Culture medium for promoting growth of AC bacteria and fermentation culture method thereof

Abstract

The invention relates to the technical field of microbial culture, and particularly provides a culture medium for promoting the growth of anaerobic corynebacterium faecalis, which is characterized in that each liter of the culture medium comprises the following components: 30-50g of a nitrogen source, 30-50g of a carbon source, 1-5g of ox liver extract powder, 1-6g of monopotassium phosphate, 0.2-0.8 g of L-cysteine, 1-6g of sodium chloride, 1-10g of soluble starch, 0.1-2g of sodium thioglycolate, 0-10g of N-acetylglucosamine and the balance of water. The solvent is purified water; the carbon source is at least one of glucose, lactose and beef powder; the nitrogen source is at least one of soybean protein isolate, tryptone, peptone, soybean peptone, yeast extract powder and digestive serum powder. The culture medium greatly improves the conditions of slow growth and low OD (optical density) value of the existing anaerobic corynebacterium faecalis, and is simple in preparation mode, convenient to operate and suitable for large-scale use.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial culture, and particularly relates to a culture medium for promoting the growth of Anaerobaculum stercoris and a fermentation culture method thereof. Background Art

[0002] Anaerobaculum stercoris ( Anaerostipes caccae ) is an anaerobic mesophilic Gram-positive bacterium isolated and identified from human feces in 2002. Currently, there is little research on this strain. In the publicly reported literature, media such as PYG, YCFA, BL, and EG are usually used for cultivation.

[0003] CN114787335A relates to a culture medium supplement for high-yield cultivation of anaerobic microorganisms containing N-acetylhexosamine, L-aspartic acid, L-cysteine, and cobalamin, a culture medium composition for high-yield cultivation of anaerobic microorganisms containing the same, and a culture method. The anaerobic microorganisms include Faecalibacterium prausnitzii ( Faecalibacterium prausnitzii ), Anaerobaculum stercoris ( Anaerostipes caccae ), Akkermansia muciniphila ( Akkermansia muciniphila ) or Bifidobacterium longum ( Bifidobacterium longum ). In its examples, Anaerobaculum stercoris ( Anaerostipes caccae ) was cultured using the culture medium of the present invention, and the OD 600 was only 0.463 ± 0.003.

[0004] During the research process, the applicant used a variety of conventional culture media (including PYG, YCFA, BL, EG, etc. and the scheme of CN114787335A) to culture Anaerobaculum stercoris ( Anaerostipes caccae ), but the effects were not ideal. Most of the solutions were clear without turbidity, that is, there was no obvious growth of bacterial cells. Therefore, it is urgent to develop a culture medium suitable for culturing Anaerobaculum stercoris ( Anaerostipes caccae ) for further research. Summary of the Invention

[0005] The purpose of the present invention is to solve the above-mentioned deficiencies of the prior art and provide a culture medium for promoting the growth of Anaerobaculum stercoris and a fermentation culture method thereof.

[0006] The present invention is achieved by the following technical solutions: A culture medium for promoting the growth of Anaerobaculum stercoris, each liter of the culture medium contains the following components: 30 - 50 g of nitrogen source, 30 - 50 g of carbon source, 1 - 5 g of beef liver extract powder, 1 - 6 g of potassium dihydrogen phosphate, 0.2 - 0.8 g of L - cysteine, 1 - 6 g of sodium chloride, 1 - 10 g of soluble starch, 0.1 - 2 g of thioglycolate, 0 - 10 g of N - acetylglucosamine, and the solvent is purified water; The carbon source is at least one of glucose, lactose, and beef powder; The nitrogen source is at least one of soy protein isolate, tryptone, peptone, soy peptone, yeast extract powder, and digested serum powder.

