A heat-resistant Phaffia rhodozyma strain and a method for producing astaxanthin using straw hydrolysate

Abstract

The present invention discloses a thermotolerant rhodozyma strain and a method for producing astaxanthin using straw hydrolyzate. The strain is classified and named as rhodozyma GF6. Xanthophyllomyces dendrorhous GF6, with a deposit number of CCTCC NO: M 2025649; the strain is inoculated into a fermentation medium, and straw hydrolyzate is used as a carbon source to efficiently produce astaxanthin at 28-30°C; the present invention utilizes agricultural waste straw hydrolyzate as a cheap crude raw material instead of the entire carbon source to produce astaxanthin, thereby increasing the fermentation temperature, reducing production costs, and being environmentally friendly.

Description

Technical Field

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

[0002] Astaxanthin, a natural carotenoid, has strong free radical scavenging activity, protecting against lipid peroxidation and oxidative damage to low-density lipoprotein cholesterol, cell membranes, cells, and tissues. In addition, astaxanthin has many essential biological functions, including preventing the oxidation of essential polyunsaturated fatty acids and the effects of ultraviolet light, supporting good vision and eye health, and enhancing immune response. Due to its high antioxidant activity, astaxanthin has been widely used as a food additive, pigment, antioxidant, and nutritional food.

[0003] The method of producing astaxanthin by fermentation of Phaffia rhodozyma has the advantages of being environmentally friendly, harmless, low-cost, and having a high safety factor, which has led to an increasing amount of attention to this method. The main factor restricting the completion of industrial production of this method is cost. First, the fermentation temperature of Phaffia rhodozyma is too low (18-22°C), so a large amount of electricity is consumed for cooling during the fermentation process. In addition, the culture medium used to cultivate Phaffia rhodozyma is relatively expensive, especially the carbon source, which consumes the most carbon source during the fermentation process. In traditional methods, glucose is used as the basic fermentation carbon source, but glucose is relatively expensive. Therefore, finding a high-quality carbon source that is relatively cheap, widely available, and more favorable for fermentation is a key issue that needs to be urgently addressed in the production of astaxanthin by Phaffia rhodozyma.

[0004] Converting straw into hydrolyzate for fermentation realizes the resource utilization of straw, reduces the environmental impact of straw waste, and has excellent environmental benefits. Straw is rich in polysaccharides such as cellulose and hemicellulose. Hydrolysis can convert these macromolecules into small-molecule sugars that can be used by microorganisms, achieving efficient utilization of biomass resources and increasing resource recycling rates, in line with the concept of sustainable development. Using straw as a raw material to prepare hydrolyzate significantly reduces fermentation costs compared to using traditional pure carbon sources such as glucose and sucrose, providing an economically viable path for large-scale industrial fermentation production. Summary of the Invention

[0005] The present invention aims to provide a thermotolerant rhodozyma strain capable of producing astaxanthin from straw hydrolyzate at 28-30°C.

[0006] The technical solution adopted in the present invention is:

[0007] A thermotolerant rhodozyma strain, designated as rhodozyma GF6 Xanthophyllomyces dendrorhous GF6 was deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China on March 31, 2025, with the deposit number CCTCC NO: M 2025649.

[0008] After 3-5 days of cultivation on solid medium, the colonies develop into round, neatly margined colonies approximately 1-3 mm in diameter. Due to the accumulation of astaxanthin, the colonies are deep coral red, with a smooth, moist surface and a viscous texture. Microscopically, the vegetative cells are oval, measuring approximately 3-6 × 5-10 μm, a relatively large size. This strain exhibits good heat resistance and can grow and ferment at temperatures between 28 and 30°C.

[0009] The thermotolerant rhodozyma is a kind of rhodozyma Xanthophyllomyces dendrorhous ) LX6 is the starting strain, which was obtained through ARTP mutagenesis, NTG mutagenesis and adaptive domestication.

[0010] The starting strain has been disclosed in the applicant's prior patent application CN119709447A, with a deposit number of CCTCC NO: M 20242814.

[0011] Application of thermotolerant Phaffia rhodozyma GF6 in fermentation production of astaxanthin.

[0012] The application is to activate the Phaffia rhodozyma GF6, culture the seeds and then inoculate them into a fermentation medium to ferment and produce astaxanthin.

