Heat-resistant phaffia rhodozyma and method for producing astaxanthin from straw hydrolysate by using heat-resistant phaffia rhodozyma

Abstract

The invention discloses heat-resistant phaffia rhodozyma and a method for producing astaxanthin from straw hydrolysate by using the heat-resistant phaffia rhodozyma. The strain is classified and named as phaffia rhodozyma GF6, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M 2025649; the strain is inoculated into a fermentation culture medium, straw hydrolysate is used as a carbon source, and astaxanthin is efficiently produced at 28-30 DEG C; according to the method, the agricultural waste straw hydrolysate is used as a cheap crude raw material to replace all carbon sources to produce astaxanthin, so that the fermentation temperature is increased, the production cost is reduced, and the method is environment-friendly.

Description

Technical Field

[0001] The present invention belongs to the field of fermentation engineering, and particularly relates to a thermotolerant Phaffia rhodozyma and a method for producing astaxanthin by using straw hydrolysate. Background Art

[0002] As a natural carotenoid, astaxanthin has strong free radical scavenging activity, and can protect against lipid peroxidation and oxidative damage to low-density lipoprotein-cholesterol, cell membranes, cells and tissues. In addition, astaxanthin also has many basic biological functions, including preventing the oxidation of essential polyunsaturated fatty acids and preventing ultraviolet effects, supporting good vision and eye health, and enhancing the immune response. Due to its high antioxidant activity, it has been widely used in food additives, pigments or antioxidants, and nutritional foods.

[0003] The method of producing astaxanthin by fermentation of Phaffia rhodozyma has the advantages of environmental protection, harmlessness, low cost, high safety factor, etc., which makes the degree of attention received by this method higher and higher. The most important factor restricting the industrial production of this method is the cost factor. Firstly, the fermentation temperature of Phaffia rhodozyma is too low (18 - 22 °C), so a large amount of electric energy is required to cool down during the fermentation process. In addition, because the cost of the culture medium used to cultivate Phaffia rhodozyma is relatively high, especially the carbon source, and the carbon source consumed during the fermentation process is the most. In traditional methods, glucose and the like are used as the basic fermentation carbon source, but the price of glucose is relatively expensive. Therefore, finding a high-quality carbon source with a relatively low price, a wide source, and being beneficial to fermentation is the key problem that urgently needs to be solved in the production of astaxanthin by Phaffia rhodozyma at present.

[0004] Converting straw into hydrolysate for fermentation realizes the resource utilization of straw, reduces the environmental pressure of straw waste, and has good environmental benefits. Straw is rich in polysaccharide substances such as cellulose and hemicellulose. Through hydrolysis treatment, these macromolecular substances can be converted into small molecular sugars that can be utilized by microorganisms, realizing the efficient utilization of biomass resources, improving the recycling rate of resources, and conforming to the concept of sustainable development. Preparing hydrolysate from straw can greatly reduce the fermentation cost compared with using traditional pure carbon sources such as glucose and sucrose, providing an economically feasible way for large-scale industrial fermentation production. Summary of the Invention

[0005] The object of the present invention is to provide a thermotolerant Phaffia rhodozyma strain that can produce astaxanthin using straw hydrolysate at 28 - 30 °C.

[0006] The technical solution adopted by the present invention is as follows: A thermotolerant Phaffia rhodozyma strain, classified and named as Phaffia rhodozyma GF6 Xanthophyllomyces dendrorhousGF6 was deposited at the China Center for Type Culture Collection on March 31, 2025. Address: Wuhan University, Wuhan, China. The deposit number is CCTCC NO: M 2025649.

[0007] After culturing on solid medium for 3 - 5 days, the colonies are round with neat edges and a diameter of about 1 - 3 mm. Due to the accumulation of astaxanthin, the colony color is dark coral red, the surface is smooth and moist, and the texture is viscous. Under the microscope, the vegetative cells are oval, and the single - cell size is about 3 - 6 × 5 - 10 μm, with a relatively large size. This strain has good heat resistance and can grow and ferment at 28 - 30 °C.

[0008] The heat - resistant Phaffia rhodozyma is obtained by using Phaffia rhodozyma ( Xanthophyllomyces dendrorhous ) LX6 as the starting strain, through ARTP mutagenesis, NTG mutagenesis, and adaptive domestication.

