Saccharomyces cerevisiae and application thereof in production of beta-phenethyl alcohol

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses saccharomycetes and application thereof in production of beta-phenethyl alcohol. The preservation number of the saccharomycetes is GDMCC No: 64846, and the saccharomycetes is named as Pichia kudriavzevii YJQ-1, and the preservation number of the saccharomycetes is GDMCC No: 64846. The specific method for producing the beta-phenethyl alcohol by the saccharomycetes comprises the following steps: inoculating the saccharomycetes into a fermentation culture medium, and fermenting at 25-35 DEG C for 60-80 hours to obtain fermentation liquor containing the beta-phenethyl alcohol after the fermentation is finished. Researches show that the Pichia kudriavzevii YJQ-1 disclosed by the invention can tolerate 8% (v / v) of ethanol and 4 g / L of beta-phenethyl alcohol, and the production of the beta-phenethyl alcohol can be carried out by utilizing a plurality of carbon sources; especially, the fermentation liquor obtained through fermentation under the fermentation condition has high content of beta-phenethyl alcohol.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a yeast and application thereof in producing beta-phenylethanol. Background Art

[0002] β-Phenylethanol (2-Phenylethanol, 2-PE), also known as β-phenylethanol, 2-phenylethanol, has a molecular formula of C8H10O and is a fatty alcohol with a rose scent. β-Phenylethanol is widely used in food, medicine, and cosmetics because of its mild and refreshing rose scent. It is often used as a preservative in cosmetics, perfumes, and medicines, as a flavor ingredient in various herbal products, and as a substrate for synthesizing other flavors or drug compounds, such as 2-phenylethyl acetate and phenylacetaldehyde; β-Phenylethanol is used as a sensory enhancer in the food and beverage industry, for example, as a flavor regulator, it is added to the ingredients of various ice creams, candies, biscuits, puddings, gelatin, cigarettes, and chewing gum. β-Phenylethanol also has antibacterial properties and can inhibit the growth of Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Candida albicans, Candida dubliniensis, Saccharomyces cerevisiae, Kluyveromyces marxianus, etc. Therefore, β-Phenylethanol is also an ideal additive for preservatives, cleaning products, and personal care products. At 20°C, the saturated solubility of β-phenylethanol in water is 19 g / L. Compared with ethanol, β-phenylethanol has higher energy, density and lower hygroscopicity, so it is considered to be a promising gasoline substitute and can also be used as an advanced biofuel.

[0003] The global production of β-phenylethanol is about 10,000 tons per year, and it can be obtained by chemical synthesis, plant extraction and microbial fermentation. β-phenylethanol produced by microbial fermentation has been recognized as safe by many international organizations, including the Food and Drug Administration (FDA), the Flavor and Extract Manufacturers Association (FEMA), the Joint Expert Committee on Food Additives (JECFA) and the European Commission (COE). Therefore, β-phenylethanol obtained by microbial fermentation is currently the most promising method.

[0004] At present, the microorganisms that synthesize β-phenylethanol are mainly bacteria and fungi. The main bacteria are Escherichia coli, and the fungi are Aspergillus niger, Aspergillus oryzae and yeast. Studies have shown that most yeasts have the ability to produce β-phenylethanol, but this production of yeast is strain-specific, that is, there may be significant differences in the production capacity of β-phenylethanol between different strains of the same genus. In addition, the current yeasts have poor tolerance to products and ethanol. For example, β-phenylethanol can inhibit the growth of yeast cells, thereby preventing the biosynthesis of β-phenylethanol. Studies have found that 2g / L to 4g / L β-phenylethanol can inhibit the growth of many yeasts. The toxicity of β-phenylethanol to yeast is strain-specific. For example, Vibrio martensii CBS 600 is completely inhibited at a concentration of 2.6g / L of β-phenylethanol; in the presence of 2.5g / L β-phenylethanol, the growth rate of the Australian ethanol-resistant Staphylococcus aureus strain used to produce cider decreases by 75%. Once the reaction system reaches a given concentration of β-phenylethanol, the growth and vitality of the yeast will decrease. In addition, yeast also produces ethanol during metabolism, and ethanol and β-phenylethanol produce a synergistic effect, increasing its toxicity to cells.

