Method for reducing lactulose-based isomers in process of producing oligosaccharides by using lactose as substrate through fermentation
By adjusting the pH before lactose sterilization, the problem of excessive lactulosyl isomers in the production of oligosaccharides by microbial fermentation is solved, and the production of lactulosyl isomers of oligosaccharides is significantly reduced, and the product meets the requirements of regulations.
Patent Information
- Application Number
- CN202411799069.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2025-03-07
AI Technical Summary
In the process of microbial fermentation and production of oligosaccharides using lactose as a substrate, it is difficult to effectively reduce the production of lactulosyl isomers, resulting in the product not meeting the requirements of regulations.
The pH adjustment is performed before lactose sterilization to ensure that the pH value of lactose is ≤5, and then sterilization is performed to reduce the production of lactulose, thereby reducing the production of oligosaccharide lactulosyl isomers during fermentation.
Through this method, the yield of oligosaccharide lactulosyl isomers during the fermentation process was significantly reduced, and the proportion was reduced from 4.45% to 1.78%, which greatly complied with the requirements of the regulations.
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Abstract
Description
Technical Field
[0001] The present application relates to the field of fermentation technology, and in particular to a method for reducing lactulose isomers in the process of producing oligosaccharides by fermentation using lactose as a substrate. Background Art
[0002] Human milk oligosaccharides are the third largest solid component in human milk after lactose and fat, with a content of about 5-15g / L. Human milk oligosaccharides contain 5 monomers, namely: D-glucose, D-galactose, N-acylglucosamine, L-fucose and N-acetylneuraminic acid (sialic acid). The above 5 monomers can be combined in different proportions to form thousands of oligosaccharides. Currently, more than 200 types have been successfully identified. Common human milk oligosaccharides include 2'-fucosyllactose (2'-fucosyllactose, 2'-FL), 3-fucosyllactose (3-fucosyllactose, 3-FL) and 6'-sialyllactose (6'-sialyllactose, 6'-SL). The reducing end of oligosaccharides is a lactose structure, on which the sugar chain is extended to form oligosaccharides with diverse structures. Based on the connected monomers, they can be divided into three main categories: (1) fucosylated neutral oligosaccharides; (2) sialylated acidic oligosaccharides; and (3) non-fucosylated neutral oligosaccharides.
[0003] At present, in addition to directly isolating oligosaccharides from breast milk, the main methods for synthesizing oligosaccharides in vitro are chemical synthesis and biosynthesis. Although chemical synthesis has made great progress in the synthesis of oligosaccharides, the use of a large amount of organic solvents, harsh reaction conditions, and lengthy reaction orders make its production cost expensive, and the presence of toxic reagents also limits the wide application of oligosaccharides in the food field.
[0004] Biosynthesis is safe, pollution-free and has a wide range of applications. Biosynthesis includes two pathways: enzymatic synthesis and microbial fermentation synthesis. Although enzymatic synthesis has a good product yield, it is not easy to obtain the necessary enzymes, and glycosyltransferase catalysis requires sugar nucleotides as receptors, which are expensive and difficult to obtain. In addition, during the enzymatic reaction, the inherent properties of the enzyme, such as stability, catalytic efficiency, and tolerance of substrates and receptors, will affect this method, making it difficult to mass produce.
[0005] With the development of metabolic engineering and microbial fermentation synthetic biology, the cloning and expression of enzymes in the microbial synthesis pathway has become more and more widespread, making the rapid development of whole-cell production possible. Microbial fermentation synthesis is a method of synthesizing oligosaccharides directly in microbial cells using their own or genetically engineered overexpressed or heterologously expressed enzymes. However, there are difficulties in the separation of metabolites in the synthesis of oligosaccharides by microbial fermentation, especially for oligosaccharides with lactose as the fermentation substrate. Various lactose isomers are easily produced in the fermentation broth, such as fucose-lactose fucose, which is a lactulose isomer.
