Method for preparing high-abundance isotope labeled microbial cells

Abstract

The invention discloses a method for preparing high-abundance isotope labeled microbial cells, which comprises the following steps: determining basic conditions of an isotope labeled culture medium based on nutritional requirements of raw material microbial cells, and preparing an initial culture medium; adjusting the substance concentration in the initial culture medium, and determining the optimal substance concentration in the initial culture medium to obtain an optimal culture medium; obtaining an initial culture environment of the raw material microbial cells based on culture environment requirements of the raw material microbial cells; based on the initial culture environment of the raw material microbial cells, the optimal culture environment of the raw material microbial cells is obtained, and optimal culture parameters are obtained; the method comprises the following steps: acquiring transferred culture microbial cells based on a transfer culture process of raw material microbial cells; the high-abundance isotope labeled microbial cell powder is obtained. The problem that in the prior art, culture parameters of microbial cells cannot be accurately adjusted, so that the isotope abundance is reduced is solved. And the effect of fully improving the isotope abundance is achieved.

Description

Technical Field

[0001] This application belongs to the field of microbial cell preparation. Specifically, it relates to a method for preparing highly enriched isotope-labeled microbial cells. Background Art

[0002] In the preparation of highly enriched isotope-labeled microbial cells, corresponding isotope substances need to be configured, and it is widely adopted to input the isotope substances as nutrients for microbial cell preparation. In the current technical solutions, for various nutrients and culture environments in the culture medium, they are mainly processed based on the general nutrient and culture environment configuration schemes. However, in terms of the effect, the isotope abundance of microbial cells cannot be comprehensively tracked. Especially for microorganisms that need to be subcultured, the isotope abundance of microbial cells at different culture stages cannot be reasonably adjusted, resulting in the fact that the isotope abundance of microbial cells cannot be most reasonably increased.

[0003] Therefore, how to improve the isotope abundance in all processes of microbial cell preparation, so as to ensure that the isotope abundance of microbial cells reaches the peak, is a technical problem that needs to be urgently solved by those skilled in the art. Summary of the Invention

[0004] In order to solve the problem of insufficient isotope abundance in the culture of microbial cells in the existing technical solutions, the following technical content is disclosed in this application to enable the isotope abundance of microbial cells to reach the peak by improving the isotope abundance in each process of microbial cell preparation. Specifically: A method for preparing highly enriched isotope-labeled microbial cells, the method comprising: Based on the nutritional requirements of the raw material microbial cells, determine the basic conditions of the isotope-labeled culture medium and configure the initial culture medium; Adjust the substance concentration in the initial culture medium, culture the raw material microbial cells, determine the optimal substance concentration in the initial culture medium, and obtain the optimal culture medium; Based on the culture environment requirements of the raw material microbial cells, obtain the initial culture environment of the raw material microbial cells; Based on the initial culture environment of the raw material microbial cells, obtain the optimal culture environment of the raw material microbial cells and obtain the optimal culture parameters; Based on the subculture process of the raw material microbial cells, obtain the subcultured microbial cells; Process the subcultured microbial cells to obtain highly enriched isotope-labeled microbial cell powder.

[0005] Optionally, based on the nutritional requirements of the raw material microbial cells, determining the basic conditions of the isotope-labeled culture medium and configuring the initial culture medium includes: Obtaining the nutritional requirements of the raw material microbial cells based on the type of the raw material microbial cells; Obtaining the types of isotope materials and non-isotope materials of the raw material microbial cells based on the nutritional requirements of the raw material microbial cells; Setting the concentrations of the isotope materials and the non-isotope materials of the raw material microbial cells based on the types of the isotope materials and the non-isotope materials of the raw material microbial cells to obtain the initial culture medium; Obtaining the isotope abundance of the initial culture medium to obtain the initial isotope abundance.

[0006] Optionally, adjusting the substance concentrations in the initial culture medium, culturing the raw material microbial cells, determining the optimal substance concentrations in the initial culture medium, and obtaining the optimal culture medium includes: Obtaining the substance concentrations of all substances in the initial culture medium, adjusting all the substances according to the corresponding substance concentration gradients to obtain staged substance concentrations; Configuring different culture media based on the staged substance concentrations to obtain candidate culture media; Inoculating the raw material microbial cells in the candidate culture media and culturing them in the same culture environment for the same time; Obtaining the raw material microbial cells in the candidate culture media to obtain amplified microbial cells; Obtaining the quantity of the amplified microbial cells to obtain an amplified microbial value; Obtaining the candidate culture medium corresponding to when the amplified microbial value is higher than a preset amplified microbial value, and the corresponding amplified microbial cells are candidate microbial cells; Obtaining the isotope utilization rate based on the isotope abundance of the candidate microbial cells; Obtaining the candidate culture medium corresponding to when the isotope utilization rate is higher than a preset isotope utilization rate to obtain the optimal culture medium.

[0007] Optionally, obtaining the isotope utilization rate based on the isotope abundance of the candidate microbial cells includes: Obtaining the substance concentration value in the candidate culture medium to obtain the initial substance concentration; Obtaining the substance concentration value in the candidate culture medium after culturing the amplified microbial cells in the candidate culture medium to obtain the final substance concentration; Obtaining the isotope utilization rate based on the initial substance concentration and the final substance concentration, and the isotope utilization rate equation is: ; Wherein, R a represents the isotope utilization rate, i represents the index of the substance corresponding to the substance concentration, j represents the total index of the substances corresponding to the substance concentration, represents the starting time, represents the end time, represents the starting substance concentration, represents the terminal substance concentration, represents the isotope abundance of the candidate microbial cells, represents the isotope abundance of the candidate culture medium.

[0008] Optionally, obtaining the initial culture environment of the raw material microbial cells based on the culture environment requirements of the raw material microbial cells includes: Obtaining the culture environment requirements of the raw material microbial cells based on the type of the raw material microbial cells; Obtaining the culture environment parameters of the raw material microbial cells based on the culture environment requirements of the raw material microbial cells; Adjusting all the culture environment parameters according to the corresponding environmental parameter gradients based on the culture environment parameters of the raw material microbial cells to obtain the initial culture environment.

[0009] Optionally, obtaining the optimal culture environment of the raw material microbial cells based on the initial culture environment of the raw material microbial cells and obtaining the optimal culture parameters includes: Obtaining all the culture environment parameters in the initial culture environment, adjusting all the culture environment parameters according to the corresponding parameter gradients to obtain the stage-by-stage culture environment parameters; Constructing different culture environments based on the stage-by-stage culture environment parameters to obtain candidate culture environments; Setting the optimal culture medium in the candidate culture environments, inoculating the raw material microbial cells on the optimal culture medium, and culturing for the same time to obtain cultured microbial cells; Obtaining the number of the cultured microbial cells to obtain the cultured microbial value; Obtaining the candidate culture environment corresponding to when the cultured microbial value is higher than the preset cultured microbial value, and the corresponding cultured microbial cells are the cultured microbial cells; Obtaining the isotope abundance change rate based on the isotope abundance of the cultured microbial cells; Obtaining the candidate culture environment corresponding to when the isotope abundance change rate is higher than the preset isotope abundance change rate to obtain the optimal culture parameters.

[0010] Optionally, obtaining the isotope abundance change rate based on the isotope abundance of the cultured microbial cells includes: Obtaining the culture environment parameters in the candidate culture environment to obtain candidate culture environment parameters; Based on the cultured microbial value and the isotope abundance of the cultured microbial cells, obtaining the isotope abundance change rate, and the isotope abundance change rate equation is: ; Wherein, represents the isotope abundance change rate, represents the isotope abundance of the candidate microbial cells, represents the isotope abundance of the candidate culture medium, M represents the cultured microbial value, m represents the inoculation quantity of the raw material microbial cells, p represents the index of the candidate culture environment parameters, q represents the total amount of the indices of the candidate culture environment parameters, represents the candidate culture environment parameters, represents the weight of the candidate culture environment parameters.

[0011] Optionally, obtaining the subcultured microbial cells based on the subculture process of the raw material microbial cells includes: Obtaining the subculture process of the raw material microbial cells, and obtaining the nutritional requirements and culture environment of the subculture process; Based on the nutritional requirements and culture environment of the subculture process, setting the initial subculture medium and the initial subculture environment for each subculture process; Obtaining the concentrations of all substances in the initial subculture medium of the subculture process, adjusting according to the corresponding concentration gradients, and formulating a culture medium to obtain a candidate subculture medium; Obtaining the initial subculture environment parameters of the subculture process, adjusting according to the corresponding environmental parameter gradients, and adjusting the culture environment to obtain a candidate subculture environment; Based on the candidate subculture medium and the candidate subculture environment, and the isotope utilization rate equation and the isotope abundance change rate equation, determining the optimal subculture medium and the optimal subculture environment; Based on the optimal subculture medium and the optimal subculture environment, subculturing the raw material microbial cells to obtain the subcultured microbial cells.

