Application of lactobacillus plantarum in fermentation production of urolithin A

Abstract

The invention discloses an application of lactobacillus plantarum in fermentation production of urolithin A. The strain of the lactobacillus plantarum is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC AB 2013128, the lactobacillus plantarum can convert ellagic acid to generate the urolithin A, and a new way is provided for preparation of the urolithin A. The invention further provides a method for producing the urolithin A through fermentation, and the yield of the urolithin A can be effectively increased.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and provides an application of Lactobacillus plantarum in producing urolithin A by fermentation. Background Art

[0002] Urolithin A (UA) is a metabolite produced by the intestinal flora from the conversion of ellagic acid (EA). 13 H8O4, relative molecular mass is 228.2. Many studies have shown that UA has excellent antioxidant activity, and the mechanism is as follows: (I) It directly removes free radicals by capturing and neutralizing reactive oxygen species such as peroxide radicals and superoxide radicals, thereby reducing cellular oxidative stress (An increased autophagic flux contributes to the anti-inflammatory potential of urolithinA in macrophages. BBA Gen. Subj. 2018, 1862, 61–70.); (II) It activates the Nrf2 antioxidant pathway and upregulates the expression of various antioxidant enzymes such as glutathione peroxidase and superoxide dismutase, thereby enhancing the cellular antioxidant defense capacity (Crosstalk of reactive oxygen species and NF-kappaB signaling. Cell Res. 2011, 21, 103–115.); (III) It inhibits the activity of enzymes involved in the generation of ROS, thereby reducing the occurrence of oxidative stress and protecting cells from oxidative damage (Effects ofUrolithinA on MitochondrialParameters in a Cellular Model ofEarlyAlzheimer Disease. Int. J. Mol. Sci. 2021, 22, 8333.). However, the intestinal flora of different human bodies is specific, and the metabolites produced vary significantly between individuals, which can be divided into three metabolic types: "phenotype 0" (no Urolithins); "phenotype A" (Uro-A); "phenotype B". At the same time, UA is easily damaged during the gastrointestinal digestion stage; studies have shown that the metabolic type is in dynamic change. Long-term intake of EA-rich foods or high-dose EA can convert some people with metabolic type 0 into metabolic type A or B.

[0003] At present, the industrial production of urolithin A mainly utilizes chemical methods, but this method has a long reaction time, high cost, high energy consumption, and expensive product price. Fermentation synthesis methods of urolithin A are reported less. Therefore, the art needs to develop suitable strains and fermentation production processes to make up for the lack of urolithin A fermentation synthesis technology and provide more options for the preparation of urolithin A. Summary of the invention

[0004] In view of the deficiencies in the prior art, the first aspect of the present invention provides a Lactobacillus plantarum obtained by screening, which can be used for fermentation production of urolithin A. The second aspect of the present invention also provides a method for fermenting urolithin A by using the Lactobacillus plantarum by optimizing process conditions.

[0005] In a first aspect, the present invention provides an application of Lactobacillus plantarum in the fermentation production of urolithin A. The Lactobacillus plantarum has a preservation number of CCTCCAB 2013128 and is preserved in the China Center for Type Culture Collection.

[0006] In a second aspect, the present invention also provides a method for producing urolithin A by fermentation, the method comprising:

[0007] Step 1: Activate the aforementioned Lactobacillus plantarum strains:

[0008] Step 2: Pick up the activated single colony and inoculate it into the seed culture medium to obtain seed liquid;

[0009] Step 3: inoculating the seed liquid into a fermentation medium containing ellagic acid for fermentation culture;

[0010] Step 4: Centrifuge the fermentation broth, separate the supernatant, and obtain the urolithin A extract.

[0011] In some specific embodiments, in step 1, the aforementioned Lactobacillus plantarum strain is activated by streaking on an MRS plate.

[0012] In some specific embodiments, in step 2, the component formula of the seed culture medium is as follows:

[0013] Rice flour 4g±10%, yeast powder 1g±10%, molasses 1g±10%, sodium acetate 1.2g±10%, copper sulfate 0.02g±10%, magnesium sulfate 0.05g±10%, malt powder 2g±10%, dipotassium hydrogen phosphate, 0.5g±10%, pH 6.7±0.2.

