Bifidobacterium adolescentis with high osmotic pressure resistance and application thereof
By screening and culturing osmotically resistant Bifidobacterium adolescentis CCFM1302, and combining it with sorbitol and glycine protectants, the problem of activity damage to lactic acid bacteria in a high osmotic pressure environment was solved, achieving high bacterial survival and product stability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2023-03-21
- Publication Date
- 2026-07-24
AI Technical Summary
Lactic acid bacteria are prone to losing their activity in high-density culture and high osmotic pressure environments, leading to damage to cell structure and physiology, which affects their application in food fermentation and cosmetics.
A strain of Bifidobacterium adolescentis, CCFM1302, was screened out. It has high osmotic pressure resistance. Its survival rate during the freeze-drying process was improved by fermenting it in a high-density fermentation medium and combining it with sorbitol and glycine as freeze-drying protectants.
Bifidobacterium adolescentis CCFM1302 can maintain high activity under an osmotic pressure of 1600 mOsm/kg, with a freeze-drying survival rate of 67.87%, achieving efficient industrial application.
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Abstract
Description
Technical Field
[0001] This invention relates to a hyperosmolar-resistant Bifidobacterium adolescentis and its applications, belonging to the field of microbial technology. Background Technology
[0002] Bifidobacterium adolescentis ( Bifidobacterium adolescentis Lactic acid bacteria (LAC) are among the most important probiotics. In the industrial production of LAC or the production of products such as acids and amino acids, high-density cultivation is a crucial way to increase the biomass of probiotics to meet industrial needs. Therefore, how to convert high-concentration substrates into products is key to improving the economic benefits of LAC products. During high-density production, there is a higher concentration of metabolic products and byproducts to accumulate. These products inevitably increase the osmotic pressure of the entire fermentation system. Since the growth density of the bacterial strain is closely related to osmotic pressure, a continuous increase in the osmotic pressure of the fermentation broth will eventually lead to cell cessation.
[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, ice crystals form as the cooling rate changes, causing mechanical freeze-drying damage to the cell structure. The increased electrolyte concentration in the unfrozen portion of the solution generates osmotic stress. As cells continuously lose water, excessively high osmotic pressure leads to structural and physiological damage to the cells, resulting in loss of activity and certain functions, affecting cell growth density, and ultimately causing bacterial cell lysis.
[0004] Furthermore, lactic acid bacteria, as an important industrial microorganism, are widely used in fermented foods, such as soy sauce, pickles, and cheese, where fermentation enhances flavor. To inhibit the growth of harmful microorganisms and extend shelf life, large amounts of salt or sugar are often added. High concentrations of sugar or salt create a high osmotic pressure environment outside the cells, causing cell shrinkage and even death. Since cells can balance the additional lactose and sucrose concentrations, the high osmotic stress exerted by sugar is much less harmful, being only a transient osmotic stress. Therefore, high salt concentration 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 this case, it is necessary to reduce 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.
[0005] Therefore, screening strains with strong osmotic pressure resistance and capable of high-density fermentation is an effective method for realizing the industrialization of Bifidobacterium adolescentis. Summary of the Invention
[0006] This invention provides a strain of Bifidobacterium adolescentis (Bifidobacterium adolescentis) Bifidobacterium adolescentis CCFM1302, the Bifidobacterium adolescentis CCFM1302, was deposited on February 16, 2023 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63176, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0007] The Bifidobacterium adolescentis ( Bifidobacterium adolescentis CCFM1302 was isolated from a fecal sample. Sequencing analysis of this strain and alignment of its 16S rDNA sequence with NCBI revealed that the strain is *Bifidobacterium adolescentis*. According to the classification of the genus *Bifidobacterium* in Bergey's Manual of Systematic Bacteriology, *Bifidobacterium adolescentis* belongs to the phylum Actinobacteria (…). Actinobacteria ), Actinomycetes ( Actinobacteria Actinomycetes ( Actinobacteridae Bifidobacteria ( ) Bifidobacteriaceae Bifidobacteriaceae ( Bifidobacteriaceae Bifidobacterium spp. Bifidobacterium Bifidobacterium adolescentis ( Bifidobacterium adolescentis It was named Bifidobacterium adolescentis (Bifidobacterium adolescentis) Bifidobacterium adolescentis (CCFM1302)
[0008] The Bifidobacterium adolescentis ( Bifidobacterium adolescentis The colonies of CCFM1302 on MRS solid medium are small, white, and opaque.
[0009] The present invention also provides a microbial preparation containing Bifidobacterium adolescentis CCFM1302.
