Fermentation method of bifidobacterium longum BBMN68 as well as fermented product, postbiotic and application of bifidobacterium longum BBMN68

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a fermentation method of bifidobacterium longum BBMN68 as well as a fermented product, a postbiotic and application of the fermented product. According to the method for fermenting the bifidobacterium longum BBMN68, a fermentation substrate comprises alkaline protease hydrolyzed milk and a pomelo peel extract; the hydrolysis rate of the alkaline protease hydrolyzed milk is 9-11%, and the water-soluble dietary fiber content of the shaddock peel extract is 75-80%; the mass ratio of the alkaline protease hydrolyzed milk to the shaddock peel extract is (10-15): (1-3). The content of active ingredients in the fermented product prepared by the method is high, and after the fermented product is prepared into the postbiotics to be taken, the immune function of an organism can be recovered, immune organ damage is reduced, inflammation is relieved, and intestinal flora is regulated and controlled.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a fermentation method for Bifidobacterium longum BBMN68, the fermentation product, post-biotics, and applications. Background Technology

[0002] A weakened immune system is prone to symptoms such as physical weakness, malnutrition, lethargy, fatigue, loss of appetite, and sleep disturbances. Furthermore, illnesses like colds, asthma, and inflammation often recur. Existing immune enhancers include biological factors (thymic factor, transfer factor, etc.), chemically synthesized substances (liposomes, polynucleotides, and surfactants, etc.), trace factors (vitamin E, vitamin A, and selenium, etc.), natural foods and medicines (propolis, ginseng, donkey-hide gelatin, codonopsis, ganoderma, angelica, rehmannia, white peony, etc.), and microorganisms (probiotics, yeast, fungi). While drugs are effective, they have issues with drug resistance and side effects. Foods are less effective and exhibit significant individual differences. Current probiotic products mainly focus on live bacteria, which suffers from limitations such as limited product variety, stringent storage requirements, and relatively simple mechanisms of action, affecting their efficacy. Therefore, how to safely and stably improve the body's immunity is a pressing issue that needs to be addressed.

[0003] In recent years, the field of epibiotics has received increasing attention. Epibiotics are bioactive substances produced during fermentation that contribute to health (including inactivated bacteria, bacterial lysates, intracellular bacterial components, cell membrane and cell wall components, and metabolites). Compared to probiotics, epibiotics have advantages in antibacterial, antioxidant, and immunomodulatory effects; moreover, they are convenient to transport and store and can be used in combination with other formulations. While exerting similar effects to probiotics, they avoid problems such as low bioavailability of live bacteria, difficulty in controlling dosage, unstable effects, and easy transmission of drug resistance genes, thus possessing strong application potential and economic value.

[0004] Bifidobacterium longum BBMN68 is a probiotic with beneficial physiological functions, isolated from the intestines of centenarians in Bama. Its probiotic functions mainly include improving intestinal digestion and enhancing the body's immunity. Yang Haiying et al. demonstrated that BBMN68 can enhance the intestinal immune barrier function by increasing the secretion of immunoglobulin A in the duodenum of mice through gavage administration of BBMN68. Simultaneously, studies found that oral administration of active BBMN68 can increase the phagocytic activity of mouse macrophages, the activity of NK cells, serum hemolysin levels, and the proliferation of splenic lymphocytes, confirming that BBMN68 can improve the body's innate and acquired immune regulatory capacity. Zhu Jun et al., after exposing mouse macrophages to intact BBMN68 cells, cell walls, and cell contents, found that BBMN68 can regulate immunity by increasing cell activity. Live Bifidobacterium longum BBMN68 and its different cellular components have been shown to enhance the body's immune function, but the efficacy, immunomodulatory effects, and functional factors of its inactivated form are still unknown, and further research is needed on how to improve its efficacy. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for enhancing the active ingredients and efficacy of Bifidobacterium longum BBMN68 fermentation products and its application in the preparation of postbiotic products.

[0006] To achieve this objective, the technical solution of the present invention is as follows:

[0007] A method for fermenting Bifidobacterium longum BBMN68, wherein the fermentation substrate includes alkaline protease hydrolyzed milk and grapefruit peel extract; the hydrolysis rate of the alkaline protease hydrolyzed milk is 9-11%, and the water-soluble dietary fiber content of the grapefruit peel extract is 75-80%; the mass ratio of the alkaline protease hydrolyzed milk to the grapefruit peel extract is (10-15):(1-3).

[0008] The present invention relates to *Bifidobacterium longum* BBMN68, accession number CGMCC No. 2265, disclosed in Chinese Patent CN101649303B. Through research, this invention has discovered that when fermenting *Bifidobacterium longum* BBMN68, adding grapefruit peel extract to the substrate can produce natural vanillin and linalyl acetate, substances that regulate immunity. Furthermore, when alkaline protease hydrolysate and grapefruit peel extract are combined in a specific ratio as a substrate, the content of vanillin and linalyl acetate in the fermentation product can be significantly increased, along with the content of various active ingredients (lactic acid, acetic acid, propionic acid, hexanoic acid, butyric acid, extracellular polysaccharides, and total short-chain fatty acids) in the fermentation product. This results in a fermentation product that enhances immunity and exhibits better biological activity.

[0009] In this invention, the main component of the grapefruit peel extract is water-soluble dietary fiber extracted from grapefruit peel. It is prepared by using grapefruit peel as raw material and employing methods known in the art to extract water-soluble dietary fiber.

[0010] The grapefruit peel extract of the present invention may specifically include: 75-80% water-soluble dietary fiber, 10-15% starch, 5-7% fat, and 5-9% protein. Preferably, it includes 75-80% water-soluble dietary fiber, 8-10% starch, 5-7% fat, and 6-8.5% protein.

[0011] For extraction methods, please refer to: Pan Wenjie, Xue Zhenglian, Zhou Hailiu. Study on ultrasonic-assisted extraction of water-soluble dietary fiber from grapefruit peel [J]. Anhui Agricultural Science Bulletin (First Half Monthly), 2011, 17(15):198-200.

[0012] The alkaline protease hydrolyzed milk of the present invention is a substance obtained by hydrolyzing animal milk with alkaline protease using a method known in the art. Specifically, the preparation method is as follows: Alkaline protease is added to animal milk at pH 8.5 at a concentration of 25 U / mL, hydrolyzed at 55°C until the hydrolysis rate reaches 9-11%, and then the enzyme is inactivated at 95°C for 5 minutes to obtain the hydrolyzed milk.

[0013] The raw materials for preparing alkaline protease hydrolyzed milk of this invention can be raw animal milk, skim milk after defatting animal milk, or an aqueous solution of animal milk powder or skim milk powder with a concentration of 10-15%. Skim milk is preferred to improve the enzymatic hydrolysis efficiency.

[0014] Furthermore, this invention found that when alkaline protease hydrolyzed milk and grapefruit peel extract are used as substrates for the fermentation of other Bifidobacteria (such as BB12), vanillin and linalyl acetate cannot be produced. Although they have a certain promoting effect on the production of other beneficial components, the promoting effect is not as good as the combined effect of the substrate of this invention and Bifidobacterium longum BBMN68.

[0015] The method for fermenting Bifidobacterium longum BBMN68 of the present invention further includes a carbon source, a nitrogen source, an antioxidant and water, or further includes a surfactant.

