Amycolatopsis thermophilus and application thereof

Abstract

The invention belongs to the technical field of food fermentation production, and particularly relates to amycolatopsis thermophilus and application thereof. The amycolatopsis thermophilus is H-1 and is preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 13, 2025, the preservation number is CGMCC No.36166, the classification name of the amycolatopsis thermophilus is Amycolatopsis thermophilus, and the preservation address of the amycolatopsis thermophilus is Beijing, China. The invention also discloses an application of the amycolatopsis thermophilus in preparation of vanillin. The highest yield of vanillin obtained by using the strain reaches 21.6 g / L.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food fermentation production, and particularly relates to a strain of Amycolatopsis thermoflava and application thereof. BACKGROUND

[0002] Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the main component of natural vanilla flavor and is the most widely used flavor in the world. It is used in many food flavorings, such as ice cream, dairy products, desserts, candies, baked products and spirits, and is also used in perfumes, medicines and personal hygiene products. Traditionally, natural plant vanillin is obtained from the fermented pods of vanilla planifolia, but factors such as difficult cultivation, high extraction cost and low yield make the natural vanillin extracted from plants expensive. Currently, vanillin is mainly synthesized by chemical route. Although its production cost is low, pollution problems and lack of substrate selectivity limit its attractiveness.

[0003] In recent years, the production of vanillin by biofermentation using renewable raw materials (such as ferulic acid derived from rice bran, cedrol from spruce lignin, corn sugar and eugenol from clove oil) has become a research hotspot. Patent CN113637607B discloses a strain of Amycolatopsis CGMCC 22871, which produces vanillin from ferulic acid as substrate at 30℃, with a molar conversion rate of 87%. Patent CN101165168B discloses a strain of Streptomyces CCTCCM 206065, which produces vanillin from ferulic acid at 30℃, with a maximum yield of 19.2 grams / liter and a molar conversion rate of 54.5%.

[0004] The production temperature of the above-mentioned vanillin-producing strains is about 30℃, at which the solubility of vanillin in aqueous solution is only 12 grams / liter. The extracted vanillin needs to be re-dissolved, which is a complex process. SUMMARY

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a strain of Amycolatopsis thermoflava, and to realize efficient production of vanillin from ferulic acid using the strain.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows: A strain of Amycolatopsis thermoflava, the mutant strain of the Amycolatopsis thermoflava is H-1, which was preserved in the China General Microbiological Culture Collection Center on October 13, 2025, with a preservation number of CGMCC No.36166, a classification and naming of Amycolatopsis thermoflava, and a preservation address of Beijing, China.

[0007] Use of the strain of Amycolatopsis sp. in the preparation of vanillin.

[0008] Use of the strain of Amycolatopsis sp. in the preparation of vanillin by converting ferulic acid as a substrate.

[0009] A preparation for producing vanillin, which contains the strain of Amycolatopsis sp.

[0010] The preparation contains the culture, the suspension of the cultured bacteria or the fermentation liquor of the strain.

[0011] A method for producing vanillin by using the strain of Amycolatopsis sp. to ferment and culture with ferulic acid as a substrate.

[0012] Specifically, the strain of Amycolatopsis sp. is cultured on a solid culture medium containing 15-20 g / L agar at 30-50 DEG C for 48-72 hours; then the seed liquid is obtained by oscillatingly culturing in a seed culture medium at 30-50 DEG C for 24-36 hours after the culture; then the seed liquid is inoculated into a fermentation culture medium at an inoculation amount of 10% (volume ratio), and the fermentation is carried out at 37-45 DEG C, with the air input amount being 1-2 vvm and the stirring speed being controlled at 300-500 rpm; after the fermentation culture for 24 hours, 20-30 g / L of ferulic acid is added, and the fermentation culture is continued until the total fermentation time reaches 48-80 hours, and then the fermentation is ended, so as to obtain the fermentation liquor containing vanillin.

[0013] The optimal growth temperature of the strain is 37-45 DEG C, and the optimal conversion temperature is 45 DEG C.

