CN111763653B - Culture method of akkermansia muciniphila based on novel electron acceptor - Google Patents
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Abstract
The invention discloses a culture method of akkermansia muciniphila based on a novel electron acceptor, which comprises the steps of adding nitrate or TMAO solution into BHI culture medium, inoculating akkermansia muciniphila (Akk bacteria) into the culture medium, and enabling the culture medium to be subjected to N-N conversion in a mixed gas ratio of 80-95% 2 、5％～20％H 2 And 0 to 3% 2 Culturing under the culture conditions of (1). The invention adopts nitrate or TMAO solution to replace the traditional gaseous CO 2 As electron acceptor of Akk bacteria, CO is avoided 2 Influence of concentration fluctuation on growth of Akk bacteria; meanwhile, the nitrate or TMAO solution can also enhance the tolerance of Akk bacteria to oxygen, thereby not only avoiding the inhibiting effect of oxygen on Akk bacteria, but also promoting the growth of Akk bacteria in a certain oxygen concentration range, thus obtaining the Akk bacteria culture method which has lower cost, higher controllability and more loose culture conditions and is beneficial to large-scale culture, and providing a new idea for the culture of Akk bacteria.
Description
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a culture method of akkermansia muciniphila based on a novel electron acceptor.
Background
Akkermansia muciniphila (Akkermansia muciniphila, hereinafter abbreviated as Akk bacterium) belongs to the phylum of verrucomicrobia and is a gram-negative bacterium. Akk bacteria are widely distributed in intestinal mucus layers of human beings and animals and are one of intestinal high-abundance symbiosis bacteria. Akk bacteria are bacteria capable of degrading mucoprotein in human intestinal tracts, can take the mucoprotein as a unique carbon source, a unique nitrogen source and a unique energy source, and can produce acetate, ethanol, propionate and sulfate in the glycolysis process. The research shows that Akk bacteria are associated with various system diseases such as obesity, diabetes, immunoregulation, alcoholic fatty liver, coronary arteriosclerosis and the like, and the content of the Akk bacteria is one of important marker microorganisms for measuring the intestinal microecological balance. The culture of Akk bacteria is particularly important as a novel candidate probiotic.
Derrien M et al (Derrien M. Akkermansia mucina gen. Nov. Sp. Nov. A human endogenous mucin-degrading bacterium [ J)]int.J.Syst.Evol.Microbiol.2004, 54.) discloses that Ackermanella mucosae can grow on a basic anaerobic medium containing porcine gastric mucin as sole carbon and nitrogen source and in a culture atmosphere of N 2 /CO 2 (80,20,v/v), chinese patent CN110551658A discloses a method of hypoxic culture of Ackermanella muciniphila by dividing N by 70 to 94% 2 、1～5％O 2 、5～25％CO 2 Culturing Akk bacteria under the gas atmosphere. Namely that CO is generally considered in the culture process of Akk bacteria at present 2 As electron acceptor, however, CO is used 2 On one hand, the cost is high, and on the other hand, because Akk bacteria can generate a small amount of CO in the culture process 2 The air environment in the culture process fluctuates to some extent, which affects the normal growth of Akk bacteria.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and nitrate or TMAO solution is added into the culture medium to replace the traditional CO 2 The culture method of Akk bacteria, which is used as an electron acceptor of Akk bacteria, has lower cost and stronger controllability.
In order to realize the purpose, the invention adopts the technical scheme that:
a process for culturing Ackermanella muciniphila based on new electron acceptor, which includes adding nitrate or TMAO solution to the culture medium, and adding N to 80-95% 2 、5～20％H 2 And 0 to 3% 2 Culturing under the culture conditions of (3).
To avoid CO during the culture of Akk bacteria 2 The influence of concentration fluctuation on the growth of Akk bacteria breaks through the conventional thinking of researchers in the field, and the nitrate or TMAO (trimethylamine oxide) solution is adopted to replace the traditionally used CO 2 As electron acceptor of Akk bacteria, akk bacteria can be made CO-free 2 The growth is normal under the existing condition, the production cost is lower, and the controllability of the concentration of the liquid solution is stronger compared with that of a gaseous electron acceptor. Meanwhile, the nitrate or TMAO solution can also enhance the oxygen tolerance of the Akk bacteria, so that the Akk bacteria can normally grow under the condition of the existence of oxygen with a certain concentration, and can properly promote the growth of the Akk bacteria under the condition of the existence of oxygen with a proper concentration.