[0006] Many current quality specifications require lactulose isomer w / % ≤ 1%. However, in the actual production process of oligosaccharides synthesized by microbial fermentation, more lactulose isomers are inevitably produced, making downstream separation and purification more difficult. The product is unqualified due to excessive lactulose isomers. Summary of the invention
[0007] The purpose of the present application is to overcome the shortcomings of the above-mentioned prior art and provide a method for reducing lactulose isomers in the process of producing oligosaccharides by fermentation using lactose as a substrate. The present application first adjusts the pH of the fermentation substrate (lactose) and then sterilizes it so that lactulose will not be produced, and lactulose isomers will not be produced during the fermentation process. The method provided by the present application greatly reduces the yield of lactulose isomers of oligosaccharides during the fermentation process.
[0008] To achieve the above purpose, the technical solution adopted by this application is:
[0009] The present application provides a method for reducing lactulose isomers in the process of producing oligosaccharides by microbial fermentation, comprising the following steps:
[0010] S1. Inoculate the seed liquid of the strain into a culture medium containing lactose and glycerol for fermentation to obtain a fermentation liquid;
[0011] S2. The pH value of the lactose contained in the culture medium in step S1 needs to be adjusted to ≤5 before sterilization, and then the lactose is sterilized and then fed;
[0012] S3. Putting the fermentation liquid treated in step S2 into a fermentation tank to obtain a fermentation liquid containing the target product, removing bacteria, removing protein and decolorizing, desalting by electrodialysis, desalting by ion exchange resin, concentrating, purifying, ultrafiltering, re-concentrating and spray drying the fermentation liquid containing the target product to obtain oligosaccharides, and detecting lactulose isomers in the oligosaccharides.
[0013] Since lactose is used as a fermentation substrate, lactulose isomers will be produced in the early treatment process. For example, oligosaccharides fermented with lactose as a substrate will basically produce lactulose isomers, because lactulose is produced during the lactose sterilization process, and lactulose combines with other oligosaccharide lactulose isomers during the fermentation process, resulting in the fermented product also containing more lactulose isomers, which does not meet regulatory requirements. In this regard, the inventor of the present application has found through a large number of experiments that the fermentation substrate lactose is first pH-adjusted (pH adjustment ≤ 5) and then sterilized. The results show that lactulose will not be produced after sterilization, and oligosaccharide lactulose isomers are not produced during the fermentation process. Using the method of the present application, the proportion of oligosaccharide lactulose isomers fermented with lactose as a substrate is only 1.78%, which is greatly reduced compared to the proportion of oligosaccharide lactulose isomers in the fermentation process performed by the existing method. Therefore, the effect of the present application is remarkable and has a high application value.
[0014] Preferably, in step S2, the pH value of the lactose contained in the culture medium in step S1 needs to be adjusted to 3-5 before sterilization.
[0015] The experiment shows that before the lactose solution was sterilized without adjusting the pH, no lactulose content was detected in it, and after sterilization, 3.96% of lactulose was detected. Before and after the lactose solution was sterilized with a pH of ≤5 (especially a pH of 3-5), no lactulose content was detected, indicating that this method can effectively reduce the lactulose production after lactose sterilization.
[0016] More preferably, in step S2, the pH value of the lactose contained in the culture medium in step S1 needs to be adjusted to 4 before sterilization.
[0017] When the pH value of lactose is adjusted to 4 before sterilization, the production of oligosaccharide lactulose isomers during the fermentation process can be better reduced. The effect is very significant and has high application value.
[0018] As a preferred embodiment of the method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation described in the present application, the initial mass concentration of lactose is 10 g / L, and the initial mass concentration of glycerol is 20 g / L.
[0019] The present application uses lactose as a substrate and adds glycerol to provide necessary nutritional conditions for the fermentation process. In addition, the glycerol concentration is controlled at a lower level during the fermentation process to increase the accumulation of the product.
[0020] As a preferred embodiment of the method for reducing lactulose isomers in the process of producing oligosaccharides by microbial fermentation described in the present application, in the fermentation culture of step S1, the mass concentration of lactose is controlled to be 9.5-10.5 g / L and the dissolved oxygen is controlled to be 25-35%.
