Method for improving yield of microbial astaxanthin by utilizing red / blue light illumination to perform double-stage regulation and control

Abstract

The invention discloses a method for improving the yield of astaxanthin synthesized by microorganisms through double-stage regulation and control by utilizing red / blue light illumination. Red light (RGB: 255, 0, 0) and blue light (RGB: 0, 0, 255) are selected as illumination sources, the illumination intensity of the red light is 1500-2000 Lux, and the illumination intensity of the blue light is 2500-3000 Lux. Red light irradiation is started for two days on the first day of phaffia rhodozyma fermentation, blue light is converted into blue light on the third day of fermentation, and after illumination is conducted for five days, the content and yield of astaxanthin are increased by 176.33% and 151.78% respectively. The research provides a simple and economic regulation and control method for efficient synthesis of astaxanthin from phaffia rhodozyma.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and particularly relates to a method for improving the yield of astaxanthin biosynthesis by strains and its application. Background Art

[0002] As a potent natural antioxidant, astaxanthin has attracted extensive attention in recent years in the fields of health products, medicine, cosmetics, and aquaculture. Astaxanthin (3,3'-dihydroxy-β,β'-carotene-4,4'-dione) is a natural keto-carotenoid, presenting an orange-red color. Its molecular structure is composed of two β-ionone rings connected by a polyene chain, with a hydroxyl group (-OH) and a keto group (=O) at each end. This unique structure endows astaxanthin with excellent antioxidant properties, and its antioxidant capacity is 550 times that of vitamin E and 54 times that of β-carotene. With the rapid development of synthetic biology, using microbial fermentation to synthesize astaxanthin is a more efficient approach. Many natural sources of astaxanthin, such as Phaffia rhodozyma, and genetically engineered yeasts, such as Saccharomyces cerevisiae, Yarrowia lipolytica, and Kluyveromyces marxianus, have been isolated and constructed. To improve the efficiency of industrial yeast fermentation for astaxanthin production, researchers have utilized various strategies such as mutagenesis, genetic modification, and fermentation regulation. Phaffia rhodozyma, as an important astaxanthin-producing strain, has made remarkable progress in aspects such as strain breeding, fermentation process optimization, and product extraction technology in recent years.

[0003] Fermentation process optimization is a key link to increase the yield. Research shows that by adopting the strategy of using a mixed carbon source (the ratio of glucose to inulin is 2:1) and stagewise supplementing nitrogen source (such as supplementing 0.75 g / L peptone at 36 hours), the astaxanthin yield can reach 83.34 mg / L. Ga et al. adding 0.2% ethanol to the culture medium can increase the specific rate of astaxanthin production, and the research by Meyea et al. shows that adding pinene at 500:1 can increase the synthesis of astaxanthin by Phaffia rhodozyma.

[0004] Hu Xiangdong et al. pointed out that astaxanthin synthesis is a photosynthetic reaction, and light intensity is one of the key factors affecting astaxanthin production. Through fermentation experiments under different light intensities, increasing the light intensity can significantly improve the productivity of astaxanthin.

[0005] "Optimization of Astaxanthin Synthesis Conditions in Phaffia rhodozyma by Laser Tweezers Raman Spectroscopy", "The Latest Literature Review of Astaxanthin", etc. These literatures have explored the conditions and related influencing factors of astaxanthin synthesis by Phaffia rhodozyma from different perspectives. Among them, light, as an important factor, has important research value in the process of optimizing astaxanthin production. Summary of the Invention

[0006] The object of the present invention is to provide a method for increasing the yield of astaxanthin produced by strain fermentation through irradiation with different light qualities, especially a method for improving the yield of microbial astaxanthin through two-stage regulation using red / blue light irradiation.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A method for producing astaxanthin by irradiating with different light qualities, in which different colors and intensities of light are selected for irradiation during the fermentation process, and a strain producing astaxanthin is used as the fermentation strain to ferment and produce astaxanthin.