[0005] In summary, most yeasts are currently intolerant to increased levels of the fermentation product β-phenylethanol, especially when the concentrations of β-phenylethanol and ethanol are increased. Therefore, developing a yeast that is tolerant to β-phenylethanol, especially to both β-phenylethanol and ethanol, has important application value. Summary of the invention

[0006] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a yeast and application thereof in producing β-phenylethanol.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention first provides a yeast, whose deposit number is GDMCC No: 64846, named

[0009] Named Pichia kudriavzevii YJQ-1.

[0010] The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 5, 2024; the collection address is No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0011] The invention provides a new yeast. Studies have shown that the Pichia kudriavzeviiYJQ-1 of the invention can tolerate 8% (v / v) ethanol and 4g / L beta-phenylethanol, and can utilize a variety of carbon sources to produce beta-phenylethanol; the yeast has important application value.

[0012] The Pichia kudriavzevii YJQ-1 (hereinafter referred to as YJQ-1) is milky white, irregularly round, small in size, with a rough, uneven surface and protruding villi.

[0013] The present invention also provides an application of the yeast in producing beta-phenylethanol.

[0014] Preferably, the specific method for producing β-phenylethanol by yeast is:

[0015] The yeast is inoculated into a fermentation medium, and fermentation is carried out at 25-35° C. for 60-80 hours. After the fermentation is completed, a fermentation liquid containing β-phenylethanol is obtained.

[0016] Preferably, before the yeast is inoculated into the fermentation medium, it is first added into the seed culture medium for activation to obtain yeast seed liquid.

[0017] Preferably, the volume addition amount of the yeast seed solution is 1% to 3% of the volume of the fermentation medium.

[0018] Most preferably, the volume of the yeast seed solution added is 2% of the volume of the fermentation medium.

[0019] Preferably, the seed culture medium is prepared with water and contains: 30-60% glucose

[0020] g / L,

[0021] KH2PO4 4~6g / L, MgSO4·7H2O 0.1~1g / L, yeast extract powder 3~6g / L.

[0022] Most preferably, the seed culture medium is prepared with water and contains: 50 g / L glucose, 5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 5 g / L yeast extract powder.

[0023] Preferably, the fermentation medium is prepared with water and contains: 80-150 g / L glucose, 0.5-5 g / L yeast extract powder, and 10-20 g / L L-Phe.

[0024] Preferably, the fermentation medium is prepared with water and contains: 90 g / L glucose, 1 g / L yeast extract powder, and 12 g / L L-Phe.

[0025] The inventors have shown in further research that in the process of fermenting β-phenylethanol using Pichia kudriavzevii YJQ-1, the composition of the fermentation medium plays an important role in the content of β-phenylethanol in the fermentation broth obtained by fermentation; the study shows that the fermentation broth obtained by fermenting Pichia kudriavzevii YJQ-1 in a fermentation medium containing glucose, yeast extract powder and L-Phe has a higher content of β-phenylethanol.

[0026] Preferably, the fermentation medium is prepared with water and further comprises a surfactant.

[0027] Preferably, the surfactant is Triton X-100.

[0028] The inventors have shown in further research that adding the surfactant Triton X-100 to the fermentation medium can significantly increase the content of β-phenylethanol in the fermentation broth. Here, the inventors need to explain that the surfactant is not added randomly to the fermentation medium, which can significantly increase the content of β-phenylethanol in the fermentation broth; improper selection of the surfactant not only fails to increase the content of β-phenylethanol in the fermentation broth, but will reduce the content of β-phenylethanol in the fermentation broth; studies have shown that only by adding the surfactant Triton X-100 to the fermentation medium can the content of β-phenylethanol in the fermentation broth be significantly increased.

[0029] Preferably, the content of Triton X-100 in the fermentation medium is 30-50 g / L.

[0030] Most preferably, the content of Triton X-100 in the fermentation medium is 42 g / L.

[0031] Preferably, the fermentation medium further comprises activated carbon.

[0032] Preferably, the activated carbon is coconut shell powdered activated carbon.

[0033] The inventors have shown in further research that adding coconut shell activated carbon to the fermentation medium can also increase the content of β-phenylethanol in the fermentation broth.

[0034] Preferably, the mass percentage of coconut shell activated carbon in the fermentation medium is 0.5-1.5%.

[0035] Most preferably, the mass percentage of coconut shell activated carbon in the fermentation medium is 1%.

