CN111909859A

CN111909859A - Pichia pastoris low-temperature culture medium

Info

Publication number: CN111909859A
Application number: CN201910373619.XA
Authority: CN
Inventors: 牛罡; 郑伟; 陶真民; 何林; 李阳; 任小飞; 张晗; 郭天罡; 张宁; 苑德禄; 徐明波
Original assignee: Beijing Shuanglu Biological Technology Co ltd; BEIJING SL PHARMACEUTICAL CO LTD
Current assignee: Beijing Shuanglu Biological Technology Co ltd; BEIJING SL PHARMACEUTICAL CO LTD
Priority date: 2019-05-07
Filing date: 2019-05-07
Publication date: 2020-11-10
Anticipated expiration: 2039-05-07
Also published as: CN111909859B

Abstract

The invention discloses a low-temperature culture medium for pichia pastoris, which is beneficial to realizing high-density fermentation of pichia pastoris while improving unit thallus expression quantity under the condition of low temperature (18-22 ℃); the protease inhibitor can also inhibit various proteases in fermentation liquor, is beneficial to maintaining the stability of the product structure and reducing the generation of related impurities of the product; when the method is applied to industrial preparation, the expression yield of the foreign protein can be obviously improved, and the downstream purification treatment difficulty is reduced. Reduces the preparation cost and processing difficulty of products such as gene engineering drugs, enzyme preparations and the like taking pichia pastoris as a host.

Description

Pichia pastoris low-temperature culture medium

Technical Field

The invention belongs to the field of biological fermentation, and relates to a culture medium suitable for culturing Pichia pastoris and expressing exogenous protein under the condition of low temperature (18-22 ℃). Comprises the components formula, pH value and culture condition of the culture medium.

Technical Field

In recent years, the application of pichia pastoris in the fields of biological pharmacy, enzyme preparations and the like is increasingly wide, and the pichia pastoris is considered to be one of the most promising heterologous protein production systems. Researchers commonly refer to Pichia pastoris fermentation handbook introduced by Invitrogen company to perform fermentation and induced expression of the engineering bacteria. BSM + PTM is given in the manual₁The culture medium of (4) and the culture temperature is preferably controlled to 30 ℃. Under the high-temperature culture condition, the density of the pichia pastoris can reach 500g/L wet cells, so that the improvement of the total expression amount of the foreign protein is facilitated, and the pichia pastoris is also the excellent point of being applied to the industrial preparation of the recombinant protein. However, the fermentation time of pichia pastoris in industrial production is often over 100 h. Therefore, at a temperature of 30 ℃, the exogenous protein obtained by expression is easy to have the phenomenon of heterogeneity of N terminal or C terminal, and further introduces related impurities with a structure highly similar to that of a target object, thereby increasing the difficulty of later purification, causing the reduction of purification yield and even causing the inaccessibility of high-purity target objects. This problem limits the efficient production of numerous recombinant products. (e.g., Zhang Pebiao et al. Pichia pastoris expression of human insulin precursor heterogeneity analysis [ J]The journal of Chinese biological products 2017,30(6): 577-581; purification of human insulin precursor expressed by Pichia pastoris and N-terminal heterogeneity analysis [ J]Flower and flowerThe university of eastern sciences, 2013,39(5), 565-; n-terminal homogeneity of Jindong goose, etc., gene engineering product and analysis of residual trace impurity protein [ J]The phenomenon was reported and studied in the publications of Biotechnology journal 1991,7(2):108-

The reason for the phenomenon of heterogeneity of the ends of the expression product is mainly as follows: the high-density fermentation obviously increases the total amount of various proteases and peptidases in the pichia pastoris, the enzyme activity of the proteases is higher under the high-temperature condition, and the foreign proteins are more easily degraded under the action of the proteases. Even if a leader peptide sequence is added in the upstream design, the foreign protein is secreted and expressed outside the cell, and the problem still cannot be avoided. Since, yeast has the property of being able to secrete small amounts of proteases to the outside of the cell; in addition, in the middle and later stages of high-temperature fermentation, the decay rate of yeast is higher, so that more protease is released in fermentation liquor, and hydrolytic damage to exogenous protein is caused.