[0016] Preferably, the fermentation substrate comprises 10-15 parts alkaline protease hydrolyzed milk, 1.5-2.0 parts yeast extract or corn steep liquor, 0.8-2.5 parts glucose or sucrose, 0.03-0.09 parts ascorbic acid or cysteine ​​hydrochloride, 1.0-3.0 parts grapefruit peel extract, 78-85 parts water, or further comprises 0.08-0.15 parts Tween-80;

[0017] More preferably, the fermentation substrate comprises 10 parts alkaline protease hydrolysate, 1.8 parts yeast extract, 2 parts glucose, 0.06 parts ascorbic acid, 0.1 parts Tween-80, 2 parts grapefruit peel extract, and 84.04 parts water.

[0018] The inoculum concentration of Bifidobacterium longum BBMN68 in the fermentation substrate of the present invention is 1–5 × 10⁻⁶. 7 CFU / mL.

[0019] The fermentation conditions of this invention are: anaerobic, 37±1℃, static culture.

[0020] The present invention also provides a fermentation product of Bifidobacterium longum BBMN68, which is prepared by the above-described method for fermenting Bifidobacterium longum BBMN68.

[0021] The fermentation product of Bifidobacterium longum BBMN68 of the present invention has a high content of multiple active ingredients, which can have better effects.

[0022] The present invention further provides the application of a composition as a fermentation substrate in increasing the content of one or more of vanillin, linaloyl acetate, lactic acid, acetic acid, propionic acid, hexanoic acid, butyric acid, extracellular polysaccharides, and total short-chain fatty acids in Bifidobacterium longum BBMN68 fermentation product; the composition comprises alkaline protease hydrolyzed milk and grapefruit peel extract; the hydrolysis rate of the alkaline protease hydrolyzed milk is 9-11%, and the water-soluble dietary fiber content of the grapefruit peel extract is 75-80%; the mass ratio of the alkaline protease hydrolyzed milk to the grapefruit peel extract is (10-15):(1-3).

[0023] The present invention also provides a method for preparing postbiotics, which is obtained by inactivating the above-mentioned Bifidobacterium longum BBMN68 fermentation product.

[0024] The postbiotic of the present invention is a postbiotic preparation with inactivated Bifidobacterium longum BBMN68 fermentation product as active ingredient. The fermentation product is at least one of Bifidobacterium longum BBMN68 fermentation culture, fermentation concentrate, fermentation dilution and crushed product, which can be liquid or solid.

[0025] The postgenetic agent of the present invention can be prepared using conventional methods in the art, such as:

[0026] (1) The strain of Bifidobacterium longum BBMN68 was activated and cultured in MRSC liquid medium and then anaerobic static cultured until the logarithmic phase.

[0027] (2) Using the culture, the concentrate obtained after concentration of the culture, or the dried matter, liquid matter, or dilution obtained after centrifugation and treatment with adjuvants as active ingredients, a postbiotic preparation containing inactivated Bifidobacterium longum BBMN68 is prepared; the number of inactivated Bifidobacterium longum BBMN68 in the postbiotic preparation is ≥50 billion / g.

[0028] Preferably, the method for preparing post-genetic agents includes:

[0029] (1) Heat the cells in the above-mentioned Bifidobacterium longum BBMN68 fermentation product at 42-45°C for 5-15 minutes (preferably, at 45°C for 10 minutes);

[0030] (2) After mixing with the inactivating protective agent, adjust the pH value to 4.3-4.8;

[0031] (3) Inactivate at 65-120℃ for 1-30 minutes.

[0032] This invention first pre-treats the bacterial cells at a low temperature, then mixes them with a protectant and adjusts the pH value to a specific level before heating to completely inactivate them (the higher the inactivation temperature, the shorter the heating time is generally). This method can better maintain the integrity of the bacterial cells, thereby improving the shelf life of the postbiotic product. If the pH value in step (2) is changed to >6, the integrity of the bacterial cells will decrease even with subsequent low-temperature heating. Performing subsequent steps without the low-temperature treatment in step (1) is also not conducive to maintaining the integrity of the bacterial cells.

[0033] More preferably, the method further includes: (4) vacuum drying at 40-60°C.

[0034] This invention provides a powdered solid formulation of post-genetic agents through vacuum drying, which is more conducive to storage and transportation.

[0035] The inactivating protective agent used in the preparation of post-genetic agents in this invention mainly serves to facilitate transportation and long-term storage. Those skilled in the art can make conventional selections based on common knowledge in the field.

[0036] Preferably, the inactivating protective agent comprises: 8-12 parts skim milk powder, 1-3 parts resistant dextrin, 4-8 parts lactose, 4-6 parts trehalose, 1-3 parts inulin, 1-3 parts maltodextrin, and 0.2-0.6 parts monosodium glutamate; or the inactivating protective agent comprises: 8-12 parts skim milk powder, 2.5-3.5 parts L-cysteine, and 8-12 parts xylan.

[0037] In the method of the present invention, the bacterial cells in the Bifidobacterium longum BBMN68 fermentation product are obtained by centrifuging the Bifidobacterium longum BBMN68 fermentation product, and the volume-to-mass ratio of the bacterial cells to the inactivation protectant is (3.8-4.2):1L / kg.

[0038] In step (2) of the method of the present invention, the pH value is adjusted by one or more of lactic acid, acetic acid or citric acid.

[0039] The postbiotic prepared by the above method of the present invention is mainly inactivated Bifidobacterium longum BBMN68 bacterial powder, which can enhance the body's immunity. It can be taken orally, and the recommended dosage is no less than 20 billion bacteria per day.

[0040] The present invention also provides an epigenetic agent, which is prepared by the above method.

[0041] The post-biotic of this invention can not only restore the body's immune function, but is also convenient to consume, easy to store, simple to treat, has a high remission rate, and has no toxic side effects.

[0042] The post-biotic of the present invention may further include at least one adjuvant suitable for post-biotic formulations, such as a protectant, excipient, etc.; the dosage form of the post-biotic may be powder, pill, capsule, granule, tablet, liquid formulation or gel.

[0043] The present invention also provides the application of the above-mentioned metagenics in the preparation of pharmaceuticals, health products or food.

[0044] The present invention also provides a product, which is a pharmaceutical, health product or food, comprising the aforementioned post-biotic.

[0045] The product of this invention can improve the immunosuppression of immune organs, repair the medullary and cortical areas of the spleen, form complete lymphoid follicles, reduce the decline in humoral immunity and the production of inflammation, regulate the composition of intestinal flora (significantly reduce the relative abundance of *Femtobacter* and *Roseidon*, and significantly increase the relative abundance of *Lactobacillus*, *Spirulina*, *Ruminococcus*, *Clostridium*-UGG-014, *Bifidobacterium*, *Dukelanthus*, and *Adlerococcus*, especially *Dukelanthus*, which can improve intestinal metabolism, immunity and reduce the occurrence of bacterial infections and other diseases), and promote the gradual recovery of immune function, thereby improving the body's immunity through a comprehensive mechanism.

[0046] The present invention provides another fermented milk, the fermented milk comprising: animal milk, sugar, protein powder, starch, pectin, agar, inactivated starter culture and the above-mentioned postbiotics, or further comprising prebiotics;

[0047] Preferably, the fermented milk comprises: 90-95 parts animal milk, 5.5-6 parts sugar, 0.6-0.7 parts protein powder, 0.38-0.42 parts starch, 0.08-0.12 parts pectin, 0.28-0.32 parts agar, 0.008-0.012 parts inactivated starter culture, and 0.08-0.12 parts post-biotic as described in claim 6, or further comprises 0.28-0.32 parts prebiotic.

[0048] More preferably, the prebiotic is inulin, and the fermentation agent includes Lactobacillus bulgaricus and Streptococcus thermophilus.

