A method for cultivating oxygen-resistant Lactobacillus acidophilus

Abstract

The present invention relates to a method for culturing an oxygen-resistant Lactobacillus acidophilus, the method comprising: culturing the activated Lactobacillus acidophilus in a microbial droplet culture instrument, using hydrogen peroxide as an oxygen stress factor in the culture to obtain Lactobacillus acidophilus droplets; purifying the Lactobacillus acidophilus droplets, inoculating them into a fermentation medium after activation, and fermenting them under the condition of introducing air. The method described in the present application can improve the domestication efficiency and accuracy of Lactobacillus acidophilus, reduce the feedback inhibition of metabolites on lactic acid bacteria, and improve the tolerance of lactic acid bacteria to oxygen, so as to achieve the purpose of high-density fermentation under aerobic conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a method for culturing oxygen-resistant Lactobacillus acidophilus. Background Art

[0002] Lactic acid bacteria (LAB) is a general term for bacteria that can produce large amounts of lactic acid using fermentable carbohydrates. It is a group of bacteria with similar metabolic types but very different morphological and physiological characteristics. It has the functions of regulating intestinal flora, improving constipation and diarrhea, and enhancing immunity.

[0003] High-density culture of lactic acid bacteria refers to the use of certain culture conditions and suitable production reactors to increase the fermentation density of the bacteria. At present, most lactic acid bacteria grow under anaerobic or facultative anaerobic conditions. Excessive oxygen has certain toxic side effects on its growth and survival. Because it lacks superoxide dismutase (SOD) and catalase (or peroxidase), its anti-oxidative stress mechanism is imperfect and cannot resist the oxidative effect of oxygen, resulting in protein and DNA damage and cell membrane damage, leading to cell death. Therefore, high-density culture of lactic acid bacteria is generally carried out under anaerobic or hypoxic conditions, which will lead to a certain restriction on the overall bacterial volume. The large amount of metabolic organic acid products (such as lactic acid, acetic acid, formic acid, etc.) produced by anaerobic fermentation will seriously inhibit the growth of the bacteria. Aerobic fermentation can change the metabolic pathway of lactic acid bacteria and reduce the production of organic acids. At present, the conventional way to improve the oxygen tolerance of lactic acid bacteria is through oxygen-assisted domestication. This method is relatively traditional, time-consuming and labor-intensive, and cannot efficiently select strains that can tolerate oxygen. At the same time, when conducting high-density aerobic fermentation, how to further reduce the damage caused by oxygen to the activity of the bacteria is still a problem that needs to be solved urgently. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for culturing oxygen-resistant Lactobacillus acidophilus.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for culturing an aerobic Lactobacillus acidophilus, the method comprising:

[0007] (1) culturing the activated Lactobacillus acidophilus in a microbial droplet culture apparatus, using hydrogen peroxide as an oxygen stress factor during the culture to obtain Lactobacillus acidophilus droplets;

[0008] (2) Purify the Lactobacillus acidophilus droplets, activate them, inoculate them into a fermentation medium, and ferment them under the condition of air flow to obtain the product.

[0009] The present application firstly passes Lactobacillus acidophilus through a high-throughput microbial droplet culture instrument to perform adaptive evolutionary domestication of the lactic acid bacteria under suitable conditions. By adopting the high-throughput microbial droplet culture instrument, the lactic acid bacteria are divided into multiple small droplets, from which the droplets with the best growth performance are screened, thereby greatly improving the domestication efficiency and saving frequent manual operations. By adding an aerobic promoter and introducing air, the accumulation of metabolic products of lactic acid bacteria can be changed, the feedback inhibition of metabolic products (such as lactic acid, acetic acid, etc.) on lactic acid bacteria can be reduced, and the tolerance of lactic acid bacteria to oxygen can be improved, and high-density fermentation can be achieved through batch or batch fed fermentation.

[0010] The method described in the present application can improve the efficiency and accuracy of lactic acid bacteria domestication, reduce the feedback inhibition of lactic acid bacteria caused by metabolites (such as lactic acid, acetic acid, etc.), and improve the tolerance of lactic acid bacteria to oxygen, so as to achieve the purpose of high-density fermentation under aerobic conditions.

[0011] Preferably, the concentration gradient of the hydrogen peroxide is 5-10, and the concentration range is 0.01-0.1%.