There are studies (e.g., Zhong Y, Yang L, Guo Y, et al. high-temperature culture of recombinant Pichia pastoris end tissue and secretion process of human interleukin-10[ J ]. Microbial Cell industries, 2014,13(1):163.) that when the secretion expression of the foreign protein is performed, the rate of newly synthesized peptide chain exceeds the folding and processing ability of endoplasmic reticulum, leading to the accumulation of immature or misfolded protein in the endoplasmic reticulum of the host bacterium in a large amount, inducing endoplasmic reticulum pressure, and further limiting the secretion expression amount of the foreign protein. Disruption of cellular metabolic homeostasis by endoplasmic reticulum pressure is also a significant cause of increased bacterial cell death rates. A number of reports have been reported in the literature that low temperature culture of Pichia pastoris leads to higher productivity per cell expression (Pingzou Li, Appl Biochem Biotechnol, 2007). The low-temperature culture can reduce the activity of various proteases and peptidases in host bacteria and is beneficial to inhibiting the terminal heterogeneity of products; can also relieve endoplasmic reticulum pressure, relieve congestion of secretion channel, improve secretion efficiency of foreign protein, reduce cell death amount, and reduce secretion amount of protease and peptidase.

But at the same time,the low fermentation temperature can directly influence the physiological process of the thalli and the activity of various enzymes related to growth, further influence the metabolic rate of the thalli and finally reduce the growth rate of the thalli in fermentation. This also makes it difficult to achieve cell densities at high temperatures, or even high densities, in an ideal fermentation cycle. Without sufficient cell number, it is difficult to ensure the total expression of the foreign protein. With BSM + PTM₁The culture medium combination of (a) cannot meet the process requirements of low-temperature fermentation.

The Chinese patent publication No. CN103923846 discloses a culture medium of Pichia pastoris. The culture medium does not contain KOH and H₃PO₄Containing NH₄H₂PO₄With KH₂PO₄Further provides a culture medium which is more beneficial to environmental protection and equipment maintenance. Does not relate to culture improvement of pichia pastoris under low temperature conditions.

The Chinese patent publication No. CN105039188A discloses a culture medium of Pichia pastoris and a culture method thereof. The culture medium contains glucose, corn steep liquor, part of inorganic salts and a small amount of yeast extract. The wet weight of the bacteria only reaches 180-220 g/L, and the culture improvement of pichia pastoris under the low-temperature condition is not involved.

The Chinese patent of publication No. CN201810081331.0 discloses a Pichia pastoris culture medium and its use, in which CaO is added₂As dissolved oxygen carrier, it can raise the fermentation dissolved oxygen level of recombinant saccharomycete and the expression level of exogenous protein. Also, improvement of culture under low temperature conditions of Pichia pastoris is not involved.

Therefore, there is a need to develop a culture medium suitable for fermentation and expression of pichia pastoris under low temperature conditions. The inhomogenous phenomenon of the tail end of the product is inhibited, the difficulty of later purification treatment is reduced, and the yield is improved; relieve endoplasmic reticulum pressure, reduce cell death rate in middle and later fermentation period, and raise the expression amount of thallus unit. While the benefits of low-temperature culture are obtained, the cell density (more than 500g/L wet cells) equivalent to that of high-temperature fermentation can be obtained, the total expression quantity of the foreign protein is greatly improved, and the production cost is reduced. And a better foundation is laid for the industrial scale application of the pichia pastoris.

The invention utilizes mathematical statistics methods such as Plackett-Burman, Central-Composite and the like and combines practical experience to carry out a large amount of culture medium formula screening and optimization tests. Finally obtaining the culture medium formula suitable for low-temperature fermentation of pichia pastoris.

Disclosure of Invention

The invention aims to provide a culture medium formula suitable for culturing Pichia pastoris and expressing recombinant protein under the condition of low temperature (18-22 ℃). To achieve the object, the present invention is characterized in that:

calcium ion (Ca) in the culture medium formula²⁺) The concentration is set at a lower level. The endoplasmic reticulum is Ca²⁺The important storage sites of (Zhengsi, etc.. inner reticulum calcium pool control calcium influx research progress) are reported]Biotechnological progress, 2017(2), Ca in endoplasmic reticulum when cells are under endoplasmic reticulum pressure²⁺Leakage into the cytoplasm occurs. Ca²⁺The increased level activates a downstream series of Ca receptors²⁺Regulated kinases are a direct factor in the disruption of cellular homeostasis and also one of the potential factors in the increase in the rate of cell death. Thus, Ca in the medium is reduced²⁺The concentration of intracellular Ca may be helpful²⁺Normal levels of maintenance, in turn, contribute to the maintenance of cellular homeostasis. Correspondingly, the concentration of the calcium sulfate dihydrate is 0.12-0.30 g/L.