Preferably, the concentration of the nitrate or TMAO in the medium is 1 to 10mM. Nitrate or TMAO is used as an electron acceptor in anaerobic respiration of Akk bacteria, and the normal physiological metabolism of the Akk bacteria can be influenced by too low concentration of the nitrate or TMAO, and the normal growth of the Akk bacteria can be influenced by too high concentration of the nitrate or TMAO. Therefore, the concentration of nitrate or TMAO most suitable for the growth of Akk bacteria is preferably 1 to 10mM.
Preferably, the concentration of said nitrate or TMAO in the culture medium is between 3 and 8mM.
Preferably, the concentration of the nitrate solution in the medium is 5mM, or the concentration of the TMAO solution in the medium is 8mM.
Preferably, the nitrate is one or more of sodium nitrate, potassium nitrate, ammonium nitrate and calcium nitrate.
Preferably, the mixing ratio of the mixed gas is 90% 2 And 10% of H 2 . Namely, after the nitrate or TMAO solution is added into the culture medium, no additional CO is needed to be introduced 2 Thus ensuring the normal growth of Akk bacteria.
Preferably, the ratio of the mixed gas is 87 to 90% 2 、8～12％H 2 And 1 to 2% of 2 . The nitrate or TMAO solution enhances the oxygen tolerance of the Akk bacteria, and when the oxygen concentration is 1-2%, the growth of the Akk bacteria can be promoted to a certain degree.
Preferably, the ratio of the mixed gas is 88.5% 2 、10％H 2 And 1.5% of 2 。
Preferably, the culture medium is a BHI culture medium, and the formula of the BHI culture medium is as follows: 37g/L of Brain-heart Infusion Extract, 5g/L of Yeast Extract, 2.5g/L of Mucin, 1mg/L of resazurin and 100mg/L of L-cysteine hydrochloride.
Compared with the prior art, the invention has the beneficial effects that:
(1) Replacing traditional CO with nitrate or TMAO solution 2 As the electron acceptor of Akk bacteria, creatively selects the concentration of nitrate or TMAO solution which is most suitable for the growth of Akk bacteria, thereby not only avoiding CO 2 The influence of concentration fluctuation on the growth of Akk bacteria is low in cost and high in controllability, large-scale culture is facilitated, and a new thought is provided for an Akk bacteria culture method.
(2) The nitrate or TMAO solution can also enhance the tolerance of Akk bacteria to oxygen, not only avoids the inhibiting effect of oxygen on the growth of Akk bacteria, but also promotes the growth of Akk bacteria in a certain oxygen concentration range.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the embodiments of the present invention, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The Akkermansia muciniphila adopted in the embodiment of the invention is Akkermansia muciniphila ATCC BAA-835, and is purchased from China center for type culture Collection.
In the embodiment of the invention, the Akk bacteria are cultured by adopting a BHI culture medium, and the preparation method of the BHI culture medium comprises the following steps:
s1, adding 37g of Brain-heart Infusion Extract, 5g of Yeast Extract and 1mL of resazurin in 0.1% (W/V) into 1L of water, uniformly mixing, stirring and dissolving, boiling to remove oxygen for 30min, then subpackaging in anaerobic vials, sealing, and sterilizing with high-pressure steam at 121 ℃ for 15min;
s2, dissolving Mucin in an anaerobic PBS buffer solution with the concentration of 1% (W/V), autoclaving, standing overnight, centrifuging at 12000rpm for 5min, and taking the supernatant to obtain a Mucin solution;
s3, dissolving L-cysteine hydrochloride in ultrapure water to obtain a 10% (W/V) L-cysteine hydrochloride solution, and filtering and sterilizing to obtain a sterile L-cysteine hydrochloride solution;
and S4, taking 375mL of the mixed solution obtained in the step S1, adding 125mL of the Mucin solution obtained in the step S2, uniformly mixing, and then adding 10mL of the L-cysteine hydrochloride solution obtained in the step S3 to obtain the BHI culture medium.
EXAMPLE 1 optimization and selection of Electron acceptors
Experimental groups: akk bacteria in logarithmic growth phase are inoculated into the BHI culture medium according to the ratio of 1 1000, and are distributed into 3 mL/tube anaerobic test tubes, and then the following electron acceptor solutions with the concentration of 5mM are respectively added: naNO 3 (nitrate), TMAO (trimethylamine oxide), naNO 2 (nitrite salt), na 2 SO 3 (sulfite) and Na 2 SO 4 (sulfate salt), na 2 S 2 O 3 (thiosulfate), feCl 3 (iron ion), naClO 2 (hypochlorite), DMSO (dimethyl sulfoxide), disodium fumarate (fumarate).
