A method for synthesizing astaxanthin by fermenting Phaffia rhodozyma

Abstract

The present invention relates to the field of microbial fermentation technology, and in particular to a method for synthesizing astaxanthin by fermentation of red Phaffia yeast. According to the difference between the optimal conditions of the growth and culture conditions of red Phaffia yeast and the conditions of astaxanthin synthesis, the present invention adopts a variable temperature and batch feeding culture strategy, and by adding a composite deep eutectic solvent to the feeding medium, the red Phaffia yeast is allowed to increase the permeability of the microbial cell membrane due to the action of the deep eutectic solution in the astaxanthin production stage, and promote the transportation of extracellular substrates to the intracellular fermentation metabolism and utilization. On the other hand, the deep eutectic solvent can also enter the cell to regulate the activity of key enzymes for astaxanthin metabolism and synthesis and cause intracellular acid-base imbalance, so as to enhance the metabolic flow of red Phaffia yeast to synthesize astaxanthin, thereby effectively increasing the biomass of red Phaffia yeast and its intracellular astaxanthin yield.

Description

Technical Field

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

[0002] Astaxanthin, chemical name is 3, 3'-dihydroxy-4, 4'-diketo-β, β'-carotene, molecular formula is C 40 H 52 O 4 , is a powerful oxygen-containing carotenoid with extremely high antioxidant capacity, and its antioxidant effect is far superior to that of β-carotene and vitamin E. The conjugated double bond chain in its molecular structure and the unsaturated ketone and hydroxyl groups at its ends give it a strong antioxidant capacity, which can scavenge free radicals, block chain reactions and inhibit lipid peroxidation, thereby protecting cells from oxidative damage. In addition to anti-aging and improving immunity, astaxanthin also has the potential to prevent and treat tumors, cardiovascular diseases and diabetes. Since the chemical synthesis of astaxanthin not only has the potential safety hazard of residual harmful substances, but also has the problem of high price, microbial fermentation is currently mainly used to produce astaxanthin, such as Haematococcus pluvialis and Phaffia rhodozyma.

[0003] Phaffia rhodozyma has the advantages of fast growth and simple culture conditions, and its fermentation has become an important way to produce astaxanthin. Phaffia rhodozyma can convert glucose and other nutrients into key metabolic intermediates, acetyl-CoA, which synthesizes isoprenyl pyrophosphate through the mevalonate pathway, and then generates farnesyl pyrophosphate under the action of isopentenyl diphosphate isomerase, which is further condensed to generate geranylgeranyl pyrophosphate, and then condensed to generate phytoene. Phaffia rhodozyma is then dehydrogenated and cyclized to generate β-carotene, and then the final product astaxanthin is generated through the action of β-hydroxylase.

[0004] However, the fermentation of astaxanthin by Phaffia rubra still has technical problems such as low carbon and nitrogen source utilization and low biomass, which seriously limit the production level of astaxanthin synthesized by Phaffia rubra. At present, researchers have obtained fermentation strains with stronger astaxanthin production ability through mutagenesis breeding and genetic engineering modification. However, research on culture medium optimization and fermentation process control for astaxanthin fermentation by microbial strains, especially fermentation process control, still has a lot of room for improvement.

[0005] Generally, there are differences between the growth and proliferation conditions of microorganisms and the synthesis conditions of secondary metabolites, such as fermentation temperature, carbon-nitrogen source ratio, etc. In addition, the synthesis stage of secondary metabolites is affected by the high density of microbial cells and the activity of key enzymes in their metabolic pathways, which limits the transfer efficiency of substrates and the conversion rate of astaxanthin. Accurately controlling the fermentation conditions for astaxanthin production and improving the transfer efficiency and conversion rate of substrates will help increase the production of astaxanthin produced by Phaffia rhodozyma fermentation.