[0009] The starting strain has been disclosed in the applicant's prior patent application CN119709447A, and the deposit number is CCTCC NO: M 20242814.

[0010] Application of the heat - resistant Phaffia rhodozyma GF6 in the fermentation production of astaxanthin.

[0011] The application is to activate the strain of Phaffia rhodozyma GF6, conduct seed culture, and then inoculate it into the fermentation medium to ferment and produce astaxanthin.

[0012] The formula of the fermentation medium is: carbon source 25 - 100 g / L, nitrogen source 5 - 30 g / L, inorganic salts 3 - 10 g / L.

[0013] The carbon source includes but is not limited to glucose, fructose, sucrose, straw hydrolysate, maltose, starch, molasses, glycerol, etc., and is further preferably straw hydrolysate.

[0014] The nitrogen source includes but is not limited to ammonium sulfate, ammonium nitrate, urea, yeast extract, corn steep liquor powder, urea, peanut cake powder, peptone, soybean powder, etc.

[0015] The inorganic salts include but are not limited to potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, sodium hydrogen phosphate, etc.

[0016] Further preferably, the fermentation medium is: glucose 25 - 50 g / L, yeast extract 1 - 3 g / L, ammonium sulfate 1 - 3 g / L, corn steep liquor powder 5 - 20 g / L, magnesium sulfate heptahydrate 1 - 3 g / L, potassium dihydrogen phosphate 1 - 3 g / L, sodium hydrogen phosphate 1 - 3 g / L.

[0017] Further preferably, the fermentation medium is: 100 g / L straw hydrolysate, 1.5 g / L ammonium sulfate, 1.5 g / L yeast extract, 10 g / L corn steep liquor powder, 1.5 g / L magnesium sulfate heptahydrate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L disodium hydrogen phosphate. Sterilize at 115 °C for 20 min.

[0018] The fermentation conditions are: fermentation temperature 28 - 30 °C, fermentation time is 7 days.

[0019] Preferably, a fermentation promoter is added at 24 - 72 h of fermentation. The promoter is a metal ion or vegetable oil, and preferably added at 48 h of fermentation.

[0020] The metal ion is copper ion, and the addition amount is 0.2 - 0.7 μmol / L; Preferably, copper sulfate is added at 24 - 72 h of fermentation, and the addition amount of copper sulfate is 0.2 - 0.7 μmol / L. More preferably, the addition amount of copper sulfate is 0.6 μmol / L.

[0021] Preferably, vegetable oil is added at 24 - 72 h of fermentation, and the addition amount of vegetable oil is 0.5 - 1.5%. More preferably, the addition amount of the vegetable oil is 0.5%.

[0022] The vegetable oil is corn oil or sunflower oil, and preferably added at 48 h of fermentation.

[0023] The straw hydrolysate is wheat straw hydrolysate, mainly containing fermentable sugars such as sucrose, glucose, and arabinose, and the content of its total reducing sugar is 200 - 500 g / L.

[0024] More specifically, it includes the following steps: (1) Inoculate Pichia rhodozyma GF6 into YM solid medium and culture at 28 °C for 2 - 3 days for strain activation; (2) Take a single colony from the YM solid medium, inoculate it into the seed medium, and culture at 28 °C and 160 rpm for 48 h to obtain the seed liquid for fermentation culture.

[0025] (3) Inoculate the seed liquid into the fermentation medium for fermentation, at 28 - 30 °C and 160 rpm, and culture for 7 days.

[0026] The fed-batch feeding method can be adopted. Specifically, after the heat-resistant Phaffia rhodozyma is subjected to seed culture, it is inoculated into a fermentation tank containing straw hydrolysate fermentation medium. The fermentation is carried out at 28 - 30 °C and 500 rpm. From the second day of fermentation, the straw hydrolysate is supplemented by the fed-batch method. The flow rate is 10 ml / h from 32 - 48 h, 6 ml / h from 48 - 72 h, and 4 ml / h from 72 - 168 h. The culture lasts for 7 days. The pH is controlled at 6.5 in the first 72 h and 5.5 after 72 h. 0.6 μmol of copper sulfate is added at 48 h of fermentation.