[0012] The concentrations can be selected from 5, 6, 7, 8, 9, 10, etc., and the concentration range can be selected from 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0013] Preferably, the number of droplets is 5-50, for example 5, 8, 10, 12, 15, 18, 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50, etc. Other specific point values ​​within the above numerical range can be selected, which will not be described here one by one.

[0014] Preferably, the number of repetitions for each concentration is 2-5 times, and the culture time for each generation is independently 8-14 h.

[0015] The number of repetitions can be selected as 2 times, 3 times, 4 times, 5 times, etc., and the time can be selected as 8 h, 8.5 h, 9 h, 9.5 h, 10 h, 10.5 h, 11 h, 11.5 h, 12 h, 12.5 h, 13 h, 13.5 h, 14 h, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0016] Preferably, the ventilation volume of the air is 0.01-0.5 vvm, for example 0.01 vvm, 0.02 vvm, 0.05 vvm, 0.08 vvm, 0.1 vvm, 0.12 vvm, 0.15 vvm, 0.18 vvm, 0.2 vvm, 0.22 vvm, 0.25 vvm, 0.28 vvm, 0.3 vvm, 0.32 vvm, 0.35 vvm, 0.38 vvm, 0.4 vvm, 0.42 vvm, 0.45 vvm, 0.48 vvm, 0.5vvm, etc. Other specific point values ​​within the above numerical range can be selected, which will not be repeated here.

[0017] Preferably, the fermentation medium in step (2) further comprises hemoglobin and vitamin K2.

[0018] Heme and vitamin K2 added to the fermentation medium act as aerobic promoters, which can enhance Lactobacillus acidophilus' tolerance to oxidative stress and the degradation of free radicals, and improve its tolerance to oxygen. Heme and vitamin K2 have potential synergistic effects in the above effects.

[0019] Preferably, the concentration of hemoglobin is 1-4 μg / mL, and the concentration of vitamin K2 is 0.5-2 μg / mL.

[0020] The concentration of hemoglobin can be selected from 1 μg / mL, 1.2 μg / mL, 1.5 μg / mL, 1.8 μg / mL, 2 μg / mL, 2.2 μg / mL, 2.5 μg / mL, 2.8 μg / mL, 3 μg / mL, 3.2 μg / mL, 3.5 μg / mL, 3.8 μg / mL, 4 μg / mL and the like, and the concentration of vitamin K2 can be selected from 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, 1.1 μg / mL, 1.2 μg / mL, 1.3 μg / mL, 1.4 μg / mL, 1.5 μg / mL, 1.6 μg / mL, 1.7 μg / mL, 1.8 μg / mL, 1.9 μg / mL, 2 μg / mL, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0021] Preferably, the pH of the fermentation in step (2) is 5.9-6.1, for example, 5.9, 5.92, 5.94, 5.96, 5.98, 6, 6.02, 6.04, 6.06, 6.08, 6.1, etc. Other specific values ​​within the above numerical range can be selected and will not be described in detail here.

[0022] Preferably, in step (2), when the mass percentage of the carbon source is lower than 0.5%, the carbon source and the nitrogen source are supplemented, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, etc. Other specific values ​​within the above numerical range can be selected and will not be described one by one here.

[0023] Preferably, the total amount of carbon source and nitrogen source added at a single time is 2-6%, for example 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8%, 5%, 5.2%, 5.4%, 5.6%, 5.8%, 6%, etc. Other specific points within the above numerical range can be selected, which will not be repeated here.

[0024] Preferably, the carbon source comprises glucose.

[0025] Preferably, the nitrogen source comprises peptone.

[0026] Preferably, the mass ratio of the additional carbon source to the nitrogen source is (1-3):(2-4).

[0027] The specific point values ​​in (1-3) can be selected as 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, etc. The specific point values ​​in (2-4) can be selected as 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, etc. Other specific point values ​​within the above numerical range can be selected and will not be listed here one by one.

[0028] Preferably, the fermentation temperature in step (2) is 30-37° C. and the fermentation time is 8-20 h.

[0029] The temperature can be selected as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, etc., and the time can be selected as 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present application firstly passes Lactobacillus acidophilus through a high-throughput microbial droplet culture instrument to perform adaptive evolutionary domestication of the lactic acid bacteria under suitable conditions. By adopting the high-throughput microbial droplet culture instrument, the lactic acid bacteria are divided into multiple small droplets, from which the droplets with the best growth performance are screened, thereby greatly improving the domestication efficiency and saving frequent manual operations. By adding an aerobic promoter and introducing air, the accumulation of metabolic products of lactic acid bacteria can be changed, the feedback inhibition of metabolic products (such as lactic acid, acetic acid, etc.) on lactic acid bacteria can be reduced, and the tolerance of lactic acid bacteria to oxygen can be improved, and high-density fermentation can be achieved through batch or batch fed fermentation.

