A culture medium for batch high-density culture of escherichia coli, a preparation method thereof, and a culture method thereof

Abstract

The application relates to a culture medium for batch high-density culture of escherichia coli, a preparation method and a culture method thereof, and relates to the technical field of microbial culture medium, which is composed of the following components: spirulina lysis solution, buffer salt 3.789-8.733 g / L, glucose 10-30 g / L, and magnesium sulfate 0.2-0.8 g / L, and the pH is 6.5-7.5. The application realizes high-density culture of escherichia coli in a batch culture mode, and the culture medium is simple in composition, low in price, and can greatly reduce the cost of escherichia coli in industrialized production application.

Description

Technical Field

[0001] This invention relates to the field of microbial culture medium technology, specifically to a culture medium for batch high-density culture of Escherichia coli, its preparation method, and its culture method. Background Technology

[0002] Bacterial culture is fundamental to both basic bacterial research and industrial applications. To maximize the yield of biomolecules, including plasmids and proteins, bacteria must first be cultured at high densities. *Escherichia coli* is both one of the most widely used model bacteria in basic research and one of the most widely used bacteria for developing engineered strains in industrial production. Therefore, high-density culture of *E. coli* is particularly important.

[0003] In actual cultivation processes, the growth of *E. coli* consumes a large amount of nutrients, and the production of various growth inhibitors (such as acetic acid) prevents the achievement of high-density cultures. To address this, several new culture modalities have been developed, such as continuous culture, fed-batch culture, and dialysis culture. These modalities achieve high-density bacterial cultivation by continuously adding fresh culture medium or removing old culture medium to eliminate the generated inhibitors. However, these modalities rely on specialized hardware or equipment, waste a significant amount of nutrients, are unsuitable for routine laboratory cultivation, and increase industrial cultivation costs.

[0004] Therefore, batch culture, which is suitable for laboratory cultivation and has low cost, has become a research focus. Batch culture refers to a culture mode in which all nutrients are added at once before cultivation, and metabolic products are not removed during the cultivation process. This culture mode does not rely on specialized equipment, does not waste culture medium, is suitable for routine laboratory cultivation, and can also reduce the cost of bacterial culture in industry. However, due to the consumption of nutrients or the accumulation of metabolic inhibitors during the cultivation process, existing culture media cannot meet the high-density cultivation of Escherichia coli under the batch culture mode. In view of this, the present invention provides a culture medium for high-density batch culture of Escherichia coli, a preparation method thereof, and a cultivation method thereof. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a culture medium, a preparation method, and a cultivation method for batch high-density culture of Escherichia coli. The aim is to achieve high-density cultivation of Escherichia coli in batch culture mode, with simple components and low cost, which can significantly reduce the cost of industrial-scale production of Escherichia coli.

[0006] In order to solve the above-mentioned technical problems, the first objective of this invention is to provide a culture medium for batch high-density culture of Escherichia coli, which is composed of the following components: Spirulina lysate, buffer salt 3.789~8.733 g / L, glucose 10~30 g / L, and magnesium sulfate 0.2~0.8 g / L, with a pH of 6.5~7.5.

[0007] The beneficial effects of this invention are: (1) The culture medium provided by the present invention can realize high-density culture of Escherichia coli in batch culture mode; (2) The culture medium provided by the present invention can rapidly obtain high-density culture of Escherichia coli; (3) The culture medium provided by the present invention has low cost and does not rely on the instruments and equipment required for other culture modes (such as continuous culture, fed batch culture and dialysis culture), which can significantly reduce the cost of Escherichia coli in laboratory or industrial production applications.

[0008] Based on the above technical solution, the present invention can be further improved as follows.

[0009] Furthermore, the buffer salt is dipotassium hydrogen phosphate and potassium dihydrogen phosphate, and the weight ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate is (2.35~5.125):(1.439~3.608).

[0010] Furthermore, each liter of the spirulina lysate is prepared by lysing 100-200g of spirulina dry powder. The culture medium consists of the following components: spirulina lysate, dipotassium hydrogen phosphate 2.35-5.125g / L, potassium dihydrogen phosphate 1.439-3.608g / L, glucose 10-30g / L, and magnesium sulfate 0.2-0.8g / L, with a pH of 6.5-7.5.

