Bifidobacterium animalis strain, probiotic formulation, and preparation method for and use of bifidobacterium animalis strain

Abstract

A bifidobacterium animalis strain, a probiotic formulation, and a preparation method for and a use of the bifidobacterium animalis strain. The bifidobacterium animalis strain has a preservation number of CGMCCNo. 25681. The bifidobacterium animalis strain and the probiotic formulation containing same can produce extracellular polysaccharides, lactic acid and short-chain fatty acid at high yield, stimulate a mouse to generate cytokines TNF-α and IL-6, have an immunity enhancement function, improve the ink propulsion rate, and have a bowel relaxing function; and the single bacterial powder of the bifidobacterium animalis strain can significantly enhance the cellular immune function and the mononuclear-macrophage function of mice.

Description

Animal Bifidobacterium strain, probiotic preparation, preparation method and use thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 202310744238.4 filed in China on June 21, 2023, and Chinese Patent Application No. 202310744483.5 filed in China on June 21, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of microbial technology, and in particular to an animal bifidobacterium strain, a probiotic preparation, and a preparation method and use thereof. Background Art

[0004] Bifidobacterium is an important component of the human intestinal flora. Its number is closely related to human health status. It has many important physiological functions for human health, such as biological barrier, nutritional effect, anti-tumor effect, improvement of gastrointestinal function, and anti-aging.

[0005] Bifidobacterium animalis is divided into two subspecies, namely Bifidobacterium animalis subsp. animalis and Bifidobacterium animalis subsp. lactis. It has the function of maintaining the balance of intestinal microecology and has good safety.

[0006] Currently, research on the effects of animal Bifidobacterium and its probiotic preparations in improving immunity and relieving constipation is insufficient. Therefore, it is crucial to screen for a strain of animal Bifidobacterium lactis that has excellent immunity-boosting and laxative properties.

[0007] Summary of the Invention

[0008] An object of the present invention is to provide a Bifidobacterium animalis strain and a probiotic preparation thereof, wherein the strain and the probiotic preparation can produce high amounts of exopolysaccharides, lactic acid and short-chain fatty acids; improve ink propulsion rate and have a laxative function.

[0009] Another object of the present invention is to provide a strain of Bifidobacterium animalis and a probiotic preparation thereof, both of which can stimulate mouse macrophage RAW264.7 cells to produce cytokines TNF-α and IL-6, thereby improving immunity. A single bacterial powder of this strain can significantly enhance the cellular immune function and monocyte-macrophage function of mice, and the powder also has an excellent laxative effect.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] In one aspect, the present invention provides a Bifidobacterium animalis strain, the deposit number of which is CGMCC No.25681.

[0012] Preferably, the Bifidobacterium animalis strain comprises the 16S rRNA gene represented by SEQ ID NO: 1.

[0013] The animal bifidobacterium strain of the present invention is derived from breast milk, can tolerate artificial gastrointestinal fluid, can adhere to intestinal epithelial cells, and has a high survival rate in the intestine, laying a foundation for its colonization in the intestine and exerting effective functions.

[0014] In one embodiment, the animal Bifidobacterium strain has the ability to produce high amounts of exopolysaccharides, lactic acid, and short-chain fatty acids, and can inhibit a variety of pathogenic bacteria.

[0015] In one embodiment, the present invention provides a probiotic preparation comprising the Bifidobacterium animalis strain as described above.

[0016] Preferably, the probiotic preparation is a live bacteria preparation, which contains 2 to 5×10 11 CFU / g of viable bacteria.

[0017] Preferably, the probiotic preparation is a dead bacteria preparation, which contains 2 to 4×10 11 CFU / g of dead bacteria.

[0018] In one embodiment, the present invention provides a food or health product comprising the probiotic preparation described above.

[0019] In one example, the food is selected from one or more of milk powder, solid beverages, fermented dairy products, milk-containing beverages and cheese.

[0020] In one example, the fermented dairy product is selected from a fermented bovine dairy product and a fermented oat dairy product.

[0021] On the other hand, the present invention provides a method for preparing the above-mentioned animal Bifidobacterium strain, comprising growing an appropriate amount of sample in a culture medium and then subjecting the sample to artificial gastric juice and artificial intestinal juice treatment, and then isolating the sample.

[0022] In another aspect, the present invention provides use of the above-mentioned Bifidobacterium animalis strain in the preparation of a laxative medicament.

[0023] The present invention also provides use of the above-mentioned Bifidobacterium animalis strain for producing exopolysaccharides, lactic acid and short-chain fatty acids.

[0024] The present invention also provides use of the above-mentioned animal Bifidobacterium strain in the preparation of a medicine for improving immunity and maintaining immune balance.

[0025] Preferably, the Bifidobacterium animalis strain is used to produce the cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

[0026] Preferably, the single bacterial powder of animal Bifidobacterium is used to enhance the cellular immune function and monocyte-macrophage function of mice.

[0027] On the other hand, the present invention also provides a freeze-dried preparation of Bifidobacterium animalis strain, which comprises the Bifidobacterium animalis strain as described above and a freeze-drying protectant.

[0028] In one example, the freeze-drying protective agent includes: 100-150 g / L skim milk powder, 20-50 g / L sucrose, 1-2 g / L vitamin C, and 0.5-1 g / L sodium L-glutamate.

[0029] In another aspect, the present invention provides a use of the freeze-dried preparation of the animal Bifidobacterium strain as described above in the preparation of a laxative drug.

[0030] The present invention also provides a use of the above-mentioned freeze-dried preparation of the animal Bifidobacterium strain for producing extracellular polysaccharides, lactic acid and short-chain fatty acids.

[0031] The present invention also provides a use of the above-mentioned freeze-dried preparation of the animal Bifidobacterium strain in the preparation of a drug for improving immunity and maintaining immune balance; optionally, the freeze-dried preparation is used to produce the cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

[0032] In another aspect, the present invention provides a method for preparing the freeze-dried preparation of the animal Bifidobacterium strain as described above, comprising the following steps:

[0033] (1) strain culture, inoculating the animal Bifidobacterium strain in a sterile liquid culture medium at an inoculum amount of 1-3% of the total amount of the culture medium, culturing for 16 to 24 hours to obtain a seed culture solution, and then inoculating the obtained seed culture solution in a fermentation medium at an inoculum amount of 1-3% of the total amount of the culture medium, fermenting and culturing to obtain a fermentation culture solution of the animal Bifidobacterium strain; and

[0034] (2) Drying.

[0035] In one example, the fermentation medium contains: 40-60 g / L glucose, 60-100 g / L yeast extract, 3-10 g / L sodium acetate trihydrate, 0.1-0.2 g / L magnesium sulfate, 0.05-0.1 g / L manganese sulfate, 1-2 g / L potassium dihydrogen phosphate, 2-4 g / L triammonium citrate, 1-2 g / L Tween 80, 0.05-0.1 g / L calcium chloride, and 0.5-1 g / L L-cysteine ​​salt.

[0036] In another example, during the fermentation process, sodium hydroxide solution is automatically added to maintain a constant pH of 5.5-6.5 until the fermentation stops producing acid, and the fermentation is terminated when sodium hydroxide is no longer added.

[0037] In another example, the preparation is a live bacterial preparation, and the method further includes preparing a freeze-drying protective agent after the strain culture step and before drying, and performing freeze drying in the drying step; preferably, the freeze-drying protective agent used comprises: 80-100 g / L skim milk powder, 40-50 g / L trehalose, 2-3 g / L vitamin C, and 4-5 g / L L-sodium glutamate protective agent; preferably, the freeze-drying conditions are: pre-freezing temperature is -40-45°C, pre-freezing time is 4-5h, primary drying temperature is -20-15°C, primary drying time is 20-25h, secondary drying temperature is 30-35°C, and secondary drying time is 6-10h.

[0038] In another example, the preparation is a dead bacteria preparation, and the drying step is to first perform heat inactivation and then dry it in a spray drying tower to obtain the dead bacteria preparation; optionally, the heat inactivation condition is 80-100°C for 10-40 minutes. Beneficial effects

[0039] The present invention has the following advantages:

[0040] 1. The animal bifidobacterium strain and the probiotic preparation (live bacteria and dead bacteria preparation) of the present invention can produce high levels of extracellular polysaccharides, lactic acid and short-chain fatty acids; can increase the ink propulsion rate, thereby helping to relieve constipation.

[0041] 2. The animal Bifidobacterium strain and its probiotic preparation (live bacteria and dead bacteria preparation) of the present invention can stimulate mouse macrophage RAW264.7 to produce cytokines TNF-α and IL-6. The animal Bifidobacterium single bacterial powder can significantly enhance the cellular immune function and monocyte-macrophage function of mice, and can significantly enhance the body's immunity.