[0012] Optionally, it further includes: Based on the transfer process of the raw material microbial cells and the transferred culture microbial cells, obtain the transfer culture steps; Establish the association relationship between the transfer culture steps, the optimal transfer culture medium, and the optimal transfer culture environment, and establish a transfer culture link; Based on the transfer culture link, obtain the substance concentration of the optimal transfer culture medium and the environmental parameters of the optimal transfer culture environment, and configure the culture medium and adjust the culture environment.

[0013] Optionally, the processing of the transferred culture microbial cells to obtain the high-abundance isotope-labeled microbial cell powder includes: Perform centrifugal enrichment on the transferred culture microbial cells to obtain the transferred culture microbial cells; Perform sterilization, freeze-drying, and grinding on the transferred culture microbial cells to obtain the high-abundance isotope-labeled microbial cell powder.

[0014] The beneficial effects of this application include: 1. Improve the isotope abundance in microbial cell culture. First, configure the culture medium and culture environment according to the general culture environment of microbial cells, and then optimize the culture medium and culture environment to adjust various culture parameters and the substance concentration of the culture medium during the microbial cell culture process. Screen the ones that can most promote the amplification efficiency of microbial cells, and on this basis, analyze the isotope abundance of microbial cells, and screen out the optimal culture medium and culture parameters, so as to ensure an increase in isotope abundance during the amplification culture of microbial cells.

[0015] 2. Achieve an increase in isotope abundance in the transfer culture process of microbial cells. During the culture of microorganisms, for the culture medium and culture environment parameters used in each amplification culture process, relevant parameters are also adjusted with high precision to ensure the accumulation of isotope substances in each transfer culture process, so that in the finally obtained transferred culture microbial cells, their isotope abundance is fully increased based on the accumulation.

[0016] 3. Realize the setting of culture environments for different types of microbial cells. Based on the transfer culture scheme for microbial cells, determine the culture medium and culture environment parameters for each transfer culture process, and establish a corresponding association relationship between these parameters and the transfer culture steps. Thus, when obtaining the raw material microbial cells and the transferred microbial products, the culture system can independently determine the decomposition steps of the transfer process, and select the culture medium and culture environment parameters for each step to achieve high-abundance isotope culture of microbial cells. Description of the Drawings

[0017] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the accompanying drawings required for use in the embodiments of the present application or the prior art. Obviously, only some embodiments of the present application are described below. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings. The drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure, but do not constitute a limitation to the present disclosure. In the drawings: Figure 1 It is a flowchart of a method for preparing highly abundant isotope-labeled microbial cells provided by an embodiment of the present application; Figure 2 It is a broken line graph of the absorption rate of microbial cells of isotope materials during the preparation of highly abundant isotope-labeled microbial cells provided by an embodiment of the present application; Figure 3 It is the isotope 13 C abundance broken line graph corresponding to the adjustment of the culture medium parameters during the preparation of highly abundant isotope-labeled microbial cells; Figure 4 It is the isotope 15 N abundance broken line graph corresponding to the adjustment of the culture environment parameters during the preparation of highly abundant isotope-labeled microbial cells; Figure 5 It is a schematic diagram showing the correlation between the subculture process and culture parameters during the preparation of highly abundant isotope-labeled microbial cells provided by an embodiment of the present application. Detailed implementation manners

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, rather than all embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application. Additionally, in the embodiments of the present application, "first", "second", etc. are used to distinguish similar objects and do not necessarily need to describe a specific order or sequence.

[0019] In the cultivation of microbial cells, there are usually two purposes. One is to simply amplify microbial cells, and the other is to screen microbial cells. Regardless of the purpose, it is necessary to appropriately adjust the culture medium and culture environment of microbial cells. Currently, in the cultivation of microbial cells for these two purposes, various environmental parameters are set, so that based on the type and concentration of substances in the culture medium and the setting of culture environment parameters, microbial cells can be amplified in this environment. However, in this method, for the purpose of screening microbial cells, an isotope labeling scheme is widely used at present, but usually only the setting of specific culture parameters is adopted. In this way, the isotope abundance of microbial cells is basically fixed, and it is difficult to further increase the isotope abundance of microbial cells. In addition, in the cultivation process of some microbial cells, multiple subculture operations are required. In the current scheme, the cultivation method based on the same parameters is adopted, and the culture environment in the subculture is not further adjusted, making it difficult to further increase the isotope abundance in the subculture process.

[0020] In the technical solution of the present application, in the cultivation of raw material microbial cells, the simultaneous adjustment of the culture medium and the culture environment is achieved, and based on multiple configuration combinations, the amplification efficiency of microbial cells is analyzed to screen out the culture medium with the highest amplification efficiency. The reason for adopting this method is that in the cultivation of microbial cells, the nutrients in the culture medium are the factors that can most affect the amplification efficiency. After determining the highest cultivation efficiency of microbial cells, the culture environment is further screened. At the same time, both of these screening processes are based on the isotope abundance as the limiting parameter, so as to ensure that in the initial cultivation, the cultivation parameters of the raw material microbial cells can ensure that the microbial cells have the highest isotope abundance. Then, in the subculture, the culture parameters are also screened in the same way to ensure that in the cultivation of microbial cells, the isotope abundance can be accumulated in each subculture process, so that the obtained microbial cell culture product has the highest isotope abundance.

[0021] The embodiment of the present application provides a method for preparing highly abundant isotope-labeled microbial cells, which improves the isotope abundance of microbial cells. Specifically: As Figure 1 shown, it is a flowchart of a method for preparing highly abundant isotope-labeled microbial cells provided by the embodiment of the present application, including: S110. Based on the nutritional requirements of raw material microbial cells, determine the basic conditions of the isotope-labeled culture medium and configure the initial culture medium; S120. Adjust the substance concentration in the initial culture medium, cultivate the raw material microbial cells, determine the optimal substance concentration in the initial culture medium, and obtain the optimal culture medium; S130. Obtain the initial culture environment of the raw material microbial cells based on the culture environment requirements of the raw material microbial cells. S140. Obtain the optimal culture environment of the raw material microbial cells based on the initial culture environment of the raw material microbial cells, and obtain the optimal culture parameters. S150. Obtain the subcultured microbial cells based on the subculture process of the raw material microbial cells. S160. Process the subcultured microbial cells to obtain high-abundance isotope-labeled microbial cell powder.

[0022] The purpose of the above steps is to reasonably screen the medium parameters and the parameters of the culture environment during the initial culture stage of the microbial cells and in each subculture step, so as to ensure that in the final obtained microbial cell culture result, the microbial cells have the highest abundance in the overall obtained culture result.

[0023] Next, the specific content of the technical solutions in each of the above steps will be analyzed and described. Specifically: As described in step S110, its purpose is to determine the nutritional requirements during the culture process based on the type of the raw material microbial cells. At the same time, in the current microbial cell culture, corresponding culture parameter standards for various types of microbial cells have been established. Based on the determination of such parameter standards, an initial medium is configured to obtain the benchmark parameters of the concentrations of various substances in the medium.

[0024] In step S110, it specifically includes: S111. Obtain the nutritional requirements of the raw material microbial cells based on the type of the raw material microbial cells.

[0025] The purpose of this step is to determine the nutrients based on the type of the raw material microbial cells and the established microbial cell nutritional requirement standards.

[0026] Among them, based on the culture requirements and culture purposes of the microbial cells, determine the nutrients required by the microbial cells during the amplification process.

[0027] Among them, based on the culture requirements and culture purposes of the microbial cells, determine the concentrations of various nutrients in the medium.

[0028] Among them, determine the concentrations of all substances in the medium, so that under such conditions, the microbial cells can be amplified and cultured.

[0029] Among them, for the nutritional requirements of microbial cells, the types of various substances and their effects on microbial cells can also be determined based on cultivation standards, and then various nutrients can be reasonably selected.

[0030] S112. Based on the nutritional requirements of the raw material microbial cells, obtain the types of isotope materials and non-isotope materials of the raw material microbial cells.

[0031] The purpose of this step is that in the cultivation process of microbial cells, different nutrients have different effects on the amplification of microbial cells. At the same time, some isotopes have a relatively serious killing effect on microbial cells, and some nutrients have low stability in microbial cells. In order to avoid reducing the detection accuracy, it is also necessary to reasonably select the isotope materials used in the cultivation process of microbial cells, and at the same time, it is necessary to analyze other nutrients without isotope labeling.