[0011] Furthermore, each liter of the spirulina lysate is prepared by lysing 120–180 g of spirulina dry powder. The culture medium consists of the following components: spirulina lysate, dipotassium hydrogen phosphate 2.95–4.525 g / L, potassium dihydrogen phosphate 1.939–3.108 g / L, glucose 14–26 g / L, and magnesium sulfate 0.3–0.7 g / L, with a pH of 6.5–7.5.

[0012] Furthermore, each liter of the spirulina lysate is prepared by lysing 140–160 g of spirulina dry powder. The culture medium consists of the following components: spirulina lysate, dipotassium hydrogen phosphate 3.55–3.925 g / L, potassium dihydrogen phosphate 2.539–2.608 g / L, glucose 18–22 g / L, and magnesium sulfate 0.4–0.6 g / L, with a pH of 6.5–7.5.

[0013] A second objective of this invention is to provide a method for preparing a culture medium for batch high-density culture of Escherichia coli, comprising the following steps: S1: Spirulina dry powder was lysed with Bacillus subtilis and a portion of buffer salts to prepare Spirulina lysate; S2: Adjust the pH of the prepared Spirulina lysate to 6.5-7.5. After adjusting the pH, add the remaining buffer salts, glucose and magnesium sulfate, shake to mix, and then sterilize under high pressure steam at at least 121°C for at least 15 minutes to obtain a culture medium for batch high-density culture of Escherichia coli.

[0014] Furthermore, the preparation of the Spirulina lysate in step S1 includes the following steps: S1-1: Inoculate the seed culture of Bacillus subtilis with an OD600 of 0.5-1.5 into NB or LB culture medium, wherein the volume ratio of the seed culture of Bacillus subtilis to the culture medium is (1-3):(10-30). Incubate in a constant temperature shaking incubator at 150-250 rpm and 32-42°C for 60-84 hours until the Bacillus subtilis reaches the plateau phase. Centrifuge to obtain the culture supernatant of Bacillus subtilis and store it for later use. S1-2: Spirulina powder is soaked in double-distilled water at a weight ratio of (5~14):(50~70) to obtain a spirulina solution; the spirulina solution is then sterilized in high-pressure steam at a temperature of at least 121℃ for at least 15 minutes to obtain a sterilized spirulina solution; then, the culture supernatant of Bacillus subtilis is added to the sterilized spirulina solution at a volume ratio of (0.1~1):(25~35) to the culture supernatant of Bacillus subtilis and double-distilled water, and the solution is placed in a constant temperature shaking incubator at 150~250 rpm and 32~42℃ for 3~7 hours to obtain spirulina lysate I; S1-3: Detect the pH of the Spirulina lysate I, adjust the pH to 6.5-7.5, and after adjusting the pH, add buffer salt and an equal amount of Bacillus subtilis culture supernatant to the Spirulina lysate I in sequence. Place it in a constant temperature shaking incubator at 170-230 rpm and 34-40°C, and continue lysis for 4-6 hours to obtain Spirulina lysate II. S1-4: Repeat the lysis step of S1-3 3 to 7 times with the lysis filtrate II of Spirulina. After the lysis is completed, centrifuge to obtain the supernatant of the lysis filtrate II of Spirulina. The supernatant of the lysis filtrate II of Spirulina is the lysis filtrate of Spirulina.

[0015] Further, the centrifugation step in S1-1 specifically involves centrifuging at 8000-12000 rpm for 15-25 min to obtain the culture supernatant of Bacillus subtilis; the centrifugation step in S1-4 specifically involves centrifuging at 8000-12000 rpm for 15-25 min to obtain the supernatant of Spirulina lysate II; The culture medium in S1-1 is either NB culture medium or LB culture medium; the NB culture medium contains 10 g / L peptone, 3 g / L beef meal and 5 g / L sodium chloride; the LB culture medium contains 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride. The buffer salts in S1-3 and S1-4 are potassium dihydrogen phosphate and dipotassium hydrogen phosphate, with a mass ratio of (1.577-2.208):(2.336-3.27) and a weight-volume ratio of the buffer salts to the spirulina lysate II of the spirulina of (3.913-5.478):(250-350). The pH adjustment steps in S1-3 and S1-4 specifically involve using 0.5~1.5 mol / L NaOH to adjust the pH of the Spirulina lysate I to 6.5~7.5.