[0009] The strain producing astaxanthin in the present invention can be Xanthophyllomyces dendrorhous or genetically engineered yeast, such as any one of Saccharomyces cerevisiae, Yarrowia lipolytica, and Kluyveromyces marxianus;

[0010] In one embodiment of the present invention, Xanthophyllomyces dendrorhous LX6 with the preservation number of CCTCC No. M 20242814 is used.

[0011] As a preferred embodiment, the present invention produces astaxanthin by microbial fermentation, including: inoculating the strain producing astaxanthin onto a YM solid medium for primary activation of the strain; picking single colonies and inoculating them into a seed medium for secondary seed culture; inoculating the seed liquid obtained from the seed culture into a fermentation medium for culture, and obtaining astaxanthin products after extraction.

[0012] The formula of the fermentation medium is: carbon source 20 - 50 g / L, nitrogen source 15 - 25 g / L, inorganic salts 1.5 - 5 g / L.

[0013] Furthermore, the carbon source for the fermentation of the used strain is glucose, and the carbon source concentration is 20 - 50 g / L.

[0014] Furthermore, the nitrogen source in the fermentation medium is at least one of ammonium sulfate, yeast powder, and corn steep liquor dry powder, and the nitrogen source is 15 - 25 g / L.

[0015] Furthermore, the inorganic salts in the fermentation medium are ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, etc.

[0016] Furthermore, the preferred formula of the fermentation medium is: glucose 30 g / L, ammonium sulfate 1.5 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, yeast powder 2 g / L, corn steep liquor powder 15 g / L.

[0017] Furthermore, the method of the two generations of seed culture is:

[0018] 1) First-generation seed culture: Pick a single colony from the YM solid medium and inoculate it into the seed medium for constant-temperature culture.

[0019] 2) Second-generation seed culture: Inoculate the first-generation seed culture solution into a new seed medium at an inoculation amount of 1-10%, and perform constant-temperature culture under the same conditions as in step (1) to obtain the seed solution for fermentation culture.

[0020] Further, the conditions for the constant-temperature culture are 22°C, 160-180 rpm, and culture for 24-36 hours.

[0021] Further, the temperature for fermentation culture is 22°C.

[0022] Further, perform fermentation culture at 500 rpm.

[0023] Further, during fermentation culture, replenish the carbon source every day starting from the second day until the initial concentration reaches 30 g / L.

[0024] Irradiate the fermentation medium of Phaffia rhodozyma on the first day of fermentation.

[0025] Select red light with a wavelength of 620 nm and blue light with a wavelength of 480 nm from different light qualities.

[0026] The light intensity range is: 1000-2500 Lux.

[0027] Adjust the light intensity of irradiation with different light qualities to: 1500-2000 Lux and 2500-3000 Lux.

[0028] The light quality is at least one of red light or blue light, and perform continuous illumination or alternating illumination; irradiating it with light during the fermentation process can promote the growth of Phaffia rhodozyma cells, thereby increasing the yield of astaxanthin.

[0029] Among the RGB values of the red light: the R value is 255, the G value is 0, and the B value is 0.

[0030] Among the RGB values of the blue light: the R value is 0, the G value is 0, and the B value is 255.

[0031] The continuous illumination time in fermentation production is 7 days.

[0032] Further preferably, in fermentation production, use a red / blue light combined illumination, first irradiate with red light for 2 days, and then irradiate with blue light for 5 days.

[0033] In fermentation production, in the combined illumination, irradiate with red light with a light intensity of 2500-3000 Lux for 2 days, and irradiate with blue light with a light intensity of 1500-2000 Lux for 5 days.

[0034] Furthermore, the fermentation broth is centrifuged to obtain the bacterial cells containing astaxanthin, and then the astaxanthin product is obtained through separation and extraction.

[0035] There are many extraction methods for astaxanthin. The extraction method of the present invention is as follows: The fermentation broth is taken, centrifuged and washed, the bacterial cells are collected, the bacterial cells are broken, extracted, and the supernatant is collected by centrifugation to obtain astaxanthin.