[0032] Among them, to determine the consumption process of various nutrients by microbial cells, the selected isotope materials need to be able to participate in the synthesis of stable proteins and genetic materials in microbial cells.

[0033] Among them, to determine the growth effect of isotope materials on microbial cells, it is necessary to select isotope materials with isotope labeling but without a serious impact on the growth of microbial cells.

[0034] Among them, for all the nutrients to be added to the culture medium, based on the consumption principle and killing ability of microbial cells for nutrients, determine the specific types of isotope materials.

[0035] In some embodiments, further screening is also carried out on various applicable isotope materials, analyze the consumption amount of such isotope materials per unit time by microbial cells, and select the isotope material with the largest consumption amount therefrom.

[0036] In some embodiments, analyze the utilization rate of various isotope materials by microbial cells, that is: analyze the cumulative amount of such isotopes in microbial cells after the microbial cells absorb the isotope materials, and select the isotope material with the highest cumulative amount therefrom.

[0037] Among them, in the cultivation of microbial cells, it is obvious that various nutrients need to be configured in the culture medium. Not all nutrients need to be configured as isotope materials, and it is also necessary to determine the types of non-isotope materials.

[0038] S113. Based on the requirements of the isotope materials and the non-isotope materials of the raw material microbial cells, set the types of the isotope materials and the non-isotope materials of the raw material microbial cells to obtain the initial culture medium.

[0039] The purpose of this step is to determine the types of isotopic materials and non-isotopic materials, determine the specific concentrations of such materials, and then configure the initial culture medium based on the concentration parameters. After inoculating microbial cells in this culture medium, the initial isotopic abundances during the cultivation process of such microbial cells can be obtained, which can be used for subsequent isotopic abundance comparison. In addition, based on the configuration of different types of initial culture media, the isotopic abundances of microbial cells after cultivation of microbial cells in different isotopic material culture environments can be further compared to screen out the most appropriate isotopic materials.

[0040] Among them, all isotopic materials and non-isotopic materials are determined, and the material concentrations are determined based on the types of such materials, so as to configure the culture medium based on the material concentration values.

[0041] Among them, the concentrations of various isotopic materials and non-isotopic materials in all culture media are recorded to obtain the initial culture medium.

[0042] In some embodiments, for different isotopic labeling materials, the isotopic materials that meet the requirements of the isotopic materials in step S112 are configured into different initial culture media, and microbial cells are cultured, and the best non-isotopic materials are screened out therefrom.

[0043] In some embodiments, for the performance analysis of different isotopic materials, the ratio of the isotopic abundance of microbial cells to the molar concentration of the isotopic materials added to the initial culture medium is calculated, and the isotopic material with the largest ratio is selected as the best isotopic material. As Figure 2 shown, the present application provides a line graph of the microbial cell absorption rate of isotopic materials during the preparation process of highly abundant isotopically labeled microbial cells. The microbial cells used are Saccharomyces cerevisiae. There are two isotopic materials, namely the nitrogen source isotopic material and the carbon source isotopic material. The nitrogen source isotopic materials are such as 15 ammonium sulfate, urea, and peptone powder labeled with 13 N, and the carbon source isotopic materials are such as Figure 2 glucose, sucrose, and corn starch labeled with Figure 2 C. Among them, the horizontal axis represents the isotopic material category, the vertical axis represents the isotopic material absorption amount and absorption rate, the bar graph part represents the isotopic material absorption amount, and the line graph part represents the absorption rate. It can be found that the overall utilization rates of the carbon source isotopic material glucose and the nitrogen source isotopic material ammonium sulfate are higher (modified corresponding 13C-labeled glucose is used as the carbon source isotope material. 15 N-labeled ammonium sulfate is used as the nitrogen source isotope-labeled material. For the preparation of the culture medium, 13 C-labeled glucose and 15 N-labeled ammonium sulfate are used as the isotope materials.

[0044] In some embodiments, the calculation method for the utilization rate is: the absorption amount of the isotope material by microbial cells per unit time / the substance concentration of the isotope material.

[0045] Among them, the nitrogen source isotope material and the carbon source isotope material used in this application are in line with the requirements of the substance concentration of nutrients in the culture medium for Saccharomyces cerevisiae. For other types of microbial cells, the absorption rate and substance concentration need to be analyzed separately to adapt to the different requirements of microbial cells for carbon source and nitrogen source isotope materials.

[0046] Among them, after determining the isotope material, the initial culture medium is prepared according to the determined type of this isotope material.

[0047] S114. Obtain the isotope abundance of the initial culture medium to obtain the initial isotope abundance.

[0048] The purpose of this step is that in the subsequent determination of the culture medium parameters, by obtaining the isotope abundance of the initial culture medium and comparing it with the isotope abundance after culturing the microbial cells, the consumption amount of the isotope substance in the culture medium can be determined, and the isotope utilization rate of the microbial cells can be obtained based on the consumption amount.

[0049] Among them, based on the detection of the initial culture medium, the isotope abundance of the initial culture medium is obtained.

[0050] In some embodiments, other methods and parameters can also be used to determine the isotope abundance parameter results of the isotope material in the initial culture medium to achieve the analysis of the isotope abundance of the isotope material.

[0051] As described in step S120, the purpose of this step is to adjust the substance concentration of all substances in the initial culture medium based on the substance concentration in the initial culture medium, so as to obtain multiple sets of data, and configure the corresponding nutrients in the culture medium according to the corresponding substance concentration, so as to obtain multiple sets of different culture media, so as to obtain multiple sets of initial culture media, and screen the culture medium with the best parameters from them based on the algorithm, so as to obtain the best culture medium.

[0052] In step S120, it specifically includes: S121. Obtain the concentrations of all substances in the initial culture medium, and adjust all the substances according to the corresponding substance concentration gradients to obtain staged substance concentrations.

[0053] The purpose of this step is that for various nutrients in the culture medium, their substance concentrations are usually different. Therefore, it is necessary to decompose all the nutrients according to the corresponding substance concentration parameters, decompose various substance parameters separately, and after obtaining the staged substance concentrations, configure the culture medium.

[0054] Among them, for all substance concentrations, determine within a certain range of upper and lower limits according to the parameters of the substance concentration, and then based on the upper and lower limit ranges, adjust the substance concentration according to the concentration gradient to obtain different groups.

[0055] In some embodiments, for the parameters of nutrients, the parameter standard itself is a substance concentration range, and then decompose within this concentration range to obtain different groups.

[0056] In some embodiments, for the nutrient concentration range, if the initial parameter is only a single data, configure this type of data according to a specific increase ratio. When it is found that the microbial cells no longer expand after a certain nutrient reaches the corresponding concentration value, these two substance concentration values are the upper and lower limits of the substance concentration.

[0057] In some embodiments, if the concentration of a nutrient is an interval, then in the configuration of the initial culture medium, when determining the parameter of this substance concentration, the median value is taken for value.

[0058] Among them, for different substance concentration ranges, the number of decomposed groups can be different. Specifically, it is necessary to decompose according to different interval parameters to obtain the corresponding data groups.

[0059] As shown in Table 1, the data table of the staged substance concentrations of various nutrients in Saccharomyces cerevisiae is as follows: ; Among them, ammonium sulfate and glucose used in the culture medium are isotope materials.

[0060] Among them, for the nutrient materials in the culture medium, based on the technical materials, the concentration of KH2PO4 is 2.5 g / L, the concentration of MgSO4 is 0.25 g / L, the concentration of ammonium sulfate is 5 g / L, and the concentration of glucose is 30 g / L. In the treatment of these nutrients, based on this substance concentration, set the upper and lower limits of the substance concentration.

[0061] Among them, for the phased labeling, the labeling can be set according to actual needs. Similarly, the substance concentration therein is also adjusted accordingly.

[0062] Among them, for all substance concentration parameters, it can be found from this table that there are multiple values. Since the obtained substance concentration values are all reasonable, the total amount of the culture medium that can be configured is d c pieces, where c represents the total amount of nutrient types and d represents the total amount of phased labels of the phased substance concentration.

[0063] S122. Configure different culture media based on the phased substance concentration to obtain candidate culture media.

[0064] The purpose of this step is to analyze the microbial cell culture parameters of different culture media in the initially configured culture medium, so as to screen out the best culture medium from the initially configured culture medium.

[0065] Among them, for the nutrient types in the initial culture medium and the phased substance concentration of different nutrients, a comprehensive configuration of the culture medium is carried out. Then, microbial cells are inoculated into all the obtained initial culture media and cultured.

