A method for high-density cultivation of Akkermansia muciniphila

Abstract

The present invention discloses a method for high-density cultivation of Akkermansia muciniphila, comprising: inoculating an Akkermansia muciniphila strain into a seed culture medium, and after two resuscitations, inoculating it into a fermenter for cultivation; using a three-stage carbon source concentration control in the fermenter cultivation. In the first stage, within 0-20.5 h of cultivation, the carbon source concentration is 14-16 g / L; in the second stage, within 20.5-31 h of cultivation, when the carbon source concentration in the fermenter decreases to 0.5-8 g / L, exponential feeding of the carbon source is started; in the third stage, within 31-34 h of cultivation, when the carbon source concentration in the fermenter is greater than 8 g / L, continue to maintain exponential feeding until the carbon source concentration in the fermenter reaches 20-25 g / L, at which time exponential feeding is stopped, and the bacteria consume the remaining carbon source until the fermentation reaches the highest biomass. The carbon source is glucose. This method can increase the biomass of Akkermansia muciniphila, and the biomass OD 600 reaches 22 in a 5 L fermenter.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial culture, and particularly relates to a method for high-density culture of Akkermansia muciniphila. Background Art

[0002] Akkermansia muciniphila is a Gram-negative bacterium, which was first isolated from human feces in 2004. Compared with other probiotics, the discovery and research of Akkermansia muciniphila are still in its infancy. Hyo Shin Yoon et al. discovered a protein secreted by Akkermansia muciniphila through liquid chromatography of proteins, named P9. Through mouse experiments, it was proved that P9 protein can induce the secretion of glucagon-like peptide-1 (GLP-1) and the thermogenesis of adipose tissue. On the basis of the study that Hubert Plovier et al. found that Akkermansia muciniphila can reduce obesity in mice, it was found that Akkermansia muciniphila inactivated by pasteurization has better efficacy and the functional protein Amuc_1100 was successfully isolated. At present, the research on Akkermansia muciniphila mainly focuses on the metabolic mechanism and the functional probiotic mechanism, exploring the functions and mechanisms of Akkermansia muciniphila from the perspective of molecular biology.

[0003] However, it cannot be ignored that the optimization of in vitro culture and high-density fermentation of Akkermansia muciniphila will bring great help to the industrial production and application of Akkermansia muciniphila. The research on the in vitro culture of Akkermansia muciniphila is relatively less. Usually, in an anaerobic bottle, BHI medium or mucin medium is used, and oxygen is removed by adding L-cysteine / cysteine salt and aeration to perform in vitro anaerobic culture of Akkermansia muciniphila. In 2021, Zhitao Li et al. constructed an in vitro bionic intestinal reactor for dynamic culture of Akkermansia muciniphila. Using BHI-pig gastric mucin, the biomass reached 1.92 g / L, which was 44.36% higher than that of static culture, while using human-derived mucin, the highest biomass reached 2.89 g / L. In 2021, Xinyue Liu et al. used BHI medium supplemented with 0.5% pig gastric mucin, and the OD 600 was about 0.22. However, the cost of mucin medium is too high and the operation of separating and purifying mucin is complex. In 2023, Wageningen University in the Netherlands used 32 g / L of pea peptone and a total concentration of mixed carbon sources of 150 mM (glucose:GlcNA = 3:1). In a 0.7 L fermenter, the pH was maintained at 7.0, and the final biomass OD 600 = 15. However, the research on high-density fermentation of Akkermansia muciniphila is currently a blank in China. Summary of the Invention

[0004] To solve the above problems, the present invention provides a method for high-density cultivation of Akkermansia muciniphila, which reduces the cultivation cost by optimizing the medium composition and performs high-density fermentation of Akkermansia muciniphila through a three-stage carbon source regulation strategy, thereby enhancing the biomass of Akkermansia muciniphila.