[0032] The method described in the present application can improve the efficiency and accuracy of lactic acid bacteria domestication, reduce the feedback inhibition of lactic acid bacteria caused by metabolites (such as lactic acid, acetic acid, etc.), and improve the tolerance of lactic acid bacteria to oxygen, so as to achieve the purpose of high-density fermentation under aerobic conditions.

[0033] Lactobacillus acidophilus Lactobacillus acidophilus LA85 strain, classified as Lactobacillus acidophilus Lactobacillus acidophilus The deposit date is July 20, 2020, the deposit number is CGMCC No.1.12735, the depositor is the General Microbiology Center of China Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0034] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0035] The hemoglobin involved in the following content is a product purchased from Xi'an Qianyecao Biotechnology Co., Ltd. under the trade name of hemoglobin.

[0036] MRS medium: dissolve 10.00 g of peptone, 10.00 g of beef extract, 5.00 g of yeast extract, 2.00 g of diammonium hydrogen citrate, 20.00 g of glucose, 1 mL of Tween-80, 2.00 g of dipotassium hydrogen phosphate, 0.58 g of manganese sulfate and 0.28 g of magnesium sulfate in 1 L of distilled water, adjust the pH to 6.4, and sterilize.

[0037] MRS solid culture medium: Add 2wt% agar powder to MRS liquid culture medium and sterilize.

[0038] The carbon source involved in the following content is glucose, and the nitrogen source is peptone.

[0039] Preparation Example 1

[0040] This preparation example provides a method for preparing Lactobacillus acidophilus droplets, the preparation method comprising:

[0041] (1) Streak Lactobacillus acidophilus LA85 into MRS solid culture medium, culture anaerobically at 37°C for 48 hours to obtain a single colony, inoculate the prepared single colony into MRS liquid culture medium, and culture at 37°C for 18 hours for activation; transfer the activated bacterial liquid to a liquid separation bottle dedicated to a high-throughput microbial droplet culture instrument by aseptic operation for standby use;

[0042] (2) Turn on the high-throughput microbial droplet culture instrument and set the acclimation conditions: 20 droplets; OD reference wavelength 600 nm; oxygen co-stimulator is hydrogen peroxide, set 8 concentration gradients, the concentration gradient is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.1%, the culture temperature is 37 °C, and culture is performed after 8 h. Each gradient is repeated 5 times, for a total of 40 times. After the culture is completed, record the OD value of each droplet of the last generation.

[0043] Preparation Example 2

[0044] This preparation example provides a method for preparing Lactobacillus acidophilus droplets, the preparation method comprising:

[0045] (1) Streak Lactobacillus acidophilus LA85 into MRS solid culture medium, culture anaerobically at 37°C for 48 hours to obtain a single colony, inoculate the prepared single colony into MRS liquid culture medium, and culture at 37°C for 18 hours for activation; transfer the activated bacterial liquid to a liquid separation bottle dedicated to a high-throughput microbial droplet culture instrument by aseptic operation for standby use;

[0046] (2) Turn on the high-throughput microbial droplet culture instrument and set the acclimation conditions: 20 droplets; OD reference wavelength 600 nm; oxygen co-stimulator is hydrogen peroxide, set 6 concentration gradients, the concentration gradient is 0.01%, 0.03%, 0.05%, 0.06%, 0.08%, 0.1%, the culture temperature is 37°C, and culture is performed after 10 h. Each gradient is repeated 4 times, and the number of subcultures is 24 times. After the culture is completed, record the OD value of each droplet of the last generation.

[0047] Preparation Example 3

[0048] This preparation example provides a method for preparing Lactobacillus acidophilus droplets, the preparation method comprising:

[0049] (1) Streak Lactobacillus acidophilus LA85 into MRS solid culture medium, culture anaerobically at 37°C for 48 hours to obtain a single colony, inoculate the prepared single colony into MRS liquid culture medium, and culture at 37°C for 18 hours for activation; transfer the activated bacterial liquid to a liquid separation bottle dedicated to a high-throughput microbial droplet culture instrument by aseptic operation for standby use;

[0050] (2) Turn on the high-throughput microbial droplet culture instrument and set the acclimation conditions: 20 droplets; OD reference wavelength 600 nm; oxygen co-stimulator is hydrogen peroxide, set 10 concentration gradients, the concentration gradients are 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, the culture temperature is 37°C, and culture is performed after 14 h. Each gradient is repeated 3 times, for a total of 30 times. After the culture is completed, record the OD value of each droplet of the last generation.