[0066] Among them, for all the initially configured culture media, all the nutrient types in the corresponding culture media and the phased substance concentration corresponding to all the nutrients need to be recorded.

[0067] Among them, for all the initially configured culture media, these initial culture media are directly converted into candidate culture media.

[0068] In some embodiments, for the initially configured culture medium, if the parameters in the initial culture medium do not exist in the phased substance concentration, an additional initial culture medium needs to be set and used as a candidate culture medium as well.

[0069] S123. Inoculate the raw material microbial cells into the candidate culture medium and place them in the same culture environment for the same period of time.

[0070] The purpose of this step is that in the screening of the culture medium, variable control needs to be carried out. Obviously, the concentration of all substances in the culture medium is the independent variable, and the culture environment of the microbial cells is obviously an irrelevant variable. Therefore, in the culture of microbial cells, it is necessary to ensure that in the culture of all microbial cells, the microbial cells are placed in the same culture environment. It should be noted that the culture environment described in this application refers to the space environment and does not include the temperature, oscillation frequency, etc. in the culture medium and culture dish.

[0071] Among them, for the culture medium inoculated with microbial cells, it is placed in the same culture environment for cultivation.

[0072] Among them, for the culture environment, the culture environment parameters are set according to the culture environment required by the microbial cells during the cultivation process, including temperature, humidity, oxygen content in the air, etc.

[0073] In some embodiments, if it is found that the total amount of microbial cell amplification in all the culture media this time is too small, the environmental parameters are adjusted.

[0074] Among them, the cultivation time of the microbial cells is set, and all the microbial cells need to ensure the same cultivation time.

[0075] In some embodiments, the total amount of microbial cell amplification is identified. When it is found that the microbial cells no longer amplify or no longer grow, it is considered that the cultivation time of the microbial cells reaches the upper limit.

[0076] S124. Obtain the raw material microbial cells in the to-be-selected culture medium to obtain amplified microbial cells.

[0077] The purpose of this step is to obtain the microbial cells that have been amplified and cultivated, laying a foundation for the subsequent microbial cell counting work.

[0078] Among them, the microbial cells are detached from the culture medium to obtain the microbial cells in different culture media.

[0079] Among them, the corresponding relationship between the currently obtained microbial cells and the culture medium is established to avoid errors in the corresponding relationship between the amplification state of the microbial cells and the culture medium.

[0080] S125. Obtain the quantity of the amplified microbial cells to obtain an amplified microbial value.

[0081] The purpose of this step is to obtain the quantity of the microbial cells corresponding to each culture medium for each type of culture medium, so as to analyze the amplification state of the microbial cells in different culture media.

[0082] Among them, the microbial cells are separated from the culture medium and counted to obtain the quantity of the microbial cells under the conditions of this culture medium.

[0083] Among them, for the counting of microbial cells, direct counting methods and indirect counting methods can be used. The direct counting methods include hemocytometer counting method, cell counter counting method, etc., and the indirect counting methods include crystal violet counting method, trypan blue counting method, etc. Flow cytometry counting method, etc. can also be used.

[0084] Among them, for each type of microbial cell, during the counting process, the obtained result is the amplified microbial value.

[0085] In some embodiments, microbial cells are also counted within different amplification time periods, and at the same time, the corresponding relationship between the amplified microbial values in different time periods and the nutrient concentration in the culture medium is established, so as to calculate the relationship between the substance concentration of different nutrients and the amplification rate.

[0086] S126. Obtain the candidate culture medium corresponding to when the amplified microbial value is higher than the preset amplified microbial value, and the corresponding amplified microbial cells are candidate microbial cells.

[0087] The purpose of this step is that after all the above steps, a large number of microbial cell amplification data sets will be obtained. However, obviously, the amplification quantity of microbial cells corresponding to some of the culture media is extremely low. No matter what the cultivation purpose is, this cultivation mode is unqualified. Therefore, by setting the preset amplified microbial value, potential culture media that meet the amplification cultivation requirements can be found more quickly, the sample data can be fully reduced, and the analysis process can be simplified.

[0088] Among them, the preset amplified microbial value can be directly set based on the work experience of technicians and cultivation requirements.

[0089] In some embodiments, for all the obtained amplified microbial values, calculate the mean value of all these values, and obtain all the amplified microbial values higher than this mean value, where this mean value is the preset amplified microbial value.

[0090] S127. Based on the isotope abundance of the candidate microbial cells, obtain the isotope utilization rate.

[0091] The purpose of this step is that after screening the amplified microbial values in different culture media through step S126, these culture media can essentially amplify and cultivate microbial cells. However, in the amplification of microbial cells, through experiments, it is found that their absorption ability for isotope materials is not completely strongly correlated with the amplified microbial value. At the same time, considering that the technical purpose of this application is to obtain highly abundant isotope-labeled microbial cells, on the basis of ensuring that microbial cells can be normally amplified and cultivated, analyze the isotope abundance to analyze whether the current culture medium can support the obtaining of highly abundant isotope-labeled microbial cells.

[0092] Among them, in the technical solution of step S127, it specifically includes: S1271. Obtain the substance concentration value in the candidate culture medium to obtain the initial substance concentration.

[0093] The purpose of this step is to determine the peak concentration of substances in the candidate culture medium, so as to better determine the consumption basis of various nutrients in the cultivation of microbial cells. That is to say, it can be used as the basic calculation value for the consumption of nutrients.

[0094] Among them, for the initial substance concentration, the substance concentrations of all nutrients in the culture medium are obtained. In the preparation of the candidate culture medium, in fact, such parameters have been recorded.

[0095] In some embodiments, instead of classifying various nutrients in the culture medium, the concentration of the nutrient mixture is directly calculated to simplify the calculation.

[0096] S1272. Obtain the substance concentration value in the candidate culture medium after culturing the amplified microbial cells in the candidate culture medium to obtain the final substance concentration.

[0097] The purpose of this step is to obtain the nutrient concentration after culturing microbial cells for a period of time, so as to better calculate the consumption of nutrients by microbial cells during the cultivation process.

[0098] Among them, the concentrations of nutrients in the candidate culture medium that have been cultured with microbial cells are measured separately to obtain the results.

[0099] In some embodiments, instead of classifying various nutrients in the candidate culture medium, the concentration of the nutrient mixture is directly calculated to simplify the calculation. However, it should be noted that this method is only applicable to the schemes with lower requirements for results, because in the cultivation of microbial cells, metabolic wastes will be produced, and the metabolic wastes will affect the calculation accuracy of the substance concentration of the mixture.

[0100] S1273. Obtain the isotope utilization rate based on the initial substance concentration and the final substance concentration. The isotope utilization rate equation is: ; Among them, R a represents the isotope utilization rate, i represents the index of the substance corresponding to the substance concentration, j represents the total index of the substances corresponding to the substance concentration, represents the starting time, represents the end time, represents the starting substance concentration, represents the final substance concentration, represents the isotope abundance of the candidate microbial cells, represents the isotope abundance of the candidate culture medium.

[0101] The purpose of this step is to analyze the total consumption of various nutrients in the current microbial cell culture process by determining the concentrations of starting substances and final substances during the microbial cell culture process. At the same time, analyze the isotope abundances of the candidate media and the isotope abundances of the microbial cells after amplification culture of the microbial cells, and combine with the consumption of nutrients to calculate the isotope utilization rate of the microbial cells.

[0102] As Figure 3 , 4 shown, the isotope abundance line graph corresponding to the adjustment of the medium parameters in the process of preparing highly abundant isotope-labeled microbial cells provided by the embodiment of the present application. After culturing the microbial cells for the candidate medium, determine the microbial cell amplification amount and the isotope abundance of the microbial cells in different groups of candidate media to determine relevant parameters.

[0103] In some embodiments, the isotope utilization rate can be determined by directly calculating the ratio of the isotope abundance of microbial cells to the number of amplified microbial cells. However, this method lacks consideration of the influence of the medium environment and can only be used for the microbial cell culture process without subculture.

[0104] Among them, for the starting time and ending time of the culture, they can be adjusted according to specific culture requirements.

[0105] In some embodiments, for the culture of microbial cells in each candidate medium, calculate the change rate of the isotope utilization rate to determine the isotope abundance growth rate of microbial cells in different culture time periods.

[0106] In some embodiments, when it is found that the isotope abundance of microbial cells no longer increases, record this culture time. The meaning of this culture time is that even if the culture time is longer, the isotope abundance of microbial cells will not increase further. In the actual microbial cell culture process, only this time length needs to be cultured.

[0107] S128. Obtain the candidate medium corresponding to the isotope utilization rate higher than the preset isotope utilization rate to obtain the optimal medium.