[0036] The inventors have shown in further research that the addition of the surfactant Triton X-100 and coconut shell activated carbon to the fermentation medium simultaneously can increase the content of β-phenylethanol in the fermentation broth to a greater extent than the fermentation broth obtained by fermenting with the surfactant Triton X-100 or coconut shell activated carbon alone.

[0037] Beneficial effects: The present invention provides a new yeast. Studies have shown that the Pichia kudriavzevii YJQ-1 of the present invention can tolerate 8% (v / v) ethanol and 4g / L β-phenylethanol, and can use a variety of carbon sources to produce β-phenylethanol. In addition, the present invention also provides a new method for producing β-phenylethanol based on Pichia kudriavzevii YJQ-1. Studies have shown that the fermentation broth obtained under the fermentation conditions of the present invention has a higher β-phenylethanol content. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the colony morphology of YJQ-1.

[0039] Figure 2 This is the cell morphology of YJQ-1 under a 1000x microscope.

[0040] Figure 3 This is an analysis chart of the tolerance of YJQ-1 to ethanol and β-phenylethanol.

[0041] Figure 4 This is the performance diagram of YJQ-1 in utilizing carbon sources and producing β-phenylethanol. DETAILED DESCRIPTION

[0042] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.

[0043] Example 1 Screening and identification of yeast

[0044] Take 1.00g of koji (baijiuqu, Angel Yeast Co., Ltd.) sample, add it to a test tube containing 9mL of sterile saline, shake at 200rpm for 30min, and mix thoroughly. Take 1mL of sample solution, add it to 50mL of M medium, and culture it at 30℃ and 120rpm for 24h. Take 200μL of bacterial solution and spread it on Bengal red medium, and culture it at 30℃ for 24h. Pick single colonies with morphological characteristics similar to yeast and repeatedly streak them in YPD culture until the colony morphology is uniform. Transfer each single colony to M medium for culture, then preserve the bacteria with glycerol at a final concentration of 25%, and store it at -80℃.

[0045] Dilute the screened strain 10 times with sterile water, draw 1 drop with a disposable dropper and place it in the center of the slide, clamp the end of the slide with tweezers and burn it 30dm above the alcohol lamp until the water has just evaporated and then remove it to fix the bacteria; then add a drop of methylene blue dye in the middle of the slide and dye for 1 minute, rinse off the dye with a washing bottle, wait for the water on the slide to dry, cover it with a coverslip, and observe the strain morphology under a 100x microscope.

[0046] The milky white, irregularly round, small, rough, uneven, and hairy strains were selected and inoculated into M medium, cultured at 30°C and 120 r / min for 24 h, and the genome was extracted using a yeast genomic DNA rapid extraction kit. The fungal 26S rDNA universal primers 26S (5`-GCATATCAATAAGCGGAGGAAAAG-3`) and 26F (5`-GGTCCGTGTTTCAAGAG-3`) were used to perform polymerase chain reaction on the selected strains for PCR amplification. The reaction system for PCR amplification is shown in Table 1, and the amplification reaction conditions are shown in Table 2. The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were uploaded to the National Center of Biotechnology Information (NCBI) for BLAST homology comparison, and sequences with high homology similarity were selected to construct a phylogenetic tree using the maximum likelihood method using MEGA 11 software.

[0047] The sequencing results were compared with the NCBI database, and the phylogenetic tree was constructed using the maximum likelihood method using the MEGA11 software. It was found that the similarity between strain YJQ-1 and Pichia kudriavzevii with accession numbers HQ262302.1, HQ262298.1, etc. was 99.83%; therefore, YJQ-1 was determined to be Pichia kudriavzevii.

[0048] Table 1. PCR reaction system

[0049]

[0050] Table 2. PCR reaction program

[0051]

[0052] Example 2 Study on the tolerance of YJQ-1 to β-phenylethanol and ethanol

[0053] β-phenylethanol M medium containing 0g / L, 1g / L, 2g / L, 3g / L, 4g / L and 5g / L was prepared respectively, and the activated YJQ-1 was inoculated into the medium containing different concentration gradients of β-phenylethanol at an inoculum of 2%, and cultured at 120rpm and 30°C. The fermentation liquid was taken every 2h to measure OD600 until the OD600 value no longer increased, and the growth curve was drawn with the culture time as the horizontal axis and OD600 as the vertical axis. Each experiment was repeated three times.

