Animal Bifidobacterium lactis subspecies capable of enhancing immunity and application thereof

Abstract

The present invention belongs to the technical field of lactic acid bacteria, and specifically relates to the preparation and application of animal Bifidobacterium lactis subspecies. The animal Bifidobacterium lactis subspecies has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on June 5, 2024, with a preservation address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC No. 30870, and is classified and named Bifidobacterium animalis subsp. lactis. The animal Bifidobacterium lactis subspecies prepared by the present invention has the function of enhancing immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of lactic acid bacteria, and particularly relates to the preparation and application of animal Bifidobacterium lactis subspecies. Background Art

[0002] Bifidobacterium animalis subsp. lactis is a lactic acid bacteria commonly used in food production. Formerly known as Bifidobacterium lactis, it is a Gram-positive rod with various morphologies, the main morphological feature of which is the bifurcated rod. Bifidobacterium animalis subsp. lactis is mainly distributed in the human intestine, and is also distributed in the digestive tract of some livestock such as pigs and dogs. Bifidobacterium animalis subsp. lactis belongs to the genus Bifidobacterium of lactic acid bacteria, and is generally believed to have the functions of promoting body growth, maintaining the balance of intestinal flora, enhancing immunity and inhibiting the growth of harmful bacteria. It is worth noting that different lactic acid strains have different focuses on functions and applications. The Bifidobacterium animalis subsp. lactis DD26 developed by us was found to be a strain with the function of enhancing immunity through animal experiments.

[0003] Lactic acid bacteria have many beneficial effects on the body, and the immunomodulation of lactic acid bacteria is one of its prominent and important functions. Studies have found that the immunomodulatory effect of lactic acid bacteria is to enhance immune function by regulating the host's immune system. Recent studies have found that several specific gene compounds in lactic acid bacteria mediate immunomodulatory effects. Studies on the effects of lactic acid bacteria on host immunobiology have shown that the mechanism of lactic acid bacteria regulating immunity is to regulate systemic and mucosal immune cells and intestinal epithelial cells. Lactic acid bacteria have a direct regulatory effect on the immune system. Lactic acid bacteria can interact with pattern recognition receptors on the surface of innate immune cells such as macrophages and dendritic cells, activate intracellular signaling pathways, promote the secretion of cytokines and enhance phagocytic function; lactic acid bacteria enhance the integrity of the intestinal mucosal barrier, promote the secretion of mucins and the expression of tight junction proteins, reduce the invasion of pathogens, induce the activation of immune cells in the intestinal mucosal lamina propria and the secretion of immunoglobulin A (IgA), and form a local immune defense. Lactic acid bacteria have an indirect regulatory effect on the immune system. The metabolites such as lactic acid and short-chain fatty acids produced by lactic acid bacteria can regulate the intestinal pH value, create a favorable environment for the growth of beneficial bacteria, and inhibit the reproduction of harmful bacteria, indirectly affecting the balance of the immune system; lactic acid bacteria can improve the composition and diversity of intestinal flora, increase the abundance of beneficial bacteria, and promote the production of flora metabolites, such as butyrate, which can affect the homeostasis of the immune system by regulating the differentiation and function of immune cells. Lactic acid bacteria can regulate gene expression and signaling pathways in host cells. Animal experiments on mice have shown that lactic acid bacteria can effectively promote the growth of mouse thymus. The thymus is one of the important immune organs of the human body, which can promote the proliferation of T lymphocytes and enhance immunity. This also shows from another perspective that lactic acid bacteria can inhibit the proliferation of pathogens by enhancing immunity. Lactic acid bacteria play an important role in maintaining the balance between necessary defense mechanisms and excessive defense mechanisms, including innate and adaptive immune responses. Lactic acid bacteria have the potential to treat diseases related to immune response, such as allergic diseases, infectious diseases, autoimmune diseases, etc. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method of animal Bifidobacterium lactis subspecies with the function of enhancing immunity and its application.

[0005] An animal Bifidobacterium lactis subspecies, wherein the animal Bifidobacterium lactis subspecies DD26 has been deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on June 5, 2024, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 30870.

[0006] Furthermore, the dried bacterial powder of Bifidobacterium animalis subspecies lactis DD26 strain was identified by the Institute of Microbiology, Chinese Academy of Sciences;

[0007] The test items include cell morphology and physical and chemical tests, molecular biological identification (16S rRNA gene sequence determination and tuf gene sequence determination), antibiotic sensitivity test, and bacterial strain stability evaluation;

[0008] The experimental results showed that the 16S rRNA gene sequence and tuf gene sequence results of F1 generation sequencing and F5 generation sequencing were the same. Figure 1 This is a comparison chart of the tuf gene sequences of the F1 and F5 generations.

