1 by gavage for 28 consecutive days. 24 hours after the last administration, the following were measured: (1) spleen index; (2) serum IgG content (ELISA method); (3) peritoneal macrophage phagocytosis rate; (4) delayed-type hypersensitivity (DTH) (DNFB-induced).

[0132] The results showed that the spleen index increased by 28%, serum IgG increased by 32%, peritoneal macrophage phagocytosis rate increased by 39%, and delayed-type hypersensitivity increased by 31%.

[0133] Example 10

[0134] The strain preparation from Example 5 was selected for mouse animal experiments. Experimental method:

[0135] Experimental animals: SPF-grade ICR mice, 18-22g, half male and half female, randomly divided into groups of 10 mice each.

[0136] Rearing environment: Temperature 22±2℃, humidity 50±5%, 12h light / 12h darkness, free access to food and water.

[0137] Control group setup:

[0138] Normal control group: 0.2 mL of 0.9% sterile saline was administered by gavage daily for 28 consecutive days.

[0139] Experimental group: 0.2 mL of sample solution was administered by gavage daily (dose 1×10⁻⁶). 9 CFU / each), for 28 consecutive days.

[0140] Spleen index, serum IgG content, peritoneal macrophage phagocytosis rate, and delayed-type hypersensitivity (DTH) were measured 24 hours after the last administration.

[0141] Detection indicators: spleen index, serum IgG, peritoneal macrophage phagocytosis rate, delayed-type hypersensitivity (DTH). The experimental group was given the Bifidobacterium live bacteria preparation of Example 1 by gavage for 28 consecutive days. 24 hours after the last administration, the following were measured: (1) spleen index; (2) serum IgG content (ELISA method); (3) peritoneal macrophage phagocytosis rate; (4) delayed-type hypersensitivity (DTH) (DNFB-induced).

[0142] The results showed that the spleen index increased by 29%, serum IgG increased by 33%, peritoneal macrophage phagocytosis rate increased by 40%, and delayed-type hypersensitivity increased by 32%.

[0143] Example 11: Long-term stability verification

[0144] The sample from Example 4 was stored at 25°C for 12 months. The test results showed a viable bacterial count of 2.1 × 10⁻⁶. 11 CFU / g, survival rate 78%, gastric acid resistance survival rate 82%, water activity 0.22, and excellent long-term stability.

[0145] Comparative Example 1: Traditional liquid oat culture medium + single fermentation process

[0146] The culture medium was prepared using traditional liquid oat flour, and a single fermentation was carried out, with the remaining conditions the same as in Example 1.

[0147] Results data: Viable bacteria count 6.2 × 10⁻⁶ 9 CFU / g, water activity 0.28, gastric acid resistance survival rate 52%, accelerated survival rate at 37℃ for 3 months 47%, macrophage phagocytosis rate increased by 11%.

[0148] Comparative Example 2: Live bacteria were prepared according to the process of Comparative Example 1.

[0149] The liquid oat fermentation process of Comparative Example 1 was used, and the bacterial cells were collected directly without lysis or enzymatic hydrolysis. The other conditions were the same as in Example 1.

[0150] Results data: Viable bacteria count 8.5 × 10⁻⁶ 9 CFU / g, gastric acid resistance survival rate 58%, accelerated survival rate at 37℃ for 3 months 53%, macrophage phagocytosis rate increased by 14%.

[0151] Comparative Example 3: Commercially available live Bifidobacterium products

[0152] Purchase commercially available Bifidobacterium triple live bacteria capsules (Peifeikang®), manufactured by Shanghai Shangyao Xinyi Pharmaceutical Co., Ltd.

[0153] Test results: viable bacteria count 1.0 × 10⁻⁶ 11 CFU / g, gastric acid resistance survival rate 65%, accelerated survival rate at 37℃ for 3 months 62%, macrophage phagocytosis rate increased by 18%.

[0154] Comparative Example 4: Yeast-free solid-state fermentation control group

[0155] Process: Unfermented ordinary oat flour culture medium was used, and the rest was the same as in Example 1.

[0156] Results: Viable bacteria count: 7.1 × 10⁻⁶ 9CFU / g, gastric acid resistance survival rate: 54%, bile salt resistance survival rate: 49%, intestinal adhesion rate: 51%, macrophage phagocytosis rate increased by 13%.