[0042] 3. The medicine comprising the freeze-dried preparation of animal Bifidobacterium, and the food or health product comprising the live or dead bacteria preparation of the animal Bifidobacterium strain of the present invention have the functions of relieving constipation and enhancing the body's immunity.

[0043] 4. The method of the present invention for preparing a freeze-dried preparation containing animal Bifidobacterium strains has simple production process parameters, is easy to control, has a short cycle, ensures a high survival rate of animal Bifidobacterium strains, and the obtained product can be stored for a long time with stable product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG1 shows a biological evolutionary tree of Bifidobacterium animalis subsp. lactis HOM2120 strain.

[0045] FIG2 shows a RAPD cluster analysis diagram of the Bifidobacterium animalis subsp. lactis HOM2120 strain of the present invention constructed based on the UPGMA method.

[0046] FIG3 shows the effect of the Bifidobacterium animalis subsp. lactis HOM2120 strain of the present invention on the secretion of TNF-α in mouse macrophages.

[0047] FIG4 shows the effect of the Bifidobacterium animalis subsp. lactis HOM2120 strain of the present invention on the IL-6 secretion of mouse macrophages.

[0048] FIG5 shows that the animal Bifidobacterium lactis subsp. lactis HOM2120 of the present invention enhances cellular immune function.

[0049] FIG6 shows that Bifidobacterium animalis subsp. lactis HOM2120 of the present invention enhances monocyte-macrophage function.

[0050] FIG7 shows the function of the animal Bifidobacterium lactis subspecies HOM2120 single bacterial powder of the present invention in improving the ink propulsion rate.

[0051] FIG8 shows the results of the present invention showing that the animal Bifidobacterium lactis subsp. lactis HOM2120 enhances the cellular immune function, humoral immune function, and monocyte-macrophage function in mice.

[0052] Microorganism Preservation Instructions

[0053] The animal Bifidobacterium (Bifidobacterium animalis) HOM2120 strain of the present invention was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on September 9, 2022, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC No. 25681.

[0054] The Bifidobacterium animalis HOM2120 strain of the present invention was sent to the Institute of Microbiology, Chinese Academy of Sciences for identification in May 2023.

[0055] The detection and identification conclusions are as follows: Under the conditions of this laboratory, based on a comprehensive analysis of experimental data such as the cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, and tuf gene sequence of the tested bacteria, and with reference to relevant research papers in the "Bergey's Manual of Systematic Bacteriology" and the International Journal of Systematic and Evolutionary Microbiology, the identification result of the tested bacteria (strain number: HOM2120) is: Bifidobacterium animalis subsp. lactis (Bifidobacterium animalis subsp. lactis).

[0056] The cell morphology of the strain is polymorphic rod-shaped; the physiological and biochemical characteristics are Gram-positive, catalase-negative (-), and oxidase-negative (-); the 16S rRNA gene sequence is shown in SEQ ID NO: 1, and the tuf gene sequence is shown in SEQ ID NO: 9. DETAILED DESCRIPTION

[0057] The present invention discloses strains, characteristics, and applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0058] To further illustrate the technical means and effects of the present invention to achieve the intended purpose, the following technical solutions of the present invention are further described in conjunction with specific embodiments, but are not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention that does not depart from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. Experimental methods for which specific conditions are not specified in the following implementations are carried out in accordance with conventional methods and conditions in the art.

[0059] Tumor necrosis factor-α (TNF-α) is primarily produced by activated macrophages, NK cells, and T lymphocytes, and regulates the body's immune response. When immunity is low, it can activate various immune cells, including lymphocytes and macrophages, enhancing their killing activity and exerting anti-infection and anti-tumor effects.

[0060] Interleukin-6 (IL-6) is primarily produced by monocytes and macrophages, Th2 cells, vascular endothelial cells, and fibroblasts, and regulates the body's immune response. IL-6 can transform B cell precursors into antibody-producing cells. It also works synergistically with colony-stimulating factor to promote the growth and differentiation of primitive bone marrow-derived cells and enhance the lytic function of natural killer cells.

[0061] As identified above, the Bifidobacterium animalis HOM2120 strain in the present invention is identified as Bifidobacterium animalis subsp. lactis. Therefore, the Bifidobacterium animalis HOM2120 strain used in the present invention refers to the Bifidobacterium animalis subsp. lactis HOM2120 strain.

[0062] In order to make the technical problems to be solved, the technical solutions adopted and the advantages of the present invention clearer, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0063] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified.

[0064] Unless otherwise specified, the reagents and materials used in the present invention were prepared using conventional methods or obtained from commercial channels.

[0065] The Bifidobacterium animalis subsp. lactis strain used in the present invention is isolated from healthy infant feces, and the specific isolation method is as described in Example 1.

[0066] Example

[0067] Example 1 Isolation and identification of Bifidobacterium animalis subspecies lactis HOM2120

[0068] (1) Preparation of artificial gastrointestinal fluid

[0069] Artificial gastric juice: Take 16.4 mL of dilute hydrochloric acid (1 mol / L), add 800 mL of water, adjust the pH to 3.0, add 10 g of pepsin, shake well, add water to 1000 mL, centrifuge at 5000 rpm for 5 min, take the supernatant, filter with 0.22 μm filter membrane for sterilization, and store at -20°C for later use.

[0070] Artificial intestinal fluid: Take 6.8 g of potassium dihydrogen phosphate, add 500 mL of water, and adjust the pH to 6.8 with 0.4% (0.1 mol / L) sodium hydroxide solution. Take 10 g of pancreatic enzyme and 3 g of porcine bile salt, add appropriate amount of water to dissolve them, mix the two liquids, add water to 1000 mL, centrifuge at 5000 rpm for 5 min, take the supernatant, filter and sterilize with a 0.22 μm filter membrane, and store at -20°C for later use.

[0071] (2) Preparation of separation culture medium

[0072] MRS-Cys liquid culture medium: Add 0.5 g of L-cysteine ​​hydrochloride per liter of MRS broth medium (catalog number: CM1163, OXOID, UK), sterilize at 121°C for 15 min, set aside, and store at 2-8°C in the dark for one week.

[0073] MRS-Cys solid medium containing bromocresol purple: Add 0.04 g of bromocresol purple per liter of MRS solid medium (Cat. No. CM1175, OXOID, UK) and stir thoroughly. Stir at 121°C for 15 min. Pour the medium into a clean plate in a laminar flow hood, pour approximately 15 mL into each culture dish, and allow to solidify before use.

[0074] (3) Isolation and screening of Bifidobacterium animalis subsp. lactis HOM2120 strain

[0075] The Bifidobacterium animalis subsp. lactis of the present invention is isolated from the feces of healthy infants.

[0076] Use sterile anaerobic tubes to collect in vitro feces and store them at low temperature under anaerobic environment. The experiment started on the day of sampling. Take about 1g of fecal sample and add it to anaerobic tube containing 9mL of MRS-Cys liquid culture medium. Incubate it anaerobically at 37℃ for 24h, centrifuge it at 8000rpm for 10min, discard the supernatant, add 10mL of artificial gastric juice, mix it, incubate it anaerobically at 37℃ for 3h, centrifuge it at 8000rpm for 10min, discard the supernatant, add 10mL of artificial intestinal juice, mix it, incubate it anaerobically at 37℃ for 3h, dilute the sample to 10 by 10-fold dilution method.-6 , from stock solution to 10 -6 100 μL of the solution was taken in sequence and spread on MRS-Cys solid culture medium plates containing bromocresol purple, and cultured anaerobically at 37° C. for 72 h.

[0077] Pick a single colony with yellowing edges, streak culture, and purify 3-4 times until the colony is single. Perform Gram staining and observe colony morphology under a microscope. Transfer the single colony to liquid culture medium for pure culture, preserve the seed in glycerol, and store at -80°C.

[0078] (4) Morphological observation of Bifidobacterium animalis subsp. lactis HOM2120 strain

[0079] Bifidobacterium animalis subsp. lactis HOM2120 was cultured anaerobically at 37°C for 48 hours on MRS agar medium to produce round, milky white colonies with a moist, smooth surface, a raised center, and neat edges. The colonies were approximately 2.5 mm in diameter. Under an optical microscope, the bacteria appeared as rods, clubs, or Y-shaped rods with swollen ends. The bacteria measured approximately 0.5-0.9 μm x 3-6 μm, were arranged singly or in pairs, were Gram-positive, and did not form spores.