[0009] The results of the detection and identification of the animal Bifidobacterium lactis subspecies DD26 strain of the present invention by the Institute of Microbiology, Chinese Academy of Sciences are as follows:

[0010] Under the conditions of this laboratory, based on a comprehensive analysis of experimental data such as cell morphology, physiological and biochemical experiments, 16S rRNA gene and tuf gene sequences of the tested strains, and with reference to the Bergey's Manual of Systematic Bacteriology, the identification result of the strain (strain number: DD26) submitted by Jiangsu Dexi Biotechnology Co., Ltd. is: Bifidobacterium animalis subsp. lactis.

[0011] The conclusion of the strain stability evaluation of the animal Bifidobacterium lactis subspecies DD26 strain of the present invention by the Institute of Microbiology, Chinese Academy of Sciences is as follows:

[0012] After five consecutive generations of subculture, comparative experiments were conducted, and no visible changes were found in the morphological characteristics, physical and chemical characteristics, classification and identification shapes, and antibiotic sensitivity of the tested strains, indicating that the characteristics of the tested strains can be stably inherited.

[0013] A method for preparing a working strain of animal Bifidobacterium lactis subspecies, comprising the following steps:

[0014] Take a sample containing animal Bifidobacterium lactis subsp. DD26 strain, add it to a liquid activation culture medium, and culture it in an anaerobic chamber at a constant temperature of 35-38°C for 10-12 hours. (2) Take the above activated bacterial solution and inoculate it into the slant of the eggplant bottle; inoculate 200μL of bacterial solution on each slant of the eggplant bottle and culture it in an anaerobic chamber at 35-38°C for 20-24 hours. (3) Use 20ml of protective agent to scrape off the bacterial moss on the slant culture medium of the eggplant bottle, mix it evenly, and divide it into sterile screw-cap cryopreservation tubes and store it at -80°C to obtain the working strain of animal Bifidobacterium lactis subsp. DD26.

[0015] Furthermore, the liquid activation culture medium formula comprises peptone, yeast powder, glucose (monohydrate), sodium chloride, anhydrous sodium acetate, L-cysteine ​​hydrochloride (monohydrate), and purified water; the pH is adjusted to 6.8±0.1, and sterilized at 121°C for 20 minutes after packaging. The eggplant bottle slant culture medium formula comprises peptone, yeast powder, glucose (monohydrate), sodium chloride, anhydrous sodium acetate, L-cysteine ​​hydrochloride (monohydrate), purified water, and agar strips; the pH is adjusted to 6.8±0.1, and sterilized at 121°C for 20 minutes after packaging. The protective agent formula comprises skim milk powder, yeast powder, L-cysteine ​​hydrochloride (monohydrate), and purified water; the pH is adjusted to 6.5±0.1, and sterilized at 115°C for 20 minutes after packaging.

[0016] A method for preparing the animal Bifidobacterium lactis subspecies freeze-dried powder is prepared by the following steps:

[0017] A method for fermenting animal Bifidobacterium lactis subspecies, comprising:

[0018] Inoculate the strain of Bifidobacterium animalis subsp. lactis on the eggplant bottle solid culture medium for activation;

[0019] Transplanting the activated bacterial strain into a primary liquid culture medium for expansion;

[0020] Transplanting the expanded primary bacterial strain into a secondary liquid culture medium for fermentation;

[0021] Transplanting the fermented secondary bacterial strain into a tertiary liquid culture medium for fermentation, and filtering the fermentation liquid to obtain animal Bifidobacterium lactis subsp.

[0022] The activated solid culture medium uses the MRS formula.

[0023] The formula of the primary liquid culture medium is 10-12.5 g / L yeast powder, 4-6 g / L glucose monohydrate, 4-6 g / L peptone, 2-4 g / L sodium chloride, and 2-4 g / L anhydrous sodium acetate. The pH is adjusted to 6.8.

[0024] The secondary liquid culture medium is formulated with 16-20 g / L yeast powder, 20-25 g / L glucose monohydrate, 16-20 g / L peptone, 2-4 g / L anhydrous sodium acetate, and 2-4 g / L galacto-oligosaccharide. The pH is adjusted to 6.8.

[0025] The formula of the three-level liquid culture medium is 16-20 g / L yeast powder, 14-18 g / L glucose monohydrate, 15-20 g / L peptone, 2-4 g / L anhydrous sodium acetate, and 3-5 g / L galacto-oligosaccharide. The pH is adjusted to 6.8.

[0026] During the fermentation process, the secondary and tertiary liquid culture media are supplemented with 3M sodium hydroxide solution; the pH of the secondary liquid culture media is 5-6.5, and the OD600 value is greater than 1.5; the pH of the tertiary liquid culture media is 5-6.5, and the OD600 value is greater than 2.