[0021] The use of the above-mentioned mass concentration range of lactose and dissolved oxygen can better promote microbial metabolic activities, avoid product synthesis restrictions, and better improve production efficiency and product quality.
[0022] As a preferred embodiment of the method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation described in the present application, in the fermentation culture of step S1, the ventilation volume is 0.5-2vvm, the rotation speed is 200-900r / min, the culture temperature is 30-40°C, and the pH of the fermentation liquid is adjusted to 6-7;
[0023] Preferably, the ventilation volume is 2 vvm, the rotation speed is 200 r / min, the culture temperature is 37° C., and the pH of the fermentation liquid is adjusted to 6.8.
[0024] When the present invention adopts the above-mentioned preferred fermentation conditions, oligosaccharides can be produced better and the lactulose isomers of oligosaccharides produced during the fermentation process can be reduced better.
[0025] Preferably, 14% by volume of NH 4 OH adjusted the pH of the fermentation broth to 6.80.
[0026] As a preferred embodiment of the method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation described in the present application, in step S1, the strain includes Escherichia coli, and the inoculation amount of the seed liquid of the strain is 1-5%, preferably the inoculation amount of the seed liquid of the strain is 5%.
[0027] As a preferred embodiment of the method for reducing lactulose isomers in the process of producing oligosaccharides by microbial fermentation described in the present application, in the step S2, the feeding rate is 8 g / L / h, and after the feeding temperature is cooled to 25-30°C and the culture is continued for 6 hours, IPTG and lactose are added for induction (400 g of lactose is mixed with IPTG and added initially, and then 400 g of lactose is added after sampling according to the test results).
[0028] As a preferred embodiment of the method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation described in the present application, the lactose is added at the 10th and 18th hours of the fermentation time.
[0029] As a preferred embodiment of the method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation described in the present application, the culture medium further comprises the following components:
[0030] 4.0g / L(NH 4 ) 2 SO 4 , 9.2g / LK 2 HPO 4 , 8.2g / L KH 2 PO4 , 0.3g / L citric acid, 6.0g / L tryptone, 2.0g / L yeast extract, 10mg / L thiamine, 2.0g / L MgSO 4 7H 2 O, 0.02g / LCaCl 2 and 10mL / L trace element solution.
[0031] The culture medium of the above formula adopted in the present application can promote the metabolic activity of microorganisms, avoid the limitation of product synthesis, and improve the production efficiency and product quality.
[0032] Compared with the prior art, this application has the following beneficial effects:
[0033] The present application provides a method for reducing lactulose isomers in the process of producing oligosaccharides by fermentation using lactose as a substrate. The present application finds that before sterilizing lactose, the pH of the fermentation liquid is adjusted to 3-5 and then sterilized, so as to reduce the production of lactulose after sterilization, and ultimately reduce the production of oligosaccharide lactulose isomers in the fermentation process. Specifically, the oligosaccharide lactulose isomers accounted for 4.45% of the oligosaccharides fermented with lactose sterilized without pH adjustment as a substrate when they were put into the tank, and the oligosaccharide lactulose isomers accounted for only 1.78% of the oligosaccharides fermented with lactose sterilized with pH adjustment as a substrate when they were put into the tank. In comparison, the proportion of oligosaccharide lactulose isomers fermented with lactose treated by the method of the present application as a substrate was reduced by 150%, and the effect of reducing the production of oligosaccharide lactulose isomers is very significant, and has a high application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is the spectrum of lactose before sterilization when the pH is adjusted to 3-5;
[0035] Figure 2 This is the spectrum of lactose after adjusting the pH to 3-5 and sterilization;
[0036] Figure 3 This is the spectrum of lactose stock solution (pH 6.18) before sterilization;
[0037] Figure 4 This is the spectrum of the lactose stock solution (pH 6.18) after sterilization;
[0038] Figure 5 is a spectrum using the method of Example 2;
[0039] Figure 6 It is the spectrum using the method of Comparative Example 1. DETAILED DESCRIPTION
[0040] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.
