A kind of anaerobic culture method of Akkermansia mucinophilus
An anaerobic culture and mucin technology, applied in the field of microbial culture, can solve the problems of difficulty in in-depth research, low bacterial activity, low bacterial abundance, etc., and achieve the effects of shortening culture time, convenient screening, and reducing culture difficulty.
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Embodiment 1
[0028] The anaerobic culture method of embodiment 1Akk bacteria
[0029] An anaerobic culture method for Akkermansia muciniphila. A single colony of Akk is taken for overnight activation, inoculated into an improved BHI medium at an inoculum of 1%, and incubated at 85-94% N 2 , 5%H 2 , 1~10%CO 2 Under anaerobic conditions, the cells were incubated at a constant temperature of 37°C.
[0030] The formula of the improved BHI medium is: Brain-heart Infusion Extract 37g / L, YeastExtract 5g / L, Mucin 2.5g / L, resazurin 1mg / L, L-cysteine hydrochloride 100mg / L.
[0031] Described improved BHI culture medium preparation method is:
[0032]S1. Preparation of Mucin solution: Dissolve Mucin in PBS buffer at a concentration of 1% (W / V), sterilize, let stand overnight, centrifuge at 12,000 rpm for 5 min, and take the supernatant for later use; in this example, weigh 1.25 g Mucin, dissolved in 125 mL PBS (pH 7.4), sterilized, let stand overnight, centrifuged at 12000 rpm for 5 min, and th...
Embodiment 2
[0036] Example 2 Culture medium optimization
[0037] Akk single colony was activated overnight, and inoculated into 1 mL of different media (medium A and B) according to 1% of the inoculum, in 90% N 2 , 5%H 2 , 5%CO 2 Under the anaerobic conditions of 37°C, the cells were incubated at 37 °C for 24 h, the samples were centrifuged at 6000 r / min for 5 min, the supernatant was discarded, and the precipitated cells were washed twice with sterilized PBS. Determination of bacterial concentration under OD 600nm , and the specific results are shown in Table 1.
[0038] Medium A: Brain-heart Infusion Extract 37g / L, Mucin 2.5g / L, Resazurin 1mg / L, L-cysteine hydrochloride 100mg / L;
[0039] Medium B (modified BHI medium): Brain-heart Infusion Extract 37g / L, YeastExtract 5g / L, Mucin 2.5g / L, Resazurin 1mg / L, L-cysteine hydrochloride 100mg / L.
[0040] Table 1 Effects of different media on the growth of Akk bacteria
[0041] Culture conditions Bacterial concentration ...
Embodiment 3
[0043] Example 3 Optimization of mixed gas for anaerobic culture
[0044] Take Akk single colony to activate overnight, inoculate 1 mL of modified BHI medium with 1% inoculum, and place it in the anaerobic system of the following mixed gas (anaerobic system A: 95% N) 2 , 5%H 2 ; Anaerobic system B: 80% N 2 , 20%CO 2 ; Anaerobic system C: 90% N 2 , 5%H 2 , 5%CO 2 ;), cultured at 37 °C for 24 h, and then streaked the activated bacterial liquid on the modified BHI solid medium, inverted at 37 °C for 24-48 h, and recorded the growth of the bacteria; the results of shaking flask culture and plate culture were respectively See figure 1 and figure 2 .
[0045] Depend on Figures 1 to 2 It can be seen that under the conditions of anaerobic system A and anaerobic system B, when the liquid level of the anaerobic bottle medium is 1 cm, the Akk bacteria basically do not grow, and only when the liquid level is not less than 2 cm, the Akk bacteria can grow. It can be cultivated i...
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