Photosensitive prodigiosin producing strain and method for improving yield of prodigiosin by utilizing light stimulation

Abstract

The invention provides a light-sensitive prodigiosin-producing strain and a method for improving the yield of prodigiosin by utilizing light stimulation, the prodigiosin-producing strain is serratia marcescens GEN-01, the preservation number is CCTCC M 2025877, the serratia marcescens GEN-01 is obtained by carrying out multiple rounds of combined mutagenesis treatment on wild serratia marcescens, the preservation number is CCTCC M 2025877, the preservation number is CCTCC M 2025877, the preservation number is CCTCC M 2025877, and the preservation number is CCTCC M 2025877. Compared with the original strain, the prodigiosin production efficiency of the obtained forward mutant strain with high yield of prodigiosin is improved by 6 times. The synthesis of the prodigiosin by the strain is influenced by illumination, the yield of the prodigiosin can be increased by 53.3% and the content is increased by 65.2% when the prodigiosin is fermented and cultured under a red light condition compared with a control group. In a 5L fermentation tank, the yield of the prodigiosin can finally reach 20.5 g / L through continuous feeding. In the fermentation process of the serratia marcescens GEN-01, the pigment production efficiency of the serratia marcescens GEN-01 is remarkably improved by continuously irradiating the serratia marcescens GEN-01 with the red light for 24-72 hours in the secondary metabolism period, and the obtained prodigiosin can be used as a natural antibacterial agent and a pigment additive and is widely applied to the fields of biological medicines, functional foods and ecological agriculture.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a photosensitive strain of bromelain-producing bromide and a method for increasing bromelain production by stimulating light. Background Technology

[0002] Prodigiosin (PG) is a red linear alkaloid compound composed of three pyrrole rings. Its chemical name is 4-methoxy-2,2′-dipyrrole-5-methylpentylpyrrole, and its molecular formula is C2. 20 H 25 N3O. It belongs to the category of microbial secondary metabolites, mainly found within the cell walls of microorganisms. It has low solubility, appearing red or pink in acidic solutions and orange or yellow in alkaline solutions. Its stability is affected by light and pH. N3O possesses various biological activities, including antibacterial, antitumor, and antimalarial effects. It can effectively inhibit the growth of various pathogenic bacteria, fungi, and cancer cells, and also shows significant effects in algae suppression and immunosuppression. Furthermore, it is used as a natural dye and cosmetic additive, possessing broad market potential.

[0003] Currently, the production of squalene mainly relies on microbial fermentation. *Serratia marcescens* was the first microorganism discovered to produce squalene and remains the most widely studied and applied production strain. Compared to other production methods, microbial fermentation offers advantages such as high production efficiency and relatively low cost, but it also presents challenges, such as optimizing fermentation conditions, increasing yield, and controlling product quality.

[0004] Studies have shown that styracin is photosensitive, and its stability is significantly affected by light exposure. Styracin undergoes photolysis under white and blue light (470 nm) irradiation, but does not degrade under red light (660 nm) and far-red light (730 nm). This photosensitivity necessitates careful attention to light-protected conditions during the fermentation process of styracin. Summary of the Invention

[0005] The purpose of this invention is to provide a high-yield Serratia marcescens strain of sclerotinib, which exhibits sensitivity to light sources, showing significant differences in sclerotinib production under different light conditions. Furthermore, this strain possesses good genetic stability, significantly improving sclerotinib production, enhancing environmental adaptability and stress resistance. Exogenous red light irradiation can further promote sclerotinib production in Serratia marcescens.

[0006] Another objective of this invention is to further increase the yield of strychnine produced by Serratia marcescens GEN-01 during fermentation by utilizing external light stimulation, so as to meet the current demand for strychnine products.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] A strain of Serratia marcescens that produces high levels of erythromycin, classified as Serratia marcescens GEN-01, was deposited on April 24, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 2025877.

[0009] The *Serratia marcescens* GEN-01 strain is a positive mutant strain that produces high levels of squalene, obtained through multiple rounds of combined mutagenesis of wild-type *Serratia marcescens*. Compared with the starting strain, the squalene production efficiency is increased by 6 times. The synthesis of squalene by this strain is affected by light; under red light conditions, the yield of squalene is increased by 53.3% and the content by 65.2% compared to the control group. In a 5L fermenter, with continuous feeding, the final yield of squalene can reach 20.5 g / L.