[0049] The animal milk of this invention can be the milk of animals such as cows, sheep, camels, deer, and horses.

[0050] This invention incorporates the aforementioned postbiotics into the preparation of fermented milk, thereby enhancing the efficacy of fermented milk. Further addition of prebiotics can yield fermented milk products with even stronger immune-enhancing effects.

[0051] This invention produces fermented milk (100g / bottle), with each bottle containing over 10 billion Bifidobacterium BBMN68 bacteria. It is recommended to take 2 bottles daily for better immune enhancement.

[0052] The fermented milk of the present invention can be prepared using methods known in the art. Preferably, the preparation method is as follows:

[0053] (1) Preheat the animal milk to 50-55℃, add white sugar, protein powder, starch, pectin, agar and inulin, and maintain the preheating temperature for 15-18 minutes.

[0054] (2) Homogenize under heating to 65℃ and 25MPa, sterilize at 135℃ for 8s after homogenization, and cool to 42℃ to obtain fermentation substrate;

[0055] (3) Add fermentation agent and Bifidobacterium longum BBMN68 inactivated bacterial powder (post-generic) to the fermentation substrate, ferment at 42℃ until the acidity reaches 70-72°T, stop fermentation, sterilize at 76-78℃ for 30s, and cool to 20℃ to obtain fermentation product.

[0056] (4) Homogenize the fermentation product and aseptically fill it.

[0057] The present invention further provides the application of the above-mentioned postbiotics or products or fermented milk in enhancing the body's immunity, reducing damage to immune organs, alleviating inflammation and / or regulating intestinal flora.

[0058] The beneficial effects of this invention are at least as follows:

[0059] This invention improves the content of multiple active ingredients in the fermentation product by specifically setting the fermentation substrate of Bifidobacterium longum BBMN68, and the obtained fermentation product can enhance immunity through a comprehensive mechanism. Attached Figure Description

[0060] Figure 1 This is an electron micrograph of the bacteria in the inactivated bacterial powder of Example 5.

[0061] Figure 2 This is an electron micrograph of the bacteria in the inactivated bacterial powder of Comparative Example 5.

[0062] Figure 3 Microscopic images (×400) of the spleen of mice in the blank control group (C) after HE staining to observe histological changes.

[0063] Figure 4 Microscopic images (×400) of the spleen of model group (M) mice after HE staining to observe histological changes.

[0064] Figure 5 Microscopic image (×400) of the spleen of mice in the levamisole-positive drug group (LMS) after HE staining to observe histological changes.

[0065] Figure 6 Microscopic images (×400) of the spleen of mice in the low-dose post-biotic group (68L) after HE staining to observe histological changes.

[0066] Figure 7 Microscopic images (×400) of histological changes observed by HE staining of the spleen of mice in the high-dose post-natal group (68H).

[0067] Figure 8 Microscopic images (×400) of the spleen of 68-year-old mice in the post-natal yogurt group, showing histological changes after HE staining.

[0068] Figure 9 Microscopic images (×400) of the spleen of mice in the post-biotic + prebiotic yogurt group (68JY) after HE staining to observe histological changes.

[0069] Figures 3 to 9 The colored arrows (if any) represent the following meanings: black arrow: necrotic cell fragments, red arrow: granulocytes, green arrow: red blood cells, yellow arrow: megakaryocytes. Detailed Implementation

[0070] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0072] In the specific embodiments of this invention, the grapefruit peel extract used in Examples 1-2 includes 75.2% water-soluble dietary fiber, 9.8% starch, 6.7% fat, and 8.3% protein. The hydrolysis rate of the alkaline protease-hydrolyzed milk is 10%. The protective agents include: 10 parts skim milk powder, 3 parts L-cysteine, and 10 parts xylan.

[0073] The grapefruit peel extract used in Examples 3-5 contained 79.9% water-soluble dietary fiber, 8.2% starch, 5.5% fat, and 6.4% protein. The hydrolysis rate of the defatted milk using alkaline protease was 10%. The preservatives included: 8 parts milk powder, 1.5 parts resistant dextrin, 2 parts lactose, 1 part trehalose, 1 part inulin, 1 part maltodextrin, and 0.5 parts monosodium glutamate.

[0074] Example 1

[0075] This embodiment provides an inactivated bacterial powder, including inactivated Bifidobacterium longum BBMN68, and the specific preparation process is as follows (each portion represents 10g):

[0076] 1. Raw materials:

[0077] Water 84.89 parts, alkaline protease hydrolyzed milk 10.0 parts, corn steep liquor 1.5 parts, sucrose 2.5 parts, cysteine ​​hydrochloride 0.06 parts, Tween-80 0.08 parts, grapefruit peel extract 1.0 part.

[0078] 2. Preparation method:

[0079] ①Preheat water to 55℃, add alkaline protease hydrolysate, sucrose, grapefruit peel extract, corn steep liquor, cysteine ​​hydrochloride, and Tween-80, and maintain the temperature while stirring for 15 minutes;

[0080] ② Heat the material obtained in step ① to 65℃ and homogenize it at 65℃ and 20MPa. After homogenization, sterilize it at 125℃ for 300s and cool it to 37℃ to obtain the fermentation substrate.

[0081] ③ Add Bifidobacterium longum BBMN68 (the fermentation strain Bifidobacterium longum BBMN68 was activated and cultured in MRSC liquid medium) to the fermentation substrate, making its concentration 1×10 7 CFU / mL, after static incubation at 37℃ under anaerobic conditions for 16 h, until the logarithmic growth phase was reached, the viable count reached 5 × 10⁻⁶. 9 CFU / mL or higher;

[0082] ④ Centrifuge the fermentation product obtained in step ③ to collect wet cells, pre-treat at 45℃ for 5 min, add protectant to the wet cells at a volume-to-mass ratio of 4:1 (wet cells: protectant = 4:1 L / kg), add lactic acid to adjust the pH to 4.3, inactivate at 120℃ for 1 min, and vacuum dry at 50℃ to obtain inactivated Bifidobacterium longum BBMN68 powder.

[0083] Example 2

[0084] This embodiment provides an inactivated bacterial powder, as detailed below:

[0085] 1. Raw materials:

[0086] Water 83.08 parts, alkaline protease hydrolyzed milk 12.0 parts, corn steep liquor 1.8 parts, sucrose 1.5 parts, cysteine ​​hydrochloride 0.03 parts, grapefruit peel extract 1.5 parts.

[0087] 2. Preparation method:

[0088] ①Preheat water to 55℃, add alkaline protease hydrolysate, sucrose, grapefruit peel extract, corn syrup, and cysteine ​​hydrochloride, and maintain the temperature while stirring for 15 minutes;

[0089] ② Heat the material obtained in step ① to 65℃ and homogenize it at 65℃ and 20MPa. After homogenization, sterilize it at 125℃ for 300s and cool it to 37℃ to obtain the fermentation substrate.

[0090] ③ Add Bifidobacterium longum BBMN68 to the fermentation substrate to a concentration of 1×10⁻⁶. 7 CFU / mL, after static incubation at 37℃ under anaerobic conditions for 16 h, until the logarithmic growth phase was reached, the viable count reached 5 × 10⁻⁶. 9 CFU / mL or higher;

[0091] ④ Centrifuge the fermentation product obtained in step ③ to collect wet cells, pre-treat at 42℃ for 15 min, add the wet cells to the protectant at a volume-to-mass ratio of 4:1 (wet cells: protectant = 4:1 L / kg), add acetic acid to adjust the pH to 4.5, inactivate at 100℃ for 3 min, and vacuum dry at 50℃ to obtain inactivated Bifidobacterium longum BBMN68 powder.