[0054] M medium containing 0%, 4%, 8%, and 10% (V / V) ethanol concentrations was prepared respectively, and the activated YJQ-1 was inoculated into the medium containing different ethanol concentration gradients at an inoculum of 2%, and cultured at 120rpm and 30°C. The fermentation liquid was taken every 2 hours to measure OD600 until the OD600 value no longer increased, with the culture time as the horizontal axis and OD600 as the vertical axis. Each experiment was repeated three times.

[0055] Depend on Figure 3 a It can be seen that with the increase of ethanol concentration, the OD600 of strain growth gradually decreased. Strain YJQ-1 can tolerate 8% ethanol concentration. When the ethanol concentration reaches 10%, yeast YJQ-1 hardly grows. Therefore, YJQ-1 can tolerate 8% ethanol concentration. During the fermentation process, excessive β-phenylethanol content will produce certain toxicity to yeast, thereby affecting the growth of yeast and the synthesis of β-phenylethanol. Therefore, the β-phenylethanol tolerance of YJQ-1 was explored. Figure 3 b It can be seen that with the increase of β-phenylethanol concentration, the OD600 of strain growth gradually decreased. When the β-phenylethanol concentration reached 5g / L, yeast YJQ-1 hardly grew. Therefore, YJQ-1 can tolerate 4g / L β-phenylethanol.

[0056] Example 3 Investigation of the carbon source utilization type and β-phenylethanol synthesis capacity of YJQ-1

[0057] The carbon source in the fermentation medium was replaced with 50 g / L glucose, fructose, sucrose, glycerol, mannitol, soluble starch, galactose or 30 g / L ethanol to prepare medium with different carbon sources, and the activated YJQ-1 was inoculated into the medium with different carbon sources at an inoculum of 2%, and cultured at 120 rpm and 30° C. The fermentation liquid was taken every 2 hours to measure OD600 until the OD600 value no longer increased, with the culture time as the horizontal axis and OD600 as the vertical axis. Each test was repeated three times. The fermentation liquid cultured for 72 hours was analyzed by gas chromatography to determine the yield of β-phenylethanol.

[0058] Depend on Figure 4It can be seen from a that except for its poor ability to utilize galactose, YJQ-1 can utilize glucose, fructose, sucrose, glycerol, soluble starch, ethanol, mannitol, and galactose as carbon sources for cell growth. After reaching the stable period, the OD600 values ​​were 11.99, 11.39, 8.88, 7.48, 3.81, 7.18, 3.33, and 1.35, respectively. The fermentation broth cultured for 72 hours was determined by gas chromatography. Figure 4 b The results showed that the contents of β-phenylethanol under glucose, fructose, sucrose, ethanol, glycerol, soluble starch, mannitol and galactose carbon sources were 2.16 g / L, 1.85 g / L, 0.62 g / L, 1.27 g / L, 0.78 g / L, 0.28 g / L, 0.15 g / L and 0.14 g / L, respectively; comprehensive analysis of the biomass and β-phenylethanol yield under different carbon sources showed that the optimal carbon source for the growth and β-phenylethanol production of YJQ-1 was glucose.

[0059] The M seed culture medium described in Examples 1 to 3 is prepared with water, comprising: 50 g / L glucose, 5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 5 g / L yeast extract powder (pH under natural conditions). The fermentation culture medium is prepared with water, comprising: 50 g / L glucose, 5 g / L KH2PO4, 0.5 g / L MgSO4·gS2O, 5 g / L yeast extract powder (pH under natural conditions), 10 g / L L-Phe. The Bengal red culture medium is prepared with water, comprising: 5 g / L peptone, 10 g / L glucose, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 15 g / L agar, 0.03 g / L Bengal red, 0.1 g / L chloramphenicol, and the final pH is 7.2.

[0060] Example 4 Method for producing β-phenylethanol by yeast

[0061] (1) Take 3 rings of YJQ-1 and inoculate them into the seed culture medium, activate them at 30°C for 24 hours, and obtain

[0062] Yeast seed liquid; the seed culture medium is prepared with water, and contains: 50 g / L glucose, 5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 5 g / L yeast extract powder;

[0063] (2) inoculating the yeast seed liquid into a fermentation medium, and fermenting at 30° C. for 72 hours to obtain a fermentation liquid containing β-phenylethanol after the fermentation is completed; wherein the volume of the yeast seed liquid added is 2% of the volume of the fermentation medium; and the fermentation medium is prepared with water and contains: 90 g / L glucose, 1 g / L yeast extract powder, and 12 g / L L-Phe.

