Bifidobacterium bifidum strain with high osmotic tolerance and application thereof

Abstract

The application discloses a high-osmotic-pressure-resistant bifidobacterium bifidum and application thereof, and belongs to the technical field of microorganisms. The application screens a bifidobacterium bifidum CCFM1301 which can resist an osmotic pressure of 1600 mOsm / kg. The fermentation density of the strain cultured in a 5L tank can reach (9.47+ / -0.30) x 10 9 CFU / mL, the viable bacterial number of the bacterial powder after freeze-drying can reach (4.02+ / -0.13) x 10 11 CFU / g, and the freeze-drying survival rate can reach (76.33+ / -0.31)%, so the strain has an industrial application prospect.

Description

Technical Field

[0001] This invention relates to a hyperosmolar-resistant Bifidobacterium bifidum and its applications, belonging to the field of microbial technology. Background Technology

[0002] Bifidobacterium bifidum is one of the most important probiotics among lactic acid bacteria. In the industrial production of lactic acid bacteria or the production of products such as acids and amino acids, high-density cultivation is a crucial method to increase probiotic biomass and substrate conversion rates to meet industrialization needs. However, in high-density production, large amounts of carbon and nitrogen sources are typically added under constant pH conditions. The accumulation of high-concentration metabolites and byproducts increases the osmotic pressure of the fermentation system, ultimately leading to cell cessation and a viable cell count in the fermentation broth of <5 × 10⁻⁶. 9 CFU / mL.

[0003] Due to the inherent characteristics of lactic acid bacteria, the bacteria are prone to losing activity during production, transportation, storage, and consumption, affecting the stability of the product. Vacuum freeze-drying technology can maintain high biological activity and stability of lactic acid bacteria inoculants during long-term storage. However, during this process, as cells continuously lose water, the electrolyte concentration in the unfrozen portion of the solution increases, causing damage to the cell structure and physiological functions, leading to the loss of activity and some functions, affecting the growth density of the bacteria after rehydration, and ultimately resulting in bacterial cell lysis. Furthermore, because Bifidobacterium fermentation can enhance the flavor of food, it is widely used in fermented foods, such as soy sauce, pickles, and cheese. To inhibit the growth of harmful microorganisms and extend shelf life, large amounts of salt or sugar are usually added. High concentrations of sugar or salt create a high osmotic pressure environment outside the cells, causing cell shrinkage or even death. Since cells can balance the additional lactose and sucrose concentrations, the high osmotic stress exerted by sugar is only transient osmotic stress, while high salt concentrations create long-term osmotic stress on the bacteria, which is a major environmental stress encountered during food fermentation. In foods, pharmaceuticals, or cosmetics containing probiotics, it is required that the microorganisms maintain a high quantity and activity in the final product. In such cases, it is necessary to reduce the water activity (A). w This helps bacteria survive and for product storage. Increased osmotic pressure due to reduced moisture can affect the physiological functions, survival ability, and expression of beneficial properties of probiotics.

[0004] During high-density fermentation, freeze-drying, and use of Bifidobacteria, osmotic stress can harm the bacteria. However, current research is limited to improving the bacteria's resistance to osmotic stress during fermentation and freeze-drying by modifying fermentation methods, culture conditions, and the types and conditions of cryoprotectants. Little attention is paid to improving the bacteria's inherent osmotic pressure tolerance. Patent CN103266076A reports a space-mutated Bifidobacterium bifidum strain S7-T5 that can tolerate the high osmotic pressure caused by a medium supplemented with 15% NaCl. However, under these high osmotic pressure conditions, Bifidobacterium bifidum S7-T5 only exhibits growth potential and its high-density fermentation performance is not disclosed. Zhang Ying et al. obtained a strain with even higher osmotic tolerance, CCFM16-1300, through successive subculturing of a highly osmotic-tolerant Bifidobacterium bifidum strain CCFM16. Although the obtained strain showed improved osmotic tolerance, its growth was inhibited, failing to meet the requirements of high-density fermentation in industrial production. Therefore, screening strains with strong osmotic pressure resistance and capable of high-density fermentation is an urgent problem to be solved in order to realize the industrialization of strains. Summary of the Invention

