Fermentation medium for improving osmotic tolerance and culture density of bifidobacterium adolescentis and application thereof
By adding isomaltooligosaccharide and uracil to the fermentation medium, the problem of low culture density of Bifidobacterium adolescentis was solved, achieving high-density culture and improved permeability resistance, thus promoting industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- INFINITUS (CHINA) CO LTD
- Filing Date
- 2023-03-17
- Publication Date
- 2026-07-10
AI Technical Summary
In existing technologies, the culture density of Bifidobacterium adolescentis is less than 1×10⁹ CFU/L, which cannot effectively improve the efficiency of industrial production. Furthermore, cell growth is damaged in a high osmotic pressure environment, affecting cell activity and functional expression.
A fermentation medium containing isomaltooligosaccharide and uracil was used, along with nitrogen sources, MgSO4, Tween and cysteine, to enhance the osmotic tolerance of Bifidobacterium adolescentis through synergistic effects and promote its high-density reproduction.
It significantly increased the culture density of Bifidobacterium adolescentis to (2.42±0.47)×10¹⁰ CFU/mL, enhanced its growth ability under high osmotic pressure, and promoted industrial application.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and in particular to a fermentation culture medium for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis and its application. Background Technology
[0002] Bifidobacteria are widely distributed in the intestines of animals and humans, playing a crucial role in maintaining host health. The adolescent-type Bifidobacterium CCFM8630 strain, isolated from the intestines of elderly individuals, has been shown in animal experiments to lower blood sugar and alleviate metabolic syndrome, delay the progression of metabolic disorders, and alleviate non-alcoholic fatty liver disease induced by high-fat, high-cholesterol diets, thus possessing potential production and application value. However, during batch culture in conventional MRS medium, the culture density of Bifidobacteria is often lower than 1 × 10⁻⁶. 9 The CFU / L density limit cannot be exceeded, which prevents the effective improvement of the industrial production efficiency of Bifidobacteria and affects the production capacity of Bifidobacteria. Therefore, there is an urgent need for a more efficient method of Bifidobacteria production.
[0003] To date, various culture methods, including dialysis culture, cell immobilization culture, and embedding methods, have been developed to address the problems associated with traditional Bifidobacterium culture. However, the industrial application of these methods is hindered by various factors. Therefore, constant pH culture remains the mainstream method. During constant pH culture, the accumulation of high concentrations of metabolites and byproducts inevitably increases the osmotic pressure of the entire fermentation system. Since the growth density of the strain is closely related to osmotic pressure, a continuous increase in the osmotic pressure of the fermentation broth will eventually lead to cell cessation. Furthermore, during freeze-drying, the increased electrolyte concentration in the unfrozen portion of the solution generates osmotic stress. As cells continuously lose water, excessively high osmotic pressure can cause structural and physiological damage to the cells, leading to loss of activity and certain functions, affecting cell growth density, and ultimately causing bacterial cell lysis. Additionally, high salt concentration is a major environmental stress encountered during food fermentation; the increased osmotic pressure caused by water reduction may affect the physiological functions, survival ability, and expression of beneficial characteristics of probiotics. Summary of the Invention
[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. To this end, the present invention provides a fermentation medium for improving the osmotic tolerance and culture density of *Bifidobacterium adolescentis*. This fermentation medium can produce *Bifidobacterium adolescentis* with strong osmotic tolerance, and it can effectively promote the reproductive capacity of *Bifidobacterium adolescentis*, enabling it to proliferate a larger number of colonies per unit area / volume, greatly increasing the culture density of *Bifidobacterium adolescentis*, thereby increasing the industrial production value of *Bifidobacterium adolescentis*.
[0005] In a first aspect, the present invention provides a culture medium specifically for Bifidobacterium adolescentis, wherein the culture medium contains isomaltooligosaccharide and uracil.
[0006] In this invention, the inventors discovered that the synergistic effect between isomaltooligosaccharide and uracil significantly enhances the osmotic tolerance of Bifidobacterium adolescentis in the culture medium and greatly increases the viable cell count in the fermentation broth, achieving a density of (2.42±0.47)×10⁻⁶. 10 CFU / mL.
[0007] In some embodiments of the present invention, the culture medium specifically for Bifidobacterium adolescentis also includes nitrogen source substances, MgSO4, Tween and cysteine.
[0008] In some embodiments of the present invention, the MgSO4 is magnesium sulfate heptahydrate (MgSO4·7H2O).