[0092] Example 3

[0093] This embodiment provides an inactivated bacterial powder, as detailed below:

[0094] 1. Raw materials

[0095] Water 78.96 parts, alkaline protease hydrolyzed fat-free milk 15.0 parts, yeast extract 2.0 parts, glucose 0.8 parts, ascorbic acid 0.09 parts, Tween-80 0.15 parts, grapefruit peel extract 3.0 parts.

[0096] 2. Preparation method:

[0097] ①Preheat water to 55℃, add alkaline protease hydrolyzed fat-free milk, glucose, grapefruit peel extract, yeast extract, ascorbic acid, and Tween-80, and maintain the temperature while stirring for 15 minutes;

[0098] ② Heat the material obtained in step ① to 65℃ and homogenize it at 65℃ and 20MPa. After homogenization, sterilize it at 125℃ for 300s and cool it to 37℃ to obtain the fermentation substrate.

[0099] ③ Add Bifidobacterium longum BBMN68 to the fermentation substrate to a concentration of 1×10⁻⁶. 7 CFU / mL, after static incubation at 37℃ under anaerobic conditions for 16 h, until the logarithmic growth phase was reached, the viable count reached 5 × 10⁻⁶. 9 CFU / mL or higher;

[0100] ④ Centrifuge the fermentation product obtained in step ③ to collect wet cells, pre-treat at 45℃ for 10 min, add the wet cells to the protectant at a volume-to-mass ratio of 4:1 (wet cells: protectant = 4:1 L / kg), add citric acid to adjust the pH to 4.8, inactivate at 85℃ for 10 min, and vacuum dry at 50℃ to obtain Bifidobacterium longum BBMN68 inactivated bacterial powder.

[0101] Example 4

[0102] This embodiment provides an inactivated bacterial powder, as detailed below:

[0103] 1. Raw materials

[0104] Water 83.86 parts, alkaline protease hydrolyzed fat-free milk 12.0 parts, yeast extract 1.5 parts, glucose 1.3 parts, ascorbic acid 0.09 parts, Tween-80 0.08 parts, grapefruit peel extract 1.0 parts.

[0105] 2. Preparation method:

[0106] ①Preheat water to 55℃, add alkaline protease hydrolyzed fat-free milk, glucose, grapefruit peel extract, yeast extract, ascorbic acid, and Tween-80, and maintain the temperature while stirring for 15 minutes;

[0107] ② Heat the material obtained in step ① to 65℃ and homogenize it at 65℃ and 20MPa. After homogenization, sterilize it at 125℃ for 300s and cool it to 37℃ to obtain the fermentation substrate.

[0108] ③ Add Bifidobacterium longum BBMN68 to the fermentation substrate to a concentration of 5×10⁻⁶. 7 CFU / mL, after static incubation at 37℃ under anaerobic conditions for 16 h, until the logarithmic growth phase was reached, the viable count reached 5 × 10⁻⁶. 9 CFU / mL or higher;

[0109] ④ Centrifuge the fermentation product obtained in step ③ to collect wet cells, pre-treat at 45℃ for 10 min, add protectant to the wet cells at a volume-to-mass ratio of 4:1 (wet cells: protectant = 4:1 L / kg), add lactic acid to adjust the pH to 4.5, inactivate at 65℃ for 30 min, and vacuum dry at 50℃ to obtain inactivated Bifidobacterium longum BBMN68 powder.

[0110] Example 5

[0111] This embodiment provides an inactivated bacterial powder, as detailed below:

[0112] 1. Raw materials

[0113] Water 84.04 parts, alkaline protease hydrolyzed fat-free milk 10.0 parts, yeast extract 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts, grapefruit peel extract 2.0 parts.

[0114] 2. Preparation method:

[0115] ①Preheat water to 55℃, add alkaline protease hydrolyzed fat-free milk, glucose, grapefruit peel extract, yeast extract, ascorbic acid, and Tween-80, and maintain the temperature while stirring for 15 minutes;

[0116] ② Heat the material obtained in step ① to 65℃ and homogenize it at 65℃ and 20MPa. After homogenization, sterilize it at 125℃ for 300s and cool it to 37℃ to obtain the fermentation substrate.

[0117] ③ Add Bifidobacterium longum BBMN68 to the fermentation substrate to a concentration of 5×10⁻⁶. 7 CFU / mL, after static incubation at 37℃ under anaerobic conditions for 16 h, until the logarithmic growth phase was reached, the viable count reached 5 × 10⁻⁶. 9 CFU / mL or higher;

[0118] ④ Centrifuge the fermentation product obtained in step ③ to collect wet cells, pre-treat at 45℃ for 10 min, add protectant to the wet cells at a volume-to-mass ratio of 4:1 (wet cells: protectant = 4:1 L / kg), add lactic acid to adjust the pH to 4.5, inactivate at 75℃ for 1 min, and vacuum dry at 50℃ to obtain inactivated Bifidobacterium longum BBMN68 powder.

[0119] Example 6

[0120] This embodiment provides an inactivated bacterial powder, which, compared to Example 5, alters the ratio of grapefruit peel extract and alkaline protease hydrolyzed fat-free milk in the fermentation substrate, as follows:

[0121] 1. Raw materials:

[0122] Water 85.54 parts, alkaline protease hydrolyzed fat-free milk 11.5 parts, yeast extract 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts, grapefruit peel extract 0.5 parts.

[0123] 2. The preparation method is the same as in Example 5.

[0124] Example 7

[0125] This embodiment provides an inactivated bacterial powder, which, compared to Example 5, alters the ratio of grapefruit peel extract and alkaline protease hydrolyzed fat-free milk in the fermentation substrate, as follows:

[0126] 1. Raw materials:

[0127] Water 82.54 parts, alkaline protease hydrolyzed fat-free milk 8.5 parts, yeast extract 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts, grapefruit peel extract 3.5 parts.

[0128] 2. The preparation method is the same as in Example 5.

[0129] Comparative Example 1

[0130] This comparative example provides an inactivated bacterial powder that, compared to Example 5, does not contain grapefruit peel extract in the fermentation substrate, as detailed below:

[0131] 1. Raw materials:

[0132] Water 86.04 parts, alkaline protease hydrolyzed fat-free milk 10.0 parts, yeast extract 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts.

[0133] 2. The preparation method is the same as in Example 5.

[0134] Comparative Example 2

[0135] This comparative example provides an inactivated bacterial powder, which, compared to Example 5, uses ordinary skim milk instead of alkaline protease hydrolyzed skim milk, as detailed below:

[0136] 1. Raw materials:

[0137] Water 84.04 parts, skim milk 10.0 parts, yeast extract 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts, grapefruit peel extract 2.0 parts.

[0138] 2. The preparation method is the same as in Example 5.

[0139] Comparative Example 3

[0140] This comparative example provides an inactivated bacterial powder, which, compared to Example 5, uses other water-soluble dietary fibers instead of grapefruit peel extract, as detailed below:

[0141] 1. Raw materials:

[0142] Water 84.04 parts, alkaline protease hydrolyzed fat-free milk 10.0 parts, yeast extract 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts, soybean polysaccharide 2.0 parts.

[0143] 2. The preparation method is the same as in Example 5.

[0144] Comparative Example 4

[0145] This comparative example provides an inactivated bacterial powder, which, compared to Example 5, uses other water-soluble dietary fibers instead of grapefruit peel extract, as detailed below:

[0146] 1. Raw materials:

[0147] Water 84.04 parts, alkaline protease hydrolyzed fat-free milk 10.0 parts, yeast extract 1.8 parts, glucose 2.0 parts, ascorbic acid 0.06 parts, Tween-80 0.1 parts, β-glucan 2.0 parts.