[0010] This invention provides a method for increasing the yield of squalene by stimulating light, including seed activation, seed culture, and fermentation culture of squalene-producing bacteria. The squalene-producing strain is Serratia marcescens GEN-01, with preservation number CCTCC M 2025877.

[0011] During fermentation, external light is used for illumination. This includes the following steps:

[0012] (1) Activate and culture Serratia marcescens GEN-01 to obtain seed culture;

[0013] (2) The seed liquid is inoculated into a fermentation medium for fermentation. During the fermentation process, light with an intensity of 100-500 Lx is used for 24-72 hours. After 72 hours of fermentation, styraxin is obtained.

[0014] In a preferred embodiment, the activation of the *Serratia marcescens* GEN-01 is performed by culturing it in LB solid medium at 30°C for 10–12 h.

[0015] In a preferred embodiment, the activated culture medium in step (1) is LB medium.

[0016] In a preferred embodiment, the culture medium for activation culture consists of: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and 20 g / L agar powder.

[0017] In a preferred embodiment, the culture medium for seed culture consists of: 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride.

[0018] In a preferred embodiment, the culture involves first inoculating the activated Serratia marcescens into a seed culture medium and culturing it at 30°C and 180 rpm for 12–24 h. The resulting culture solution is then inoculated into a new seed culture medium at an inoculation rate of 8–10% (v / v) and cultured for another 10–12 h under the same conditions to obtain a seed solution with an OD value of 2–3.

[0019] In a preferred embodiment, the volume ratio of the seed liquid to the fermentation medium in step (2) is 1:25, and the fermentation is carried out at 25-37℃ for 24-72h, preferably at 30℃ and 180rpm for 24-72h.

[0020] In a preferred embodiment, the fermentation culture medium in step (2) consists of: glucose 16-25 g / L, peptone 10-20 g / L, CaCl2 7.5-12.5 g / L, L-proline 5-10 g / L, MgSO4·7H2O 0.1-0.2 g / L, FeSO4·7H2O 0.03-0.09 g / L, water as solvent, and pH 5.0-6.5.

[0021] As a preferred embodiment, in step (2), after 24 hours of fermentation, the shaker is irradiated with red light of 100 Lx intensity at a distance of 5 cm for 48 hours.

[0022] Our research team obtained a high-yielding mutant strain of *Serratia marcescens* GEN-01 through targeted screening. In a 30 mL shake-flask fermentation system, its squalene yield reached 2.1 g / L, a six-fold increase compared to the wild-type strain. Notably, experimental data showed that the secondary metabolite synthesis pathway of this strain was significantly sensitive to light conditions: after continuous irradiation with 100 Lx red light for 48 h, the yield and content of squalene increased by 53.3% and 65.2%, respectively. In a 5 L fermenter, by utilizing a light-controlled and continuously fed strategy, the final squalene yield reached 20.5 g / L. Further research indicated a synergistic regulatory effect between spectral wavelength and intensity on metabolic flux.

[0023] This discovery provides a new perspective for elucidating the molecular mechanism by which environmental factors regulate the biosynthesis of erythromycin. This invention not only expands the theoretical framework of light-controlled microbial secondary metabolism but also provides experimental evidence for developing industrial fermentation processes based on photobioreactors. Further research into the relationship between light and pigment synthesis can provide important theoretical basis and application value for agriculture, industry, and medicine.

[0024] Beneficial effects:

[0025] This invention yielded a novel *Serratia marcescens* strain, GEN-01, through combined mutagenesis screening. This strain exhibits good genetic stability and photosensitivity, and a higher yield of squalene. Fermentation of the strain in a 5L bioreactor, with irradiation using 100Lx red light during fermentation, promoted squalene synthesis. The squalene yield reached 20.5 g / L after 72 hours of fermentation in a 5L fermenter. The extracted squalene can be used in the pharmaceutical, agricultural, and food industries. Attached Figure Description

[0026] Figure 1 The fermentation results in Example 2 are shown below after irradiation for 72 hours under different light conditions: dark, white light, red light, blue light, orange light, and green light. Blue light represents the fermentation OD of *Serratia marcescens*. 600nm Growth status; Pink: succinyl rubigin yield; Orange: succinyl rubigin content.