[0014] The solid culture medium contains 4 g / L of glucose, 4 g / L of yeast powder, 10 g / L of malt extract powder and 15-20 g / L of agar powder, and the pH is 7.0-7.2; The seed culture medium contains 10 g / L of glucose, 10 g / L of peptone, 5 g / L of yeast powder, 5 g / L of beef extract and 2 g / L of sodium chloride, and the pH is 6.5-7.0; The fermentation culture medium contains 32 g / L of glucose, 8 g / L of yeast powder, 0.8 g / L of magnesium sulfate heptahydrate, 2 g / L of sodium chloride and 0.01 g / L of calcium chloride, and the pH is 6.5-7.0.

[0015] The application has the following advantages: The application screens a mutant strain of Amycolatopsis sp., and uses the strain to produce vanillin by taking ferulic acid as a substrate; the optimal conversion temperature of the strain is 45 DEG C, the solubility of vanillin at the temperature is more than 20 g / L, and the highest yield of vanillin obtained by using the strain reaches 21.6 g / L. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Comparison of growth ability of the Amycolatopsis thermoflava mutant H-1 at different temperatures.

[0017] Figure 2 Comparison of vanillin production ability of the Amycolatopsis thermoflava mutant H-1 at different temperatures.

[0018] Figure 3 Vanillin change curve during substrate conversion at different fermentation culture temperatures in the preparation process of the present application.

[0019] Figure 4 Solubility of vanillin in aqueous solution at different temperatures. DETAILED DESCRIPTION

[0020] The specific embodiments of the present application are further described in conjunction with the examples below, and it should be noted that the specific embodiments described herein are only for the purpose of illustration and explanation of the present application, and are not limited to the present application.

[0021] The experimental materials not specially indicated herein can be commercially available.

[0022] The ISP2 solid culture medium in the following examples contains: glucose 4 g / L, yeast powder 4 g / L, malt extract powder 10 g / L, agar powder 20 g / L, pH 7.0-7.2; The seed culture medium contains: glucose 10 g / L, protein peptone 10 g / L, yeast powder 5 g / L, beef extract 5 g / L, sodium chloride 2 g / L, pH 6.5-7.0; The fermentation culture medium contains: glucose 32 g / L, yeast powder 8 g / L, magnesium sulfate heptahydrate 0.8 g / L, sodium chloride 2 g / L, calcium chloride 0.01 g / L, pH 6.5-7.0.

[0023] Example 1 Isolation, mutagenesis, evolution and identification of Amycolatopsis thermoflava.

[0024] 1. Isolation The soil sample was obtained from Fenghuang Mountain in Laishan District of Yantai City. 1 gram of the soil sample was added into 50 ml of ISP2 medium (4 g / L glucose, 4 g / L yeast extract, 10 g / L malt extract, pH 7.0) and enriched at 45°C for 48 hours. Then, the sample was diluted and coated on the culture dish containing ISP2 solid medium (4 g / L glucose, 4 g / L yeast extract, 10 g / L malt extract, 15 g / L agar, pH 7.0), and cultured at 45°C for 3 days. After single colonies were formed, 200 colonies with actinomycete morphology were selected and inoculated into fermentation medium (32 g / L glucose, 8 g / L yeast extract, 0.8 g / L magnesium sulfate heptahydrate, 2 g / L sodium chloride, 0.01 g / L calcium chloride, pH 7.0). After 24 hours of culture at 45°C, 10 g / L ferulic acid was added for further culture for 24 hours. The yield of vanillin was determined, and the strain with the highest yield was selected.

[0025] 2. Identification The strain is gram-positive and aerobic, can grow at 50°C, can utilize xylose, arabinose, cellobiose, lactose and mannitol, and cannot utilize sucrose. The genomic GC content is 71.9%. The genomic DNA of the strain was extracted, and the 16S rDNA sequence was amplified using 27F / 1492R primers. The sequence of the 27F primer is 5'-AGAGTTTGATCCTGGCTCAG-3', and the sequence of the 1492R primer is 5'-TACGGTTACCTTGTTACGACTT-3'. The obtained DNA fragment was subjected to sequence determination, and then submitted to NCBI for Blast analysis. The results showed that the 16S rDNA sequence of the strain is 100% identical to the 16S rDNA sequence of Amycolatopsis thermoflava. Based on the above characteristics, the above strain with high vanillin yield is identified as Amycolatopsis thermoflava.