[0148] 2. The preparation method is the same as in Example 5.

[0149] Comparative Example 5

[0150] This comparative example provides an inactivated bacterial powder, which is basically the same as the preparation method in Example 5, except that in step ④ of the preparation, the step of pretreatment at 45°C for 10 min is omitted, and subsequent steps are performed directly. The results showed an increased bacterial cell breakage rate (approximately 10%), and a large number of bacterial cells exhibited severe shrinkage (see [link to example]). Figure 2 (This makes it more prone to breakage later.)

[0151] Experimental Example 1

[0152] In this experimental example, the inactivated bacterial powder prepared in the above examples was observed by electron microscopy and viable bacterial count was detected. It was found that the bacterial cells were intact and unbroken (see electron micrograph of representative example 5). Figure 1 Furthermore, plate counting showed no viable bacteria.

[0153] Methods for observing bacterial strains using scanning electron microscopy:

[0154] Weigh 0.1g of bacterial powder and add it to 10mL of PBS. Vortex to mix thoroughly, centrifuge at 8000rpm for 3-5min, discard the supernatant, and pour in 2.5% glutaraldehyde fixative. Dehydrate using an alcohol gradient of 30%, 50%, 60%, 70%, 80%, 90%, 95%, and 100%, for 15min per gradient. Perform critical point drying using a critical point dryer. Fix the dried bacterial powder onto the sample stage using carbon conductive double-sided tape, ensuring uniform dispersion and firm adhesion. Remove any excess unfixed bacterial cells from the sample stage using a suction bulb. Spray with gold and observe under a scanning electron microscope.

[0155] Methods for detecting viable bacteria by plate counting:

[0156] Refer to the Bifidobacterium count section of GB 4789.35-2023 National Food Safety Standard for Microbiological Examination of Food (Lactic Acid Bacteria Examination).

[0157] Based on the estimated Bifidobacterium content in the sample, select 2-3 consecutive suitable dilutions. For each dilution, pipette 1 mL of the homogenate into a sterile Petri dish, preparing two Petri dishes for each dilution. After transferring the dilution into the Petri dishes, pour 15-20 mL of modified MRS agar medium (mupirocin lithium and cysteine ​​hydrochloride, cooled to 48-50℃) into each dish, rotating to ensure thorough mixing. After the medium solidifies, invert the dish for anaerobic incubation at 36℃±1℃. Based on the growth characteristics of Bifidobacterium, incubation for 48 hours is generally recommended. If no colonies grow or the growth is small, incubation can be extended to 72 hours. After incubation, count all colonies on the plate. The entire process from sample dilution to plate pouring should be completed within 15 minutes.

[0158] Experiment Example 2

[0159] This experiment tested the active metabolites in the inactivated powders prepared in the above examples and comparative examples. The specific results are shown in Tables 1 and 2.

[0160] Table 1. Active metabolites in fermentation products of the examples.

[0161] Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Linaloyl acetate (μg / g) 910 890 939 920 950 702 610 Extracellular polysaccharides (μg / g) 256 253 261 272 268 264 262 Vanillin (μg / g) 506 512 503 530 546 266 231 Lactic acid (mg / g) 148.5 139.8 128.5 140.8 156.8 135.5 140.1 Acetic acid (mg / g) 48.71 48.82 46.98 47.89 50.17 43.61 42.24 Propionic acid (mg / g) 0.35 0.41 0.49 0.39 0.48 0.29 0.36 Butyric acid (mg / g) 3.01 2.88 3.12 3.18 3.35 2.22 1.835 Hexanoic acid (mg / g) 1.82 2.56 1.83 1.95 2.02 1.71 1.49 Total short-chain fatty acids (mg / g) 54.57 55.78 53.75 54.06 57.51 48.85 46.07

[0162] Table 2. Active metabolites in comparative fermentation products

[0163] Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Linaloyl acetate (μg / g) 0 156 22 109 Extracellular polysaccharides (μg / g) 210 190 197 188 Vanillin (μg / g) 0 107 41 80 Lactic acid (mg / g) 130.8 122.9 129.8 131.5 Acetic acid (mg / g) 41.05 40.25 38.88 40.11 Propionic acid (mg / g) 0.27 0.15 0.19 0.18 Butyric acid (mg / g) 0.81 1.89 1.35 1.18 Hexanoic acid (mg / g) 1.23 1.33 1.25 1.58 Total short-chain fatty acids (mg / g) 43.89 44.06 42.01 43.62

[0164] For the detection methods of linalyl acetate and vanillin, please refer to "GB 1886.126-2015 National Food Safety Standard - Linalyl Acetate as a Food Additive" and "GB 5009.284-2021 National Food Safety Standard - Determination of Vanillin, Methyl Vanillin, Ethyl Vanillin and Coumarin in Food - Method III".

[0165] Total short-chain fatty acids are fatty acids with a chain length of 1-6 carbon atoms, specifically including: acetic acid, propionic acid, isobutyric acid, valeric acid, isovaleric acid, n-butyric acid, hexanoic acid, etc.

[0166] The test results show that, after fermentation, compared with the control group, the fermentation product of this invention contains a large number of active metabolites, including natural vanillin, linalyl acetate, lactic acid, acetic acid, propionic acid, hexanoic acid, butyric acid, and extracellular polysaccharides, which help regulate immunity. Linalyl acetate belongs to the terpenoid class of compounds, and studies have shown that it has anti-inflammatory effects in mouse allergic asthma models, rat inflammatory edema models, and endothelial cell and epithelial cell models. Vanillin has antibacterial activity, certain antioxidant and anti-cancer effects, and can participate in signal transduction between bacterial cells. Extracellular polysaccharides have antioxidant, immunomodulatory, antitumor, and anti-ulcer biological activities.

[0167] Experimental Example 3

[0168] This experimental example verifies the effect of the inactivated bacterial powder prepared in this invention and the yogurt containing the bacterial powder on immune recovery.

[0169] 1.1 Laboratory Animals

[0170] Purchase 120 healthy male Balb / c mice of SPF grade, aged 5-6 weeks and weighing 20±2g, and house them in standard mouse cages in the animal room. During the acclimatization period, allow them free access to food and water. During the experiment, mark them with numbers and observe and record their food intake, mental state, etc. Other management measures are in accordance with the mouse breeding and management manual.

[0171] 1.1.1 Establishment and grouping of animal models

[0172] SPF-grade male Balb / c mice (weighing 20±2g) were randomly divided into 10 groups of 12 mice each, as follows:

[0173] (1) C: Blank control group;

[0174] (2) M: CTX cyclophosphamide module;

[0175] (3) LMS: Levamisole positive drug group;

[0176] (4) 68L: The low-dose post-biotic group of Example 5 (BBMN68: 1×10 8 (units / (kg·d));

[0177] (5) 68H: The high-dose post-biotic group of Example 5 (BBMN68: 1×10⁻⁶) 10 (units / (kg·d));

[0178] (6) 68Y: Postbiotic yogurt group (BBMN68: 5×108 (units / (kg·d));

[0179] (7) 68JY: Postbiotic + Prebiotic Yogurt Group (BBMN68: 5×10 8 (each / (kg·d) + 25g / (kg·d) of inulin).