[0006] Deep eutectic solvents are a type of liquid formed by hydrogen bonding interactions between two or more components in a specific ratio, with a low melting point. They are often used as green solvents to replace traditional solvents for biocatalytic conversion, increase the solubility of substrates, thereby enhancing the mass transfer efficiency of substrates and the target product synthesis metabolic flow, and improve biocatalytic conversion efficiency and yield.

[0007] However, the application and related effects of deep eutectic solvents in astaxanthin fermentation are still unknown. Therefore, this application combines temperature-variable fermentation and fed-batch fermentation containing deep eutectic solvents as an important technical means to increase the yield of astaxanthin synthesized by Phaffia rhodozyma. Summary of the invention

[0008] The main purpose of the present invention is to provide a method for increasing the yield of astaxanthin synthesized by Phaffia rhodozyma fermentation, aiming to solve the problem of low yield of astaxanthin synthesized by Phaffia rhodozyma in the existing fermentation technology.

[0009] In order to achieve the above-mentioned object, the present invention provides a method for increasing the yield of astaxanthin synthesized by red Phaffia yeast fermentation, the method comprising: combining temperature-variable fermentation and batch-fed fermentation methods to efficiently produce astaxanthin using red Phaffia yeast, wherein the temperature-variable fermentation and batch-fed fermentation processes include accurately controlling the temperature of the fermentation system and adding a carbon-nitrogen source feed medium containing a deep eutectic solvent, the deep eutectic solvent can increase the permeability and enzyme activity of the yeast cell membrane and stimulate the stress response of the yeast cell, promote the entry of nutrients into the cell and enhance the metabolic flow of the astaxanthin synthesis pathway to increase the yield of astaxanthin in red Phaffia yeast; specifically, the method for synthesizing astaxanthin by fermenting red Phaffia yeast is as follows:

[0010] The red Phaffia yeast seed liquid is inoculated into the fermentation medium at an inoculation rate of 5-8% (v / v) for the first stage of fermentation, the culture temperature is 25-30°C, the stirring speed is 100-200 rpm, and the first stage of fermentation time is 36-60h; after completing the 36-60h fermentation of the first stage, the second stage of variable temperature and batch feeding fermentation is carried out, wherein: the temperature is adjusted to 15-20°C, the other conditions remain unchanged, and feeding is carried out once when entering the second stage, and then feeding is carried out once every 12h, and the feeding volume each time is 50% (v / v) of the initial culture medium, and feeding is carried out 3 times in total;

[0011] Furthermore, during the whole fermentation process, ammonia water is used to control the pH to 5.0-6.5, the fermentation tank aeration volume is controlled to 50-200 mL / min, and the dissolved oxygen is controlled to 20-60%;

[0012] Further, the feed medium used in the feed is a carbon and nitrogen source medium containing a deep eutectic solvent, and the feed medium includes, in terms of g / L concentration: 60-150 g / L of glycerol, 30-60 g / L of yeast powder, 15-30 g / L of deep eutectic solvent A, 15-30 g / L of deep eutectic solvent B, and the rest is water, pH 6.0-6.5;

[0013] The deep eutectic solvent A is composed of choline chloride and urea, and the molar ratio of choline chloride to urea is 1:2; the deep eutectic solvent B is composed of choline chloride and citric acid, and the molar ratio of choline chloride to citric acid is 1:1;

[0014] Further, according to the g / L concentration, the components of the fermentation medium include: maltose 10-50 g / L, glycerol 20-50 g / L, yeast powder 10-20 g / L, soy peptone 5-10 g / L, potassium dihydrogen phosphate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, magnesium sulfate heptahydrate 1-2 g / L, calcium chloride 0.2-0.5 g / L, biotin 0.5-1 g / L, and the rest is water, pH 6.0-6.5;

[0015] Further, the fermentation temperature in the fermentation system is controlled by a temperature-controlled jacket of the fermentation tank;

[0016] Furthermore, after the above fermentation, the biomass of Phaffia rhodozyma was 143.4-178.5 g / L, and the astaxanthin production was 667.2-725.8 mg / L.