[0010] In one embodiment of the present invention, the microbial preparation is a liquid or solid preparation containing Bifidobacterium adolescentis CCFM1302.
[0011] In one embodiment of the present invention, the microbial preparation contains ≥2.1 × 10⁻⁶ viable Bifidobacterium adolescentis CCFM1302 bacteria. 11 CFU / mL or ≥7.1×10 11 CFU / g.
[0012] In one embodiment of the present invention, the microbial preparation further contains sorbitol and glycine.
[0013] The present invention also provides a method for preparing the microbial preparation, wherein Bifidobacterium adolescentis CCFM1302 is inoculated into a high-density culture medium for fermentation.
[0014] In one embodiment of the present invention, the high-density culture medium comprises tryptone, glucose, cysteine, MgSO4·7H2O, and Tween 80.
[0015] Preferably, the high-density culture medium comprises 22 g / L tryptone, 61 g / L glucose, 1 g / L cysteine, 0.5 g / L MgSO4·7H2O, and 1 mL / L Tween 80.
[0016] In one embodiment of the present invention, the fermentation is a high-density fermentation, wherein the inoculum amount of the high-density fermentation is 3~5% (v / v), the fermentation temperature is 30~40℃, the pH is 5.5~6.5, and the fermentation time is 16~18 h.
[0017] The present invention also provides the application of Bifidobacterium adolescentis CCFM1302, or a microbial preparation thereof, or a method for preparing a microbial preparation containing Bifidobacterium adolescentis CCFM1302 in the high-density fermentation preparation of Bifidobacterium adolescentis or products containing Bifidobacterium adolescentis, the application including food, pharmaceuticals, and cosmetics.
[0018] Beneficial effects
[0019] (1) This invention screened out a strain of Bifidobacterium adolescentis (Bifidobacterium adolescentis) Bifidobacterium adolescentis CCFM1302 is a strain that can tolerate hyperosmolarity, with an osmotic pressure of 1600 mOsm / kg, which is the high osmotic pressure caused by a culture medium containing 3.6% NaCl solution.
[0020] (2) The Bifidobacterium adolescentis CCFM1302 selected in this invention was inoculated into a high-density fermentation medium and fermented for 18 hours to achieve a fermentation density of (1.82±0.08)×10⁻⁶. 10 CFU / mL.
[0021] (3) The Bifidobacterium adolescentis CCFM1302 screened in this invention exhibits good resistance to osmotic stress during freeze-drying, with a freeze-drying survival rate of (67.87±0.35)%, which can be efficiently industrialized.
[0022] Preservation of biological materials
[0023] A strain of Bifidobacterium adolescentis ( Bifidobacterium adolescentis (CCFM1302, taxonomically named) Bifidobacterium adolescentis It was deposited on February 16, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63176, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description
[0024] Figure 1 Bifidobacterium adolescentis ( Bifidobacterium adolescentis Colony characteristics of CCFM1302.
[0025] Figure 2 Growth curves and generation times of Bifidobacterium adolescentis CCFM1302 under different osmotic pressures.
[0026] Figure 3 Growth curves and generation times of Bifidobacterium adolescentis CCFM8630 under different osmotic pressures. Detailed Implementation
[0027] The present invention will be further described below with reference to specific embodiments and accompanying drawings.
[0028] 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.
[0029] The culture media involved in the following examples are as follows:
[0030] 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.
[0031] 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.
[0032] MRS medium for osmotic adjustment: Osmotic pressure is adjusted by adding NaCl to MRS liquid medium. Adding 3 g of NaCl to 1 L 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.)
[0033] High-density fermentation medium: tryptone 22 g / L, glucose 61 g / L, cysteine 1 g / L, MgSO4·7H2O 0.5 g / L, Tween 80 1 mL / L.
[0034] The detection methods involved in the following embodiments are as follows:
[0035] Determination of viable lactic acid bacteria count:
[0036] The national standard GB 4789.35-2016, "National Food Safety Standard - Microbiological Testing of Food - Lactic Acid Bacteria Detection," was adopted.
[0037] Determination of the survival rate of lyophilized lactic acid bacteria:
[0038] The freeze-dried survival rate of Bifidobacterium was calculated using the following formula:
[0039] Freeze-dried survival rate (%) =
[0040] Glucose content detection: Measured using a glucose assay kit (product number: Beyotime S0201M).
[0041] Osmotic pressure testing: The osmotic pressure was measured using a Löserom 806m freezing point osmotic pressure meter.
[0042] Rehydration conditions: After lyophilization, remove the corresponding sample and rehydrate it with room temperature physiological saline to the original volume before lyophilization.