[0180] Among them, group 68Y is a fermented dairy product containing Bifidobacterium longum BBMN68 prepared in Example 5, and the preparation method is as follows:

[0181] (1) Preheat 92.55kg of raw milk to 50℃, add 6.0kg of white sugar, 0.65kg of protein powder, 0.4kg of starch, 0.1kg of pectin and 0.3kg of agar, and maintain the preheating temperature for 15min;

[0182] (2) Homogenize under heating to 65℃ and 25MPa, sterilize at 135℃ for 8s after homogenization, and cool to 42℃ to obtain fermentation substrate;

[0183] (3) Add 0.01 kg of starter culture (containing Lactobacillus bulgaricus and Streptococcus thermophilus, with a live count ratio of 1:2) and 0.1 kg of Bifidobacterium longum BBMN68 inactivated powder (post-generic) prepared in Example 5 to the fermentation substrate, ferment at 42°C until the acidity reaches 70°T, stop the fermentation, sterilize at 76°C for 30 s, and cool to 20°C to obtain the fermentation product;

[0184] (4) Homogenize the fermentation product and aseptically fill it. The protein content in the final product is 3.2%.

[0185] Among them, group 68JY is a fermented dairy product containing Bifidobacterium longum BBMN68 prepared in Example 5 and prebiotics, and its preparation method is as follows:

[0186] (1) Preheat 92.5kg of raw milk to 55℃, add 5.8kg of white sugar, 0.6kg of protein powder, 0.4kg of starch, 0.1kg of pectin, 0.3kg of agar and 0.3kg of inulin, and maintain the preheating temperature for 18 minutes;

[0187] (2) Homogenize under heating to 65℃ and 25MPa, sterilize at 135℃ for 8s after homogenization, and cool to 42℃ to obtain fermentation substrate;

[0188] (3) Add 0.01 kg of starter culture (containing Lactobacillus bulgaricus and Streptococcus thermophilus, with a live count ratio of 1:2) and 0.1 kg of Bifidobacterium longum BBMN68 inactivated powder (post-generic) prepared in Example 5 to the fermentation substrate, ferment at 42°C until the acidity reaches 72°T, stop the fermentation, sterilize at 78°C for 30 s, and cool to 20°C to obtain the fermentation product;

[0189] (4) Homogenize the fermentation product and aseptically fill it. The protein content in the final product is 3.1%.

[0190] Except for the blank control group, the other 9 groups were injected with cyclophosphamide 50 mg / kg / day for 5 consecutive days to establish an immunosuppression (low immunity) model. Except for the blank control group and the model group (CTX cyclophosphamide modeling group), which were administered 0.1 mL / 10 g of normal saline by gavage, and the levamisole positive drug group, which were administered levamisole 50 mg / (kg·day) by gavage, the other 7 groups were administered the corresponding dosage of their respective target substances by gavage for 15 consecutive days.

[0191] During the experiment, the health status of mice in each group was observed, including development, diet, mood, coat color, and mental state. The body weight of mice in each group was measured daily between 8:00 and 10:00 AM. The food intake of mice in each group was measured every three days, and cage bedding was changed to ensure a clean and tidy environment. Data were recorded and statistically analyzed. Mice were fasted and deprived of water for 12 hours before sacrifice to ensure the recovery of their intestines for subsequent experiments.

[0192] 1.1.2 Collection of blood, tissue, and intestinal contents samples

[0193] After enucleating the mice, 70 μL of the blood solution was pipetted into a 1.5 mL anticoagulant tube for whole blood leukocyte count analysis. The remaining blood was left at room temperature until serum separated, then centrifuged at 4500 rpm for 15 min and aliquoted for subsequent experimental analysis.

[0194] Mice were euthanized by enucleation and cervical dislocation. Their bodies were disinfected with 75% ethanol. The spleen, thymus, mesenteric lymph nodes, and inguinal lymph nodes were dissected and removed. After washing with PBS, the organs were weighed, and their organ indices were calculated. The spleen tissue was removed, slowly rinsed with ice-cold PBS, and divided into two portions. One portion was placed in a 2mL centrifuge tube containing 4% paraformaldehyde solution, fixed at room temperature for 20 hours, and then embedded in paraffin. The other portion was slowly rinsed with ice-cold PBS, rapidly frozen in liquid nitrogen, and stored in a -80°C cryogenic freezer for subsequent analysis.

[0195] The contents near the cecum were rinsed slowly with ice-cold PBS and then placed into 1.5 mL cryovials for 16S rDNA analysis of changes in gut microbiota diversity.

[0196] 1.2 Animal test indicators

[0197] 1.2.1 Detection of organ index in mice

[0198] Mice were fasted for 12 hours after the last administration, blood was collected by enucleation, and they were euthanized by cervical dislocation. The thymus and spleen were then removed and weighed for wet weight. The thymus index and spleen index of the mice were calculated.

[0199] Thymus index = thymus weight (mg) / body weight (10g);

[0200] Spleen index = spleen weight (mg) / body weight (10g).

[0201] 1.2.2 Measurement of peripheral immune organ lymph nodes in mice

[0202] Mice were fasted for 12 hours after the last administration, their eyeballs were removed to collect blood, and they were euthanized by cervical dislocation. Mesenteric lymph nodes and inguinal lymph nodes were removed and weighed as wet weight.

[0203] 1.2.3 Effects on whole blood immune cells in cyclophosphamide-induced immunosuppressed mice

[0204] After the last administration, the mice were fasted for 12 hours, and then 70 μL of whole blood was collected by removing the eyeballs of the mice. The contents of white blood cells, neutrophils, lymphocytes, eosinophils and monocytes in the mouse blood were detected by a fully automated blood cell analyzer.

[0205] 1.2.4 Pathological observation of mouse spleen

[0206] The tissues were embedded in paraffin, and the paraffin blocks were cut into 4 μm thick sections using a microtome before hematoxylin / eosin HE staining. Images of the sections were acquired using a 3DHISTECH (Hungary) Pannoramic 250 digital slide scanner. For each section, the gross lesions of the entire tissue were first observed at low magnification, and then 400x images were acquired for the specific lesions of the areas to be observed.

[0207] 1.2.5 Measurement of physiological and biochemical indicators

[0208] The levels of IL-2 and TNF-α in serum, as well as the levels of serum immunoglobulins IgA, IgM, and IgG, were determined according to the kit instructions.

[0209] 1.2.6 Intestinal Microbiota Analysis

[0210] High-throughput sequencing of gut microbiota was performed on cecal contents. Samples underwent DNA extraction, PCR amplification, quantitative PCR, and MiSeq library construction before MiSeq sequencing. The MiSeq sequencing data were then subjected to bioinformatics analysis. First, PE reads were assembled based on overlap relationships, and sequence quality was controlled and filtered. After sample differentiation, OTU clustering and taxonomic analysis were performed. Based on the OTU clustering results, various diversity indices and sequencing depth could be analyzed. Based on taxonomic information, statistical analysis of community structure at various taxonomic levels could be conducted. Building upon these analyses, a series of in-depth statistical and visualization analyses, including multivariate analysis and significance testing, could be performed on the community composition and phylogenetic information of multiple samples.

[0211] 2 Results

[0212] 2.1 Effects on mouse immune organs

[0213] The immune system is an essential defense mechanism for living organisms, composed of immune organs, immune cells, and immune molecules. The spleen, thymus, and lymph nodes are the main immune organs in animals. The thymus directly regulates cellular immunity and also indirectly plays a role in humoral immunity. The spleen contains abundant lymphocytes and macrophages, which are closely related to the body's specific immunity. Therefore, the levels of spleen and thymus indices can, to some extent, reflect the body's immune function.

