Method for promoting phaffia rhodozyma to produce astaxanthin

Abstract

The invention provides a method for promoting phaffia rhodozyma to produce astaxanthin, and belongs to the technical field of microbial fermentation. The invention provides a phaffia rhodozyma LX6, the phaffia rhodozyma LX6 is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC No. M 20242814, the phaffia rhodozyma LX6 is classified and named as the phaffia rhodozyma LX6, and the preservation time is December 16, 2024. The phaffia rhodozyma LX6 is named as the phaffia rhodozyma LX6. According to the fermentation method for improving the yield of the astaxanthin, provided by the invention, the yield of the biologically produced astaxanthin is promoted to be improved, the astaxanthin extracted by fermentation can be used for feeds, health-care products, cosmetics and the like, and the extracted thalli can be used as proteins to be added into animal feeds, so that the quality of the feeds is improved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to a method for promoting Phaffia rhodozyma to produce astaxanthin. Background Art

[0002] Astaxanthin is a natural keto-carotenoid 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 has many basic biological functions, including preventing the oxidation of essential polyunsaturated fatty acids and preventing the effects of ultraviolet rays, supporting good vision and eye health, and enhancing immune response. Due to its high antioxidant activity, it has been widely used in food additives, medicines, pigments or antioxidants, and nutritional foods.

[0003] Phaffia rhodozyma is currently the only native strain producing astaxanthin and is also one of the high-yield strains. The technical problem to be solved by the present invention is to further increase the production of astaxanthin by using Phaffia rhodozyma to meet the current demand for astaxanthin products. Summary of the invention

[0004] The object of the present invention is to provide a method for promoting the production of astaxanthin by the red yeast Phaffia rubrum by utilizing Tween-80.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A red Phaffia yeast (Xanthophyllomyces dendrorhous) LX6, the red Phaffia yeast LX6 is deposited in the China Center for Type Culture Collection (CCTCC), the address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the deposit number is CCTCC No. M 20242814, and the classification name is red Phaffia yeast LX6 Xanthophyllomyces dendrorhous LX6, preservation date is December 16, 2024.

[0006] The present invention also provides an application of the rhodozyma yeast LX6 in producing astaxanthin.

[0007] The present invention also provides a method for promoting the production of astaxanthin by the rhodozyma yeast LX6 by using Tween-80, characterized in that it comprises the following steps: (1) activating and culturing Phaffia rhodozyma LX6 to obtain seed liquid; (2) inoculating the seed liquid into a fermentation medium for fermentation, adding Tween-80 during the fermentation process, and obtaining astaxanthin after fermentation.

[0008] Preferably, the activation of Phaffia rhodozyma LX6 is carried out in YM solid medium at 23-27° C. for 2-3 days.

[0009] Preferably, the YM solid culture medium is formulated as follows: glucose 18-22 g / L, peptone 4-6 g / L, Yeast powder 2~4 g / L, malt extract 2~4 g / L, agar powder 18~22 g / L, and the rest is water.

[0010] Preferably, the culture is to first inoculate the activated Phaffia rhodozyma into a seed culture medium, The culture was carried out at 23-27° C. and 130-180 rpm for 36-72 hours, and the obtained culture solution was inoculated into a new seed culture medium at an inoculation rate of 8-12% (v / v). The culture was continued for 36-72 hours under the same conditions to obtain a seed solution having an OD value of 12-20.

[0011] Preferably, the volume ratio of the seed liquid to the fermentation medium in step (2) is 1:8-12. The fermentation is carried out at 23-27°C and 130-180 rpm for 5-8 days.

[0012] Preferably, the formula of the fermentation medium in step (2) is: glucose 18-22 g / L, sulfuric acid 1-2 g / L of ammonium, 1-2 g / L of potassium dihydrogen phosphate, 1-2 g / L of magnesium sulfate heptahydrate, 1-3 g / L of yeast powder, and 13-17 g / L of corn syrup powder. The formula of the fermentation medium is the same as the formula of the seed culture medium.

[0013] Preferably, the Tween-80 in step (2) is added at 0-72h of fermentation, and the added amount is 0.7~0.9% of the liquid volume.

[0014] Preferably, during the fermentation process of step (2), grapes are added daily from the 12th to the 24th hour. Sugar to concentration of 18~22 g / L.

[0015] The fermentation method for increasing astaxanthin production provided by the present invention promotes the improvement of astaxanthin production in biological production. The strain is fermented in a 2 L biological culture vessel, and after Tween-80 is added during the fermentation process, the permeability of the yeast strain is changed, the synthesis of astaxanthin is promoted, and the culture is continued while keeping other conditions unchanged. The astaxanthin content in the obtained product is 1123.162 mg / L, and the cell dry weight reaches 44 g / L. The astaxanthin extracted by fermentation can be used for feed, health products, cosmetics, etc., and the extracted bacteria can be added to animal feed as protein, which improves the quality of feed and alleviates the huge demand pressure for food in feed production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The content of astaxanthin produced by the red Phaffia yeast strain in Example 1 using different concentrations of Tween-80 shake flask fermentation; Figure 2 The content of astaxanthin produced by shaking flask fermentation of Phaffia rhodozyma after adding Tween-80 at different times in Example 2; Figure 3 The content of astaxanthin produced by the red Phaffia yeast strain in Example 3 using Tween-80 in a fermenter.