[0036] Beneficial effects:

[0037] The present invention provides a fermentation method that can increase the yield of astaxanthin, which promotes the improvement of the astaxanthin yield in biological production. The strain is fermented in a 2L fermenter, and red light and blue light illumination fermentation are carried out during the fermentation process. Cultivation is continued while keeping other conditions unchanged. The astaxanthin content in the obtained product is significantly increased under the combined red / blue light illumination, which is 176.33% higher than the control, the yield is 1582.54 mg / L, and the content is 151.78% higher than the control; the extracted astaxanthin can be used in feeds, health products, cosmetics, etc., and the bacterial cells after extraction can be added as protein to animal feeds, improving the quality of the feeds and alleviating the huge demand pressure of feed production on grains. Description of the drawings

[0038] Figure 1 The influence of different light qualities on the astaxanthin yield;

[0039] Figure 2 The influence of red light RGB shake flasks on the astaxanthin yield in shake flask fermentation;

[0040] Figure 3 The influence of blue light RGB shake flasks on the astaxanthin yield in shake flask fermentation;

[0041] Figure 4 The influence of the red light illumination intensity in shake flask fermentation on the astaxanthin yield;

[0042] Figure 5 The influence of the blue light illumination intensity in shake flask fermentation on the astaxanthin yield;

[0043] Figure 6 The influence of the red light illumination time on the astaxanthin yield in shake flask fermentation;

[0044] Figure 7 The influence of the blue light illumination time on the astaxanthin yield in shake flask fermentation;

[0045] Figure 8 The influence of the combined red / blue light illumination time on the astaxanthin yield in shake flask fermentation;

[0046] Figure 9 The influence of the combined red / blue light illumination intensity on the astaxanthin yield in shake flask fermentation;

[0047] Figure 10 Fermentation tank yield under red / blue light combination illumination;

[0048] Figure 11 Fermentation tank yield without illumination. Detailed implementation manners

[0049] The present invention will be further described in detail below in conjunction with examples, but the implementation manners of the present invention are not limited thereto. In addition, what is described in the examples is only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0050] The culture media used in the examples are as follows:

[0051] YM solid culture medium: glucose 10 g / L, peptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L, agar powder 20 g / L, and the rest is water.

[0052] YM culture medium: glucose 30 g / L, peptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L, and the rest is water.

[0053] The preparation method is as follows: Weigh 2 g of glucose, 0.5 g of peptone, 0.3 g of yeast extract, and 0.3 g of malt extract, dissolve them in 100 mL of distilled water, and adjust the pH to 5.5 ± 0.2 with 4M NaOH and 4M H3PO4. Add 2 g of agar powder to the YM solid culture medium, shake well, and sterilize at 115 °C for 20 min.

[0054] Fermentation culture medium: glucose 30 g / L, ammonium sulfate 1.5 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, yeast extract 2 g / L, corn steep liquor powder 15 g / L. Glucose is prepared into a mother liquor and sterilized separately.

[0055] The seed culture medium is the same as the fermentation culture medium.

[0056] The different light quality irradiation conditions in the examples are as follows:

[0057] The wavelength of red light is selected at 620 nm, and the light intensity is 2500 - 3000 Lux.

[0058] The wavelength of blue light is selected at 480 nm, and the light intensity is 1500 - 2000 Lux.

[0059] The methods for measuring the dry weight of microbial cells and the astaxanthin content in the examples are as follows:

[0060] Method for measuring the dry weight of microbial cells: Take 1 ml of fermentation broth with a pre-weighed centrifuge tube, centrifuge and wash twice, and then place it in an oven at 65 °C to dry to a constant weight.

[0061] Extraction and determination method of astaxanthin: The content of astaxanthin was determined by ultraviolet spectrophotometry.