[0026] 16S rDNA sequence 3. Mutagenesis The initially selected strain was inoculated into fermentation medium and cultured at 45°C on a shaker until the mid-log phase. After washing with physiological saline, the bacteria were suspended to 10 8 milliliter, 5 milliliters of which were inoculated into a 9-centimeter petri dish at a distance of 30 centimeters from the ultraviolet lamp for 15 seconds, 30 seconds, 1 minute, and 5 minutes, respectively. 100 microliters of the mutagenized bacteria obtained from each different mutagenesis condition were inoculated onto ISP2 solid medium and cultured at 45°C in the dark for 7-8 days. The single colonies were selected and inoculated into fermentation medium, and 10 grams of ferulic acid per liter was added after 24 hours of culture at 45°C on a shaker. The vanillin yield was determined, and the strain with the highest yield was selected for the next evolution.

[0027] 4. Evolution The strain obtained by mutagenesis screening was inoculated into evolution medium No. 1 (32 grams of glucose, 8 grams of yeast powder, 0.8 grams of magnesium sulfate heptahydrate, 2 grams of sodium chloride, 0.01 grams of calcium chloride, 6 grams of ferulic acid, pH 7.0), and cultured at 45°C on a shaker for 24 hours. Then, the bacteria were inoculated into fresh evolution medium No. 1 at a concentration of 1%, and cultured again at 45°C on a shaker for 24 hours. This process was repeated 5 times. Then, the bacteria were inoculated into evolution medium No. 2 (32 grams of glucose, 8 grams of yeast powder, 0.8 grams of magnesium sulfate heptahydrate, 2 grams of sodium chloride, 0.01 grams of calcium chloride, 12 grams of ferulic acid, pH 7.0), and the operation was repeated 5 times again. Then, the bacteria were inoculated into evolution medium No. 3 (32 grams of glucose, 8 grams of yeast powder, 0.8 grams of magnesium sulfate heptahydrate, 2 grams of sodium chloride, 0.01 grams of calcium chloride, 18 grams of ferulic acid, pH 7.0), and the operation was repeated 5 times again. Then, the bacteria were inoculated into evolution medium No. 4 (32 grams of glucose, 8 grams of yeast powder, 0.8 grams of magnesium sulfate heptahydrate, 2 grams of sodium chloride, 0.01 grams of calcium chloride, 24 grams of ferulic acid, pH 7.0), and the operation was repeated 5 times again. Then, the bacteria were inoculated into evolution medium No. 5 (32 grams of glucose, 8 grams of yeast powder, 0.8 grams of magnesium sulfate heptahydrate, 2 grams of sodium chloride, 0.01 grams of calcium chloride, 30 grams of ferulic acid, pH 7.0), and the operation was repeated 5 times again. The final bacteria were inoculated onto ISP2 solid medium in a petri dish and cultured at 45°C for 4 days. After single colonies were formed, the colonies were selected and inoculated into fermentation medium (32 grams of glucose, 8 grams of yeast powder, 0.8 grams of magnesium sulfate heptahydrate, 2 grams of sodium chloride, 0.01 grams of calcium chloride, pH 7.0). After 24 hours of culture, 30 grams of ferulic acid was added, and the bacteria were cultured at 45°C on a shaker for 72 hours. The vanillin yield was determined, and the strain with the highest yield was selected, which was named H-1 in the laboratory.

[0028] Amycolatopsis thermoflava is H-1, which was preserved in China General Microbiological Culture Collection Center on October 13, 2025, and the preservation number is CGMCC No. 36166, the classification and naming is Amycolatopsis thermoflava, and the preservation address is Beijing, China.

[0029] Example 2 Using Amycolatopsis thermoflava H-1 to transform ferulic acid to produce vanillin under the culture condition of 37℃.