[0007] Preferably, each liter of the culture medium contains the following components: 8 - 15 g of peptone, 1 - 5 g of soy peptone, 2 - 8 g of yeast extract powder, 1 - 5 g of beef powder, 10 - 15 g of digested serum powder, 0.5 - 3 g of beef liver extract powder, 3 - 33 g of glucose, 2 - 5 g of potassium dihydrogen phosphate, 2 - 5 g of sodium chloride, 2 - 8 g of soluble starch, 0.3 - 0.8 g of L - cysteine, 0.1 - 0.5 g of thioglycolate, 0 - 30 g of lactose, and 2 - 8 g of N - acetylglucosamine.

[0008] Furthermore, each liter of the culture medium contains the following components: 10.0 g of peptone, 3.0 g of soy peptone, 5.0 g of yeast extract powder, 2.2 g of beef powder, 13.5 g of digested serum powder, 1.2 g of beef liver extract powder, 18.0 g of glucose, 2.5 g of potassium dihydrogen phosphate, 3.0 g of sodium chloride, 5.0 g of soluble starch, 0.5 g of L - cysteine, 0.3 g of thioglycolate, 15.0 g of lactose, and 5.0 g of N - acetylglucosamine.

[0009] The fermentation culture method using the above - mentioned culture medium for promoting the growth of Clostridium perfringens anaerobe includes the following steps: S1. Inoculate Clostridium perfringens anaerobe into the above - mentioned culture medium for strain activation; S2. When OD 600 reaches 1 - 3, end the seed culture and conduct microscopic examination; S3. Pour the seed culture solution without contaminants detected by microscopic examination into the fermenter of the above - mentioned culture medium under the protection of the inoculation loop flame, and carry out anaerobic fermentation with pressure - maintaining and tank - sealing.

[0010] Preferably, the inoculation amount of Clostridium perfringens anaerobe in step S1 is 0.25 - 2%.

[0011] Preferably, the fermentation parameters in step S3 are: temperature 35 - 42 °C, pH 6 - 8, and stirring speed 80 - 120 rpm.

[0012] Preferably, during the fermentation process, if no white bubbles are found, add a carbon source to the fermenter.

[0013] Furthermore, the carbon source is a 50 m / v% glucose solution.

[0014] Preferably, an aqueous ammonia solution is used to adjust the pH of the fermentation broth to be constant during the fermentation process.

[0015] Furthermore, the concentration of the aqueous ammonia is 8-15 v / v%.

[0016] The beneficial effects of the present invention are as follows: The present invention provides a culture medium and a fermentation culture method that are beneficial to the growth of Corynebacterium anaerobium, which can greatly shorten the culture time of Corynebacterium anaerobium, enable the rapid proliferation of the bacteria in a short time, obtain a large amount of high-density fermentation broth, and have a high viable bacteria count in the bacterial sludge. After centrifugation, more bacterial powder can be prepared, providing raw materials for freeze-dried bacterial powder, and greatly improving the current situation of slow growth and low OD value of Corynebacterium anaerobium. The preparation method of the culture medium is simple, the operation is convenient, and it is suitable for large-scale use. Description of the Drawings

[0017] Figure 1 It is a result diagram of the screening culture of amino acid types and concentrations in Example 1 of the present invention (wherein, 1A is the result diagram of the screening culture of amino acid types, and 1B is the result diagram of the screening culture of amino acid concentrations).

[0018] Figure 2 It is a result diagram of the screening culture of carbon source types and concentrations in Example 2 of the present invention (wherein, 1A is the result diagram of the screening culture of carbon source types, and 1B is the result diagram of the screening culture of carbon source concentrations).

[0019] Figure 3 It is a result diagram of the screening culture of prebiotics in Example 3 of the present invention.

[0020] Figure 4 It is a diagram of the enlarged culture state in the fermenter in Example 4 of the present invention.