[0043] HPLC conditions for proline detection: Mobile phase A (pH=7.2): 27.6 mmol / L sodium acetate-triethylamine-tetrahydrofuran (v / v ratio 500 : 0.11 : 2.5); Mobile phase B (pH=7.2): 80.9 mmol / L sodium acetate-methanol-acetonitrile (v / v ratio 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; 20.1 min, 100% B; 24.0 min, 0% B; mobile phase flow rate: 1.0 mL / min; column temperature: 40°C; UV detector (VWD) wavelength: 262 nm.
[0044] 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.
[0045] Example 1: Screening and identification of Bifidobacterium adolescentis CCFM1302
[0046] 1. Screening
[0047] Using adult fecal samples, the samples were serially diluted 10-fold with sterile physiological saline to 10⁻⁶. -6 Then take 100 μL of each diluted by a factor of 10. -4 10 -5 10 -6 The diluted solution was plated on MRS solid medium and incubated upside down at 37°C for 48 h. The 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 h, 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 of MRS liquid medium and incubated at 37°C for 18 h. Then, at an inoculation rate of 5%, strains with osmotic pressures of 700, 1000, 1300, and 1600 mOsm / kg were inoculated into MRS liquid medium, respectively, to screen for strains with high osmotic tolerance, resulting in strain CCFM1302.
[0048] 2. Identification
[0049] The genome of strain CCFM1302 was extracted, and the 16S rDNA of strain CCFM1302 was amplified and sequenced (by Shanghai Meiji Biotechnology Co., Ltd.). The sequence was then compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Bifidobacterium adolescentis, and it was named Bifidobacterium adolescentis. Bifidobacterium adolescentis (CCFM1302)
[0050] 3. Save
[0051] Pick Bifidobacterium adolescentis ( Bifidobacterium adolescentis A single colony of CCFM1302 was inoculated into MRS liquid medium and cultured at 37°C for 18 h to obtain bacterial suspension. 1 mL of bacterial suspension was placed in 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°C.
[0052] Example 2: Permeability, culture and freeze-drying of Bifidobacterium adolescentis CCFM1302
[0053] The specific steps are as follows:
[0054] (1) Preparation of Bifidobacterium seed culture: Take the Bifidobacterium adolescentis obtained in Example 1 ( Bifidobacterium of a young man CCFM1302 was streaked on MRS solid medium and incubated at 37°C for 36 h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and incubated at 37°C for 24 h to obtain seed culture.
[0055] (2) The prepared seed culture 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.
[0056] (3) The culture medium prepared in step (2) was inoculated into MRS liquid medium with osmotic pressure of 700, 1000, 1300 and 1600 mOsm / kg at an inoculation rate of 5% (v / v) and cultured anaerobically at 37℃ for 24 h. OD was measured every 2 h. 600 Once the stabilization period is reached, the generation time is calculated. The results are shown in Table 1.
[0057] (4) Prepare high-density fermentation medium. Place 3 L of fermentation medium in a 5 L fermenter, sterilize at 115℃ for 15 min, and cool under nitrogen pressure of 0.1 MPa.
[0058] (5) The culture medium prepared in step (2) was inoculated into the fermentation medium in step (4) at an inoculation rate of 5% (v / v), and cultured at a constant temperature of pH 5.5 and 37℃. Samples were taken every 2 h to measure OD. 600 Fermentation continued until the stationary phase, at which point it ended, yielding the fermentation broth. The fermentation broth was centrifuged, and the bacterial cells were collected. The glucose content, osmotic pressure, and viable cell count at the fermentation endpoint were measured. The results are shown in Table 2.
[0059] (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.
[0060] Table 1. Results of osmosis resistance of Bifidobacterium adolescentis CCFM1302
[0061]
[0062] The results showed that the initial inhibition osmotic pressure (osmotic pressure at which the growth rate begins to be inhibited) of Bifidobacterium adolescentis CCFM1302 was 1000 mOsm / kg, and the complete inhibition osmotic pressure (osmotic pressure at which the growth rate is completely inhibited) was 1600 mOsm / kg.
[0063] Table 2 Results of fermentation and freeze-drying of Bifidobacterium adolescentis CCFM1302 in this example.
[0064]
[0065] 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 (1.82±0.08)×10⁻⁶. 10 The CFU / mL count of the lyophilized bacterial powder was (7.13±0.80)×10⁻¹⁰. 11 CFU / g, viable bacteria count after freeze-drying and rehydration was (2.14±0.24)×10⁻¹⁰. 11 CFU / ml, lyophilized survival rate (67.87±0.35)%.