[0027] Beneficial effects:

[0028] In the present invention, a heat-resistant Phaffia rhodozyma strain is screened, which can use straw hydrolysate, an agricultural waste, as a carbon source to produce astaxanthin at 28 - 30 °C. It uses inexpensive crude raw materials to replace all carbon sources for astaxanthin production, raises the fermentation temperature, reduces production costs, and is environmentally friendly. The fermentation temperature of this strain is 3 - 5 °C higher than that of the original strain, achieving the efficient synthesis of astaxanthin at 28 - 30 °C. While the temperature is raised, the synthesis ability of astaxanthin by the strain under high-temperature conditions is enhanced. The astaxanthin yield and content in the shake flask scale are 5.4 times and 2.2 times higher than those of the original strain at 28 °C respectively. Through fermentation tank scale-up, the astaxanthin production level is further improved, realizing the efficient synthesis of astaxanthin under high-temperature conditions.

[0029] In the present invention, an efficient astaxanthin production process for a heat-resistant Phaffia rhodozyma strain at 28 - 30 °C is established by exogenous addition of astaxanthin production promoters (copper ions, corn oil) and process optimization. Based on this process, pilot-scale fermentation production is carried out, and the astaxanthin yield and content reach 1744.5 mg / L and 20.5 mg / g respectively. Description of the drawings

[0030] Figure 1 It is a graph showing the improvement of the dry weight, astaxanthin yield and content of the mutant strain with the acclimation time through adaptive acclimation; Figure 2 It is a graph showing the final strain stability test of the mutant strain after adaptive acclimation; Figure 3 It is a comparative graph of the shake flask fermentation results of the heat-resistant strain GF6 using glucose and straw hydrolysate respectively; Figure 4 It is a comparison result of the shake flask fermentation production of astaxanthin by the heat-resistant strain GF6 with different metal ions added exogenously at 28 °C; Figure 5 It is the optimization of the copper ion addition amount added exogenously to the heat-resistant strain GF6 at 28 °C; Figure 6 It is the optimization of the copper ion addition time added exogenously to the heat-resistant strain GF6 at 28 °C; Figure 7Comparison of shake - flask fermentation of heat - resistant strain GF6 for astaxanthin production with exogenous addition of vegetable oil at 28°C; Figure 8 Optimization of the addition amount of corn oil added exogenously to heat - resistant strain GF6 at 28°C; Figure 9 Optimization of the addition time of corn oil added exogenously to heat - resistant strain GF6 at 28°C; Figure 10 Results of small - scale fermentation of heat - resistant strain GF6 in a 5 - L fermenter at 28°C; Figure 11 Results of pilot - scale amplification fermentation of heat - resistant strain GF6 in a 50 - L fermenter at 30°C. Specific implementation mode

[0031] The following examples illustrate the present invention in detail.

[0032] In the examples, the methods for measuring the dry weight of microbial cells and the astaxanthin content are as follows: Method for measuring the dry weight of microbial cells: Take an appropriate amount of fermentation broth with a pre - weighed centrifuge tube, centrifuge and wash twice, then place it in an oven at 65°C and dry to a constant weight.

[0033] Method for extraction and determination of astaxanthin in the examples: The content of astaxanthin is determined by ultraviolet spectrophotometry.

[0034] Take 1 mL of fermentation broth, centrifuge at 12000 rpm for 2 min, wash twice with deionized water, collect the cells, dry the water droplets on the tube wall with filter paper, add 1 mL of dimethyl sulfoxide (DMSO) pre - heated to 55°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, centrifuge at 4°C and 12000 r / min for 2 min, and measure the OD474 of the supernatant with a spectrophotometer. If the cell pellet is still colored, repeat the extraction until it becomes white. The calculation formula for the astaxanthin content is as follows: Astaxanthin (mg / L)=(A×V1×10000) / (2150×V2 ) Where: A—OD474 value; V1—Total volume of organic solvent (mL); 2150—Specific extinction coefficient; V2—Volume of fermentation broth (mL); 10000: Unit conversion coefficient (mg / L).

[0035] The straw hydrolysis broth described in the following examples is provided by Suzhou Polyway Original Company.