[0051] Preparation Example 4

[0052] This preparation example provides a preparation method of Lactobacillus acidophilus droplets, which differs from Preparation Example 1 only in that step (2) is "turn on a high-throughput microbial droplet culture instrument and set the acclimation conditions: 20 droplets; OD reference wavelength 600 nm; oxygen coercion factor hydrogen peroxide, 3 concentration gradients of 0.01%, 0.05%, and 0.1%; culture temperature 37°C; culture for 8 hours before subculturing; each gradient is repeated 5 times, for a total of 15 times; after the culture is completed, record the OD value of each droplet of the last generation". Other operations remain unchanged.

[0053] Example 1

[0054] This embodiment provides a method for culturing Lactobacillus acidophilus, the method comprising:

[0055] (1) Collect the droplet with the highest OD value obtained in Preparation Example 1, perform aseptic streaking operation on MRS solid culture medium, anaerobically culture at 37°C for 48 hours to obtain a single colony, inoculate the prepared single colony into MRS liquid culture medium, culture at 37°C for 18 hours for activation, transfer the activated bacterial liquid to MRS seed culture medium at an inoculum volume of 2%, and culture for 10 hours;

[0056] (2) The seed liquid was inoculated at an inoculum size of 2% into MRS medium containing 2.5 μg / mL heme and 1 μg / mL vitamin K2. The fermentation was carried out under the condition of aeration of 0.3 vvm of air and the stirring speed was 50 rpm. When the fermentation pH dropped below 6, 20% sodium hydroxide solution was added to adjust the pH to 6±0.1. When the carbon source content was lower than 0.5%, the carbon source and nitrogen source were supplemented in a mass ratio of 3:4. The total amount of carbon source and nitrogen source added at a single time was 3%. The fermentation temperature was 37°C and the fermentation time was 18 h.

[0057] Example 2

[0058] This embodiment provides a method for culturing Lactobacillus acidophilus, the method comprising:

[0059] (1) Collect the droplet with the highest OD value obtained in Preparation Example 1, perform aseptic streaking operation on MRS solid culture medium, anaerobically culture at 37°C for 48 hours to obtain a single colony, inoculate the prepared single colony into MRS liquid culture medium, culture at 37°C for 18 hours for activation, transfer the activated bacterial liquid to MRS seed culture medium at an inoculum volume of 2%, and culture for 10 hours;

[0060] (2) The seed liquid was inoculated at an inoculum size of 2% into MRS medium containing 4 μg / mL heme and 0.5 μg / mL vitamin K2. The fermentation was carried out under the condition of aeration of 0.1 vvm of air and the stirring speed was 50 rpm. When the fermentation pH dropped below 6, 20% sodium hydroxide solution was added to adjust the pH to 6±0.1. When the carbon source content was lower than 0.5%, the carbon source and nitrogen source were supplemented in a mass ratio of 2:3. The total amount of carbon source and nitrogen source added at a single time was 3%. The fermentation temperature was 30°C and the fermentation time was 20 h.

[0061] Example 3

[0062] This embodiment provides a method for culturing Lactobacillus acidophilus, the method comprising:

[0063] (1) Collect the droplet with the highest OD value obtained in Preparation Example 1, perform aseptic streaking operation on MRS solid culture medium, anaerobically culture at 37°C for 48 hours to obtain a single colony, inoculate the prepared single colony into MRS liquid culture medium, culture at 37°C for 18 hours for activation, transfer the activated bacterial liquid to MRS seed culture medium at an inoculum volume of 2%, and culture for 10 hours;

[0064] (2) The seed liquid was inoculated at an inoculum size of 2% into MRS medium containing 1 μg / mL heme and 2 μg / mL vitamin K2. The fermentation was carried out under the condition of 0.5 vvm air and the stirring speed was 50 rpm. When the fermentation pH dropped below 6, 20% sodium hydroxide solution was added to adjust the pH to 6±0.1. When the carbon source content was lower than 0.5%, the carbon source and nitrogen source were supplemented in a mass ratio of 1:2. The total amount of carbon source and nitrogen source added at a single time was 3%. The fermentation temperature was 35°C and the fermentation time was 10 h.