[0016] The third objective of this invention is to provide a method for batch high-density culture of Escherichia coli, comprising the following steps: inoculating a seed culture of Escherichia coli with an OD600 of 0.5 to 1.5 onto any of the above-mentioned culture media, wherein the volume ratio of the seed culture of Escherichia coli to the culture media is (1 to 9): 100; and placing the inoculated culture media in a constant temperature shaking incubator and culturing it for 100 to 140 hours at a temperature of 37±5℃ and a rotation speed of 150 to 250 rpm.

[0017] Further, the method includes the following steps: inoculating the seed culture of Escherichia coli with an OD600 of 1 onto the culture medium as described above, wherein the volume ratio of the seed culture of Escherichia coli to the culture medium during inoculation is 5:100, and placing the inoculated culture medium in a constant temperature shaking incubator and culturing it at a temperature of 37°C and a rotation speed of 200 rpm for 100-140 h. Attached Figure Description

[0018] Figure 1 Examples 1 to 3 of this invention are comparative graphs showing the effect of different concentrations of buffer salts on the proliferation of Escherichia coli. *** P <0.001, **, P <0.01; Figure 2 Examples 4 to 6 of this invention are comparative graphs showing the effect of different concentrations of glucose on the proliferation of Escherichia coli. ***P <0.001, **, P <0.01, *, P <0.05; Figure 3 Examples 7 to 9 of this invention show a comparison of the effects of different concentrations of magnesium sulfate on the proliferation of *Escherichia coli*. P <0.001, **, P <0.01, *, P <0.05; Figure 4 This is a comparison chart showing the culture effect of Escherichia coli in Example 10 of the present invention and in Comparative Example 1 LB medium. *** P <0.001; Figure 5 This is a comparison diagram of Escherichia coli colonies cultured in the culture medium of Example 10 of the present invention and in the LB culture medium of Comparative Example 1. Figure 6 This is a colony count of *E. coli cultured in the culture medium of Example 10 of the present invention and the LB culture medium of Comparative Example 1. P <0.05. Detailed Implementation

[0019] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0020] Explanation of the source of the medicine or material: Spirulina powder was purchased from Hunan Zhengdi Biological Resources Development Co., Ltd. Escherichia coli K-12 was purchased from Guangdong Huankai Microbial Technology Co., Ltd. Bacillus subtilis ATCC6633 was purchased from Guangdong Huankai Microbial Technology Co., Ltd.

[0021] Example 1 This embodiment relates to a culture medium for batch high-density culture of Escherichia coli, which consists of the following components: 100 mL of Spirulina lysate, 0.3737 g / L of dipotassium hydrogen phosphate, 0.2524 g / L of potassium dihydrogen phosphate, and pH 7.

[0022] This embodiment relates to a method for preparing a culture medium for batch high-density culture of Escherichia coli, comprising the following steps: S1: Spirulina dry powder was lysed with Bacillus subtilis to prepare Spirulina lysate; S2: Adjust the pH of the prepared Spirulina lysate to 7 with 1 mol / L sodium hydroxide. After pH adjustment, add 0.3737 g / L dipotassium hydrogen phosphate and 0.2524 g / L potassium dihydrogen phosphate, shake to mix, and then sterilize under high pressure steam at at least 121°C for at least 15 minutes. Cool to obtain a culture medium for batch high-density culture of Escherichia coli.