[0005] This invention provides a strain of Bifidobacterium bifidum CCFM1301, which was deposited on February 16, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:63175, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] The *Bifidobacterium bifidum* CCFM1301 strain was isolated from infant fecal samples from Shanghai. Sequencing analysis of the strain and alignment of its 16S rDNA sequence with NCBI confirmed that the strain is *Bifidobacterium bifidum*. According to the classification of the genus *Bifidobacterium* in Bergey's Manual of Systematic Bacteriology, *Bifidobacterium bifidum* belongs to the phylum Actinobacteria, class Actinobacteria, subclass Actinobacteridae, order Bifidoacteriales, family Bifidoobacteriaceae, and genus *Bifidobacterium*. The species *Bifidobacterium bifidum* is named *Bifidobacterium bifidum* CCFM1301.

[0007] The colonies of Bifidobacterium bifidum CCFM1301 on MRS solid medium were small, white, and opaque.

[0008] The present invention also provides a microbial preparation containing Bifidobacterium bifidum CCFM1301.

[0009] In one embodiment of the present invention, the microbial preparation is a liquid or solid preparation containing Bifidobacterium bifidum CCFM1301.

[0010] In one embodiment of the present invention, the microbial preparation contains a viable count of Bifidobacterium bifidum CCFM1301 ≥ 1.21 × 10⁻⁶. 11 CFU / mL or ≥4.02×10 11 CFU / g.

[0011] In one embodiment of the present invention, the microbial preparation further contains sorbitol and glycine.

[0012] The present invention also provides a method for preparing the microbial preparation, wherein Bifidobacterium bifidum CCFM1301 is inoculated into a high-density culture medium for fermentation.

[0013] In one embodiment of the present invention, the high-density culture medium comprises tryptone, glucose, cysteine, MgSO4·7H2O, and Tween 80.

[0014] Preferably, the high-density culture medium comprises 37 g / L tryptone, 55 g / L glucose, 1 g / L cysteine, 1.25 g / L MgSO4·7H2O, and 1 mL / L Tween 80.

[0015] In one embodiment of the present invention, the fermentation is a high-density fermentation, wherein the inoculum amount is 3-5% (v / v), the fermentation temperature is 30-40°C, the pH is 5.5-6.5, and the fermentation time is 16-18 hours.

[0016] The present invention also provides the application of Bifidobacterium bifidum CCFM1301, or a microbial preparation thereof, or a method for preparing a microbial preparation containing Bifidobacterium bifidum CCFM1301 in the high-density fermentation preparation of Bifidobacterium bifidum or products containing Bifidobacterium bifidum, the products including food, pharmaceuticals, and cosmetics.

[0017] Beneficial effects

[0018] (1) The present invention screened out a strain of Bifidobacterium bifidum CCFM1301. This strain is resistant to hyperosmolarity and can withstand an osmotic pressure of 1600 mOsm / kg, that is, the high osmotic pressure caused by the culture medium with 3.6% NaCl solution added.

[0019] (2) The Bifidobacterium bifidum CCFM1301 selected in this invention was inoculated into a high-density fermentation medium and fermented for 18 hours to achieve a fermentation density of (9.47±0.30)×10⁻⁶. 9 CFU / mL.

[0020] (3) The Bifidobacterium bifidum CCFM1301 screened in this invention exhibits good resistance to osmotic stress during freeze-drying, with a freeze-drying survival rate of (76.33±0.31)%, which can be efficiently industrialized.

[0021] Preservation of biological materials

[0022] A strain of Bifidobacterium bifidum (CCFM1301), taxonomically named Bifidobacterium bifidum, was deposited on February 16, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO:63175), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0023] Figure 1 Colony characteristics of Bifidobacterium bifidum CCFM1301.

[0024] Figure 2 Growth curves and generation times of Bifidobacterium bifidum CCFM1301 under different osmotic pressures.

[0025] Figure 3 Growth curves and generation times of Bifidobacterium bifidum CCFM16 under different osmotic pressures. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0027] The peptone, beef extract, glucose, sodium acetate, diammonium citrate, agar powder, casein peptone, magnesium sulfate heptahydrate, manganese sulfate monohydrate, Tween 80, and cysteine ​​salts involved in the following examples were purchased from Shanghai Chuangsai Technology Co., Ltd.