[0013] The fermentation medium formula is: carbon source 25-100 g / L, nitrogen source 5-30 g / L, and inorganic salt 3-10 g / L.

[0014] The carbon source includes but is not limited to glucose, fructose, sucrose, straw hydrolyzate, maltose, starch, molasses, glycerol, etc., and is more preferably straw hydrolyzate.

[0015] The nitrogen source includes but is not limited to ammonium sulfate, ammonium nitrate, urea, yeast extract, corn steep liquor powder, urea, peanut meal, peptone, soybean meal and the like.

[0016] The inorganic salts include but are not limited to potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, ammonium sulfate, disodium hydrogen phosphate, and the like.

[0017] Further preferably, the fermentation medium is: 25-50 g / L glucose, 1-3 g / L yeast extract, 1-3 g / L ammonium sulfate, 5-20 g / L corn steep liquor powder, 1-3 g / L magnesium sulfate heptahydrate, 1-3 g / L potassium dihydrogen phosphate, and 1-3 g / L disodium hydrogen phosphate.

[0018] More preferably, the fermentation medium comprises: 100 g / L straw hydrolyzate, 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, and 1.5 g / L sodium dihydrogen phosphate. Sterilize at 115°C for 20 min.

[0019] The fermentation culture conditions are: fermentation temperature 28-30° C., and fermentation time 7 days.

[0020] Preferably, a fermentation promoter is added at 24 to 72 hours of fermentation, wherein the promoter is a metal ion or a vegetable oil, and is preferably added at 48 hours of fermentation.

[0021] The metal ion is copper ion, and the addition amount is 0.2-0.7 μmol / L;

[0022] Preferably, copper sulfate is added at 0.2-0.7 μmol / L during fermentation for 24-72 hours, and more preferably, copper sulfate is added at 0.6 μmol / L.

[0023] Preferably, the vegetable oil is added 24 to 72 hours after fermentation, and the amount of the vegetable oil added is 0.5 to 1.5%. More preferably, the amount of the vegetable oil added is 0.5%.

[0024] The vegetable oil is corn oil or sunflower oil, and is preferably added at the 48th hour of fermentation.

[0025] The straw hydrolyzate is wheat straw hydrolyzate, which mainly contains fermentable sugars such as sucrose, glucose, and arabinose, and the total reducing sugar content thereof is 200-500 g / L.

[0026] More specifically, the following steps are included:

[0027] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28°C for 2-3 days to activate the strain;

[0028] (2) Take a single colony from the YM solid culture medium and inoculate it into the seed culture medium. Incubate it at 28°C and 160 rpm for 48 hours to obtain the seed solution for fermentation.

[0029] (3) Inoculate the seed liquid into the fermentation medium and ferment at 28-30°C, 160 rpm for 7 days.

[0030] A fed-batch method can be used, specifically, the thermotolerant red yeast is inoculated into a fermentation tank filled with straw hydrolyzate fermentation medium after seed culture, 28~30℃, 500rpm, and straw hydrolyzate is supplemented by fed-batch method from the second day of fermentation, with a flow rate of 10 ml / h for 32-48h, 6 ml / h for 48-72h, and 4 ml / h for 72-168h. The culture is carried out for 7 days, the pH is controlled at 6.5 for the first 72h, and the pH is controlled at 5.5 after 72h. 0.6μmol copper sulfate is added after 48h of fermentation.

[0031] Beneficial effects:

[0032] The present invention screened a thermotolerant Phaffia rhodozyma strain that can produce astaxanthin at 28-30°C using agricultural waste straw hydrolyzate as a carbon source. This replaces the entire carbon source with inexpensive crude raw materials to produce astaxanthin, thereby increasing the fermentation temperature, reducing production costs, and being environmentally friendly. The fermentation temperature of this strain is 3-5°C higher than that of the starting strain, achieving efficient synthesis of astaxanthin at 28-30°C. As the temperature is increased, the strain's ability to synthesize astaxanthin under high temperature conditions is enhanced. The astaxanthin yield and content at 28°C on a shake flask scale are 5.4 times and 2.2 times higher than those of the starting strain, respectively. The astaxanthin production level is further increased by fermentation tank amplification, achieving efficient synthesis of astaxanthin under high temperature conditions.