[0030] Amycolatopsis thermoflava H-1 preserved in a -80℃ ultra-low temperature refrigerator was streaked on ISP2 solid culture medium (glucose 4 g / L, yeast extract 4 g / L, malt extract powder 10 g / L, agar powder 15 g / L, pH 7.2), and cultured at 37℃ for 2-3 days. After the culture medium surface was covered with yellow spores, the spores were collected from the plate and inoculated into conical flasks containing 50 ml of seed culture medium (glucose 10 g / L, protein peptone 10 g / L, yeast extract 5 g / L, beef extract 5 g / L, sodium chloride 2 g / L, pH 7) at an inoculation amount of 10% (v / v). The seed culture liquid was cultured at 37℃ and 220 rpm for 24 hours to obtain a seed liquid. The cultured seed culture liquid was inoculated into a fermentation tank containing fresh fermentation medium (glucose 32 g / L, yeast extract 8 g / L, magnesium sulfate heptahydrate 0.8 g / L, sodium chloride 2 g / L, calcium chloride 0.01 g / L, pH 7) at an inoculation amount of 10% (v / v). The fermentation temperature was controlled at 37℃, the air input amount was 1 vvm, the stirring speed was adjusted to 500 rpm, and the pH and dissolved oxygen were not controlled during fermentation. After 24 hours of fermentation, the optical density value of the strain was measured at 600 nm (see Figure 1 ). At this time, 24 g / L of substrate ferulic acid was added, and the fermentation was continued. After 72 hours of fermentation, the fermentation was stopped, and the yield concentration of vanillin in the system was detected by high performance liquid chromatography (see Figure 2 ).

[0031] Example 3 Using Amycolatopsis thermoflava H-1 to transform ferulic acid to produce vanillin under the culture condition of 42℃.

[0032] The Amycolatopsis thermoflava H-1 preserved in an ultra-low temperature refrigerator at -80°C was streaked on ISP2 solid medium (glucose 4 g / L, yeast extract 4 g / L, malt extract powder 10 g / L, agar powder 15 g / L, pH 7.2) and cultured at 42°C for 3 days. After the culture medium was covered with yellow spores, the spores were collected from the plate and inoculated into conical flasks containing 50 mL of seed culture medium (glucose 10 g / L, protein peptone 10 g / L, yeast extract 5 g / L, beef extract 5 g / L, sodium chloride 2 g / L, pH 7) at 42°C and 220 rpm for 24 hours to obtain a seed solution. The cultured seed solution was inoculated into a fermentation tank containing fresh fermentation medium (glucose 32 g / L, yeast extract 8 g / L, magnesium sulfate heptahydrate 0.8 g / L, sodium chloride 2 g / L, calcium chloride 0.01 g / L, pH 7) at a 10% (v / v) inoculation amount, and the fermentation temperature was controlled at 42°C, the air input amount was 1 vvm, the stirring speed was adjusted to 500 rpm, and the pH and dissolved oxygen were not controlled during the fermentation. After 24 hours of fermentation, the optical density of the strain was measured at 600 nm using a spectrophotometer (see Figure 1 ). At this time, 24 g / L of the substrate ferulic acid was added, and the fermentation was continued. After a total fermentation time of 72 hours, the fermentation was terminated, and the yield concentration of vanillin in the system was detected by high-performance liquid chromatography (see Figure 2 ).

[0033] Example 4 Amycolatopsis thermoflava H-1 was used to transform ferulic acid to produce vanillin under a 45°C culture condition.

[0034] The thermophilic *Amyces flavovirens* H-1, stored at -80℃, was streaked onto ISP2 solid medium (4 g / L glucose, 4 g / L yeast extract, 10 g / L malt extract, 15 g / L agar, pH 7.2) and incubated at 45℃ for 3 days. Once the medium surface was covered with yellow spores, the spores were collected from the plates and inoculated into Erlenmeyer flasks containing 50 mL of seed culture medium (10 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 5 g / L beef extract, 2 g / L sodium chloride, pH 7) and incubated at 45℃. Seed culture was obtained by shaking at 220 rpm for 24 hours. The prepared seed culture was then inoculated at a rate of 10% (v / v) into fermenters containing fresh fermentation medium (32 g / L glucose, 8 g / L yeast extract, 0.8 g / L magnesium sulfate heptahydrate, 2 g / L sodium chloride, 0.01 g / L calcium chloride, pH 7). The fermentation temperature was controlled at 45°C, the air flow rate at 1 vvm, and the stirring speed at 500 rpm. pH and dissolved oxygen were not controlled during fermentation. After 24 hours of fermentation, the optical density of the strain was measured at 600 nm using a spectrophotometer (see [reference]). Figure 1 At this point, 24 g / L of the substrate ferulic acid was added, and fermentation continued until the total fermentation time reached 72 hours, at which point fermentation was terminated. The yield and concentration of vanillin in the system were detected by high-performance liquid chromatography (see [link to relevant documentation]). Figure 2 ).

