Method for increasing yield of astaxanthin by utilizing flavonoid compounds

Abstract

The invention discloses a method for increasing the yield of astaxanthin by utilizing a flavonoid compound, which comprises the following steps: taking phaffia rhodozyma as a fermentation strain, and adding the flavonoid compound for fermentation to produce astaxanthin. Quercetin and baicalein are selected as sources of flavonoid compounds; the concentration of the flavonoid compound mother liquor is 2-5g / L, and the flavonoid compound is added into the fermentation culture medium when the phaffia rhodozyma is fermented for 2-3 days. Flavonoid compounds are added in the fermentation process, so that phaffia rhodozyma cell membrane fluidity is promoted, and the yield of astaxanthin is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and particularly relates to a method for increasing the yield of astaxanthin synthesized by bacterial strains and an application thereof. Background Art

[0002] Astaxanthin is a natural ketocarotenoid with an orange-red color. It has a highly efficient singlet oxygen quenching ability and strong antioxidant capacity, which can protect lipid peroxidation and oxidative damage to low-density lipoprotein-cholesterol, cell membranes, cells and tissues. In addition, astaxanthin is widely used in cosmetics, feed additives and medical industries due to its antioxidant, coloring and immunity-enhancing properties. The traditional production process of astaxanthin mainly includes natural extraction and chemical synthesis; however, they cannot meet the growing market demand and consumer demand for natural products. With the rapid development of synthetic biology, the use of microbial fermentation to synthesize astaxanthin is a more efficient approach. Many natural sources of astaxanthin, such as Phaffia rhodozyma and genetically engineered yeast, such as Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus, have been isolated and constructed. In order to improve the efficiency of industrial yeast fermentation to produce astaxanthin, researchers have used a variety of strategies such as mutagenesis, genetic modification and fermentation regulation.

[0003] Ga et al. added 0.2% ethanol to the culture medium to increase the specific rate of astaxanthin production. Meyea et al. showed that adding 500:1 pinene can increase the synthesis of astaxanthin in Phaffia rubra. However, when the concentration of pinene exceeds 120:1, the specific growth rate drops rapidly. Most of the biological activity of astaxanthin is due to its own targeting to mitochondria, where it inserts itself into the cell membrane. Astaxanthin is inserted into the cell membrane of Phaffia rubra. On the one hand, astaxanthin is a powerful antioxidant that protects mitochondria and other sensitive organelles from damage by reactive oxygen species; on the other hand, astaxanthin hinders cell membrane flow, thereby affecting the growth and development of Phaffia rubra and its activity, which is not conducive to the accumulation of astaxanthin in the later stage of Phaffia rubra, resulting in a decrease in the production and content of Phaffia rubra. Now, solving the problem of astaxanthin affecting cell membrane fluidity can greatly increase the production of astaxanthin. Summary of the invention

[0004] The purpose of the present invention is to provide a method for improving the yield of astaxanthin biosynthesized by strain fermentation by utilizing flavonoid compounds.

[0005] The purpose of the present invention is achieved through the following technical solutions: A method for producing astaxanthin using flavonoid compounds comprises preparing the flavonoid compounds into a mother solution of a certain concentration, adding the mother solution into a fermentation medium, and using red Phaffia yeast as a fermentation strain to ferment and produce astaxanthin.

[0006] The astaxanthin-producing strain of the present invention can be any one of Phaffia rhodozyma and genetically engineered yeast, such as Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus; In one embodiment of the present invention, the red yeast ( Xanthophyllomyces dendrorhous )DW6.

[0007] As a preferred embodiment, the present invention produces astaxanthin by microbial fermentation, including: taking the oil-producing yeast strain and inoculating it on YM solid culture medium for initial activation of the strain; picking a single colony and inoculating it on a seed culture medium for second-generation seed culture; inoculating the seed liquid obtained by the seed culture into a fermentation culture medium for culture, and obtaining the astaxanthin product after extraction.

[0008] The fermentation medium formula is: carbon source 20~50 g / L, nitrogen source 15~25 g / L, inorganic salt 1.5~5 g / L.

[0009] Furthermore, the carbon source for fermentation of the strain used is glucose, and the carbon source concentration is 20-50 g / L.