[0108] The purpose of this step is that by setting a preset isotope utilization rate, media with an isotope utilization rate higher than this value can be applied. At the same time, candidate media that meet the requirements can be further screened out, and based on the analysis of various requirements, the optimal medium can be selected.

[0109] Among them, for the preset isotope utilization rate, it can be directly set based on the needs and relevant requirements of technicians.

[0110] In some embodiments, for all the acquired isotopes, the mean value is calculated numerically, and the obtained mean value is the preset isotope utilization rate.

[0111] Among them, obviously, there will be multiple candidate culture media that meet the requirements. Then, further screening is carried out based on requirements such as the cost of various isotope materials and the consumption cost of all nutrients, so as to obtain the optimal culture medium.

[0112] As described in step S130, the purpose of this step is that, on the basis of having determined the optimal culture medium, actually only one task has been completed. Because in the cultivation of microbial cells, the culture environment will also have a great impact on the cultivation state of microbial cells. In this step, the culture environment for microbial cells can be further analyzed, and the influence of the culture environment on the isotope abundance of microbial cells can be analyzed, so as to ensure that the set culture environment can improve the isotope abundance of microbial cells.

[0113] In step S130, it specifically includes: S131. Based on the type of the raw material microbial cells, obtain the culture environment requirements of the raw material microbial cells.

[0114] The purpose of this step is that in the cultivation of microbial cells, according to the cultivation standard, the basic culture environment is determined to avoid the mismatch between microbial cells and the culture environment, resulting in the inability to amplify and cultivate microbial cells.

[0115] Among them, based on the type of microbial cells, the culture environment requirements of the microbial cells are determined.

[0116] Among them, for the culture environment requirements of microbial cells, all the culture environment parameters are set.

[0117] S132. Based on the culture environment requirements of the raw material microbial cells, obtain the culture environment parameters of the raw material microbial cells.

[0118] The purpose of this step is that for the culture environment requirements of microbial cells, by determining the culture environment parameters, the initial culture environment can be set, thereby constructing the culture environment of microbial cells.

[0119] Among them, for the culture environment parameters of microbial cells, all the parameters are set.

[0120] S133. Based on the culture environment parameters of the raw material microbial cells, adjust all the culture environment parameters according to the corresponding environmental parameter gradients to obtain the initial culture environment.

[0121] The purpose of this step is to adjust all the culture environment parameters of microbial cells according to the gradient parameters of the values of various culture environment parameters, so as to construct different initial culture environments and lay a foundation for the subsequent determination of the optimal culture environment.

[0122] Among them, for all the culture environment parameters, the upper and lower limits are determined within a certain range according to the culture environment parameters. Then, based on the upper and lower limit ranges, the culture environment parameters are adjusted according to the environmental parameter gradient to obtain different groups.

[0123] In some embodiments, for the culture environment parameters, the parameter standard itself is the interval of the culture environment parameters. Then, it is decomposed within this interval of the culture environment parameters to obtain different groups.

[0124] In some embodiments, for the interval of the culture environment parameters, if the initial parameter is only one data, this kind of data is configured according to a specific increase ratio. When it is found that the microbial cells no longer expand after a certain culture environment reaches the corresponding culture environment value, these two culture environment parameters are the upper and lower limits of the culture environment parameters.

[0125] In some embodiments, if the culture environment parameter is an interval, then in the setting of the initial culture environment, for the determination of this culture environment parameter, the median value is taken for value.

[0126] Among them, for different intervals of the culture environment parameters, the number of decomposed groups can be different. Specifically, it needs to be decomposed according to different interval parameters to obtain the corresponding data groups.

[0127] As shown in Table 2, it is a data table of the phased culture environment parameters of various culture environments in the process of microbial cell culture, which is: ; Among them, the phased label can be increased or decreased accordingly based on the culture requirements of the microbial cells. Correspondingly, the amount of data corresponding to the phased label also needs to be adjusted.

[0128] Among them, for all the culture environment parameters, it can be found from this table that there are multiple values. Since the obtained culture environment parameters are all reasonable, the total number of combinations of the settable culture environment parameters is species, where e represents the total amount of culture environment types, and f represents the total amount of phased labels of the phased culture environment parameters.

[0129] As described in step S140, the purpose of this step is to adjust the parameters of all culture environments based on the initial culture environment, so as to obtain multiple sets of data, and set up the culture environments to obtain various culture environments, and screen the culture environment with the best parameters from them based on the algorithm, so as to obtain the best culture parameters.

[0130] In step S140, it specifically includes: S141. Obtain all the culture environment parameters in the initial culture environment, and adjust all the culture environment parameters according to the corresponding parameter gradients to obtain phased culture environment parameters.

[0131] The purpose of this step is to culture based on such parameters after the culture environment parameters that can be constructed have been determined, so as to determine the influence on the culture of microbial cells under different culture environments, and lay a foundation for the subsequent construction of the culture environment.

[0132] Among them, for all the obtained phased culture environment parameters, for different environment types, randomly select one environment parameter to obtain all the culture environment parameters in the shaping of the culture environment.

[0133] In some embodiments, if it is found that the values in the initial culture environment do not exist in the culture environment parameters in Table 2, it is also necessary to set up the initial culture environment and analyze the isotope abundance of microbial cells in this environment.

[0134] S142. Construct different culture environments based on the phased culture environment parameters to obtain candidate culture environments.

[0135] The purpose of this step is to set up the culture environment based on the obtained phased culture environment parameters and analyze the isotope abundance of microbial cells in different culture environments.

[0136] Among them, for the types of environment parameters in the initial culture environment and the phased environment parameters of different culture environment parameters, a comprehensive configuration of the culture environment is carried out. Then, microbial cells are inoculated in all the obtained initial culture environments and cultured.

[0137] Among them, for all the configured initial culture environments, it is necessary to record all the types of culture environments in the corresponding culture environment and the phased culture environment parameters corresponding to all the types of culture environments.

[0138] Among them, for all the configured initial culture environments, these initial culture environments are directly converted into candidate culture environments.

[0139] In some embodiments, for the configured initial culture environment, if the parameters in the initial culture environment do not exist in the staged culture environment parameters, it is necessary to additionally set the initial culture environment and also use it as an alternative culture environment.

[0140] S143. Set the optimal culture medium in the alternative culture environment, inoculate the raw material microbial cells on the optimal culture medium, and culture for the same time to obtain cultured microbial cells.

[0141] The purpose of this step is to ensure that the parameters of the culture medium are exactly the same. Then, when setting the culture environment, it can fully guarantee the limitation of the irrelevant variable of the culture medium, so as to analyze the influence of the culture environment on the microbial cell culture.

[0142] Among them, for the culture medium of microbial cells, the already obtained optimal culture medium is selected.

[0143] Among them, the culture time of microbial cells is set, and all microbial cells need to ensure the same culture time.

[0144] In some embodiments, the total amplification amount of microbial cells is identified. When it is found that the microbial cells no longer amplify or no longer grow, it is considered that the culture time of the microbial cells reaches the upper limit.

[0145] In some embodiments, in steps S120 and S140, all the parameter analysis steps in the culture medium and the culture environment are integrated to obtain a joint analysis scheme of the relevant parameters of "culture medium - culture environment", and the best "culture medium - culture environment" scheme is screened out from it. It should be noted that this method can only be applied to the case where the gradient settings of all parameters are relatively large, otherwise it will lead to an excessive amount of data obtained, making the screening difficulty and calculation amount of various parameters too large. In addition, the defect of this method is that it can only form a binding relationship between two culture elements, namely the culture medium and the culture environment. In some cases, some real elements may need to be considered, and some of the parameters need to be adjusted separately to achieve decoupled adjustment of the culture medium parameters and the culture environment parameters. Obviously, this integrated analysis scheme cannot adapt to this situation.

[0146] S144. Obtain the number of the cultured microbial cells to obtain a cultured microbial value.

[0147] The purpose of this step is to obtain the number of microbial cells in different culture environments, so that subsequent microbial cell counting can be carried out to analyze the promoting effect of the current microbial cell culture environment on the culture of microorganisms.

[0148] Among them, microbial cells are separated from the culture medium and counted to obtain the number of microbial cells under the culture environmental conditions.

[0149] Among them, for the counting of microbial cells, direct counting methods and indirect counting methods can be used. The direct counting methods include the hemocytometer counting method, the cell counter counting method, etc., and the indirect counting methods include the crystal violet counting method, the trypan blue counting method, etc. The flow cytometer counting method can also be used.

[0150] Among them, for each type of microbial cell, during the counting process, the obtained result is the amplified microorganism value.