[0062] Take 1 mL of the fermentation broth and centrifuge it at 12,000 rpm for 1 min, wash it twice with deionized water, collect the thalli, dry the water droplets on the tube wall with filter paper, add 1 mL of dimethyl sulfoxide (DMSO) preheated at 65 °C, oscillate to suspend, add 2 mL of acetone, vortex oscillate for 20 - 30 s, then let it stand in the dark for 15 minutes at 4 °C, centrifuge at 12,000 rpm for 2 min, and measure the OD of the supernatant with a spectrophotometer 474 If the bacterial sludge is still colored, repeat the extraction until it becomes white. The calculation formula for the astaxanthin content is as follows:

[0063]

[0064] In the formula: A—OD 478mm value

[0065] V1—Total volume of organic solvent (mL);

[0066] 2150—Specific extinction coefficient;

[0067] V2—Volume of fermentation broth (mL);

[0068] 10000: Value after unit conversion (mg / L)

[0069] The strains used in the following examples are:

[0070] Xanthophyllomyces dendrorhous LX6, with the preservation number of CCTCC No. M20242814, has been disclosed in the patent literature CN119709447A and is an existing strain.

[0071] In the following examples, the wavelength of red light is selected at 620 nm; the wavelength of blue light is selected at 480 nm.

[0072] Example 1

[0073] The Xanthophyllomyces dendrorhous strain was used for light-shaking flask fermentation to produce astaxanthin using different colors of light.

[0074] (1) Inoculate Xanthophyllomyces dendrorhous, using the publicly available existing strain LX6, into the YM solid medium and culture it at 22 °C for 2 - 3 days for strain activation;

[0075] (2) Seed culture, including:

[0076] a. Primary seed culture: Pick a single colony from the YM solid medium and inoculate it into the seed culture medium. Incubate at a constant temperature of 22°C and 160 rpm for 48 h.

[0077] b. Secondary seed culture: Take the culture solution from the primary seed culture and inoculate it into a new seed culture medium at an inoculation amount of 10%. Incubate at a constant temperature under the same conditions as in a to obtain the seed solution for fermentation culture.

[0078] (3) Inoculate the seed solution obtained from the seed culture into a triangular flask containing the fermentation medium and ferment at 22°C and 160 rpm. Starting from the second day of fermentation, supplement glucose every day until the concentration reaches 30 g / L. At the 0 h of fermentation, use red light (RGB: 255, 0, 0), blue light (RGB: 0, 0, 255), orange light (RGB: 255, 95, 0), green light (RGB: 0, 255, 0), pink light (RGB: 255, 0, 169), purple light (RGB: 140, 0, 255), and white light (RGB: 255, 255, 255) with a light intensity of 2500 - 3000 Lux for illumination respectively, and culture for seven days. Measure the dry cell weight, extract astaxanthin from the fermentation broth, and measure the astaxanthin content. Repeat the experiment. As Figure 1 shown, after seven days of fermentation under blue light illumination, the astaxanthin yield can reach 137.00 mg / L (the amount of astaxanthin produced per liter of fermentation broth), which is significantly increased by 25.51% compared with the control group, and the content is significantly increased by 27.78% compared with the control group; after seven days of fermentation under red light illumination, the astaxanthin yield reaches 109.30 mg / L, which is not significantly increased compared with non-illuminated group, only increased by 0.13%, but under red light illumination, the dry cell weight can reach 16.8 g / L (the amount of astaxanthin produced per liter of fermentation broth), which is significantly increased by 25.69% compared with the control group, while the yields after illumination with other colors of light have no obvious changes. It shows that blue light illumination will increase the astaxanthin yield; red light illumination will increase the dry cell weight.

[0079] Example 2

[0080] The Xanthophyllomyces dendrorhous strain is used for shake flask fermentation of astaxanthin production with different light qualities of different light intensities.

[0081] (1) Inoculate Xanthophyllomyces dendrorhous LX6 into the YM solid medium and culture at 22°C for 2 - 3 days for strain activation.