[0027] Figure 2 The fermentation results in Example 3 are shown in the shake flask after irradiation with 100 Lx red light for 24 h, 48 h, and 72 h, respectively. Light purple indicates the fermentation OD of *Serratia marcescens*. 600nm Growth status; Pink: styracin production; Light green: styracin content.

[0028] Figure 3 The fermentation results in Example 4 are shown in the shake flask after irradiation with 500 Lx red light for 24 h, 48 h, and 72 h, respectively. Light purple indicates the fermentation OD of *Serratia marcescens*. 600nm Growth status; Pink: styracin production; Light green: styracin content.

[0029] Figure 4 The fermentation results of the shake flask in Example 5 under 100 Lx red light intensity at different time periods are shown below. Light day 1 to 2: irradiation on days 1-2 of fermentation; Light day 2 to 3: irradiation on days 2-3 of fermentation; Light day 1, 3: irradiation on days 1 and 3 of fermentation. Light purple indicates the fermentation OD of *Serratia marcescens*. 600nm Growth status; Pink: styracin production; Light green: styracin content.

[0030] Figure 5 The OD values ​​of the original strain and the mutant strain Serratia marcescens GEN-01 in Example 6 are... 600nm Growth status and styrax production.

[0031] Figure 6 The OD of *Serratia marcescens* GEN-01 in Example 7 was measured in a 5L fermenter over 0–72 h. 600nm Growth status, styrax erythrin production, and glucose content in the fermentation broth. Detailed Implementation

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] The culture medium formulations in the following examples are as follows:

[0034] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar powder 20 g / L, sterilized at 115℃ for 20 min;

[0035] LB medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, sterilized at 115℃ for 20 min;

[0036] Fermentation medium: glucose 20 g / L, peptone 16 g / L, CaCl2 5 g / L, L-proline 7.5 g / L, MgSO4·7H2O 0.2 g / L, FeSO4·7H2O 0.06 g / L, sterilized at 115℃ for 20 min. Glucose was prepared as a stock solution and sterilized separately.

[0037] The method for determining the yield and content of styraxin used in this invention is as follows:

[0038] Method for determining the yield of styraxin: Ultraviolet spectrophotometry was used to determine the yield of styraxin (OD). 535nm ).

[0039] After fermentation, the bacterial culture was added to anhydrous methanol solution at pH 3 at a volume ratio of 1:99, and then extracted with shaking in the dark for 6 hours. The mixture was then centrifuged at 8000 rpm / min and 4℃ for 10 minutes, and the supernatant was collected for OD measurement using a spectrophotometer. 535nm (If pigment remains in the precipitate, extraction can be repeated until white.) Substitute the readings into the styraxin-absorbance standard curve equation to obtain the styraxin yield (C) (mg / L). The formula for calculating styraxin yield is as follows:

[0040]

[0041] In the formula: A——OD 535nm reading;

[0042] V1 — Volume of organic solvent + volume of fermentation broth (mL);

[0043] V2 — Fermentation broth volume (mL).

[0044] Method for determining the content of squalene: Add 2 mL of fermentation broth to a pre-dried centrifuge tube of known weight, centrifuge at 4000 rpm / min for 4 min, wash with ultrapure water, centrifuge again, repeat the washing process twice, and then dry in an oven at 65℃ until constant weight. Obtain the cell dry weight (M) (g / L), and then calculate the squalene content. The formula for calculating the squalene content is as follows:

[0045]

[0046] In the formula: C—the yield of styracil-containing erythromycin (mg / L);

[0047] M — Mass of fermentation broth after drying / Volume of fermentation broth * 10 3 (g / L).

[0048] The technical solution of the present invention is further described in detail below with reference to specific embodiments.