[0151] In some embodiments, the microbial cells in different amplification time periods are also counted, and the corresponding relationship between the amplified microorganism values in different time periods and the nutrient concentration in the culture medium is established, so as to calculate the relationship between the substance concentration of different nutrients and the amplification rate.

[0152] S145. Obtain the candidate culture environment corresponding to when the culture microorganism value is higher than the preset culture microorganism value, and the corresponding culture microbial cells are culture microbial cells.

[0153] The purpose of this step is to initially screen the obtained large number of microorganism values by setting the preset culture microorganism value and comparing the culture microorganism value with it, so as to fully reduce the screening difficulty and complexity of the culture environment.

[0154] Among them, the preset culture microorganism value can be directly set based on the work experience of technicians and culture requirements.

[0155] In some embodiments, for all the obtained culture microorganism values, calculate the mean value of all these values, and obtain all the culture microorganism values higher than this mean value, where this mean value is the preset culture microorganism value.

[0156] S146. Obtain the isotope abundance change rate based on the isotope abundance of the culture microbial cells.

[0157] The purpose of this step is to further analyze the influence of the current culture environment on the isotope abundance of microbial cells on the basis of determining the optimal culture medium, so as to ensure that in the subsequent analysis, in the configured culture environment, it can be fully ensured that under the action of the corresponding culture environment, the microbial cells can also ensure high isotope abundance.

[0158] Among them, in the technical solution of step S146, it specifically includes: S1461. Obtain the culture environment parameters in the candidate culture environment to obtain the candidate culture environment parameters.

[0159] The purpose of this step is to determine the culture environment parameters in the candidate culture environment. Considering that in the setting of the culture environment, this parameter does not change, so in the specific process, the direct culture environment parameters can be adjusted to the candidate culture environment parameters, so as to determine the influence of various culture environment parameters on the culture of microbial cells.

[0160] Among them, for the culture environment parameters, in the setting of the culture environment, in fact, such parameters have been recorded.

[0161] S1462. Based on the culture microorganism value and the isotope abundance of the cultured microbial cells, obtain the isotope abundance change rate. The isotope abundance change rate equation is: ; Among them, R b represents the isotope abundance change rate, represents the isotope abundance of the candidate microbial cells, represents the isotope abundance of the candidate culture medium, M represents the culture microorganism value, m represents the inoculation quantity of the raw material microbial cells, p represents the index of the candidate culture environment parameters, q represents the total amount of the indexes of the candidate culture environment parameters, represents the candidate culture environment parameters, represents the weight of the candidate culture environment parameters.

[0162] The purpose of this step is to analyze the isotope abundance change rate of microbial cells under the action of the current culture environment by obtaining the culture environment parameters.

[0163] Such as Figure 3 、 4As shown, it is a broken line graph of isotope abundances corresponding to the adjustment of culture environment parameters in the preparation process of highly abundant isotope-labeled microbial cells provided by an embodiment of the present application. Among them, in this embodiment, only the amplification quantity of microbial cells in culture environments based on different temperatures is analyzed, and the isotope abundances of microbial cells in such environments are analyzed. It should be noted that for the technical solution of the present application, the parameters shown here are only for the convenience of writing. In specific analyses, the amplification quantity of microbial cells and the isotope abundances of microbial cells can also be analyzed by establishing groups. For example: One of the groups is [28.4, 5.7, 7%, 230] - stable stage, which respectively represents that the culture of Saccharomyces cerevisiae is in the stable stage, the environmental temperature is 28.4 °C; the pH value is 5.7; the dissolved oxygen concentration is 7%; the oscillation speed is 230 rpm. Of course, corresponding groups are also established for other culture stages of Saccharomyces cerevisiae.

[0164] Among them, for the inoculation quantity of raw material microbial cells, it can be adjusted according to specific culture requirements.

[0165] In some embodiments, for the culture of microbial cells in each candidate culture environment, the value of the isotope abundance change rate is calculated to determine the isotope abundance growth rate of microbial cells within different culture time periods.

[0166] In some embodiments, when it is found that the isotope abundance of microbial cells no longer increases, record this culture time. The meaning of this culture time is that even if the culture time is longer, the isotope abundance of microbial cells will not increase further. In the actual culture process of microbial cells, it is only necessary to culture for this time length.

[0167] S147. Obtain the candidate culture environment corresponding to when the isotope abundance change rate is higher than the preset isotope abundance change rate to obtain the optimal culture parameters.

[0168] The purpose of this step is that by setting the preset isotope abundance change rate, the culture environment with an isotope abundance change rate higher than this value can be applied. At the same time, candidate culture environments that meet the requirements can be further screened out from them, and based on the analysis of various requirements, the optimal culture environment can be selected.

[0169] Among them, for the preset isotope abundance change rate, it can be directly set based on the needs and related requirements of technicians.

[0170] In some embodiments, for all the obtained isotope abundance change rate values, calculate the mean value, and the obtained mean value is the preset isotope abundance change rate.

[0171] Among them, there will obviously be multiple candidate culture environments that meet the requirements. Then, based on various culture speeds and requirements for microbial values, etc., further screening is carried out to obtain the optimal culture environment.

[0172] As described in step S150, the purpose of this step is that in the cultivation of microbial cells, in some cases, subculture of microbial cells is required. In each subculture process, various parameters may need to be adjusted to promote the differentiation of microbial cells. By analyzing the subculture process of microbial cells and obtaining the subcultured microbial cells, personalized adjustment of the culture medium and culture environment in each culture step of the subculture process can be achieved.

[0173] In step S150, it specifically includes: S151. Obtain the subculture process of the raw material microbial cells, and obtain the nutritional requirements and culture environment of the subculture process.

[0174] The purpose of this step is that only when the subculture process of the raw material microbial cells is determined can the nutritional requirements and culture environment of each subculture step in the whole process be based on, and subsequent parameter adjustment can be carried out based on these parameters.

[0175] Among them, based on the technical solution, after obtaining the raw material microbial cells and the subculture process, the microbial cells obtained are used to determine all the subculture processes that can be set therein, and the subculture steps are obtained.

[0176] Among them, determine the culture medium and culture environment standards in all subculture steps, and then set these parameters, so as to set the corresponding parameters and carry out step-by-step subculture of microbial cells.

[0177] S152. Based on the nutritional requirements and culture environment of the subculture process, set the initial culture medium and initial culture environment for each subculture process.

[0178] The purpose of this step is to determine the relevant nutrients and culture environment parameters in each subculture step, and then adjust the culture medium and culture environment after obtaining these parameters for phased adjustment.

[0179] Among them, configure the culture medium and set the culture environment according to all the parameters therein to carry out the subculture of microbial cells.

[0180] Among them, for all the parameters, the parameter setting for the culture medium is the same as that in step S110, and the setting for the culture environment is the same as that in step S140, which will not be elaborated here.

[0181] Among them, according to the substance concentration of the culture medium and the culture environment parameters, the initial culture medium is configured, and the initial culture environment is set.

[0182] Among them, based on the initial culture medium and the initial culture environment, the microbial cells are cultured. It is necessary to inoculate the microbial cells on the initial culture medium and configure them into the corresponding initial culture environment.

[0183] S153. Obtain all the substance concentrations in the initial transfer culture medium of the transfer culture process, adjust them according to the corresponding concentration gradients, and configure the culture medium to obtain the candidate transfer culture media.

[0184] The purpose of this step is to adjust the substance concentrations of all substances based on the substance concentrations in the initial culture medium, so as to obtain multiple sets of data, and configure the corresponding nutrients in the culture medium according to the corresponding substance concentrations, so as to obtain multiple sets of different culture media, so as to obtain multiple sets of candidate transfer culture media, and thus establish the selection basis for the optimal transfer culture medium.

[0185] Among them, for the configuration of the candidate transfer culture medium, it is the same as steps S121 - S122, which will not be elaborated here.

[0186] S154. Obtain the initial transfer culture environment parameters of the transfer culture process, adjust them according to the corresponding environmental parameter gradients, and adjust the culture environment to obtain the candidate transfer culture environment.

[0187] The purpose of this step is to determine the culture environment parameters during the transfer culture process, and based on the initial culture environment parameters, all the parameters can be adjusted to obtain the candidate transfer culture environment, laying a foundation for the selection of the culture environment parameters during the subsequent transfer culture process.

[0188] Among them, for the determination of the candidate transfer culture environment, it is the same as steps S141 - S142, which will not be elaborated here.

[0189] S155. Based on the candidate transfer culture medium, the candidate transfer culture environment, the isotope utilization rate equation, and the isotope abundance change rate equation, determine the optimal transfer culture medium and the optimal transfer culture environment.