[0041] In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.
[0042] Example 1
[0043] In this example, a lactose stock solution (500 g of lactose sample was weighed, dissolved in 100°C water, and fixed to 1.2 L) was taken, the pH was adjusted to 4, and a treated lactose solution was obtained, which was then sterilized (boiling water bath for 10 min). The spectra of the lactose solution before and after sterilization are shown in FIG. Figure 1-Figure 2 shown.
[0044] In this example, lactose stock solution (pH 6.18) was directly sterilized (boiling water bath for 10 min) without adjusting the pH, to obtain the sterilized lactose stock solution (pH 6.18). The spectra of the lactose stock solution (pH 6.18) before and after sterilization are shown in FIG. Figure 3-Figure 4 shown.
[0045] The lactulose content in the lactose stock solution (pH 6.18) was not detected before the pH was adjusted for sterilization. After sterilization, it was detected that the lactulose content was 3.96%.
[0046] The lactulose content was not detected in the lactose solution before and after the pH was adjusted to 3-5 for sterilization, indicating that this method can effectively reduce the lactulose production after lactose sterilization.
[0047] Example 2
[0048] This embodiment provides a method for reducing lactulosyl isomers in the process of producing oligosaccharide fucose lactose by fermentation using lactose as a substrate, comprising the following steps:
[0049] S1. The seed liquid of the strain was inoculated into a fermentation tank medium containing lactose and glycerol (initial lactose mass concentration was 10 g / L, initial glycerol mass concentration was 20 g / L) at an inoculum size of 5% for fermentation and culture, with a ventilation volume of 2 vvm, a rotation speed of 200 r / min, and a culture temperature of 37°C. During the fermentation process, the pH of the fermentation tank liquid was adjusted to 6.80 using 14% NH4OH by volume. During the fermentation process, the glycerol concentration was controlled at a low level to increase the accumulation of the product, the substrate lactose mass concentration was controlled at about (10±0.5) g / L, and the dissolved oxygen in the fermentation tank was (30±5)%, to obtain a fermentation liquid;
[0050] S2, adjusting the pH value of the fermentation broth in step S1 to 4, then sterilizing the fermentation broth with lactose (boiling water bath for 10 min), continuing to culture for 6 h after cooling, and then feeding (feeding rate of 8 g / L / h), adding IPTG and lactose for induction, and then adding 400 g of lactose after sampling according to the test results;
[0051] S3. The fermentation liquid treated in step S2 is subjected to bacterial removal, protein removal and decolorization, electrodialysis desalination, ion exchange resin desalination, concentration, purification, ultrafiltration, re-concentration and spray drying to obtain oligosaccharides, and the lactulose isomers in the oligosaccharide fucose lactose are detected.
[0052] S31, bacteria removal: the fucose lactose fermentation liquid released from the fermentation tank was added into the 30nm ceramic composite membrane for sterilization, the membrane flux was 20L / h, and the membrane area was 1.7m 2 , membrane temperature <40℃.
[0053] S32, protein removal and decolorization: the sterilized fermentation liquid is concentrated and added into a heating tank, heated to 60-90°C and kept warm for 30-60 minutes, 1-4% (Jiangsu Zhuxi plant activated carbon) 305 wet carbon is added, and the mixture is kept warm and stirred for 30 minutes. The decolorized liquid is passed through a multi-layer filter to remove the activated carbon to obtain a decolorized liquid.
[0054] S33, electrodialysis desalination: add the decolorizing liquid into the electrodialyser for desalination, set the voltage to 39V, and end the electrodialysis when the liquid conductivity drops below 2000μs / cm.
[0055] S34, ion exchange resin desalination: the above electrodialysis desalination liquid is passed through anion and cation exchange resin chromatography columns at a flow rate of 1.5BV / h, first through the cation resin and then through the anion resin. The conductivity of the collected sample solution should be below 20μs / cm.