[0036] The method for measuring the total reducing sugar in the straw hydrolysis broth in the present invention is: DNS method, and the specific steps are as follows: First, draw a glucose standard curve: Take 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose standard solution (1 mg / mL) into 20 mL EP tubes respectively. Accurately add 2.5 mL of DNS reagent to each tube. Boil in a water bath at 100°C for 5 min. After cooling, make up to 20 mL with pure water. Measure the absorbance at a wavelength of 540 nm. The obtained glucose standard curve is: y = 3.008x - 0.1653, R2 = 0.9906; Then, measure the supernatant of the fermentation broth. Dilute the fermentation broth supernatant 100 times. Take 1.0 mL of the diluted sugar solution into a 20 mL EP tube, add 2.5 mL of DNS reagent, boil in a water bath at 100°C for 5 min. After cooling, make up to 20 mL with water. Measure the absorbance at a wavelength of 540 nm. Substitute it into the glucose standard curve. By calculation, the total reducing sugar content in the straw hydrolysate is 251 g / L.

[0037] The supernatant obtained by centrifuging the straw hydrolysate at 10000 rpm for 8 min is used as the culture medium solvent. Add ammonium sulfate, yeast extract, corn steep liquor powder, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, and disodium hydrogen phosphate to prepare the fermentation medium.

[0038] Example 1

[0039] Use Rhodotorula rubra LX6 to mutagenize and breed a heat-resistant and high-yield astaxanthin-producing mutant strain of Rhodotorula rubra (1) Cultivate the starting strain LX6 of Rhodotorula rubra until the OD600 of the bacterial liquid is 1.8 - 2.2. Collect the bacterial cells for ARTP mutagenesis. When performing ARTP mutagenesis, the power of helium is 100 - 150 w, the gas flow rate is 8 - 12 SLM, the mutagenesis distance is 1.5 - 2.5 mm, and the mutagenesis time is 60 - 70 s. Collect the mutagenized strains and culture them overnight at 27°C. Further screen out the mutant strains that can synthesize astaxanthin at 27°C by flow cytometry. The sorting conditions of the flow cytometry are: forward scatter (FSC, 175), side scatter (SSC, 211), screening channels FL1 (568 nm) and FL2 (569 nm).

[0040] (2) Cultivate the sorted mutant strains at 27°C until the OD600 of the bacterial liquid is 1.8 - 2.2. Collect the bacterial cells for NTG mutagenesis. The concentration of NTG is 1.8 - 2.3 mmol / L, and the treatment time of NTG is 13 - 18 min. Culture overnight at 28°C, and screen out the mutant strains that can produce astaxanthin at 28°C by flow cytometry.

[0041] (3) Adaptation and domestication at 28°C: Continuously subculture the mutant strains selected at 28°C in the presence of 10 mmol / L H2O2 at 28°C to obtain a high-yield astaxanthin mutant strain at 28°C. As Figure 1The adaptive domestication shown above further enhanced the growth of the strain at 28 °C and the production of astaxanthin. After 960 hours of adaptive domestication, the astaxanthin yield increased from 118.9 mg / L to 161.9 mg / L. The mutagenized strain was named Phaffia rhodozyma GF6 Xanthophyllomyces dendrorhous GF6 has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2025649

[0042] (3)Stability test of mutagenized strain at 28 °C Inoculate the selected mutagenized strain GF6 onto YM solid medium and culture it at 25 °C for 3 days for strain activation; pick a single colony with a darker color from the YM solid medium and inoculate it into liquid YM medium at 28 °C and culture it at a constant temperature of 200 rpm for 48 h; inoculate it into a 500 mL flask containing 50 mL of YM liquid medium at an inoculation amount of 10% and culture it at 28 °C and 200 rpm for 48 h. Each time, inoculate the bacterial liquid into fresh YM liquid medium at an inoculation amount of 10% and subculture it continuously for 8 times to test the production and growth performance of the mutagenized strain. As Figure 2 shown, the heat-resistant strain GF6 can stably produce about 160 mg / L of astaxanthin at the shake-flask scale at 28 °C

[0043] The starting strain hardly grew at 28 °C, and the biomass could only reach 2.1 g / L after five days of shake-flask fermentation, with an astaxanthin yield of 11.3 mg / L. After mutagenesis combined with adaptive domestication, its biomass and astaxanthin yield were increased to 13.5 g / L and 161.9 mg / L respectively