[0190] The purpose of this step is to calculate the isotope abundance during the transfer culture process of the microbial cells, and thus analyze based on the transfer culture medium and the transfer culture environment to analyze the isotope abundance of each step in the transfer culture process. Therefore, in this case, it can be ensured that in all culture parameter situations of the transfer culture process, all transfer culture processes can comprehensively achieve high - abundance accumulation of isotopes.

[0191] Among them, the method for determining the optimal subculture medium is the same as that in steps S123 to S128, which will not be elaborated here.

[0192] Among them, the method for determining the optimal subculture environment is the same as that in steps S143 to S147, which will not be elaborated here.

[0193] S156. Based on the optimal subculture medium and the optimal subculture environment, subculture the raw material microbial cells to obtain the subcultured microbial cells.

[0194] The purpose of this step is that for the subculture process of microbial cells, since all subculture steps can ensure the highest isotope abundance accumulation effect of microbial cells, therefore, the finally obtained subcultured microbial cells can be directly obtained, and this microbial cell is the subcultured microbial cell.

[0195] Among them, after completing the subculture operation, separate the subcultured microbial cells from the medium, and based on actual needs, count and perform related processing on the subcultured microbial cells.

[0196] Among them, for the counting of microbial cells, direct counting methods and indirect counting methods can be used. Direct counting methods include hemocytometer counting method, cell counter counting method, etc., and indirect counting methods include crystal violet counting method, trypan blue counting method, etc., and flow cytometer counting method, etc. can also be used.

[0197] After obtaining the optimal subculture medium and the optimal subculture environment for each step in the microbial cell subculture process, essentially the various culture parameters in each subculture process have been determined, and in the culture of the same type of microbial cells, these obtained culture parameters can be directly used for culture. In order to more conveniently perform high-abundance isotope culture on the same type of microbial cells, the following steps can also be set, such as Figure 5 As described, a schematic diagram of the association between the subculture process and culture parameters in the process of preparing high-abundance isotope-labeled microbial cells provided by the embodiment of the present application is specifically as follows: S157. Based on the subculture process of the raw material microbial cells and the subcultured microbial cells, obtain the subculture steps.

[0198] The purpose of this step is to obtain the subculture process of microbial cells, so as to obtain all subculture steps therein, and based on this method, set automatic control nodes, and after the automation control system reaches the nodes, adjust the medium parameters and culture environment parameters in the subculture.

[0199] Among them, for the determination of the subculture process, all the culture steps therein are set as culture parameter adjustment nodes.

[0200] Among them, in each subculture process, it is determined based on the culture time of microbial cells, so as to establish a culture time discontinuous curve. Being in the curve means that no adjustment of culture parameters is required during this time period, and only at the discontinuous points is it necessary to adjust the culture parameters.

[0201] In some embodiments, the discontinuous points are determined based on the nutrient change curve in the optimal subculture medium to set the discontinuous points.

[0202] In some embodiments, based on the requirements of different subculture processes for the culture environment, the adjustment change points of the culture environment are used as discontinuous points.

[0203] In some embodiments, based on both the nutrients in the medium and the culture environment, the discontinuous points in the subculture process are obtained to establish specific subculture steps.

[0204] In some embodiments, serial numbers are set for all the discontinuous points. In addition to establishing the subculture steps, in an automated control system, based on the human-machine interface, the current subculture process being in is displayed, and an alarm is issued after the corresponding subculture steps are completed. And based on the requirements of the next subculture process, the culture parameters that need to be adjusted are directly displayed by the human-machine interface. The culture parameters include the nutrient types and concentrations of the medium, culture environment parameters, etc.

[0205] S158. Establish the association relationship between the subculture steps, the optimal subculture medium, and the optimal subculture environment, and establish a subculture link.

[0206] The purpose of this step is to connect all the parts that need to adjust the culture parameters during the subculture process of microbial cells, so as to achieve the correct connection of the subculture process, realize the visual display of the subculture link, and lay a foundation for subsequent data addition work.

[0207] As Figure 5 shown in (a), for the entire subculture process, the culture parameters that need to be adjusted for each subculture step are marked to obtain the subculture process.

[0208] Among them, based on the subculture steps, the adjacent subculture connection nodes are connected, and the subculture steps are determined and identified.

[0209] In some embodiments, since the subculture steps have been numbered, during the specific processing, adjacent numbers are constructed. Based on this method, the subculture link can be established more quickly.

[0210] S159. Based on the subculture link, obtain the substance concentration of the optimal subculture medium and the environmental parameters of the optimal subculture environment, and configure the medium and adjust the culture environment.

[0211] The purpose of this step is to add the medium and culture environment parameters of the corresponding subculture steps to the obtained subculture link, which means that in the specific subculture link, the corresponding culture parameter adjustment content can be set based on the subculture link steps.

[0212] As Figure 5 shown in (b), after determining the subculture process, based on the setting requirements and configuration standards of the culture parameters, adjust the required adjustment content marked in the subculture process to specific culture parameters, so as to perform corresponding subculture on the microbial cells based on the parameters set therein.

[0213] Among them, directly add the corresponding medium and culture environment parameters to the subculture link.

[0214] In some embodiments, the medium and culture environment parameters are set simultaneously for each subculture step in the subculture link.

[0215] In some embodiments, for the subculture steps in the subculture link, in addition to numbering, the types of parameters to be adjusted for each subculture step are also marked, such as: the parameters to be adjusted are divided into only adjusting the medium, only adjusting the culture environment, and adjusting both the medium and the culture environment simultaneously.

[0216] As described in step S160, the purpose of this step is to process the obtained subculture microbial cells, and based on the subsequent processing of the subculture microbial cells, obtain the final product and apply it.

[0217] In step S160, it specifically includes: S161. Perform centrifugal enrichment on the subculture microbial cells to obtain the subculture microbial cells.

[0218] The purpose of this step is to enrich all the generated subculture microbial cells, so that based on this processing method, it is easier to extract all the microbial cells.

[0219] Among them, for the enrichment treatment of subcultured microbial cells, a centrifugation treatment method is adopted, and then the subcultured microbial cells are extracted from the enriched culture medium.

[0220] In some embodiments, other methods can also be used to enrich the subcultured microbial cells, so as to make it more convenient to extract the obtained subcultured microbial cells.

[0221] S162. Sterilize, freeze-dry and grind the subcultured microbial cells to obtain the high-abundance isotope-labeled microbial cell powder.

[0222] The purpose of this step is to obtain a finished product through corresponding treatment of the subcultured microbial cells, so as to obtain results according to specific subculture purposes.

[0223] Among them, for the treatment process of subcultured microorganisms, the obtained microbial cells are sterilized, freeze-dried and ground to obtain a powder finished product.

[0224] Among them, according to the treatment process of microbial cells, the subcultured microbial cells can also be correspondingly treated according to other requirements.

[0225] The present application discloses a method for preparing high-abundance isotope-labeled microbial cells. The beneficial effects of the present application include: 1. Improve the isotope abundance in microbial cell culture. First, configure the culture medium and culture environment according to the general culture environment of microbial cells, and then optimize the culture medium and culture environment to adjust various culture parameters and the substance concentration of the culture medium during the microbial cell culture process, and screen out the ones that can most promote the amplification efficiency of microbial cells. On this basis, analyze the isotope abundance of microbial cells, and screen out the best culture medium and culture parameters, so as to ensure an increase in isotope abundance during the amplification culture of microbial cells.

[0226] 2. Achieve an increase in isotope abundance in the subculture process of microbial cells. During the culture of microorganisms, for the culture medium and culture environment parameters used in each amplification culture process, relevant parameters are also adjusted with high precision to ensure the accumulation of isotope substances in each subculture process, so that in the finally obtained subcultured microbial cells, their isotope abundance is fully increased based on the accumulation.

[0227] 3. The culture environment settings for different microbial cell types are realized. Based on the subculture protocol of microbial cells, the parameters of the culture medium and culture environment for each subculture process are determined, and a corresponding association relationship is established between such parameters and the subculture steps. Thus, when the raw material microbial cells and the subcultured microbial products are obtained, the culture system can independently determine the decomposition steps of the subculture process, and select the parameters of the culture medium and culture environment at each step to achieve the high-abundance isotope culture of microbial cells.