[0023] The preparation of the Spirulina lysate in step S1 includes the following steps: S1-1: Inoculate the seed culture of Bacillus subtilis with OD600≈1 into the culture medium. Add 1 ml of fresh seed culture of Bacillus subtilis (OD600≈1) to 10 ml of NB medium (the NB medium contains 10 g / L peptone, 3 g / L beef meal and 5 g / L sodium chloride). Incubate in a constant temperature shaking incubator at 200 rpm and 35°C for 72 hours. When the Bacillus subtilis reaches the plateau phase, centrifuge at 10,000 rpm for 20 min to obtain the culture supernatant of Bacillus subtilis. Seal and store in a refrigerator at 4°C for later use. S1-2: Soak 15g of spirulina powder in 100ml of double-distilled water to obtain a spirulina solution; then sterilize the spirulina solution in high-pressure steam at 121℃ for 15min to obtain a sterilized spirulina solution; after cooling, add 1ml of Bacillus subtilis culture supernatant to the sterilized spirulina solution, and lyse it in a constant temperature shaker (37℃, 200 rpm) for 5 hours to obtain spirulina lysate I; S1-3: Detect the pH of the Spirulina lysate I, adjust the pH to 7 using 1 mol / L sodium hydroxide solution, and then add 1.893 g of potassium dihydrogen phosphate, 2.803 g of dipotassium hydrogen phosphate and an equal amount of the Bacillus subtilis culture supernatant from S1-2 to the Spirulina lysate I in sequence. Continue lysis in a constant temperature shaker (37°C, 200 rpm) for 5 hours to obtain Spirulina lysate II. S1-4: Repeat the pH adjustment process of S1-3 on the spirulina lysate II, add buffer salt and the culture supernatant of Bacillus subtilis in sequence, and lyse again 4 times. After lysis, centrifuge at 10,000 rpm for 20 min to obtain the supernatant of the spirulina lysate II as spirulina lysate.

[0024] This embodiment relates to a method for batch high-density culture of Escherichia coli, comprising the following steps: inoculating the seed culture of Escherichia coli with an OD600 of 1 onto the aforementioned culture medium, wherein the volume ratio of the seed culture of Escherichia coli to the culture medium during inoculation is 5:100; placing the inoculated culture medium in a constant temperature shaking incubator and culturing it for 120 hours at a temperature of 37°C and a rotation speed of 200 rpm; and taking samples every 12 hours to measure the OD600 value.

[0025] The OD600 values ​​mentioned are all the OD600 obtained from the sample minus the OD600 of the corresponding culture medium, reflecting the growth of Escherichia coli. They are measured using a Shimadzu UV-2700 with a wavelength of 600 nm.

[0026] Example 2 Compared with Example 1, this embodiment is the same as Example 1 except that 0.235g of dipotassium hydrogen phosphate and 0.3608g of potassium dihydrogen phosphate are added.

[0027] Example 3 Compared with Example 1, this embodiment is the same as Example 1 except that 0.5125g of dipotassium hydrogen phosphate and 0.1439g of potassium dihydrogen phosphate are added.

[0028] according to Figure 1 It can be seen that from Examples 1 to 3, the maximum OD600 of Escherichia coli growth was highest when the buffer was 0.3737 g of dipotassium hydrogen phosphate and 0.2524 g of potassium dihydrogen phosphate.

[0029] Example 4 Compared with Example 1, this embodiment adds 1g of glucose to the culture medium used for batch high-density culture of Escherichia coli, while all other aspects are the same as in Example 1.

[0030] Example 5 Compared with Example 4, the amount of glucose added in this example is 2g, while everything else is the same as in Example 4.

[0031] Example 6 Compared with Example 4, the amount of glucose added in this example is 3g, while everything else is the same as in Example 4.

[0032] according to Figure 2 It can be seen that from Examples 4 to 6, the culture effect was optimal when the buffer was 0.3737g of dipotassium hydrogen phosphate and 0.2524g of potassium dihydrogen phosphate, and 1g of glucose was added.

[0033] Example 7 Compared with Example 4, this embodiment adds 1g of glucose and 50mg of magnesium sulfate to the culture medium used for batch high-density culture of Escherichia coli, while the rest is the same as in Example 1.

[0034] Example 8 Compared with Example 7, the amount of magnesium sulfate added in this example is 20 mg, while everything else is the same as in Example 7.

[0035] Example 9 Compared with Example 7, the amount of magnesium sulfate added in this example is 80 mg, while everything else is the same as in Example 7.