[0021] Figure 5 It is a result diagram of the viable bacteria detection in Example 4 of the present invention. Embodiment

[0022] To better understand the present invention, the present invention will be further described below in conjunction with the embodiments and the drawings. The following embodiments are only for illustrating the present invention rather than limiting it. The experimental materials described in the patent are all conventional commercially available products. Example 1 Screening of Amino Acid Types and Concentrations

[0023] Prepare 6 culture media containing different amino acid components according to the formula and concentration in Table 1 (the raw materials are purchased from Qingdao Haibo), and sterilize them at 121 °C under high pressure for 15 minutes. Respectively take 300 μL of Corynebacterium anaerobium Anaerostipes caccae) Inoculate into the above 6 kinds of culture media, and culture at 37°C for 12 h in an anaerobic workbench, then measure its OD 600 value, and obtain the Figure 1 results shown in Figure 1 A. It can be seen from the data in Anaerostipes caccae A that the growth OD value of the culture medium with additional L-cysteine for the strain of Anaerobaculum stercoricanum (

[0024] Table 1 Formulation of culture media for screening amino acid types Medium components (g / L) Formula 1 Formula 2 Formula 3 Formula 4 Formula 5 Formula 6 Peptone 10.0 10.0 10.0 10.0 10.0 10.0 Soy peptone 3.0 3.0 3.0 3.0 3.0 3.0 Yeast extract powder 5.0 5.0 5.0 5.0 5.0 5.0 Beef powder 2.2 2.2 2.2 2.2 2.2 2.2 Digestive serum powder 13.5 13.5 13.5 13.5 13.5 13.5 Ox liver extract powder 1.2 1.2 1.2 1.2 1.2 1.2 Glucose 3.0 3.0 3.0 3.0 3.0 3.0 Potassium dihydrogen phosphate 2.5 2.5 2.5 2.5 2.5 2.5 Sodium chloride 3.0 3.0 3.0 3.0 3.0 3.0 Soluble starch 5.0 5.0 5.0 5.0 5.0 5.0 L-cysteine 0.5 0.3 0.3 0.3 0.3 0.3 Sodium thioglycollate 0.3 0.3 0.3 0.3 0.3 0.3 Aspartic acid 0 0.2 0 0 0 0 Proline 0 0 0.2 0 0 0 Histidine 0 0 0 0.2 0 0 Sodium glutamate 0 0 0 0 0.2 0 Subsequently, prepare 5 portions of culture media with different concentrations of L-cysteine according to the formulations and concentrations in Table 2, and sterilize them at 121°C under high pressure for 15 minutes. Then, take 300 μL of Anaerobaculum stercoricanum ( Anaerostipes caccae ), respectively, and inoculate them into the above 5 kinds of culture media. Culture at 37°C for 15 h in an anaerobic workbench, and measure its OD 600 value, and obtain the Figure 1 results shown in Figure 1 B. It can be seen from the data in Anaerostipes caccae B that the addition of 0.6 g / L L-cysteine to the culture medium significantly promotes the growth of the strain of Anaerobaculum stercoricanum (

[0025] Table 2 Formulation of culture media for screening amino acid concentrations Medium components (g / L) Formula 7 Formula 8 Formula 9 Formula 10 Formula 11 Peptone 10.0 10.0 10.0 10.0 10.0 Soy peptone 3.0 3.0 3.0 3.0 3.0 Yeast extract powder 5.0 5.0 5.0 5.0 5.0 Beef powder 2.2 2.2 2.2 2.2 2.2 Digestive serum powder 13.5 13.5 13.5 13.5 13.5 Ox liver extract powder 1.2 1.2 1.2 1.2 1.2 Glucose 3.0 3.0 3.0 3.0 3.0 Potassium dihydrogen phosphate 2.5 2.5 2.5 2.5 2.5 Sodium chloride 3.0 3.0 3.0 3.0 3.0 Soluble starch 5.0 5.0 5.0 5.0 5.0 L-cysteine 0.4 0.5 0.6 0.7 0.8 Sodium thioglycollate 0.3 0.3 0.3 0.3 0.3 Example 2 Screening of carbon source types and concentrations