[0035] Comparative Example 1 Vanillin was produced by converting ferulic acid using Amycolatopsis thermoflava H-1 under different temperature (30℃) culture conditions.

[0036] The thermophilic *Amyces flavonoides* mutant strain H-1, stored at -80℃, was streaked onto ISP2 solid medium (4 g / L glucose, 4 g / L yeast extract, 10 g / L malt extract, 15 g / L agar powder, pH 7.2) and incubated at 30℃ for 3 days. Once the medium surface was covered with yellow spores, the spores were collected from the plates and inoculated into 50 mL of seed culture medium (10 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 5 g / L beef extract). Seed culture was obtained by shaking a conical flask containing 2 g / L sodium chloride (pH 7) at 30°C and 220 rpm for 24 hours. The seed culture was then inoculated at a volume ratio of 10% into fermenters containing fresh fermentation medium (32 g / L glucose, 8 g / L yeast extract, 0.8 g / L magnesium sulfate heptahydrate, 2 g / L sodium chloride, 0.01 g / L calcium chloride, pH 7). The fermentation temperature was controlled at 30°C, the air flow rate at 1 vvm, and the stirring speed at 500 rpm. pH and dissolved oxygen were not controlled during fermentation. After 24 hours of fermentation, the optical density of the strain was measured at 600 nm using a spectrophotometer (see [reference]). Figure 1 At this point, 24 g / L of the substrate ferulic acid was added, and fermentation continued until the total fermentation time reached 72 hours, at which point fermentation was terminated. The yield and concentration of vanillin in the system were detected by high-performance liquid chromatography (see [link to relevant documentation]). Figure 2 ).

[0037] Comparative Example 2 Vanillin was produced by converting ferulic acid using Amycolatopsis thermoflava H-1 under culture conditions at 50℃.

[0038] The thermophilic *Amyces flavonoides* H-1, stored at -80℃, was streaked onto ISP2 solid medium (4 g / L glucose, 4 g / L yeast extract, 10 g / L malt extract, 15 g / L agar, pH 7.2) and incubated at 50℃ for 3 days. Once the medium surface was covered with yellow spores, the spores were collected from the plates and inoculated into 50 mL of seed culture medium (10 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 5 g / L beef extract, chlorine...). Seed culture was obtained by shaking a conical flask containing 2 g / L sodium chloride (pH 7) at 50°C and 220 rpm for 24 hours. The seed culture was then inoculated at a 10% (v / v) inoculation rate into fermenters containing fresh fermentation medium (32 g / L glucose, 8 g / L yeast extract, 0.8 g / L magnesium sulfate heptahydrate, 2 g / L sodium chloride, 0.01 g / L calcium chloride, pH 7). The fermentation temperature was controlled at 50°C, the air flow rate at 1 vvm, and the stirring speed at 500 rpm. pH and dissolved oxygen were not controlled during fermentation. After 24 hours of fermentation, the optical density of the strain was measured at 600 nm using a spectrophotometer and found to be 9.7 (see [reference needed]). Figure 1 At this point, 24 g / L of the substrate ferulic acid was added, and fermentation continued until the total fermentation time reached 72 hours, at which point fermentation was terminated. The yield and concentration of vanillin in the system were detected by high-performance liquid chromatography (see [link to relevant documentation]). Figure 2 ).

[0039] Depend on Figure 1 It is evident that the growth ability of the thermophilic adenophora mutant strain H-1 varies at different temperatures. The strain grows vigorously at 37-45℃, with the highest optical density reaching 18.2 at 45℃. While the strain also grows further at 30℃, its growth is not particularly strong, and at 50℃, its growth is significantly slow. Figure 2 The vanillin production capacity of the thermophilic adenophora mutant strain H-1 at different temperatures was significantly enhanced at 37-45℃, with a yield of 21.6 g / L at 45℃. However, at 30℃, the vanillin production capacity of the strain was only at the existing level, and at 50℃, the vanillin production capacity of the strain was significantly reduced.