[0028] The culture media involved in the following examples are as follows:

[0029] MRS solid medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, agar 20 g / L, cysteine ​​0.5 g / L.

[0030] MRS liquid culture medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, cysteine ​​0.5 g / L.

[0031] MRS medium for osmotic pressure adjustment: Osmotic pressure is adjusted by adding NaCl to MRS liquid medium. Adding 3g of NaCl to 1L of MRS liquid medium results in an average osmotic pressure increase of 100 mOsm / kg. (The osmotic pressure of MRS liquid medium is 300 mOsm / kg.)

[0032] High-density fermentation medium: tryptone 37 g / L, glucose 55 g / L, cysteine ​​1 g / L, MgSO4·7H2O 1.25 g / L, Tween 80 1 mL / L.

[0033] The detection methods involved in the following embodiments are as follows:

[0034] Determination of viable lactic acid bacteria count:

[0035] The national standard GB 4789.35-2016, "National Food Safety Standard: Microbiological Examination of Food - Examination of Lactic Acid Bacteria," was adopted.

[0036] Determination of the survival rate of lyophilized lactic acid bacteria:

[0037] The freeze-dried survival rate of Bifidobacterium was calculated using the following formula:

[0038]

[0039] Glucose content detection: Measured using a glucose assay kit (product number: Beyotime S0201M).

[0040] Osmotic pressure testing: using The freezing point osmotic pressure was measured using a type of freezing point osmotic pressure meter.

[0041] Rehydration conditions: After lyophilization, remove the corresponding sample and rehydrate it with room temperature physiological saline to the original volume before lyophilization.

[0042] HPLC conditions for proline detection: Mobile phase A (pH = 7.2): 27.6 mmol / L sodium acetate-triethylamine-tetrahydrofuran (v / v 500:0.11:2.5); Mobile phase B (pH = 7.2): 80.9 mmol / L sodium acetate-methanol-acetonitrile (v / v 1:2:2). Agilent Hypersil ODS column (5 μm, 4.0 mm x 250 mm); gradient elution was used with the following program: 0 min, 8% B; 17 min, 50% B; 20 min, 100% B; 24 min, 0% B; mobile phase flow rate: 1.0 mL / min; column temperature: 40°C; UV detector (VWD) wavelength: 262 nm.

[0043] ICPMS detection K + The instrument conditions were optimized using a 1 μg / L tuning solution. The optimized parameters were: RF power: 1320 W; carrier gas flow rate: 1.15 L / min; sampling depth: 7 mm; S / C temperature: 2 °C; peristaltic pump speed: 0.1 r / min; sampling cone and retrieval cone type: nickel cone; sample lifting speed: 0.50 r / min; number of repeated samplings: 3.

[0044] Example 1: Screening and identification of Bifidobacterium bifidum CCFM1301

[0045] 1. Screening

[0046] Using infant feces from Shanghai as samples, sterile saline was serially diluted 10-fold to 10000. -6 Then take 100 μL of each diluted 10. -4 10 -5 10 -6 The diluted solution was plated on MRS solid medium and incubated upside down at 37°C for 48 hours. Colony morphology was observed and recorded. Colonies of different morphologies on the MRS solid medium were streaked and isolated. After incubation at 37°C for 48 hours, single colonies of different morphologies on the MRS solid medium were streaked and isolated again until pure single colonies with consistent morphology were obtained. Single colonies on the MRS solid medium were inoculated into 5 ml MRS liquid medium and incubated at 37°C for 18 hours. Then, 5% of the inoculum was inoculated into MRS liquid medium with osmotic pressures of 700, 1000, 1300, and 1600 mOsm / kg, respectively, to screen for strains with high osmotic tolerance, resulting in strain CCFM1301.

[0047] 2. Identification

[0048] The genome of strain CCFM1301 was extracted, and the 16S rDNA of strain CCFM1301 was amplified and sequenced (by Invesco Great Trading Co., Ltd.). The sequence was compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Bifidobacterium bifidum, and it was named Bifidobacterium bifidum CCFM1301.