[0228] Those of ordinary skill in the art can understand that all or part of the steps of implementing the above method embodiments can be completed by hardware related to computer program instructions. The foregoing computer program can be stored in a non-volatile storage medium. When the computer program is executed, it performs the steps including the above method embodiments. Alternatively, if the above integrated unit of the present invention is implemented in the form of a software functional module and sold or used as an independent product, it can also be stored in a non-volatile storage medium. Based on such understanding, the technical solution of the embodiments of the present invention, in essence, or the part that contributes to the prior art can be embodied in the form of a software product. The computer software product is stored in a non-volatile storage medium and includes several instructions for causing an electronic device (which can be a personal computer, a server, a network device, etc.) to execute all or part of the methods described in the various embodiments of the present invention.

[0229] As described above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention.

Claims

1. A method for preparing highly abundant isotope-labeled microbial cells, characterized in that, The method includes: Based on the nutritional requirements of the raw material microbial cells, determining the basic conditions of the isotope-labeled culture medium and preparing an initial culture medium; Adjusting the substance concentrations in the initial culture medium, culturing the raw material microbial cells, determining the optimal substance concentrations in the initial culture medium, and obtaining an optimal culture medium; Based on the culture environment requirements of the raw material microbial cells, obtaining the initial culture environment of the raw material microbial cells; Based on the initial culture environment of the raw material microbial cells, obtaining the optimal culture environment of the raw material microbial cells and obtaining optimal culture parameters; Based on the subculture process of the raw material microbial cells, obtaining subcultured microbial cells; Processing the subcultured microbial cells to obtain high-abundance isotope-labeled microbial cell powder.

2. The method for preparing highly abundant isotope-labeled microbial cells according to claim 1, wherein The determining the basic conditions of the isotope-labeled culture medium based on the nutritional requirements of the raw material microbial cells and preparing the initial culture medium includes: Based on the type of the raw material microbial cells, obtaining the nutritional requirements of the raw material microbial cells; Based on the nutritional requirements of the raw material microbial cells, obtaining the types of isotope materials and non-isotope materials of the raw material microbial cells; Based on the types of the isotope materials and non-isotope materials of the raw material microbial cells, setting the concentrations of the isotope materials and non-isotope materials of the raw material microbial cells to obtain the initial culture medium; Obtaining the isotope abundance of the initial culture medium to obtain an initial isotope abundance.

3. The method for preparing highly abundant isotope-labeled microbial cells according to claim 1, characterized in that, The adjusting the substance concentrations in the initial culture medium, culturing the raw material microbial cells, determining the optimal substance concentrations in the initial culture medium, and obtaining the optimal culture medium includes: Obtaining the substance concentrations of all substances in the initial culture medium, adjusting all substances according to corresponding substance concentration gradients to obtain staged substance concentrations; Preparing different culture media based on the staged substance concentrations to obtain candidate culture media; Inoculating the raw material microbial cells in the candidate culture media and culturing them in the same culture environment for the same time; Obtaining the raw material microbial cells in the candidate culture media to obtain amplified microbial cells; Obtaining the quantity of the amplified microbial cells to obtain an amplified microbial value; Obtaining the candidate culture medium corresponding to when the amplified microbial value is higher than a preset amplified microbial value, and the corresponding amplified microbial cells are candidate microbial cells; Based on the isotope abundance of the candidate microbial cells, obtaining an isotope utilization rate; Obtaining the candidate culture medium corresponding to when the isotope utilization rate is higher than a preset isotope utilization rate to obtain the optimal culture medium.

4. The method for preparing highly abundant isotope-labeled microbial cells according to claim 3, characterized in that, The obtaining the isotope utilization rate based on the isotope abundance of the candidate microbial cells includes: Obtaining the substance concentration value in the candidate culture medium to obtain an initial substance concentration; Obtaining the substance concentration value in the candidate culture medium after culturing the amplified microbial cells in the candidate culture medium to obtain a final substance concentration; Obtaining the isotope utilization rate based on the initial substance concentration and the final substance concentration, and the isotope utilization rate equation is: ； Among them, represents the isotope utilization rate, i represents the index of the substance corresponding to the substance concentration, j represents the total amount of the index of the substance corresponding to the substance concentration, represents the starting time, represents the end time, represents the starting substance concentration, represents the final substance concentration, represents the isotope abundance of the candidate microbial cells, represents the isotope abundance of the candidate culture medium.

5. The method for preparing highly abundant isotope-labeled microbial cells according to claim 1, wherein Based on the culture environment requirements of the raw material microbial cells, obtaining the initial culture environment of the raw material microbial cells, including: Based on the type of the raw material microbial cells, obtaining the culture environment requirements of the raw material microbial cells; Based on the culture environment requirements of the raw material microbial cells, obtaining the culture environment parameters of the raw material microbial cells; Based on the culture environment parameters of the raw material microbial cells, adjusting all the culture environment parameters according to the corresponding environmental parameter gradients to obtain the initial culture environment.

6. The method for preparing highly abundant isotope-labeled microbial cells according to claim 1, characterized in that, Based on the initial culture environment of the raw material microbial cells, obtaining the optimal culture environment of the raw material microbial cells to obtain the optimal culture parameters, including: Obtaining all the culture environment parameters in the initial culture environment, adjusting all the culture environment parameters according to the corresponding parameter gradients to obtain the stage-by-stage culture environment parameters; Constructing different culture environments based on the stage-by-stage culture environment parameters to obtain the candidate culture environments; Setting the optimal culture medium in the candidate culture environments, inoculating the raw material microbial cells on the optimal culture medium, and culturing for the same time to obtain the cultured microbial cells; Obtaining the quantity of the cultured microbial cells to obtain the cultured microbial value; Obtaining the candidate culture environment corresponding to when the cultured microbial value is higher than the preset cultured microbial value, and the corresponding cultured microbial cells are the cultured microbial cells; Based on the isotope abundance of the cultured microbial cells, obtaining the isotope abundance change rate; Obtaining the candidate culture environment corresponding to when the isotope abundance change rate is higher than the preset isotope abundance change rate to obtain the optimal culture parameters.

7. The method for preparing highly abundant isotope-labeled microbial cells according to claim 6, characterized in that, The obtaining the isotope abundance change rate based on the isotope abundance of the cultured microbial cells includes: Obtaining the culture environment parameters in the candidate culture environment to obtain the candidate culture environment parameters; Based on the cultured microbial value and the isotope abundance of the cultured microbial cells, obtaining the isotope abundance change rate, and the isotope abundance change rate equation is: ； Wherein, represents the isotope abundance change rate, represents the isotope abundance of the candidate microbial cells, represents the isotope abundance of the candidate culture medium, M represents the value of the cultured microorganisms, m represents the inoculation quantity of the raw material microbial cells, p represents the index of the candidate culture environment parameters, q represents the total amount of the indexes of the candidate culture environment parameters, represents the candidate culture environment parameters, represents the weight of the candidate culture environment parameters.

8. The method for preparing highly abundant isotope-labeled microbial cells according to any one of claims 1 to 7, characterized in that, Based on the subculture process of the raw material microbial cells, obtaining the subcultured microbial cells, including: Obtaining the subculture process of the raw material microbial cells, and obtaining the nutritional requirements and culture environment of the subculture process; Based on the nutritional requirements and culture environment of the subculture process, setting the initial subculture medium and the initial subculture environment for each subculture process; Obtaining all the substance concentrations in the initial subculture medium of the subculture process, adjusting according to the corresponding concentration gradients, and configuring the culture medium to obtain the candidate subculture medium; Obtaining the initial subculture environment parameters of the subculture process, adjusting according to the corresponding environmental parameter gradients, and adjusting the culture environment to obtain the candidate subculture environment; Based on the candidate subculture medium and the candidate subculture environment, and the isotope utilization rate equation and the isotope abundance change rate equation, determining the optimal subculture medium and the optimal subculture environment; Based on the optimal subculture medium and the optimal subculture environment, subculture the microbial cells of the raw material to obtain the subcultured microbial cells.

9. The method for preparing highly abundant isotope-labeled microbial cells according to claim 8, wherein It further includes: Based on the subculture process of the microbial cells of the raw material and the subcultured microbial cells, obtain the subculture steps; Establish the association relationship between the subculture steps, the optimal subculture medium and the optimal subculture environment, and establish a subculture link; Based on the subculture link, obtain the substance concentration of the optimal subculture medium and the environmental parameters of the optimal subculture environment, and configure the medium and adjust the culture environment.

10. The method for preparing highly abundant isotope-labeled microbial cells according to claim 1, characterized in that, The processing of the subcultured microbial cells to obtain the high-abundance isotope-labeled microbial cell powder includes: Perform centrifugal enrichment on the subcultured microbial cells to obtain the subcultured microbial cells; Perform sterilization, freeze-drying and grinding on the subcultured microbial cells to obtain the high-abundance isotope-labeled microbial cell powder.

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