[0065] Example 4

[0066] The present embodiment provides a method for culturing Lactobacillus acidophilus, which differs from the embodiment 1 only in that step (2) is "inoculating the seed liquid at an inoculum amount of 2% into an MRS medium containing 3.5 μg / mL heme, fermenting under the condition of passing 0.3 vvm of air, with a stirring speed of 50 rpm, and when the fermentation pH drops below 6, adding 20% ​​sodium hydroxide solution to adjust the pH to 6±0.1, and when the carbon source content is lower than 0.5%, supplementing the carbon source and nitrogen source in a mass ratio of 3:4, the total amount of the carbon source and nitrogen source supplemented at a single time is 3%, the fermentation temperature is 37°C, and the fermentation time is 18 h". Other operations remain unchanged.

[0067] Example 5

[0068] The present embodiment provides a method for culturing Lactobacillus acidophilus, which differs from the embodiment 1 only in that step (2) is "inoculating the seed liquid at an inoculum amount of 2% into an MRS medium containing 3.5 μg / mL vitamin K2, fermenting under the condition of passing 0.3 vvm air, stirring at a speed of 50 rpm, and when the fermentation pH drops below 6, adding 20% ​​sodium hydroxide solution to adjust the pH to 6±0.1, when the carbon source content is lower than 0.5%, supplementing the carbon source and nitrogen source in a mass ratio of 3:4, the total amount of carbon source and nitrogen source supplemented at a single time is 3%, the fermentation temperature is 37°C, and the fermentation time is 18 h". Other operations remain unchanged.

[0069] Example 6

[0070] This embodiment provides a method for culturing Lactobacillus acidophilus, which differs from Embodiment 1 only in that step (2) is "inoculating the seed liquid at an inoculum amount of 2% into an MRS medium containing 2.5 μg / mL heme and 1 μg / mL vitamin K2, fermenting under the condition of passing 0.3 vvm air, stirring at a speed of 50 rpm, and when the fermentation pH drops below 6, adding 20% ​​sodium hydroxide solution to adjust the pH to 6±0.1, the fermentation temperature is 37°C, and the fermentation time is 18 h". Other operations remain unchanged.

[0071] Example 7

[0072] This embodiment provides a method for culturing Lactobacillus acidophilus, which differs from Embodiment 1 only in that step (2) is "inoculating the seed liquid at an inoculum amount of 2% into an MRS medium containing 4 μg / mL heme and 2 μg / mL vitamin K2, fermenting under the condition of passing 0.3 vvm air, stirring at a speed of 50 rpm, and when the fermentation pH drops below 6, adding 20% ​​sodium hydroxide solution to adjust the pH to 6±0.1, the fermentation temperature is 37°C, and the fermentation time is 18 h". Other operations remain unchanged.

[0073] Comparative Example 1

[0074] This comparative example provides a method for culturing Lactobacillus acidophilus, the method comprising:

[0075] (1) Collect the droplet with the highest OD value obtained in Preparation Example 1, perform aseptic streaking operation on MRS solid culture medium, anaerobically culture at 37°C for 48 hours to obtain a single colony, inoculate the prepared single colony into MRS liquid culture medium, and culture at 37°C for 18 hours for activation; transfer the activated bacterial liquid to MRS seed culture medium at an inoculum volume of 2% and culture for 10 hours;

[0076] (2) The seed liquid was inoculated into MRS medium at an inoculum size of 2%, and fermentation was carried out under the condition of passing 0.3 vvm of air. The stirring speed was 50 rpm. When the fermentation pH dropped below 6, 20% sodium hydroxide solution was added to adjust the pH to 6±0.1. When the carbon source content was lower than 0.5%, the carbon source and nitrogen source were supplemented in a mass ratio of 3:4. The total amount of carbon source and nitrogen source added at a single time was 3%. The fermentation temperature was 37°C and the fermentation time was 18 h.

[0077] Comparative Example 2

[0078] This comparative example provides a method for culturing Lactobacillus acidophilus, the method comprising:

[0079] Lactobacillus acidophilus LA85 was streaked into MRS solid culture medium, and anaerobically cultured at 37°C for 48 hours to obtain a single colony. The prepared single colony was inoculated into MRS liquid culture medium and cultured at 37°C for 18 hours for activation. The activated bacterial liquid was inoculated with an inoculum of 2% in MRS culture medium containing 2.5 μg / mL heme and 1 μg / mL vitamin K2, and fermented under the condition of 0.3 vvm air and a stirring speed of 50 rpm. When the fermentation pH dropped below 6, 20% sodium hydroxide solution was added to adjust the pH to 6±0.1. When the carbon source content was lower than 0.5%, the carbon source and nitrogen source were supplemented with a mass ratio of 3:4, and the total amount of carbon source and nitrogen source added at a single time was 3%. The fermentation temperature was 37°C and the fermentation time was 18 hours.