[0026] Prepare 5 portions of culture media containing different carbon source components according to the formulations and concentrations in Table 3, and sterilize them at 121°C under high pressure for 15 minutes (glucose, fructose, maltose, and sucrose solutions are prepared separately and sterilized at 115°C under high pressure for 30 minutes). Take 300 μL of Anaerobaculum stercoricanum ( Anaerostipes caccae ), respectively, and inoculate them into the above 5 kinds of culture media. Culture at 37°C for 12 h in an anaerobic workbench and measure the growth OD value at 600 nm, and obtain the Figure 2 results shown in Figure 2 A. It can be seen from the data in

[0027] Table 3 Formulation of culture media for screening carbon source types Medium components (g / L) Formula 12 Formula 13 Formula 14 Formula 15 Formula 16 Peptone 10.0 10.0 10.0 10.0 10.0 Soy peptone 3.0 3.0 3.0 3.0 3.0 Yeast extract powder 5.0 5.0 5.0 5.0 5.0 Beef powder 2.2 2.2 2.2 2.2 2.2 Digestive serum powder 13.5 13.5 13.5 13.5 13.5 Ox liver extract powder 1.2 1.2 1.2 1.2 1.2 Glucose 33.0 3.0 3.0 3.0 3.0 Potassium dihydrogen phosphate 2.5 2.5 2.5 2.5 2.5 Sodium chloride 3.0 3.0 3.0 3.0 3.0 Soluble starch 5.0 5.0 5.0 5.0 5.0 L-cysteine 0.3 0.3 0.3 0.3 0.3 Sodium thioglycollate 0.3 0.3 0.3 0.3 0.3 Lactose 0 30.0 0 0 0 Fructose 0 0 30.0 0 0 Maltose 0 0 0 30.0 0 Sucrose 0 0 0 0 30.0 Therefore, continue the experiment on the concentration ratios of glucose and lactose as shown in Table 4, and obtain the Figure 2The growth curve shown in Figure B. It can be seen from the experimental results that the effect of adding 18 g / L glucose + 15 g / L lactose is the best, which makes the growth of Clostridium perfringens ( Anaerostipes caccae ) reach a relatively high level at 18 h and continue to grow steadily until 26 h; followed by the medium with 33 g / L glucose added, which makes the growth of Clostridium perfringens ( Anaerostipes caccae ) reach a relatively high level at 18 h, and then gradually stabilizes, with only a slight decline in growth at 26 h; again, the media with 13 g / L glucose + 20 g / L lactose and 23 g / L glucose + 10 g / L lactose added, although the growth is relatively slow in the initial stage (18 h), but the cell growth also reaches a relatively high level in the later stage (26 h); the worst is the medium with 30 g / L lactose added, which is basically stable at OD 600 around 4.0 after 18 h, but there is a slight decline in the later stage.

[0028] Table 4 Formulas of carbon source concentration screening media Medium components (g / L) Formula 17 Formula 18 Formula 19 Formula 20 Formula 21 Peptone 10.0 10.0 10.0 10.0 10.0 Soy peptone 3.0 3.0 3.0 3.0 3.0 Yeast extract powder 5.0 5.0 5.0 5.0 5.0 Beef powder 2.2 2.2 2.2 2.2 2.2 Digestive serum powder 13.5 13.5 13.5 13.5 13.5 Ox liver extract powder 1.2 1.2 1.2 1.2 1.2 Glucose 33.0 3.0 13.0 18.0 23.0 Potassium dihydrogen phosphate 2.5 2.5 2.5 2.5 2.5 Sodium chloride 3.0 3.0 3.0 3.0 3.0 Soluble starch 5.0 5.0 5.0 5.0 5.0 L-cysteine 0.3 0.3 0.3 0.3 0.3 Sodium thioglycollate 0.3 0.3 0.3 0.3 0.3 Lactose 0 30.0 20.0 15.0 10.0 From Figure 2 the combination of Figure A and Figure 2B, it can be seen that when glucose and / or lactose are added additionally to the medium, the growth of the cells can be promoted, and the best effect is achieved when the addition amounts of glucose and lactose are nearly 1:1. Example 3 Screening of prebiotics