[0027] In a specific embodiment, it includes:

[0028] S1. Inoculate the strain of Bifidobacterium animalis subspecies lactis DD26 on a solid culture medium at a volume ratio of 1:(8-10), and then activate it in an anaerobic environment at 37°C for 24 hours;

[0029] S2, the activated bacteria were placed at 60cm 2 The solid culture medium is inoculated with (200-300) mL of liquid culture medium and then transferred to the primary liquid culture medium, and then placed in an oxygen-free environment at 37°C for 12 hours;

[0030] S3, transplanting the expanded bacterial strain into the secondary liquid culture medium at an inoculation rate of (2-3) vol%, and then fermenting it in an anaerobic environment at 37°C for 8-12 hours; during the fermentation process, nitrogen is introduced into the fermentation tank, and 3M sodium hydroxide is added to the secondary liquid culture medium to control the pH of the secondary liquid culture medium at 5-6.5;

[0031] S4, when the fermentation time is greater than 8h, the fermentation liquid is sampled and the OD600 is tested. When the OD600 is greater than 1.5, the secondary fermentation is considered to be complete.

[0032] S5, transplanting the fermented secondary strain into the tertiary liquid culture medium at an inoculation rate of (4-5) vol%, and then fermenting it in an anaerobic environment at 37°C for 10-14 hours; during the fermentation process, nitrogen is introduced into the fermentation tank, and 3M sodium hydroxide is added to the tertiary liquid culture medium to control the pH of the secondary liquid culture medium at 5-6.5;

[0033] S6. When the fermentation time is greater than 10 hours, take a sample of the fermentation broth and test the OD600. When the OD600 is greater than 2.0 and no longer increases significantly, it is considered the end point of the fermentation and the fermentation is stopped. The number of viable bacteria in the fermentation broth is tested.

[0034] By adopting the above technical scheme, the fermentation and cultivation process of animal Bifidobacterium lactis subspecies is optimized, which is conducive to obtaining an ideal fermentation and cultivation effect of animal Bifidobacterium lactis subspecies.

[0035] In a specific embodiment, in S3, the secondary liquid culture medium is stirred at a speed of 60-100 rpm during fermentation; in S4, the tertiary liquid culture medium is stirred at a speed of 60-100 rpm during fermentation.

[0036] A centrifugation and freeze-drying method for animal Bifidobacterium lactis subspecies, comprising:

[0037] S7, collecting bacteria: centrifuging the fermentation liquid of Bifidobacterium animalis subsp. lactis, discarding the supernatant, and obtaining bacterial mud of Bifidobacterium animalis subsp. lactis;

[0038] S8. Prepare protective agent: the protective agent is prepared and sterilized by the following raw materials in parts by weight: 4-6 parts of maltodextrin, 1-3 parts of trehalose, 2-4 parts of resistant dextrin and 0.5-1 parts of sodium D ascorbate, and the balance is purified water;

[0039] S9, vacuum freeze drying: the animal Bifidobacterium lactis subsp. lactis mud is mixed with a protective agent to prepare an emulsion; the emulsion is vacuum freeze dried until the moisture content of the bacterial powder is less than 5%, and the freeze-dried bacterial powder is crushed with a 60-mesh sieve to prepare the freeze-dried bacterial powder, and the powder is vacuum packed for storage.

[0040] By adopting the above technical solution and adding a protective agent during vacuum freeze drying, the bacterial strains can be protected from low temperature damage, thereby further enhancing the survival rate of the bacterial strains.

[0041] In a specific embodiment, the mass ratio of the animal Bifidobacterium lactis subsp. lactis slurry to the protective agent is 1:(1-2).

[0042] By adopting the above technical solution, the mass ratio of animal Bifidobacterium lactis subspecies mud to the protective agent is optimized, which is beneficial to enhancing the survival rate of lactic acid bacteria after freeze-drying.

[0043] In a specific embodiment, the animal Bifidobacterium lactis subsp. lactis centrifugation process further includes cleaning the bacterial sludge, specifically: washing the bacterial sludge obtained by pipeline centrifugation with an equal volume of sterile physiological saline, centrifuging at a speed of 3000-5000 r / min for 3-5 minutes, and discarding the supernatant to obtain Bifidobacterium lactis subsp. lactis bacterial sludge.

[0044] The animal Bifidobacterium lactis subspecies DD26 of the present invention has the function of enhancing immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a comparison chart of the tuf gene sequences of the F1 and F5 generations.

[0046] Figure 2 Cell morphology and physicochemical test results.

[0047] Figure 3 These are the results of the acid and bile salt resistance tests.

[0048] Figure 4 is the body weight of mice before and after the experiment.

[0049] Figure 5 It is the description of immune index. DETAILED DESCRIPTION

[0050] The present invention will be further described by the following embodiments and test examples.

[0051] Embodiment 1:

[0052] A kind of separation and identification of animal Bifidobacterium lactis subspecies DD26 strain:

[0053] Strain collection: The samples were collected from the feces of healthy infants in Suzhou, Jiangsu Province, and diluted with sterile physiological saline to different concentration gradients; the samples were spread on MRS plate culture medium using a spreader, and cultured under anaerobic conditions at 35-38℃ for 36-48h; single colonies with smooth and plump colonies and complete milky white edges were selected on the culture medium; microscopic examination was performed; the selected single colonies were inoculated onto MRS plate culture medium using the streak method, and cultured under anaerobic conditions at 35-38℃ for 36-48h, and single colonies were picked and placed in glycerol tubes for storage at -80℃.

