Rhodotorula mucilaginosa and application thereof

Abstract

The application provides a torulopsis utmi and a method and application for preparing glyceryl shark acid ester by fermentation of the torulopsis utmi, and belongs to the field of biotechnology and food engineering. The torulopsis utmi is a torulopsis utmi obtained through multiple rounds of traditional mutagenesis breeding, and the torulopsis utmi is used to prepare glyceryl shark acid ester. The obtained product glyceryl shark acid ester has high fermentation titer, low content of erucic acid in the product and high biological safety. The method has the advantages of low oxygen demand of the bacterial cells, fast growth speed of the bacterial cells, fast metabolic carbon source speed and high yield of glyceryl shark acid ester, and is beneficial to industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and food engineering, and relates to a Rhodotorula mucilaginosa and application thereof. BACKGROUND

[0002] Squalene, scientific name 24-carbon-cis-15-enoic acid, is rich in animal brain white matter and peripheral nerve tissue, and is the main component of brain lipids and nerve fiber myelin sheath. It accounts for as high as 40% of the total fatty acids in brain lipids, and its role in maintaining the normal function of the nervous system is attracting more and more attention. Studies have shown that squalene is a necessary nutrient element for brain development and normal function maintenance. The fact that breast milk contains rich squalene suggests that squalene may play an important role in the development of the nervous system of infants and young children. Squalene also has potential application prospects in athlete nutrition enhancement due to its dual functions of neuroprotection and energy supplementation. In addition to the health care effect, squalene also shows great application prospects in medicine. Studies have shown that squalene can be used to treat nervous system disorders such as multiple sclerosis, schizophrenia, Alzheimer's disease, Parkinson's syndrome, and can also be used to treat some genetic lipid metabolism disorders such as Zellweger syndrome and adrenoleukodystrophy.

[0003] Squalene can be synthesized by chemical carbonation of cheap erucic acid. However, chemically synthesized squalene has potential safety risks due to the use of a large amount of toxic reagents, and is not suitable for application in the food field.

[0004] Natural squalene has high safety. Studies have found that a variety of plants, marine fish, microalgae, and mold oils contain squalene, among which microalgae and mold oil have lower squalene content and less research. Plant oil is currently the main source of natural squalene, among which Allium sativum oil has the highest squalene content, which can reach 40%-60% of the total fatty acids. However, Allium sativum is an endangered plant, and combined with factors such as difficulty in artificial cultivation and slow growth, the production capacity is extremely low, which cannot meet the market demand. Currently, commercially available squalene products are mainly derived from Acer truncatum Batal. oil, but Acer truncatum also has problems such as long cultivation cycle and unstable seed oil yield, which makes its production cost high, limiting the large-scale application of squalene. More importantly, Acer truncatum oil contains a large amount of erucic acid, which may have adverse effects on heart health.

[0005] Microbial fermentation production of shark acid is not affected by season and environment, and the growth and metabolism of microorganisms is fast, with high space-time productivity, which is an ideal way for large-scale production of shark acid. However, the fermentation titer of shark acid using wild type strains is generally low, such as the mold Mortierella capitata RD000969 can only produce 186.3 mg / L of shark acid; the microalgae Mychonastes afer HSO-3-1 can only produce 61.56 mg / L of shark acid. The use of genetically engineered strains can greatly improve the fermentation titer of shark acid, such as Wang Shi'an et al. reported that the genetically engineered Yarrowia lipolytica can ferment 25.7 g / L of shark acid from glucose, which is the highest level of microbial fermentation production of shark acid at present. However, from the perspective of biological safety and consumer acceptance, the use of natural strains or mutant strains selected by traditional mutagenesis for fermentation production of shark acid has more advantages in the field of food.

[0006] Therefore, there is an urgent need for a method for producing shark acid or shark acid glyceride with high yield, low erucic acid content, and without using genetically engineered strains for fermentation. SUMMARY

[0007] To solve the above technical problems, the present application uses a round red yeast selected by multiple rounds of traditional mutagenesis, and a method for preparing shark acid glyceride by fermentation of the mutagenized round red yeast. The method has high shark acid fermentation titer, low erucic acid content in the product, and high biological safety.

[0008] In a first aspect, the present application provides a round red yeast.

[0009] A round red yeast (Latin name: Rhodotorula toruloides), which was deposited with the China Center for Type Culture Collection on October 13, 2022, and has the accession number CCTCC NO: M 20221564. The round red yeast can be referred to as Rhodotorula toruloides OMK-87 in the present application.

[0010] In a second aspect, the present application provides the use of the round red yeast of the first aspect.

[0011] The round red yeast of the first aspect is used in the preparation of shark acid or shark acid glyceride.

[0012] In a third aspect, the present application provides a method for preparing shark acid glyceride.