Blank control group: taking Akk bacteria in a logarithmic phase of growth, inoculating the Akk bacteria into the BHI culture medium according to a ratio of 1.
The experimental group and the blank control group were simultaneously in the absence of CO 2 (the mixing ratio of the mixed gas is 90% by volume N 2 、10％H 2 ) Or with CO 2 (the mixing ratio of the mixed gas is 80% 2 、10％H 2 、10％CO 2 ) The culture temperature was 37 ℃. Then, samples were taken every 24 hours, and the OD value (denoted as OD) at a wavelength of 600nm of each sample was measured by a microplate reader 600 ) The total number of bacterial colonies per ml of sample (denoted as CFU value) was also determined by plate count. The results are shown in tables 1-2. Wherein Table 1 is CO-free 2 Under the condition, different electron acceptors influence the growth of Akk bacteria; table 2 shows the presence of CO 2 Effect of each electron acceptor on Akk bacterial growth under conditions.
TABLE 1 Effect of different electron acceptors on Akk bacterial growth (CO-free) 2 )
TABLE 2 different electron acceptor pairs AkkInfluence of bacterial growth (with CO) 2 )
Since the initial inoculum size of Akk bacteria was the same, the initial Akk bacteria concentration was the same in each sample. Due to the influence of the color of the reagent and the oxidation of the reagent during the detection process, the OD in part of the sample is caused 600 Higher values (e.g. with FeCl addition) 3 And NaClO 2 Sample of (2) and the growth rate of Akk bacteria is different in the presence of different electron receptors, some bacteria die during the determination process, and the spectrophotometer cannot accurately distinguish the survival state of the bacteria, resulting in the determined OD 600 Since the value is high, the growth of Akk bacteria is determined by using the total number of bacterial colonies (i.e., CFU value) as a main index.
Based on the above measurement results, it was found that in the absence of CO 2 In the presence of the enzyme, the bacteria died gradually due to the lack of electron acceptor in the placebo group, naNO compared to the placebo group 2 、FeCl 3 And NaClO 2 The CFU data measured at 24h were significantly lower than the blank control, i.e., naNO 2 、FeCl 3 And NaClO 2 The normal growth of Akk bacteria is obviously inhibited; na (Na) 2 SO 4 、Na 2 S 2 O 3 Although DMSO and disodium fumarate properly prolong the survival time of Akk bacteria, the electron acceptor can not maintain normal growth and proliferation of Akk bacteria from the viewpoint of CFU value, i.e., akk bacteria cannot utilize the electron acceptor; and NaNO 3 TMAO can make Akk bacteria normally proliferate, that is, akk bacteria can utilize NaNO 3 TMAO grows and proliferates as an electron acceptor. In CO 2 In the presence of the carrier, akk bacteria in the blank control group can utilize CO 2 Normal growth and proliferation as electron acceptor, and CO 2 Can be added with Na 2 SO 4 、Na 2 S 2 O 3 Akk bacteria with DMSO and disodium fumarate grow back to normal level but cannot compensate for NaNO 2 、FeCl 3 And NaClO 2 Inhibition ofWhile CO is present 2 Does not affect NaNO 3 And growth of Akk bacteria in TMAO, even if part of CO is mixed in the culture environment 2 The growth of Akk bacteria can not be influenced, and the culture environment is more loose. At the same time compared with CO 2 As electron donor, naNO 3 Or TMAO as electron donor can promote Akk bacteria proliferation to some extent.
EXAMPLE 2 optimization of Electron acceptor concentration
With NaNO 3 Or taking TMAO as an electron donor, inoculating Akk bacteria in the logarithmic growth phase into the BHI culture medium according to the ratio of 1 3 And TMAO as electron acceptor, sequentially added to each test tube at concentration gradients of 0.5mM, 1mM, 3mM, 5mM, 8mM, 10mM, and 12mM, respectively, and mixed gas ratio of 90% 2 、10％H 2 The culture was carried out at 37 ℃. Samples were taken every 24h and the OD value (denoted as OD) of each sample was determined by a microplate reader at a wavelength of 600nm 600 ) The total number of bacterial colonies per ml of sample (denoted as CFU value) was also determined by plate count. The results are shown in tables 3 to 4. Wherein Table 3 is NaNO 3 Influence of concentration on growth of Akk bacteria; table 4 shows the effect of TMAO concentration on Akk bacteria growth.