[0080] (5) Identification of Bifidobacterium animalis subsp. lactis HOM2120

[0081] 16S rRNA gene identification: DNA was extracted from the preserved strains and 16S rRNA gene amplification was performed using universal primers 27F: 5'-AGTTTGATCMTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3' (see Table 1) for PCR amplification and agarose gel electrophoresis. The gel was then excised, recovered, and sequenced. The isolated strains were subjected to 16S rRNA gene sequencing. Based on the 16S rRNA gene sequence, the BLAST tool was used to compare the strains in the NCBI database, and a biological evolutionary tree was drawn using Mega 7.0 software. As shown in Figure 1, the identification result was Bifidobacterium animalis subsp. lactis, named HOM2120. The 16S rRNA gene sequence is shown in SEQ ID NO: 1:

[0082] (6) Random amplified polymorphic DNA (RAPD) strain identification: DNA was extracted from the preserved strains. Using the strain DNA as a template, five primers, OPA-02: TGC CGA GCT G; OPA-18: AGG TGA CCG T; OPL-07: AGG CGG GAA C; OPL-16: AGG TTG CAG G; and OPM-05: GGG AAC GTG T, were used to amplify DNA fragments that could show polymorphisms by PCR. After gel electrophoresis, different DNA differences were observed. Cluster analysis software was used for analysis, and the results are shown in Figure 2. As shown in Figure 2, animal Bifidobacterium lactis subsp. lactis HOM2120 is different from some commercial animal Bifidobacterium lactis subsp. lactis strains and is unique.

[0083] Table 1

[0084] (7) Genome resequencing: DNA was extracted from the preserved strain and randomly fragmented. DNA fragments of the desired length were recovered by electrophoresis, and adapters were added for cluster preparation. The fragments were then sequenced on an Illumina platform. Data analysis and assembly were performed, and the GC content was determined to be 60.44%. Using comparative genomics methods, the resequenced whole genome of Bifidobacterium animalis subsp. lactis HOM2120 was compared with the reference genome of Bifidobacterium animalis subsp. lactis published on the NCBI website for single nucleotide polymorphism (SNP) analysis. The results are shown in Table 2.

[0085] Table 2 SNP analysis results of Bifidobacterium animalis subsp. lactis HOM2120

[0086] As shown in Table 2, the SNP differences between animal Bifidobacterium lactis subsp. lactis HOM2120 and other animal Bifidobacterium lactis subsp. lactis reference genomes are no less than 34. The results show that animal Bifidobacterium lactis subsp. lactis HOM2120 is well distinguished from other Bifidobacteria and is unique.

[0087] Example 2 Test on the ability to inhibit common pathogenic bacteria

[0088] (1) Activation of indicator bacteria

[0089] The indicator strains Escherichia coli ATCC8739, Staphylococcus aureus ATCC6538, Salmonella typhimurium ATCC14028, Pseudomonas aeruginosa ATCC9027, Listeria monocytogenes ATCC19111, and Clostridium difficile ATCC9689 were all purchased from the China Industrial Microorganism Collection Center. Indicator bacteria (Escherichia coli ATCC8739; Staphylococcus aureus ATCC6538; Salmonella typhimurium ATCC14028; Pseudomonas aeruginosa ATCC9027; Listeria monocytogenes ATCC19111) were inoculated into TSB medium at an inoculum size of 1% of the total culture volume and cultured aerobically at 37°C, 180 rpm, for 24 hours before use. Clostridium difficile ATCC9689 was inoculated into BHI medium at an inoculum size of 1% of the total culture volume and cultured anaerobically at 37°C for 24 hours before use.

[0090] (2) Activation of Bifidobacterium animalis subsp. lactis

[0091] The -80°C-stored Bifidobacterium animalis subsp. lactis HOM2120 and a control strain prepared in Example 1 were inoculated at 1% of the total culture volume into sterilized MRS-Cys liquid culture medium described in Example 1. The culture was anaerobically incubated at 37°C for 24 hours and activated twice to obtain a fermentation broth. The culture was then centrifuged at 8000 rpm for 10 minutes, and the supernatant was used for antibacterial testing.

[0092] (3) Plate preparation

[0093] Heat the sterilized TSA medium until completely melted, pour it into a petri dish, place it on a horizontal surface to form an even agar layer, and wait for it to solidify. Add the indicator bacteria to the TSA medium, shake it evenly, then pour it into a pre-prepared TSA blank agar plate and let it stand to solidify.

[0094] (4) Antibacterial experiment

[0095] Using sterile tweezers, gently place the Oxford cup on a flat plate, maintaining a certain distance between them. Add 0.2 mL of the lactic acid bacteria fermentation broth supernatant to each sample. Allow the mixture to diffuse in a 4°C refrigerator for 24 hours. Then, incubate in a 37°C incubator for at least 18 hours and observe for the appearance of an inhibition zone. Once the inhibition zone forms, measure with a ruler. Using the liquid culture medium (MRS-Cys) from Example 1 as a negative control, perform three replicates for each sample. The inhibition results are shown in Table 3.

[0096] Table 3 Inhibitory effect of animal Bifidobacterium lactis subspecies HOM2120 on pathogenic bacteria Note: “–” no antibacterial activity; “+” 11-16mm; “++” 17-22mm; “+++” ≥ 23mm

[0097] As shown in Table 3, animal Bifidobacterium lactis subspecies HOM2120 has inhibitory effects on all six pathogenic bacteria. Compared with the other two strains of animal Bifidobacterium lactis subspecies, it has better antibacterial effects on Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa and Clostridium difficile, indicating that it has a better ability to inhibit pathogenic bacteria.

[0098] Example 3 Gastrointestinal transit capacity test

[0099] (1) Activation of strains

[0100] The animal Bifidobacterium lactis subsp. lactis HOM2120 and the control strain prepared in Example 1 and stored at -80°C were inoculated at an inoculum rate of 1% of the total culture medium into the sterilized MRS-Cys liquid culture medium described in Example 1, and cultured anaerobically at 37°C for 24 hours. After activation twice, the fermentation broth of the strain was obtained.

[0101] (2) Preparation of artificial gastric juice

[0102] Take 16.4 ml of dilute hydrochloric acid, add about 800 ml of water and 10 g of pepsin, shake well, adjust the pH to 3.0, add water to 1000 ml, filter with a 0.2 μm microporous membrane and set aside.

[0103] (3) Preparation of artificial intestinal fluid

[0104] Take 6.8g of potassium dihydrogen phosphate, add 500mL of water to dissolve, adjust the pH to 6.8, add 10g of trypsin and 3g of porcine bile salt, mix the two solutions after dissolution, add water to 1000mL, filter with a 0.22um sterile filter membrane under sterile environment, and set aside.

[0105] (4) Evaluation of the survival ability of strains in the simulated gastrointestinal tract

[0106] Take 1 mL of activated bacterial suspension of the strain to be tested and add the cells to 9 mL of artificial gastric fluid (pH 3.0). Mix thoroughly and count the viable cells. Place the culture in a 37°C incubator for 3 hours and then count the viable cells. After 3 hours of incubation in artificial gastric fluid, transfer all cells to an equal volume of artificial intestinal fluid (pH 6.8), mix thoroughly, and incubate at 37°C. Count the viable cells on plates using MRS medium at 3 and 24 hours. Calculate the survival rate using the following formula:

[0107] Gastric juice 3-hour survival rate (%) = [logCFUN1 / logCFUN0] × 100%

[0108] 3-hour survival rate in intestinal fluid (%) = [logCFUN2 / logCFUN0] × 100%

[0109] 24-hour survival rate in intestinal fluid (%) = [logCFUN3 / logCFUN0] × 100%

[0110] N0 = number of live bacteria of animal Bifidobacterium lactis subsp. lactis before treatment, N1 = number of live bacteria of animal Bifidobacterium lactis subsp. lactis 3 hours after treatment with gastric juice, N2 = number of live bacteria of animal Bifidobacterium lactis subsp. lactis 3 hours after treatment with intestinal juice, N3 = number of live bacteria of animal Bifidobacterium lactis subsp. lactis 24 hours after treatment with intestinal juice.

[0111] The survival rates of Bifidobacterium animalis subsp. lactis HOM2120 in simulated gastrointestinal fluid are shown in Table 4 .

[0112] Table 4 Note: The numbers with different capital letters in the same column are significantly different (p<0.01).

[0113] As shown in Table 4, after 3 hours of treatment with simulated gastric fluid, the survival rate of Bifidobacterium animalis subsp. lactis HOM2120 exceeded 98%. After 24 hours of treatment with simulated intestinal fluid, its survival rate remained above 91%. Its intestinal fluid tolerance was significantly (p<0.01) superior to that of the other two strains of Bifidobacterium animalis subsp. lactis. This indicates that Bifidobacterium animalis subsp. lactis HOM2120 has a high survival rate in the intestine, laying the foundation for its colonization and effective function in the intestine.