[0009] In some embodiments of the present invention, the Tween includes Tween 80, Tween 60 and Tween 20.
[0010] In some embodiments of the present invention, each L of the Bifidobacterium adolescentis-specific culture medium contains the following proportions: isomaltooligosaccharide: nitrogen source: MgSO4: cysteine: uracil: Tween = 60-70g: 20-30g: 0.1-0.5g: 0.5-1g: 5-15g: 0.5-1mL.
[0011] In some embodiments of the present invention, each L of the Bifidobacterium adolescentis-specific culture medium contains isomaltooligosaccharide: nitrogen source: MgSO4: cysteine: uracil: Tween = 70g: 28g: 0.25g: 1.0g: 10.0g: 1mL.
[0012] In some embodiments of the present invention, the nitrogen source includes at least one of peptone and yeast extract.
[0013] In some embodiments of the present invention, the nitrogen source also includes beef extract powder and whey protein powder.
[0014] In some embodiments of the present invention, the peptone includes fish peptone, soybean peptone, and trypsin peptone.
[0015] In some embodiments of the present invention, the yeast extract includes yeast extract powder and yeast extract.
[0016] In some embodiments of the present invention, the nitrogen source is peptone and yeast extract.
[0017] In some embodiments of the present invention, the yeast extract is yeast powder, i.e., powdered yeast extract.
[0018] In some embodiments of the present invention, the mass ratio of peptone to yeast extract is 0.9 to 1.2:1.
[0019] In some embodiments of the present invention, the mass ratio of peptone to yeast extract is 1:1.
[0020] In some embodiments of the present invention, the culture medium specifically for Bifidobacterium adolescentis uses water as a solvent.
[0021] In some embodiments of the present invention, the Bifidobacterium adolescentis includes Bifidobacterium adolescentis CCFM8630. Of course, those skilled in the art may also use other non-standard strains of Bifidobacterium adolescentis.
[0022] A second aspect of the present invention provides a method for preparing the Bifidobacterium adolescentis culture medium described in the first aspect of the present invention, comprising the following steps:
[0023] The isomaltooligosaccharide, uracil, nitrogen source, MgSO4, Tween, and cysteine are mixed with water according to the proportions described in the first aspect of this invention, sterilized at 110–120°C for 12–17 minutes, and after sterilization, pressurized with inert nitrogen gas to obtain the desired product.
[0024] In some embodiments of the present invention, the inert nitrogen gas includes nitrogen gas.
[0025] In some embodiments of the present invention, the pressure range of the pressure holding is 0.02 to 0.04 MPa.
[0026] In some embodiments of the present invention, the pressure range for the pressure holding is 0.03 MPa.
[0027] In some embodiments of the present invention, the Bifidobacterium adolescentis is pre-activated using MRS solid medium for anaerobic culture.
[0028] In some embodiments of the present invention, the gaseous atmosphere of the anaerobic culture is nitrogen, carbon dioxide and hydrogen.
[0029] In some embodiments of the present invention, the anaerobic culture atmosphere is 80% (v / v) nitrogen, 10% (v / v) carbon dioxide and 10% (v / v) hydrogen.
[0030] In some embodiments of the present invention, the specific operation of the activation treatment is as follows: after inoculating Bifidobacterium adolescentis onto a plate, it is placed upside down in an anaerobic workstation containing 80% nitrogen, 10% carbon dioxide and 10% hydrogen and cultured at 37°C for 24-48 hours.
[0031] A third aspect of the present invention provides the application of the Bifidobacterium adolescentis-specific culture medium described in the first aspect of the present invention in the cultivation and / or screening of Bifidobacterium adolescentis.
[0032] In this invention, by culturing Bifidobacterium adolescentis using the special culture medium for Bifidobacterium adolescentis described in the first aspect of this invention, the permeability resistance of the cultured Bifidobacterium adolescentis can be effectively improved, thereby obtaining Bifidobacterium adolescentis with permeability resistance.
[0033] In this invention, by culturing Bifidobacterium adolescentis using the special culture medium for Bifidobacterium adolescentis described in the first aspect of this invention, the yield of Bifidobacterium adolescentis per unit area / volume can be increased, thereby obtaining Bifidobacterium adolescentis with higher reproductive capacity.