[0014] In some specific embodiments, in step 2, the component formula of the seed culture medium is as follows:

[0015] Rice flour 4g±5%, yeast powder 1g±5%, molasses 1g±5%, sodium acetate 1.2g±5%, copper sulfate 0.02g±5%, magnesium sulfate 0.05g±5%, malt powder 2g±5%, dipotassium hydrogen phosphate, 0.5g±5%, pH 6.7±0.2.

[0016] In some specific embodiments, in step 2, the component formula of the seed culture medium is as follows:

[0017] Rice flour 4g±3%, yeast powder 1g±3%, molasses 1g±3%, sodium acetate 1.2g±3%, copper sulfate 0.02g±3%, magnesium sulfate 0.05g±3%, malt powder 2g±3%, dipotassium hydrogen phosphate, 0.5g±3%, pH 6.7±0.2.

[0018] In some specific embodiments, in step 2, the component formula of the seed culture medium is as follows:

[0019] Rice flour 4g±2%, yeast powder 1g±2%, molasses 1g±2%, sodium acetate 1.2g±2%, copper sulfate 0.02g±2%, magnesium sulfate 0.05g±2%, malt powder 2g±2%, dipotassium hydrogen phosphate, 0.5g±2%, pH 6.7±0.2.

[0020] In some specific embodiments, in step 2, the component formula of the seed culture medium is as follows:

[0021] Rice flour 4g±1%, yeast powder 1g±1%, molasses 1g±1%, sodium acetate 1.2g±1%, copper sulfate 0.02g±1%, magnesium sulfate 0.05g±1%, malt powder 2g±1%, dipotassium hydrogen phosphate, 0.5g±1%, pH 6.7±0.1.

[0022] In some specific embodiments, in step 2, the component formula of the seed culture medium is as follows:

[0023] Rice flour 4g, yeast powder 1g, molasses 1g, sodium acetate 1.2g, copper sulfate 0.02g, magnesium sulfate 0.05g, malt powder 2g, dipotassium hydrogen phosphate 0.5g, pH 6.7.

[0024] In some specific embodiments, in step 3, the inoculation amount of the seed solution is 0.1-10%.

[0025] In some specific embodiments, in step 3, the inoculation amount of the seed solution is 1-5%.

[0026] In some specific embodiments, in step 3, the fermentation culture conditions are: culture temperature: 37±0.5°C, culture time: 80±5h, culture pH 7.1±0.1.

[0027] In some specific embodiments, in step 3, the fermentation culture conditions are: culture temperature: 37±0.3°C, culture time: 80±3h, culture pH 7.1±0.1.

[0028] In some specific embodiments, in step 3, the fermentation culture conditions are: culture temperature: 37±0.2°C, culture time: 80±2h, culture pH 7.1±0.05.

[0029] In some specific embodiments, in step 3, the fermentation culture conditions are: culture temperature: 37±0.1°C, culture time: 80±1h, culture pH 7.1±0.05.

[0030] In some specific embodiments, in step 3, the fermentation culture conditions are: culture temperature: 37° C., culture time: 80 h, culture pH 7.1.

[0031] In a third aspect, the present invention further provides a seed culture medium, wherein the component formula of the seed culture medium is as follows:

[0032] Rice flour 4g±10%, yeast powder 1g±10%, molasses 1g±10%, sodium acetate 1.2g±10%, copper sulfate 0.02g±10%, magnesium sulfate 0.05g±10%, malt powder 2g±10%, dipotassium hydrogen phosphate 0.5g±10%, pH 6.7±0.2.

[0033] In some specific embodiments, the seed culture medium has the following components:

[0034] Rice flour 4g±5%, yeast powder 1g±5%, molasses 1g±5%, sodium acetate 1.2g±5%, copper sulfate 0.02g±5%, magnesium sulfate 0.05g±5%, malt powder 2g±5%, dipotassium hydrogen phosphate 0.5g±5%, pH 6.7±0.2.

[0035] In some specific embodiments, the seed culture medium has the following components:

[0036] Rice flour 4g±3%, yeast powder 1g±3%, molasses 1g±3%, sodium acetate 1.2g±3%, copper sulfate 0.02g±3%, magnesium sulfate 0.05g±3%, malt powder 2g±3%, dipotassium hydrogen phosphate 0.5g±3%, pH 6.7±0.2.