[0082] (2) Seed culture, including:

[0083] a. Primary seed culture: Pick a single colony from the YM solid medium and inoculate it into the seed culture medium. Incubate at a constant temperature of 22°C and 160 rpm for 48 h.

[0084] b. Secondary seed culture: Inoculate the culture solution from the first-generation seed culture into a new seed medium at an inoculation amount of 10%, and perform constant-temperature culture under the same conditions as in a to obtain the seed solution for fermentation culture.

[0085] (3) Inoculate the seed solution obtained from seed culture into a triangular flask containing fermentation medium for fermentation at 22 °C and 160 rpm. From the second day of fermentation, supplement glucose every day until the concentration reaches 30 g / L, culture for seven days, measure the dry cell weight, extract astaxanthin from the fermentation broth, measure the astaxanthin content, and repeat the experiment. Starting from the first day of fermentation, use red light with different RGB values and intensities of 2500 - 3000 Lux, namely standard red (RGB: 255, 0, 0), dark red (RGB: 139, 0, 0), light red (RGB: 255, 102, 102), and use blue light with different RGB values and intensities of 2500 - 3000 Lux, namely standard blue (RGB: 0, 0, 255), dark blue (RGB: 0, 0, 139), light blue (RGB: 135, 206, 235) for illumination. As Figure 2 and Figure 3 shown, after seven days of fermentation with red light of RGB (255, 0, 0), the astaxanthin yield can reach 105.23 mg / L (the amount of astaxanthin produced per liter of fermentation broth), which is not significantly improved compared to non-illuminated conditions, with an increase of 0.17%, but the DCW increases by 27.58%. As the illumination intensity decreases, both the yield and biomass will gradually decrease, and the decrease in illumination intensity will inhibit the growth and metabolic activities of the strain. After seven days of fermentation with blue light of RGB (0, 0, 255), the astaxanthin yield can reach 126.14 mg / L (the amount of astaxanthin produced per liter of fermentation broth), which is significantly increased by 25.34% compared to non-illuminated conditions, and the content is significantly increased by 27.58% compared to non-illuminated conditions. As the illumination intensity increases, both the yield and biomass will gradually decrease, and the increase in illumination intensity will inhibit the growth and metabolic activities of the yeast strain.

[0086] Example 3

[0087] The Xanthophyllomyces dendrorhous strain is used for flask fermentation of astaxanthin production with different light qualities at different illumination intensities.

[0088] (1) Inoculate Xanthophyllomyces dendrorhous LX6 onto YM solid medium and culture at 22 °C for 2 - 3 days for strain activation;

[0089] (2) Seed culture, including:

[0090] a. First-generation seed culture: Take a single colony from the YM solid medium and inoculate it into the seed medium for constant-temperature culture at 22 °C and 160 rpm for 48 h;

[0091] b. Secondary seed culture: Inoculate the culture solution from the primary seed culture into a new seed medium at an inoculation amount of 10%, and perform constant-temperature culture under the same conditions as in a to obtain the seed solution for fermentation culture.

[0092] (3) Inoculate the seed solution obtained from seed culture into a triangular flask containing fermentation medium for fermentation at 22 °C and 160 rpm. Starting from the second day of fermentation, supplement glucose every day until the concentration reaches 30 g / L, culture for seven days, measure the dry cell weight, extract astaxanthin from the fermentation broth, measure the astaxanthin content, and repeat the experiment. Starting from the first day of fermentation, irradiate with red light and blue light with intensities of 1500 - 2000 Lux and 2500 - 3000 Lux respectively, as Figure 4 and Figure 5 shown, after seven days of fermentation with red light with an illumination intensity of 2500 - 3000 Lux, the astaxanthin yield can reach 107.19 mg / L (the amount of astaxanthin produced per liter of fermentation broth), which is not significantly improved compared to without illumination, an increase of 0.37%, but the DCW increases by 26.74%. As the illumination intensity decreases, both the yield and biomass will gradually decrease, and the decrease in illumination intensity will inhibit the growth and metabolic activities of the strain. After seven days of fermentation with blue light with an illumination intensity of 1500 - 2000 Lux, the astaxanthin yield can reach 136.54 mg / L (the amount of astaxanthin produced per liter of fermentation broth), which is significantly increased by 36.13% compared to without illumination, and the content is significantly increased by 37.95% compared to without illumination. As the illumination intensity increases, both the yield and biomass will gradually decrease, and the increase in illumination intensity will inhibit the growth and metabolic activities of the yeast strain.