[0017] Further, Phaffia rhodozyma was inoculated into the fermentation medium from the preserved slant medium, the culture temperature was 25-30°C, the shaking speed was 200-400 rpm, the culture time was 48-60h, the medium pH was 6.0-6.5, and the primary seed solution was obtained.

[0018] Further, the first-level seed solution is inoculated into the fermentation medium at an inoculation rate of 0.5-1.0% (v / v), the culture temperature is 25-30°C, the shaking speed is 200-400 rpm, the culture time is 48-60h, and the medium pH is 6.0-6.5 to obtain the second-level seed solution.

[0019] Preferably, the red yeast is red yeast ( Paffia rhodozyma )NCUPFR001, the deposit number is CGMCC No.31951.

[0020] Another technical solution provided by the present invention is the use of a deep eutectic solvent in increasing the yield of astaxanthin produced by Phaffia rhodozyma;

[0021] Further, it is used in increasing the permeability of microbial cell membranes;

[0022] Further, it is an application in regulating the activity of key enzymes in astaxanthin metabolism and synthesis and causing intracellular acid-base imbalance, and further, the key enzymes include but are not limited to acetyl coenzyme A, isoprene pyrophosphate isomerase, and β-hydroxylase;

[0023] Furthermore, the deep eutectic solvent comprises a deep eutectic solvent A and a deep eutectic solvent B; the deep eutectic solvent A is composed of choline chloride and urea, and the molar ratio of choline chloride to urea is 1:2; the deep eutectic solvent B is composed of choline chloride and citric acid, and the molar ratio of choline chloride to citric acid is 1:1.

[0024] By implementing the technical solution of the present invention, the present invention has the following beneficial effects:

[0025] According to the different optimum conditions for the growth and culture conditions of Phaffia rhodozyma and the synthesis conditions of astaxanthin, the present invention adopts a variable temperature and fed-batch culture strategy, and by adding a composite deep eutectic solvent to the feed medium, the deep eutectic solution can increase the permeability of the microbial cell membrane of Phaffia rhodozyma in the astaxanthin production stage, thereby promoting the transportation of extracellular substrates to the intracellular fermentation metabolism and utilization, and the deep eutectic solvent can also enter the cell to regulate the activity of key enzymes for astaxanthin metabolism and synthesis and cause intracellular acid-base imbalance, thereby enhancing the metabolic flow of Phaffia rhodozyma to synthesize astaxanthin, thereby effectively increasing the biomass of Phaffia rhodozyma and its intracellular astaxanthin yield. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below in conjunction with embodiments.

[0027] The red Phaffia yeast used in the present invention and the embodiments is the red Phaffia yeast ( Paffia rhodozyma ) NCUPFR001, the strain NCUPFR001 was deposited on September 12, 2024 in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No.31951.

[0028] Example 1 A method for increasing the yield of astaxanthin synthesized by Phaffia rhodozyma fermentation

[0029] Primary seed culture: Use an inoculation loop to pick an appropriate amount of strain from a single colony of Phaffia rhodozyma NCUPFR001 preserved on a solid slant and inoculate it into sterile fermentation medium (30 mL / 100 mL conical flask, according to g / L concentration, maltose 10 g / L, glycerol 20 g / L, yeast powder 20 g / L, soy peptone 10 g / L, potassium dihydrogen phosphate 1 g / L, potassium hydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 1 g / L, calcium chloride 0.2 g / L, biotin 0.5 g / L, pH 6.5), culture temperature 25 ° C, shaker speed 200 rpm, culture for 48 h, to obtain the primary seed culture solution.

[0030] Secondary seed culture: Under sterile conditions, inoculate the primary seeds into the culture medium (100mL / 500mL conical flask) at a 1% inoculation rate. The culture medium composition is the same as the primary seed culture medium. The culture temperature is 25°C, the shaker speed is 200rpm, and the culture is carried out for 48 hours to obtain the secondary seed culture solution.