[0037] In some specific embodiments, the seed culture medium has the following components:

[0038] Rice flour 4g±2%, yeast powder 1g±2%, molasses 1g±2%, sodium acetate 1.2g±2%, copper sulfate 0.02g±2%, magnesium sulfate 0.05g±2%, malt powder 2g±2%, dipotassium hydrogen phosphate 0.5g±2%, pH 6.7±0.2.

[0039] In some specific embodiments, the seed culture medium has the following components:

[0040] Rice flour 4g±1%, yeast powder 1g±1%, molasses 1g±1%, sodium acetate 1.2g±1%, copper sulfate 0.02g±1%, magnesium sulfate 0.05g±1%, malt powder 2g±1%, dipotassium hydrogen phosphate 0.5g±1%, pH 6.7±0.1.

[0041] In some specific embodiments, the seed culture medium has the following components:

[0042] Rice flour 4g, yeast powder 1g, molasses 1g, sodium acetate 1.2g, copper sulfate 0.02g, magnesium sulfate 0.05g, malt powder 2g, dipotassium hydrogen phosphate 0.5g, pH 6.7.

[0043] Beneficial effects of the present invention: The present invention unexpectedly discovered a strain of Lactobacillus plantarum that can convert ellagic acid to produce urolithin A by screening a large number of existing strains, providing a new way for the preparation of urolithin A. The present invention also provides a method for fermenting urolithin A using the Lactobacillus plantarum, which can effectively increase the yield of urolithin A by optimizing the seed culture medium and fermentation process conditions, and the yield can reach 29.58 μM. The seed culture medium for culturing the Lactobacillus plantarum provided by the present invention can significantly increase the growth rate and viability of the bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Results of strain screening test.

[0045] Figure 2 Comparison results of the total number of Lactobacillus plantarum colonies in each culture medium after 8 hours of fermentation.

[0046] Figure 3 Comparison results of the total colonies of Lactobacillus plantarum in each culture medium after 10 hours of fermentation.

[0047] Figure 4 This is a comparison of the total number of Lactobacillus plantarum colonies in each culture medium at the end of fermentation.

[0048] Figure 5 The results of the single-factor experiment show the effect of culture temperature on the production of urolithin A.

[0049] Figure 6This is the result of the effect of culture time on urolithin A production in a single factor experiment.

[0050] Figure 7 The results are shown in the single factor experiment showing the effect of culture pH on urolithin A production.

[0051] Figure 8 Fermentation product detection chart, where part A shows the standard sample detection chart of ellagic acid and urolithin A, and part B shows the fermentation product detection chart. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials involved in the present invention can be obtained from commercial channels unless otherwise specified.

[0054] Example 1

[0055] 1 Materials and methods

[0056] 1.1 Materials

[0057] Strains: The strains used in this example were purchased from a strain collection center, as shown in Table 1 below:

[0058] Table 1 Detailed information of strains

[0059]

[0060]

[0061] Reagents: Culture medium components (glucose, peptone, etc.) were from Sinopharm Group; Urolithin A (HPLC ≥ 99%, CAS # 1143-70-0) standard, Aladdin Co., Ltd.; Chloramphenicol, Sangon Biotech (Shanghai) Co., Ltd.; Dichloromethane and acetone were analytical grade, methanol was chromatographic grade, and other reagents were from Sinopharm Group.

[0062] 1.2 Instruments and Equipment

[0063] Nanodrop 2000c ultra-micro-nucleic acid and protein quantification instrument, Thermo Corporation, USA; refrigerated centrifuge, Sigma, Germany; spectrophotometer DU-800, Beckman, USA; biochemical incubator, Shanghai Boxun Industrial Co., Ltd.; Waters e2695 high-performance liquid chromatography, Waters Technology Co., Ltd. (Shanghai, China)

[0064] 1.3 Initial screening of strains

[0065] Each strain was fermented and the product was determined using the following method, the steps are as follows:

[0066] A. Activation of strains: Activate the Lactobacillus plantarum stored in a -80°C refrigerator by streaking on an MRS plate;

[0067] B. Seed culture: Pick a single colony after activation and inoculate it into MRS seed culture medium to obtain seed liquid;

[0068] C. Fermentation culture: inoculate the seed liquid into the fermentation medium supplemented with EA for fermentation culture;

[0069] D. Separation and extraction: Centrifuge the fermentation broth and the resulting supernatant is the crude UA extract.