[0005] An embodiment of the present invention provides a method for high-density cultivation of Akkermansia muciniphila, which comprises the following steps:

[0006] (1) Inoculate the Akkermansia muciniphila strain into a seed medium, and after two resuscitations, inoculate it into a fermenter for cultivation;

[0007] (2) Use three-stage carbon source concentration control in the fermenter cultivation. In the first stage, from 0 to 20.5 h of cultivation, the carbon source concentration is 14 - 16 g / L; in the second stage, from 20.5 to 31 h of cultivation, when the carbon source concentration in the fermenter decreases to 0.5 - 8 g / L, start carbon source exponential feeding; in the third stage, from 31 to 34 h of cultivation, when the carbon source concentration in the fermenter is greater than 8 g / L, continue to maintain exponential feeding until the carbon source concentration in the fermenter reaches 20 - 25 g / L, at which point stop exponential feeding, and the cells consume the remaining carbon source until the fermentation reaches the highest biomass. The carbon source is glucose.

[0008] According to an embodiment of the present invention, a method for high-density cultivation of Akkermansia muciniphila divides the control of the carbon source concentration into three stages. While reducing the impact of too high carbon source concentration on cell growth, exponentially feeding the glucose carbon source according to the cell growth law can ensure that the cells grow and metabolize in the best state, thereby obtaining higher biomass and product yield. The biomass OD 600 reaches 22 in a 5 L fermenter, which is the highest level reported so far; and it can effectively avoid carbon source waste and the generation of by-products during the fermentation process, further improve the sustainability of the entire fermentation process, reduce production and operation costs, and is suitable for industrial production.

[0009] Optionally, the medium in the fermenter is 35 - 38 g / L of tryptone, 0.7 - 0.9 g / L of cysteine, 3 - 5 g / L of NaHCO3, 0.3 - 0.5 g / L of KH2PO4, and 1.3 - 1.4 g / L of Na2HPO4·12H2O. This medium is beneficial for reducing the lag phase of Akkermansia muciniphila growth and promoting rapid cell growth to reach a higher biomass in the stationary phase. Compared with the traditional fermentation medium, this medium has higher bioconversion efficiency and lower cost, bringing significant benefits to industrial production.

[0010] Optionally, in step (2), the inoculation amount of the fermenter is 8-16%, the temperature of the fermenter is 35-38°C, the rotation speed is 90-110 rpm, and the initial pH is controlled at 7.5-7.9.

[0011] Furthermore, when the pH of the fermenter drops to 7.0, the lye in the feeding bottle is automatically added to the fermenter through a peristaltic pump to control the pH of the fermenter at 6.5-7.2.

[0012] Optionally, in step (2), the specific growth rate of the bacteria in the second stage is 0.05-0.3.

[0013] Optionally, in step (2), the formula for exponential feeding is: F M,t S i =V M X t V t =17.5*e 0.15t ; where F M,t is the glucose feeding rate, S i is the glucose concentration in the feeding bottle, X t and V t are the dry cell density and the volume of the fermentation broth at the glucose feeding time t respectively, and V M is the current specific glucose consumption rate.

[0014] Furthermore, in step (2), the glucose concentration in the feeding bottle is 400-800 g / L.

[0015] Optionally, step (1) is: inoculating the preserved strain into BHI medium, culturing at 35-38°C and 90-110 rpm for 15-24 h to obtain the first-stage seed liquid, taking the first-stage seed liquid and inoculating it into BHI medium, culturing at 35-38°C and 90-110 rpm for 15-24 h to obtain the second-stage seed liquid, and inoculating the second-stage seed liquid into the fermenter for culture.

[0016] The additional aspects and advantages of the present invention will be partially given in the following description, partially become apparent from the following description, or be understood through the practice of the present invention. Description of the Drawings

[0017] Figure 1 It is the cell growth curve when the initial carbon source concentration of the medium in the fermenter is 15.0 g / L;

[0018] Figure 2 It is the cell growth curve when the initial carbon source concentration of the medium in the fermenter is 35.0 g / L;

[0019] Figure 3 It is the cell growth curve when the initial carbon source concentration of the medium in the fermenter is 55.0 g / L;

[0020] Figure 4 The cell growth curve after optimizing other components of the culture medium in the fermenter;

[0021] Figure 5 The cell growth curve of carbon source fed-batch culture for Akkermansia muciniphila in the fermenter before medium optimization;

[0022] Figure 6 The cell growth curve of carbon source fed-batch culture for Akkermansia muciniphila in the fermenter after medium optimization;

[0023] Figure 7 The effect of three-stage control of the fermenter carbon source on the growth of Akkermansia muciniphila in the fermenter. Detailed implementation mode

[0024] The technical solutions of the present invention are described below through specific examples. It should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combined steps or the insertion of other method steps between these clearly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Moreover, unless otherwise stated, the numbers of each method step are only convenient tools for identifying each method step, rather than limiting the arrangement order of each method step or the scope of implementation of the present invention. The change or adjustment of their relative relationship, without substantial change in technical content, should also be regarded as the scope of implementation of the present invention.