[0114] Example 4 Intestinal epithelial cell adhesion ability test

[0115] (1) Preparation of cell culture medium

[0116] Complete culture medium: high-glucose DMEM medium supplemented with 10% inactivated fetal bovine serum (FBS) and 1% (v / v) antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin), mixed and stored at 4°C.

[0117] Incomplete culture medium: high-glucose DMEM medium to which 10% inactivated fetal bovine serum (FBS) was added, mixed, and stored at 4°C.

[0118] (2) Cell recovery and culture

[0119] Human colorectal adenocarcinoma Caco-2 cells were purchased from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences. The cells were resuspended in fresh culture medium and evenly dispersed in a culture flask. The cells were cultured at 37°C in an atmosphere of 5% CO2 and 95% air. The culture medium was replaced every 48 hours during recovery. When the cells were well grown (80% confluence), the Caco-2 cells were digested with trypsin-EDTA solution at 37°C. After digestion, the cell concentration was adjusted to 2 × 10 5 The cells were seeded into 24-well plates and cultured until the confluence reached 80%.

[0120] (3) Activation of Bifidobacterium animalis subsp. lactis

[0121] The animal Bifidobacterium lactis subsp. lactis HOM2120 prepared in Example 1 and stored at -80°C was inoculated into the sterilized MRS-Cys liquid culture medium described in Example 1 at an inoculum amount of 1% of the total culture medium, and anaerobically cultured at 37°C for 24 hours. After activation twice, the fermentation broth of the strain was obtained.

[0122] (4) Adhesion test

[0123] The cells grown in the corresponding culture medium were collected by centrifugation at 8000 rpm for 10 min; the cells were washed three times with DPBS and resuspended in incomplete culture medium, and the cell concentration was adjusted to 10 8 cfu / mL; 1 mL of the above bacterial suspension was added to a 24-well plate containing Caco-2 cells grown to a monolayer and incubated at 37°C in a 5% CO2 incubator for 2 h; after incubation, the cells were washed three times with sterile DPBS; the Caco-2 cells were digested with trypsin-EDTA solution at 37°C, and the cell number and the number of viable bacteria before and after adhesion were counted. Three parallels were made for each sample. The results are shown in Table 5.

[0124] Table 5 Adhesion ability of Bifidobacterium animalis subsp. lactis HOM2120 to Caco-2 cells Note: The numbers with different capital letters in the same column are extremely significantly different (p<0.01), and the numbers with different lowercase letters are significantly different (p<0.05)

[0125] The results showed that the adhesion rate of animal Bifidobacterium lactis subspecies HOM2120 to human colon cancer cells Caco-2 was 3.78 and the adhesion index was 3.29, which were better than the other two strains of animal Bifidobacterium lactis subspecies and had a better ability to adhere to intestinal epithelial cells.

[0126] Adhesion index = number of bacteria after adhesion / number of cells per dish

[0127] Adhesion rate = number of bacteria after adhesion / number of bacteria before adhesion

[0128] Example 5 Extracellular polysaccharide (EPS) production capacity test

[0129] The animal Bifidobacterium lactis subsp. HOM2120 prepared in Example 1 and stored at -80°C was inoculated into the sterilized MRS-Cys liquid culture medium described in Example 1 at an inoculum size of 1% of the total culture medium. The culture was placed in an anaerobic state at 37°C for 24 hours and activated twice to obtain a fermentation liquid. The supernatant was then centrifuged at 8000rpm for 10 minutes and the content of exopolysaccharides was detected by the phenol-sulfuric acid method. The MRS-Cys liquid culture medium described in Example 1 was a negative control. Three replicates were performed for each sample. The data were analyzed using SPSS software for single-factor ANOVA. The results are shown in Table 6.

[0130] Table 6 Extracellular polysaccharide (EPS) production capacity of Bifidobacterium animalis subsp. lactis HOM2120 Note: The numbers with different capital letters in the same column are significantly different (p<0.01).

[0131] It can be seen that compared with other animal Bifidobacterium lactis subspecies, animal Bifidobacterium lactis subspecies HOM2120 has a stronger ability to produce extracellular polysaccharides (EPS).

[0132] Example 6 Lactic acid and short-chain fatty acid production capacity test

[0133] The animal bifidobacterium lactis subspecies HOM2120 of-80 ℃ of freezing preservations prepared among the embodiment 1 was accessed in the sterilized MRS-Cys liquid nutrient medium described in the embodiment 1 with an inoculum size accounting for 1% of the total culture medium amount, and 37 ℃ were left to stand for anaerobic cultivation for 24 hours, and the bacterial strain fermentation liquid was obtained after activation twice. Then centrifugal 10min under the 8000rpm condition was used, and the content of lactic acid and short-chain fatty acids was detected by gas chromatograph of the supernatant. The MRS-Cys liquid nutrient medium described in the embodiment 1 was a negative control, and other 2 strains of animal bifidobacterium lactis subspecies were as positive controls, and 3 parallels were made for each sample, and data adopted SPSS25.0 software to carry out one-way analysis of variance (ANOVA) and Duncan's multiple comparisons, and the results are shown in Table 7.

[0134] Table 7 Lactic acid and short-chain fatty acid production capacity of Bifidobacterium animalis subsp. lactis HOM2120 Note: The numbers with different capital letters in the same column are significantly different (p<0.01).

[0135] As shown in Table 7, compared with other Bifidobacterium animalis subsp. lactis, Bifidobacterium animalis subsp. lactis HOM2120 has a stronger ability to produce lactic acid and short-chain fatty acids (acetic acid, formic acid, propionic acid, butyric acid and isobutyric acid).

[0136] Example 7 In vitro cytokine secretion test

[0137] The animal Bifidobacterium lactis subsp. lactis HOM2120 prepared in Example 1 and stored at -80°C was inoculated into the sterilized MRS-Cys liquid culture medium described in Example 1 at an inoculum rate of 1% of the total culture medium. The culture was anaerobically cultured at 37°C for 24 hours and activated for two generations for viable counts and cell experiments. The cells were collected by centrifugation at 8000 rpm for 10 minutes and then adjusted to a working concentration of approximately 5.0 × 10 5 CFU / mL) was the live bacteria group. After the bacteria were collected, they were heated at 100°C for 30 min, and then the bacterial count was adjusted to about 5.0×10 5 CFU / mL is the low-dose group with dead bacteria, about 5.0×10 6 CFU / mL is the dead bacteria medium dose group, about 5.0×10 7 CFU / mL was the high-dose group of killed bacteria; mouse macrophage RAW264.7 (about 5×10 5 4.After 2h attachment, discard culture medium, every hole adds 1mL containing bacteria culture medium; Establish blank control group, add 1mL DMEM culture medium; Collect cell supernatant after cultivating together 24h, adopt enzyme-linked immunosorbent assay (ELISA), according to the content of TNF-α and IL-6 in the test kit specification sheet measuring cell supernatant.Be negative control with the MRS-Cys liquid nutrient medium described in embodiment 1, the BH13-25 and BH13-33 bifidobacterium lactis subspecies viable bacteria of same dosage are as positive control, and each sample is done 3 parallels, and each sample is done 3 parallels, and data adopt GraphPad prism9 software to carry out one-way variance (ANOVA) and Dunnett's t test i.e. multiple experimental groups and a control group between mean two two comparative analysis, the results are shown in Figure 3 and Figure 4.

[0138] Figures 3 and 4 show that compared with Bifidobacterium animalis subsp. lactis BH13-25 and BH13-33, both live and killed strains of Bifidobacterium animalis subsp. lactis HOM2120 significantly (p<0.001) increased TNF-α and IL-6 secretion in RAW264.7 cells. At the same dose, live strains were superior to killed strains; immune activity increased with increasing dose. This suggests that Bifidobacterium animalis subsp. lactis HOM2120 has potential immunomodulatory abilities.

[0139] Example 8 Antibiotic Sensitivity Test

[0140] The antimicrobial susceptibility test was carried out according to the ISO10932-2010 Milk and dairy products. Antibiotic minimum inhibitory concentration (MIC) determination method for bifidobacteria and non-enterococcal lactic acid bacteria (LAB) developed by the International Organization for Standardization.

[0141] (1) Culture medium preparation

[0142] MRS-Cys liquid culture medium: same as Example 1

[0143] LSM-Cys liquid medium: Weigh 21.06 g of Iso-Sensitest medium (Cat. No. CM0473B, OXOID, UK), 5.2 g of MRS Broth, and 0.3 g of L-cysteine ​​hydrochloride, add water to 0.5 L, adjust the pH to 6.85 ± 0.1, sterilize at 121°C for 15 min (the pH should be 6.7 ± 0.1), set aside, and store in the dark at 2-8°C for one week.