[0049] 3. Save

[0050] Single colonies of Bifidobacterium bifidum (CCFM1301) were picked and inoculated into MRS liquid medium and cultured at 37℃ for 18 h to obtain bacterial suspension. 1 mL of bacterial suspension was taken into a sterile centrifuge tube, centrifuged at 8000 r / min for 3 min, and the upper culture medium was discarded. The bacterial sludge was resuspended in 30% glycerol solution and stored at -80℃.

[0051] Example 2: Permeability, culture and freeze-drying of Bifidobacterium bifidum CCFM1301

[0052] The specific steps are as follows:

[0053] (1) Preparation of Bifidobacterium seed culture: Bifidobacterium bifidum CCFM1301 obtained in Example 1 was streaked on MRS solid medium and cultured at 37℃ for 36h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and cultured at 37℃ for 24h to obtain seed culture.

[0054] (2) The prepared seed solution was inoculated into an Erlenmeyer flask containing MRS liquid culture medium at an inoculation rate of 5% (v / v) and cultured anaerobically at 37°C for 24 h to obtain the culture medium.

[0055] (3) The culture medium prepared in step (2) was inoculated into 5 mL MRS liquid medium with osmotic pressures of 300, 700, 1000, 1300, and 1600 mOsm / kg at an inoculation rate of 5% (v / v). The medium was then anaerobic at 37°C for 24 h, and samples were taken every 2 h to measure OD. 600 Once the stabilization period is reached, the generation time is calculated. The results are shown in Table 1.

[0056] (4) Prepare high-density fermentation medium. Place 3L of fermentation medium in a 5L triple in-situ fermenter, sterilize at 115℃ for 15min, and cool by filling with nitrogen and maintaining pressure at 0.1Mpa.

[0057] (5) The culture medium prepared in step (2) was inoculated into the high-density fermentation medium in step (4) at an inoculation rate of 5% (v / v), and cultured at a constant temperature of pH 6.5 and 37℃. Samples were taken every 2 hours to measure OD. 600 Fermentation continued until the stationary phase, at which point the fermentation ended, yielding the fermentation broth. The glucose content, osmotic pressure, and viable cell count of the fermentation broth were measured at the fermentation endpoint. The results are shown in Table 2.

[0058] (6) Centrifuge the fermentation broth obtained in step (5), collect the cells, and prepare a freeze-drying protectant solution (solution concentration 40%) with sorbitol and glycine at a mass ratio of 1:1. Shake until fully homogeneous, and freeze-dry the resuspension. Freeze-drying process: Pre-freeze, control the temperature of the plate to decrease from room temperature to -4℃ within 10 min and maintain for 1 h, then decrease to -50℃ within 1 h and maintain for 1 h; in the first drying stage, reduce the vacuum degree and adjust the plate temperature to increase to -30℃ within 1 h, and maintain at a vacuum degree of 200 μbar for 18 h to remove free water; in the second drying stage, control the plate temperature to increase to 25℃ within 1 h and maintain at a vacuum degree of 20 μbar for 16 h; prepare bacterial powder; detect the number of viable bacteria after rehydration of bacterial powder and the number of viable bacteria in the resuspension before freeze-drying, and calculate the freeze-drying survival rate. The results are shown in Table 2.

[0059] Table 1. Results of the osmosis resistance of Bifidobacterium bifidum CCFM1301

[0060]

[0061] The results showed that the initial inhibition osmotic pressure (osmotic pressure when the growth rate begins to be inhibited) of Bifidobacterium bifidum CCFM1301 was 1000 mOsm / kg, and the complete inhibition osmotic pressure (osmotic pressure when the growth rate is completely inhibited) was 1600 mOsm / kg.

[0062] Table 2 Results of fermentation and freeze-drying of Bifidobacterium bifidum CCFM1301 in this example.