Synchronously, adding magnesium ion (Mg) in the culture medium²⁺) The concentration is set at a lower level. Mg (magnesium)²⁺Is a cofactor of key enzyme reactions in a plurality of physiological processes in cells; plays an important role in energy metabolism, active transport and maintenance of electrolyte balance; is an essential factor for maintaining the stability of biological membranes and genomes; and is involved in the transmission and expression of genetic information. Mg (magnesium)²⁺Homeostasis in a cell is an important factor in maintaining cell homeostasis. Reduction of Mg in culture media²⁺The concentration of intracellular Mg under low temperature condition may be favorable²⁺In turn, helps in the maintenance of cellular homeostasis. Accordingly, the number of the first and second electrodes,the concentration of the magnesium sulfate heptahydrate is 2.0-5.0 g/L.

Synchronously, potassium ion (K) in the culture medium composition⁺) The concentration is set at a lower level. K⁺Is an inorganic ion essential for the survival and growth of microorganisms, can promote the metabolism of carbohydrate, and maintain the intracellular and extracellular osmotic pressure and acid-base balance. The literature shows (Sun Tinghong et al, the influence of several inorganic ions on the physiological metabolism of beer yeast and the acid production mechanism during fermentation [ J]The university of Dalian industry, 2002,21(1):29-32.), has obvious effect of promoting the growth of yeast by potassium ion concentration in a certain range. In experimental research, the low potassium ion concentration in the culture medium is maintained under the low temperature condition, so that the growth and the activity of the pichia pastoris are more favorably realized. Correspondingly, the concentration of the potassium sulfate in the invention is 4.0-6.0 g/L.

Synchronously, adding zinc ion (Zn) in the culture medium²⁺) The concentration is set at a higher level. It is known that zinc ion is essential for the growth and development of organisms, is a key element involved in the synthesis of DNA and RNA, and has other various biological functions. Numerous documents show that yeasts have high bearing capacity and tolerance capacity to zinc ions, and the zinc content of some zinc-rich yeasts can reach over 9mg/g (Guo Xuena et al. Zinc-rich yeast breeding and culture condition research [ J]Microbiology report, 2004, 44(2). Our experimental study finds that under the low temperature condition, the concentration of zinc ions in the culture medium is improved, and the growth and the activity of the pichia pastoris are more facilitated. Correspondingly, the concentration of zinc chloride in the trace element solution is 100-160 g/L, and the concentration of zinc chloride in the mixed culture medium is 0.35-0.51 g/L.

Simultaneously, a small amount of Yeast Extract (Yeast Extract) solution is fed at a constant speed during the fermentation process, or quantitatively supplemented in stages. The organic nitrogen source is mainly from microorganisms or animals and plants, such as peptone, yeast extract, soybean hydrolysate, casein and the like, contains abundant small peptides, amino acids or protein components, and is a common culture medium component. When using nitrogen element in ammonia water or ammonium salt, yeast needs to synthesize inorganic nitrogen into amino acid first. The organic nitrogen component is easier to utilize for yeast, and part of the component can be directly utilized and belongs to a rapid nitrogen source. The experimental research of the inventor finds that the introduction of the organic nitrogen source is beneficial to the growth of the pichia pastoris under the low-temperature condition, but the addition of the organic nitrogen source can introduce exogenous impurities which form a complex structure, is not beneficial to a downstream purification process, and has the problem of overhigh cost under the industrial scale. In order to solve the defects caused by introducing the organic nitrogen source, the yeast extract homologous with pichia pastoris is selected, the introduced exogenous impurities are similar to the impurities of the fermentation system, and the influence on downstream purification is small. The addition amount and the addition mode are further optimized and adjusted. Through comparison tests, the addition amount of the yeast extract is reduced to about 1.8g/L, and the yeast extract is fed at a constant speed (or quantitatively supplemented by stages) in the whole fermentation process. Thus, the organic nitrogen component added at a low speed can be utilized by the bacteria in time without causing accumulation of related impurities, and the lower adding amount also brings less related impurities and controls the cost. The test results show that the addition of small amounts of yeast extract does not affect the downstream purification process under the regulation and control method established in the present invention. Correspondingly, the total adding amount of the yeast extract in the culture medium is 1.0-3.0 g/L.