[0144] (2) Activation and proliferation of Bifidobacterium animalis subsp. lactis

[0145] The -80°C frozen Bifidobacterium animalis subsp. lactis HOM2120 prepared in Example 1 was inoculated into the sterilized liquid culture medium (MRS-Cys) described in Example 1 at an inoculum size of 1% of the total culture medium, incubated anaerobically at 37°C for 24 hours, and activated twice to obtain a fermentation broth. The activated Bifidobacterium animalis subsp. lactis HOM2120 was propagated on MRS-Cys agar medium and incubated anaerobically at 37°C for 48 hours.

[0146] (3) Preparation of antibiotic microdilution plates

[0147] Weigh 0.0512 g of antibiotics and add 10 mL of solvent. Dissolve chloramphenicol and erythromycin in ethanol (without filtration), ampicillin in phosphate buffer (pH 8.0, 0.1 mol / L), and other antibiotics in water. After vortexing, filter through a 0.22 μm filter and aliquot into EP tubes at a concentration of 5120 μg / mL (5.12 mg / mL). Store at -20°C until needed. Dilute the antibiotic stock solution with water (phosphate buffer for ampicillin) to the appropriate concentration range. Add 50 μL of the dilution to the wells of a microdilution plate.

[0148] (4) Preparation of bacterial suspension

[0149] Pick up a single colony from the agar plate. Suspend the colony in a sterile culture tube filled with 2 mL to 5 mL of sterile saline. Suspend the colony in pre-reduced LSM-Cys liquid medium. Suspend the colony until the turbidity of the solution reaches McFarland standard 1 or the optical density at 625 nm is 0.16 to 0.2 using a spectrophotometer. The suspension is approximately equivalent to 3 × 10 8 CFU / mL. Dilute the bacterial suspension with the recommended culture medium. Dilute the bacterial suspension 500-fold with MRS-Cys liquid medium because the antibiotic solution will dilute the culture medium twice. Dispense the diluted bacterial suspension within 30 minutes of preparation. When using frozen plates, thaw the frozen antibiotic solution under anaerobic conditions immediately before use. Dispense 50 μL of the diluted suspension into each well of the microdilution plate (approximately 3×10 4 CFU / well). Incubate the plates at 37°C under static anaerobic conditions for 48 hours. When using anaerobic jars, cover the plates with lids between each plate to create a uniform environment within the jar. Each experiment is repeated three times, with both positive and negative control groups. Positive control wells contain no antibiotic but instead contain the test strain and culture medium containing the solvent containing the highest concentration of antibiotic. Negative control wells contain no test strain or antibiotic but instead contain culture medium.

[0150] (5) Read the MIC result

[0151] After 48 hours of incubation, read the MIC visually. After incubation, inspect the negative control wells for visible bacterial growth. If contamination is observed, reject all data generated for that strain. Note: Lack of growth in the positive control wells indicates that the strain being tested is sensitive to the solvent used to dissolve the antibiotic. In this case, reading the MIC for that particular antibiotic is meaningless. If the negative and positive controls are normal, determine bacterial growth visually for each antibiotic by comparison with the positive control. Ideally, place the microdilution plate on top of a magnifying glass rack with a bench lamp providing indirect light for easy reading. Bacterial growth is easily detected under a magnifying glass as a pellet at the bottom of the well. Discard any series of wells where discontinuous growth is observed (e.g., growth at 16 μg / mL and 64 μg / mL, but no growth at 32 μg / mL). The endpoint is defined as the lowest antibiotic concentration at which no growth is observed visually. This concentration is the MIC for that antibiotic for that particular strain. Three replicates are performed for each sample, with Lactobacillus plantarum ATCC14917 serving as the positive control strain. The antibiotic susceptibility results for animal Bifidobacterium lactis subsp. lactis HOM2120 are shown in Table 8. As can be seen from Table 8, the MIC results for Lactobacillus plantarum ATCC14917 are consistent with those shown in the appendix to ISO 10932-2010, demonstrating that this experimental method is accurate. According to the 2012 European Food Safety Authority (EFSA) standards for antibiotic resistance of bacteria used in food, animal Bifidobacterium lactis subsp. lactis HOM2120 is sensitive to erythromycin, chloramphenicol, vancomycin, ampicillin, clindamycin, streptomycin, and kanamycin. Therefore, animal Bifidobacterium lactis subsp. lactis HOM2120 products are relatively safe.

[0152] Table 8 Antibiotic sensitivity results of Bifidobacterium animalis subspecies lactis HOM2120

[0153] Example 9 Preparation process 1 of active bacterial powder of animal Bifidobacterium lactis subspecies HOM2120

[0154] (1) Culture of bacterial strains

[0155] The animal bifidobacterium lactis subspecies HOM2120 prepared in Example 1 at -80°C cryopreservation was inoculated into sterile MRS-Cys liquid medium at an inoculum size of 1% of the total culture medium, and cultured at 37°C for 16-24 hours. The activated seed culture liquid was obtained by passage culture twice. The seed culture liquid was inoculated into fermentation medium M416 at an inoculum size of 1% of the total culture medium, and cultured at 37°C for anaerobic conditions at a speed of 50 rpm. During the fermentation, sodium hydroxide solution was added automatically to maintain a constant pH of 5.0. The culture was fermented to a tank under jars for 16-24 hours to obtain a high-density culture liquid of animal bifidobacterium lactis subspecies HOM2120, with a viable count of up to 22.4 billion CFU / mL. The fermentation medium M416 formula is shown in Table 9.

[0156] Table 9 Fermentation medium M416 formula

[0157] (2) Preparation of lyophilization protective agent

[0158] Sterile water was mixed with the protective agent raw materials to prepare a protective agent containing 100 g / L skim milk powder, 50 g / L sucrose, 2 g / L vitamin C, and 1 g / L L-cysteine ​​salt.

[0159] (3) Freeze-drying

[0160] The fermented bacterial liquid of animal Bifidobacterium lactis subsp. HOM2120 after culturing was centrifuged at 2-8°C and 6500 rpm for 15 min, the supernatant was discarded, the bacterial sludge was collected, and the bacterial sludge was washed 1-2 times with 0.9% sterile saline, and the washed bacterial sludge was mixed with the above protective agent to make the bacterial concentration of the mixed bacterial liquid reach 10 10 CFU / mL or above, freeze-dry in a freeze dryer, pre-freeze at -40 degrees Celsius for 4 hours, vacuumize, raise the temperature to -12 degrees Celsius, dry for 22 hours, raise the temperature to 35 degrees Celsius, and dry for 7 hours. After freeze-drying, use a fine grinder to crush the cake to obtain the freeze-dried powder. The number of viable bacteria in the freeze-dried powder reaches 4.3×10 11 CFU / g.

[0161] Example 10 Preparation process 2 of active bacterial powder of animal Bifidobacterium lactis subspecies HOM2120

[0162] (1) Culture of bacterial strains

[0163] The animal Bifidobacterium lactis subsp. HOM2120 prepared in Example 1 and stored frozen at -80°C was inoculated into sterile MRS-Cys liquid medium at an inoculum size of 3% of the total medium amount, cultured at 37°C for 16-24 hours, and subcultured twice to obtain an activated seed culture liquid. The seed culture liquid was inoculated into fermentation medium M453 at an inoculum size of 3% of the total medium amount, cultured anaerobically at 35°C at a rotation speed of 50 rpm, and sodium hydroxide solution was automatically added during the fermentation process to maintain a constant pH of 5.5. The fermentation was continued for 16-24 hours to obtain a high-density culture liquid of animal Bifidobacterium lactis subsp. HOM2120, with a viable count of up to 15.3 billion CFU / mL.

[0164] The formula of fermentation medium M453 is shown in Table 10.

[0165] Table 10

[0166] (2) Preparation of lyophilization protective agent

[0167] Sterile water was mixed with the protective agent raw materials to prepare a protective agent containing 150 g / L skim milk powder, 20 g / L sucrose, 1 g / L vitamin C, and 0.5 g / L sodium L-glutamate.

[0168] (3) Freeze-drying

[0169] The fermented bacterial liquid of animal Bifidobacterium lactis subsp. HOM2120 after culturing was centrifuged at 2-8°C and 6500 rpm for 15 min, the supernatant was discarded, the bacterial sludge was collected, and the bacterial sludge was washed 1-2 times with 0.9% sterile saline, and the washed bacterial sludge was mixed with the above protective agent to make the bacterial concentration of the mixed bacterial liquid reach 10 10 CFU / mL or above, freeze-dry in a freeze dryer, pre-freeze at -35 degrees Celsius for 5 hours, vacuumize, raise the temperature to -10 degrees Celsius, dry for 15 hours, raise the temperature to 30 degrees Celsius, and dry for 10 hours. After freeze-drying, crush the cake with a fine grinder to obtain the freeze-dried powder. The number of viable bacteria in the freeze-dried powder reaches 3.7×10 11 CFU / g.