[0063]

[0064] The results showed that the glucose content of the fermentation broth at the end of the culture was ≥3.00 g / L, indicating that the glucose content in the fermentation medium of the present invention was sufficient; the osmotic pressure of the fermentation broth at the end of fermentation was close to complete osmotic pressure suppression; and the viable cell count of the fermentation broth was (9.47±0.30)×10⁻⁶. 9 CFU / mL, viable bacterial count after lyophilization and rehydration was (1.21±0.12)×10⁻¹⁰. 11 The CFU / ml count of the freeze-dried bacterial powder was (4.02±0.13)×10⁻¹⁰. 11 CFU / g, freeze-dried survival rate (76.33±0.31)%.

[0065] Example 3: Bifidobacterium bifidum CCFM1301 under osmotic stress K + Content change

[0066] (1) Preparation of Bifidobacterium seed culture: Bifidobacterium bifidum CCFM1301 obtained in Example 1 was streaked on MRS solid medium and cultured at 37℃ for 36h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and cultured at 37℃ for 24h to obtain seed culture.

[0067] (2) The seed culture was inoculated at a rate of 5% into ordinary MRS (osmolarity 300 mOsm / kg) and MRS liquid medium (osmolarity 1000 mOsm / kg), respectively. The culture was anaerobic at 37°C for 24 h. After harvesting the bacteria at 0 h and the early logarithmic phase, the bacterial concentration was adjusted to achieve the desired OD. 600 The bacterial cells were collected after centrifugation at 10000g for 10 minutes until the saturation reached 0.45.

[0068] (3) Add 1 mL of bacterial sludge and 5 mL of nitric acid to a 50 mL centrifuge tube that has been acid-soaked in an acid tank. Digest at 160 °C for 40 min on a graphite digester. Then add 1 mL of hydrogen peroxide and continue digesting for another 30 min. Finally, bring the volume to 50 mL, take 10 mL and pass it through a membrane. Measure the K using an inductively coupled plasma mass spectrometer (ICPMS). + Content. The results are shown in Table 3.

[0069] Table 3. Bifidobacterium bifidum CCFM1301 under different osmotic pressure stress K + Content (mg / g) results

[0070]

[0071] The results showed that, under an osmotic pressure of 1000 mOsm / kg, Bifidobacterium bifidum CCFM1301 fermented from 0 h to the early logarithmic phase K + The increase in content was 25 times higher than that at an osmotic pressure of 300 mOsm / kg, indicating that K + It regulates the bacterial osmotic pressure balance to a certain extent.

[0072] Example 4: Proline content of Bifidobacterium bifidum CCFM1301 under osmotic stress

[0073] (1) Preparation of Bifidobacterium seed culture:

[0074] Bifidobacterium bifidum CCFM1301 obtained in Example 1 was streaked on MRS solid medium and cultured at 37°C for 36 h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 24 h to obtain seed culture.

[0075] (2) Bifidobacterium bifidum was inoculated into 100 mL of MRS medium with salt added to 1000 mOsm / kg and without salt (osmotic pressure 300 mOsm / kg), respectively, and cultured at 37°C for 24 h. The bacterial concentration was adjusted to OD using sterilized MRS liquid medium. 600 It reaches 0.45.

[0076] (4) Take an equal volume of culture medium for sample processing: centrifuge at 5000 r / min for 20 min at 4℃, collect the bacterial cells, and then wash with PBS. Repeat the above process 3 times. Collect the bacterial cells and disrupt them in an ultrasonic cell disruptor according to the following procedure: 7.6 min, work / interval = 5 s / 5 s. Then freeze and thaw repeatedly at -80℃ 5-6 times, sonicate repeatedly 5-6 times, centrifuge (4000 r / min, 20 min), collect the supernatant, dilute with an equal volume of 10 g / 100 mL trichloroacetic acid (TCA), record the dilution factor, and let stand for 1 h to ensure that the final concentration of TCA in the solution system is 5%. After filtering with double-layer filter paper, take 1 mL of the clear filtrate into a 1.5 mL centrifuge tube, label it, and centrifuge at 15000 rpm for 30 min. After filtering again with a 0.22 μm water membrane, take 400 μL of the supernatant into a liquid phase sample bottle. Determine the change in proline content of Bifidobacterium bifidum CCFM1301 under osmotic pressure stress by high performance liquid chromatography (HPLC). The results are shown in Table 4.