[0049] Example 1

[0050] *Serratia marcescens* GEN-01 is a high-yield positive mutant strain of styraxanthin obtained through multiple rounds of combined mutagenesis of wild-type *Serratia marcescens*. The wild-type *Serratia marcescens* was collected in our laboratory from soil near Junzi Lake at Nanjing University of Technology. The mutagenesis conditions were as follows: *Serratia marcescens* was cultured at 30℃ until the bacterial culture reached OD500. 600 When the concentration reached 0.6-1.0, the bacterial cells were collected and mutagenized using ambient pressure room temperature plasma for 25 seconds. Under the same temperature conditions, the cells were treated with 4% ethyl methanesulfonate or nitrosoguanidine at 180 rpm for 30 and 6 minutes respectively. The cells were then plated on LB agar plates and incubated at 30°C for 24-48 hours. By using a combination of ambient pressure isothermal plasma (ARTP), nitrosoguanidine (NTG), and ethyl methanesulfonate (EMS) mutagenesis, a red strain that was sensitive to light, genetically stable, and produced the highest yield was obtained and named *Serratia marcescens* GEN-01, with the preservation number CCTCC M 2025877.

[0051] Physiological characteristics of strain Serratia marcescens GEN-01:

[0052] (1) Strain identification: PCR experiment was conducted to detect the Serratia marcescens strain identification gene. The strain was identified as Serratia marcescens. The GEN-01 mutant strain showed enhanced growth rate and improved fermentation efficiency. It was also sensitive to light and could promote the production of serotonin under red light.

[0053] (2) Under the microscope, the bacterial cells appear as red straight rods, with no obvious changes in cell morphology compared to wild-type bacteria;

[0054] (3) The colonies formed on solid culture medium have smooth, moist surfaces, neat edges, and are pink or red.

[0055] (4) The GEN-01 mutant strain has enhanced tolerance to temperature, pH and other conditions and can maintain activity in a wider range of temperature and pH. The strain Serratia marcescens GEN-01 can grow and produce styraxin at temperatures of 25-37℃ and pH of 6.0-8.0.

[0056] Example 2

[0057] The yield of strychnine produced by fermentation of Serratia marcescens GEN-01 was measured under conditions of light avoidance, white light, red light, blue light, orange light, and green light with an intensity of 100 Lx for 72 h.

[0058] (1) Activation of bacterial strain: Inoculate the bacterial strain into LB solid medium and incubate at 30°C for 12 hours;

[0059] (2) Seed culture: Take a single colony from LB solid medium, inoculate it into seed medium, and incubate at 30℃ and 180rpm for 12h to obtain seed liquid;

[0060] (3) The seed culture solution obtained from seed culture was inoculated into Erlenmeyer flasks containing fermentation medium at an inoculation rate of 2% (v / v). Fermentation was carried out at 30℃ and 180 rpm. At the beginning of fermentation, different light conditions were used to irradiate the flasks at a distance of 5 cm for 72 h. After fermentation, the cell dry weight was measured, and styraxin was extracted from the fermentation broth. The production and content of styraxin were measured, and the experiment was repeated.

[0061] Three parallel experiments were conducted under the same conditions, and the results are as follows: Figure 1 As shown, different light conditions have significantly different effects on cell growth and metabolite production. The red light-treated group of *Serratia marcescens* GEN-01 exhibited a significant growth advantage and increased metabolite production, with its OD... 600 The red light group showed significantly higher levels of styraxin (2959.8 mg / L) and styraxin (348.1 mg / g) than the control and white light groups, indicating that red light significantly promoted cell proliferation. Simultaneously, the red light group exhibited significantly higher yields (2959.8 mg / L) and concentrations (348.1 mg / g) of styraxin compared to other groups, exceeding the control group by 42.1% and 44.2%, respectively. In contrast, the white light group showed significantly higher OD values. 600 Both the production of cymoxanil and cymoxanil was at the lowest level, indicating that they have an inhibitory effect on cell growth and metabolite accumulation. OD values ​​for the blue and green light groups were also low. 600The yield and content of styracil were similar to those of the control group, with no significant promoting or inhibiting effect observed. The OD of the orange light group... 600 The levels of styracil were similar to the control group, but the yield and content of styracil were increased compared to the control group, though significantly lower than those in the red light group. These results indicate that red light has a significant advantage in promoting cell growth and metabolite accumulation, while the promoting effects of other light conditions (such as white, blue, orange, and green light) are relatively weak.