[0170] Example 11 Preparation process of animal Bifidobacterium lactis subspecies HOM2120 dead bacteria powder

[0171] (1) Culture of bacterial strains

[0172] Bacteria culture as in Example 9 or Example 10

[0173] (2) Preparation of bacterial powder

[0174] The fermentation liquid of Bifidobacterium animalis subsp. lactis HOM2120 obtained by high-density fermentation was treated at 100°C for 10 minutes, and 100g of maltodextrin was added per liter of fermentation liquid. The powder was dried in a spray dryer. The number of dead bacteria counted by blood cell counting plate under a microscope reached 2.2×10 11 CFU / g.

[0175] Example 12 Animal Test on Immunity Enhancement

[0176] (1) Experimental animals and grouping: 192 18g-20g SPF grade KM female mice bred by Beijing Huafukang Biotechnology Co., Ltd. were selected and randomly divided into four batches after animal adaptation and observation, with 4 groups in each batch and 12 mice in each group. The experimental group was divided into three dose groups, the low dose group (5×10 9 CFU / Kg BW, medium dose group (2.5×10 10 CFU / Kg BW) and high-dose group (5×10 10 CFU / kg BW) were administered orally daily with the active bacterial powder of Bifidobacterium animalis subsp. lactis HOM2120 prepared in Example 9, using normal saline as the solvent. Various parameters were measured by oral gavage for 32 consecutive days. The mice were gavaged at a volume of 10 mL / kg BW. The control group received a high-dose of the bacterial powder supplemented with normal saline solution of equal mass. All dose groups were fed a maintenance feed. Experimental group 1 underwent a carbon clearance experiment; experimental group 2 underwent organ / body weight ratio determination and delayed-type hypersensitivity experiments; experimental group 3 underwent an experiment on phagocytosis of chicken erythrocytes by mouse peritoneal macrophages; and experimental group 4 underwent a ConA-induced mouse lymphocyte transformation experiment.

[0177] (2) Experimental methods

[0178] A. Determination of organ-to-body weight ratio

[0179] The mice were weighed and killed by cervical dislocation. The spleen and thymus were removed, the fascia was removed, and the blood stains on the surface of the organs were absorbed with filter paper. The mice were weighed, and the spleen / body weight ratio and thymus / body weight ratio were calculated.

[0180] B. Delayed-type hypersensitivity (DTH) test (plantar thickening method)

[0181] Sheep blood was collected and washed three times with saline. Each mouse was then injected intraperitoneally with 0.2 mL of a 2% (v / v, prepared with saline) sheep red blood cell (SRBC) suspension (2000 rpm, 10 minutes). Four days after sensitization, the thickness of the left hind paw was measured. Then, 20 μL of a 20% (v / v, prepared with saline) SRBC suspension was injected subcutaneously at the measurement site. The thickness of the left hind paw was measured 24 hours after injection. Both measurements were performed three times at the same site, and the average was taken. The difference in paw thickness before and after challenge was used to indicate the degree of DTH. If the difference in the test sample group was significantly higher than that in the control group, the test result was considered positive.

[0182] C. ConA-induced mouse lymphocyte transformation experiment (MTT method)

[0183] Remove the spleen aseptically and place it in a small dish containing an appropriate amount of sterile Hank's solution. Use forceps to gently grind the spleen to prepare a single cell suspension. Filter through a 200-mesh sieve to prepare a cell suspension. Wash twice with Hank's solution, centrifuging each time for 5 minutes (1000 rpm). Then suspend the cells in 1 ml of complete culture medium and count them under a microscope. Adjust the cell concentration to 3×10 6 The spleen cell suspension was then divided into two wells of a 24-well culture plate, 1.0 mL per well. 75 μL of ConA solution (equivalent to 7.5 μg / mL) was added to one well, while the other well served as a control. The cells were incubated in a CO2 incubator at 37°C in a 5% CO2 atmosphere for 72 hours. Four hours before the end of the incubation period, 0.7 mL of supernatant was gently aspirated from each well, and 0.7 mL of RPMI-1640 culture medium without calf serum was added. MTT (5 mg / mL) was also added at 50 μL / well, and the cells were incubated for another 4 hours. After the incubation period, 1.0 mL of acidic isopropanol was added to each well and mixed thoroughly by pipetting to completely dissolve the purple crystals. This solution was then transferred to a cuvette and colorimetrically measured on a spectrophotometer at a wavelength of 570 nm. The proliferation capacity of lymphocytes was determined by subtracting the optical density of the well without ConA from the optical density of the well with ConA. A positive result was determined if the difference in optical density between the test sample group and the control group was significantly higher.

[0184] D. Determination of Antibody-Producing Cells (PFC) (Jerne Modified Slide Method)

[0185] Sheep blood was collected and washed three times with saline. Each mouse was then intraperitoneally injected with 0.2 mL of a 2% (v / v, prepared in saline) SRBC suspension. Five days after SRBC immunization, mice were sacrificed by cervical dislocation. The spleens were removed and placed in a small dish containing an appropriate amount of sterile Hank's solution. The spleens were gently ground to prepare a cell suspension. The cells were centrifuged (1000 rpm) for 5 minutes, washed twice with Hank's solution, and finally suspended in 8.0 mL of Hank's solution. After dissolving the agarose by heating, mix it with an equal amount of 2x Hank's solution and dispense it into small test tubes, 0.5 mL per tube. Add 50 μL of a 10% (v / v) SRBC suspension prepared with SA buffer and 8 μL of a spleen cell suspension to the tubes. Mix quickly and pour the mixture onto a glass slide coated with a thin layer of agarose. Create parallel sections. After the agar solidifies, place the slide horizontally on a slide rack and incubate in a CO2 incubator at 37°C for 1 hour. Then, add complement diluted in SA buffer (1:8) into the grooves of the slide rack. After a further 1.5 hours of incubation, count the hemolytic plaques. This is expressed as the number of plaques per total splenocyte count. If the number of plaques in the test sample group is significantly higher than that in the control group, the test result is considered positive.

[0186] E. Determination of half hemolysis value (HC50)

[0187] Sheep blood was collected and washed three times with saline. Each mouse was immunized with 0.2 mL of a 2% (v / v, prepared in saline) SRBC suspension intraperitoneally. Five days later, the eyeballs were enucleated and blood was collected in a centrifuge tube. The tube was allowed to stand for approximately 1 hour, and the coagulated blood was separated from the tube wall to allow the serum to fully precipitate. The tube was centrifuged at 3000 rpm for 10 minutes, and the serum was collected. The serum was diluted 300-fold with SA buffer, and 1.0 mL was placed in a test tube. 0.5 mL of a 10% (v / v, prepared in SA buffer) SRBC suspension and 1.0 mL of complement (diluted 1:8 with SA buffer) were then added. A control tube without serum was also prepared (SA buffer was used instead). The tube was incubated in a 37°C water bath for 15 minutes, and the reaction was terminated by ice bath. The tube was centrifuged at 2000 rpm for 10 minutes, and 1.0 mL of the supernatant was collected and 3.0 mL of Hb diluent was added. Simultaneously, take 0.25 mL of a 10% (v / v, prepared in SA buffer) SRBC suspension and add Hb diluent to 4.0 mL in a separate test tube. Mix thoroughly and allow to stand for 10 minutes. Then, measure the optical density of each tube at 540 nm, using a control tube as a blank. The amount of hemolysin is expressed as the hemolysis value at half maximum (HC50), calculated as follows: HC50 = Optical density of sample / Optical density of SRBC at half maximum hemolysis × dilution factor. If the HC50 of the test sample group is significantly higher than that of the control group, the test result is considered positive.

[0188] F. Carbon clearance experiment in mice

[0189] Inject 4 times diluted India ink (0.05mL / 10gBW) into the tail vein of the mouse according to body weight. Start the timer immediately after the ink is injected. 2 minutes and 10 minutes after the ink injection, take 20μL of blood from the epicanthus venous plexus and add it to 2.0mL of 0.1% Na2CO3 solution. Measure the optical density (OD) at a wavelength of 600nm using a spectrophotometer, and use 0.1% Na2CO3 solution as a blank control. Sacrifice the mouse, and weigh the liver and spleen. The phagocytic index (a) is used to represent the carbon clearance ability of the mouse. Calculate a according to the following formula:

[0190] k = (1gOD1 - 1gOD2) / (t2 - t1) a = body weight ÷ (liver weight + spleen weight) × k 1 / 3

[0191] The phagocytic index of the test sample group was significantly higher than that of the control group, and the experimental result was determined to be positive.