Synchronously, on the basis of the above formula setting, the fermentation pH value is controlled at a higher level. The test result shows that under the condition of low temperature and the condition of the composition of the culture medium, the higher pH value of the fermentation liquor is more beneficial to the growth and expression of the pichia pastoris. Correspondingly, the pH value of the culture medium is controlled to be 6.2-6.8.

Based on the above, the technical scheme adopted by the invention is as follows: a low-temperature culture medium for Pichia pastoris, comprising the following components in concentration:

the formula of the trace element solution is as follows:

preferably, the pichia pastoris low temperature type culture medium comprises the following components in concentration:

the formula of the trace element solution is as follows:

advantageous effects

The invention provides a culture medium which is suitable for culture of pichia pastoris and expression of exogenous proteins under the condition of low temperature (18-22 ℃). The density of the bacteria reaches the level of high-temperature (28-32 ℃) fermentation under the condition of considerable time consumption, namely the weight of wet bacteria is more than 500 g/L. Thereby the total expression amount of the foreign protein is equivalent to or exceeds the level under the high temperature condition. Meanwhile, the phenomenon of recombinant protein terminal heterogeneity is inhibited under the condition of low-temperature culture, the proportion of impurities related to terminal heterogeneity is obviously reduced, and the level is obviously superior to that in the case of high-temperature culture. Taking fermentation expression of insulin analogue-insulin aspart precursor as an example, the culture medium provided by the invention is used for fermentation and expression of pichia pastoris engineering bacteria (containing insulin aspart precursor genes) under a low-temperature condition, and the fermentation density can reach more than 510g/L and is equivalent to the level during high-temperature fermentation; the whole expression quantity can reach about 5.0g/L, which is slightly superior to the level of high-temperature fermentation; the terminal heterogeneity impurity of the expression product is less than 6%, which is obviously better than about 17% under high temperature condition.

The invention solves the problems of higher expression rate of recombinant protein unit thalli and lower total expression amount under the low temperature condition, improves the purity of recombinant protein products, greatly reduces the downstream purification treatment difficulty, improves the overall yield and reduces the industrial production cost. Expands the application conditions of the pichia pastoris and lays a better foundation for the application of the pichia pastoris in an industrial scale.

Drawings

FIG. 1 is a graph showing the increase of wet weight of cells in BSM medium in example 2

FIG. 2 is a graph showing the detection results of 108h recombinant protein induced by BSM medium in example 2

FIG. 3 is a graph showing the increase of wet weight of cells in BSM, modified I, modified II and modified III media in example 3

FIG. 4 is a graph showing the detection results of 108h recombinant protein induced by BSM medium in example 3

FIG. 5 is a graph showing the results of detection of 108h recombinant protein induced by modified I medium in example 3

FIG. 6 is a graph showing the results of detection of 108h recombinant protein induced by modified II medium in example 3

FIG. 7 is a graph showing the results of detection of 108h recombinant protein induced by modified III medium in example 3

FIG. 8 is a graph showing the comparison of the wet weight increase of 108h cells induced by BSM medium in example 2 and by modified III medium in example 3

Detailed Description

The present invention is further illustrated by the following examples, and those skilled in the art can make routine alternatives to the existing techniques based on the technical idea of the present invention for specific methods or materials used in the examples, and not limited to the specific descriptions of the examples of the present invention.

The test methods used in the examples are, unless otherwise specified, regarded as conventional methods; the materials, reagents and the like used are commercially available unless otherwise specified.

In the following examples of the invention, the recombinant pichia pastoris used was from the beijing dilu pharmaceutical shards limited company. The strain contains an insulin aspart precursor gene, and can secrete and express the insulin aspart precursor after induction.

1) YPG medium:

yeast extract 10.0g/L

Tryptone 20.0g/L

Glycerol 10.0g/L

2) BSM medium:

3) PTM1 solution:

the PTM1 solution is sterilized by filtration.

4) A supplemented medium: purified aqueous glycerol solution (50% V/V) containing 1.2% trace elements; methanol containing 1.2% trace elements;

contains 100g/L of purified aqueous solution of yeast extract.