[0056] S35, concentration: concentrate the desalted liquid after ion exchange, the concentration temperature is 55-65°C, the pressure in the tank is 0.09MPa, each concentration time does not exceed 3h, and the concentration is concentrated to 200-300g / L.
[0057] S36, purification: the above concentrate is purified by moving bed chromatography (SSMB) to collect the required components.
[0058] SSMB chromatography stands for Sequential Simulated Moving Bed. It uses the differences in the forces between different solutes and the stationary phase and the mobile phase, such as distribution, adsorption, and ion exchange. When the two phases move relative to each other, each solute is balanced multiple times between the two phases, so that each solute is separated from each other to obtain the target substance. The operation of the equipment is divided into four steps: A, B, C, and D. Steps A and D run separately, and steps B and C run simultaneously. Step B feeds out fast components, step C feeds in water and produces slow components, and step D feeds in water and produces fast components. Manually set the running time of the four steps A, B, C, and D on the operation interface, A (339s), B (169s), C (169s), and D (106s). Manually set the flow rate of each step on the high-pressure infusion pump, including the circulation pump (50mL / min), the volumetric pump (50mL / min), and the sample pump (25mL / min). After all parameters are set correctly, switch to automatic mode and click System Start to run. The feed is an oligosaccharide solution with a pH of 6-8.
[0059] S37, ultrafiltration: The collected components after SSMB were added to the multifunctional membrane device (2000D) for ultrafiltration, and the membrane clear liquid was collected. Membrane flux 35L / h, membrane area 1.7m 2 , membrane temperature <35℃, membrane inlet pressure 4.2MPa, heat exchanger pressure 0.4MPa, and membrane permeate pressure -2.3MPa.
[0060] S38. Concentration: Concentrate the membrane clear liquid after ultrafiltration in batches to 200g / L~300g / L, the concentration temperature is 50~60℃, the pressure in the tank is 0.09MPa, and the concentration time each time does not exceed 3h. Release the concentrated liquid for sampling and testing and prepare for spray drying.
[0061] S39, spray drying: spray drying the above concentrate, with an inlet air temperature of 150°C, an outlet air temperature of 98-100°C, a feed speed of 30 rpm, and collecting and weighing samples approximately every 40 minutes.
[0062] S40, Detection: Take a small amount of sample for lactulose isomer detection.
[0063] Example 3
[0064] Compared with Example 2, the difference is that in step S2, the pH value of the fermentation liquid in step S1 is adjusted to 3, and the remaining steps are the same as Example 2.
[0065] Example 4
[0066] Compared with Example 2, the difference is that in step S2, the pH value of the fermentation liquid in step S1 is adjusted to 5, and the remaining steps are the same as in Example 2.
[0067] Comparative Example 1
[0068] Compared with Example 2, the difference is that in step S2, the fermentation liquid of step S1 is directly subjected to lactose sterilization (boiling water bath for 10 minutes) without adjusting the pH value. The remaining steps are the same as in Example 2.
[0069] Comparative Example 2
[0070] Compared with Example 2, the difference lies in step S2. The difference lies in step S2, in which the pH value of the fermentation liquid in step S1 is adjusted to 8. The remaining steps are the same as those in Example 2.
[0071] The above examples and comparative examples were subjected to fucose lactose detection and lactulose isomer detection, and the specific detection conditions were as follows:
[0072] Instrument: Shimadzu high performance liquid chromatograph, UV detector;
[0073] Column temperature: 60°C;
[0074] Mobile phase: acetonitrile: 10 mM ammonium formate aqueous solution (adjusted to pH 4.5 with formic acid) mixed in a volume ratio of 7:3;
[0075] Flow rate: 1ml / min;
[0076] Column model: TSKgel Amide-80 5μm 4.6mmI.D.*25cm;
[0077] Lactulosyl isomer detection:
[0078] Instrument: Thermo Fisher high performance liquid chromatograph, charged aerosol detector (CAD);
[0079] Column temperature: 25°C; Nebulizer temperature: 35°C; Data acquisition rate: 20 Hz; Power function: 1; Filters: 5;
[0080] Flow rate: 1.1 mL / min;
[0081] Chromatographic column model: amino polymer column, 250mm×4.6mm, 5μm;
[0082] Mobile phase: A: water, B: acetonitrile (gradient elution);
[0083] The gradient elution conditions are shown in Table 1:
[0084] Table 1
[0085]
[0086] The lactulose isomer ratios of the fermented liquids of the above-mentioned examples and comparative examples when released from the tank are shown in Table 2.