[0033] This study established a highly efficient astaxanthin production process at 28-30°C using a thermotolerant strain of Phragmites rubrum by exogenously adding astaxanthin production promoters (copper ions and corn oil) and optimizing the process. Pilot fermentation production based on this process achieved astaxanthin yield and content of 1744.5 mg / L and 20.5 mg / g, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a graph showing the improvement of the dry weight, astaxanthin production and content of the mutagenic strain over time through adaptive acclimation;

[0035] Figure 2 This is a diagram showing the stability test of the final strain after adaptive domestication of the mutagenic strain;

[0036] Figure 3 The comparison chart shows the fermentation results of heat-resistant strain GF6 with glucose and straw hydrolysate in shake flasks;

[0037] Figure 4 Comparison of astaxanthin production results by shake flask fermentation of thermotolerant strain GF6 at 28℃ with exogenous addition of different metal ions;

[0038] Figure 5 Optimization of the amount of exogenously added copper ions for the thermotolerant strain GF6 at 28°C;

[0039] Figure 6 Optimization of the time of exogenous copper ion addition to the thermotolerant strain GF6 at 28°C;

[0040] Figure 7 Comparison of astaxanthin production by shake flask fermentation of thermotolerant strain GF6 at 28℃ with exogenous addition of vegetable oil;

[0041] Figure 8 Optimization of the amount of exogenously added corn oil for the thermotolerant strain GF6 at 28°C;

[0042] Figure 9 Optimization of the time of exogenous addition of corn oil to the thermotolerant strain GF6 at 28°C;

[0043] Figure 10 The results of a small-scale fermentation of the heat-resistant strain GF6 at 28°C in a 5L fermenter.

[0044] Figure 11 The results of the pilot-scale fermentation of the heat-resistant strain GF6 in a 50 L fermenter at 30°C were shown. DETAILED DESCRIPTION

[0045] The following examples illustrate the present invention in detail.

[0046] The method for determining the dry weight of microbial cells and the astaxanthin content in the embodiment is:

[0047] Method for determining the dry weight of microbial cells: take an appropriate amount of fermentation liquid into a pre-weighed centrifuge tube, centrifuge and wash twice, and then place it in an oven at 65°C to dry to constant weight.

[0048] Extraction and determination method of astaxanthin in the examples: The astaxanthin content was determined by ultraviolet spectrophotometry.

[0049] Take 1mL of fermentation broth and centrifuge at 12000rpm for 2min, wash twice with deionized water, collect the bacteria, use filter paper to absorb the water droplets on the wall of the tube, add 1mL of dimethyl sulfoxide (DMSO) preheated at 55℃, oscillate and resuspend, add 2mL of acetone, vortex and oscillate for 20-30s, then stand in the dark for 15 minutes, centrifuge at 4℃, 12000r / min for 2min, and measure the OD474 of the supernatant with a spectrophotometer. If the bacterial mud is still colored, repeat the extraction until it turns white. The astaxanthin content is calculated as follows:

[0050] Astaxanthin (mg / L) = (A × V1 × 10000) / (2150 × V2)

[0051] Where: A—OD474 value;

[0052] V1—total volume of organic solvent (mL);

[0053] 2150——Specific extinction coefficient;

[0054] V2——fermentation broth volume (mL);

[0055] 10000: Unit conversion factor (mg / L).

[0056] The straw hydrolyzate described in the following examples was provided by Suzhou Juwei Original Technology Co., Ltd.

[0057] The method for determining the total reducing sugar in the straw hydrolyzate in the present invention is: DNS method, and the specific steps are as follows:

[0058] First, draw a glucose standard curve: Dispense 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose standard solution (1 mg / mL) into a 20 mL EP tube. Accurately add 2.5 mL of DNS reagent to each tube. Boil in a boiling water bath for 5 minutes. After cooling, add pure water to a total of 20 mL. Measure the absorbance at 540 nm. The resulting glucose standard curve is: y = 3.008x - 0.1653 R² = 0.9906;

[0059] The fermentation supernatant was then diluted 100-fold, and 1.0 mL of the diluted sugar solution was placed in a 20 mL EP tube. 2.5 mL of DNS reagent was added and the tube was boiled in boiling water for 5 minutes. After cooling, the volume was filled up to 20 mL with water. The absorbance was measured at 540 nm. Substituting this into the glucose standard curve, the total reducing sugar content in the straw hydrolyzate was calculated to be 251 g / L.

