Method for culturing and screening animal bifidobacterium subsp. Lactis and incubator suitable for method
Through the step-by-step domestication culture screening method, the oxygen tolerance of Bifidobacteria lactis subspecies in animals was improved, and the problem of difficulty in survival in an aerobic environment was solved, and the production and storage process was simplified.
Patent Information
- Application Number
- CN202510050966.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-13
- Publication Date
- 2025-04-18
AI Technical Summary
Due to the lack of catalase, the animal Bifidobacterium milk subspecies is difficult to tolerate oxygen, which makes it difficult to survive during production and storage and needs to be isolated from oxygen.
The oxygen tolerance of animal Bifidobacteria milk subspecies is gradually improved by culturing and screening in multiple culture environments with low to high oxygen content.
The successful acquisition of Bifidobacteria lactis subspecies strain with significantly improved oxygen resistance solved the problem of its survival in an aerobic environment and simplified the production and storage process.
Smart Images
Figure CN119842587A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of bifidobacteria, and more specifically, to a method for culturing and screening animal bifidobacterium lactis subspecies and a culture box suitable for the method. Background Art
[0002] Bifidobacterium is a common physiological lactic acid bacteria in the human body. It plays an important role in maintaining and regulating the intestinal flora in the human body and promoting human health.
[0003] The genus Bifidobacterium is divided into 34 different species, among which Bifidobacterium animalis subsp. lactis is an important species in the genus Bifidobacterium. Bifidobacterium animalis subsp. lactis can act as a biological barrier and has the effects of anti-tumor, enhancing immunity, improving gastrointestinal function, and anti-aging. Therefore, Bifidobacterium animalis subsp. lactis is widely used in the production of lactic acid bacteria products.
[0004] However, animal Bifidobacterium lactis subsp. lactis is an anaerobic bacterium that lacks catalase, which results in the oxidation of O2 in animal Bifidobacterium lactis subsp. lactis. 2 - OH - , H 2 O 2 Metabolites such as oxidative stress and oxygen will accumulate in cells and lead to cell death; therefore, during production and storage, isolation from oxygen is one of the keys to ensure the survival of animal Bifidobacterium lactis subsp.; this has brought a huge burden to the production and storage of animal Bifidobacterium lactis subsp. Therefore, if the oxygen tolerance of animal Bifidobacterium lactis subsp. can be improved, it will be of great significance for the production and storage of animal Bifidobacterium lactis subsp. Summary of the invention
[0005] In order to improve the oxygen resistance of animal Bifidobacterium lactis subspecies, the present application provides a culture and screening method for animal Bifidobacterium lactis subspecies and a culture box suitable for the method.
[0006] In the first aspect, a method for culturing and screening animal Bifidobacterium lactis subspecies is provided, which adopts the following technical scheme: The method for culturing and screening animal Bifidobacterium lactis subspecies comprises: S1, anaerobic culture of Bifidobacterium animalis subsp. lactis; S2, dividing the bacterial solution of animal Bifidobacterium lactis subsp. obtained in S1 into multiple portions and culturing them in an aerobic culture environment; selecting the bacterial solution of animal Bifidobacterium lactis subsp. with good growth, making agar plates after gradient dilution, and then culturing them in the same aerobic culture environment as before in this step; then selecting a single colony of animal Bifidobacterium lactis subsp. with good growth, and culturing them again in the same aerobic culture environment as before in this step; S3, dividing the bacterial solution of animal Bifidobacterium lactis subsp. obtained in the previous step into multiple portions and culturing them in a culture environment with an oxygen content higher than that in the previous step; selecting the bacterial solution of animal Bifidobacterium lactis subsp. with good growth, making agar plates after gradient dilution, and then culturing them in the same aerobic culture environment as before in this step; then selecting a single colony of animal Bifidobacterium lactis subsp. with good growth, and culturing them again in the same aerobic culture environment as before in this step; S4, repeating S3 several times to obtain a strain of Bifidobacterium animalis subsp. lactis with obvious oxygen resistance.
[0007] By adopting the above technical solution, animal Bifidobacterium lactis subsp. is gradually domesticated by culturing and screening in multiple culture environments with oxygen content ranging from low to high, and finally a strain of animal Bifidobacterium lactis subsp. is obtained with obvious oxygen tolerance. Compared with screening and domestication in a single oxygen-containing environment, the oxygen tolerance of animal Bifidobacterium lactis subsp. obtained by the step-by-step domestication method is more ideal.
[0008] In a specific possible implementation scheme, it includes: S0, prepare liquid culture medium; S1, inoculating animal Bifidobacterium lactis subsp. lactis into the liquid culture medium obtained in S0; and then culturing in an anaerobic environment for 10-14 hours; S2, the bacterial solution of animal Bifidobacterium lactis subsp. obtained in S1 is divided into multiple portions and inoculated into the liquid culture medium obtained in S0; then placed in a culture environment with an oxygen content of 2-5 vol% and cultured for 12-20 hours; the bacterial solution of animal Bifidobacterium lactis subsp. with good growth is selected, and agar plates are prepared after gradient dilution, and then placed in the same environment as the liquid culture medium in this step and cultured for 8-18 hours; then a single colony of animal Bifidobacterium lactis subsp. with good growth is selected, transferred into the liquid culture medium obtained in S0, and then placed in the same environment as the agar plate culture in this step and cultured for 6-15 hours; S3, the bacterial solution of animal Bifidobacterium lactis subsp. obtained in the previous step is divided into multiple portions and inoculated into the liquid culture medium obtained in the S0; then cultured in a culture environment with an oxygen content higher than that in the previous step for 12-20 hours; the bacterial solution of animal Bifidobacterium lactis subsp. with good growth is selected, and an agar plate is prepared after gradient dilution, and then placed in the same environment as the liquid culture medium in this step for 8-18 hours; then a single colony of animal Bifidobacterium lactis subsp. with good growth is selected, and moved into the liquid culture medium obtained in S0, and then placed in the same environment as the agar plate culture in this step for 6-15 hours; S4, repeating S3 several times to obtain a strain of Bifidobacterium animalis subsp. lactis with obvious oxygen resistance.