[0044] Example 2

[0045] Fermentation production of astaxanthin by the mutagenized strain GF6 of Phaffia rhodozyma using straw hydrolysate at the shake-flask scale (1)Inoculate Phaffia rhodozyma GF6 onto YM solid medium and culture it at 28 °C for 2 - 3 days for strain activation (2)Seed culture, including a. First-generation seed culture: Pick a single colony from the YM solid medium and inoculate it into the seed medium at 28 °C and culture it at a constant temperature of 160 rpm for 48 h b. Second-generation seed culture: Take the culture solution of the first-generation seed culture and inoculate it into a new seed medium at an inoculation amount of 10%, and culture it at the same conditions as in a to obtain the seed liquid for fermentation culture

[0046] (3)Inoculate the seed liquid obtained from seed culture into a triangular flask containing straw hydrolysate fermentation medium and ferment it at 28 °C and 160 rpm for 7 days. Determine the dry cell weight, extract astaxanthin from the fermentation broth, and determine the astaxanthin content. Repeat the experiment, as Figure 3As shown, the astaxanthin content can reach 160.8 mg / L after 7 days of fermentation, and the astaxanthin content can also reach 12.8 mg / g, which is comparable to that using glucose as the carbon source. Therefore, the straw hydrolysate can be used as a carbon source to replace glucose for fermentative production of astaxanthin.

[0047] The fermentation medium using the straw hydrolysate as the carbon source is as follows: 100 g / L straw hydrolysate, 1.5 g / L ammonium sulfate, 1.5 g / L yeast extract, 10 g / L corn steep liquor powder, 1.5 g / L magnesium sulfate heptahydrate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L disodium hydrogen phosphate. Sterilize at 115 °C for 20 min.

[0048] Example 3

[0049] Comparison of astaxanthin production by shake flask fermentation of the mutant strain GF6 of Phaffia rhodozyma at 28 °C with different metal ions added externally (1) Inoculate Phaffia rhodozyma GF6 into the YM solid medium and culture at 28 °C for 2 - 3 days for strain activation. (2) Seed culture, including: a. First-generation seed culture: Take single colonies from the YM solid medium and inoculate them into the seed medium. Incubate at 28 °C and 160 rpm for 48 h. b. Second-generation seed culture: Inoculate the culture solution from the first-generation seed culture into a new seed medium at an inoculation amount of 10% and incubate under the same conditions as in a to obtain the seed solution for fermentation culture.

[0050] (3) Inoculate the seed solution obtained from seed culture into a triangular flask containing a 100 g / L straw hydrolysate fermentation medium (100 g / L straw hydrolysate, 1.5 g / L ammonium sulfate, 1.5 g / L yeast extract, 10 g / L corn steep liquor powder, 1.5 g / L magnesium sulfate heptahydrate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L disodium hydrogen phosphate, sterilized at 115 °C for 20 min) for fermentation. Add 1% of cobalt ions (cobalt chloride), calcium ions (calcium chloride), barium ions (barium chloride), ferrous ions (ferrous sulfate), ferric ions (ferric chloride), manganese ions (manganese chloride), zinc ions (zinc chloride), copper ions (copper chloride), nickel ions (nickel chloride) respectively at 24 h. Extract astaxanthin from the fermentation broth and measure the astaxanthin content. Repeat the experiment. As Figure 4 shown, the experimental group with copper ions added has the best results after 7 days of fermentation. The astaxanthin yield can reach 185.5 mg / L and the content can reach 14.6 mg / g, which are increased by 19.7% and 18.7% respectively compared with the control group without adding metal ions, effectively promoting the synthesis of astaxanthin.

[0051] Example 4

[0052] Optimization of the Addition Amount of Exogenous Copper Ions in Mutant Strain GF6 of Phaffia rhodozyma at 28°C (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture it at 28°C for 2 - 3 days for strain activation; (2) Seed culture, including: a. Primary seed culture: Pick single colonies from the YM solid medium, inoculate them into the seed medium, and culture them at a constant temperature of 28°C and 160 rpm for 48 h; 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 culture it at a constant temperature under the same conditions as in a to obtain the seed solution for fermentation culture.