[0060] The supernatant obtained by centrifuging the straw hydrolyzate at 10,000 rpm for 8 minutes is used as a culture medium solvent, and ammonium sulfate, yeast extract, corn steep liquor powder, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, and disodium hydrogen phosphate are added to prepare the fermentation medium.

[0061] Example 1

[0062] Breeding of a thermotolerant and high-astaxanthin-producing Phaffia rhodozyma mutant strain using Phaffia rhodozyma LX6 mutagenesis

[0063] (1) The starting strain of Phaffia rhodozyma LX6 was cultured to an OD600 of 1.8-2.2. The cells were collected and subjected to ARTP mutagenesis. During ARTP mutagenesis, the helium power was 100-150W, the gas volume was 8-12 SLM, the mutagenesis distance was 1.5-2.5mm, and the mutagenesis time was 60-70s. The collected mutagenized strains were cultured overnight at 27℃. The mutant strains that could synthesize astaxanthin at 27℃ were further screened by flow cytometry. The flow cytometer was used for sorting under the following working conditions: forward scatter (FSC, 175), side scatter (SSC, 211), and screening channels FL1 (568nm) and FL2 (569nm).

[0064] (2) The selected 27°C mutant strains were cultured to an OD600 of 1.8-2.2, and the cells were collected for NTG mutagenesis at a concentration of 1.8-2.3 mmol / L for 13-18 min. The strains were cultured overnight at 28°C, and the mutant strains capable of producing astaxanthin at 28°C were screened by flow cytometry.

[0065] (3) Adaptation at 28℃: The mutant strain selected at 28℃ was cultured continuously at 28℃ with 10mmol / LH2O2 added to obtain a mutant strain with high astaxanthin production at 28℃. Figure 1 The adaptive acclimation further enhanced the growth of the strain at 28°C and the production of astaxanthin. After 960 hours of adaptive acclimation, the astaxanthin production increased from 118.9 mg / L to 161.9 mg / L. The induced strain was named Rhodozyma GF6. Xanthophyllomyces dendrorhous GF6 has been deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 2025649.

[0066] (3) Stability test of mutagenic bacteria at 28°C

[0067] The selected mutant bacteria GF6 were inoculated into YM solid medium and cultured at 25℃ for 3 days to activate the strain; a single colony with darker color was taken from the YM solid medium and inoculated into liquid YM medium at 28℃ and 200 rpm for 48h; a 10% inoculation amount was inoculated into a 500mL shake flask containing 50mL YM liquid medium and cultured at 28℃ and 200 rpm for 48h. Each time, the bacterial liquid was inoculated into fresh YM liquid medium at 10% inoculation amount, and the culture was continuously subcultured 8 times to test the production and growth performance of the mutant bacteria. Figure 2 The heat-resistant strain GF6 was able to stably produce about 160 mg / L astaxanthin at 28°C in a shake flask scale.

[0068] The starting strain hardly grows at 28°C. After five days of shake flask fermentation, the biomass can only reach 2.1 g / L and the astaxanthin production is 11.3 mg / L. After mutagenesis combined with adaptability acclimation, its biomass and astaxanthin production are increased to 13.5 g / L and 161.9 mg / L, respectively.

[0069] Example 2

[0070] Production of astaxanthin by shake flask-scale fermentation of straw hydrolysate using the mutant strain GF6 of Phaffia rhodozyma

[0071] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28°C for 2-3 days to activate the strain;

[0072] (2) Seed cultivation, including:

[0073] a. First-generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 28°C and 160 rpm for 48 hours;

[0074] 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% inoculum volume. Incubate at a constant temperature under the same conditions as a to obtain the seed fluid for fermentation culture.

[0075] (3) The seed liquid obtained from seed culture was inoculated into a triangular flask containing straw hydrolyzate fermentation medium for fermentation at 28°C and 160 rpm for 7 days. The cell dry weight was determined, astaxanthin in the fermentation liquid was extracted, and the astaxanthin content was determined. The experiment was repeated. Figure 3 As shown in the results, 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 equivalent to glucose as a carbon source. Therefore, straw hydrolyzate can be used as a carbon source to replace glucose for fermentation to produce astaxanthin.