[0009] By adopting the above technical scheme, the culture and screening method of animal Bifidobacterium lactis subspecies is optimized, which is conducive to obtaining animal Bifidobacterium lactis subspecies with ideal oxygen resistance.
[0010] In a specific embodiment, in the S0, the liquid culture medium includes 8-10 parts by mass of hydrolyzed casein, 5-8 parts by mass of peptone, 3.5-6.5 parts by mass of glucose, 0.2-0.8 parts by mass of L-cysteine, 4-8 parts by mass of Moringa seed extract, 2-6 parts by mass of triammonium citrate, 0.5-2 parts by mass of dipotassium bicarbonate, 0.3-0.8 parts by mass of magnesium sulfate, 0.4-1 parts by mass of calcium chloride, 0.1-0.4 parts by mass of potassium carbonate, 0.8-1.3 parts by mass of Tween 80 and 1000 parts by mass of water; the pH value of the liquid culture medium is 6.5-7.0.
[0011] By adopting the above technical solution, the liquid culture medium is optimized, which is conducive to the domestication of animal Bifidobacterium lactis subspecies with better oxygen resistance.
[0012] In a specific embodiment, in S1, the culture is carried out in a static manner at a culture temperature of 36.5-37.5°C.
[0013] In a specific implementation scheme, in S2-3, the culture is carried out by shaking, the shaking speed is 80-120r / min, and the culture temperature is 36.5-37.5°C.
[0014] In a specific embodiment, in S1-4, the inoculation amount of the bacterial liquid of Bifidobacterium animalis subsp. lactis is 3-8 vol%.
[0015] In a specific implementation scheme, in S4, S3 is repeated twice; The oxygen content of the S2 culture environment, the oxygen content of the S3 culture environment and the oxygen content of the S4 culture environment are arranged in a gradient from low to high.
[0016] By adopting the above technical solution, the oxygen content of the culture environment is arranged in a gradient from low to high, and this setting is conducive to the gradual domestication of animal Bifidobacterium lactis subspecies, thereby obtaining animal Bifidobacterium lactis subspecies with more ideal performance.
[0017] In a specific implementation scheme, the oxygen content of the S2 culture environment, the oxygen content of the S3 culture environment and the oxygen content of the S4 culture environment are 5 vol%, 10 vol%, 15 vol%, 21 vol% respectively; or are 3 vol%, 9 vol%, 15 vol%, 21 vol% respectively; or are 2 vol%, 8 vol%, 14 vol%, 21 vol% respectively.
[0018] In a second aspect, an incubator suitable for the above-mentioned method for culturing and screening animal Bifidobacterium lactis subspecies is provided, which adopts the following technical solution: The incubator comprises a box body, an air inlet pipeline, an air outlet pipeline and a temperature and humidity controller; the box body has a culture chamber; The air inlet pipeline and the air outlet pipeline are respectively connected to the culture chamber; one end of the air inlet pipeline away from the culture chamber is connected to a plurality of branch pipes, a partition is arranged in the air inlet pipeline, and a through hole is provided on the partition; The temperature and humidity controller is used to control the temperature and humidity in the culture room.
[0019] By adopting the above technical solution, various oxygen-containing environments can be provided, which is conducive to the domestication of animal Bifidobacterium lactis subspecies. At the same time, a partition with through holes is arranged in the air intake pipeline, which is conducive to the uniform mixing of gases from different branches in the air intake pipeline, so that the atmosphere entering the culture room is uniform, ensuring the effect of cultivating and domesticating animal Bifidobacterium lactis subspecies.
[0020] In a specific implementation manner, the box body is further provided with an operation hole for gloves.
[0021] By adopting the above technical solution, the operator can operate the sample placed in the culture chamber without opening the box, thereby improving the effect of culturing and domesticating animal Bifidobacterium lactis subspecies.
[0022] In summary, the present application has at least one of the following beneficial technical effects: 1. The animal Bifidobacterium lactis subspecies obtained in the present application has obvious growth properties in an aerobic environment, indicating that the oxygen tolerance of the animal Bifidobacterium lactis subspecies obtained in the present application is significantly improved.
[0023] 2. The present application provides step-by-step acclimation of animal Bifidobacterium lactis subsp. in an environment with gradually increasing oxygen content, so that the obtained animal Bifidobacterium lactis subsp. strain has obvious oxygen tolerance. Compared with screening and acclimation in a single oxygen-containing environment, the oxygen tolerance of animal Bifidobacterium lactis subsp. obtained by step-by-step acclimation is more ideal.