[0034] A fourth aspect of the present invention provides a method for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis, comprising the following steps:
[0035] Bifidobacterium adolescentis is inoculated into the special culture medium for Bifidobacterium adolescentis described in the first aspect of the present invention, and fermented and cultured under stirring, pH 5-6, and temperature 35-39°C until the stable phase is reached.
[0036] In some embodiments of the present invention, the stirring rate is 180 to 250 rpm.
[0037] In some embodiments of the present invention, the stirring rate is 200 rpm.
[0038] In some embodiments of the present invention, the inoculation amount of Bifidobacterium adolescentis is 3-7%.
[0039] In some embodiments of the present invention, the inoculum amount of Bifidobacterium adolescentis is 5%.
[0040] In some embodiments of the present invention, the bacterial concentration is 3.5–4.5 × 10⁻⁶. 7 CFU / L.
[0041] In some embodiments of the present invention, the bacterial concentration is 4 × 10⁻⁶. 7 CFU / L.
[0042] In some embodiments of the present invention, the bacterial solution is a seed culture of Bifidobacterium adolescentis. The specific preparation method is as follows: the activated Bifidobacterium adolescentis is inoculated into MRS liquid culture medium and anaerobically cultured at 37±2℃ for 24-48h. When the culture medium changes from transparent to turbid, it is centrifuged at 3000g and 4℃ for 5min, the bacterial sludge is collected, washed with sterile distilled water, and suspended in MRS culture medium to obtain the solution.
[0043] In some embodiments of the present invention, the pH is 5.5.
[0044] In some embodiments of the present invention, the pH is maintained by adding alkali solution.
[0045] A fifth aspect of the invention provides the application of the method described in the fourth aspect of the invention in the cultivation and / or screening of Bifidobacterium adolescentis.
[0046] In some embodiments of the present invention, the culture density of the cultivated and / or screened Bifidobacterium adolescentis is greater than or equal to 1.95 × 10⁻⁶. 10 The concentration of CFU / mL and the osmotic pressure of the culture medium after fermentation is greater than or equal to 1100 mOsm / kg.
[0047] In some embodiments of the present invention, the osmotic pressure of the culture medium after fermentation is greater than or equal to 1583 mOsm / kg.
[0048] In some embodiments of the present invention, the culture density of the cultivated and / or screened Bifidobacterium adolescentis is 2.42 ± 0.47 × 10⁻⁶. 10 The concentration of CFU / mL was 1604±21 mOsm / kg after fermentation.
[0049] The beneficial effects of this invention are:
[0050] 1. This invention has developed a culture medium capable of increasing the fermentation density of Bifidobacterium adolescentis. While enhancing the osmotic tolerance of Bifidobacterium adolescentis, it also enables high-density propagation of the bacteria, achieving a fermentation density of (2.42±0.47)×10⁻⁶. 10 The extent of CFU / mL.
[0051] 2. The fermentation culture method of Bifidobacterium adolescentis based on the above-mentioned culture medium in this invention can effectively improve the osmotic tolerance of Bifidobacterium adolescentis, thereby increasing the upper limit of fermentation cessation due to hyperosmotic stress. Compared with the conventional fermentation method using MRS, it has a higher fermentation density, thus effectively promoting the industrial development of Bifidobacterium adolescentis. Attached Figure Description
[0052] Figure 1The following describes the culture of Bifidobacterium adolescentis using the special culture medium for Bifidobacterium adolescentis in this embodiment of the invention. Here, a represents the effect of culture medium with different osmotic pressures on the proliferation of Bifidobacterium adolescentis, and b represents the effect of culture medium with different osmotic pressures on the generation time of Bifidobacterium adolescentis; the same letter represents the same significance. Detailed Implementation
[0053] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.
[0054] In the following examples, the Bifidobacterium adolescentis used was Bifidobacterium adolenscentis CCFM 8630, which was obtained from the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC NO.14395.
[0055] Example 1: A fermentation medium for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis.
[0056] This embodiment provides a fermentation medium for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis, as well as a method for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis based on the medium.
[0057] The specific steps are as follows:
[0058] (1) Activation of Bifidobacterium adolescentis:
[0059] Remove Bifidobacterium adolescentis CCFM 8630 from the -80℃ freezer, allow it to thaw, and then shake well. Use a disposable sterile inoculation loop to take a small amount of bacterial solution and streak it onto an MRS solid medium plate for purification. After static solidification, invert the plate and incubate it in an anaerobic workstation containing 80% nitrogen, 10% carbon dioxide, and 10% hydrogen at 37℃ for 24-48 hours to obtain activated Bifidobacterium adolescentis CCFM 8630.