[0192] G. Experiment on phagocytosis of chicken red blood cells by mouse peritoneal macrophages (ex vivo method)

[0193] Mice were intraperitoneally injected with 1 mL of 20% (v / v, prepared with normal saline) chicken red blood cell suspension (2000 r / min, 10 min). After 30 min, the mice were killed by cervical dislocation and fixed on a mouse board in a supine position. 2 mL of normal saline was injected intraperitoneally and the abdomen was gently massaged 20 times. 1 mL of peritoneal macrophage wash was taken and dropped onto 2 glass slides respectively. The slides were placed in an enamel box lined with wet gauze and moved to a 37°C incubator for incubation for 30 min. After incubation, the slides were rinsed in normal saline to remove unattached cells. The slides were dried, fixed with a 1:1 acetone-methanol solution, stained with Giemsa-phosphate buffer, and then rinsed with distilled water and dried. The macrophages were counted under an oil immersion lens, and 100 macrophages were counted per slide. The phagocytic rate and phagocytic index were calculated according to the following formula:

[0194] Phagocytic percentage (%) = number of macrophages phagocytosing chicken red blood cells / number of macrophages counted × 100% Phagocytic index = total number of chicken red blood cells phagocytosed / number of macrophages counted

[0195] The obtained phagocytosis percentage is then converted into data according to the following formula: Where P is the phagocytosis percentage, expressed as a decimal. The data obtained are quantitative data. The phagocytosis percentage and phagocytosis index of the test sample group are significantly higher than those of the control group, which can be considered a positive result of the experiment.

[0196] H. Determination of NK cell activity (lactate dehydrogenase LDH assay)

[0197] The target cells YAC-1 were subcultured 24 h before the experiment. Before use, they were washed 3 times with Hank's solution, and the cell concentration was adjusted to 4×10 5 cells / mL with RPMI1640 complete culture medium containing 10% calf serum. The experimental mice were sacrificed by cervical dislocation. The spleen was aseptically removed and made into a spleen cell suspension, which was washed 2 times with Hank's solution, and centrifuged at 1000 r / min for 10 min each time. The supernatant was discarded, the cell mass was resuspended, 0.5 mL of sterilized water was added for 20 s, and after lysing red blood cells, 0.5 mL of 2-fold Hank's solution and 8.0 mL of Hank's solution were added, centrifuged at 1000 r / min for 10 min, resuspended with 1.0 mL of RPMI1640 complete culture medium containing 10% calf serum, counted under a microscope, and the cell concentration was adjusted to 2×10 7 cells / mL with RPMI1640 complete culture medium. The effector-to-target ratio was 50∶1. 100 μL of target cells and effector cells were each taken and added to a U-shaped 96-well culture plate; 100 μL of target cells and culture medium were added to the natural release wells of target cells, and 100 μL of target cells and 1% NP40 were added to the maximum release wells of target cells; three parallel wells were set for each of the above items. They were placed in a carbon dioxide incubator and cultured at 37℃ and 5% CO2 for 4 h. The 96-well plate was centrifuged at 1500 r / min for 5 min, 100 μL of the supernatant from each well was aspirated and placed in a flat-bottom 96-well culture plate, 100 μL of LDH substrate solution was added, reacted for 10 min, and then 30 μL of 1 mol / L HCl solution was added to each well to terminate the reaction. The optical density value (OD) was measured at 490 nm with an enzyme-labeled instrument. Calculate the NK cell activity: NK cell activity (%) = (OD of reaction wells - OD of natural release wells) / (OD of maximum release wells - OD of natural release wells) × 100%

[0198] The obtained NK cell activity was subjected to data conversion according to the following formula, where P is the NK cell activity, expressed as a decimal. The obtained data are measurement data. The NK cell activity of the test sample group is significantly higher than that of the control group, and the positive result of this experiment can be determined.

[0199] (3) Data processing

[0200] SPSS software was used for data processing. Analysis of variance was adopted, but the homogeneity of variance test should be carried out first according to the procedure of analysis of variance. If the variances are homogeneous, the F value is calculated. When the F value < F0.05, the conclusion is that there is no significant difference among the means of each group; when the F value ≥ F0.05 and P ≤ 0.05, the Duncan multiple comparison method was used for statistics; for non-normal or heterogeneous variance data, appropriate variable transformation was carried out. After meeting the requirements of normality or homogeneity of variance, the transformed data were used for statistics; if the purpose of normality or homogeneity of variance was still not achieved after variable transformation, the rank sum test was used for statistics.

[0201] (4) Experimental results

[0202] Table 11 shows the differences in body weight of mice in the high-, medium-, and low-dose groups of Bifidobacterium animalis subsp. lactis HOM2120 and the control group before and after the experiment.

[0203] Table 11 Note: P>0.05 compared with negative control group

[0204] The results in Table 11 show that compared with the control group, there was no significant difference in body weight of mice in the high, medium and low dose groups of Bifidobacterium lactis HOM2120 before and after the experiment (p>0.05), which indicates that the active bacterial powder of Bifidobacterium lactis HOM2120 has no effect on the body weight of mice.

[0205] Table 12 shows the effect of animal Bifidobacterium lactis subsp. HOM2120 powder on the organ / body ratio of mice

[0206] Table 12 Note: P>0.05 compared with the negative control group

[0207] As can be seen from the results in Table 12, compared with the control group, there were no significant differences in the spleen / body and thymus / body of mice in the high, medium and low dose groups of active bacterial powder of animal Bifidobacterium lactis subspecies HOM2120 (p>0.05), which indicates that the active bacterial powder of animal Bifidobacterium lactis subspecies HOM2120 has no effect on the weight of the spleen and thymus of mice.

[0208] Table 13 and Figure 5 show the effects of animal Bifidobacterium lactis subsp. lactis HOM2120 powder on sheep red blood cell (SRBC)-induced DTH in mice and on ConA-induced proliferation of mouse spleen lymphocytes.

[0209] Table 13 Note: *p<0.05 compared with the control group, **p<0.01 compared with the control group, ***p<0.001 compared with the control group.

[0210] As shown in Table 13 and Figure 5, compared with the control group, the high-dose group of active bacterial powder of animal Bifidobacterium lactis subsp. lactis HOM2120 significantly increased the degree of thickening of the paw pads of mice induced by sheep red blood cells (p < 0.05); the low-dose group of active bacterial powder significantly increased the degree of thickening of the paw pads of mice induced by sheep red blood cells (p < 0.01). The high-dose group of active bacterial powder of animal Bifidobacterium lactis subsp. lactis HOM2120 significantly increased the proliferation capacity of mouse spleen lymphocytes induced by ConA (p < 0.001). This indicates that the active bacterial powder of animal Bifidobacterium lactis subsp. lactis HOM2120 significantly enhances the cellular immune function of mice.

[0211] Table 14 shows the effect of Bifidobacterium animalis subsp. lactis HOM2120 powder on the number of antibody-producing cells and serum half-hemolysis value

[0212] Table 14

[0213] The results in Table 14 show that compared with the control group, there was no significant difference in the number of hemolytic plaques in mice in each dose group of Bifidobacterium animalis subsp. lactis HOM2120 active bacterial powder (p>0.05), and there was no significant difference in the half-maximal hemolysis value of mice serum (p>0.05).

[0214] Table 15 and Figure 6 show the effects of animal Bifidobacterium lactis subsp. lactis HOM2120 powder on carbon clearance capacity and the phagocytic rate and phagocytic index of chicken erythrocytes by mouse macrophages.

[0215] Table 15 Note: *p<0.05 compared with G1 group, **p<0.01 compared with G1 group, ***p<0.001 compared with G1 group

[0216] The results in Table 15 and Figure 6 show that compared with the control group, both the low- and high-dose active bacterial powder groups of Bifidobacterium animalis subsp. lactis HOM2120 significantly improved the carbon clearance ability of mice (p < 0.01); the medium-dose group significantly improved the carbon clearance ability of mice (p < 0.05). Both the low- and high-dose active bacterial powder groups significantly increased the phagocytic rate (p < 0.01) and phagocytic index (p < 0.01) of chicken erythrocytes by mouse macrophages; the medium-dose group significantly increased the phagocytic rate (p < 0.05) and phagocytic index (p < 0.05) of chicken erythrocytes by mouse macrophages. This indicates that the active bacterial powder of Bifidobacterium animalis subsp. lactis HOM2120 significantly enhances the function of mouse monocyte-macrophage cells.