[0031] 1L fermentation tank culture: inoculate 5-8% of the inoculum into sterile fermentation medium (400 mL / 1L fermentation tank) under sterile conditions, the culture temperature is 25-30°C, the stirring speed is 100-200 rpm, the fermentation tank ventilation is 50-200mL / min, the dissolved oxygen is 20-60%, the fermentation time is 36-60h, and ammonia water is added during the fermentation process to control the pH at 5.0-6.5. Specific culture conditions are shown in Table 1.

[0032] The composition of the above-mentioned sterile fermentation medium is: maltose 10-50 g / L, glycerol 20-50 g / L, yeast powder 10-20 g / L, soy peptone 5-10 g / L, potassium dihydrogen phosphate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, magnesium sulfate heptahydrate 1-2 g / L, calcium chloride 0.2-0.5 g / L, biotin 0.5-1 g / L, pH 6.0-6.5. The specific composition of the medium is shown in Table 1.

[0033] Variable temperature and fed-batch fermentation: After fermentation for 36-60 hours in a 1L fermenter, the fermentation temperature was adjusted to 15-20°C, and feeding was carried out once. Thereafter, feeding medium was added once every 12 hours, 200 mL each time, for a total of 3 feedings. The stirring speed, ventilation volume, dissolved oxygen, and pH of the fermentation liquid remained unchanged. During the fermentation process, ammonia water was added to control the pH at 5.0-6.5.

[0034] The composition of the above-mentioned fed-batch fermentation medium is as follows: in terms of g / L concentration, 60-150 g / L of glycerol, 30-60 g / L of yeast powder, 15-30 g / L of deep eutectic solvent A (choline chloride / urea (molar ratio 1:2)), and 15-30 g / L of deep eutectic solvent B (choline chloride / citric acid (molar ratio 1:1)), wherein the deep eutectic solvent is obtained by mixing the corresponding components in a specific molar ratio and heating them at 80°C to form a uniform liquid.

[0035] According to the different fermentation medium composition, feed medium and fermentation conditions of each batch of experiments, the experiments are divided into Experimental Examples 1-5 and Comparative Examples. The specific conditions of each experimental example and comparative example are shown in Table 1.

[0036] After fermentation, the cells were collected by centrifugation and the biomass and astaxanthin production were 150.7-178.5 g / L and 684.4-725.8 mg / L, which were significantly better than those of the feed medium without choline chloride / urea and choline chloride / citric acid.

[0037] After the fermentation, the bacteria were collected and total RNA was extracted to analyze the relative content of mRNA of key enzymes for astaxanthin synthesis. The cell membrane permeability of Phaffia rhodozyma was analyzed by flow cytometry, and the pH difference between the intracellular and extracellular environments (△pH) was determined using an intracellular pH test kit.

[0038] The cell membrane integrity and relative content of key enzyme mRNA of the experimental example were calculated with the results of the comparative example as 100%, and the intracellular and extracellular △pH was determined. The results showed that the red yeast Phaffia ovata fed with feed medium containing choline chloride / urea (molar ratio 1:2) and choline chloride / citric acid (molar ratio 1:1) showed higher transcription of key enzymes and better cell membrane permeability, and an imbalance of intracellular and extracellular pH occurred, which was conducive to the entry of substrates into the cell membrane and enhanced astaxanthin synthesis.

[0039] Table 1

[0040]

[0041] Example 2 A method for increasing the yield of astaxanthin synthesized by Phaffia rhodozyma fermentation

[0042] Primary seed culture: Use an inoculating loop to pick an appropriate amount of strain from a single colony of Phaffia rhodozyma NCUPFR001 preserved on a solid slant and inoculate it into a sterile fermentation medium (30 mL / 100 mL conical flask, according to g / L concentration, 50 g / L maltose, 50 g / L glycerol, 10 g / L yeast powder, 5 g / L soy peptone, 3 g / L potassium dihydrogen phosphate, 3 g / L potassium hydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, 0.2 g / L calcium chloride, 0.5 g / L biotin, pH 6.5), culture temperature was 30 ° C, shaker speed was 400 rpm, culture was carried out for 60 h to obtain the primary seed culture solution.