[0024] 3. In the present application, the oxygen content of the culture environment is set to be arranged in a gradient from low to high, which is conducive to the gradual domestication of animal Bifidobacterium lactis subspecies, thereby obtaining animal Bifidobacterium lactis subspecies with more ideal performance.
[0025] 4. The incubator of the present application can provide various oxygen-containing environments, which are conducive to the domestication of animal Bifidobacterium lactis subspecies. At the same time, a partition with through holes is arranged in the air inlet pipeline of the incubator, which is conducive to the uniform mixing of gases from different branches in the air inlet pipeline, so that the atmosphere entering the culture room is uniform, ensuring the effect of cultivating and domesticating animal Bifidobacterium lactis subspecies. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 This is a schematic diagram of the appearance of the incubator of Example 11 of the present application.
[0027] Figure 2 It is a cross-sectional schematic diagram of the intake pipe of Example 11 of the present application.
[0028] Explanation of the reference numerals: 1. Box; 11. Culture chamber; 12. Operation hole; 13. Shaker; 14. Sample access door; 15. Observation window; 2. Air inlet pipe; 21. Partition; 22. Through hole; 23. Nitrogen branch pipe; 24. Oxygen branch pipe; 25. Air branch pipe; 26. Flow meter; 3. Air outlet pipe; 4. Temperature and humidity controller; 41. Humidifier. DETAILED DESCRIPTION
[0029] The following is combined with Figure 1-2 The present application is further described in detail with reference to the accompanying drawings and embodiments.
[0030] The materials used in the embodiments and comparative examples are: The original Bifidobacterium animalis subsp. Lactis is a lactic acid bacterium isolated from the feces of healthy infants; the strain was deposited in the General Microbiology Center of the China Culture Collection Administration on June 5, 2024, with the deposit number CGMCC No. 30870, and the classification name is Bifidobacterium animalis subsp. Lactis, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101.
[0031] Moringa seed extract was purchased from Shaanxi Chenxi Biotechnology Co., Ltd.; peptone was derived from potato and the brand was Millipore.
[0032] Example 1 This embodiment discloses a method for culturing and screening animal Bifidobacterium lactis subspecies, comprising the following steps: S0: Take 8 g of hydrolyzed casein, 8 g of peptone, 3.5 g of glucose, 0.8 g of L-cysteine, 8 g of Moringa seed extract, 6 g of triammonium citrate, 2 g of dipotassium bicarbonate, 0.8 g of magnesium sulfate, 0.4 g of calcium chloride, 0.4 g of potassium carbonate, and 1.3 g of Tween 80, add them to 1 L of deionized water, stir evenly, and adjust the pH value to 6.5 to obtain a liquid culture medium.
[0033] S1: Take a blue-mouth bottle, add 100 mL of the liquid culture medium obtained in S0, and inoculate the original animal Bifidobacterium lactis subsp. lactis strain into the liquid culture medium at an inoculation rate of 5 vol%; then place the liquid culture medium inoculated with animal Bifidobacterium lactis subsp. lactis in an incubator in a culture room with a working atmosphere of nitrogen (i.e., anaerobic), and culture it at 37°C (±0.5°C) for 12 hours.
[0034] S2: Take a 96-well plate and add 1 mL of the liquid culture medium obtained in S0 to each well; then inoculate the animal Bifidobacterium lactis subsp. lactis obtained in S1 into each well of the 96-well plate at an inoculation rate of 5 vol%; then place the 96-well plate in an incubator with an oxygen content of 5 vol% in the culture chamber, and culture at 37°C (±0.5°C) and 100 r / min for 12 h; then detect the OD of each well 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4 After gradient dilution, the samples were mixed with 45°C agar (inoculation amount 5 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator with an oxygen content of 5 vol% in the culture room, and cultured at 37°C (±0.5°C) and 100 r / min for 8 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing the liquid culture medium obtained from S0 (inoculation amount 5 vol%), and then placed in an incubator with an oxygen content of 5 vol% in the culture room, and cultured at 37°C (±0.5°C) and 100 r / min for 6 h.
[0035] S3: The animal Bifidobacterium lactis subsp. lactis obtained in S2 was inoculated into each well of a 96-deep-well plate at an inoculation volume of 5 vol%, and each well contained 1 mL of liquid culture medium; the 96-deep-well plate was then placed in an incubator with an oxygen content of 10 vol% in the culture chamber, and cultured at 37°C (±0.5°C) and 100 r / min for 12 h; the OD of each well was then detected. 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4After gradient dilution, the samples were mixed with 45°C agar (inoculation amount 5 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator with an oxygen content of 10 vol% in the culture room, and cultured at 37°C (±0.5°C) and 100 r / min for 8 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing a liquid culture medium obtained from S0 (inoculation amount 5 vol%), and then placed in an incubator with an oxygen content of 10 vol% in the culture room, and cultured at 37°C (±0.5°C) and 100 r / min for 6 h.
[0036] S4: S4-1. Inoculate the animal Bifidobacterium lactis subsp. lactis obtained in S3 into each well of a 96-deep-well plate at an inoculation volume of 5 vol%, with each well containing 1 mL of liquid culture medium; then place the 96-deep-well plate in an incubator with an oxygen content of 15 vol% in the culture chamber, and culture at 37°C (±0.5°C) and 100 r / min for 12 h; then detect the OD of each well. 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4 After gradient dilution, the samples were mixed with 45°C agar (inoculation amount 5 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator with an oxygen content of 15 vol% in the culture room, and cultured at 37°C (±0.5°C) and 100 r / min for 8 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing the liquid culture medium obtained from S0 (inoculation amount 5 vol%), and then placed in an incubator with an oxygen content of 15 vol% in the culture room, and cultured at 37°C (±0.5°C) and 100 r / min for 6 h.