[0029] In order to investigate the effect of N-acetylglucosamine on the growth of the cells, 5 media were prepared according to the formulas and concentrations in Table 5 and sterilized at 121 °C under high pressure for 15 minutes. 300 μL of Clostridium perfringens ( Anaerostipes caccae ) were respectively inoculated into the above 5 media and cultured at 37 °C for 20 h in an anaerobic workstation, and the OD 600 values were measured at different times to obtain the results shown in Figure 3 . It can be seen from the data in Figure 3 that N-acetylglucosamine can significantly promote the growth of the Clostridium perfringens ( Anaerostipes caccae ) strain.

[0030] Table 5 Formulas of prebiotic screening media Medium components (g / L) Formula 22 Formula 23 Formula 24 Formula 25 Formula 26 Peptone 10.0 10.0 10.0 10.0 10.0 Soy peptone 3.0 3.0 3.0 3.0 3.0 Yeast extract powder 5.0 5.0 5.0 5.0 5.0 Beef powder 2.2 2.2 2.2 2.2 2.2 Digestive serum powder 13.5 13.5 13.5 13.5 13.5 Ox liver extract powder 1.2 1.2 1.2 1.2 1.2 Glucose 3.0 3.0 3.0 18.0 18.0 Potassium dihydrogen phosphate 2.5 2.5 2.5 2.5 2.5 Sodium chloride 3.0 3.0 3.0 3.0 3.0 Soluble starch 5.0 5.0 5.0 5.0 5.0 L-cysteine 0.3 0.5 0.5 0.5 0.5 Sodium thioglycollate 0.3 0.3 0.3 0.3 0.3 Lactose 0 0 30.0 15.0 15.0 N-acetylglucosamine 0 0 0 0 5.0 Example 4 Scale-up culture in a fermenter

[0031] Using Formula 26 to conduct high-density aseptic culture of Clostridium perfringens ( Anaerostipes caccae ) in a 5 L fermenter and detecting its viable cell count. The specific operations are as follows: Take 300 μL of Clostridium perfringens ( Anaerostipes caccae ) and inoculate it into 300 mL of the medium with Formula 26, and place it in an anaerobic workstation for primary seed culture. After culturing for 12 h, measure the OD 600When it reaches 2.5, end the cultivation. After examining under the microscope and finding no contaminants, prepare for fermentation in the fermenter.

[0032] Sterilize the fermenter containing 3 L of Medium 26. Introduce nitrogen gas, and pour the above-mentioned qualified seed culture solution into the fermenter under the protection of the inoculation loop flame. Keep the pressure and ferment in a sealed tank. Set the fermentation parameters: fermentation temperature 37 °C, constant pH 6.5 (adjusted with 10% ammonia water), stirring speed 100 rpm, and start the cultivation.

[0033] During the fermentation process, take samples for measurement every 2 h to monitor the off-line pH, sugar content (add 50% glucose when there is no white foam), OD 600 value, etc. The fermenter during the cultivation is as Figure 4 shown. When fermenting for 9 h, measure the biomass OD 600 and when the growth is gentle, empty the tank.

[0034] Use the viable count method to detect the viable bacteria count in the emptied tank. Dilute the bacteria solution in the emptied tank (diluted test solution) with normal saline. Take 100 μL of the diluted test solutions at three gradients of 10 -6 , 10 -7 , 10 -8 respectively, and evenly spread them on the surface of GAM agar medium (purchased from Qingdao Haibo, product number HB8518-1) with a sterile spreading rod, avoiding spreading near the edge of the petri dish. For each gradient, make three parallel petri dishes. After all spreading is completed, invert and culture at 37 °C for 24 h, and observe and count the colony growth.