[0070] The results of the initial screening test of the strains are as follows Figure 1 As shown. The initial screening results showed that most strains did not have the effect of converting ellagic acid (EA) to urolithin A (UA), for example, the UA production of strains No. 2, 3, 9, 12, and 14 was less than 1 μM; most strains had a weak effect of converting ellagic acid (EA) to urolithin A (UA), for example, the UA production of strains No. 1, 4, 5, 6, 7, 10, 11, and 13 was less than 4 μM; however, it was unexpectedly found that the strain No. 8 had a strong ability to convert ellagic acid (EA) to urolithin A (UA), and its UA production reached about 10 μM. Therefore, this strain No. 8 was selected as the research object for the development of proliferation culture medium and process optimization.

[0071] 1.4 Optimization of fermentation medium of Lactobacillus plantarum CCTCCAB 2013128

[0072] Different fermentation media (as shown in Table 2) were used to test the culture effect of Lactobacillus plantarum.

[0073] Table 2 Screening of different fermentation media

[0074]

[0075] Fermentation medium development test results such as Figure 2 to Figure 4 As shown. Figure 2 to Figure 4, at 8h, 10h and the end of fermentation, by comparing the data of each experimental group, it can be seen that the culture medium of experimental group 1 can effectively proliferate plant lactobacillus, and its bacterial growth rate and bacterial viability reach more than three times of experimental group 2 and experimental group 3, while the fermentation culture effect of plant lactobacillus in experimental group 4 and experimental group 5 is crossed. And it is also found through microscopic examination that the bacterial cell obtained by the culture medium of experimental group 1 is obviously more robust than other experimental groups. The strain obtained by culturing the optimized culture medium is used to optimize the fermentation process conditions in the next step.

[0076] 1.5 Optimization of fermentation conditions of Lactobacillus plantarum

[0077] Using the strain obtained from the optimization of the fermentation medium in 1.4 above, further process condition optimization experiments were carried out.

[0078] (1) Single factor experiment

[0079] The UA mass concentration was used as the evaluation index to optimize the culture temperature, culture time and initial pH. The culture time was fixed at 60h, the initial pH was 6.9, and the optimized culture temperature levels were 35, 36, 37, 38, and 39°C; the initial pH was fixed at 6.9, the culture temperature was 37°C, and the optimized culture time levels were 40, 50, 60, 70, and 80h; the culture time was fixed at 60h, the culture temperature was 37°C, and the optimized initial pH levels were 6.5, 6.7, 6.9, 7.1, and 7.3.

[0080] UA Detection Method

[0081] HPLC analysis conditions: Chromatographic separation and analysis of samples was performed on a 250×4.6 mm, 5 μm particle size ZORBAX SB-C18 column (Agilent, USA). Acetonitrile and 1% formic acid were used as the mobile phase, the injection volume was 20 μL, and the flow rate was 1 mL / min. Recording was performed at 305 nm. The elution gradient was: 0-15 minutes, 0-20% acetonitrile; 15-20 minutes, 20-70% acetonitrile; 20-21 minutes, 70-95% acetonitrile; 21-24 minutes, 95-100% acetonitrile; 24-25 minutes, 100-20% acetonitrile.

[0082] The results of the single factor experiment are as follows Figure 5 to Figure 7 As shown. Figure 4 As shown in the figure, the production of urolithin A first increased and then decreased with the increase of temperature. When the fermentation temperature was 36°C, 37°C, and 38°C, the mass concentration of urolithin A was at a relatively high level (15.62-18.22 μM). When the culture time was 70 h and 80 h, the mass concentration of urolithin A was the highest (20.57 μM). When the culture pH was 7.1, the mass concentration of urolithin A was the highest, reaching 19.85 μM.

[0083] (2) Response surface experiment

[0084] A three-factor three-level response surface experiment was conducted with UA mass concentration as the response value to optimize the three process parameters of culture temperature, culture time and initial pH to determine the optimal culture method for Lactobacillus. The experimental design is shown in Table 3.

[0085] Table 3 Parameter settings for response surface experiment

[0086]

[0087] On the basis of the single factor experiment, the yield of urolithin A was taken as the response value, the culture temperature (A), culture time (B), and initial pH (C) were selected as three factors, and the Box-Behnken design scheme was used to conduct a three-factor three-level response surface experiment. The results are listed in Table 3. The results show that the experimental group 12 can achieve 29.58μM. In addition, after multiple regression fitting, the regression equation is: UA = 19.03 + 2.13 × A + 2.75 × B - 0.905 × C + 0.875 × AB - 0.308AC + 2.00 × BC - 4.05 × A 2 +4.98×B 2 +0.58×C 2 .