[0037] S4-2, inoculate the animal Bifidobacterium lactis subsp. lactis obtained in S4-1 into each well of a 96-deep-well plate at an inoculation volume of 5 vol%, each well containing 1 mL of liquid culture medium; then place the 96-deep-well plate in an incubator in a culture room with an air working atmosphere (i.e., an oxygen content of 21 vol%), and culture at 37°C (±0.5°C) and 100 r / min for 12 h; then detect the OD of each well 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4After gradient dilution, the obtained products were mixed with 45°C agar (inoculation amount 5 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained. The agar plate was placed in an incubator in a culture room with a working atmosphere of air (i.e., an oxygen content of 21 vol%), and cultured at 37°C (±0.5°C) and 100 r / min for 8 h. After that, a single colony of animal Bifidobacterium lactis subsp. lactis with good growth potential was selected and placed in a plate containing a liquid culture medium obtained from S0 (inoculation amount 5 vol%), and then placed in an incubator in a culture room with a working atmosphere of air (i.e., an oxygen content of 21 vol%), and cultured at 37°C (±0.5°C) and 100 r / min for 6 h.
[0038] Embodiment 2-4 Example 2-4 is basically the same as Example 1, except that in the method for culturing and screening animal Bifidobacterium lactis subsp. lactis, the culturing time of S2-4 is different, as shown in Table 1 for details.
[0039] Table 1 Cultivation time of S2-4 in the cultivation and screening method of Examples 1-4 Example 5 This embodiment is basically the same as the embodiment 3, except that in the method for culturing and screening animal Bifidobacterium lactis subsp. lactis, the composition ratio of the liquid culture medium S0 is different.
[0040] Specifically: S0: Take 10 g of hydrolyzed casein, 5 g of peptone, 6.5 g of glucose, 0.2 g of L-cysteine, 4 g of Moringa seed extract, 2 g of triammonium citrate, 0.5 g of dipotassium bicarbonate, 0.3 g of magnesium sulfate, 1 g of calcium chloride, 0.1 g of potassium carbonate, and 0.8 g of Tween 80, add them to 1 L of deionized water, stir well, and adjust the pH value to 7.0 to obtain a liquid culture medium.
[0041] Example 6 This embodiment is basically the same as the embodiment 3, except that in the method for culturing and screening animal Bifidobacterium lactis subsp. lactis, the composition ratio of the liquid culture medium S0 is different.
[0042] Specifically: S0: Take 9 g of hydrolyzed casein, 7 g of peptone, 5 g of glucose, 0.5 g of L-cysteine, 6 g of Moringa seed extract, 4 g of triammonium citrate, 1.2 g of dipotassium bicarbonate, 0.6 g of magnesium sulfate, 0.7 g of calcium chloride, 0.3 g of potassium carbonate, and 1 g of Tween 80, add them to 1 L of deionized water, stir evenly, and adjust the pH value to 7.0 to obtain a liquid culture medium.
[0043] Example 7 This embodiment is basically the same as embodiment 6, except that in the method for culturing and screening animal Bifidobacterium lactis subsp. lactis, some process parameters of S1 are different.
[0044] Specifically: S1: Take a blue-mouth bottle, add 100 mL of the liquid culture medium obtained in S0, and inoculate the original animal Bifidobacterium lactis subsp. lactis strain into the liquid culture medium at an inoculation rate of 3 vol%; then place the liquid culture medium inoculated with animal Bifidobacterium lactis subsp. lactis in an incubator in a culture room with a working atmosphere of nitrogen (i.e., anaerobic), and culture it at 37°C (±0.5°C) for 14 hours.
[0045] Example 8 This embodiment is basically the same as embodiment 6, except that in the method for culturing and screening animal Bifidobacterium lactis subsp. lactis, some process parameters of S1 are different.
[0046] Specifically: S1: Take a blue-mouth bottle, add 100 mL of the liquid culture medium obtained in S0, and inoculate the original animal Bifidobacterium lactis subsp. lactis strain into the liquid culture medium at an inoculation rate of 8 vol%; then place the liquid culture medium inoculated with animal Bifidobacterium lactis subsp. lactis in an incubator in a culture room with a working atmosphere of nitrogen (i.e., anaerobic), and culture it at 37°C (±0.5°C) for 10 hours.
[0047] Example 9 This embodiment is basically the same as embodiment 6, except that in the method for culturing and screening animal Bifidobacterium lactis subsp. lactis, some process parameters of S2-4 are different.
[0048] Specifically: S2: Take a 96-well plate and add 1 mL of the liquid culture medium obtained in S0 to each well; then inoculate the animal Bifidobacterium lactis subsp. lactis obtained in S1 into each well of the 96-well plate at an inoculation rate of 3 vol%; then place the 96-well plate in an incubator with an oxygen content of 3 vol% in the culture chamber, and culture at 37°C (±0.5°C) and 120 r / min for 18 h; then detect the OD of each well. 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4After gradient dilution, the samples were mixed with 45°C agar (inoculation amount 3 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator with an oxygen content of 3 vol% in the culture room, and cultured at 37°C (±0.5°C) and 120 r / min for 16 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing a liquid culture medium obtained from S0 (inoculation amount 3 vol%), and then placed in an incubator with an oxygen content of 3 vol% in the culture room, and cultured at 37°C (±0.5°C) and 120 r / min for 12 h.