[0054] Strain identification: The animal Bifidobacterium lactis subspecies DD26 strain was detected and identified by the Institute of Microbiology, Chinese Academy of Sciences.

[0055] Cell morphology and physicochemical experimental results are shown in Figure 2 .

[0056] Molecular biological identification results: the 16S rRNA sequence is shown in SEQ ID No: 1, and the tuf gene sequence is shown in SEQ ID No: 2.

[0057] Based on a comprehensive analysis of experimental data such as cell morphology, physiological and biochemical experiments, 16S rRNA gene and tuf gene sequences of the tested bacteria, and with reference to the Bergey's Manual of Systematic Bacteriology, the Institute of Microbiology, Chinese Academy of Sciences identified the DD26 strain as Bifidobacterium animalis subsp. lactis.

[0058] Embodiment 2:

[0059] A method for preparing a working strain of animal Bifidobacterium lactis subspecies, comprising the following steps:

[0060] Take a sample containing animal Bifidobacterium lactis subsp. DD26 strain, add it to the liquid activation culture medium, and culture it in an anaerobic chamber at a constant temperature of 35-38°C for 10 hours. (2) Take the above activated bacterial solution and inoculate it into the slant of the eggplant bottle; inoculate 200μL of bacterial solution on each slant of the eggplant bottle and culture it in an anaerobic chamber at 35-38°C for 20 hours. (3) Use 20ml of protective agent to scrape off the bacterial moss on the slant culture medium of the eggplant bottle, mix it evenly, and divide it into sterile screw-cap cryopreservation tubes and store it at -80°C to obtain the working strain of animal Bifidobacterium lactis subsp. DD26.

[0061] Furthermore, the liquid activation culture medium formula comprises peptone, yeast powder, glucose (monohydrate), sodium chloride, anhydrous sodium acetate, L-cysteine ​​hydrochloride (monohydrate), and purified water; the pH is adjusted to 6.8±0.1, and sterilized at 121°C for 20 minutes after packaging. The eggplant bottle slant culture medium formula comprises peptone, yeast powder, glucose (monohydrate), sodium chloride, anhydrous sodium acetate, L-cysteine ​​hydrochloride (monohydrate), purified water, and agar strips; the pH is adjusted to 6.8±0.1, and sterilized at 121°C for 20 minutes after packaging. The protective agent formula comprises skim milk powder, yeast powder, L-cysteine ​​hydrochloride (monohydrate), and purified water; the pH is adjusted to 6.5±0.1, and sterilized at 115°C for 20 minutes after packaging.

[0062] A method for preparing the animal Bifidobacterium lactis subspecies freeze-dried powder is prepared by the following steps:

[0063] A method for fermenting animal Bifidobacterium lactis subspecies, comprising:

[0064] Inoculate the strain of Bifidobacterium animalis subsp. lactis on the eggplant bottle solid culture medium for activation;

[0065] Transplanting the activated bacterial strain into a primary liquid culture medium for expansion;

[0066] Transplanting the expanded primary bacterial strain into a secondary liquid culture medium for fermentation;

[0067] Transplanting the fermented secondary bacterial strain into a tertiary liquid culture medium for fermentation, and filtering the fermentation liquid to obtain animal Bifidobacterium lactis subsp.

[0068] The activated solid culture medium uses the MRS formula.

[0069] The formula of the primary liquid culture medium is 10 / L yeast powder, 4g / L glucose monohydrate, 4g / L peptone, 2g / L sodium chloride, and 2g / L anhydrous sodium acetate. The pH is adjusted to 6.8.

[0070] The secondary liquid culture medium is formulated with 16 g / L yeast powder, 20 g / L glucose monohydrate, 16 g / L peptone, 2 g / L anhydrous sodium acetate, and 2 g / L galacto-oligosaccharide. The pH is adjusted to 6.8.

[0071] The formula of the three-level liquid culture medium is 16 g / L yeast powder, 14 g / L glucose monohydrate, 15 g / L peptone, 2 g / L anhydrous sodium acetate, and 3 g / L galacto-oligosaccharide. The pH is adjusted to 6.8.

[0072] During the fermentation process, the secondary and tertiary liquid culture media are supplemented with 3M sodium hydroxide solution; the pH of the secondary liquid culture media is 5-6.5, and the OD600 value is greater than 1.5; the pH of the tertiary liquid culture media is 5-6.5, and the OD600 value is greater than 2.