[0010] Furthermore, the nitrogen source in the fermentation medium is ammonium sulfate, yeast powder and corn steep liquor powder, and the nitrogen source is 15-25 g / L.

[0011] Furthermore, the nitrogen source in the fermentation medium is ammonium sulfate, potassium dihydrogen phosphate, etc.

[0012] Furthermore, the fermentation medium formula is preferably: 30 g / L glucose, 1.5 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 2 g / L yeast powder, and 15 g / L corn syrup powder.

[0013] Furthermore, the two-generation seed culture method is: 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; 2) Second generation seed culture: Take the first generation seed culture solution and inoculate it into a new seed culture medium at a rate of 1-10%, and culture it at a constant temperature under the same conditions as step (1) to obtain a seed solution for fermentation culture.

[0014] Furthermore, the constant temperature culture conditions are 25°C, 160-180rpm, and culture for 24-36 hours.

[0015] Furthermore, the fermentation temperature is 25°C.

[0016] Further, the fermentation culture was carried out at 500 rpm.

[0017] Furthermore, during the fermentation culture, the carbon source was supplemented every day from the second day to an initial concentration of 30 g / L.

[0018] Flavonoids were added to the fermentation medium on the 2nd to 3rd day of the Phaffia rhodozyma fermentation.

[0019] The flavonoids are preferably added within 48 to 72 hours; more preferably within 72 hours.

[0020] The concentration of flavonoid stock solution is 2~5g / L.

[0021] Dissolve the flavonoids in anhydrous ethanol to prepare a mother solution with a concentration of 2-5 g / L; The flavonoid compound is at least one of quercetin and baicalein; adding the flavonoid compound during the fermentation process can promote the fluidity of the cell membrane of Phaffia rhodozyma, thereby increasing the yield of astaxanthin.

[0022] The concentration of the quercetin mother solution is 2 g / L, and the amount of quercetin added is 10-40 mg / L; more preferably, the amount of quercetin added is 20 mg / L.

[0023] The mother solution concentration of baicalein is 5 g / L, the added amount of baicalein is 20-50 mg / L, more preferably the added amount of baicalein is 40-50 mg / L, and more preferably the added amount of baicalein is 50 mg / L.

[0024] Furthermore, the fermentation liquid is centrifuged to obtain astaxanthin-containing bacteria, which are then separated and extracted to obtain an astaxanthin product.

[0025] There are many ways to extract astaxanthin. The extraction method of the present invention is: taking the fermentation liquid for centrifugal washing, collecting the bacterial cells, breaking the bacterial cells, extracting, centrifuging and collecting the supernatant to obtain astaxanthin.

[0026] Compared with the prior art, the present invention has the following advantages and effects: The invention provides a fermentation method capable of increasing the yield of astaxanthin, and promotes the increase of the yield of astaxanthin in biological production. The strain is fermented in a 2 L fermenter, and after quercetin and baicalein are added during the fermentation process, the culture is continued while keeping other conditions unchanged. The astaxanthin content in the obtained product is 46.14% higher than that of the control in the fermenter with quercetin added, and the yield is 551.90 mg / L; the yield in the fermenter with baicalein added is 25.62% higher than that of the control, and is 474.39 mg / L; the extracted astaxanthin can be used in feed, health products, cosmetics, etc., and the extracted bacteria can be added into animal feed as protein, thereby improving the quality of the feed and alleviating the huge demand pressure on food for feed production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Effects of different flavonoids on astaxanthin production; Figure 2 Effect of baicalein addition concentration on astaxanthin production in shake flask fermentation; Figure 3 Effect of quercetin concentration on astaxanthin production in shake flask fermentation; Figure 4 Effect of baicalein addition time on astaxanthin production in shake flask fermentation; Figure 5 Effect of quercetin addition time on astaxanthin production in shake flask fermentation; Figure 6 The yield of fermenters supplemented with baicalein; Figure 7 Fermentation tank yields with quercetin addition; Figure 8 Fermenter yields without added flavonoids. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. In addition, the embodiments are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0029] The culture medium used in the examples is as follows: YM solid culture medium: 10 g / L glucose, 5 g / L peptone, 3 g / L yeast powder, 3 g / L malt extract, 20 g / L agar powder, and the rest water.