[0088] Through software analysis, we can know that the correlation coefficient R of this model is 2 =0.918, close to 1, indicating that the data has a high degree of fitting and reliability. Therefore, the culture temperature: 37°C, culture time: 80h, culture pH: 7.1 were selected as the subsequent fermentation process.

[0089] Table 4 Response surface design scheme and experimental results

[0090]

[0091]

[0092] Example 2 Fermentation production of urolithin A

[0093] The strain of Lactobacillus plantarum (preservation number is CCTCCAB 2013128) is fermented by the following method, the steps are as follows:

[0094] 1. Activation of strains: Activate the Lactobacillus plantarum kept in a -80°C refrigerator by streaking on an MRS plate.

[0095] 2. Seed culture: Pick the activated single colony and inoculate it into the seed culture medium, and obtain the seed solution after 24 hours of culture; the component formula of the seed culture medium is: 4g rice flour, 1g yeast powder, 1g molasses, 1.2g sodium acetate, 0.02g copper sulfate, 0.05g magnesium sulfate, 2g malt powder, 0.5g dipotassium hydrogen phosphate, pH 6.7.

[0096] 3. Fermentation culture: Inoculate the seed liquid at an inoculum of 1.5% into a fermentation medium supplemented with ellagic acid for fermentation culture; the fermentation culture conditions are: culture temperature: 37° C., culture time: 80 h, culture pH 7.1.

[0097] 4. Separation and extraction: Centrifuge the fermentation broth (10,000 × g for 15 min) and the resulting supernatant is the UA extract.

[0098] The fermentation process products are shown in Figure Figure 8 shown. Figure 8 Part A shows the standard test graph of urolithin A and ellagic acid. It can be seen that the peak time of ellagic acid is 6-7 minutes, and the peak time of urolithin A is 9-10 minutes. The two can be completely distinguished. Figure 8 It can be seen from part B that after transformation by Lactobacillus plantarum of this example, ellagic acid is converted into a variety of metabolites, among which urolithin A is one of the main products. After separation, the yield of urolithin A in the UA extract was calculated to be 29.58 μM.

[0099] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of Lactobacillus plantarum in the fermentation production of urolithin A, characterized in that: The Lactobacillus plantarum has a preservation number of CCTCCAB 2013128 and is preserved in the China Center for Type Culture Collection.

2. A method for producing urolithin A by fermentation, the method comprising: Step 1: activating Lactobacillus plantarum strains, wherein the Lactobacillus plantarum strain has a deposit number of CCTCCAB 2013128 and is deposited in China Center for Type Culture Collection; Step 2: Pick a single activated colony and inoculate it into a seed culture medium to obtain a seed solution; Step 3: inoculating the seed liquid into a fermentation medium containing ellagic acid for fermentation culture; Step 4: Centrifuge the fermentation broth, separate the supernatant, and obtain the urolithin A extract.

3. The method according to claim 2, characterized in that In the step 1, the plant lactobacillus strain is activated by streaking on an MRS plate.

4. The method according to claim 2, characterized in that In step 2, the component formula of the seed culture medium is as follows: rice flour 4g±10%, yeast powder 1g±10%, molasses 1g±10%, sodium acetate 1.2g±10%, copper sulfate 0.02g±10%, magnesium sulfate 0.05g±10%, malt powder 2g±10%, dipotassium hydrogen phosphate, 0.5g±10%, pH 6.7±0.2; Preferably, in step 2, the component formula of the seed culture medium is as follows: rice flour 4g±5%, yeast powder 1g±5%, molasses 1g±5%, sodium acetate 1.2g±5%, copper sulfate 0.02g±5%, magnesium sulfate 0.05g±5%, malt powder 2g±5%, dipotassium hydrogen phosphate, 0.5g±5%, pH 6.7±0.2; Preferably, in step 2, the component formula of the seed culture medium is as follows: rice flour 4g±3%, yeast powder 1g±3%, molasses 1g±3%, sodium acetate 1.2g±3%, copper sulfate 0.02g±3%, magnesium sulfate 0.05g±3%, malt powder 2g±3%, dipotassium hydrogen phosphate, 0.5g±3%, pH 6.7±0.2; Further preferably, in step 2, the component formula of the seed culture medium is as follows: rice flour 4g±2%, yeast powder 1g±2%, molasses 1g±2%, sodium acetate 1.2g±2%, copper sulfate 0.02g±2%, magnesium sulfate 0.05g±2%, malt powder 2g±2%, dipotassium hydrogen phosphate, 0.5g±2%, pH 6.7±0.2; More preferably, in step 2, the component formula of the seed culture medium is as follows: rice flour 4g±1%, yeast powder 1g±1%, molasses 1g±1%, sodium acetate 1.2g±1%, copper sulfate 0.02g±1%, magnesium sulfate 0.05g±1%, malt powder 2g±1%, dipotassium hydrogen phosphate, 0.5g±1%, pH 6.7±0.