[0080] Comparative Example 3

[0081] This comparative example provides a method for culturing Lactobacillus acidophilus, the method comprising:

[0082] (1) Collect the droplet with the highest OD value obtained in Preparation Example 1, perform aseptic streaking operation on MRS solid culture medium, anaerobically culture at 37°C for 48 hours to obtain a single colony, inoculate the prepared single colony into MRS liquid culture medium, culture at 37°C for 18 hours for activation, transfer the activated bacterial liquid to MRS seed culture medium at an inoculum volume of 2%, and culture for 10 hours;

[0083] (2) The seed liquid was inoculated into MRS medium at an inoculum size of 2%, and fermentation was carried out under anaerobic conditions with a stirring speed of 50 rpm. When the fermentation pH dropped below 6, 20% sodium hydroxide solution was added to adjust the pH to 6±0.1. When the carbon source content was lower than 0.5%, the carbon source and nitrogen source were supplemented in a mass ratio of 3:4. The total amount of carbon source and nitrogen source added at a single time was 3%. The fermentation temperature was 37°C and the fermentation time was 18 h.

[0084] Test Example 1

[0085] OD value test

[0086] Test method: The samples were tested using a spectrophotometer at 600nm.

[0087] OD 600 The test results are shown in Table 1.

[0088] Table 1

[0089]

[0090] It can be seen from the data in Table 1 that the concentration gradient of hydrogen peroxide cannot be too small, and a large span of hydrogen peroxide concentration will affect the growth of the strain.

[0091] Test Example 2

[0092] Test for live bacteria count in bacterial powder: refer to GB 4789.35-2016 "National Food Safety Standard - Microbiological Examination of Foods - Examination of Lactic Acid Bacteria".

[0093] The results of viable bacterial counts are shown in Table 2.

[0094] Table 2

[0095]

[0096] From the data in Table 2, it can be seen that when heme and vitamin K2 are added simultaneously under aerobic fermentation conditions, the effect is better than the addition of the two alone when the total amount is constant, indicating that heme and vitamin K2 can cooperate with each other as aerobic promoters and have potential synergistic effects. The addition of nitrogen source and carbon source will also affect the number of viable bacteria in the fermentation broth, and hydrogen peroxide stress and aerobic promoter stress can also cooperate with each other. Both are indispensable, and the effect of anaerobic fermentation is inferior to that of aerobic fermentation.

[0097] The applicant declares that the present invention illustrates the method for cultivating an oxygen-resistant Lactobacillus acidophilus of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0098] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0099] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for culturing an oxygen-tolerant Lactobacillus acidophilus, characterized in that: The culture method comprises: (1) culturing the activated Lactobacillus acidophilus in a microbial droplet culture apparatus, using hydrogen peroxide as an oxygen stress factor during the culture to obtain Lactobacillus acidophilus droplets; (2) Purifying the Lactobacillus acidophilus droplets, activating them and inoculating them into a fermentation medium, and fermenting them under the condition of introducing air to obtain; The fermentation medium in step (2) further comprises heme at a concentration of 1-4 μg / mL and vitamin K2 at a concentration of 1-2 μg / mL; The concentration gradient of the hydrogen peroxide is 5-10, and the concentration range is 0.01-0.1%; The fermentation temperature in step (2) is 30-37°C and the fermentation time is 8-20 h; The Lactobacillus acidophilus is the Lactobacillus acidophilus LA85 strain with a preservation number of CGMCC No.1.12735.

2. The culture method according to claim 1, characterized in that The hydrogen peroxide gradient was repeated 2-5 times, and the culture time of each generation was independently 8-14 h.

3. The culture method according to claim 1, characterized in that The ventilation volume of the air is 0.01-0.5 vvm.

4. The culture method according to claim 1, characterized in that The pH of the fermentation in step (2) is 5.9-6.

1.

5. The culture method according to claim 1, characterized in that In step (2), when the mass percentage of the carbon source is lower than 0.5%, the carbon source and the nitrogen source are added.

6. The culture method according to claim 5, characterized in that: The mass ratio of the added carbon source and nitrogen source is (1-3):(2-4).

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