[0073] In a specific possible implementation scheme, it includes:

[0074] S1. Inoculate animal Bifidobacterium lactis subspecies DD26 on a solid culture medium at a volume ratio of 1:8, and then activate it in an anaerobic environment at 37°C for 24 hours;

[0075] S2, the activated bacteria were placed at 60cm 2 The solid culture medium was inoculated with 200 mL of liquid culture medium in the same proportion and then inoculated into the primary liquid culture medium, and then incubated in an oxygen-free environment at 37°C for 12 hours;

[0076] S3, transplanting the expanded strain into the secondary liquid culture medium at an inoculation rate of 2 vol%, and then fermenting it in an anaerobic environment at 37°C for 8 hours; during the fermentation process, nitrogen was introduced into the fermentation tank, and 3M sodium hydroxide was added to the secondary liquid culture medium to control the pH of the secondary liquid culture medium at 5-6.5;

[0077] S4, when the fermentation time is greater than 8h, the fermentation liquid is sampled and the OD600 is tested. When the OD600 is greater than 1.5, the secondary fermentation is considered to be complete.

[0078] S5, transplanting the fermented secondary strain into the tertiary liquid culture medium at an inoculation rate of 4 vol%, and then fermenting it in an anaerobic environment at 37°C for 10-14 hours; during the fermentation process, nitrogen is introduced into the fermentation tank, and 3M sodium hydroxide is added to the tertiary liquid culture medium to control the pH of the secondary liquid culture medium at 5-6.5;

[0079] S6. When the fermentation time is greater than 10 hours, take a sample of the fermentation broth and test the OD600. When the OD600 is greater than 2.0 and no longer increases significantly, it is considered the end point of the fermentation and the fermentation is stopped. The number of viable bacteria in the fermentation broth is tested.

[0080] By adopting the above technical scheme, the fermentation and cultivation process of animal Bifidobacterium lactis subspecies is optimized, which is conducive to obtaining an ideal fermentation and cultivation effect of animal Bifidobacterium lactis subspecies.

[0081] In a specific embodiment, in S3, the secondary liquid culture medium is stirred at a speed of 60-100 rpm during fermentation; in S4, the tertiary liquid culture medium is stirred at a speed of 60-100 rpm during fermentation.

[0082] A centrifugation and freeze-drying method for animal Bifidobacterium lactis subspecies, comprising:

[0083] S7, collecting bacteria: centrifuging the fermentation liquid of Bifidobacterium animalis subsp. lactis, discarding the supernatant, and obtaining bacterial mud of Bifidobacterium animalis subsp. lactis;

[0084] S8. Prepare protective agent: the protective agent is prepared and sterilized by the following raw materials in parts by weight: 4 parts of maltodextrin, 1 part of trehalose, 2 parts of resistant dextrin and 0.5 parts of sodium D ascorbate, and the balance is purified water;

[0085] S9, vacuum freeze drying: the animal Bifidobacterium lactis subsp. lactis mud is mixed with a protective agent to prepare an emulsion; the emulsion is vacuum freeze dried until the moisture content of the bacterial powder is less than 5%, and the freeze-dried bacterial powder is crushed with a 60-mesh sieve to prepare the freeze-dried bacterial powder, and the powder is vacuum packed for storage.

[0086] By adopting the above technical solution and adding a protective agent during vacuum freeze drying, the bacterial strains can be protected from low temperature damage, thereby further enhancing the survival rate of the bacterial strains.

[0087] In a specific embodiment, the mass ratio of the animal Bifidobacterium lactis subspecies slurry to the protective agent is 1:1.

[0088] By adopting the above technical solution, the mass ratio of animal Bifidobacterium lactis subspecies mud to the protective agent is optimized, which is beneficial to enhancing the survival rate of lactic acid bacteria after freeze-drying.

[0089] In a specific embodiment, the animal Bifidobacterium lactis subsp. lactis centrifugation process further includes cleaning the bacterial sludge, specifically: washing the bacterial sludge obtained by pipeline centrifugation with an equal volume of sterile physiological saline, centrifuging at a speed of 3000-5000 r / min for 3-5 minutes, and discarding the supernatant to obtain Bifidobacterium lactis subsp. lactis bacterial sludge.

[0090] The animal Bifidobacterium lactis subspecies DD26 of the present invention has the function of enhancing immunity.

[0091] Embodiment 3:

[0092] A method for preparing a working strain of animal Bifidobacterium lactis subspecies, comprising the following steps:

[0093] Take a sample containing animal Bifidobacterium lactis subsp. DD26 strain, add it to the liquid activation culture medium, and culture it in an anaerobic chamber at a constant temperature of 35-38°C for 12 hours. (2) Take the above activated bacterial solution and inoculate it into the slant of the eggplant bottle; inoculate 200μL of bacterial solution on each slant of the eggplant bottle and culture it in an anaerobic chamber at 35-38°C for 24 hours. (3) Use 20ml of protective agent to scrape off the bacterial moss on the slant culture medium of the eggplant bottle, mix it evenly, and divide it into sterile screw-cap cryopreservation tubes and store it at -80°C to obtain the working strain of animal Bifidobacterium lactis subsp. DD26.