[0025] In order to better understand the above technical solutions, the exemplary embodiments of the present invention are described in more detail below. Although the exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0026] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0027] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0028] Example 1 Initial carbon source concentration in the fermenter and optimization of the culture medium in the shaker flask

[0029] The resuscitation medium for Akkermansia muciniphila uses BHI medium (brain heart infusion medium). The Akkermansia muciniphila strain cryopreserved with glycerol is inoculated into a headspace bottle with a 10 mL, 37 g / L BHI system at an inoculation amount of 1% (v / v). After culturing for 24 h, it is inoculated into a serum bottle with a 50 mL, 37 g / L BHI system at an inoculation amount of 4%. After culturing until OD 600 = 0.7, it is inoculated into a fermenter at an inoculation amount of 10%. The fermenter uses a 3 L system and requires 300 mL of secondary seeds.

[0030] The initial medium for the fermenter is as follows: tryptone 37 g / L, NaHCO3 4 g / L, KH2PO4 0.4 g / L, Na2HPO4·12H2O 1.34 g / L, NH4Cl 0.3 g / L, NaCl 0.3 g / L, MgCl·6H2O 0.1 g / L, CaCl2 0.11 g / L, L-cysteine 0.4 g / L, 1 mL of vitamin complex solution, 1 mL of trace element acid trace solution, and 1 mL of trace element base trace solution. Among them, the composition of the acid trace solution: H3BO3 0.618 g / L, ZnCl2 0.682 g / L, CuCl2·2H2O 0.170 g / L, MnCl2·4H2O 0.990 g / L, CoCl2·6H2O 1.190 g / L, NiCl2·6H2O 0.238 g / L, made up to 1 L with deionized water. Take 10 mL of the above solution, add FeCl2·4H2O 0.149 g / L, 0.42 mL of concentrated hydrochloric acid, and make up to 100 mL with deionized water. The composition of the base trace solution: Na2SeO3·5H2O 0.262 g / L, Na2WO·2H2O 0.330 g / L, Na2MoO4·2H2O 0.242 g / L, NaOH 4.0 g / L, made up to 1 L with deionized water. Take 10 mL of the above solution and make up to 100 mL with deionized water.

[0031] Optimization of the initial carbon source concentration in the fermenter: The initial carbon source (glucose) concentration in the fermenter is selected as 15.0 g / L, 35.0 g / L, 55.0 g / L, 100.0 g / L; after inoculation in the fermenter, samples are taken to measure the biomass, glucose concentration, and organic acid content at different time points. The biomass is measured by the absorbance value at 600 nm using an ultraviolet spectrophotometer, that is, OD 600 ; the glucose concentration uses DNS reagent; the organic acid detection uses gas chromatography.

[0032] The results are as Figures 1-4 shown, Figures 1-3They corresponded to carbon source concentrations of 15.0 g / L, 35.0 g / L, and 55.0 g / L respectively. A carbon source concentration of 100 g / L did not support the growth of the strain. The results showed that when the residual glucose concentration was greater than or equal to 4.0 g / L, the growth of Akkermansia muciniphila might be inhibited to some extent, mainly manifested as an increase in the lag phase. Therefore, to achieve high-density fermentation of Akkermansia muciniphila, it was necessary to maintain a low glucose concentration for cultivation. Although the biomass was the highest at a glucose concentration of 55 g / L, it could be achieved by feeding glucose later to avoid the inhibition caused by too high a carbon source concentration.