[0064] Example 5 Method for producing β-phenylethanol by yeast

[0065] The difference between Example 5 and Example 4 is that the composition of the fermentation medium is different; in Example 5, a surfactant Triton X-100 is also added to the fermentation medium;

[0066] The fermentation medium is prepared with water and contains: 90 g / L glucose, 1 g / L yeast extract powder, 12 g / L L-Phe, and 42 g / L Triton X-100.

[0067] Example 6 Method for producing β-phenylethanol by yeast

[0068] The difference between Example 6 and Example 4 is that the composition of the fermentation medium is different; in Example 6, coconut shell activated carbon is also added to the fermentation medium;

[0069] The fermentation medium is prepared with water and contains: 90 g / L glucose, 1 g / L yeast extract powder, 12 g / L L-Phe, and 1 (mass) % coconut shell activated carbon.

[0070] Example 7 Method for producing β-phenylethanol by yeast

[0071] The difference between Example 7 and Example 4 is that the composition of the fermentation medium is different; in Example 7, a surfactant Triton X-100 and coconut shell activated carbon are also added to the fermentation medium;

[0072] The fermentation medium is prepared with water and contains: 90 g / L glucose, 1 g / L yeast extract powder, 12 g / L L-Phe, 42 g / L Triton X-100, and 1 (mass) % coconut shell activated carbon.

[0073] Comparative Example 1 Method for producing β-phenylethanol by yeast

[0074] The difference between Comparative Example 1 and Example 5 is that the composition of the fermentation medium is different; in Comparative Example 1, a surfactant Triton-114 is added to the fermentation medium;

[0075] The fermentation medium is prepared with water and contains: 90 g / L glucose, 1 g / L yeast extract powder, 12 g / L L-Phe, and 42 g / L Triton-114.

[0076] Comparative Example 2 Method for producing β-phenylethanol by yeast

[0077] The difference between Comparative Example 2 and Example 5 is that the composition of the fermentation medium is different; in Comparative Example 2, a surfactant Tween-20 is added to the fermentation medium;

[0078] The fermentation medium is prepared with water and contains: 90 g / L glucose, 1 g / L yeast extract powder, 12 g / L L-Phe, and 42 g / L Tween-20.

[0079] The content of β-phenylethanol in the fermentation broth obtained by fermentation in Examples 4 to 7 and Comparative Examples 1 and 2 was determined by gas chromatography; the test results are shown in Table 3.

[0080] The specific method is as follows:

[0081] (1) The standard curve of β-phenylethanol was determined by the external standard method: standard samples with different mass concentrations of 0.2, 0.4, 0.6, 0.8, and 1 g / L were prepared, and the peak areas of the corresponding concentrations were obtained by gas chromatography. The standard curve of β-phenylethanol was drawn with the concentration of β-phenylethanol as the horizontal axis and the peak area of ​​β-phenylethanol as the vertical axis.

[0082] (2) GC conditions: Chromatographic column: DB-WAX UI gas chromatography column (30m×0.25mm×0.25μm); Inlet temperature: 220℃. Detector temperature: 230℃. Program temperature: initial temperature 40℃, hold for 6min, program temperature rise to 120℃ at a rate of 5℃ / min, then program temperature rise to 210℃ at a rate of 50℃ / min, hold for 10min. Carrier gas: N2; flow rate: 1.0mL / min; air flow rate: 300mL / min; H2 flow rate: 35mL / min; injection mode: split injection, split ratio of 25:1, injection volume: 1μL.

[0083] Table 3. Determination results of β-phenylethanol content in fermentation broth

[0084]

[0085]

[0086] From the experimental results in Table 1, it can be seen that the fermentation broth containing β-phenylethanol obtained by fermentation in Example 4 has a β-phenylethanol content of 3.13 g / L, which is significantly higher than the highest value of 2.53 g / L in Example 3. This shows that in the process of fermenting β-phenylethanol by using Pichiakudriavzevii YJQ-1, the composition of the fermentation medium plays an important role in the content of β-phenylethanol in the fermentation broth obtained by fermentation. The above experimental results show that the fermentation broth obtained by fermenting Pichiakudriavzevii YJQ-1 in a fermentation medium containing glucose, yeast extract powder and L-Phe can significantly increase the content of β-phenylethanol in the fermentation broth compared with other fermentation mediums, so that the fermentation broth obtained by fermentation has a higher content of β-phenylethanol.