[0030] YM medium: 30 g / L glucose, 5 g / L peptone, 3 g / L yeast powder, 3 g / L malt extract, and the rest water.

[0031] The preparation method is as follows: weigh 2 g of glucose, 0.5 g of peptone, 0.3 g of yeast powder, and 0.3 g of malt extract, dissolve in 100 mL of distilled water, and adjust the pH to 5.5±0.2 with 4 M NaOH and 4 M H3PO4. Add 2 g of agar powder to the YM solid medium, shake well, and sterilize at 115°C for 20 min.

[0032] Fermentation medium: 30 g / L glucose, 1.5 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 2 g / L yeast powder, 15 g / L corn syrup powder. Glucose was prepared into mother liquor and sterilized separately.

[0033] The seed medium and fermentation medium are the same.

[0034] The formula of the foreign aid additive in the embodiment is as follows: Baicalein mother solution: weigh 0.5±0.01g baicalein, add 100mL of anhydrous ethanol to make the mother solution concentration 5g / L, filter through an organic phase filter membrane for later use.

[0035] Quercetin mother solution: weigh 0.25±0.01g quercetin, add 100mL of anhydrous ethanol to make the mother solution concentration 2.5g / L, filter through an organic phase filter membrane for later use.

[0036] Naringenin mother solution: weigh 0.25±0.01g naringenin, add 100mL of anhydrous ethanol to make the mother solution concentration 2.5g / L, filter through an organic phase filter membrane for later use.

[0037] The method for determining the dry weight of microbial cells and the astaxanthin content in the embodiment is: Method for determining the dry weight of microbial cells: Take 1 ml of fermentation liquid in a pre-weighed centrifuge tube, wash it twice by centrifugation, and then place it in an oven at 65°C to dry to constant weight.

[0038] Astaxanthin extraction and determination method: Astaxanthin content was determined by UV spectrophotometry.

[0039] Take 1 mL of fermentation broth and centrifuge at 12000 rpm for 1 min, wash twice with deionized water, collect the bacteria, use filter paper to absorb the water droplets on the tube wall, add 1 mL of dimethyl sulfoxide (DMSO) preheated at 65 ℃, oscillate and suspend, add 2 mL of acetone, vortex and oscillate for 20-30 s, then stand in the dark for 15 minutes, centrifuge at 4 ℃, 12000 rpm for 2 min, and measure the OD of the supernatant with a spectrophotometer. 474 If the bacterial sludge is still colored, it can be repeatedly extracted until it turns white. The astaxanthin content is calculated as follows:

[0040] Where: A—OD 478mm value V1—total volume of organic solvent (mL); 2150——Specific extinction coefficient V2——fermentation broth volume (mL); 10000: The number after unit conversion (mg / L) The strains used in the following examples are: Phaffia rhodozyma Xanthophyllomyces dendrorhous ) DW6, with the deposit number of CCTCC M 2021732, has been disclosed in patent document CN118222560A and is an existing strain. Example 1

[0041] Production of astaxanthin by Phaffia rhodozyma strains using different flavonoids in shake flask fermentation.

[0042] (1) Phaffia rhodozyma Xanthophyllomyces dendrorhous ), the publicly available strain DW6 was inoculated into YM solid medium and cultured at 25°C for 2-3 days to activate the strain; (2) Seed cultivation, including: a. First generation seed culture: Take a single colony from YM solid medium and inoculate it into seed culture medium at 25°C and 160 rpm for 48 h; b. Second generation seed culture: Take the culture fluid of the first generation seed culture and inoculate it into a new seed culture medium at a 10% inoculation rate, and culture it at a constant temperature under the same conditions as a to obtain the seed solution for fermentation culture.