[0043] Secondary seed culture: Under sterile conditions, inoculate the primary seeds at a rate of 0.5% in the culture medium (100mL / 500mL conical flask). The composition of the culture medium is the same as that of the primary seed culture medium. The culture temperature is 30°C, the shaking speed is 400rpm, and the culture is carried out for 48 hours to obtain the secondary seed culture solution.

[0044] 1L fermenter culture: Under sterile conditions, inoculate 5-8% of the inoculum into sterile fermentation medium (400 mL / 1L fermenter), the culture temperature is 25-30°C, the stirring speed is 100-200 rpm, the fermenter ventilation volume is 50-200mL / min, the dissolved oxygen is 20-60%, the fermentation time is 36-60h, and ammonia water is added during the fermentation process to control the pH at 5.0-6.5.

[0045] The composition of the above-mentioned sterile fermentation medium is: maltose 10-50 g / L, glycerol 20-50 g / L, yeast powder 10-20 g / L, soy peptone 5-10 g / L, potassium dihydrogen phosphate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, magnesium sulfate heptahydrate 1-2 g / L, calcium chloride 0.2-0.5 g / L, biotin 0.5-1 g / L, pH 6.0-6.5. The specific composition of the medium is shown in Table 2.

[0046] Variable temperature and fed-batch fermentation: After fermentation in a 1L fermenter for 36-60h, the fermentation temperature is adjusted to 15-20℃, and feeding is performed once. Then, feed medium is added once every 12h, 200mL each time, and a total of 3 feedings. The stirring speed, ventilation volume, dissolved oxygen, and pH of the fermentation liquid remain unchanged, and ammonia water is added during the fermentation process to control the pH at 5.0-6.5.

[0047] The composition of the above-mentioned fed-batch fermentation medium is as follows: in terms of g / L concentration, 60-150 g / L of glycerol, 30-60 g / L of yeast powder, 15-30 g / L of deep eutectic solvent A (choline chloride / urea (molar ratio 1:2)), and 15-30 g / L of deep eutectic solvent B (choline chloride / citric acid (molar ratio 1:1)), wherein the deep eutectic solvent is obtained by mixing the corresponding components in a specific molar ratio and heating them at 80°C to form a uniform liquid.

[0048] According to the different fermentation medium composition, feed medium and fermentation conditions of each batch of experiments, the experiments are divided into Experimental Examples 1-5 and Comparative Examples. The specific conditions are shown in Table 2.

[0049] After fermentation, the cells were collected by centrifugation and the biomass was 143.3-172.4 g / L and the astaxanthin production was 667.2-718.6 mg / L, which were significantly better than the biomass and astaxanthin of the feed medium without the addition of choline chloride / urea and choline chloride / citric acid.

[0050] After the fermentation, the total RNA was extracted from the cells to analyze the relative content of mRNA of key enzymes for astaxanthin synthesis, and the cell membrane permeability of Phaffia rhodozyma was analyzed by flow cytometry, and the pH difference between the intracellular and extracellular environments (△pH) was measured by the intracellular pH test kit. The cell membrane integrity and relative content of key enzyme mRNA of the experimental example were calculated with the indicators of the comparative example as 100%, and the intracellular and extracellular △pH was measured.

[0051] The results showed that the red yeast P. rhodozyma fed with feed medium containing choline chloride / urea (molar ratio of 1:2) and choline chloride / citric acid (molar ratio of 1:1) showed higher transcription of key enzymes and better cell membrane permeability, and an imbalance of intracellular and extracellular pH, which was beneficial for the substrate to enter the cell membrane and enhance astaxanthin synthesis.