[0094] Furthermore, the liquid activation culture medium formula comprises peptone, yeast powder, glucose (monohydrate), sodium chloride, anhydrous sodium acetate, L-cysteine ​​hydrochloride (monohydrate), and purified water; the pH is adjusted to 6.8±0.1, and sterilized at 121°C for 20 minutes after packaging. The eggplant bottle slant culture medium formula comprises peptone, yeast powder, glucose (monohydrate), sodium chloride, anhydrous sodium acetate, L-cysteine ​​hydrochloride (monohydrate), purified water, and agar strips; the pH is adjusted to 6.8±0.1, and sterilized at 121°C for 20 minutes after packaging. The protective agent formula comprises skim milk powder, yeast powder, L-cysteine ​​hydrochloride (monohydrate), and purified water; the pH is adjusted to 6.5±0.1, and sterilized at 115°C for 20 minutes after packaging.

[0095] A method for preparing the animal Bifidobacterium lactis subspecies freeze-dried powder is prepared by the following steps:

[0096] A method for fermenting animal Bifidobacterium lactis subspecies, comprising:

[0097] Inoculate the strain of Bifidobacterium animalis subsp. lactis on the eggplant bottle solid culture medium for activation;

[0098] Transplanting the activated bacterial strain into a primary liquid culture medium for expansion;

[0099] Transplanting the expanded primary bacterial strain into a secondary liquid culture medium for fermentation;

[0100] Transplanting the fermented secondary bacterial strain into a tertiary liquid culture medium for fermentation, and filtering the fermentation liquid to obtain animal Bifidobacterium lactis subsp.

[0101] The activated solid culture medium uses the MRS formula.

[0102] The formula of the primary liquid culture medium is 12.5 g / L yeast powder, 6 g / L glucose monohydrate, 6 g / L peptone, 4 g / L sodium chloride, and 4 g / L anhydrous sodium acetate. The pH is adjusted to 6.8.

[0103] The secondary liquid culture medium is formulated with 20 g / L yeast powder, 25 g / L glucose monohydrate, 20 g / L peptone, 4 g / L anhydrous sodium acetate, and 4 g / L galacto-oligosaccharide. The pH is adjusted to 6.8.

[0104] The formula of the three-level liquid culture medium is 20 g / L yeast powder, 18 g / L glucose monohydrate, 20 g / L peptone, 4 g / L anhydrous sodium acetate, and 5 g / L galacto-oligosaccharide. The pH is adjusted to 6.8.

[0105] During the fermentation process, the secondary and tertiary liquid culture media are supplemented with 3M sodium hydroxide solution; the pH of the secondary liquid culture media is 5-6.5, and the OD600 value is greater than 1.5; the pH of the tertiary liquid culture media is 5-6.5, and the OD600 value is greater than 2.

[0106] In a specific possible implementation scheme, it includes:

[0107] S1. Inoculate animal Bifidobacterium lactis subspecies DD26 on a solid culture medium at a volume ratio of 1:10, and then activate it in an anaerobic environment at 37°C for 24 hours;

[0108] S2, the activated bacteria were placed at 60cm 2 The solid culture medium was inoculated with 300 mL of liquid culture medium in the same proportion and then transferred to the primary liquid culture medium, which was then incubated at 37°C in an anaerobic environment for 12 hours.

[0109] S3, transplanting the expanded strain into the secondary liquid culture medium at an inoculation rate of 3 vol%, and then fermenting it in an anaerobic environment at 37°C for 8-12 hours; during the fermentation process, nitrogen is introduced into the fermentation tank, and 3M sodium hydroxide is added to the secondary liquid culture medium to control the pH of the secondary liquid culture medium at 5-6.5;

[0110] S4, when the fermentation time is greater than 8h, the fermentation liquid is sampled and the OD600 is tested. When the OD600 is greater than 1.5, the secondary fermentation is considered to be complete.

[0111] S5, transplanting the fermented secondary strain into the tertiary liquid culture medium at an inoculation rate of 5 vol%, and then fermenting it in an anaerobic environment at 37°C for 10-14 hours; during the fermentation process, nitrogen is introduced into the fermentation tank, and 3M sodium hydroxide is added to the tertiary liquid culture medium to control the pH of the secondary liquid culture medium at 5-6.5;

[0112] S6. When the fermentation time is greater than 10 hours, take a sample of the fermentation broth and test the OD600. When the OD600 is greater than 2.0 and no longer increases significantly, it is considered the end point of the fermentation and the fermentation is stopped. The number of viable bacteria in the fermentation broth is tested.

[0113] By adopting the above technical scheme, the fermentation and cultivation process of animal Bifidobacterium lactis subspecies is optimized, which is conducive to obtaining an ideal fermentation and cultivation effect of animal Bifidobacterium lactis subspecies.

[0114] In a specific embodiment, in S3, the secondary liquid culture medium is stirred at a speed of 60-100 rpm during fermentation; in S4, the tertiary liquid culture medium is stirred at a speed of 60-100 rpm during fermentation.