[0053] (3) Inoculate the seed solution obtained from seed culture into a triangular flask containing 100 g / L straw hydrolysate fermentation medium (100 g / L straw hydrolysate, 1.5 g / L ammonium sulfate, 1.5 g / L yeast extract, 10 g / L corn steep liquor dry powder, 1.5 g / L magnesium sulfate heptahydrate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L disodium hydrogen phosphate, sterilized at 115°C for 20 min) for fermentation. Add 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 μM copper ions respectively when culturing for 24 h, extract astaxanthin from the fermentation broth, and measure the astaxanthin content. Repeat the experiment. As Figure 5 , the results show that the effect of adding 0.6 μM copper ions is the best, and the astaxanthin yield and content reach 197.4 mg / L and 15.3 mg / g respectively, which are 22.5% and 24.4% higher than those of the control group without adding metal ions, further improving the level of astaxanthin production by heat-resistant strain GF6 at 28°C.

[0054] Example 5

[0055] Optimization of the Addition Time of Exogenous Copper Ions in Mutant Strain GF6 of Phaffia rhodozyma at 28°C (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture it at 28°C for 2 - 3 days for strain activation; (2) Seed culture, including: a. Primary seed culture: Pick single colonies from the YM solid medium, inoculate them into the seed medium, and culture them at a constant temperature of 28°C and 160 rpm for 48 h; 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 culture it at a constant temperature under the same conditions as in a to obtain the seed solution for fermentation culture.

[0056] (3)The seed liquid obtained by seed culture was inoculated into a triangular flask containing a 100 g / L straw hydrolysate fermentation medium (100 g / L straw hydrolysate, 1.5 g / L ammonium sulfate, 1.5 g / L yeast extract, 10 g / L corn steep liquor powder, 1.5 g / L magnesium sulfate heptahydrate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L disodium hydrogen phosphate, sterilized at 115 °C for 20 min) for fermentation. Cupric ions were added at 0, 24, 48, 72, 96, and 120 h of fermentation respectively. Astaxanthin was extracted from the fermentation broth, and the astaxanthin content was measured. The experiment was repeated. As Figure 6 , the addition time of cupric ions also had a significant effect on the astaxanthin production of the mutant strain GF6. The optimal addition time was after 48 h of fermentation. The yield and content of astaxanthin of the strain reached 210.2 mg / L and 16.3 mg / g respectively. On the basis of optimizing the addition amount, further improvement was obtained, which was 30.4% and 33.6% higher than that of the control group without adding metal ions respectively. Thus, we determined that cupric ions could be used as an astaxanthin production promoter to improve the astaxanthin production level of the heat-resistant strain GF6 at 28 °C, and the addition of 0.6 μM at 48 h of fermentation was the best.

[0057] Example 6

[0058] Comparison of astaxanthin production by shake flask fermentation of the mutant strain GF6 of Phaffia rhodozyma with exogenous addition of vegetable oil at 28 °C (1)Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28 °C for 2 - 3 days for strain activation; (2)Seed culture, including: a. Primary seed culture: Pick a single colony from the YM solid medium and inoculate it into the seed medium, and culture at 28 °C and 160 rpm for 48 h; b. Secondary seed culture: Inoculate the culture solution of the primary seed culture into a new seed medium at an inoculation amount of 10%, and culture at the same conditions as a to obtain the seed liquid for fermentation culture.

[0059] (3)The seed liquid obtained by seed culture was inoculated into a triangular flask containing a 100 g / L straw hydrolysate fermentation medium (100 g / L straw hydrolysate, 1.5 g / L ammonium sulfate, 1.5 g / L yeast extract, 10 g / L corn steep liquor powder, 1.5 g / L magnesium sulfate heptahydrate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L disodium hydrogen phosphate, sterilized at 115 °C for 20 min) for fermentation. 1% of corn oil, sunflower oil, and soybean oil were added at 24 h respectively. Astaxanthin was extracted from the fermentation broth, and the astaxanthin content was measured. The experiment was repeated. As Figure 7As shown in the figure, the experimental group with corn oil added 7 days after fermentation had the best results. The astaxanthin yield reached 195.8 mg / L and the content reached 15.4 mg / g, which were 21.5% and 26.2% higher than those of the control group without added vegetable oil, respectively. This indicates that corn oil can effectively promote the production of astaxanthin by the heat-resistant strain GF6 at 28°C as a plant oil.