[0033] Optimization of medium components: The seed resuscitation steps of Akkermansia muciniphila were as described above. The secondary resuscitation seeds with an OD 600 = 0.7 were inoculated into 50 mL shake flasks at an inoculation amount of 8%. The optimized glucose concentration for the components was 15 g / L. The other components of the Control medium in the control group were the same as those of the above fermenter medium. In the experimental group Without inorganic salt, other components except NH4Cl, NaCl, MgCl·6H2O, CaCl2, trace element acid trace, and alkali trace solutions were the same as those of the control group Control. In the experimental group Without inorganic salt and vitamin, the vitamin complex solution was further removed on the basis of the experimental group Without inorganic salt, and the remaining components were the same. After inoculation, the biomass (OD 600 ) was measured at different time points.

[0034] The results were as Figure 4 shown. The initial medium contained many unoptimized components such as inorganic salts, vitamins, and trace elements. After removing these elements uniformly in the shake flasks, there was not much impact on the biomass of Akkermansia muciniphila.

[0035] Example 2 Carbon source fed-batch for fermenter cultivation of Akkermansia muciniphila: Before and after medium optimization

[0036] The optimized fermentation medium in the shake flask was used for scale-up cultivation in the fermenter. The composition of the fermenter medium for Akkermansia muciniphila was 37 g / L tryptone, 0.4 g / L cysteine, 4 g / L NaHCO3, 0.4 g / L KH2PO4, 1.34 g / L Na2HPO4·12H2O, and the glucose concentration was 15 g / L. After the medium and the fermenter were sterilized at 121 / 40 °C, the inoculation amount was 10% inoculated into the fermenter. The temperature of the fermenter was 37 °C, the rotation speed was 100 rpm, and the initial pH was controlled at 7.5 - 7.9. The glucose feeding method used fed-batch fermentation. When the initial glucose in the fermenter was consumed, the carbon source was manually fed into the fermenter until the glucose concentration recovered to 15 g / L.

[0037] From Figure 5 (before medium optimization) and Figure 6 (after medium optimization), it can be seen that the medium without inorganic salts, vitamins, and trace elements has improved the maximum biomass in fermenter culture, promoted the faster growth of the bacteria into the stationary phase, and increased the space-time yield.

[0038] Example 3 Application of Akkermansia muciniphila Fermenter Medium Combined with Three-Stage Carbon Source Control

[0039] The composition of the Akkermansia muciniphila fermenter medium is 37 g / L of tryptone, 0.4 g / L of cysteine, 4 g / L of NaHCO3, 0.4 g / L of KH2PO4, 1.34 g / L of Na2HPO4·12H2O, and the glucose concentration is 15 g / L. After the medium and the fermenter are sterilized at 121 / 40 °C, the inoculation amount is inoculated into the fermenter at 10%, the fermenter temperature is 37 °C, the rotation speed is 100 rpm, and the initial pH is controlled at 7.8 (adjusted with 1-3 M Na2CO3). When the fermenter pH drops to 7.0, NaOH is used to adjust the pH of the fermentation broth to maintain it at 6.5 - 7.2, and the adjustment is carried out with reference to Patent CN 116970506 A.

[0040] In the fermenter culture, a three-stage carbon source control strategy is used, as Figure 7 shown. In the first stage, from 0 - 20.5 h, the initial glucose concentration in the fermenter is 14 - 16 g / L, and as the bacteria grow, the glucose concentration in the fermenter naturally decreases. In the second stage, from 20.5 - 31 h, when the glucose concentration in the fermenter decreases to 0.5 - 8 g / L, carbon source exponential feeding starts. The carbon source exponential feeding calculates the exponential feeding according to the bacterial growth kinetics: F M,t S i = V M X t V t = 17.5 * e 0.15t , F M,t (mL / h) is the glucose feeding rate, S i is the glucose concentration in the feeding bottle (500 g / L), X t (g / L) and V t (L) are the dry cell density and the fermentation broth volume (3 L) at the glucose feeding time t respectively, V Mis the current specific glucose consumption rate. The carbon source in the feeding bottle is automatically added to the fermenter using peristaltic pump B. The carbon source concentration in the feeding bottle is 500 g / L, and the specific growth rate limiting the growth of the bacteria is 0.15. By adjusting the rotation speed of peristaltic pump B, the feeding rate can be precisely controlled. In the third stage, from 31 to 34 h, when the carbon source concentration in the fermenter starts to accumulate (greater than 8 g / L), although the bacterial growth kinetics in this stage do not conform to the kinetic model of exponential feeding, exponential feeding is still continued. The feeding formula is the same as that in the second stage until the carbon source concentration in the fermenter reaches 20 - 25 g / L. At this time, exponential feeding is stopped, and the bacteria consume the remaining carbon source until the fermentation reaches the highest biomass. The glucose concentration has three stages of change throughout the whole stage. From 0 to 20.5 h, it naturally drops from 15 g / L to 0.5 - 8 g / L. From 20.5 to 31 h, exponential feeding maintains a low glucose level (0 - 8 g / L). From 31 to 34 h, the glucose concentration in the tank increases to 20 - 25 g / L. After completing the three-stage regulation of the carbon source, the biomass in the fermenter will reach the highest level.