[0115] A centrifugation and freeze-drying method for animal Bifidobacterium lactis subspecies, comprising:

[0116] S7, collecting bacteria: centrifuging the fermentation liquid of Bifidobacterium animalis subsp. lactis, discarding the supernatant, and obtaining bacterial mud of Bifidobacterium animalis subsp. lactis;

[0117] S8. Prepare protective agent: the protective agent is prepared and sterilized by the following raw materials in parts by weight: 6 parts of maltodextrin, 3 parts of trehalose, 4 parts of resistant dextrin and 1 part of sodium D ascorbate, and the balance is purified water;

[0118] S9, vacuum freeze drying: the animal Bifidobacterium lactis subsp. lactis mud is mixed with a protective agent to prepare an emulsion; the emulsion is vacuum freeze dried until the moisture content of the bacterial powder is less than 5%, and the freeze-dried bacterial powder is crushed with a 60-mesh sieve to prepare the freeze-dried bacterial powder, and the powder is vacuum packed for storage.

[0119] By adopting the above technical solution and adding a protective agent during vacuum freeze drying, the bacterial strains can be protected from low temperature damage, thereby further enhancing the survival rate of the bacterial strains.

[0120] In a specific embodiment, the mass ratio of the animal Bifidobacterium lactis subspecies slurry to the protective agent is 1:2.

[0121] By adopting the above technical solution, the mass ratio of animal Bifidobacterium lactis subspecies mud to the protective agent is optimized, which is beneficial to enhancing the survival rate of lactic acid bacteria after freeze-drying.

[0122] In a specific embodiment, the animal Bifidobacterium lactis subsp. lactis centrifugation process further includes cleaning the bacterial sludge, specifically: washing the bacterial sludge obtained by pipeline centrifugation with an equal volume of sterile physiological saline, centrifuging at a speed of 3000-5000 r / min for 3-5 minutes, and discarding the supernatant to obtain Bifidobacterium lactis subsp. lactis bacterial sludge.

[0123] The animal Bifidobacterium lactis subspecies DD26 of the present invention has the function of enhancing immunity.

[0124] Embodiment 4:

[0125] A lactic acid bacteria solid beverage prepared from the animal Bifidobacterium lactis subspecies DD26, the formula is prepared from the following raw materials by weight: resistant dextrin 15 parts, oligofructose 10 parts, oligogalactose 10 parts, inulin 20 parts, trehalose 30 parts, polydextrose 10 parts, animal Bifidobacterium lactis subspecies DD26 (live bacteria 2×10 11 CFU / g) lyophilized powder 10 parts, flavoring agent 5 parts.

[0126] The solid beverage preparation method comprises the following process steps: mixing animal Bifidobacterium lactis subspecies DD26 freeze-dried powder (passed through a 60-mesh sieve), resistant dextrin, oligofructose, oligogalactose, inulin, trehalose, polydextrose, and flavoring agent uniformly, and pressing into 1g or 1.5g strips. The live bacteria content of each 1g strip is 20 billion of animal Bifidobacterium lactis subspecies DD26, and the live bacteria content of each 1.5g strip is 30 billion of animal Bifidobacterium lactis subspecies DD26.

[0127] The flavoring agent in the formula is mainly used to improve or mask the fermentation taste in the freeze-dried powder of animal bifidobacterium lactis subsp., and give the solid beverage a specific flavor. Generally, natural flavoring agents are used, including one or two of cranberry fruit extract, blueberry fruit extract or elderberry fruit extract.

[0128] The animal Bifidobacterium lactis subspecies DD26 strain of the present invention has the function of enhancing immunity. When used, it is often used with other lactic acid bacteria such as Bifidobacterium longum subspecies longum, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus casei to prepare a composite strain lactic acid bacteria agent, thereby giving the product new functions.

[0129] Test Example 5:

[0130] Animal Bifidobacterium lactis subspecies DD26 acid and bile salt resistance test:

[0131] Bifidobacterium animalis subsp. lactis DD26 (live bacteria 2×10 11 CFU / g) were inoculated at 1% (W / V) in MRS medium at pH 6.0, pH 4.0 and pH 2.0 under anaerobic conditions for 3 h, and the plate count method was used to detect live bacteria. The results are shown in the table. Figure 3 .

[0132] The experimental results showed that most of the live bacteria of animal Bifidobacterium lactis subspecies DD26 survived for 3 hours under pH 4.0, and there was still a sufficient number of live bacteria of animal Bifidobacterium lactis subspecies DD26 to survive for 3 hours under pH 2.0, indicating that most of the live bacteria of animal Bifidobacterium lactis subspecies DD26 can pass through the acidic environment of the stomach.

[0133] Bifidobacterium animalis subsp. lactis DD26 (live bacteria 2×10 11CFU / g) were inoculated with 0.1% bile salt concentration, 0.4% bile salt concentration and MRS medium without bile salt at an inoculum of 1% (W / V) and cultured under anaerobic conditions for 3 h. The plate count method was used to detect viable bacteria. The results are shown in the table. Figure 3 .