[0049] S3: The animal Bifidobacterium lactis subsp. lactis obtained in S2 was inoculated into each well of a 96-deep-well plate at an inoculation volume of 3 vol%, and each well contained 1 mL of liquid culture medium; the 96-deep-well plate was then placed in an incubator with an oxygen content of 9 vol% in the culture chamber, and cultured at 37°C (±0.5°C) and 120 r / min for 18 h; the OD of each well was then detected. 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4 After gradient dilution, the samples were mixed with 45°C agar (inoculation amount 3 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator with an oxygen content of 9 vol% in the culture room, and cultured at 37°C (±0.5°C) and 120 r / min for 16 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing a liquid culture medium obtained from S0 (inoculation amount 3 vol%), and then placed in an incubator with an oxygen content of 9 vol% in the culture room, and cultured at 37°C (±0.5°C) and 120 r / min for 12 h.
[0050] S4: S4-1. Inoculate the animal Bifidobacterium lactis subsp. lactis obtained in S3 into each well of a 96-deep-well plate at an inoculation volume of 3 vol%, with each well containing 1 mL of liquid culture medium; then place the 96-deep-well plate in an incubator with an oxygen content of 15 vol% in the culture chamber, and culture at 37°C (±0.5°C) and 120 r / min for 18 h; then detect the OD of each well. 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4After gradient dilution, the samples were mixed with 45°C agar (inoculation amount 3 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator with an oxygen content of 15 vol% in the culture room, and cultured at 37°C (±0.5°C) and 120 r / min for 16 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing the liquid culture medium obtained from S0 (inoculation amount 3 vol%), and then placed in an incubator with an oxygen content of 15 vol% in the culture room, and cultured at 37°C (±0.5°C) and 120 r / min for 12 h.
[0051] S4-2, inoculate the animal Bifidobacterium lactis subsp. lactis obtained in S4-1 into each well of a 96-deep-well plate at an inoculation volume of 3 vol%, each well containing 1 mL of liquid culture medium; then place the 96-deep-well plate in an incubator in a culture room with an air working atmosphere (i.e., an oxygen content of 21 vol%), and culture at 37°C (±0.5°C) and 120 r / min for 18 h; then detect the OD of each well 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4 After gradient dilution, the mixture was mixed with 45°C agar (inoculation amount 3 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator in a culture room with a working atmosphere of air (i.e., an oxygen content of 21 vol%), and cultured at 37°C (±0.5°C) and 120 r / min for 16 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing a liquid culture medium obtained from S0 (inoculation amount 3 vol%), and then placed in an incubator in a culture room with a working atmosphere of air (i.e., an oxygen content of 21 vol%), and cultured at 37°C (±0.5°C) and 120 r / min for 12 h.
[0052] Example 10 This embodiment is basically the same as embodiment 6, except that in the method for culturing and screening animal Bifidobacterium lactis subsp. lactis, some process parameters of S2-4 are different.
[0053] Specifically: S2: Take a 96-well plate and add 1 mL of the liquid culture medium obtained in S0 to each well; then inoculate the animal Bifidobacterium lactis subsp. lactis obtained in S1 into each well of the 96-well plate at an inoculation rate of 8 vol%; then place the 96-well plate in an incubator with an oxygen content of 2 vol% in the culture chamber, and culture at 37°C (±0.5°C) and 80 r / min for 18 h; then detect the OD of each well. 600 , select the hole with the highest OD value;-6 , 10 -5 , 10 -4 After gradient dilution, the mixture was mixed with 45°C agar (inoculation amount 8 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator with an oxygen content of 2 vol% in the culture room, and cultured at 37°C (±0.5°C) and 80 r / min for 16 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing the liquid culture medium obtained from S0 (inoculation amount 8 vol%), and then placed in an incubator with an oxygen content of 2 vol% in the culture room, and cultured at 37°C (±0.5°C) and 80 r / min for 12 h.
[0054] S3: The animal Bifidobacterium lactis subsp. lactis obtained in S2 was inoculated into each well of a 96-deep-well plate at an inoculation volume of 8 vol%, and each well contained 1 mL of liquid culture medium; the 96-deep-well plate was then placed in an incubator with an oxygen content of 8 vol% in the culture chamber, and cultured at 37°C (±0.5°C) and 80 r / min for 18 h; the OD of each well was then detected. 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4 After gradient dilution, the mixture was mixed with 45°C agar (inoculation amount 8 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator with an oxygen content of 8 vol% in the culture room, and cultured at 37°C (±0.5°C) and 80 r / min for 16 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing the liquid culture medium obtained from S0 (inoculation amount 8 vol%), and then placed in an incubator with an oxygen content of 8 vol% in the culture room, and cultured at 37°C (±0.5°C) and 80 r / min for 12 h.