[0013] A method for preparing shark acid glyceride, comprising: fermenting the round red yeast of claim 1 in a fermentation medium to obtain shark acid glyceride.

[0014] In some embodiments, the method comprises: after the first aspect of the Rhodotorula mucilaginosa is cultured in a large scale, the Rhodotorula mucilaginosa is fermented in a fermentation medium to obtain the glyceryl shark acid ester.

[0015] In some embodiments, the fermentation medium comprises a carbon source, a nitrogen source, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, calcium sulfate, sodium sulfate, trace elements, and vitamins.

[0016] In some embodiments, the carbon source comprises at least one of glucose, sucrose, fructose, sorbitol, and glycerol, and preferably is glucose. The carbon source comprising glucose is beneficial to reduce the oxygen demand of the bacteria and is beneficial to industrial fermentation.

[0017] In some embodiments, the nitrogen source comprises at least one of ammonium sulfate, urea, yeast extract, and soybean peptone, and preferably is ammonium sulfate. The nitrogen source comprising ammonium sulfate is beneficial to increase the yield of the product and the fermentation titer of the glyceryl shark acid ester.

[0018] In some embodiments, the trace elements comprise at least one of iron, boron, copper, manganese, molybdenum, and zinc. In some embodiments, the trace elements comprise iron, and the use of iron is beneficial to increase the growth rate of the bacteria, the carbon source metabolic rate, and the yield of the glyceryl shark acid ester. In some embodiments, the trace elements comprise iron, boron, copper, manganese, molybdenum, and zinc.

[0019] In some embodiments, the trace elements comprise at least one of ferric chloride, boric acid, copper sulfate, manganese sulfate, sodium molybdate, and zinc sulfate. In some embodiments, the trace elements comprise ferric chloride, boric acid, copper sulfate, manganese sulfate, sodium molybdate, and zinc sulfate.

[0020] In some embodiments, the vitamins comprise at least one of biotin, pantothenic acid or a salt thereof, folic acid, inositol, nicotinic acid, pyridoxine, riboflavin, and thiamine. In some embodiments, the vitamins comprise at least one of biotin, calcium pantothenate, folic acid, inositol, nicotinic acid, pyridoxine, riboflavin, and thiamine. In some embodiments, the vitamins preferably comprise pantothenic acid or a salt thereof (such as calcium pantothenate), and the use of the pantothenic acid or the salt thereof is beneficial to increase the growth rate of the bacteria, the carbon source metabolic rate, and the yield of the glyceryl shark acid ester. In some embodiments, the vitamins comprise biotin, calcium pantothenate, folic acid, inositol, nicotinic acid, pyridoxine, riboflavin, and thiamine.

[0021] In some embodiments, the pH of the fermentation medium is 4.5-7.0. In some embodiments, the pH of the fermentation medium is 5.5-6.5. In some embodiments, the pH of the fermentation medium is preferably 6.0. A pH of 6.0 in the fermentation medium is beneficial for improving the growth and metabolism of the strain and the yield of squalene.

[0022] In some embodiments, the fermentation temperature is 25°C-30°C, preferably 28°C. A fermentation temperature of 28°C is beneficial for increasing the growth and metabolism of the bacterial strain and the yield of squalene.

[0023] In some embodiments, the fermentation medium comprises, based on the total volume of the fermentation medium, 15 g / L-25 g / L ammonium sulfate, 30 g / L-600 g / L glucose, 13 g / L-22 g / L potassium dihydrogen phosphate, 3 g / L-8 g / L magnesium sulfate, 10 mg / L-40 mg / L ferrous sulfate, 0.2 g / L-2 g / L calcium sulfate, 0.2 g / L-2 g / L sodium sulfate, 0.5 ml / L-3 ml / L trace element solution, and 0.5 ml / L-3 ml / L vitamin mixture, as well as a solvent, wherein the solvent is water;

[0024] The trace element solution comprises boric acid 2.23 g / L-5.67 g / L, copper sulfate 102.2 mg / L-309.6 mg / L, ferric chloride 1.22 g / L-4.55 g / L, manganese sulfate 0.32 g / L-1.53 ​​g / L, sodium molybdate 0.95 g / L-2.31 g / L, and zinc sulfate 1.23 g / L-4.87 g / L, as well as a solvent, which is water.

[0025] The vitamin mixture contains biotin 2.3 mg / L-21.2 mg / L, calcium pantothenate 1.00 g / L-40.56 g / L, folic acid 2.6 mg / L-23.4 mg / L, inositol 4.3 g / L-23.1 g / L, niacin 1.02 g / L-3.56 g / L, pyridoxine 1.26 g / L-3.78 g / L, riboflavin 0.54 g / L-3.45 g / L, and thiamine 0.66 g / L-3.89 g / L, as well as a solvent, which is water.