[0066] Example 3: Bifidobacterium adolescentis CCFM1302 under osmotic stress K + Content change
[0067] (1) Preparation of Bifidobacterium seed culture: Take the Bifidobacterium adolescentis obtained in Example 1 ( Bifidobacterium of a young manCCFM1302 was streaked on MRS solid medium and incubated at 37°C for 36 h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and incubated at 37°C for 24 h to obtain seed culture.
[0068] (2) Inoculate the seed liquid at a rate of 5% into ordinary MRS (osmotic pressure of 300 mOsm / kg) and MRS liquid medium (osmotic pressure of 1000 mOsm / kg), respectively, and culture anaerobicly at 37℃ for 24 h. After harvesting the bacteria at 0 h and the early stage of the logarithmic phase, adjust the bacterial concentration to make OD600 reach 0.45, centrifuge at 10000g for 10 min, and collect the bacterial cells.
[0069] (3) Add 1 mL of bacterial sludge and 5 mL of nitric acid to a 50 mL centrifuge tube that has been soaked in an acid tank, and digest it at 160 °C for 40 min on a graphite digester. Then add 1 mL of hydrogen peroxide and continue digesting for 30 min. Finally, bring the volume to 50 mL, take 10 mL and pass it through a membrane, and determine the K+ content by inductively coupled plasma mass spectrometry (ICPMS). The results are shown in Table 3.
[0070] Table 3. Bifidobacterium adolescentis CCFM1302 under different osmotic stress conditions. + Content (mg / g) results
[0071]
[0072] The results showed that, under an osmotic pressure of 1000 mOsm / kg, *Bifidobacterium adolescentis* CCFM1302 fermented from 0 h to the early logarithmic phase K... + The increase in content was 19 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.
[0073] Example 4: Proline content of Bifidobacterium adolescentis CCFM1302 under osmotic stress
[0074] (1) Preparation of Bifidobacterium seed culture: Take the Bifidobacterium adolescentis obtained in Example 1 ( Bifidobacterium of a young man CCFM1302 was streaked on MRS solid medium and incubated at 37°C for 36 h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and incubated at 37°C for 24 h to obtain seed culture.
[0075] (2) Inoculate Bifidobacterium adolescentis into 100 mL of MRS medium with salt added to 1000 mOsm / kg and without salt (osmotic pressure of 300 mOsm / kg) and culture for 24 h. Adjust the bacterial concentration to achieve the desired OD. 600 It reaches 0.45.
[0076] (4) Take an equal amount of culture medium for sample processing: centrifuge at 5000 r / min for 20 min at 4℃, collect the bacterial cells and 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, working / 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), take the supernatant, dilute with an equal volume of 10 g / 100 mL trichloroacetic acid, record the dilution factor, and let stand for 1 h to ensure that the final TCA concentration 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 adolescentis CCFM1302 under osmotic pressure stress by high performance liquid chromatography (HPLC). The results are shown in Table 4.
[0077] Table 4. Proline content of Bifidobacterium adolescentis CCFM1302 under osmotic stress
[0078]
[0079] The results showed that the intracellular proline concentration of Bifidobacterium adolescentis CCFM1302 was about 2.8 times higher at an osmotic pressure of 1000 mOsm / kg compared to that at an osmotic pressure of 300 mOsm / kg, indicating that proline regulated the bacterial osmotic pressure balance to some extent.
[0080] Comparative Example 1: Osmotic tolerance, culture, and freeze-drying of Bifidobacterium adolescentis CCFM8630
[0081] The specific implementation method is the same as in Example 2, except that Bifidobacterium adolescentis (…) is used. Bifidobacterium of a young man CCFM8630 replaces Bifidobacterium adolescentis ( Bifidobacterium adolescentis CCFM1302 and Bifidobacterium adolescentis CCFM8630 have been disclosed in patent CN107699517A. The stationary phase OD of Bifidobacterium adolescentis CCFM8630 was determined. 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 osmotic resistance of Bifidobacterium adolescentis CCFM8630
[0083]
[0084] The results showed that the initial inhibition osmotic pressure of Bifidobacterium adolescentis CCFM8630 was 1000 mOsm / kg, and the complete inhibition osmotic pressure was 1200 mOsm / kg, which was much lower than that of Bifidobacterium adolescentis CCFM1302.
[0085] Table 6 Results of Fermentation and Freeze-Drying of Bifidobacterium adolescentis CCFM8630 in this Example
[0086]
[0087] The results showed that the glucose content of the fermentation broth at the end of the culture was 34.42 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 did not reach the complete inhibition osmotic pressure, possibly due to the excessively high osmotic pressure of the initial medium; the viable cell count of the fermentation broth was (3.74±0.24)×10⁻⁶. 9 The CFU / mL and the freeze-dried survival rate were (51.38±0.47)%, both significantly lower than those of Bifidobacterium adolescentis CCFM1302.