TABLE 3 NaNO 3 Effect of concentration on Akk bacterial growth
TABLE 4 Effect of TMAO concentration on Akk bacteria growth
From the above measurement results, naNO was found 3 And TMAO concentration of 1 to 10mMIn support of the normal growth and proliferation of Akk bacteria, the normal physiological metabolism of Akk bacteria is influenced due to the reduction of the content of an electron acceptor when the concentration is lower than 1mM, and the cell osmotic pressure and the enzyme activity are influenced when the concentration is higher than 10mM, so that the normal growth of Akk bacteria is influenced. Further, naNO 3 And TMAO at a concentration of 3-8 mM is more favorable for proliferation of Akk bacteria, and 5mM NaNO 3 Or 8mM TMAO can maximally promote the growth and proliferation of Akk bacteria.
EXAMPLE 3 optimization of mixture gas ratio
Akk bacteria in the log phase of growth are inoculated into the BHI medium according to the ratio of 1 3 (nitrate) and 8mM TMAO were added as electron acceptors to the respective test tubes, and then the respective test tubes were divided into 7 groups, and cultured in mixed gas at 37 ℃ in the following ratio, respectively.
Samples were taken every 24h and the OD of each sample (denoted as OD) was determined by spectrophotometry at a wavelength of 600nm 600 ) The total number of bacterial colonies per ml of sample (denoted as CFU value) was also determined by plate count. The results are shown in tables 5 to 6. Wherein Table 5 is NaNO 3 When the compound gas is used as an electron donor, the mixed gas with different proportions has influence on the growth of Akk bacteria; table 6 shows the effect of mixed gases of different proportions on the growth of Akk bacteria when TMAO is used as an electron donor.
TABLE 5 influence of mixed gas of different proportions on the growth of Akk bacteria (NaNO) 3 )
TABLE 6 influence of mixed gas of different proportions on Akk bacteria growth (TMAO)
As is clear from the above measurement results, naNO was used as the indicator 3 And TMAO is used as an electron donor, so that the tolerance of the Akk bacteria of anaerobic bacteria to oxygen can be enhanced, the Akk bacteria can normally grow and proliferate when the oxygen concentration is 0-3%, and the inhibition effect of the oxygen to the Akk bacteria is enhanced when the oxygen concentration is higher than 3%, so that the growth of the Akk bacteria is inhibited. Furthermore, the growth of Akk bacteria can be remarkably promoted when the oxygen concentration is 1% -2%, and the promoting effect of oxygen on the growth of Akk bacteria is strongest when the oxygen concentration is 1.5%.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. The method for culturing Ackermanella muciniphila based on the novel electron acceptor is characterized in that nitrate or TMAO solution is added into a culture medium, the final concentration of the nitrate or TMAO solution is 1-10 mM, and the N percent is calculated according to the proportion of mixed gas of 80-95% 2 、5～20％H 2 And 0 to 3% 2 Culturing under the culture conditions of (1).
2. The method for culturing akkermansia muciniphila based on a novel electron acceptor according to claim 1, wherein the final concentration of the nitrate or TMAO solution is 3 to 8mM.
3. The method for culturing akkermansia muciniphila based on a novel electron acceptor according to claim 1, wherein the final concentration of the nitrate solution is 5mM or the final concentration of the TMAO solution is 8mM.
4. The method for culturing akkermansia muciniphila based on a novel electron acceptor according to any one of claims 1 to 3, wherein the nitrate is one or more of sodium nitrate, potassium nitrate, ammonium nitrate and calcium nitrate.
5. The method for culturing Akkermansia muciniphila based on a novel electron acceptor according to claim 1, wherein the ratio of said mixed gas is 90% by weight N 2 And 10% of H 2 。
6. The method for culturing akkermansia muciniphila based on a novel electron acceptor according to claim 1, wherein the ratio of the mixed gas is 87 to 90% 2 、8～12％H 2 And 1 to 2% 2 。
7. The method for culturing Ackermanella muciniphila according to claim 6, wherein the ratio of said mixed gas is 88.5% by weight N 2 、10％H 2 And 1.5% of 2 。
8. The method for culturing akkermansia muciniphila based on the novel electron acceptor according to claim 1, wherein the culture medium is a BHI culture medium, and the formula of the BHI culture medium is as follows:
37g/L of Brain-heart infusion extract, 5g/L of Yeast extract, 2.5g/L of Mucin, 1mg/L of resazurin and 100mg/L of L-cysteine hydrochloride.
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