A method for promoting the growth of Clostridium scindens ATCC 35704

Abstract

The application discloses a method for promoting growth of Clostridium scindens ATCC 35704. It is found that culture supernatant of Bacteroides ovatus ATCC 8483 can effectively promote growth of Clostridium scindens ATCC 35704, and can improve the efficiency of obtaining Clostridium scindens ATCC 35704, thereby providing a new method for efficiently culturing Clostridium scindens ATCC 35704.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and relates to a method for promoting the growth of Clostridium scindens ATCC 35704. Background Art

[0002] Clostridium scindens ATCC 35704 is a Gram-positive bacterium with a round, neatly marginated, opaque, off-white front, a bright surface, a moist texture, and is easily provoked. Studies have shown that Clostridium scindens ATCC 35704 can prevent and treat Clostridium difficile infection, improve Staphylococcus aureus-induced mastitis, convert glucocorticoids into androgens, and is negatively correlated with obesity.

[0003] It can be seen that Clostridium scindens ATCC 35704 has very high application value.

[0004] In order to promote the growth of Clostridium scindens ATCC 35704 and improve the efficiency of obtaining Clostridium scindens ATCC 35704, the present invention is specially proposed. Summary of the Invention

[0005] The object of the present invention is to provide a method for promoting the growth of Clostridium scindens ATCC 35704.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A method for promoting the growth of Clostridium scindens ATCC 35704 comprises adding an appropriate amount of culture supernatant of Bacteroides ovatus ATCC 8483 to a culture medium of Clostridium scindens ATCC 35704.

[0008] Preferably, the culture medium of Clostridium scindens ATCC 35704 is DSM Medium 110.

[0009] Preferably, the culture supernatant of Bacteroides ovatus ATCC 8483 is the culture supernatant of Bacteroides ovatus ATCC 8483 after culturing in BHI medium for an appropriate period of time.

[0010] Beneficial effects:

[0011] The present invention finds that the culture supernatant of Bacteroides ovatus ATCC 8483 can effectively promote the growth of Clostridium scindens ATCC 35704 and improve the efficiency of obtaining Clostridium scindens ATCC 35704, which provides a new method for efficiently culturing Clostridium scindens ATCC 35704. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Figure 2 is the growth curve of Clostridium scindens under different culture conditions. DETAILED DESCRIPTION

[0013] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0014] 1. Experimental Materials

[0015] Clostridium scindens ATCC 35704 was purchased from ATCC and cultured strictly according to the ATCC culture instructions.

[0016] Bacteroides ovatus ATCC 8483 was purchased from ATCC and cultured strictly according to the culture instructions of ATCC.

[0017] DSM Medium 110 was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0018] Brain heart infusion broth (BHI) was purchased from Solebro.

[0019] 2. Experimental Methods

[0020] 1. Activation of Clostridium scindens ATCC 35704 strain

[0021] Prepare DSM Medium 110 ground meat medium using 500 g of lean meat or horse meat. Remove fat and connective tissue before grinding. Combine the meat, 1 L of water, and 25 mL of sodium hydroxide, then stir and cook for 15 minutes. Cool to room temperature, skim off the fat, filter, and retain the meat pellets and filtrate. Add water to the filtrate to a final volume of 1000 mL, then add: tryptone, yeast extract, potassium phosphate (K₂HPO₄), resazurin, D-glucose, cellobiquinone, maltose, and soluble starch. Finally, sterilize at 121°C for 15 minutes, immediately evacuate the anaerobic glove box, and allow to cool to room temperature before use. To resuspend the bacteria: Take a glycerol stock frozen at -80°C, thaw in a 37°C water bath with rapid shaking, transfer to an anaerobic incubator, and inoculate with sterilized, aliquoted culture medium. Incubate at 37°C on a shaker for 48 hours until the bacteria reach the third generation.

[0022] 2. Activation of Bacteroides ovatus ATCC 8483 strain

[0023] To prepare Brain Heart Infusion Broth (BHI): Place 1.15g of BHI powder into an Erlenmeyer flask, add 30mL of grade tertiary water, seal with a breathable membrane, and evacuate the flask in a glove box for 72 hours. Sterilize at 121°C for 15 minutes, remove from the flask, evacuate the flask, and cool it down before use. To rehydrate bacteria: Transfer glycerol stock frozen at -80°C to a 37°C water bath and shake rapidly until thawed. Transfer the glycerol stock to an anaerobic incubator and inoculate with sterilized, aliquoted culture medium. Incubate in a 37°C shaker for 36 hours. Allow the bacteria to grow to the third generation before use.

[0024] 3. Fermentation culture and growth curve

[0025] Bacteroides ovatus ATCC 8483 bacteria solution was cultured in brain heart infusion broth (BHI) for 36 h, then 1.4 mL of B.O bacteria solution was taken in an EP tube. After centrifugation at 4°C, 8000 rpm for 10 min, 1.2 mL of supernatant was taken and filtered with a 0.22 μm filter to obtain the supernatant of the bacteria solution culture. 300 μL of Clostridium scindens ATCC 35704 was cultured in 5 mL of DSM Medium 110 medium, and 1 mL of BHI medium supernatant was added to the control group, and 1 mL of Bacteroides ovatus ATCC 8483 supernatant after culture was added to the experimental group, and three groups were repeated. From hour 0, 200 μL of suspension was taken from each group and added to a 96-well plate, which was sealed with sterile PBS, and the OD600 value was measured. Clostridium scindens ATCC 35704 was returned for further culture, and the suspension was taken every two hours thereafter to measure OD600. The OD values of each time period were counted and a growth curve was made, and the OD600 at the platform stage was finally obtained at 36 h.

[0026] 4. Statistical analysis

[0027] The data are expressed as mean ± standard deviation, and statistical analysis was performed using GraphPad Prism 8 software. The difference between the two groups was compared using t test. When P<0.05, the difference was considered statistically significant.

[0028] III. Experimental results

[0029] Figure 1 The growth curve of Clostridium scindens in different culture environments, and the growth rate is represented by OD600. By comparing the OD600 values of the two groups at each time point, it can be seen that Figure 1 It can be seen that from the 18 h logarithmic growth phase of the bacteria, the growth rate of Clostridium scindens with B.O culture supernatant was significantly higher than that of the BHI supernatant group, and the final concentration of Clostridium scindens with B.O culture supernatant was also significantly higher than that of the BHI supernatant group. The separation degree of the two curves P<0.0001, with *** significance.

[0030] The above experimental results show that the culture supernatant of Bacteroides ovatus ATCC 8483 can effectively promote the growth of Clostridium scindens ATCC 35704 and improve the efficiency of obtaining Clostridium scindens ATCC 35704, which provides a new method for the efficient cultivation of Clostridium scindens ATCC 35704.

[0031] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A promotion Clostridium difficile A method for growing ATCC 35704, characterized in that: exist Clostridium difficile Add appropriate amount of ATCC 35704 culture medium Bacteroides ovatus The culture supernatant of ATCC 8483; wherein, Clostridium difficile The culture medium of ATCC 35704 is DSM Medium 110 culture medium, Bacteroides ovatus The culture supernatant of ATCC 8483 was Bacteroides ovatus The supernatant of the culture medium after ATCC8483 was cultured in BHI medium for an appropriate period of time.

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