[0043] (3) The seed liquid obtained from the seed culture was inoculated into a triangular flask containing fermentation medium and fermented at 25°C, 160rpm. Glucose was added daily to a concentration of 30 g / L from the second day of fermentation. At the 48th hour of fermentation, 20 mg / L of quercetin, naringenin and baicalein were added respectively. The culture was continued for seven days, the cell dry weight was determined, astaxanthin in the fermentation broth was extracted, and the astaxanthin content was determined. The experiment was repeated. Figure 1 As shown, after seven days of fermentation with quercetin added, the astaxanthin production can reach 111.64 mg / L (the amount of astaxanthin produced per L of fermentation liquid), which is a significant increase of 23.55% compared with no addition; after seven days of fermentation with baicalein added, the astaxanthin production can reach 97.89 mg / L (the amount of astaxanthin produced per L of fermentation liquid), which is a significant increase of 8.33% compared with no addition, while the production of naringenin decreased. Example 2

[0044] Phaffia rhodozyma strains were used to produce astaxanthin using shake flask fermentation with different concentrations of flavonoids.

[0045] (1) Phaffia rhodozyma Xanthophyllomyces dendrorhous ) DW6 was inoculated into YM solid medium and cultured at 25°C for 2-3 days to activate the strain; (2) Seed cultivation, including: a. First generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 25°C and 160 rpm for 48 h; b. Second generation seed culture: Take the culture fluid of the first generation seed culture and inoculate it into a new seed culture medium at a 10% inoculation rate, and culture it at a constant temperature under the same conditions as a to obtain the seed solution for fermentation culture.

[0046] (3) The seed liquid obtained from the seed culture was inoculated into a Erlenmeyer flask containing fermentation medium for fermentation at 25°C and 160 rpm. Glucose was added daily from the second day of fermentation to a concentration of 30 g / L. The culture was continued for seven days, and the cell dry weight was determined. Astaxanthin in the fermentation liquid was extracted and the astaxanthin content was determined. The experiment was repeated.

[0047] On the second day of fermentation, 10-60 mg / L of baicalin and 10-40 mg / L of quercetin were added to the fermentation medium. Figure 2 As shown in the figure, after adding 50 mg / L of baicalin for seven days of fermentation, the astaxanthin production can reach 96.72 mg / L (the amount of astaxanthin produced per L of fermentation liquid), which is significantly increased by 16.4% compared with no addition, and the content is significantly increased by 38.5% compared with no addition. As the concentration of baicalin increases, the yield and biomass will gradually decrease. The increase in the concentration of baicalin will inhibit the growth and metabolic activity of the yeast strain. Figure 3 As shown, after seven days of fermentation with 20 mg / L of quercetin, the astaxanthin production can reach 106.95 mg / L (the amount of astaxanthin produced per L of fermentation liquid), which is 28.8% higher than without addition, and the content is 36.4% higher than without addition. With the increase of quercetin concentration, the yield and biomass will gradually decrease. The increase of quercetin addition concentration will inhibit the growth and metabolic activity of yeast strains. Example 3

[0048] Astaxanthin was produced by shake flask fermentation using Phaffia rhodozyma strain by adding baicalein and quercetin at different times.

[0049] (1) Phaffia rhodozyma Xanthophyllomyces dendrorhous ) DW6 was inoculated into YM solid medium and cultured at 25°C for 2-3 days to activate the strain; (2) Seed cultivation, including: a. First generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 25°C and 160 rpm for 48 h; b. Second generation seed culture: Take the culture fluid of the first generation seed culture and inoculate it into a new seed culture medium at a 10% inoculation rate, and culture it at a constant temperature under the same conditions as a to obtain the seed solution for fermentation culture.