[0087] From the experimental results in Table 1, it can be seen that the fermentation broth containing β-phenylethanol obtained by fermentation in Example 5 has a β-phenylethanol content significantly higher than that in Example 4; this indicates that in the process of fermenting β-phenylethanol by Pichia kudriavzevii YJQ-1, adding the surfactant Triton X-100 to the fermentation medium can significantly increase the β-phenylethanol content in the fermentation broth.

[0088] From the experimental results in Table 1, it can be seen that the fermentation broth containing β-phenylethanol obtained by fermentation in Comparative Examples 1 and 2 has a β-phenylethanol content that is not significantly increased, or even decreased, compared with that in Example 4; this indicates that in the process of using Pichiakudriavzevii YJQ-1 to ferment and produce β-phenylethanol, surfactants are not added randomly to the fermentation medium, which can significantly increase the content of β-phenylethanol in the fermentation broth; improper selection of surfactants not only fails to increase the content of β-phenylethanol in the fermentation broth, but also reduces the content of β-phenylethanol in the fermentation broth; the above experimental results show that only by adding the surfactant Triton X-100 to the fermentation medium can the content of β-phenylethanol in the fermentation broth be significantly increased.

[0089] From the experimental results in Table 1, it can be seen that the fermentation broth containing β-phenylethanol obtained by fermentation in Example 6 has a β-phenylethanol content significantly higher than that in Example 4; this indicates that in the process of producing β-phenylethanol by fermentation using Pichia kudriavzevii YJQ-1, adding coconut shell activated carbon to the fermentation medium can also increase the β-phenylethanol content in the fermentation broth.

[0090] From the experimental results in Table 1, it can be seen that the fermentation broth containing β-phenylethanol obtained by fermentation in Example 7 has a higher degree of improvement in the content of β-phenylethanol than that in Example 4, which means that in the process of fermenting β-phenylethanol by using Pichiakudriavzevii YJQ-1, adding the surfactant Triton X-100 and coconut shell activated carbon to the fermentation medium at the same time can improve the content of β-phenylethanol in the fermentation broth to a greater extent than the fermentation broth obtained by fermenting with the surfactant Triton X-100 or the coconut shell activated carbon alone.

Claims

1. A yeast, whose deposit number is GDMCC No: 64846, and is named Pichia kudriavzevii YJQ-1.

2. Use of the yeast according to claim 1 in producing β-phenylethanol.

3. The use according to claim 2, characterized in that: The specific method for producing β-phenylethanol by yeast is as follows: The yeast of claim 1 is inoculated into a fermentation medium, and fermented at 25 to 35° C. for 60 to 80 hours. After the fermentation is completed, a fermentation liquid containing β-phenylethanol is obtained.

4. The use according to claim 3, characterized in that: Before the yeast is inoculated into the fermentation medium, it is first added into the seed culture medium for activation to obtain yeast seed liquid.

5. The use according to claim 3, characterized in that: The volume of the yeast seed solution added is 1% to 3% of the volume of the fermentation medium; Most preferably, the volume of the yeast seed solution added is 2% of the volume of the fermentation medium.

6. The use according to claim 3, characterized in that: The seed culture medium is prepared with water and contains: 30-60 g / L glucose, 4-6 g / L KH2PO4, 0.1-1 g / L MgSO4·7H2O, and 3-6 g / L yeast extract powder; Most preferably, the seed culture medium is prepared with water and contains: 50 g / L glucose, 5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 5 g / L yeast extract powder.

7. The use according to claim 3, characterized in that: The fermentation medium is prepared with water and contains: 80-150 g / L of glucose, 0.5-5 g / L of yeast extract powder, and 10-20 g / L of L-Phe.

8. The use according to claim 3, characterized in that: The fermentation medium is prepared with water and also contains a surfactant.

9. The use according to claim 3, characterized in that: The surfactant is Triton X-100. Preferably, the content of Triton X-100 in the fermentation medium is 30-50 g / L. Most preferably, the content of Triton X-100 in the fermentation medium is 42 g / L.

10. The use according to claim 3, characterized in that: The fermentation medium also contains activated carbon.

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