[0036] according to Figure 3 It can be seen that from Examples 7 to 9, when the buffer is 0.3737g dipotassium hydrogen phosphate and 0.2524g potassium dihydrogen phosphate, and 1g glucose, the addition of 50mg magnesium sulfate results in faster growth and higher density of Escherichia coli.

[0037] Example 10 This embodiment relates to a culture medium for batch high-density culture of Escherichia coli, which consists of the following components: 100 mL of Spirulina lysate, 0.3737 g / L of dipotassium hydrogen phosphate, 0.2524 g / L of potassium dihydrogen phosphate, 1 g / L of glucose, and 50 mg / L of magnesium sulfate, with a pH of 7.

[0038] The preparation method of the culture medium for batch high-density culture of Escherichia coli and the culture method for batch high-density culture of Escherichia coli in this embodiment are the same as those in Example 1.

[0039] Comparative Example 1 This comparative example relates to a method for culturing Escherichia coli in LB medium, comprising the following steps: Step 1: Preparation of LB medium: Add 10g tryptone, 5g yeast extract and 10g sodium chloride to double-distilled water to 1000ml, mix well and sterilize at 121℃ for 15min. Step 2: Inoculate the seed culture of Escherichia coli with an OD600 of 1 into LB culture medium at a volume ratio of 5:100. Step 3: Incubate in a constant temperature shaking incubator at 200 rpm and 37°C, taking samples every 12 hours to measure the OD600 value, for a total of 120 hours.

[0040] The results of comparing the culture of Escherichia coli in Example 10 with those in Comparative Example 1 are as follows: Figure 4 As shown. By Figure 4It can be seen that the culture medium provided by the present invention only requires 36 hours to reach the maximum culture volume, while the LB culture medium requires 48 hours to reach the maximum culture volume; the OD600 value of Escherichia coli at the maximum culture volume of the culture medium provided by the present invention is about 16.7, which is much higher than the maximum culture volume of Escherichia coli on the LB culture medium (OD600 value is about 6.5).

[0041] Experimental Example: Evaluation of Escherichia coli activity 1. Evaluation of Escherichia coli activity based on the dilution plating method 1.1 Method for evaluating the viability of Escherichia coli based on the dilution plating method This test example evaluates the activity of Escherichia coli in Example 10 and Comparative Example 1 using the dilution plating method.

[0042] The dilution-spread plate method includes the following steps: For the 36-hour culture medium obtained in Example 10 and Comparative Example 1, take 1 ml of bacterial suspension from each. The bacterial suspension obtained in Example 10 is diluted 17-fold (OD600 approximately equal to 1), and then serially diluted 10-fold six times. Using a glass spreader, evenly spread 20 μL of the bacterial suspension onto a plate. Similarly, the bacterial suspension obtained in Comparative Example 1 is diluted 5-fold (OD600 approximately equal to 1), and then serially diluted 10-fold five times. Using a glass spreader, evenly spread 20 μL of the bacterial suspension onto a plate. After incubation at 37°C for 16 hours, count the number of colonies.

[0043] Note: The above experiment was repeated 3 times.

[0044] 1.2 Results Example 10 and Comparative Example 1 were compared after culturing E. coli for 36 hours, and the number of viable E. coli was compared between the two. Figure 5 It can be seen that the *E. coli* cultured using the medium of this invention, when cultured to its highest density, showed colony counts exceeding 100 on diluted plating plates, slightly higher than those cultured in LB medium. This indicates that the *E. coli* cultured using the medium of this invention has similar or slightly higher activity than *E. coli* cultured in LB medium. Figure 6 It can be seen that the number of viable Escherichia coli cultured in the culture medium of the present invention is significantly higher than that of Escherichia coli cultured in LB medium.

[0045] In summary, the culture medium provided by this invention can rapidly obtain high-density culture of Escherichia coli in batch culture mode, and its components are simple and inexpensive, which can significantly reduce the cost of Escherichia coli in laboratory or industrial production applications.