[0093] Example 4

[0094] Perform illumination at different times and use the Phaffia rhodozyma strain for shake-flask fermentation to produce astaxanthin.

[0095] (1) Inoculate Xanthophyllomyces dendrorhous LX6 into YM solid medium and culture at 22 °C for 2 - 3 days for strain activation;

[0096] (2) Seed culture, including:

[0097] a. Primary seed culture: Take a single colony from the YM solid medium and inoculate it into the seed medium, and perform constant-temperature culture at 22 °C and 160 rpm for 48 h;

[0098] b. Secondary seed culture: Inoculate the culture solution from the primary seed culture into a new seed medium at an inoculation amount of 10%, and perform constant-temperature culture under the same conditions as in a to obtain the seed solution for fermentation culture.

[0099] (3) The seed liquid obtained by seed culture is inoculated into a triangular flask containing fermentation medium and fermented at 22°C and 160 rpm. From the second day of fermentation, glucose is supplemented every day until the concentration reaches 30 g / L. Light is applied starting from the 0th day, 1st day, 2nd day, 3rd day, 4th day, 5th day, and 6th day of fermentation respectively. Red light with a light intensity of 2500 - 3000 Lux and blue light with a light intensity of 1500 - 2000 Lux are used respectively, and cultured for seven days. The dry cell weight is measured, astaxanthin in the fermentation broth is extracted, and the astaxanthin content is measured. The experiment is repeated. As Figure 6 shown, when red light illumination is carried out on it from the 0th day to the 3rd day, although the astaxanthin yield has no obvious change compared with that without illumination, the DCW of the cells has increased compared with that without illumination. It is preferably illuminated with red light from the 0th day to the 3rd day; more preferably, red light illumination with a light intensity of 2500 - 3000 Lux is carried out on the 0th day, and fermentation continues. After seven days, the fermentation ends. The astaxanthin yield can reach 118.71 mg / L (the amount of astaxanthin produced per liter of fermentation broth), which is 1.91% higher than that without illumination, and the increase is not obvious, but the DCW can reach 15.27 g / L, which is significantly increased by 15.95% compared with that without illumination. As the red light illumination time shortens, both the yield and biomass will gradually decrease. Illumination in the later stage of fermentation will inhibit the growth and metabolic activities of yeast strains. As Figure 7 shown, when blue light illumination is carried out on it from the 4th to the 6th day of fermentation, the astaxanthin yield has increased compared with that without addition. It is preferably illuminated with blue light from the 4th to the 6th day; more preferably, blue light illumination is carried out on the 4th day; blue light illumination with a light intensity of 1500 - 2000 Lux is carried out on the 4th day of fermentation, and fermentation continues. After seven days, the fermentation ends. The astaxanthin yield can reach 140.99 mg / L (the amount of astaxanthin produced per liter of fermentation broth), which is significantly increased by 43.67% compared with that without addition, and the content is significantly increased by 35.43% compared with that without addition. As the blue light time shortens, both the yield and biomass will gradually decrease. Starting illumination will inhibit the growth and metabolic activities of yeast strains.

[0100] Example 5

[0101] Under different times, the Xanthophyllomyces dendrorhous strain uses red / blue light combined illumination in shake flask fermentation to produce astaxanthin.