[0060] Example 7

[0061] Optimization of the addition amount of exogenous corn oil for the mutant strain GF6 of Phaffia rhodozyma at 28°C (1) Inoculate the Phaffia rhodozyma GF6 into the YM solid medium and culture it at 28°C for 2 - 3 days for strain activation. (2) Seed culture, including: a. First-generation seed culture: Take a single colony from the YM solid medium and inoculate it into the seed medium at 28°C and culture it at a constant temperature of 160 rpm for 48 h. b. Second-generation seed culture: Inoculate the culture solution from the first-generation seed culture into a new seed medium at an inoculation amount of 10% and culture it at a constant temperature under the same conditions as in a to obtain the seed solution for fermentation culture.

[0062] (3) Inoculate the seed solution obtained from seed culture into a triangular flask containing a fermentation medium of 100 g / L straw hydrolysate (100 g / L straw hydrolysate, 1.5 g / L ammonium sulfate, 1.5 g / L yeast extract, 10 g / L corn steep liquor dry powder, 1.5 g / L magnesium sulfate heptahydrate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L disodium hydrogen phosphate, sterilized at 115°C for 20 min) and ferment. Add 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3% (v / v) of corn oil at 24 h, as Figure 8 , and the results show that the addition amount of corn oil has a significant impact on the production of astaxanthin. Adding too much corn oil has a negative impact on astaxanthin production, and the effect of adding 0.5% (v / v) corn oil is the best. The yield and content of astaxanthin have been further improved, reaching 210.1 mg / L and 16.8 mg / g respectively, which are 30.3% and 38% higher than those of the control group without added vegetable oil.

[0063] Example 8

[0064] Optimization of the addition time of exogenous corn oil for the mutant strain GF6 of Phaffia rhodozyma at 28°C (1) Inoculate the Phaffia rhodozyma GF6 into the YM solid medium and culture it at 28°C for 2 - 3 days for strain activation. (2) Seed culture, including: a. First-generation seed culture: Take a single colony from the YM solid medium and inoculate it into the seed medium at 28°C and culture it at a constant temperature of 160 rpm for 48 h. b. Secondary seed culture: Inoculate the culture broth 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 liquid for fermentation culture.

[0065] (3) Inoculate the seed liquid obtained from seed culture into a triangular flask containing a fermentation medium of 100 g / L straw hydrolysate (100 g / L straw hydrolysate, 1.5 g / L ammonium sulfate, 1.5 g / L yeast extract, 10 g / L corn steep liquor powder, 1.5 g / L magnesium sulfate heptahydrate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L disodium hydrogen phosphate, sterilized at 115 °C for 20 min) for fermentation. Add 0.5% corn oil at 0, 24, 48, 72, 96, and 120 h of fermentation respectively, measure the astaxanthin content, and repeat the experiment. As Figure 9 shown, adding at 48 h is the optimal time. Through the optimization of the addition time, the production level of astaxanthin is further improved, and the yield and content of astaxanthin reach 223.3 mg / L and 18.1 mg / g respectively, which are 38.5% and 48.1% higher than those of the control group without adding vegetable oil. Thus, we determined that corn oil can be used as an astaxanthin production promoter to improve the astaxanthin production level of the thermotolerant strain GF6 at 28 °C, and adding 0.5% (v / v) at 48 h of fermentation is the best.

[0066] Example 9

[0067] Establishment of the process for fermentative production of astaxanthin by the thermotolerant strain GF6 of Phaffia rhodozyma using straw hydrolysate and exogenous additives in a fermenter.

[0068] (1) Inoculate Phaffia rhodozyma GF6 into the YM solid medium and culture at 28 °C for 2 - 3 days for strain activation; (2) Seed culture, including: a. Primary seed culture: Take a single colony from the YM solid medium and inoculate it into the seed medium at 28 °C and perform constant-temperature culture at 160 rpm for 48 h; b. Secondary seed culture: Inoculate the culture broth 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 liquid for fermentation culture.