[0062] Example 3

[0063] The yield of strychnine produced by fermentation of Serratia marcescens GEN-01 under red light irradiation at 100 Lx intensity for 24 h, 48 h, and 72 h was determined.

[0064] Following the steps in Example 2, the seed culture was obtained and inoculated at an inoculation rate of 2% (v / v) into Erlenmeyer flasks containing fermentation medium. Fermentation was carried out at 30°C and 180 rpm. At the beginning of fermentation, the flasks were irradiated with red light at an intensity of 100 Lx at a distance of 5 cm for 24 h, 48 h, and 72 h. After fermentation, the cell dry weight was measured, and styracil was extracted from the fermentation broth. The yield and content of styracil were determined, and the experiment was repeated.

[0065] Three parallel experiments were conducted under the same conditions, and the results are as follows: Figure 2 As shown, the growth of *Serratia marcescens* was similar under different light exposure times, and the production of squalene under 100 Lx red light for 48 h and 72 h was significantly higher than that of the control group without light. The squalene content after 48 h of light exposure was 374.8 mg / g, and after 72 h of light exposure was 371.6 mg / g, while the squalene content under no light exposure was only 238.3 mg / g, representing increases of 57.3% and 55.9%, respectively. However, due to the energy consumption of light, the 48 h light exposure was chosen for subsequent experiments when the difference in squalene content between 48 h and 72 h of light exposure was not significant.

[0066] Example 4

[0067] The only difference between Example 4 and Example 3 is that the intensity of the red light is 500 Lx, while all other operations during fermentation are the same.

[0068] Three parallel experiments were conducted under the same conditions, and the results are as follows: Figure 3As shown, the growth of *Serratia marcescens* GEN-01 was slightly improved compared to the control group under different light exposure times. Furthermore, the yield of squalene under 500 Lx red light for 48 h and 72 h was significantly higher than that under the unexposed control group. The squalene content after 48 h of light exposure was 344.6 mg / g, and after 72 h of light exposure it was 330.8 mg / g, while the squalene content under unexposed light was only 236.1 mg / g, representing increases of 41.7% and 40.1%, respectively. The difference in squalene content between 48 h and 72 h of 500 Lx red light exposure was not significant, but the increase in squalene content was less pronounced compared to 100 Lx red light exposure. Therefore, 100 Lx red light exposure for 48 h was selected for subsequent experiments.

[0069] Example 5

[0070] The yield of sparganin in Serratia marcescens GEN-01 was determined under 100 Lx red light intensity, after 1-2 days of light exposure, 2-3 days of light exposure, and light exposure at intervals (days 1 and 3 of fermentation).

[0071] The seed culture was obtained according to the steps in Example 1. The seed culture was inoculated at an inoculation rate of 2% (v / v) into Erlenmeyer flasks containing fermentation medium and fermented at 30°C and 180 rpm. Irradiation was performed on days 1 and 2, days 2 and 3, and days 1 and 3 of fermentation, for a duration of 48 hours for each group. The irradiation method was the same as in Example 2. After fermentation, the cell dry weight was measured, and styraxin was extracted from the fermentation broth. The production and content of styraxin were determined, and the experiment was repeated.

[0072] Three parallel experiments were conducted under the same conditions, and the results are as follows: Figure 4 As shown, under 100 Lx red light irradiation, different light duration arrangements significantly affected the yield and content of squalene. Under the light conditions of day 2-3 of fermentation, the yield and content of squalene reached their highest levels, at 3162.8 mg / L and 385.7 mg / g, respectively, representing increases of 51% and 63.3% compared to the control group. This indicates that light irradiation on day 2-3 of fermentation maximally promotes squalene synthesis, maintaining high biomass while maximally activating the product synthesis pathway. Light irradiation on days 1-2 of fermentation or intermittent light irradiation (days 1 and 3) was relatively less effective than light irradiation on days 2-3, but still showed a significant improvement compared to the control group.