[0060] The composition of the MRS solid culture medium is as follows (final concentration): 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 1.0 g / L, and agar powder 20 g / L.
[0061] (2) Preparation of Bifidobacterium adolescentis seed culture:
[0062] A single colony of the activated Bifidobacterium adolescentis CCFM8630 from step (1) was picked and inoculated into a test tube containing MRS liquid medium. The culture was then anaerobically cultured at 37°C for 24-48 hours. Once the medium changed from clear to turbid, it was centrifuged at 3000g and 4°C for 5 minutes to collect the bacterial sludge. The sludge was washed twice with sterile distilled water and then resuspended in MRS liquid medium. This bacterial suspension serves as the seed culture for subsequent fermentation experiments.
[0063] The composition of the MRS liquid culture medium is as follows (final concentration): 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, and cysteine 1.0 g / L.
[0064] (3) Prepare a special fermentation medium to improve the osmotic tolerance and culture density of Bifidobacterium adolescentis:
[0065] Based on the final concentration, 70 g / L of isomaltooligosaccharide, 28 g / L of a complex nitrogen source (a 1:1 mass ratio of fish peptone and yeast extract FM528), 0.25 g / L of MgSO4·7H2O, 1 mL / L of Tween 80, 1.0 g / L of cysteine, and 10.0 g / L of uracil (osmotic pressure 616 mOsm / kg) were mixed and placed in a 5 L fermenter (using water as the solvent). The mixture was then sterilized at 115 °C for 15 min. After sterilization, nitrogen gas was introduced to maintain a pressure of 0.03 MPa. After cooling, a special fermentation medium was obtained to improve the osmotic tolerance and culture density of Bifidobacterium adolescentis.
[0066] (4) pH-controlled batch culture of Bifidobacterium adolescentis:
[0067] Take the seed solution from step (2) and add it at 5% (v / v) (concentration controlled at 4×10). 7 An inoculum of approximately CFU / L was added to the special fermentation medium prepared in step (3), and the medium was cultured anaerobically at a constant temperature of 200 rpm, pH 5.5, and 37°C. OD was measured every 2 hours. 600 After the Bifidobacterium adolescentis reached the stationary phase (16-20h), the fermentation was stopped, and the fermentation broth was obtained, thus obtaining Bifidobacterium adolescentis with osmotic resistance and completing its high-density culture.
[0068] Example 2: A fermentation medium for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis.
[0069] This embodiment provides a fermentation medium for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis, as well as a method for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis based on the medium.
[0070] The specific steps are as follows:
[0071] (1) Activation of Bifidobacterium adolescentis:
[0072] Remove Bifidobacterium adolescentis CCFM 8630 from the -80℃ freezer, allow it to thaw, and then shake well. Use a disposable sterile inoculation loop to take a small amount of bacterial solution and streak it onto an MRS solid medium plate for purification. After static solidification, invert the plate and incubate it in an anaerobic workstation containing 80% nitrogen, 10% carbon dioxide, and 10% hydrogen at 37℃ for 24-48 hours to obtain activated Bifidobacterium adolescentis CCFM 8630.
[0073] The composition of the MRS solid culture medium is as follows (final concentration): 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 1.0 g / L, and agar powder 20 g / L.
[0074] (2) Preparation of Bifidobacterium adolescentis seed culture:
[0075] A single colony of the activated Bifidobacterium adolescentis CCFM8630 from step (1) was picked and inoculated into a test tube containing MRS liquid medium. The culture was then anaerobically cultured at 37°C for 24-48 hours. Once the medium changed from clear to turbid, it was centrifuged at 3000g and 4°C for 5 minutes to collect the bacterial sludge. The sludge was washed twice with sterile distilled water and then resuspended in MRS liquid medium. This bacterial suspension serves as the seed culture for subsequent fermentation experiments.
[0076] The composition of the MRS liquid culture medium is as follows (final concentration): 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, and cysteine 1.0 g / L.