[0087] Table 2
[0088] The ratio of lactulose isomer to total sugar area (%) Example 2 0.78 Example 3 0.83 Example 4 1.21 Comparative Example 1 4.45 Comparative Example 2 6.82
[0089] The oligosaccharide lactulose isomers accounted for 4.45% when the oligosaccharide was fermented with lactose sterilized without pH adjustment as the substrate, and the oligosaccharide lactulose isomers accounted for only 1.78% when the oligosaccharide was fermented with lactose sterilized after pH adjustment as the substrate. In comparison, the oligosaccharide lactulose isomers accounted for 150% less when the lactose treated by the method of the present application was fermented as the substrate. The spectrum of the method of Example 2 is shown in FIG. Figure 5 As shown, the spectrum using the method of Comparative Example 1 is as follows Figure 6 shown.
[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.
Claims
1. A method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation, characterized in that: The following steps are involved: S1. Inoculate the seed liquid of the strain into a culture medium containing lactose and glycerol for fermentation to obtain a fermentation liquid; S2. The pH value of the lactose contained in the culture medium in step S1 needs to be adjusted to ≤5 before sterilization, and then the lactose is sterilized and then fed; S3. Putting the fermentation liquid treated in step S2 into a fermentation tank to obtain a fermentation liquid containing the target product, removing bacteria, removing protein and decolorizing, desalting by electrodialysis, desalting by ion exchange resin, concentrating, purifying, ultrafiltering, re-concentrating and spray drying the fermentation liquid containing the target product to obtain oligosaccharides, and detecting lactulose isomers in the oligosaccharides.
2. The method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation according to claim 1, characterized in that: In step S2, the pH value of the lactose contained in the culture medium in step S1 needs to be adjusted to 3-5 before sterilization.
3. The method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation according to claim 2, characterized in that: In step S2, the pH value of the lactose contained in the culture medium in step S1 needs to be adjusted to 4 before sterilization.
4. The method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation according to claim 1, characterized in that: The initial mass concentration of the lactose is 10 g / L, and the initial mass concentration of the glycerol is 20 g / L.
5. The method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation according to claim 1, characterized in that: In the fermentation culture of step S1, the mass concentration of lactose is controlled to be 9.5-10.5 g / L, and the dissolved oxygen is controlled to be 25-35%.
6. The method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation according to claim 1, characterized in that: In the fermentation culture of step S1, the ventilation volume is 0.5-2vvm, the rotation speed is 200-900r / min, the culture temperature is 30-40°C, and the pH of the fermentation liquid is adjusted to 6-7.
7. The method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation according to claim 1, characterized in that: In the step S1, the strain includes Escherichia coli, and the inoculation amount of the seed liquid of the strain is 1-5%.
8. The method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation according to claim 1, characterized in that: In step S2, the feeding rate is 8 g / L / h, and after the feeding temperature is lowered to 25-30° C., the culture is continued for 6 hours, and then IPTG and lactose are added for induction.
9. The method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation according to claim 1, characterized in that: The lactose is added at the 10th and 18th hours of the fermentation time.
10. The method for reducing lactulosyl isomers in the process of producing oligosaccharides by microbial fermentation according to claim 1, characterized in that: The culture medium also includes the following components: 4.0g / L(NH4)2SO4, 9.2g / L K2HPO4, 8.2g / L KH2PO4, 0.3g / L citric acid, 6.0g / L tryptone, 2.0g / L yeast extract, 10mg / L thiamine, 2.0g / L MgSO4·7H2O, 0.02g / LCaCl2 and 10mL / L trace element solution.