[0077] Table 4. Proline content of Bifidobacterium bifidum CCFM1301 under osmotic stress

[0078]

[0079] The results showed that the intracellular proline concentration of Bifidobacterium bifidum CCFM1301 increased at an osmotic pressure of 1000 mOsm / kg, which was 2.2 times that at an osmotic pressure of 300 mOsm / kg, indicating that proline regulated the bacterial osmotic pressure balance to a certain extent.

[0080] Control Example 1: Osmotic tolerance, culture, and freeze-drying of Bifidobacterium bifidum CCFM16

[0081] The specific implementation method is the same as in Example 2, except that Bifidobacterium bifidum CCFM16 is used instead of Bifidobacterium bifidum CCFM1301. Bifidobacterium bifidum CCFM16 is disclosed in patent CN106834187A. The stationary period OD of Bifidobacterium bifidum CCFM16 was measured. 600 The fermentation time was calculated, and the results are shown in Table 5. The glucose content, osmotic pressure, and viable cell count of the fermentation broth at the fermentation endpoint were measured, and the results are shown in Table 6. The cells were freeze-dried, and the viable cell count after rehydration and in the resuspension before freeze-drying were measured to calculate the freeze-drying survival rate, and the results are shown in Table 6.

[0082] Table 5. Results of the osmosis resistance of Bifidobacterium bifidum CCFM16

[0083]

[0084] The results showed that the initial inhibition osmotic pressure of Bifidobacterium bifidum CCFM16 was 700 mOsm / kg, and the complete inhibition osmotic pressure was 900 mOsm / kg, which was much smaller than that of Bifidobacterium bifidum CCFM1301.

[0085] Table 6. Results of fermentation and freeze-drying of Bifidobacterium bifidum CCFM16 using this example.

[0086]

[0087] The results showed that the glucose content of the fermentation broth at the end of the culture was 29.20 g / L, indicating that the glucose content in the fermentation medium of this invention was sufficient. The osmotic pressure of the fermentation broth at the end of fermentation did not reach complete osmotic inhibition, possibly due to the excessively high osmotic pressure of the initial medium. The viable cell count in the fermentation broth was (5.42 ± 0.4) × 10⁻⁶. 8 The CFU / mL and the freeze-dried survival rate were (34.42±0.26)%, both significantly lower than those of Bifidobacterium bifidum CCFM1301.

[0088] Control Example 2: Bifidobacterium bifidum CCFM16 under osmotic stress K + Content change

[0089] The specific implementation method is the same as in Example 3, except that Bifidobacterium bifidum CCFM1301 is replaced with Bifidobacterium bifidum CCFM16, and the K of Bifidobacterium bifidum CCFM16 under initial inhibition of osmotic pressure stress is measured. + Content. The results are shown in Table 7.

[0090] Table 7. Bifidobacterium bifidum CCFM16 under different osmotic pressure stress. + Content (mg / g) results

[0091]

[0092] The results showed that, under an initial suppressive osmotic pressure of 700 mOsm / kg, *Bifidobacterium bifidum* CCFM1301 fermented from 0 h to the early logarithmic phase K... + The increase in content was 5.8 times higher than that at an osmotic pressure of 300 mOsm / kg, but the increase was significantly lower than that of Bifidobacterium bifidum CCFM1301, indicating that Bifidobacterium bifidum CCFM16 is less capable of maintaining ion balance than Bifidobacterium bifidum CCFM1301 when subjected to osmotic pressure stress.

[0093] Control Example 3: Proline content of Bifidobacterium bifidum CCFM16 under osmotic stress

[0094] The specific implementation method is the same as in Example 4, except that Bifidobacterium bifidum CCFM16 is used instead of Bifidobacterium bifidum CCFM1301, and the change in proline content of Bifidobacterium bifidum CCFM16 under initial osmotic pressure suppression is measured. The results are shown in Table 8.