[0077] (3) Prepare a special fermentation medium to improve the osmotic tolerance and culture density of Bifidobacterium adolescentis:
[0078] Based on the final concentration, 60 g / L of isomaltooligosaccharide, 24 g / L of a complex nitrogen source (a 1:1 mass ratio of fish peptone and yeast extract FM528), 0.25 g / L of MgSO4·7H2O, 0.5 mL / L of Tween 80, 1.0 g / L of cysteine, and 15.0 g / L of uracil (osmotic pressure 528 mOsm / kg) were mixed and placed in a 5 L fermenter (using water as the solvent). The mixture was then sterilized at 115 °C for 15 min. After sterilization, nitrogen gas was introduced to maintain a pressure of 0.03 MPa. After cooling, a special fermentation medium was obtained to improve the osmotic tolerance and culture density of Bifidobacterium adolescentis.
[0079] (4) pH-controlled batch culture of Bifidobacterium adolescentis:
[0080] Take the seed liquid from step (2) and add it at 5% (v / v) (concentration controlled at 4×10). 7 An inoculum of approximately CFU / L was added to the special fermentation medium prepared in step (3), and the medium was cultured anaerobically at a constant temperature of 200 rpm, pH 5.5, and 37°C. OD was measured every 2 hours. 600 After the Bifidobacterium adolescentis reached the stationary phase, the fermentation was stopped, and the fermentation broth was obtained, thus obtaining Bifidobacterium adolescentis with osmotic resistance, and its high-density culture was completed.
[0081] Example 3: A fermentation medium for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis.
[0082] This embodiment provides a fermentation medium for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis, as well as a method for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis based on the medium.
[0083] The specific steps are as follows:
[0084] (1) Activation of Bifidobacterium adolescentis:
[0085] Remove Bifidobacterium adolescentis CCFM 8630 from the -80℃ freezer, allow it to thaw, and then shake well. Use a disposable sterile inoculation loop to take a small amount of bacterial solution and streak it onto an MRS solid medium plate for purification. After static solidification, invert the plate and incubate it in an anaerobic workstation containing 80% nitrogen, 10% carbon dioxide, and 10% hydrogen at 37℃ for 24-48 hours to obtain activated Bifidobacterium adolescentis CCFM 8630.
[0086] The composition of the MRS solid culture medium is as follows (final concentration): 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 1.0 g / L, and agar powder 20 g / L.
[0087] (2) Preparation of Bifidobacterium adolescentis seed culture:
[0088] A single colony of the activated Bifidobacterium adolescentis CCFM8630 from step (1) was picked and inoculated into a test tube containing MRS liquid medium. The culture was then anaerobically cultured at 37°C for 24-48 hours. Once the medium changed from clear to turbid, it was centrifuged at 3000g and 4°C for 5 minutes to collect the bacterial sludge. The sludge was washed twice with sterile distilled water and then resuspended in MRS liquid medium. This bacterial suspension serves as the seed culture for subsequent fermentation experiments.
[0089] The composition of the MRS liquid culture medium is as follows (final concentration): 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, and cysteine 1.0 g / L.
[0090] (3) Prepare a special fermentation medium to improve the osmotic tolerance and culture density of Bifidobacterium adolescentis:
[0091] Based on the final concentration, 65 g / L of isomaltooligosaccharide, 26 g / L of a complex nitrogen source (a 1:1 mass ratio of fish peptone and yeast extract FM528), 0.25 g / L of MgSO4·7H2O, 0.5 mL / L of Tween 80, 1.0 g / L of cysteine, and 12.5 g / L of uracil (osmotic pressure 572 mOsm / kg) were mixed and placed in a 5 L fermenter (using water as the solvent). The mixture was then sterilized at 115 °C for 15 min. After sterilization, nitrogen gas was introduced to maintain a pressure of 0.03 MPa. After cooling, a special fermentation medium was obtained to improve the osmotic tolerance and culture density of Bifidobacterium adolescentis.
[0092] (4) pH-controlled batch culture of Bifidobacterium adolescentis:
[0093] Take the seed liquid from step (2) and add it at 5% (v / v) (concentration controlled at 4×10). 7An inoculum of approximately CFU / L was added to the special fermentation medium prepared in step (3), and the medium was cultured anaerobically at a constant temperature of 200 rpm, pH 5.5, and 37°C. OD was measured every 2 hours. 600 After the Bifidobacterium adolescentis reached the stationary phase, the fermentation was stopped, and the fermentation broth was obtained, thus obtaining Bifidobacterium adolescentis with osmotic resistance, and its high-density culture was completed.