[0076] The fermentation medium using straw hydrolyzate as a carbon source comprises: 100 g / L straw hydrolyzate, 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, and 1.5 g / L disodium hydrogen phosphate. Sterilize at 115°C for 20 min.

[0077] Example 3

[0078] Comparison of astaxanthin production by shake flask fermentation of Phaffia rhodozyma GF6 at 28℃ with exogenous addition of different metal ions

[0079] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28°C for 2-3 days to activate the strain;

[0080] (2) Seed cultivation, including:

[0081] a. First-generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 28°C and 160 rpm for 48 hours;

[0082] 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% inoculum volume. Incubate at a constant temperature under the same conditions as a to obtain the seed fluid for fermentation culture.

[0083] (3) The seed liquid obtained by seed culture was inoculated into a triangular flask containing 100 g / L straw hydrolyzate fermentation medium (100 g / L straw hydrolyzate, 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 sodium hydrogen phosphate, sterilized at 115 ° C for 20 min) for fermentation. 1% of cobalt ion (cobalt chloride), calcium ion (calcium chloride), barium ion (barium chloride), divalent iron ion (ferrous sulfate), trivalent iron ion (ferric chloride), manganese ion (manganese chloride), zinc ion (zinc chloride), copper ion (copper chloride), nickel ion (nickel chloride) was added for 24 h, and astaxanthin in the fermentation liquid was extracted and the astaxanthin content was determined. The experiment was repeated. Figure 4 As shown in the figure, the experimental group that added copper ions after 7 days of fermentation had the best results. The astaxanthin production could reach 185.5 mg / L and the content could reach 14.6 mg / g, which were 19.7% and 18.7% higher than the control group without adding metals, respectively, effectively promoting the synthesis of astaxanthin.

[0084] Example 4

[0085] Optimization of the amount of exogenously added copper ions to the mutagenized Phaffia rhodozyma strain GF6 at 28℃

[0086] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28°C for 2-3 days to activate the strain;

[0087] (2) Seed cultivation, including:

[0088] a. First-generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 28°C and 160 rpm for 48 hours;

[0089] 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% inoculum volume. Incubate at a constant temperature under the same conditions as a to obtain the seed fluid for fermentation culture.

[0090] (3) The seed liquid obtained by seed culture was inoculated into a triangular flask containing 100 g / L straw hydrolyzate fermentation medium (100 g / L straw hydrolyzate, 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 sodium hydrogen phosphate, sterilized at 115°C for 20 min) for fermentation. After culturing for 24 h, 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 μM copper ions were added, respectively, to extract astaxanthin from the fermentation liquid, determine the astaxanthin content, and repeat the experiment. Figure 5The results showed that adding 0.6 μM copper ions had the best effect, with the astaxanthin production and content reaching 197.4 mg / L and 15.3 mg / g, respectively, which were 22.5% and 24.4% higher than the control group without adding metal ions, further improving the level of astaxanthin production by the heat-resistant strain GF6 at 28°C.

[0091] Example 5

[0092] Optimization of the time of exogenous copper ion addition to the mutagenic strain GF6 of Phaffia rhodozyma at 28℃

[0093] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28°C for 2-3 days to activate the strain;

[0094] (2) Seed cultivation, including:

[0095] a. First-generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 28°C and 160 rpm for 48 hours;

[0096] 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% inoculum volume. Incubate at a constant temperature under the same conditions as a to obtain the seed fluid for fermentation culture.

[0097] (3) The seed liquid obtained by seed culture was inoculated into a triangular flask containing 100 g / L straw hydrolyzate fermentation medium (100 g / L straw hydrolyzate, 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 sodium hydrogen phosphate, sterilized at 115 ° C for 20 min) and fermented. 0.6 μM copper ion was added at 0, 24, 48, 72, 96, and 120 h of fermentation, and astaxanthin in the fermentation liquid was extracted. The astaxanthin content was determined and the experiment was repeated. Figure 6 The timing of copper ion addition also significantly affected astaxanthin production in the induced strain GF6, with the optimal addition occurring 48 hours after fermentation, reaching astaxanthin production and content of 210.2 mg / L and 16.3 mg / g, respectively. This was further improved by optimizing the addition amount, reaching increases of 30.4% and 33.6%, respectively, compared to the control group without metal ion addition. Thus, we have determined that copper ions can act as an astaxanthin production promoter, increasing astaxanthin production in the heat-resistant strain GF6 at 28°C, and that the optimal addition is 0.6 μM at 48 hours of fermentation.