[0035] The results are as Figure 5 shown. The OD Anaerostipes caccae value of Clostridium perfringens ( 600 ) in the 5 L fermenter reaches above 20 and the viable bacteria count in the emptied tank reaches 4.9×10 10 CFU / mL.

[0036] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A culture medium for promoting the growth of Corynebacterium perfringens anaerobically, characterized in that, Each liter of the culture medium contains the following components: 30 - 50 g of nitrogen source, 30 - 50 g of carbon source, 1 - 5 g of beef liver extract powder, 1 - 6 g of potassium dihydrogen phosphate, 0.2 - 0.8 g of L-cysteine, 1 - 6 g of sodium chloride, 1 - 10 g of soluble starch, 0.1 - 2 g of thioglycolate, 0 - 10 g of N-acetylglucosamine, and the solvent is purified water; The carbon source is at least one of glucose, lactose, and beef powder; The nitrogen source is at least one of soy protein isolate, tryptone, peptone, soy peptone, yeast extract powder, and digested serum powder.

2. A culture medium for promoting the growth of Anaerobacillus stercorican, according to claim 1, characterized in that, Each liter of the culture medium contains the following components: 8 - 15 g of peptone, 1 - 5 g of soy peptone, 2 - 8 g of yeast extract powder, 1 - 5 g of beef powder, 10 - 15 g of digested serum powder, 0.5 - 3 g of beef liver extract powder, 3 - 33 g of glucose, 2 - 5 g of potassium dihydrogen phosphate, 2 - 5 g of sodium chloride, 2 - 8 g of soluble starch, 0.3 - 0.8 g of L-cysteine, 0.1 - 0.5 g of thioglycolate, 0 - 30 g of lactose, 2 - 8 g of N-acetylglucosamine.

3. A medium for promoting the growth of Anaerobacillus stercorican, according to claim 1, characterized in that, Each liter of the culture medium contains the following components: 10.0 g of peptone, 3.0 g of soy peptone, 5.0 g of yeast extract powder, 2.2 g of beef powder, 13.5 g of digested serum powder, 1.2 g of beef liver extract powder, 18.0 g of glucose, 2.5 g of potassium dihydrogen phosphate, 3.0 g of sodium chloride, 5.0 g of soluble starch, 0.5 g of L-cysteine, 0.3 g of thioglycolate, 15.0 g of lactose, 5.0 g of N-acetylglucosamine.

4. A fermentation culture method using the culture medium for promoting the growth of Anaerobaculum stercoricanum according to any one of claims 1-3, characterized in that, It includes the following steps: S1. Inoculate Clostridium perfringens into the culture medium described in any one of claims 1 - 3 for strain activation; S2. When OD 600 reaches 1 to 3, end the seed culture and conduct microscopic examination; S3. Pour the seed culture solution without detected contaminants under the protection of the inoculation loop flame into a fermenter containing the culture medium described in any one of claims 1 - 3, and carry out anaerobic fermentation under pressure and sealed tank.

5. The fermentation culture method according to claim 4, wherein The inoculation amount of Clostridium perfringens described in step S1 is 0.25 - 2%.

6. The fermentation culture method according to claim 4, characterized in that, The fermentation parameters described in step S3 are: temperature 35 - 42 °C, pH 6 - 8, stirring speed 80 - 120 rpm.

7. The fermentation culture method according to claim 6, wherein During the fermentation process, if no white bubbles are found, add carbon source to the fermenter.

8. The fermentation culture method according to claim 7, wherein The carbon source is a 50 m / v% glucose solution.

9. The fermentation culture method according to claim 6, wherein During the fermentation process, use ammonia water solution to adjust the fermentation broth to keep its pH constant.

10. The fermentation culture method according to claim 9, wherein The concentration of the ammonia water is 8 - 15 v / v%.

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