[0017] Deposit certificate

[0018] The red yeast LX6 is deposited in the China Center for Type Culture Collection (CCTCC), with the address of Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number of CCTCC No. M 20242814 and the classification name of red yeast LX6. Xanthophyllomyces dendrorhous LX6, preservation date is December 16, 2024. DETAILED DESCRIPTION

[0019] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0020] The culture medium formulas in the following examples are as follows: YM medium: 20 g / L glucose, 5 g / L peptone, 3 g / L yeast powder, 3 g / L malt extract, and the rest is water; YM solid medium is supplemented with 20 g / L agar powder. The preparation method is as follows: weigh 2 g glucose, 0.5 g peptone, 0.3 g yeast powder, and 0.3 g malt extract, dissolve in 100 mL distilled water, and adjust the pH to 5.5 ± 0.2 with 4M NaOH and 4M H3PO4. Add 2 g agar powder to the YM solid medium, shake well, and sterilize at 115 °C for 20 min.

[0021] Fermentation medium: glucose 20 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 syrup powder 15 g / L. Glucose is prepared into mother solution and sterilized separately.

[0022] The seed culture medium and fermentation culture medium have the same formula.

[0023] Phaffia rhodozyma strains ( Xanthophyllomyces dendrorhous ) LX6 from Phaffia rhodozyma ( Xanthophyllomyces dendrorhous)DW6 (accession number: CCTCC M 2021732), the red yeast LX6 is deposited in the China Center for Type Culture Collection (CCTCC), the address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the accession number is CCTCC No. M 20242814, and the classification name is red yeast LX6 Xanthophyllomyces dendrorhous LX6, preservation date is December 16, 2024.

[0024] The physical and chemical properties of the red yeast strain LX6 were tested in the following steps: (1) Microscopic examination: observe whether the yeast cell morphology undergoes obvious changes. If no obvious changes occur, it can be identified as strain LX6. (2) Product extraction, liquid phase qualitative and quantitative detection of the product, verifying the yield of the metabolite. When the yield of the mutant strain exceeds 10% of the original strain, it can be identified as the fermentation strain LX6. After comparison, the concentration of astaxanthin produced by DW6 was 330 mg / L, and the yield of the LX6 mutant strain was 420 mg / l, which was 30% higher than that of DW6, and was determined to be strain LX6; (3) Strain identification: Perform a strain PCR experiment to detect the identification gene of the red yeast strain. Use the universal primers ITS1 and ITS4 to send the sample for testing and identify the bacterium as the red yeast strain.

[0025] The above mutagenesis process was repeated 10 times, and the detection was performed according to the above steps. The physicochemical properties of the obtained Phaffia rhodozyma strain LX6 were the same, indicating that the above mutagenesis method can repeatedly obtain a stable target strain.

[0026] Example 1

[0027] Phaffia rhodozyma strains were used to produce astaxanthin by shake flask fermentation using different concentrations of Tween-80.

[0028] (1) Activation of the bacteria: Phaffia rhodozyma Xanthophyllomyces dendrorhous ) LX6 was inoculated into YM solid medium and cultured at 25 °C for 3 days; (2) Seed cultivation: the specific steps are as follows: a. First-generation seed culture: Take a single colony from the YM solid medium, inoculate it into the seed medium, and culture it at 25°C and 160rpm for 48 hours to obtain the first-generation seed solution; b. Second generation seed culture: Take the first generation seed solution and inoculate it into a new seed culture medium at a 10% (v / v) inoculation rate, and culture it at a constant temperature under the same conditions as a to obtain a seed solution for fermentation culture with an OD value of 15.

[0029] (3) The seed liquid obtained from the seed culture was inoculated into a triangular flask containing fermentation medium at a volume ratio of 1:10 for fermentation. The culture was carried out at 25°C and 160 rpm. Glucose was added daily to a concentration of 20 g / L starting from 24 hours of fermentation. Different concentrations of Tween-80 were added at the 48th hour of fermentation and cultured for seven days. The dry cell weight (DCW) was determined, astaxanthin in the fermentation liquid was extracted, and the astaxanthin content (Content) and yield (Production) were determined. The experiment was repeated.

[0030] The method for determining the dry weight of microbial cells and astaxanthin content is as follows: 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.

[0031] Astaxanthin extraction and determination method: Astaxanthin content was determined by UV spectrophotometry.