[0217] Table 16 shows the effect of animal Bifidobacterium lactis subsp. lactis HOM2120 powder on NK cell activity assay

[0218] Table 16 Note: P>0.05 compared with the negative control group

[0219] As shown in the results of Table 16, compared with the control group, there was no significant difference in the NK cell activity of mice in each dose group of Bifidobacterium animalis subsp. lactis HOM2120 (p>0.05).

[0220] In summary, as shown in Figure 8, compared with the control group, active bacterial powder of Bifidobacterium animalis subsp. lactis HOM2120 significantly enhanced the cellular immune function and monocyte-macrophage function of mice, while having no effect on the body weight, thymus and spleen weights, humoral immune function, or NK cell activity of mice. In summary, active bacterial powder of Bifidobacterium animalis subsp. lactis HOM2120 has the function of enhancing immunity.

[0221] Example 13 Laxative Function Animal Test

[0222] (1) Experimental animals and grouping: A total of 36 18g-20g SPF grade KM male mice bred by Beijing Huafukang Biotechnology Co., Ltd. were selected and randomly divided into four groups of 12 mice each after animal adaptation and feeding observation for small intestine animal test; the experimental group was the single bacterial powder group (animal Bifidobacterium lactis subsp. lactis HOM2120, dosed at 5×10 10 CFU / Kg BW).

[0223] (2) Experimental methods

[0224] The mice were gavaged daily with normal saline as the solvent, and were orally administered once a day. After 15 consecutive days of gavage, the mice in each group were fasted for 16 hours but not water. The model control group and the experimental group were gavaged with compound diphenoxylate (5 mg / Kg BW), and the blank control group was gavaged with distilled water. 0.5 hours after the administration of compound diphenoxylate, each sample dose group was given ink (containing 5% activated carbon and 10% gum arabic) containing the corresponding test sample, and the blank control group and the model control group were gavaged with ink. After 25 minutes, the animals were immediately killed by cervical dislocation, the intestinal cavity was opened to separate the mesentery, and the intestinal tube from the pylorus at the upper end to the ileocecal region at the lower end was cut and placed on a tray. The small intestine was gently pulled into a straight line, and the intestinal length was measured as the "total length of the small intestine", and the length from the pylorus to the ink front was the "ink propulsion length". The ink propulsion rate was calculated according to the following formula:

[0225] Ink propagation rate (%) = ink propagation length (cm) / total length of small intestine (cm) × 100%.

[0226] (3) Data processing

[0227] The ink propagation rate required data conversion: X = Sin-1√p (p is the ink propagation rate, expressed as a decimal). Data were processed using SPSS software. Independent sample t-tests were used. Statistical results were divided into two parts: 1. Levenes' test for homogeneity of variance; 2. T-test for equality of means. When the homogeneity of variance test result (Sig) > 0.05, the variances were considered homogeneous, and the Sig value in the first column of the t-test was used. When Sig ≤ 0.05, the statistical results were considered different between the two groups. When the homogeneity of variance test result (Sig) ≤ 0.05, the variances were considered unequal, and the Sig value in the second column of the t-test was used. When Sig ≤ 0.05, the statistical results were considered different between the two groups.

[0228] (4) Experimental results

[0229] Table 17 and Figure 7 show the effect of animal Bifidobacterium lactis subsp. HOM2120 single bacterial powder on ink propulsion rate

[0230] Table 17 Note: # Compared with the blank control group, p<0.05, ## Compared with the blank control group, p<0.01, ### Compared with the blank control group, p<0.001. *Compared with the model control group, p<0.05, **Compared with the model control group, p<0.01, ***Compared with the model control group, p<0.001.

[0231] As shown in the result of Table 17 and Fig. 7, after the test substance 15d of the single bacterial powder of Bifidobacterium lactis HOM2120 of mice was orally administered, mice constipation model was caused with compound diphenoxylate. Compared with the blank control group, the Chinese ink propulsion rate of model control group decreased, and the p value showed that there was significant difference, which showed that the model was successfully established. Compared with the model control group, the Chinese ink propulsion rate of Bifidobacterium lactis HOM2120 single bacterial powder group of Bifidobacterium lactis HOM2120 improved, and the p value showed that there was significant difference, which illustrates that the single bacterial powder of Bifidobacterium lactis HOM2120 of animals has laxative function potential.

[0232] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

[0233] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the details shown and described herein.

Claims

1. A strain of Bifidobacterium animalis, the deposit number of which is CGMCC No.25681.

2. The animal Bifidobacterium strain according to claim 1, wherein The Bifidobacterium animalis strain comprises the 16S rRNA gene represented by SEQ ID NO:

1.

3. A probiotic preparation comprising the Bifidobacterium animalis strain according to claim 1.

4. The probiotic preparation according to claim 3, wherein The probiotic preparation is a live bacteria preparation, and the live bacteria preparation contains 2 to 5×10 11 CFU / g of live bacteria.

5. The probiotic preparation according to claim 3, wherein The probiotic preparation is a dead bacteria preparation, and the dead bacteria preparation contains 2 to 4×10 11 CFU / g of dead bacteria.

6. A food or health product comprising the probiotic preparation according to any one of claims 3 to 5.

7. The food or health product according to claim 6, wherein: The food is selected from one or more of milk powder, solid beverage, fermented dairy product, milk-containing beverage and cheese.

8. The food or health product according to claim 7, wherein: The fermented dairy product is selected from the group consisting of fermented cow's dairy products and fermented oat dairy products.

9. Use of the animal Bifidobacterium strain according to claim 1 in the preparation of a laxative medicine.

10. Use of the animal Bifidobacterium strain according to claim 1 for producing exopolysaccharides, lactic acid and short-chain fatty acids.

11. Use of the animal Bifidobacterium strain according to claim 1 in the preparation of a drug for improving immunity and maintaining immune balance; Optionally, the Bifidobacterium animalis strain is used to produce the cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

12. The use according to claim 11, wherein The single bacterial powder of animal bifidobacterium is used for enhancing the cellular immune function and the mononuclear-macrophage function of mice.

13. A freeze-dried preparation of Bifidobacterium animalis, comprising the Bifidobacterium animalis according to claim 1 and a freeze-drying protectant.

14. The animal Bifidobacterium strain freeze-dried preparation according to claim 12, wherein The freeze-drying protective agent comprises: 100-150 g / L skim milk powder, 20-50 g / L sucrose, 1-2 g / L vitamin C, and 0.5-1 g / L L-sodium glutamate protective agent.

15. A method for preparing the freeze-dried preparation of animal Bifidobacterium strain according to claim 13, comprising the following steps: (1) strain culture, inoculating the animal Bifidobacterium strain in a sterile liquid culture medium at an inoculum amount of 1-3% of the total amount of the culture medium, culturing for 16 to 24 hours to obtain a seed culture solution, and then inoculating the obtained seed culture solution in a fermentation medium at an inoculum amount of 1-3% of the total amount of the culture medium, fermenting and culturing to obtain a fermentation culture solution of the animal Bifidobacterium strain; and (2) Drying.

16. The method according to claim 15, wherein: The fermentation medium comprises: 40-60 g / L of glucose, 60-100 g / L of yeast extract, 3-10 g / L of sodium acetate trihydrate, 0.1-0.2 g / L of magnesium sulfate, 0.05-0.1 g / L of manganese sulfate, 1-2 g / L of dipotassium hydrogen phosphate, 2-4 g / L of triammonium citrate, 1-2 g / L of Tween 80, 0.05-0.1 g / L of calcium chloride, and 0.5-1 g / L of L-cysteine ​​salt.

17. The method according to claim 15, wherein: During the fermentation process, sodium hydroxide solution is automatically added to maintain a constant pH of 5.5-6.5 until the fermentation stops producing acid, and the fermentation is terminated when sodium hydroxide is no longer added.

18. The method according to claim 15, wherein: The preparation is a live bacteria preparation, and the method further comprises preparing a lyophilization protective agent after the strain culturing step and before drying, and freeze-drying is performed in the drying step; preferably, the lyophilization protective agent used comprises: 80-100 g / L skim milk powder, 40-50 g / L trehalose, 2-3 g / L vitamin C, and 4-5 g / L L-sodium glutamate protective agent; preferably, the lyophilization conditions are: pre-freezing temperature of -40--45°C, pre-freezing time of 4-5 h, primary drying temperature of -20--15°C, primary drying time of 20-25 h, secondary drying temperature of 30-35°C, and secondary drying time of 6-10 h.

19. The method according to claim 15, wherein: The preparation is a dead bacteria preparation, and the drying step is to first perform heat inactivation and then dry it in a spray drying tower to obtain the dead bacteria preparation; optionally, the heat inactivation condition is 80-100°C for 10-40 minutes.

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