[0055] S4: S4-1. Inoculate the animal Bifidobacterium lactis subsp. lactis obtained in S3 into each well of a 96-deep-well plate at an inoculation volume of 8 vol%, with each well containing 1 mL of liquid culture medium; then place the 96-deep-well plate in an incubator with an oxygen content of 14 vol% in the culture chamber, and culture at 37°C (±0.5°C) and 80 r / min for 18 h; then detect the OD of each well. 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4After gradient dilution, the mixture was mixed with 45°C agar (inoculation amount 8 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator with an oxygen content of 14 vol% in the culture room, and cultured at 37°C (±0.5°C) and 80 r / min for 16 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing the liquid culture medium obtained from S0 (inoculation amount 8 vol%), and then placed in an incubator with an oxygen content of 14 vol% in the culture room, and cultured at 37°C (±0.5°C) and 80 r / min for 12 h.
[0056] S4-2, inoculate the animal Bifidobacterium lactis subsp. lactis obtained in S4-1 into each well of a 96-deep-well plate at an inoculation volume of 8 vol%, each well containing 1 mL of liquid culture medium; then place the 96-deep-well plate in an incubator in a culture room with an air working atmosphere (i.e., an oxygen content of 21 vol%), and culture at 37°C (±0.5°C) and 80 r / min for 18 h; then detect the OD of each well. 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4 After gradient dilution, the mixture was mixed with 45°C agar (inoculation amount 8 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained; the agar plate was placed in an incubator in a culture room with a working atmosphere of air (i.e., an oxygen content of 21 vol%), and cultured at 37°C (±0.5°C) and 80 r / min for 16 h; then a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing a liquid culture medium obtained from S0 (inoculation amount 8 vol%), and then placed in an incubator in a culture room with a working atmosphere of air (i.e., an oxygen content of 21 vol%), and cultured at 37°C (±0.5°C) and 80 r / min for 12 h.
[0057] Comparative Example This comparative example discloses a method for culturing and screening animal Bifidobacterium lactis subspecies, comprising the following steps: S0: Take 8 g of hydrolyzed casein, 8 g of peptone, 3.5 g of glucose, 0.8 g of L-cysteine, 8 g of Moringa seed extract, 6 g of triammonium citrate, 2 g of dipotassium bicarbonate, 0.8 g of magnesium sulfate, 0.4 g of calcium chloride, 0.4 g of potassium carbonate, and 1.3 g of Tween 80, add them to 1 L of deionized water, stir evenly, and adjust the pH value to 6.5 to obtain a liquid culture medium.
[0058] S1: Take a blue-mouth bottle, add 100 mL of the liquid culture medium obtained in S0, and inoculate the original animal Bifidobacterium lactis subsp. lactis strain into the liquid culture medium at an inoculation rate of 5 vol%; then place the liquid culture medium inoculated with animal Bifidobacterium lactis subsp. lactis in an incubator in a culture room with a working atmosphere of nitrogen (i.e., anaerobic), and culture it at 37°C (±0.5°C) for 12 hours.
[0059] S2: Take a 96-well plate and add 1 mL of the liquid culture medium obtained in S0 to each well; then inoculate the animal Bifidobacterium lactis subsp. lactis obtained in S1 into each well of the 96-well plate at an inoculation rate of 5 vol%; then place the 96-well plate in an incubator filled with air in the culture chamber, and culture at 37°C (±0.5°C) and 100 r / min for 12 h; then detect the OD of each well 600 , select the hole with the highest OD value; -6 , 10 -5 , 10 -4 After gradient dilution, the samples were mixed with 45°C agar (inoculation amount 5 vol%), and then poured into a sterilized culture dish. After the agar solidified, an agar plate was obtained. The agar plate was placed in an incubator filled with air in the culture room, and cultured at 37°C (±0.5°C) with shaking at 100 r / min for 8 h. Then, a single colony of animal Bifidobacterium lactis subsp. lactis with good growth was selected and placed in a plate containing the liquid culture medium obtained from S0 (inoculation amount 5 vol%), and then placed in an incubator filled with air in the culture room, and cultured at 37°C (±0.5°C) with shaking at 100 r / min for 6 h.
[0060] Performance Testing The animal Bifidobacterium lactis subspecies obtained in Examples 1-10 and the comparative example and the unscreened original animal Bifidobacterium lactis subspecies (recorded as blank samples) were inoculated into the liquid culture medium disclosed in Example 6 at an inoculation amount of 5 vol%, and then the liquid culture medium was placed in an incubator filled with air in the culture chamber, and cultured at 37°C (±0.5°C) and 100 r / min for 36 hours; the obtained bacterial liquid was tested, and the test results are listed in Table 2.
[0061] Absorbance (OD value) determination: 200 μL of each bacterial solution was taken into a 96-well ELISA plate, and the absorbance of the bacterial solution was measured at 600 nm using an ultraviolet spectrophotometer. The higher the absorbance, the higher the bacterial density in the bacterial solution, that is, the better the growth of animal Bifidobacterium lactis subsp.
[0062] Table 2 OD values of bacterial solutions obtained in Examples 1-10 and Comparative Examples and original bacterial solutions project OD value Example 1 0.428 Example 2 0.563 Example 3 0.652 Example 4 0.683 Example 5 0.623 Example 6 0.696 Example 7 0.677 Example 8 0.683 Example 9 0.661 Example 10 0.685 Comparative Example 0.319 Blank 0.026 Referring to Table 2, it can be found from the test results of Examples 1-10 that the animal Bifidobacterium lactis subspecies obtained by culturing and screening in each embodiment of the present application has obvious growth in an aerobic environment; in particular, compared with the original animal Bifidobacterium lactis subspecies strain, the aerobic growth of the animal Bifidobacterium lactis subspecies obtained in the present application is significantly higher, indicating that the oxygen tolerance of the animal Bifidobacterium lactis subspecies obtained by culturing and screening in each embodiment of the present application is significantly improved.