[0046] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0047] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A culture medium for batch high-density culture of *Escherichia coli*, characterized in that, It consists of the following components: Spirulina lysate, buffer salts 3.789~8.733 g / L, glucose 10~30 g / L, and magnesium sulfate 0.2~0.8 g / L, with a pH of 6.5~7.5; The buffer salt is dipotassium hydrogen phosphate and potassium dihydrogen phosphate, and the weight ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate is (2.35~5.125):(1.439~3.608). The preparation of the Spirulina lysate includes the following steps: S1-1: Bacillus subtilis with an OD600 of 0.5–1.5 ( Bacillus subtilis The seed culture of Bacillus subtilis is inoculated into NB or LB culture medium, with the volume ratio of the seed culture of Bacillus subtilis to the culture medium being (1~3):(10~30). The culture is carried out in a constant temperature shaking incubator at 150~250 rpm and 32~42℃ for 60~84 hours until the Bacillus subtilis reaches the plateau phase. After centrifugation, the culture supernatant of Bacillus subtilis is obtained and stored for later use. S1-2: Spirulina powder is soaked in double-distilled water at a weight ratio of (5~14):(50~70) to obtain a spirulina solution; the spirulina solution is then sterilized in high-pressure steam at a temperature of at least 121℃ for at least 15 minutes to obtain a sterilized spirulina solution; then, the culture supernatant of Bacillus subtilis is added to the sterilized spirulina solution at a volume ratio of (0.1~1):(25~35) to the culture supernatant of Bacillus subtilis for at a volume of (0.1~1):(25~35), and the solution is placed in a constant temperature shaking incubator at 150~250 rpm and 32~42℃ for 3~7 hours to obtain the spirulina lysate I; S1-3: Detect the pH of the Spirulina lysate I, adjust the pH to 6.5-7.5, and after adjusting the pH, add buffer salt and an equal amount of Bacillus subtilis culture supernatant to the Spirulina lysate I in sequence. Place it in a constant temperature shaking incubator at 170-230 rpm and 34-40°C, and continue lysis for 4-6 hours to obtain Spirulina lysate II. S1-4: Repeat the lysis step of S1-3 3 to 7 times with the lysis filtrate II of Spirulina. After the lysis is completed, centrifuge to obtain the supernatant of the lysis filtrate II of Spirulina. The supernatant of the lysis filtrate II of Spirulina is the lysis filtrate of Spirulina. The Bacillus subtilis strain is Bacillus subtilis ATCC6633.

2. The culture medium for batch high-density culture of *Escherichia coli* according to claim 1, characterized in that, Each liter of the spirulina lysate is prepared by lysing 100-200g of spirulina dry powder. The culture medium consists of the following components: spirulina lysate, dipotassium hydrogen phosphate 2.35-5.125g / L, potassium dihydrogen phosphate 1.439-3.608g / L, glucose 10-30g / L, and magnesium sulfate 0.2-0.8g / L, with a pH of 6.5-7.

5.

3. The culture medium for batch high-density culture of *Escherichia coli* according to claim 1, characterized in that, Each liter of the spirulina lysate is prepared by lysing 120–180 g of spirulina dry powder. The culture medium consists of the following components: spirulina lysate, dipotassium hydrogen phosphate 2.95–4.525 g / L, potassium dihydrogen phosphate 1.939–3.108 g / L, glucose 14–26 g / L, and magnesium sulfate 0.3–0.7 g / L, with a pH of 6.5–7.

5.

4. The culture medium for batch high-density culture of *Escherichia coli* according to claim 1, characterized in that, Each liter of the spirulina lysate is prepared by lysing 140–160 g of spirulina dry powder. The culture medium consists of the following components: spirulina lysate, dipotassium hydrogen phosphate 3.55–3.925 g / L, potassium dihydrogen phosphate 2.539–2.608 g / L, glucose 18–22 g / L, and magnesium sulfate 0.4–0.6 g / L, with a pH of 6.5–7.

5.

5. A method for preparing a culture medium for batch high-density culture of *Escherichia coli* according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1: Spirulina dry powder was lysed with Bacillus subtilis and a portion of buffer salt to prepare Spirulina lysate; S2: Adjust the pH of the prepared Spirulina lysate to 6.5-7.