[0050] (3) The seed solution obtained by seed culture was inoculated into a triangular flask containing fermentation medium for fermentation at 25°C and 160rpm. From the second day of fermentation, glucose was added every day to a concentration of 30 g / L. At the 0th, 24th, 48th, 72nd and 96th hours of fermentation, 50 mg / L baicalein and 20 mg / L quercetin were added. The culture was carried out for seven days, the cell dry weight was determined, astaxanthin in the fermentation broth was extracted, and the astaxanthin content was determined. The experiment was repeated. Figure 4As shown in the figure, when baicalein is added at 48h-96h of fermentation, the astaxanthin production is higher than that without addition. Preferably, baicalein is added at 48h-72h; more preferably, baicalein is added at 72h. When 50mg / L of baicalein is added at 72h of fermentation and fermentation is continued and terminated after seven days, the astaxanthin production can reach 105.45 mg / L (the amount of astaxanthin produced per L of fermentation liquid), which is significantly increased by 20.60% and the content is significantly increased by 35.18% compared with that without addition. With the increase of baicalein time, the production and biomass will gradually decrease. Adding baicalein at the beginning or in the late stage of fermentation will inhibit the growth and metabolic activity of yeast strains. Figure 5 As shown in the figure, when quercetin is added at 48h~96h of fermentation, the astaxanthin production is increased compared with that without addition. It is preferred to add quercetin at 48h~72h; more preferably, 20mg / L of quercetin is added at 72h, and fermentation is continued. After seven days, the fermentation is terminated, and the astaxanthin production can reach 117.16mg / L (the amount of astaxanthin produced per L of fermentation liquid), which is significantly increased by 33.98% compared with no addition, and the content is significantly increased by 34.59% compared with no addition. With the increase of quercetin addition time, the yield and biomass will gradually decrease. The addition of quercetin at the beginning (before 24h) or in the late fermentation (after 96h) will inhibit the growth and metabolic activity of the yeast strain. Example 4

[0051] The rhodozyma yeast strain produces astaxanthin using flavonoid fermentation tank fermentation.

[0052] (1) Phaffia rhodozyma Xanthophyllomyces dendrorhous ) DW6 was inoculated into YM solid medium and cultured at 25°C for 2-3 days to activate the strain; (2) Seed cultivation, including: a. First generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 25°C and 160 rpm for 48 h; b. Second generation seed culture: Take the culture fluid of the first generation seed culture and inoculate it into a new seed culture medium at a 10% inoculation rate, and culture it at a constant temperature under the same conditions as a to obtain the seed solution for fermentation culture.

[0053] (3) The seed solution obtained from seed culture was inoculated into a 2 L fermentation tank filled with fermentation medium at 25 °C and 500 rpm. From the second day of fermentation, glucose was added every day to a concentration of 30 g / L. From the 72nd hour to the 96th hour, 50 mg / L baicalein and 20 mg / L quercetin were added respectively. The culture was carried out for seven days, the cell dry weight was determined, and astaxanthin in the fermentation broth was extracted. The astaxanthin content was determined. The experiment was repeated. Figure 6 The yield in the fermentation tank with baicalein added was 809.61 mg / L, which was 41.37% higher than that of the control. Figure 7 The yield in the fermentation tank with quercetin added was 1324.97 mg / L, an increase of 131.31% over the control.

Claims

1. A method for producing astaxanthin using flavonoid compounds, characterized in that: The flavonoid compounds are prepared into a mother solution of a certain concentration, added into a fermentation medium, and the red Phaffia yeast is used as a fermentation strain to produce astaxanthin.

2. The method for producing astaxanthin using flavonoids according to claim 1, characterized in that: The flavonoid compound is at least one of quercetin and baicalein.

3. The method for producing astaxanthin using flavonoids according to claim 1, characterized in that: The red yeast is red yeast ( Xanthophyllomyces dendrorhous )DW6, whose accession number is CCTCC M2021732.

4. The method for producing astaxanthin using flavonoids according to claim 1, characterized in that: The concentration of flavonoid stock solution is 2~5g / L.

5. The method for producing astaxanthin using flavonoids according to claim 1, characterized in that: The flavonoid compound is added to the fermentation medium on the 2nd to 3rd day of the fermentation of Phaffia rhodozyma.

6. The method for producing astaxanthin using flavonoids according to claim 1, characterized in that: The flavonoids are dissolved in anhydrous ethanol to prepare a mother solution with a concentration of 2-5 g / L.

7. The method for producing astaxanthin using flavonoids according to claim 2, characterized in that: The added amount of quercetin was 20 mg / L.

8. The method for producing astaxanthin using flavonoids according to claim 2, characterized in that: The added amount of baicalein was 50 mg / L.

9. The method for producing astaxanthin using flavonoids according to claim 1, characterized in that: The fermentation medium formula is: 20-50 g / L carbon source, 15-25 g / L nitrogen source, and 1.5-5 g / L inorganic salt.

10. The method for producing astaxanthin using flavonoids according to claim 1, characterized in that: The fermentation conditions were: 25°C, 500 rpm for 7 days.

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