[0214] The experimental results are shown in Table 3. Compared with the blank control group, the thymus index and lymph node wet weight of the model group mice were significantly reduced, indicating immunosuppression, while the spleen index was significantly increased. This finding may be due to the compensatory enlargement of the spleen caused by metabiotics and their products under immunodeficiency conditions, and this compensatory effect may improve the host's immunity (P<0.05). The intervention of metabiotics and their products in this invention can increase the thymus index and lymph node wet weight and reduce the spleen index in immunosuppressed mice (P<0.05). This indicates that the intervention of metabiotics and their products can alleviate the immune organ damage caused by cyclophosphamide and improve the non-specific immunity of mice.

[0215] Table 3 Effects on peripheral immune organs in immunosuppressed mice

[0216]

[0217] Different lowercase letters in the numbers in the table indicate significant differences (P < 0.05).

[0218] 2.2 Effects on the pathological characteristics of organs and tissues in immunized mice

[0219] Microscopic images of histological changes in the spleens of mice from each group after HE staining are shown below. Figures 3 to 9 As shown in the figure, compared with the blank control group, the model group mice showed an unclear boundary between the white and red pulp, varying degrees of white pulp atrophy, smaller or even absent lymphoid nodules, and significant extramedullary hematopoiesis within the red pulp, with sheet-like distribution of erythrocytes and granulocytes at various stages, interspersed with scattered megakaryocytes and cell debris, and inflammatory cell infiltration. In the intervention group of the post-biotic and its products of this invention, the spleen tissue capsule of mice was intact, with no hyperplasia observed in the capsule and fibrous connective tissue of the splenic trabeculae, a clear boundary between the white and red pulp, and relatively intact and clear periarterial lymphatic sheaths and lymphoid nodules within the white pulp, with no significant atrophy or hyperplasia observed in the white pulp. The results indicate that intervention with the post-biotic and its products can alleviate the damage to the spleen tissue, an immune organ, caused by cyclophosphamide.

[0220] 2.3 Effects on whole blood immune cells in immunosuppressed mice

[0221] The number of lymphocytes and leukocytes in the blood reflects the overall level of the body's immune response. The experimental results are shown in Table 4. Compared with the blank control group, the model group significantly increased the proportion of leukocytes and eosinophils, and decreased the proportion of neutrophils, lymphocytes, and monocytes. Compared with the model group, gavage administration of the post-biotic and its products of this invention significantly reduced the proportion of leukocytes and peripheral blood eosinophils in mice, and increased the proportion of neutrophils, lymphocytes, and monocytes (P<0.05). Studies have shown that an increase in the proportion of eosinophils is commonly associated with inflammation, due to the use of the drug-induced myelosuppressant cyclophosphamide, which leads to an increase in the proportion of eosinophils related to allergies. The post-biotic and its products of this invention alleviated the inhibitory effect of cyclophosphamide on humoral immune function in mice, reduced inflammation, and promoted the gradual recovery of immune function in mice.

[0222] Table 4 Effects on immunosuppressed peripheral blood cells

[0223] Grouping leukocyte(%) Neutrophils (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) C <![CDATA[5.200±0.337 a ]]> <![CDATA[76.340±5.092 a ]]> <![CDATA[60.780±8.210 a ]]> <![CDATA[0.720±0.303 a ]]> <![CDATA[0.240±0.015 a ]]> M <![CDATA[10.728±0.353 c ]]> <![CDATA[44.033±7.559 c ]]> <![CDATA[32.317±6.513 c ]]> <![CDATA[0.517±0.047 c ]]> <![CDATA[0.583±0.028 c ]]> LMS <![CDATA[7.633±0.454 b ]]> <![CDATA[47.550±6.335 c ]]> <![CDATA[45.000±5.469 ab ]]> <![CDATA[0.600±0.020 b ]]> <![CDATA[0.200±0.054 ab ]]> 68L <![CDATA[5.113±0.784 a ]]> <![CDATA[54.475±6.382 bc ]]> <![CDATA[42.350±5.211 b ]]> <![CDATA[0.650±0.031 b ]]> <![CDATA[0.280±0.041 ab ]]> 68H <![CDATA[5.740±0.321 a ]]> <![CDATA[60.560±4.778 b ]]> <![CDATA[46.560±5.146 ab ]]> <![CDATA[0.920±0.069 a ]]> <![CDATA[0.340±0.061 b ]]> 68Y <![CDATA[5.634±0.864 ab ]]> <![CDATA[57.580±6.718 b ]]> <![CDATA[48.920±4.001 ab ]]> <![CDATA[1.260±0.141 a ]]> <![CDATA[0.290±0.045 ab ]]> 68JY <![CDATA[5.874±0.115 a ]]> <![CDATA[59.780±3.883 b ]]> <![CDATA[53.120±6.143 ab ]]> <![CDATA[0.860±0.076 a ]]> <![CDATA[0.250±0.062 ab ]]>

[0224] Different lowercase letters in the numbers in the table indicate significant differences (P < 0.05).

[0225] 2.4 Effects on serum cytokines in immunosuppressed mice

[0226] Cytokines are potential therapeutic targets for regulating immune responses. Th cells are the initial CD4 cells. +T cells are classified into different subtypes based on their functions, with Th1 and Th2 being the most common. To ensure normal immunity and overall health, the secretion of cytokines maintains a balance between Th1 and Th2 cells. IL-2 is a T cell growth factor that significantly influences T cell proliferation and the differentiation of effector and memory cells, promoting T cell immune responses. TNF-α plays a crucial role in innate, acquired humoral, and cellular immune responses, and is one of the most important cytokines in the body, serving as a prototype cytokine for maintaining immune homeostasis.

[0227] The experimental results are shown in Table 5. Compared with the blank control group, the model group significantly reduced the serum levels of TNF-α and IL-2 in mice. Compared with the model group, gavage administration of the post-biotic and its products of this invention increased the serum cytokine content (P<0.05). This indicates that the post-biotic and its products of this invention alleviated the inhibitory effect of cyclophosphamide on humoral immune function in mice, and in terms of immune cells, the post-biotic and its products of this invention exhibited enhanced Th1 cell immunomodulatory capacity.

[0228] Table 5 Effects on serum cytokines

[0229] Cytokines C M LMS 68L 68H 68Y 68JY TNF-α (pg / mL) <![CDATA[163.47±8.92 a ]]> <![CDATA[81.20±4.89 d ]]> <![CDATA[161.53±6.88 a ]]> <![CDATA[111.34±3.32 c ]]> <![CDATA[150.59±3.50 b ]]> <![CDATA[151.11±3.46 ab ]]> <![CDATA[157.71±6.61 ab ]]> IL-2 (pg / mL) <![CDATA[29.22±3.22 a ]]> <![CDATA[15.80±3.06 d ]]> <![CDATA[18.37±1.34 c ]]> <![CDATA[23.09±1.28 b ]]> <![CDATA[23.47±0.84 b ]]> <![CDATA[22.21±1.43 b ]]> <![CDATA[23.58±1.13 b ]]>

[0230] Different lowercase letters in the numbers in the table indicate significant differences (P < 0.05).

[0231] 2.5 Effects on serum immunoglobulins in immunosuppressed mice

[0232] Immunoglobulins (Ig) are animal proteins with antibody activity, mainly found in plasma, body fluids, tissues, and some secretions. In humans, IgA exists primarily in monomeric and dimer forms, classified into serum and secretory types. Secretory IgA is a major component of the body's mucosal defense system, forming the first line of immune defense against pathogens and harmful substances in the respiratory, digestive, and genitourinary tracts, and is the most important antibody in mucosal immunity. The experimental results are shown in Table 6. The table shows that the IgA level in mice treated with cyclophosphamide was significantly lower than that in the blank control group (P<0.05). Compared with the model group, the post-biotic and its products of this invention increased serum IgA secretion (P<0.05), indicating that they can enhance the humoral immune response in immunosuppressed mice.