5. After pH adjustment, add the remaining buffer salts, glucose, and magnesium sulfate, shake to mix, and then sterilize under high pressure steam at at least 121°C for at least 15 minutes to obtain a culture medium for batch high-density culture of Escherichia coli. The preparation of the Spirulina lysate in step S1 includes the following steps: S1-1: Inoculate the seed culture of Bacillus subtilis with an OD600 of 0.5-1.5 into NB or LB culture medium, wherein the volume ratio of the seed culture of Bacillus subtilis to the culture medium is (1-3):(10-30). Incubate in a constant temperature shaking incubator at 150-250 rpm and 32-42°C for 60-84 hours until the Bacillus subtilis reaches the plateau phase. Centrifuge to obtain the culture supernatant of Bacillus subtilis and store it for later use. S1-2: Spirulina powder is soaked in double-distilled water at a weight ratio of (5~14):(50~70) to obtain a spirulina solution; the spirulina solution is then sterilized in high-pressure steam at a temperature of at least 121℃ for at least 15 minutes to obtain a sterilized spirulina solution; then, the culture supernatant of Bacillus subtilis is added to the sterilized spirulina solution at a volume ratio of (0.1~1):(25~35) to the culture supernatant of Bacillus subtilis for at a volume of (0.1~1):(25~35), and the solution is placed in a constant temperature shaking incubator at 150~250 rpm and 32~42℃ for 3~7 hours to obtain the spirulina lysate I; S1-3: Detect the pH of the Spirulina lysate I, adjust the pH to 6.5-7.5, and after adjusting the pH, add buffer salt and an equal amount of Bacillus subtilis culture supernatant to the Spirulina lysate I in sequence. Place it in a constant temperature shaking incubator at 170-230 rpm and 34-40°C, and continue lysis for 4-6 hours to obtain Spirulina lysate II. S1-4: Repeat the lysis step of S1-3 3 to 7 times with the lysis filtrate II of Spirulina. After the lysis is completed, centrifuge to obtain the supernatant of the lysis filtrate II of Spirulina. The supernatant of the lysis filtrate II of Spirulina is the lysis filtrate of Spirulina.

6. The method for preparing a culture medium for batch high-density culture of Escherichia coli according to claim 5, characterized in that, The centrifugation step in S1-1 specifically involves centrifuging at 8000–12000 rpm for 15–25 min to obtain the culture supernatant of Bacillus subtilis; the centrifugation step in S1-4 specifically involves centrifuging at 8000–12000 rpm for 15–25 min to obtain the supernatant of Spirulina lysate II. The culture medium in S1-1 is either NB culture medium or LB culture medium; the NB culture medium contains 10 g / L peptone, 3 g / L beef meal and 5 g / L sodium chloride; the LB culture medium contains 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride. The buffer salts in S1-3 and S1-4 are potassium dihydrogen phosphate and dipotassium hydrogen phosphate, with a mass ratio of (1.577-2.208):(2.336-3.27) and a weight-volume ratio of the buffer salts to the spirulina lysate II of the spirulina of (3.913-5.478):(250-350). The pH adjustment steps in S1-3 and S1-4 specifically involve using 0.5~1.5 mol / L NaOH to adjust the pH of the Spirulina lysate I to 6.5~7.

5.

7. A method for batch high-density culture of Escherichia coli, characterized in that, The procedure includes the following steps: inoculating a seed culture of Escherichia coli with an OD600 of 0.5 to 1.5 onto the culture medium as described in any one of claims 1 to 4, wherein the volume ratio of the seed culture of Escherichia coli to the culture medium during inoculation is (1 to 9): 100; and placing the inoculated culture medium in a constant temperature shaking incubator and culturing it for 100 to 140 hours at a temperature of 37±5℃ and a rotation speed of 150 to 250 rpm.

8. The method for batch high-density culture of Escherichia coli according to claim 7, characterized in that, The procedure includes the following steps: inoculating the seed culture of Escherichia coli with an OD600 of 1 onto the culture medium, wherein the volume ratio of the seed culture of Escherichia coli to the culture medium is 5:100; and placing the inoculated culture medium in a constant temperature shaking incubator and culturing it at a temperature of 37°C and a rotation speed of 200 rpm for 100-140 hours.

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