[0040] Therefore, the suitable temperature for vanillin production by the strain of the present invention is 37-45℃, and the optimal temperature is 45℃.

[0041] Example 5 Vanillin was produced by converting ferulic acid using Amycolatopsis thermoflava H-1 at 45℃ under different culture times.

[0042] The thermophilic *Amyces flavovirens* H-1, stored at -80℃, was streaked onto ISP2 solid medium (4 g / L glucose, 4 g / L yeast extract, 10 g / L malt extract, 15 g / L agar, pH 7.2) and incubated at 45℃ for 3 days. Once the medium surface was covered with yellow spores, the spores were collected from the plates and inoculated into Erlenmeyer flasks containing 50 mL of seed culture medium (10 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 5 g / L beef extract, 2 g / L sodium chloride, pH 7) and incubated at 45℃. Seed culture was obtained by shaking at 220 rpm for 24 hours. The prepared seed culture was then inoculated at a rate of 10% (v / v) into fermenters containing fresh fermentation medium (32 g / L glucose, 8 g / L yeast extract, 0.8 g / L magnesium sulfate heptahydrate, 2 g / L sodium chloride, 0.01 g / L calcium chloride, pH 7). The fermentation temperature was controlled at 45°C, the air flow rate at 1 vvm, and the stirring speed at 500 rpm. pH and dissolved oxygen were not controlled during fermentation. After 24 hours of fermentation, ferulic acid was added, and fermentation continued. The yield and concentration of vanillin in the fermentation broth were measured at different total fermentation times (see [reference]). Figure 3 ).

[0043] Depend on Figure 3 It is evident that the yield of vanillin varies with different total fermentation times, reaching a maximum of 21.6 g / L when the total fermentation time reaches 72 h.

[0044] The relative solubility of the products prepared using the examples and comparative examples is shown in the figure. Figure 4 visible.

Claims

1. A thermophilic amylopectin bacterium, characterized by: The thermophilic amycolatopsis thermoflava is identified as H-1, which was deposited on October 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36166 and classified as Amycolatopsis thermoflava. The deposit address is Beijing, China.

2. The use of the thermophilic adenophora stricta bacterium described in claim 1 in the preparation of vanillin.

3. The application of *Amycium xanthophyte* according to claim 2 in the preparation of vanillin, characterized in that: Application of the thermophilic xanthophylloides in the conversion of ferulic acid as a substrate to prepare vanillin.

4. A formulation for producing vanillin, characterized in that: The formulation contains the thermophilic adenophorauric acid bacterium described in claim 1.

5. The formulation for producing vanillin according to claim 4, characterized in that: The formulation contains a culture, culture suspension or fermentation broth of the thermophilic adenophora stricta.

6. A method for producing vanillin, characterized in that: Vanillin can be obtained by fermentation culture using ferulic acid as a substrate with thermophilic adenophora as described in claim 1.

7. The method for producing vanillin according to claim 6, characterized in that: The thermophilic adenophora stricta bacteria described in claim 1 were cultured on a solid medium containing 15-20 g / L agar at 30-50°C for 48-72 hours. After culture, the culture was transferred to a seed culture medium at 30-50°C with shaking for 24-36 hours to obtain a seed culture. After culturing, the seed culture was inoculated at a volume ratio of 10% into a fermentation medium at 37-45°C with an air flow rate of 1-2 vvm and a stirring speed controlled at 300-500 rpm. After 24 hours of fermentation, 20-30 g / L of ferulic acid substrate was added, and fermentation continued until the total fermentation time reached 48-80 hours, at which point fermentation was terminated, yielding a fermentation broth containing vanillin.

8. The method for producing vanillin according to claim 7, characterized in that: The solid culture medium contains: 4 g / L glucose, 4 g / L yeast powder, 10 g / L malt extract powder, 15-20 g / L agar powder, and a pH of 7.0-7.2; The seed culture medium contains: 10 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 5 g / L beef extract, 2 g / L sodium chloride, and a pH of 6.5–7.

0. The fermentation medium contains 32 g / L glucose, 8 g / L yeast extract, 0.8 g / L magnesium sulfate heptahydrate, 2 g / L sodium chloride, and 0.01 g / L calcium chloride, with a pH of 6.5–7.0.

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