[0041] In the second stage of carbon source control, the method of anaerobic exponential feeding is used. In a 5 L fermenter, the biomass OD 600 reaches 22, which is the highest level reported so far. Compared with fed-batch fermentation, the strategy of exponential feeding is more convenient in operation and does not require continuous monitoring of the residual sugar concentration in the culture medium.

[0042] In summary, according to the embodiments of the present invention, the present application optimizes the carbon source concentration of the initial medium in the fermenter, which is beneficial to the improvement of the bacterial biomass while reducing the bacterial growth lag phase. Combining with the three-stage control strategy of the carbon source, it significantly improves the highest biomass of Akkermansia muciniphila in the fermenter. At the same time, it does not require frequent sampling to monitor the carbon source concentration in the tank, reducing the operation cost.

[0043] In the description of this specification, the descriptions with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification.

[0044] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for high-density cultivation of Akkermansia muciniphila, characterized in that, It includes the following steps: (1) Inoculate Akkermansia muciniphila strain into the seed medium, and after two resuscitations, inoculate it into the fermenter for cultivation; the medium in the fermenter is tryptone 35 - 38 g / L, cysteine 0.7 - 0.9 g / L, NaHCO3 3 - 5 g / L, KH2PO4 0.3 - 0.5 g / L, and Na2HPO4·12H2O 1.3 - 1.4 g / L; (2) Use three-stage carbon source concentration control in fermentor culture. In the first stage, from 0 to 20.5 h of culture, the carbon source concentration is 14 - 16 g / L. In the second stage, from 20.5 to 31 h of culture, when the carbon source concentration in the fermentor decreases to 0.5 - 8 g / L, start exponential feeding of the carbon source. In the third stage, from 31 to 34 h of culture, when the carbon source concentration in the fermentor is greater than 8 g / L, continue to maintain exponential feeding until the carbon source concentration in the fermentor reaches 20 - 25 g / L, then stop exponential feeding, and the cells consume the remaining carbon source until the fermentation reaches the highest biomass. The carbon source is glucose. The formula for exponential feeding is: F M,t S i = V M X t V t = 17.5*e 0.15t ; where F M,t is the glucose feeding rate, in mL / h, S i is the glucose concentration in the feeding bottle, with a concentration of 400 - 800 g / L, X t and V t are the dry cell density and the fermentation broth volume at the glucose feeding time t respectively, X t is in g / L, V t is in L, and V M is the current specific glucose consumption rate.

2. The method according to claim 1, wherein In step (2), the inoculation amount of the fermenter is 8 - 16%, the temperature of the fermenter is 35 - 38 °C, the rotation speed is 90 - 110 rpm, and the initial pH is controlled at 7.5 - 7.

9.

3. The method according to claim 2, wherein When the pH of the fermenter drops to 7.0, the lye in the feeding bottle is automatically added into the fermenter through a peristaltic pump to control the pH of the fermenter at 6.5 - 7.

2.

4. The method according to claim 1, characterized in that, In step (2), the specific growth rate of the bacteria in the second stage is 0.05 - 0.

3.

5. The method according to claim 1, characterized in that, Step (1) is: inoculate the preserved strain into the BHI medium, cultivate it at 35 - 38 °C and 90 - 110 rpm for 15 - 24 h to obtain the primary seed liquid, take the primary seed liquid and inoculate it into the BHI medium, cultivate it at 35 - 38 °C and 90 - 110 rpm for 15 - 24 h to obtain the secondary seed liquid, and inoculate the secondary seed liquid into the fermenter for cultivation.

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