[0063] From the test results of Example 1 and Comparative Example 1, it can be found that the oxygen tolerance of the obtained animal Bifidobacterium lactis subsp. lactis is more ideal when the oxygen content of the culture environment is gradually increased by adopting a step-by-step gradient acclimation method compared with direct air acclimation.
[0064] From the test results of Examples 1-4, it can be found that the extension of the culture time is conducive to obtaining Bifidobacterium animalis subsp. lactis with more ideal performance.
[0065] Embodiment 11 This embodiment discloses an incubator suitable for embodiments 1-10, referring to Figure 1 The incubator includes a box body 1, an air inlet pipeline 2, an air outlet pipeline 3 and a temperature and humidity controller 4.
[0066] Reference Figure 1 The box 1 has a closed culture chamber 11 inside. A shaking table 13 is provided in the culture chamber 11 for placing a culture container containing the strain to be cultured. At the same time, a sample access door 14 is provided on the side wall of the box 1 to connect the culture chamber 11 with the outside world, and is used to put the sample into the culture chamber 11 or take the sample out of the culture chamber 11; an observation window 15 is also provided on the side wall of the box 1 to facilitate real-time observation of the culture of the strain.
[0067] Reference Figure 1 The air inlet pipeline 2 and the air outlet pipeline 3 are respectively arranged on opposite sides of the box body 1 and are both connected to the culture chamber 11; the air inlet pipeline 2 is used to introduce the working atmosphere into the culture chamber 11, and the air outlet pipeline 3 is connected to the external fan to exhaust the working atmosphere from the culture chamber 11.
[0068] See also Figure 1 One end of the air inlet pipe 2 is connected to the culture chamber 11; the other end is connected to the nitrogen branch pipe 23, the oxygen branch pipe 24 and the air branch pipe 25 at the same time; the nitrogen branch pipe 23, the oxygen branch pipe 24 and the air branch pipe 25 are connected to the nitrogen source, the oxygen source and the air source respectively. At the same time, in order to accurately control the flow rate of each gas, a flow meter 26 is provided on the nitrogen branch pipe 23, the oxygen branch pipe 24 and the air branch pipe 25.
[0069] Reference Figure 2, a plurality of partitions 21 are arranged inside the air intake pipeline 2 to cut off the air intake pipeline 2 transversely, and a plurality of through holes 22 are provided on the partitions 21. Through the above arrangement, the instantaneous speed of the gas from each branch pipe will be accelerated when passing through the through hole 22; according to the Bernoulli principle, the instantaneous pressure of this part of the gas will drop, so that there will be a pressure difference between this part of the gas and the gas in other parts of the air intake pipeline 2, so that the gas will have convection, thereby increasing the agitation of the gas in the air intake pipeline 2, so that the gas from different branches is evenly mixed in the air intake pipeline 2, and the culture effect is guaranteed.
[0070] Reference Figure 1 The temperature and humidity controller 4 is installed on the box body 1 to control the heating wire (not shown) installed in the wall of the box body 1, thereby controlling the temperature in the culture chamber 11; at the same time, the temperature and humidity controller 4 can also control the humidifier 41 installed on the inner wall of the culture chamber 11, thereby adjusting the humidity in the culture chamber 11 when necessary.
[0071] The implementation principle of the incubator of this embodiment is: The strain to be cultured is placed in the culture chamber 11 through the sample access door 14 .
[0072] The gas in the culture chamber 11 is evacuated through the gas outlet pipe 3; then, according to the requirement for the oxygen content in the working atmosphere in the culture chamber 11, a corresponding amount of oxygen, nitrogen or air is introduced; these gases enter the culture chamber 11 after being fully mixed in the gas inlet pipe 2.
[0073] At the same time, the temperature and humidity controller 4 is started to make the culture chamber 11 reach the ideal temperature.
[0074] Thereafter, as required, the shaking table 13 is turned on for shaking culture or the shaking table 13 is kept off for static culture.
[0075] In addition, refer to Figure 1 , so that the operator can operate the sample placed in the culture chamber 11 without opening the box 1, thereby improving the culture and acclimatization effect; in some embodiments, the side wall of the box 1 is also symmetrically provided with two operation holes 12 with gloves.
[0076] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.
Claims
1. A method for culturing and screening animal Bifidobacterium lactis subspecies, characterized in that: include: S1, anaerobic culture of Bifidobacterium animalis subsp. lactis; S2, dividing the bacterial solution of animal Bifidobacterium lactis subsp. obtained in S1 into multiple portions and culturing them in an aerobic culture environment; selecting the bacterial solution of animal Bifidobacterium lactis subsp. with good growth, making agar plates after gradient dilution, and then culturing them in the same aerobic culture environment as before in this step; then selecting a single colony of animal Bifidobacterium lactis subsp. with good growth, and culturing them again in the same aerobic culture environment as before in this step; S3, dividing the bacterial solution of animal Bifidobacterium lactis subsp. obtained in the previous step into multiple portions and culturing them in a culture environment with an oxygen content higher than that in the previous step; selecting the bacterial solution of animal Bifidobacterium lactis subsp. with good growth, making agar plates after gradient dilution, and then culturing them in the same aerobic culture environment as before in this step; then selecting a single colony of animal Bifidobacterium lactis subsp. with good growth, and culturing them again in the same aerobic culture environment as before in this step; S4, repeating S3 several times to obtain a strain of Bifidobacterium animalis subsp. lactis with obvious oxygen resistance.