[0098] Example 6

[0099] Comparison of astaxanthin production by shake flask fermentation of Phaffia rhodozyma GF6 with exogenous vegetable oil at 28℃

[0100] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28°C for 2-3 days to activate the strain;

[0101] (2) Seed cultivation, including:

[0102] a. First-generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 28°C and 160 rpm for 48 hours;

[0103] 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% inoculum volume. Incubate at a constant temperature under the same conditions as a to obtain the seed fluid for fermentation culture.

[0104] (3) The seed liquid obtained by seed culture was inoculated into a triangular flask containing 100 g / L straw hydrolyzate fermentation medium (100 g / L straw hydrolyzate, 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 sodium hydrogen phosphate, sterilized at 115 ° C for 20 min) for fermentation. After 24 hours, 1% corn oil, sunflower oil, and soybean oil were added respectively to extract astaxanthin from the fermentation liquid and determine the astaxanthin content. The experiment was repeated. Figure 7 As shown in the results, the experimental group with corn oil added after 7 days of fermentation had the best results, with astaxanthin production reaching 195.8 mg / L and content reaching 15.4 mg / g, which were 21.5% and 26.2% higher than the control group without vegetable oil, respectively. This shows that corn oil can be used as a vegetable oil to effectively promote the production of astaxanthin by the heat-resistant strain GF6 at 28°C.

[0105] Example 7

[0106] Optimization of the amount of exogenous corn oil added to the mutagenized strain GF6 of Phaffia rhodozyma at 28℃

[0107] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28°C for 2-3 days to activate the strain;

[0108] (2) Seed cultivation, including:

[0109] a. First-generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 28°C and 160 rpm for 48 hours;

[0110] 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% inoculum volume. Incubate at a constant temperature under the same conditions as a to obtain the seed fluid for fermentation culture.

[0111] (3) The seed liquid obtained by seed culture was inoculated into a triangular flask containing 100 g / L straw hydrolyzate fermentation medium (100 g / L straw hydrolyzate, 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 sodium hydrogen phosphate, sterilized at 115 °C for 20 min) for fermentation. 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% (v / v) corn oil were added for 24 h, respectively. Figure 8 The results showed that the amount of corn oil added significantly affected astaxanthin production, with excessive corn oil addition having a negative impact. Adding 0.5% (v / v) corn oil had the best effect. The astaxanthin production and content were further enhanced, reaching 210.1 mg / L and 16.8 mg / g, respectively, which were 30.3% and 38% higher than the control group without vegetable oil.

[0112] Example 8

[0113] Optimization of the time of adding exogenous corn oil at 28℃ to the mutagenized strain GF6 of Phaffia rhodozyma

[0114] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28°C for 2-3 days to activate the strain;

[0115] (2) Seed cultivation, including:

[0116] a. First-generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 28°C and 160 rpm for 48 hours;

[0117] 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% inoculum volume. Incubate at a constant temperature under the same conditions as a to obtain the seed fluid for fermentation culture.

[0118] (3) The seed liquid obtained by seed culture was inoculated into a triangular flask containing 100 g / L straw hydrolyzate fermentation medium (100 g / L straw hydrolyzate, 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 sodium hydrogen phosphate, sterilized at 115 ° C for 20 min) and fermented. 0.5% corn oil was added at 0, 24, 48, 72, 96, and 120 h of fermentation, and the astaxanthin content was determined. The experiment was repeated. Figure 9As shown, 48 hours of addition was the optimal time. After optimizing the addition time, astaxanthin production levels were further improved, reaching 223.3 mg / L and 18.1 mg / g, respectively, representing increases of 38.5% and 48.1% compared to the control group without vegetable oil. This confirms that corn oil can be used as an astaxanthin production promoter to enhance astaxanthin production in the thermotolerant GF6 strain at 28°C, with the optimal addition of 0.5% (v / v) at 48 hours of fermentation.