[0026] In some embodiments, the fermentation medium comprises, based on the total volume of the fermentation medium, 16 g / L ammonium sulfate, 300 g / L glucose, 14 g / L potassium dihydrogen phosphate, 4 g / L magnesium sulfate, 20 mg / L ferrous sulfate, 0.5 g / L calcium sulfate, 0.8 g / L sodium sulfate, 1 ml / L trace element solution, and 1 ml / L vitamin mixture, as well as a solvent, which is water.

[0027] The trace element solution contains 3.89 g / L boric acid, 205.9 mg / L copper sulfate, 3.22 g / L ferric chloride, 0.98 g / L manganese sulfate, 1.86 g / L sodium molybdate, and 3.73 g / L zinc sulfate, as well as a solvent, which is water.

[0028] The vitamin mixture contains 16.3 mg / L biotin, 20.66 g / L calcium pantothenate, 16.3 mg / L folic acid, 18.3 g / L inositol, 2.56 g / L niacin, 2.56 g / L pyridoxine, 1.23 g / L riboflavin, and 1.36 g / L thiamine, as well as a solvent, which is water.

[0029] In some embodiments, the expanded culture includes culture using solid slant culture medium and seed culture medium.

[0030] In some embodiments, the fermentation pH is 2-8. In some embodiments, the fermentation pH is 4.5-7.0. In some embodiments, the fermentation pH is 5.5-6.5. In some embodiments, the fermentation pH is preferably 6.0.

[0031] The fermentation pH is controlled using ammonia water or NaOH aqueous solution.

[0032] In some embodiments, the fermentation temperature is 18°C-37°C. In some embodiments, the fermentation temperature is 20°C-35°C. In some embodiments, the fermentation temperature is 25°C-30°C. In some embodiments, the fermentation temperature is preferably 28°C.

[0033] In some embodiments, the fermentation is carried out at a dissolved oxygen content of 5%-60%. In some embodiments, the fermentation is preferably carried out at a dissolved oxygen content of 15%-60%.

[0034] In some embodiments, the fermentation process further includes introducing air.

[0035] In some embodiments, the fermentation aeration ratio is 1.0 VVM-1.5 VVM.

[0036] In some embodiments, the method further includes adding glucose solution after the dissolved oxygen content rebounds during fermentation.

[0037] In some embodiments, the glucose content in the glucose solution is 30wt%-70wt%.

[0038] In some embodiments, the flow rate of the glucose solution is 200 g / L fermentation broth / day.

[0039] In some embodiments, the fermentation process is further characterized by stirring at a speed of 100 rpm to 600 rpm.

[0040] In some embodiments, the fermentation time is 60-210 hours.

[0041] In some embodiments, the method includes: expanding the Rhodotorula glutinis described in the first aspect using a solid slant culture medium and a seed culture medium, then fermenting it in a fermentation medium to obtain squalene; the fermentation medium, calculated by its total volume, comprises 16 g / L ammonium sulfate, 300 g / L glucose, 14 g / L potassium dihydrogen phosphate, 4 g / L magnesium sulfate, 20 mg / L ferrous sulfate, 0.5 g / L calcium sulfate, 0.8 g / L sodium sulfate, 1 ml / L trace element solution, and 1 ml / L vitamin mixture, as well as a solvent, water; the trace element solution comprises 3.89 g / L boric acid, 205.9 mg / L copper sulfate, 3.22 g / L ferric chloride, 0.98 g / L manganese sulfate, 1.86 g / L sodium molybdate, and 3.7 g / L zinc sulfate. 3 g / L; the vitamin mixture contains 16.3 mg / L biotin, 20.66 g / L calcium pantothenate, 16.3 mg / L folic acid, 18.3 g / L inositol, 2.56 g / L niacin, 2.56 g / L pyridoxine, 1.23 g / L riboflavin, and 1.36 g / L thiamine, and a solvent, water; the fermentation pH is 6.0; the fermentation temperature is 28°C; the fermentation process also includes aeration, with an aeration ratio of 1.0 VVM-1.5 VVM; the pH is maintained at 6.0 with ammonia (ammonia is used to control the pH to provide sufficient nitrogen for cell growth); fermentation is carried out with dissolved oxygen content >40%; when the dissolved oxygen content rebounds, a 70% glucose solution is added to maintain the dissolved oxygen content at 15%-60%; at OD 600 When the pH is approximately 300, replace the ammonia with NaOH aqueous solution to adjust the pH (limiting the nitrogen source to stimulate the accumulation of lipids and squalene in the bacterial cells).

[0042] Beneficial effects

[0043] Compared with the prior art, one embodiment of the present invention includes at least one of the following beneficial effects:

[0044] (1) Compared with other trace elements, the addition of iron to the fermentation medium described in this invention is beneficial to improve the growth rate of the cells, the rate of carbon source metabolism, and the yield of squalene.