1.

5. The method according to claim 2, characterized in that In step 2, the component formula of the seed culture medium is as follows: 4g rice flour, 1g yeast powder, 1g molasses, 1.2g sodium acetate, 0.02g copper sulfate, 0.05g magnesium sulfate, 2g malt powder, 0.5g dipotassium hydrogen phosphate, pH 6.

7.

6. The method according to claim 2, characterized in that In step 3, the inoculation amount of the seed solution is 0.1-10%; preferably, the inoculation amount of the seed solution is 1-5%.

7. The method according to claim 2, characterized in that In step 3, the fermentation culture conditions are: culture temperature: 37±0.5°C, culture time: 80±5h, culture pH 7.1±0.1; Preferably, in step 3, the fermentation culture conditions are: culture temperature: 37±0.3°C, culture time: 80±3h, culture pH 7.1±0.1; Preferably, in step 3, the fermentation culture conditions are: culture temperature: 37±0.2°C, culture time: 80±2h, culture pH 7.1±0.05; Further preferably, in step 3, the fermentation culture conditions are: culture temperature: 37±0.1°C, culture time: 80±1h, culture pH 7.1±0.

05.

8. The method according to claim 2, characterized in that In step 3, the fermentation culture conditions are: culture temperature: 37° C., culture time: 80 h, and culture pH 7.

1.

9. A seed culture medium, the component formula of the seed culture medium is as follows: Rice flour 4g±10%, yeast powder 1g±10%, molasses 1g±10%, sodium acetate 1.2g±10%, copper sulfate 0.02g±10%, magnesium sulfate 0.05g±10%, malt powder 2g±10%, dipotassium hydrogen phosphate 0.5g±10%, pH 6.7±0.2; Preferably, the seed culture medium has the following component formula: rice flour 4g±5%, yeast powder 1g±5%, molasses 1g±5%, sodium acetate 1.2g±5%, copper sulfate 0.02g±5%, magnesium sulfate 0.05g±5%, malt powder 2g±5%, dipotassium hydrogen phosphate 0.5g±5%, pH 6.7±0.2; Preferably, the seed culture medium has the following component formula: rice flour 4g±3%, yeast powder 1g±3%, molasses 1g±3%, sodium acetate 1.2g±3%, copper sulfate 0.02g±3%, magnesium sulfate 0.05g±3%, malt powder 2g±3%, dipotassium hydrogen phosphate 0.5g±3%, pH 6.7±0.2; Further preferably, the component formula of the seed culture medium is as follows: rice flour 4g±2%, yeast powder 1g±2%, molasses 1g±2%, sodium acetate 1.2g±2%, copper sulfate 0.02g±2%, magnesium sulfate 0.05g±2%, malt powder 2g±2%, dipotassium hydrogen phosphate 0.5g±2%, pH 6.7±0.2; More preferably, the component formula of the seed culture medium is as follows: rice flour 4g±1%, yeast powder 1g±1%, molasses 1g±1%, sodium acetate 1.2g±1%, copper sulfate 0.02g±1%, magnesium sulfate 0.05g±1%, malt powder 2g±1%, dipotassium hydrogen phosphate 0.5g±1%, pH 6.7±0.

1.

10. The seed culture medium as claimed in claim 9, wherein the component formula of the seed culture medium is as follows: 4g rice flour, 1g yeast powder, 1g molasses, 1.2g sodium acetate, 0.02g copper sulfate, 0.05g magnesium sulfate, 2g malt powder, 0.5g dipotassium hydrogen phosphate, pH 6.7.

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