[0233] Most antibacterial and antiviral antibodies are currently of the IgG type, playing a major role in the body's cellular immunity. They promote the phagocytic activity of mononuclear macrophages, neutralize the toxicity of bacterial toxins, and bind to viral antigens, rendering viruses unable to infect host cells. The experimental results are shown in Table 6. Compared with the model group, the serum IgG levels in immunosuppressed mice were significantly upregulated after gavage administration of the probiotic and its products (P<0.05), indicating that the probiotic and its products of this invention have a positive regulatory effect on IgG levels in intestinal tissues. Furthermore, the probiotic and its products of this invention have a certain improving effect on the immune function of immunosuppressed mice treated with cyclophosphamide.

[0234] Immunoglobulin M (IgM) is the largest immunoglobulin in molecular weight, mainly synthesized and secreted by plasma cells in the spleen and lymph nodes. It is primarily distributed in serum, accounting for 5%–10% of total serum Ig. IgM possesses potent bactericidal, complement-activating, immunomodulatory, and agglutinating effects, and also participates in the pathological processes of certain autoimmune diseases and hypersensitivity reactions. The IgM level in mice treated with cyclophosphamide was significantly lower than that in the blank control group (P<0.05). Compared with the model group, the post-biotic and its products of this invention increased serum IgM secretion (P<0.05), indicating that they can enhance the humoral immune response in immunosuppressed mice.

[0235] Table 6. Effects on serum immunoglobulins

[0236]

[0237] Different lowercase letters in the numbers in the table indicate significant differences (P < 0.05).

[0238] 2.6 Effects on the gut microbiota of immunosuppressed mice

[0239] The study of the effects of the postbiotic of this invention on the characteristic flora of immunocompromised mice revealed changes in 7 beneficial and 2 harmful genera out of 9 immune-related bacterial genera. The abundance of 7 beneficial bacteria associated with immunity and metabolic status was increased, while the abundance of 2 harmful bacteria associated with intestinal diseases and inflammation was decreased. These results indicate that the postbiotic of this invention has a regulatory effect on the intestinal flora.

[0240] Table 7. Effects of the product on the characteristic microbiota of immunocompromised mice.

[0241]

[0242] The inactivated bacterial powder (post-biotic) prepared in the other embodiments of the present invention also have similar effects to the inactivated bacterial powder of Example 5, which can enhance the immunity of mice, reduce damage to immune organs, alleviate inflammation, and regulate intestinal flora. Among all embodiments, Example 5 has the best overall effect.

[0243] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for fermenting Bifidobacterium longum BBMN68, characterized in that, The fermentation substrate includes alkaline protease hydrolyzed milk and grapefruit peel extract; the hydrolysis rate of the alkaline protease hydrolyzed milk is 9-11%, and the water-soluble dietary fiber content of the grapefruit peel extract is 75-80%; the mass ratio of the alkaline protease hydrolyzed milk to the grapefruit peel extract is (10-15):(1-3).

2. The method for fermenting Bifidobacterium longum BBMN68 according to claim 1, characterized in that, The fermentation substrate also includes a carbon source, a nitrogen source, an antioxidant, and water, or may further include a surfactant; Preferably, the fermentation substrate comprises 10-15 parts alkaline protease hydrolyzed milk, 1.5-2.0 parts yeast extract or corn steep liquor, 0.8-2.5 parts glucose or sucrose, 0.03-0.09 parts ascorbic acid or cysteine ​​hydrochloride, 1.0-3.0 parts grapefruit peel extract, 78-85 parts water, or further comprises 0.08-0.15 parts Tween-80; And / or, the inoculum concentration of Bifidobacterium longum BBMN68 in the fermentation substrate is 1–5 × 10⁻⁶. 7 CFU / mL; And / or, the fermentation conditions are: anaerobic, 37±1℃, static culture.

3. A fermentation product of Bifidobacterium longum BBMN68, characterized in that, It is prepared by the method of fermenting Bifidobacterium longum BBMN68 as described in any one of claims 1-2.

4. The use of a composition as a fermentation substrate in increasing the content of one or more of vanillin, linaloyl acetate, lactic acid, acetic acid, propionic acid, hexanoic acid, butyric acid, extracellular polysaccharides, and total short-chain fatty acids in Bifidobacterium longum BBMN68 fermentation product; said composition comprising alkaline protease hydrolyzed milk and grapefruit peel extract; said alkaline protease hydrolyzed milk having a hydrolysis rate of 9-11%, said grapefruit peel extract having a water-soluble dietary fiber content of 75-80%; said alkaline protease hydrolyzed milk and grapefruit peel extract having a mass ratio of (10-15):(1-3).

5. A method for preparing post-genetic agents, characterized in that, Obtained by inactivating the Bifidobacterium longum BBMN68 fermentation product according to claim 3; Preferably, the method includes: (1) Heat the cells of Bifidobacterium longum BBMN68 fermentation product according to claim 3 at 42-45°C for 5-15 minutes; (2) After mixing with the inactivating protective agent, adjust the pH value to 4.3-4.8; (3) Inactivate at 65-120℃ for 1-30 minutes; More preferably, the method further includes: (4) vacuum drying at 40-60°C; And / or, the inactivating protective agent comprises: 8-12 parts skim milk powder, 1-3 parts resistant dextrin, 4-8 parts lactose, 4-6 parts trehalose, 1-3 parts inulin, 1-3 parts maltodextrin, and 0.2-0.6 parts monosodium glutamate; or the inactivating protective agent comprises: 8-12 parts skim milk powder, 2.5-3.5 parts L-cysteine, and 8-12 parts xylan; And / or, the bacterial cells in the Bifidobacterium longum BBMN68 fermentation product are obtained by centrifuging the Bifidobacterium longum BBMN68 fermentation product, and the volume-to-mass ratio of the bacterial cells to the inactivation protectant is (3.8-4.2):1L / kg; And / or, in step (2), the pH value is adjusted with one or more of lactic acid, acetic acid or citric acid.

6. An epigenetic agent, characterized in that, It is prepared by the method described in claim 5.

7. The use of the post-biotic as described in claim 6 in the preparation of pharmaceuticals, health products or food.

8. A product, wherein the product is a pharmaceutical, health product, or food, characterized in that, Includes the postgenetic agent as described in claim 6.

9. A fermented milk, characterized in that, The fermented milk comprises: animal milk, sugar, protein powder, starch, pectin, agar, inactivated starter culture, and the post-biotic as described in claim 6, or further comprises prebiotics; Preferably, the fermented milk comprises: 90-95 parts animal milk, 5.5-6 parts sugar, 0.6-0.7 parts protein powder, 0.38-0.42 parts starch, 0.08-0.12 parts pectin, 0.28-0.32 parts agar, 0.008-0.012 parts inactivated starter culture, and 0.08-0.12 parts postbiotic as described in claim 6, or further comprises 0.28-0.32 parts prebiotic, more preferably, the prebiotic is inulin, and the starter culture comprises Lactobacillus bulgaricus and Streptococcus thermophilus.

10. The use of the postbiotic of claim 6, the product of claim 8, or the fermented milk of claim 9 in enhancing the body's immunity, reducing damage to immune organs, alleviating inflammation, and / or regulating the intestinal flora.

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