[0094] Verification Example
[0095] The fermentation broth obtained in Example 1 was centrifuged to collect the bacterial sludge. The bacterial sludge was diluted with CYS physiological saline and the viable count was determined on MRS solid medium using the pour count method (refer to the national standard GB 4789.35-2016 Food Safety National Standard for Microbiological Detection of Lactic Acid Bacteria).
[0096] The aforementioned CYS saline solution contains, per liter, 6g of disodium hydrogen phosphate, 4.5g of potassium dihydrogen phosphate, 4g of sodium chloride, 0.5g of L-cysteine hydrochloride, and 0.6g of Tween 60. CYS saline solution is more effective than ordinary saline solution in counting Bifidobacteria.
[0097] The results are shown in Table 1.
[0098] Table 1. Fermentation results of Bifidobacterium adolescentis using CCFM 8630
[0099]
[0100] The residual sugar content in the culture medium was determined using the detection method in the national standard GB / T 20881-2017 isomaltooligosaccharide.
[0101] The above results indicate that the residual sugar content in the fermentation broth at the end of the culture was 5.95 g / L, suggesting that the carbon source in the fermentation medium of this embodiment had a high utilization rate at the fermentation endpoint. Furthermore, the osmotic pressure of the fermentation broth at the end of fermentation was greater than the complete inhibition osmotic pressure (the complete inhibition pressure of Bifidobacterium adolescentis CCFM8630 is 1100 mOsm / kg), indicating that the osmotic tolerance of Bifidobacterium adolescentis CCFM 8630 was significantly improved. The determination of viable cell count confirmed that this specialized fermentation medium increased the viable cell count in the fermentation broth, achieving a density of (2.42 ± 0.47) × 10⁻⁶. 10 CFU / mL.
[0102] Meanwhile, the effect of the special fermentation medium in Example 1 on the osmotic pressure resistance of Bifidobacterium adolescentis was determined. The specific determination method was as follows: take the seed liquid from step (2) of the example, and add it at 5% (v / v) (concentration controlled at 4×10). 7Inoculation amounts (approximately CFU / L) were inoculated into the aforementioned specialized fermentation medium and MRS liquid medium, respectively, with osmotic pressures adjusted to 500 mOsm / kg, 900 mOsm / kg, and 1300 mOsm / kg using NaCl. The media were then anaerobically cultured under the same conditions for 24–36 hours, with OD values measured every 2 hours. 600 The logarithmic generation time was calculated based on the growth curve, and the viable cell count was determined.
[0103] The results are shown in Table 2 and Figure 1 As shown.
[0104] Table 2. Results of osmotic pressure resistance test of Bifidobacterium adolescentis CCFM 8630
[0105]
[0106]
[0107] The above results indicate that the specialized fermentation medium in this embodiment of the invention (i.e., the fermentation medium using isomaltooligosaccharide as a carbon source and supplemented with uracil) can increase the stationary OD of Bifidobacterium adolescentis CCFM8630 under the same osmotic pressure conditions compared to conventional media. 600 The value of is reduced, and the generation time is decreased at the same time.
[0108] The same tests were performed on Examples 2 and 3 above, and the results were similar to those of Example 1.
[0109] Comparative Example 1: Dedicated fermentation medium without uracil
[0110] Fermentation of Bifidobacterium adolescentis CCFM 8630 was carried out according to the steps in Example 1. The difference between Comparative Example 1 and Example 1 is that uracil was not added to the special fermentation medium in Comparative Example 1.
[0111] The specific composition of the special fermentation medium in Comparative Example 1 is as follows: based on the final concentration, isomaltooligosaccharide 70 g / L, compound nitrogen source (fish peptone and yeast extract FM528 mixed in a 1:1 mass ratio) 28 g / L, MgSO4·7H2O 0.25 g / L, Tween 80 1 mL / L, and cysteine 1.0 g / L.
[0112] The fermentation broth in Comparative Example 1 was tested using the method described in the validation example.
[0113] The results are shown in Table 3.