[0119] Example 9

[0120] A fermentation process for producing astaxanthin by the thermotolerant rhodozyma strain GF6 using straw hydrolysate and exogenously added promoters was established.

[0121] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28°C for 2-3 days to activate the strain;

[0122] (2) Seed cultivation, including:

[0123] a. First-generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 28°C and 160 rpm for 48 hours;

[0124] 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% inoculum volume. Incubate at a constant temperature under the same conditions as a to obtain the seed fluid for fermentation culture.

[0125] (3) The seed liquid obtained from seed culture was inoculated into a 5L fermentation tank filled with straw hydrolyzate fermentation medium. The culture temperature was 28°C and 500 rpm. Straw hydrolyzate was added by flow addition. The flow rate was 10 ml / h for 32-48h, 6 ml / h for 48-72h, and 4 ml / h for 72-168h. The culture was continued for 7 days. 0.6 μM copper ions and 0.5% corn oil were added at 48h of fermentation to promote astaxanthin production. During the fermentation process, the pH was controlled at 6.5 for the first 72h and at 5.5 after 72h. The fermentation was carried out using a two-stage pH method, such as Figure 10 Its astaxanthin production reached 828.2 mg / L, content reached 20.2 mg / g, and biomass reached 41g / L.

[0126] Example 10

[0127] The thermotolerant rhodozyma strain GF6 was used to produce astaxanthin by fermentation using straw hydrolysate and exogenously added promoters.

[0128] (1) Inoculate Phaffia rhodozyma GF6 into YM solid medium and culture at 28°C for 2-3 days to activate the strain;

[0129] (2) Seed cultivation, including:

[0130] a. First-generation seed culture: Take a single colony from YM solid medium and inoculate it into seed medium at 28°C and 160 rpm for 48 hours;

[0131] 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% inoculum volume. Incubate at a constant temperature under the same conditions as a to obtain the seed fluid for fermentation culture.

[0132] (3) The seed liquid obtained from seed culture was inoculated into a 50 L fermentation tank filled with straw hydrolyzate fermentation medium at 30°C, DO was controlled at 30-40%, and straw hydrolyzate was supplemented by flow addition. The flow rate was 20 ml / h for 12-48h, 15 ml / h for 48-72h, and 10 ml / h for 72-168h. 5 g / L yeast powder and 10 g / L corn steep liquor powder were added at 48h of fermentation. 0.6 μM copper ions and 0.5% corn oil were added exogenously at 48h of fermentation to promote astaxanthin production. The culture was carried out for 7 days, with the pH controlled at 6.5 for the first 72h and at 5.5 after 72h. The fermentation was carried out using a two-stage pH method, as shown in Figure 2. Figure 11 The astaxanthin production reached 1744.5 mg / L, the content reached 20.5 mg / g, and the biomass reached 85.1 g / L. The pilot test results showed that the heat-resistant strain has the potential for industrial scalability, and the astaxanthin level has met the requirements of industrial production.

Claims

1. A thermotolerant rhodozyma yeast, characterized in that: Its classification is named as Phaffia rhodozyma ( Xanthophyllomyces dendrorhous ) GF6, deposit number is CCTCC NO: M 2025649.

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

3. The use according to claim 2, characterized in that Thermotolerant rhodozyma yeast is inoculated into the nutrient medium and fermented to produce astaxanthin.

4. The use according to claim 3, characterized in that The nutrient medium comprises: 100 g / L straw hydrolyzate, 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, and 1.5 g / L disodium hydrogen phosphate.

5. The use according to claim 3, characterized in that The fermentation temperature is 28-30° C., and the fermentation time is 5-10 days.

6. The use according to claim 4, characterized in that Metal ions or vegetable oil are added during fermentation for 24 to 72 hours, wherein the metal ions are copper ions and the vegetable oil is corn oil or sunflower oil.

7. The use according to claim 6, characterized in that The added amount of the metal ion is 0.5-0.7 μmol / L, and the added amount of the vegetable oil is 0.5-1.5%.

8. The use according to claim 7, characterized in that The added amount of the metal ions is 0.6 μmol / L, and the added amount of the vegetable oil is 0.5%.

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