[0102] (1) Inoculate Xanthophyllomyces dendrorhous LX6 into YM solid medium and culture at 22°C for 2 - 3 days for strain activation;

[0103] (2) Seed culture, including:

[0104] a. Primary seed culture: Take a single colony from the YM solid medium and inoculate it into the seed medium, and culture at a constant temperature of 22°C and 160 rpm for 48 h;

[0105] b. Secondary seed culture: Inoculate the culture solution from the primary seed culture into a new seed medium at an inoculation amount of 10%, and perform constant-temperature culture under the same conditions as in a to obtain the seed solution for fermentation culture.

[0106] (3) Inoculate the seed solution obtained from seed culture into a triangular flask containing fermentation medium for fermentation at 22 °C and 160 rpm. Starting from the second day of fermentation, supplement glucose every day until the concentration reaches 30 g / L, culture for seven days, measure the dry cell weight, extract astaxanthin from the fermentation broth, measure the astaxanthin content, and repeat the experiment. Starting from the first day of fermentation, perform light fermentation under red light for a certain period of time and then transfer to light fermentation under blue light for a combined light fermentation for a total of seven days. Select to perform light fermentation under red light with an intensity of 2500 - 3000 Lux for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days and then transfer to light fermentation under blue light with an intensity of 2500 - 3000 Lux for 6 days, 5 days, 4 days, 3 days, 2 days, 1 day. As Figure 8 shown, first perform light fermentation under red light for 2 days, then perform light fermentation under blue light for 5 days. After seven days of fermentation, the astaxanthin yield can reach 144.31 mg / L (the amount of astaxanthin produced per liter of fermentation broth), which is significantly increased by 48.13% compared to non-light fermentation, and the content is significantly increased by 39.99% compared to the control group; it can be seen that the dry cell weight is significantly increased with the increase of the red light illumination time. When performing light fermentation under red light for 6 days and then performing light fermentation under blue light for 1 day, the DCW after seven days of fermentation is significantly increased by 21.65% compared to the control group.

[0107] Example 6

[0108] Under different light intensities, the Xanthophyllomyces dendrorhous strain uses combined red / blue light shaking flask fermentation to produce astaxanthin.

[0109] (1) Inoculate Xanthophyllomyces dendrorhous LX6 onto YM solid medium and culture at 22 °C for 2 - 3 days for strain activation;

[0110] (2) Seed culture, including:

[0111] a. Primary seed culture: Take a single colony from the YM solid medium and inoculate it into the seed medium, and perform constant-temperature culture at 22 °C and 160 rpm for 48 h;

[0112] b. Secondary seed culture: Inoculate the culture solution from the primary seed culture into a new seed medium at an inoculation amount of 10%, and perform constant-temperature culture under the same conditions as in a to obtain the seed solution for fermentation culture.

[0113] (3) The seed solution obtained from seed cultivation was inoculated into an Erlenmeyer flask containing a fermentation medium and fermented at 22°C and 160 rpm. From the second day of fermentation, glucose was supplemented daily until the concentration reached 30 g / L. The cultivation was carried out for seven days. The dry cell weight was measured, astaxanthin was extracted from the fermentation broth, and the astaxanthin content was measured. The experiment was repeated. Starting from the first day of fermentation, red light with different light intensities was used for light fermentation for two days, and then transferred to blue light with different light intensities for light fermentation, for a total of seven days of combined light fermentation. Red light illumination fermentation was carried out at light intensities of 1500 - 2000 Lux (weak) and 2500 - 3000 Lux (strong) for 2 days respectively, and then transferred to blue light illumination fermentation at light intensities of 1500 - 2000 Lux and 2500 - 3000 Lux for 5 days respectively. As Figure 9 shown, first, red light illumination fermentation was carried out at a light intensity of 2500 - 3000 Lux (strong) for 2 days, and then blue light illumination fermentation was carried out at a light intensity of 1500 - 2000 Lux (weak) for 5 days. After seven days of fermentation, the astaxanthin yield could reach 153.97 mg / L (the amount of astaxanthin produced per liter of fermentation broth), which was significantly increased by 55.15% compared with non - illuminated fermentation, and the content was significantly increased by 49.35% compared with the control group.