[0032] Take 1 mL of fermentation liquid 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 oscillation for 20-30s, then stand in the dark for 15 minutes, centrifuge at 4 ℃, 12000 rpm for 2 min, and measure the OD478 of the supernatant with a spectrophotometer. If the bacterial mud is still colored, it can be repeatedly extracted until it turns white. The astaxanthin content is calculated as follows: Astaxanthin (mg / L) =

[0033] Where: A—OD478nm 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 results are as follows Figure 1 As shown, after seven days of fermentation with the addition of 0.8% Tween-80, the astaxanthin content can reach 328.7 mg / L (the amount of astaxanthin produced per L of fermentation liquid). With the increase of Tween-80 concentration, the yield and content will gradually decrease; the difference in biomass is small, therefore, Tween-80 concentration exceeding 0.8% will inhibit the growth and metabolic activity of yeast strains.

[0034] Example 2

[0035] Astaxanthin was produced by shaking flask fermentation of Phaffia rhodozyma LX6 by adding Tween-80 at different times.

[0036] According to the steps of Example 1, a seed solution (OD value of 15) was obtained, and the seed solution was inoculated into a triangular shake flask filled with fermentation medium at a volume ratio of 1:10 for fermentation, and cultured at 25°C and 160 rpm. 0.8% Tween-80 was added at 0, 24, 48, 72, 96, and 120 h of fermentation, respectively. The fermentation culture was carried out for 7 days, and glucose was added every day from 24 h of fermentation to a concentration of 20 g / L. The fermentation liquid on the 7th day was taken for extraction treatment. The cell dry weight was determined, astaxanthin in the fermentation liquid was extracted, and the astaxanthin content was determined. The experiment was repeated, and the results were as follows: Figure 2 As shown, after 48 hours of fermentation, the astaxanthin content reached 563.287 mg / L (the amount of astaxanthin produced per L of fermentation liquid).

[0037] Example 3

[0038] The seed solution (OD value is 15) was obtained according to the steps of Example 1, and the seed solution was inoculated into a 2 L fermentation tank filled with fermentation medium at a volume ratio of 1:10, 25 ° C, 500 rpm, and the glucose concentration in the fermentation liquid was ensured to be not less than 10 g / L every day during the fermentation process. 0.8% Tween-80 was added at 48 h of fermentation to continue fermentation and culture. The cell dry weight was determined for seven days, astaxanthin in the fermentation liquid was extracted, and the astaxanthin content was determined. The experiment was repeated, and the results were as follows: Figure 3 As shown, the astaxanthin production can reach 1.123 g / L (the amount of astaxanthin produced per L of fermentation liquid).

[0039] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A red yeast ( Xanthophyllomyces dendrorhous ) LX6, characterized in that The red yeast LX6 is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC No. M 20242814 and is classified as red yeast LX6. Xanthophyllomyces dendrorhous LX6, preservation date is December 16, 2024.

2. Use of the rhodozyma yeast LX6 according to claim 1 in producing astaxanthin.

3. A method for promoting the production of astaxanthin by the rhodozyma yeast LX6 according to claim 1 by using Tween-80, characterized in that: The steps include: (1) activating and culturing Phaffia rhodozyma LX6 to obtain seed liquid; (2) The seed liquid is inoculated into a fermentation medium for fermentation, Tween-80 is added during the fermentation process, and astaxanthin is obtained after fermentation.

4. The method according to claim 3, characterized in that The activation of Phaffia rhodozyma LX6 is carried out in YM solid culture medium at 23-27° C. for 2-3 days.

5. The method according to claim 4, characterized in that The formula of the YM solid culture medium is: 18-22 g / L of glucose, 4-6 g / L of peptone, 2-4 g / L of yeast powder, 2-4 g / L of malt extract, 18-22 g / L of agar powder, and the rest is water.

6. The method according to claim 5, characterized in that The culture is to first inoculate the activated red Phaffia yeast into a seed culture medium, culture it at 23-27° C. and 130-180 rpm for 36-72 hours, and then inoculate the obtained culture solution into a new seed culture medium at an inoculation amount of 8-12% (v / v), and continue to culture for 36-72 hours under the same conditions to obtain a seed solution, wherein the OD value of the seed solution is 12-20.

7. The method according to claim 6, characterized in that In step (2), the volume ratio of the seed liquid to the fermentation medium is 1:8-12, and the fermentation is carried out at 23-27°C and 130-180 rpm for 5-8 days.

8. The method according to claim 7, characterized in that The formula of the fermentation medium in step (2) is: 18-22 g / L of glucose, 1-2 g / L of ammonium sulfate, 1-2 g / L of potassium dihydrogen phosphate, 1-2 g / L of magnesium sulfate heptahydrate, 1-3 g / L of yeast powder, and 13-17 g / L of corn syrup powder. The formula of the fermentation medium is the same as the formula of the seed culture medium.

9. The method according to claim 8, characterized in that The Tween-80 in step (2) is added between the 0th and 72nd hours of fermentation, and the added amount is 0.7-0.9% of the volume of the fermentation liquid.

10. The method according to claim 9, characterized in that During the fermentation process of step (2), glucose is supplemented every day from the 12th to the 24th hour to a concentration of 18 to 22 g / L.

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