[0114] Table 3. Fermentation results of Bifidobacterium adolescentis using CCFM 8630
[0115]
[0116] It can be observed that when Bifidobacterium adolescentis CCFM 8630 was fermented using the dedicated fermentation medium in Comparative Example 1 under the same conditions, the residual sugar content of the fermentation broth at the end of the culture in Comparative Example 1 was 30.77 g / L, indicating that the carbon source content in the medium of Comparative Example 1 was sufficient. After the fermentation of Comparative Example 1 was completed, the osmotic pressure of the fermentation broth was close to complete osmotic pressure suppression, indicating that the fermentation of Comparative Example 1 stopped due to hyperosmotic stress. Compared with Example 1, the number of viable bacteria in the fermentation broth of Comparative Example 1 was significantly reduced. Therefore, it can be concluded that the dedicated fermentation medium with 10 g / L uracil added in the embodiments of the present invention can effectively improve the osmotic tolerance of Bifidobacterium adolescentis CCFM8630, thereby increasing the fermentation density.
[0117] Comparative Example 2: Specialized fermentation media with different carbon sources
[0118] Fermentation of Bifidobacterium adolescentis CCFM 8630 was carried out according to the steps in Example 1. The difference between Comparative Example 2 and Example 1 is that the special fermentation medium in Comparative Example 2 did not contain a different carbon source than that in Example 1. Specifically, isomaltooligosaccharide in the special fermentation medium of Example 1 was replaced with anhydrous glucose at final concentrations of 40 g / L, 50 g / L, and 60 g / L (referred to as experimental groups A, B, and C, respectively).
[0119] The fermentation broth in Comparative Example 2 was tested using the method described in the validation example.
[0120] The results are shown in Table 4.
[0121] Table 4. Fermentation results of Bifidobacterium adolescentis using CCFM 8630
[0122]
[0123] It can be observed that when isomaltooligosaccharide in the special fermentation medium of Example 1 of this invention is replaced with glucose as a carbon source, the residual sugar content of the fermentation broth at the end of the culture is >4.00 g / L, indicating that the carbon source content in the medium of Comparative Example 2 is sufficient. After the fermentation of Comparative Example 2, the osmotic pressure of the fermentation broth is close to complete osmotic pressure suppression, indicating that fermentation stops due to hyperosmotic stress. Moreover, compared with Example 1, the number of viable bacteria in the fermentation broth of Comparative Example 2 is significantly reduced, indicating that the synergistic effect of uracil and isomaltooligosaccharide is lacking, and it is unable to significantly increase the fermentation density of Bifidobacterium adolescentis CCFM8630.
[0124] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A fermentation medium for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis, characterized in that, The fermentation medium contains isomaltooligosaccharide, uracil, nitrogen source, MgSO4, Tween, and cysteine; The fermentation medium contained in each L of the medium contains the following proportions: isomaltooligosaccharide: nitrogen source: MgSO4: cysteine: uracil: Tween = 60~70 g: 20~30 g: 0.1~0.5 g: 0.5~1 g: 5~15 g: 0.5~1 mL; The Bifidobacterium adolescentis is Bifidobacterium adolescentis (… Bifidobacterium adolenscentis CCFM 8630, accession number CGMCC NO.14395.
2. The fermentation medium according to claim 1, characterized in that, The nitrogen source includes at least one of peptone and yeast extract.
3. The method for preparing the fermentation medium according to any one of claims 1 to 2 comprises the following steps: mixing isomaltooligosaccharide, uracil, nitrogen source, MgSO4, Tween and cysteine with water in the proportions specified in claim 1, sterilizing at 110-120°C for 12-17 min, and then pressurizing with inert nitrogen gas after sterilization to obtain the medium.
4. The application of the fermentation medium according to any one of claims 1 to 2 in the cultivation of Bifidobacterium adolescentis.
5. A method for improving the osmotic tolerance and culture density of Bifidobacterium adolescentis, comprising the following steps: inoculating Bifidobacterium adolescentis into the fermentation medium according to any one of claims 1 to 2, and fermenting and culturing it to the stationary phase under stirring, pH 5 to 6, and temperature 35 to 39°C; The Bifidobacterium adolescentis is Bifidobacterium adolescentis (… Bifidobacterium adolenscentis CCFM 8630, accession number CGMCC NO.14395.
6. The method according to claim 5, characterized in that, The inoculum size of *Bifidobacterium adolescentis* was 3-7%, and the bacterial concentration was 3.5-4.5 × 10⁻⁶. 7 CFU / L.
7. The method according to claim 5, characterized in that, The culture density of the cultured Bifidobacterium adolescentis was greater than or equal to 1.95 × 10⁻⁶. 10 The concentration of CFU / mL and the osmotic pressure of the culture medium after fermentation is greater than or equal to 1100 mOsm / kg.