[0088] Control Example 2: Bifidobacterium adolescentis CCFM8630 under osmotic stress K + Content change
[0089] The specific implementation method is the same as in Example 3, except that Bifidobacterium adolescentis (…) is used. Bifidobacterium of a young man CCFM8630 replaces Bifidobacterium adolescentis ( Bifidobacterium adolescentis CCFM1302 was used to determine the K+ levels of Bifidobacterium adolescentis CCFM8630 under initial inhibitory osmotic stress. + Content. The results are shown in Table 7.
[0090] Table 7. K+ content (mg / g) of Bifidobacterium adolescentis CCFM8630 under different osmotic stresses.
[0091]
[0092] The results showed that, under an osmotic pressure of 700 mOsm / kg, *Bifidobacterium adolescentis* CCFM8630 fermented from 0 h to the early logarithmic phase K... + The increase in content was 11.7 times higher than that at an osmotic pressure of 300 mOsm / kg, but the increase was significantly lower than that of Bifidobacterium adolescentis CCFM1302, which was 0.4 times the increase of Bifidobacterium adolescentis CCFM1302. This indicates that Bifidobacterium adolescentis CCFM8630 is weaker than Bifidobacterium adolescentis CCFM1302 in maintaining ion balance under osmotic pressure stress.
[0093] Comparative Example 3: Proline content of Bifidobacterium adolescentis CCFM8630 under osmotic stress
[0094] (The specific implementation method is the same as in Example 4, except that Bifidobacterium adolescentis CCFM8630 is used instead of Bifidobacterium adolescentis CCFM1302, and the change in proline content of Bifidobacterium adolescentis CCFM8630 under initial osmotic pressure suppression is measured. The results are shown in Table 8.)
[0095] Table 8. Proline content of Bifidobacterium adolescentis CCFM8630 under osmotic stress
[0096]
[0097] The results showed that the intracellular proline concentration of Bifidobacterium adolescentis CCFM8630 increased by 6.15 mg / g at an osmotic pressure of 700 mOsm / kg, which was only 0.5 times that of Bifidobacterium adolescentis CCFM1302. This indicates that when Bifidobacterium adolescentis CCFM8630 is subjected to osmotic stress, the regulatory ability of proline as a compatible solute is weaker than that of Bifidobacterium adolescentis CCFM1302.
[0098] Comparative Example 4: Fermentation densities of Bifidobacterium adolescentis CCFM1302 and CCFM8630 under different osmotic pressures
[0099] The specific steps are as follows:
[0100] (1) Preparation of Bifidobacterium seed culture: The Bifidobacterium adolescentis CCFM1302 obtained in Example 1 and the control strain CCFM8630 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 61 g / L, tryptone 22 g / L, MgSO4·7H2O 0.5 g / L, cysteine 1 g / L (osmotic pressure 520 mOsm / kg); Group B: glucose 41 g / L, tryptone 18 g / L, MgSO4·7H2O 0.5 g / L, cysteine 1 g / L (osmotic pressure 372 mOsm / kg). The prepared 3 L fermentation media was placed in a 5 L triple in-situ fermenter, sterilized at 115℃ 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 h 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 adolescentis under different osmotic pressures
[0104]
[0105] The results showed that the fermentation density of CCFM1302 was significantly higher than that of CCFM8630.
[0106] 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 strain of Bifidobacterium adolescentis resistant to hyperosmolarity ( Bifidobacterium adolescentis CCFM1302, characterized in that, The aforementioned Bifidobacterium adolescentis was deposited at the Guangdong Provincial Center for Microbial Culture Collection on February 16, 2023, with accession number GDMCC No: 63176.
2. A microbial preparation containing the Bifidobacterium adolescentis CCFM1302 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 adolescentis CCFM1302.
4. The microbial preparation as described in claim 3, characterized in that, The microbial preparation contains ≥2.1 × 10⁻⁶ viable Bifidobacterium adolescentis CCFM1302 bacteria. 11 CFU / mL or ≥7.1×10 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 adolescentis CCFM1302 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 a fermentation temperature of 30~40℃, pH 5.5~6.5, and a fermentation time of 16~18 h.
9. The use of the Bifidobacterium adolescentis CCFM1302 of claim 1, or the microbial preparation of any one of claims 2-5, or the method of any one of claims 6-8 in the high-density fermentation preparation of food containing Bifidobacterium adolescentis.