Claims

1. A process for preparing live Bifidobacterium bacteria that regulate immunity, characterized in that, Includes the following steps: (1) Strain selection: One or more compound strains of Bifidobacterium animalis, Bifidobacterium breve, and Bifidobacterium longum were selected as production strains; the Bifidobacterium animalis was Bifidobacterium animalis subsp. lactis V9, classified as Bifidobacterium animalis subsp. lactis, with a deposit date of April 8, 2025 and a deposit number of CGMCC No. 34121; the Bifidobacterium longum was Bifidobacterium longum BB36, classified as Bifidobacterium longum, with a deposit date of April 8, 2025 and a deposit number of CGMCC. No. 34123; The Bifidobacterium breve mentioned is Bifidobacterium breve BB-K9, classified and named Bifidobacterium breve, with a deposit date of April 14, 2025, and deposit number CGMCC No. 34184. (2) Preparation of yeast solid fermentation oat culture medium: After the oats are peeled and soaked, yeast is inoculated for solid fermentation. The fermentation product is crushed, water extracted and filtered, and glucose and agar are added to obtain oat culture medium. (3) Activation of strain: The strain from step (1) is anaerobic cultured at 36-38℃, with pH controlled at ≤5.0, until OD 600 ≥1, no bacteria detected under microscopic examination; (4) Graded expansion culture: The activated strain is subjected to first-level, second-level and third-level anaerobic expansion culture to obtain high-vitality seed liquid; (5) Primary fermentation: After inoculating the seed liquid into the culture medium, anaerobic fermentation is carried out at 36-38℃ until the total sugar is ≤0.5wt% and the pH is ≤4.5 to complete the primary fermentation; (6) Secondary fermentation: Add glucose, stachyose and seed liquid from the same plant to the primary fermentation liquid, and anaerobic ferment at 36-38℃ until pH≥4.5 to complete the feed recovery fermentation; (7) Low-temperature high-speed centrifugation concentration: control the centrifugation temperature at 4-10℃ and collect the bacterial cells; (8) Emulsification: Add a compound stress protectant to the collected bacterial cells and emulsify thoroughly; (9) Gradient temperature freeze drying: control the final product temperature ≥25℃, and the water activity of the dried product ≤0.22; (10) Sterilely pulverize and package to obtain a live Bifidobacterium preparation.

2. The process for preparing live Bifidobacterium bacteria according to claim 1, characterized in that, In step (2), the solid-state fermentation conditions are: after inoculating with yeast, solid-state fermentation is carried out at 20-30℃ for 5-10 days; the water extraction conditions are: after the fermented product is crushed, deionized water is added at a material-to-liquid ratio of 1:50-100, and the mixture is kept warm at 85-90℃, stirred, and filtered.

3. The process for preparing live Bifidobacterium bacteria according to claim 1, characterized in that, In step (4), during the anaerobic amplification process: the primary seed inoculation amount is 1%–2%, and the culture is carried out anaerobically at 36–38℃ for 10–12 hours until OD. 600 ≥1.0; secondary seed inoculation amount 5%~10%, anaerobic culture at 36~38℃ for 8~10h until OD 600 ≥1.5; 10%–15% of the seed was used as a third-grade seed inoculation, and the seed was anaerobically cultured at 36–38℃ for 6–8 hours until the OD reached its maximum. 600 ≥2.0, viable count ≥1.0×10 9 CFU / mL was used to obtain a highly viable seed culture.

4. The process for preparing live Bifidobacterium bacteria according to claim 1, characterized in that, In step (5), the culture medium for the primary fermentation consists of: 40-80 wt% oat culture medium, 0.1-0.2 wt% compound vitamins, 0.1-0.2 wt% urea, and the remainder is seed liquid.

5. The process for preparing live Bifidobacterium bacteria according to claim 1, characterized in that, In step (6), the added glucose, stachyose and seed liquid from the same plant are calculated based on the total weight of the primary fermentation liquid as follows: glucose 0.5-2 wt%, stachyose 0.05-0.1 wt%, and seed liquid from the same plant 2-20 wt%.

6. The process for preparing live Bifidobacterium bacteria according to claim 1, characterized in that, In step (8), the composite stress protectant is composed of the following components: trehalose, skim milk powder, sodium ascorbate, fructooligosaccharides, and γ-aminobutyric acid; wherein, based on the total weight of the bacterial cells, the components are: trehalose 10-12 wt%, skim milk powder 6-8 wt%, sodium ascorbate 0.8-1.0 wt%, fructooligosaccharides 4.0-5.0 wt%, and γ-aminobutyric acid 0.3-0.5 wt%.

7. The process for preparing live Bifidobacterium bacteria according to claim 1, characterized in that, In step (9), the conditions for gradient temperature freeze drying are: pre-freezing at -40℃ for 3 to 6 hours, first sublimation at -10℃ for 6 to 10 hours, and desorption drying at 25℃ for 5 to 8 hours.

8. The process for preparing live Bifidobacterium bacteria according to claim 1, characterized in that, The viable count of the obtained Bifidobacterium live bacteria preparation is ≥2.0×10⁻⁶. 11 CFU / g, water activity ≤0.22, gastric acid resistance survival rate ≥85%, bile resistance survival rate ≥80%, 37℃ accelerated survival rate for 3 months ≥85%, and cell integrity rate ≥98%.

9. The use of a Bifidobacterium live bacteria preparation obtained by the preparation process of Bifidobacterium live bacteria as described in any one of claims 1 to 8 in the preparation of health foods, functional foods, capsules, tablets, and oral liquids that regulate immunity, regulate intestinal flora, assist in protecting gastric mucosa, and have antioxidant properties.