[0134] The experimental results showed that most of the live bacteria of animal Bifidobacterium lactis subspecies DD26 survived for 3 hours at a bile salt concentration of 0.1%, and some live bacteria of animal Bifidobacterium lactis subspecies DD26 still survived for 3 hours at a bile salt concentration of 0.4%, indicating that animal Bifidobacterium lactis subspecies DD26 has good bile salt tolerance.

[0135] Test Example 6:

[0136] Effect of animal Bifidobacterium lactis subspecies DD26 on body weight and immune organ indexes of immunocompromised mice:

[0137] KM mice, all male, 6 weeks old, weighing (20±2) g. The laboratory temperature was maintained at 20-22°C, the humidity was 40-60%, and the standard light cycle was 12 hours of light and 12 hours of dark. Standard laboratory diet and water were provided after autoclaving, and adaptive feeding was carried out for one week. The mice were divided into 3 groups, 20 in each group, and the weight of the mice before and after the experiment was recorded. The normal group, the control group, the lactic acid bacteria group (Bifidobacterium animalis subsp. lactis DD26, 1×10 9 CFU / d). The normal group and the control group were gavaged with sterile saline, and the lactic acid bacteria group was gavaged with 0.05g of the solid beverage prepared in Experimental Example 3, once a day for 17 consecutive days. Except for the mice in the normal group, the mice in the control group and the lactic acid bacteria group were intraperitoneally injected with 75mg / kg / d of cyclophosphamide on the 8th, 9th and 10th days. On the last day of the experiment, the mice were weighed and blood was collected from their orbits. The mice were killed by dislocation, and the thymus and spleen were weighed to calculate the spleen index and thymus index. The levels of IgG, IgA and IgM in mouse serum were determined using an immunoglobulin detection kit. The experimental results are shown in Figure 5 .

[0138] Reference Figure 4 The weight of mice before and after the experiment: on the 10th day of the experiment, after 3 consecutive days of cyclophosphamide injection, the weight of mice in the control group and lactic acid bacteria group decreased significantly compared with the normal group. On the 17th day, the weight of mice in the control group decreased significantly compared with the normal group, and the weight of mice in the lactic acid bacteria group increased significantly compared with the control group.

[0139] Reference Figure 5 The results of the mouse immune index statistics thymus index and spleen index test showed that the thymus index of the lactic acid bacteria group was significantly higher than that of the control group, but still lower than that of the normal group, indicating that lactic acid bacteria may have a certain protective effect on the thymus. The spleen index of the lactic acid bacteria group was close to that of the normal group and significantly higher than that of the control group, indicating that lactic acid bacteria may help restore spleen function.

[0140] Reference Figure 5 Mouse immune index statistics of mouse serum immunoglobulin content, lactic acid bacteria group IgG, IgA and IgM content are significantly higher than the control group, close to the normal group level, indicating that lactic acid bacteria can help restore the normal level of immunoglobulin. These results show that lactic acid bacteria has a certain immunomodulatory effect on cyclophosphamide-induced immunosuppressed mice.

[0141] The above experimental results indicate that animal Bifidobacterium lactis subspecies DD26 has good tolerance to gastric acid and bile salts, and has the function of enhancing immunity, and can be used in the development of products that enhance immune function.

Claims

1. A culture of animal Bifidobacterium lactis subspecies DD26 having the function of enhancing immunity, characterized in that: The Bifidobacterium animalis subsp. lactis DD26 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 5, 2024, with a deposit number of CGMCC No. 30870; the 16S rRNA sequence of the Bifidobacterium animalis subsp. lactis is shown in SEQ ID No: 1; the method for preparing the culture comprises: inoculating the Bifidobacterium animalis subsp. lactis DD26 strain into the following culture medium and culturing at 35-38° C. for 10-14 hours; the formula of the culture medium comprises: yeast powder, glucose monohydrate, peptone, anhydrous sodium acetate, and galacto-oligosaccharide.

2. A lactic acid bacteria agent having the function of enhancing immunity, characterized in that: The lactic acid bacteria agent having the function of enhancing immunity comprises the Bifidobacterium animalis subspecies lactis DD26 strain according to claim 1.

3. The lactic acid bacteria agent having the ability to enhance immunity according to claim 2, characterized in that: In the lactic acid bacteria agent, the viable count of the animal Bifidobacterium lactis subspecies DD26 strain is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g; the solid form of the lactic acid bacteria agent includes freeze-dried powder or granular form; the liquid form of the lactic acid bacteria agent includes drops with edible oil as a carrier.

4. The lactic acid bacteria agent having the function of enhancing immunity according to claim 2, characterized in that: The lactic acid bacteria agent with the function of enhancing immunity also contains prebiotics; the prebiotics include any one of oligofructose, oligogalactose, inulin, trehalose, resistant dextrin, and polydextrose, or a combination of two or more thereof.

Images