[0073] Example 6

[0074] OD after 72 hours of fermentation of the original strain and the mutant strain Serratia marcescens GEN-01600nm Growth status and styrax production.

[0075] Following the steps in Example 2, the seed culture was obtained and inoculated into Erlenmeyer flasks containing fermentation medium at an inoculation rate of 2% (v / v). Fermentation was carried out at 30°C and 180 rpm for 72 hours. After fermentation, styracil was extracted from the fermentation broth, and the styracil yield was determined. The experiment was repeated.

[0076] Three parallel experiments were conducted under the same conditions, and the results are as follows: Figure 5 As shown, the biomass (OD) of the original strain after 72 hours of fermentation 600 The yields of styracil and cymoxanil were 30.6 and 351.2 mg / L, respectively. Meanwhile, the biomass (OD) of the mutant strain *Serratia marcescens* GEN-01 was... 600 The yields of styracil and cymoxanil were 62.8 and 2127.6 mg / L, respectively. Compared with the starting strain, the styracil production efficiency was increased by 6 times.

[0077] Example 7

[0078] OD of Serratia marcescens GEN-01 in a 5L fermenter from 0 to 72 h 600nm Growth status, styrax erythrin production, and glucose content in the fermentation broth.

[0079] Following the steps in Example 2, the seed culture was obtained and inoculated at an inoculation rate of 2% (v / v) into a 5L fermenter containing fermentation medium. Fermentation was carried out at 30°C for 72 hours. During the process, dissolved oxygen was controlled to 30% by adjusting the aeration rate and stirring speed, and the glucose concentration in the fermentation broth was ensured to be no less than 1 g / L daily. After 24 hours of fermentation, a 100 Lx red light strip was wrapped around the bottom of the 5L fermenter to continuously irradiate the fermentation broth for 48 hours. The results are as follows... Figure 6 As shown, by utilizing light regulation and continuous feeding strategies, the final yield of lecithin can reach 20.5 g / L.

[0080] The examples provide a new strategy for the efficient production of styraxin, which not only increases yield but also optimizes the content within the bacteria, providing higher quality raw materials for subsequent extraction and purification. The operation is simple, easy to apply industrially, and has significant economic value and market prospects.

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A photosensitive bacterium producing erythromycin is classified as *Serratia marcescens*. Serratia marcescens GEN-01 was deposited at the China Center for Type Culture Collection on April 24, 2025, with accession number CCTCC NO: M 2025877.

2. The application of the pyrifos-producing strain according to claim 1 in the production of pyrifos.

3. A method for increasing the yield of erythromycin using light stimulation, characterized in that, After activating and culturing Serratia marcescens to obtain seed liquid, it is inoculated into fermentation medium to produce serotonin. During the fermentation process, it is irradiated with light of 100-500 Lx for 24-72 h.

4. The method for increasing the yield of erythromycin using light stimulation according to claim 3, characterized in that, The *Serratia marcescens* is *Serratia marcescens* (… Serratia marcescens The preservation number of Serratia marcescens GEN-01 is CCTCC M 2025877.

5. A method for increasing the yield of erythromycin using light stimulation according to claim 3, characterized in that, The activation involves inoculating *Serratia marcescens* into LB solid medium and culturing it at 28–30°C for 10–12 h.

6. The method for increasing the yield of erythromycin using light stimulation according to claim 3, characterized in that, The fermentation medium consists of 16-25 g / L glucose, 10-20 g / L peptone, 7.5-12.5 g / L CaCl2, 5-10 g / L L-proline, 0.1-0.2 g / L MgSO4·7H2O, and 0.03-0.09 g / L FeSO4·7H2O, with water as the solvent and a pH of 5.0-6.

5.

7. A method for increasing the yield of erythromycin using light stimulation according to claim 3, characterized in that, The fermentation was carried out at 28-30℃ for 24-72 h.

8. A method for increasing the yield of erythromycin using light stimulation according to claim 3, characterized in that, After 24 hours of fermentation, the mixture was exposed to light.

9. The method according to claim 8, characterized in that, The illumination is performed using red light with an intensity of 100-500 Lx.

10. The method according to claim 9, characterized in that, Irradiate with red light of 100 Lx for 48 h during fermentation.

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