[0114] Example 7

[0115] The Xanthophyllomyces dendrorhous strain was used to ferment and produce astaxanthin using a red / blue light illumination fermenter.

[0116] (1) The Xanthophyllomyces dendrorhous LX6 was inoculated onto YM solid medium and cultured at 22°C for 2 - 3 days for strain activation.

[0117] (2) Seed cultivation includes:

[0118] a. First - generation seed cultivation: A single colony was taken from the YM solid medium and inoculated into the seed medium. It was cultured at a constant temperature of 22°C and 160 rpm for 48 h.

[0119] b. Second - generation seed cultivation: The culture solution from the first - generation seed cultivation was inoculated into a new seed medium at an inoculation amount of 10% and cultured at the same conditions as in a to obtain the seed solution for fermentation culture.

[0120] (3) The seed solution obtained from seed cultivation was inoculated into a 2 - L fermenter containing a fermentation medium. Fermentation was carried out at 22°C and 500 rpm. From the second day of fermentation, glucose was supplemented daily until the concentration reached 30 g / L. Red light illumination fermentation at a light intensity of 2500 - 3000 Lux was carried out starting at the 0 h for 2 days, and the light intensity was adjusted to 1500 - 2000 Lux of blue light for continued light fermentation for five days. The total cultivation was carried out for seven days. The dry cell weight was measured, astaxanthin was extracted from the fermentation broth, and the astaxanthin content was measured. The results are asFigure 10 As shown, repeated experiments were carried out, and no light was applied under the same conditions as a control. The yield results of the fermenter without light are as Figure 11 shown, as Figure 10 shown, the yield in the fermenter with red / blue light illumination fermentation was 1582.53 mg / L, which was 176.33% higher than that of the control, and the content was 151.78% higher.

Claims

1. A method for increasing astaxanthin production by irradiating with different light qualities, characterized in that, Using Xanthophyllomyces dendrorhous as the fermentation strain, different light qualities are selected during its fermentation process, and within a certain range of light intensities, irradiation is carried out for the fermentation production of astaxanthin.

2. The method for increasing astaxanthin production by irradiating with different light qualities according to claim 1, wherein The light quality is at least one of red light or blue light.

3. The method for increasing astaxanthin production by irradiating with different light qualities according to claim 1, characterized in that, The illumination methods include one or a combination of continuous illumination, light / dark alternation, or irradiation with different light qualities at intervals.

4. The method for increasing astaxanthin production by irradiating with different light qualities according to claim 1, characterized in that, The Rhodotorula rubra yeast is Rhodotorula rubra ( Xanthophyllomyces dendrorhous Xanthophyllomyces dendrorhous ) LX6, and its preservation number is CCTCC No. M 20242814.

5. The method for increasing astaxanthin production by irradiating with different light qualities according to claim 1, wherein The light intensity range is: 1000 - 2500 Lux.

6. The method for increasing astaxanthin production by irradiating with different light qualities according to claim 1, characterized in that, The different light qualities start irradiating the fermentation medium on the first day of the fermentation of Xanthophyllomyces dendrorhous.

7. The method for increasing astaxanthin production by irradiating with different light qualities according to claim 2, wherein The illumination wavelength of the red light is 620 nm. In the RGB values of the red light: the R value is 255, the G value is 0, and the B value is 0.

8. The method for increasing astaxanthin production by irradiating with different light qualities according to claim 2, wherein The illumination wavelength of the blue light is 480 nm. In the RGB values of the blue light: the R value is 0, the G value is 0, and the B value is 255.

9. The method for increasing astaxanthin production by irradiating with different light qualities according to claim 3, characterized in that, The continuous illumination time is 7 days in the fermentation production.

10. The method for increasing astaxanthin production by irradiating with different light qualities according to claim 3, wherein In the fermentation production, using a combination of red / blue light illumination, first irradiate with red light for 2 days, and then irradiate with blue light for 5 days.

Images