[0052] Table 2

[0053]

[0054] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A method for synthesizing astaxanthin by fermentation of Phaffia rhodozyma, characterized in that: The details are as follows: The red Phaffia yeast seed liquid is inoculated into the fermentation medium at an inoculation rate of 5-8% for the first stage of fermentation, the culture temperature is 25-30°C, the stirring speed is 100-200 rpm, and the first stage of fermentation time is 36-60h; after completing the 36-60h fermentation of the first stage, the second stage of variable temperature and batch feeding fermentation is carried out, wherein: the temperature is adjusted to 15-20°C, and the other conditions remain unchanged, and feeding is carried out once when entering the second stage, and then feeding is carried out once every 12h, and the feeding volume each time is 50% of the initial culture medium, and feeding is carried out 3 times in total; The feed medium used in the feed is a carbon and nitrogen source medium containing a deep eutectic solvent. According to the g / L concentration, the feed medium includes: 60-150 g / L glycerol, 30-60 g / L yeast powder, 15-30 g / L deep eutectic solvent A, 15-30 g / L deep eutectic solvent B, and the rest is water, pH 6.0-6.5; The deep eutectic solvent A is composed of choline chloride and urea, and the molar ratio of choline chloride to urea is 1:2; the deep eutectic solvent B is composed of choline chloride and citric acid, and the molar ratio of choline chloride to citric acid is 1:1; The red yeast is red yeast ( Phaffia rhodozyma )NCUPFR001, the deposit number is CGMCCNo.31951.

2. The method for synthesizing astaxanthin by fermentation of Phaffia rhodozyma as claimed in claim 1, characterized in that: During the whole fermentation process, ammonia water is used to control the pH at 5.0-6.5, the fermentation tank ventilation volume is controlled at 50-200 mL / min, and the dissolved oxygen is controlled at 20-60%.

3. The method for synthesizing astaxanthin by fermentation of Phaffia rhodozyma as claimed in claim 1, characterized in that: The fermentation medium comprises: 10-50 g / L maltose, 20-50 g / L glycerol, 10-20 g / L yeast powder, 5-10 g / L soy peptone, 1-3 g / L potassium dihydrogen phosphate, 1-3 g / L dipotassium hydrogen phosphate, 1-2 g / L magnesium sulfate heptahydrate, 0.2-0.5 g / L calcium chloride, 0.5-1 g / L biotin, and the rest is water, with a pH of 6.0-6.

5.

4. The method for synthesizing astaxanthin by fermentation of Phaffia rhodozyma as claimed in claim 1, characterized in that: The seed solution preparation method is as follows: The red Phaffia yeast was inoculated into the fermentation medium from the preserved slant medium, the culture temperature was 25-30°C, the shaking speed was 200-400 rpm, the culture time was 48-60h, and the medium pH was 6.0-6.5 to obtain the first-level seed solution; The first-level seed liquid is inoculated into the fermentation medium at an inoculation rate of 0.5-1.0%, the culture temperature is 25-30°C, the shaking speed is 200-400 rpm, the culture time is 48-60h, and the medium pH is 6.0-6.5 to obtain the second-level seed liquid.

5. Application of a deep eutectic solvent in the production of astaxanthin by Phaffia rhodozyma, characterized in that the deep eutectic solvent comprises a deep eutectic solvent A and a deep eutectic solvent B; the deep eutectic solvent A is composed of choline chloride and urea, and the molar ratio of choline chloride to urea is 1:2; the deep eutectic solvent B is composed of choline chloride and citric acid, and the molar ratio of choline chloride to citric acid is 1:1; The red yeast is red yeast ( Phaffia rhodozyma )NCUPFR001, the deposit number is CGMCCNo.31951.

6. The use according to claim 5, characterized in that It is used to increase the permeability of microbial cell membranes; or to regulate the activity of key enzymes in astaxanthin metabolism and synthesis; or to cause intracellular acid-base imbalance.

7. The use according to claim 6, characterized in that The key enzymes include but are not limited to acetyl-CoA, isoprene pyrophosphate isomerase, and β-hydroxylase.

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