[0069] (3) The seed liquid obtained from seed culture was inoculated into a 5 L fermenter containing a straw hydrolysate fermentation medium. At 28 °C and 500 rpm, the straw hydrolysate was supplemented by a feeding method. The flow rate was 10 ml / h from 32 - 48 h, 6 ml / h from 48 - 72 h, and 4 ml / h from 72 - 168 h, and the culture was carried out for 7 days. At 48 h of fermentation, 0.6 μM copper ions and 0.5% corn oil were added respectively to promote astaxanthin production. During the fermentation process, the pH was controlled at 6.5 in the first 72 h and at 5.5 after 72 h, and a two-stage pH method was used for fermentation, as Figure 10 Its astaxanthin yield reached 828.2 mg / L, the content reached 20.2 mg / g, and the biomass reached 41 g / L.

[0070] Example 10 Industrial pilot test on the fermentation production of astaxanthin by the thermotolerant strain GF6 of Phaffia rhodozyma using straw hydrolysate and exogenous additives.

[0071] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28 °C for 2 - 3 days for strain activation; (2) Seed culture, including: 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 28 °C and 160 rpm for 48 h; b. Secondary seed culture: Take the culture solution from the primary seed culture and inoculate it into a new seed medium at an inoculation amount of 10%, and culture at a constant temperature under the same conditions as in a to obtain the seed liquid for fermentation culture.

[0072] (3) The seed liquid obtained from seed culture was inoculated into a 50 L fermenter containing a straw hydrolysate fermentation medium. At 30 °C, the DO was controlled at 30 - 40%. The straw hydrolysate was supplemented by a feeding method. The flow rate was 20 ml / h from 12 - 48 h, 15 ml / h from 48 - 72 h, and 10 ml / h from 72 - 168 h. At 48 h of fermentation, 5 g / L yeast powder and 10 g / L corn steep liquor dry powder were supplemented. At 48 h of fermentation, 0.6 μM copper ions and 0.5% corn oil were added externally to promote astaxanthin production. The culture was carried out for 7 days. The pH was controlled at 6.5 in the first 72 h and at 5.5 after 72 h, and a two-stage pH method was used for fermentation, as Figure 11 Its astaxanthin yield reached 1744.5 mg / L, the content reached 20.5 mg / g, and the biomass reached 85.1 g / L. The pilot test results show that the thermotolerant strain has the potential for industrial scale-up, and this astaxanthin level has met the requirements of industrial production.

Claims

1. A heat-resistant Phaffia rhodozyma, characterized in that, Its classification name is Phaffia rhodozyma GF6 Xanthophyllomyces dendrorhous GF6, and its deposit number is CCTCC NO: M 2025649.

2. Use of the thermotolerant Phaffia rhodozyma according to claim 1 in the fermentation production of astaxanthin.

3. The application according to claim 2, characterized in that, Inoculate the thermotolerant Phaffia rhodozyma into a nutrient medium for fermentation production of astaxanthin.

4. The application according to claim 3, wherein The nutrient medium comprises a carbon source at 25 - 100 g / L, a nitrogen source at 5 - 30 g / L, and inorganic salts at 3 - 10 g / L; the carbon source is at least one of glucose, fructose, sucrose, straw hydrolysate, maltose, starch, molasses, glycerol; the nitrogen source is at least one of ammonium sulfate, ammonium nitrate, urea, yeast extract, corn steep liquor powder, urea, peanut cake powder, peptone, soybean powder; the inorganic salts are at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, sodium hydrogen phosphate.

5. The application according to claim 3, wherein The fermentation temperature is 28 - 30 °C and the fermentation time is 5 - 10 days.

6. The application according to claim 4, characterized in that The nutrient medium is: 100 g / L straw hydrolysate, 1.5 g / L ammonium sulfate, 1.5 g / L yeast extract, 10 g / L corn steep liquor powder, 1.5 g / L magnesium sulfate, 1.5 g / L potassium dihydrogen phosphate, 1.5 g / L dipotassium hydrogen phosphate.

7. The application according to claim 4, characterized in that, Add a promoter 24 - 72 h after fermentation, and the promoter is a metal ion or vegetable oil.

8. The application according to claim 7, characterized in that The metal ion is copper ion, and the vegetable oil is corn oil or sunflower oil.

9. The application according to claim 7, characterized in that The addition amount of the metal ion is 0.5 - 0.7 μmol / L, and the addition amount of the vegetable oil is 0.5 - 1.5%.

10. The application according to claim 9, characterized in that The addition amount of the metal ion is 0.6 μmol / L, and the addition amount of the vegetable oil is 0.5%.

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