Sterilizing the purified water solution of the glycerol and the purified water solution of the yeast extract at the high temperature of 121-124 ℃ for 30-35 min;

the methanol was filtered through 0.45 μm.

EXAMPLE 1 preparation of culture Medium

In the following examples of the present invention, the culture medium used was composed of the following components in terms of the total volume of the culture medium:

low-temperature modified medium:

the formula of the low-temperature type improved culture medium trace element solution comprises the following components:

on the basis of the above-mentioned culture media, three different modified culture media, named modified I, modified II and modified III, were obtained by preparing the culture media according to the formulations of Table 1, respectively.

TABLE 1

The preparation process comprises dissolving the components in the table with 80-90% of purified water, stirring, and fixing the volume to the final volume with purified water; after the prepared culture medium is sterilized at high temperature, a PTM1 solution is added, the pH value is detected, and ammonia water is used for adjusting the pH value to the required pH value.

Example BSM + PTM at 228-32 deg.C₁Fermentation test of culture Medium

Adopting a BSM culture medium as a fermentation culture medium, and carrying out culture and expression of recombinant insulin analogue precursor Pichia pastoris engineering bacteria in a 1.5T fermentation scale, wherein the method comprises the following steps:

step one, taking recombinant insulin analogue precursor recombinant pichia pastoris engineering bacteria, adding 1000 mu l of working strain into a 500ml triangular flask filled with 100ml YPG culture medium, and performing shake culture at 28-32 ℃ and 220rpm/min for 52 hours; 750 μ L of bacterial liquid cultured for 52 hours was added to a 500ml flask containing 150ml of YPG medium in 40 flasks, and shake-cultured at 28-32 ℃ and 220rpm/min for 16 hours to obtain 6L of secondary seed liquid.

Step two, preparing 54L of fermentation medium (containing 60L of medium components), adding 6L of secondary seed liquid into the fermentation medium according to the inoculation amount of 10%, carrying out tertiary seed culture, controlling the temperature to be 30 ℃, leading in air at a ratio of 1.0vvm, and culturing for 6-10 h, wherein the dissolved oxygen value is not less than 30%, and the OD value is 6-10.

And step three, preparing 540L of fermentation medium (containing 600L of medium components), adding 60L of secondary seed liquid into the fermentation medium according to the inoculation amount of 10%, and performing fermentation culture.

Controlling the temperature at 28-32 ℃, controlling the air inlet ratio to be 1.0vvm, controlling the dissolved oxygen value to be more than or equal to 30% by stirring, and beginning to supplement the purified glycerol aqueous solution after the dissolved oxygen value rebounds to 80-100%; setting the initial replenishing rate F of the glycerol solution₀Adjusting the glycerol solution replenishment rate in real time (namely the wet weight growth rate is 10-15 g/h) based on the cell density growth rate to obtain a large amount of cells, wherein the temperature is controlled to be 30 ℃, the air introduction ratio is 1.0vvm, and the dissolved oxygen value is more than or equal to 30%; when the wet weight of the thalli reaches 200g/L, the glycerol solution is stopped being supplemented.

Step five, after glycerol is stopped being supplemented for 1 hour, methanol solution is started to be supplemented for induction, and the initial methanol solution supplementing speed F is set₀And (2) adjusting the methanol solution replenishment rate in real time based on the recombinant protein expression amount (namely the recombinant protein expression amount increase rate is 0.02-0.05 g/L) to obtain a certain amount of recombinant protein, controlling the temperature to be 28-32 ℃, controlling the air introduction ratio to be 2.5vvm, and controlling the dissolved oxygen value to be more than or equal to 20%.

Step six, the induction time is 108h, the fermentation culture is stopped, and the wet weight of the thalli and the content of the recombinant protein are detected.

Sampling every 12h from the beginning of induction to detect the wet weight of the thalli, namely the wet weight of the thalli at time points of 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h and 108 h; taking the fermentation liquor for centrifugation for 108h, and detecting the content of the recombinant protein in the supernatant.

In example 2, the wet weight growth of the cells in the BSM medium is shown in FIG. 1, the detection of the recombinant protein in 108h is shown in FIG. 2, and the results of induction to the wet weight of the cells in 108h, the expression level of the recombinant protein and the proportion of heterogeneous impurities are shown in Table 2.