[0095] Table 8. Proline content of Bifidobacterium bifidum CCFM16 under osmotic stress

[0096]

[0097] The results showed that the intracellular proline concentration of Bifidobacterium bifidum CCFM16 during the stationary phase was 10.19 mg / g at an osmotic pressure of 700 mOsm / kg, which was 1.6 times lower than that of Bifidobacterium bifidum CCFM1301. This indicates that when Bifidobacterium bifidum CCFM16 is subjected to osmotic stress, the regulatory ability of proline as a compatible solute is weaker than that of Bifidobacterium bifidum CCFM1301.

[0098] Control Example 4: Fermentation density of Bifidobacterium bifidum CCFM1301, CCFM16, and CCFM16-1300

[0099] The specific steps are as follows:

[0100] (1) Preparation of Bifidobacterium seed culture: Bifidobacterium bifidum CCFM1301 obtained in Example 1 and control strains CCFM16 and CCFM16-1300 were streaked on MRS solid medium and cultured at 37℃ for 36 h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and cultured at 37℃ for 24 h to obtain seed culture;

[0101] (2) Two groups of fermentation media with different osmotic pressures were prepared: Group A: glucose 30 g / L, tryptone 26 g / L, MgSO4·7H2O 1.25 g / L, cysteine ​​1 g / L (osmotic pressure 360 ​​mOsm / kg); Group B: glucose 55 g / L, tryptone 37 g / L, MgSO4·7H2O 1.25 g / L, cysteine ​​1 g / L (osmotic pressure 590 mOsm / kg). 3 L of the prepared fermentation media was placed in a 5 L triple in-situ fermenter, sterilized at 115 °C for 15 min, and then cooled under nitrogen pressure of 0.1 MPa.

[0102] (3) The culture medium prepared in step (1) was inoculated into the high-density fermentation medium in step (2) at an inoculation rate of 5% (v / v), and cultured at a constant temperature of pH 6.5 and 37℃. Samples were taken every 2 hours to measure OD. 600 Fermentation continued until the stabilization period, at which point the fermentation ended, yielding the fermentation broth. The glucose content, osmotic pressure, and viable cell count of the fermentation broth were measured at the fermentation endpoint. The results are shown in Table 9.

[0103] Table 9 Fermentation density of Bifidobacterium bifidum under different osmotic pressures

[0104]

[0105]

[0106] The results showed that the fermentation density of CCFM1301 was significantly higher than that of CCFM16 and CCFM16-1300.

[0107] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A osmotically resistant strain of Bifidobacterium bifidum ( Bifidobacterium bifidum CCFM1301, characterized in that, The Bifidobacterium bifidum CCFM1301 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 16, 2023, with accession number GDMCC No: 63175.

2. A microbial preparation containing the Bifidobacterium bifidum CCFM1301 as described in claim 1.

3. The microbial preparation as described in claim 2, characterized in that, The microbial preparation is a liquid or solid preparation containing Bifidobacterium bifidum CCFM1301.

4. The microbial preparation as described in claim 3, characterized in that, The microbial preparation contains ≥1.21 × 10⁻⁶ viable Bifidobacterium CCFM1301 bacteria. 11 CFU / mL or ≥4.02×10 11 CFU / g.

5. The microbial preparation as described in claim 4, characterized in that, The microbial preparation also contains sorbitol and glycine.

6. A method for preparing the microbial preparation according to any one of claims 2-5, characterized in that, The Bifidobacterium bifidum CCFM1301 of claim 1 was fermented in a culture medium.

7. The method as described in claim 6, characterized in that, The culture medium is a high-density culture medium; the high-density culture medium contains: tryptone 20~30 g / L, glucose 60~70 g / L, cysteine ​​0.1~1.0 g / L, MgSO4·7H2O 0.1~1.0 g / L, and Tween 80 1~2 mL / L.

8. The method as described in claim 7, characterized in that, The fermentation is a high-density fermentation, with an inoculum amount of 3-5% by volume, a fermentation temperature of 30-40℃, a pH of 5.5-6.5, and a fermentation time of 16-18 hours.

9. The use of the *Bifidobacterium bifidum* CCFM1301 according to claim 1, or the microbial preparation according to any one of claims 2-5, in the high-density fermentation preparation of *Bifidobacterium bifidum* or products containing *Bifidobacterium bifidum*, characterized in that, The products include food or medicine.

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