2. The method for culturing and screening animal Bifidobacterium lactis subspecies according to claim 1, characterized in that: include: S0, prepare liquid culture medium; S1, inoculating animal Bifidobacterium lactis subsp. lactis into the liquid culture medium obtained in S0; Then, culture in an anaerobic environment for 10-14 hours; S2, the bacterial solution of animal Bifidobacterium lactis subsp. obtained in S1 is divided into multiple portions and inoculated into the liquid culture medium obtained in S0; then placed in a culture environment with an oxygen content of 2-5 vol% and cultured for 12-20 h; the bacterial solution of animal Bifidobacterium lactis subsp. with good growth is selected, and agar plates are prepared after gradient dilution, and then placed in the same environment as the liquid culture medium in this step and cultured for 8-18 h; then a single colony of animal Bifidobacterium lactis subsp. with good growth is selected, moved into the liquid culture medium obtained in S0, and then placed in the same environment as the agar plate culture in this step and cultured for 6-15 h; S3, the bacterial solution of animal Bifidobacterium lactis subsp. obtained in the previous step is divided into multiple portions and inoculated into the liquid culture medium obtained in the S0; then cultured in a culture environment with an oxygen content higher than that in the previous step for 12-20 hours; the bacterial solution of animal Bifidobacterium lactis subsp. with good growth is selected, and an agar plate is prepared after gradient dilution, and then placed in the same environment as the liquid culture medium in this step for 8-18 hours; then a single colony of animal Bifidobacterium lactis subsp. with good growth is selected, and moved into the liquid culture medium obtained in S0, and then placed in the same environment as the agar plate culture in this step for 6-15 hours; S4, repeating S3 several times to obtain a strain of Bifidobacterium animalis subsp. lactis with obvious oxygen resistance.
3. The method for culturing and screening animal Bifidobacterium lactis subspecies according to claim 2, characterized in that: In the S0, the liquid culture medium includes 8-10 parts by mass of hydrolyzed casein, 5-8 parts by mass of peptone, 3.5-6.5 parts by mass of glucose, 0.2-0.8 parts by mass of L-cysteine, 4-8 parts by mass of Moringa seed extract, 2-6 parts by mass of triammonium citrate, 0.5-2 parts by mass of dipotassium bicarbonate, 0.3-0.8 parts by mass of magnesium sulfate, 0.4-1 parts by mass of calcium chloride, 0.1-0.4 parts by mass of potassium carbonate, 0.8-1.3 parts by mass of Tween 80 and 1000 parts by mass of water; The pH value of the liquid culture medium is 6.5-7.
0.
4. The method for culturing and screening animal Bifidobacterium lactis subspecies according to claim 2, characterized in that: In the S1, the culture is carried out in a static manner, and the culture temperature is 36.5-37.5°C.
5. The method for culturing and screening animal Bifidobacterium lactis subspecies according to claim 2, characterized in that: In the S2-3, the culture is carried out by shaking, the shaking speed is 80-120r / min, and the culture temperature is 36.5-37.5°C.
6. The method for culturing and screening animal Bifidobacterium lactis subspecies according to claim 2, characterized in that: In S1-4, the inoculation amount of the animal Bifidobacterium lactis subsp. lactis liquid is 3-8 vol%.
7. The method for culturing and screening animal Bifidobacterium lactis subspecies according to any one of claims 1 to 6, characterized in that: In S4, S3 is repeated twice; The oxygen content of the S2 culture environment, the oxygen content of the S3 culture environment and the oxygen content of the S4 culture environment are arranged in a gradient from low to high.
8. The method for culturing and screening animal Bifidobacterium lactis subspecies according to claim 7, characterized in that: The oxygen content of the S2 culture environment, the oxygen content of the S3 culture environment and the oxygen content of the S4 culture environment are 5 vol%, 10 vol%, 15 vol%, 21 vol% respectively; or 3 vol%, 9 vol%, 15 vol%, 21 vol% respectively; or 2 vol%, 8 vol%, 14 vol%, 21 vol% respectively.
9. An incubator suitable for the method for culturing and screening animal Bifidobacterium lactis subspecies according to any one of claims 1 to 8, characterized in that: It comprises a box (1), an air inlet pipeline (2), an air outlet pipeline (3) and a temperature and humidity controller (4); the box (1) has a culture chamber (11); The air inlet pipeline (2) and the air outlet pipeline (3) are respectively connected to the culture chamber (11); one end of the air inlet pipeline (2) away from the culture chamber (11) is connected to a plurality of branch pipes; a partition (21) is provided in the air inlet pipeline (2), and a through hole (22) is provided on the partition (21); The temperature and humidity controller (4) is used to control the temperature and humidity in the culture chamber (11).
10. The incubator according to claim 9, characterized in that: The box body (1) is also provided with an operating hole (12) for gloves.