TABLE 2

Fermentation medium	Wet weight of thallus	Amount of recombinant protein expressed	Non-uniform N-terminal or C-terminal ratio
				BSM	525g/L	5.06g/L	17.677％

Example BSM + PTM at 318-22 deg.C₁Fermentation test of culture medium and low-temperature type improved I, II and III culture media

BSM, an improved I culture medium, an improved II culture medium and an improved III culture medium are respectively used as fermentation culture media to carry out fermentation culture on recombinant insulin analogue pre-recombinant Pichia pastoris engineering bacteria, and the method comprises the following steps:

And step three, preparing 540L of fermentation medium (containing 600L of medium components), adding 60L of third-grade seed liquid into the fermentation medium according to the inoculation amount of 10%, and starting fermentation culture. While adding yeast extract solutions to the modified I, modified II and modified III media at 5ml/h, 14ml/h and 9ml/h, respectively.

Step (ii) ofFourthly, controlling the temperature to be 18-22 ℃, controlling the air inlet ratio to be 1.0vvm, controlling the dissolved oxygen value to be more than or equal to 30% by stirring, and beginning to supplement the purified glycerol aqueous solution after the dissolved oxygen value rebounds to 80-100%; setting the initial replenishing rate F of the glycerol solution₀Adjusting the glycerol solution replenishment rate in real time (namely the wet weight growth rate is 10-15 g/h) based on the cell density growth rate to obtain a large amount of cells, wherein the temperature is controlled to be 18-22 ℃, the air introduction ratio is 1.0vvm, and the dissolved oxygen value is more than or equal to 30%; when the wet weight of the thalli reaches 200g/L, the glycerol solution is stopped being supplemented.

Step five, after glycerol is stopped being supplemented for 1 hour, methanol solution is started to be supplemented for induction, and the initial methanol solution supplementing speed F is set₀And (2) adjusting the methanol solution replenishment rate in real time based on the recombinant protein expression amount (namely the recombinant protein expression amount increase rate is 0.02-0.05 g/L) to obtain a certain amount of recombinant protein, controlling the temperature to be 18-22 ℃, controlling the air introduction ratio to be 2.5vvm, and controlling the dissolved oxygen value to be more than or equal to 20%.

And step six, terminating the culture expression when the induction time is 108h, and detecting the wet weight of the thalli and the content of the target protein.

In example 3, the increase of wet weight of cells in the BSM, modified I, modified II and modified III media is shown in FIG. 3, the detection of recombinant proteins in the BSM, modified I, modified II and modified III media for 108h is shown in FIG. 4, FIG. 5, FIG. 6 and FIG. 7, and the wet weight of cells and the content of recombinant proteins and the heterogeneous impurity ratio induced in each media for 108h are shown in Table 3.

TABLE 3

Fermentation medium	Wet weight of thallus	Amount of recombinant protein expressed	N-terminal heterogeneous impurity ratio
				BSM	390g/L	3.95g/L	8.013％
Improvement I	503g/L	4.79g/L	6.735％
				Improvement II	532g/L	4.88g/L	7.298％
Improvement III	524g/L	5.01g/L	5.747％

The invention repeatedly verifies the three types of Pichia pastoris engineering bacteria GS115, X-33 and SMD1168 according to the methods in the embodiments 2 and 3, and similar experimental results are obtained. The low-temperature culture medium disclosed by the invention has universality for pichia pastoris.

Claims

1. A low-temperature culture medium for Pichia pastoris, which is characterized by consisting of the following components in percentage by volume of the total culture medium:

the formula of the trace element solution is as follows:

2. the culture medium according to claim 1, consisting of the following components, based on the total volume of the culture medium:

the formula of the trace element solution is as follows:

3. the use of the culture medium formulation of claims 1-2 for the culture of pichia pastoris.

4. The use of claim 3, wherein the culturing comprises fermentation and seed broth culturing.

5. The culture medium of claim 1-2, wherein the Pichia pastoris is a wild type strain or a modified recombinant engineered strain containing a foreign gene.

6. The medium according to claim 1 to 2, wherein hydrates having different numbers of water molecules are used, and the metal ion molar concentrations of the metal salt components are the same.

7. The yeast extract in the culture medium of claim 1-2 is added in a manner of feeding at or near constant speed during the whole fermentation process, or quantitatively supplemented in stages during the whole fermentation process after being prepared into a solution.