[0045] (2) Compared with other carbon sources, the carbon source in the fermentation medium of the present invention includes glucose, which is beneficial to reducing the oxygen demand of the cells and is beneficial to industrial fermentation.

[0046] (3) Compared with other nitrogen sources, the nitrogen source in the fermentation medium of the present invention includes ammonium sulfate, which is beneficial to improving product yield and fermentation potency of squalene.

[0047] (4) Compared with other vitamins, the addition of pantothenic acid or its salt to the fermentation medium described in this invention is beneficial to improve the growth rate of the cells, the rate of carbon source metabolism, and the yield of squalene.

[0048] (5) The Rhodotorula glutinis OMK-87 described in this invention is a traditionally induced Rhodotorula glutinis, a non-transgenic engineered strain. The preparation of squalene glycerides using the Rhodotorula glutinis OMK-87 has a high consumer acceptance.

[0049] (6) Using the OMK-87 round red yeast provided by the present invention, the fermentation titer of squalene glycerol is high, the erucic acid content in the product is low, and the biosafety is high.

[0050] (7) The preparation method of shark glycerol provided by the present invention yields shark glycerol with high fermentation potency, low erucic acid content in the product, and high biosafety, making it an excellent dietary supplement. Attached Figure Description

[0051] Figure 1 This is a statistical chart showing the effects of different carbon sources on the growth, oil production, and accumulation of squalene glycerol in Rhodotorula glutinis OMK-87 in Example 3 of the present invention.

[0052] Figure 2 This is a statistical chart showing the effects of different nitrogen sources on the growth, oil production, and accumulation of squalene glycerol in Rhodotorula glutinis OMK-87 in Example 3 of the present invention.

[0053] Figure 3 This is a statistical chart showing the effects of iron and calcium pantothenate on the growth, oil production, and accumulation of squalene glycerol in Rhodotorula glutinis OMK-87 in Example 3 of the present invention.

[0054] Figure 4 This is a statistical chart showing the effect of pH on the growth, oil production, and accumulation of squalene glycerol in Rhodotorula glutinis OMK-87 in Example 3 of the present invention.

[0055] Figure 5 This is a statistical chart showing the effect of temperature on the growth, oil production, and accumulation of squalene glycerol in Rhodotorula glutinis OMK-87 in Example 3 of the present invention.

[0056] Figure 6 The growth curves and oil and squalene potency curves of Rhodotorula glutinis OMK-87 in a 30L fermenter are shown in Example 4 of this invention.

[0057] Figure 7This is a gas chromatogram of the squalene obtained in Example 4 of the present invention after derivatization with KOH methanol solution. In each figure, "squalene potency" indicates the potency of squalene (calculated as squalene).

[0058] Terminology Explanation

[0059] In the foregoing description of this invention, all figures disclosed herein, whether or not the words “approximately” or “about” are used, are approximate values. Based on the disclosed figures, the value of each figure may vary by less than ±10% or by a difference that is considered reasonable by those skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0060] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0061] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0062] The term "VVM" stands for air volume / culture volume / min, which is the ratio of the air volume per minute to the actual liquid volume in the tank.

[0063] The term "wt%" indicates a percentage by mass.

[0064] The term "rpm" indicates rotational speed: revolutions per minute. Specific Implementation

[0065] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0066] Examples are listed below to describe the present invention. However, it should be understood that the present invention is not limited to these examples, but merely provides a method for practicing the present invention.

[0067] Example 1: Isolation and screening of microorganisms producing high levels of squalene glycerol

[0068] Samples were collected from various natural environments, such as marine sediments, coastal mangroves, oil refineries, vegetable garden soil, and the surfaces of food products like apples and cheese. Environmental samples were collected directly from deep soil, while food samples, such as apple peels, were scraped off. 1.5g of each sample was added to 100mL of enrichment medium (using 2g / L olive oil as the sole carbon source) for cultivation. Bacterial enrichment was achieved using olive oil M9 medium (formulation: 2g / L olive oil (PVA emulsified), 12.8g / L Na2HPO4·7H2O, 3g / L KH2PO4, 0.5g / L NaCl, 1g / L NH4Cl, 0.24g / L MgSO4, and CaCl2). 0.011 g / L (balance: water); Actinomycetes were enriched using olive oil Gao's No. 1 medium (formulation: olive oil 2 g / L (PVA emulsified), potassium nitrate 1.0 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.5 g / L, ferrous sulfate 0.01 g / L, sodium chloride 0.5 g / L, and potassium dichromate 0.1 g / L, balance: water); yeasts and molds were enriched using olive oil PDB medium (formulation: olive oil 2 g / L (PVA emulsified) and potato extract 4 g / L, pH 5.6±0.2, balance: water). After one week of culture, primary cultures were obtained. 1 mL of the primary culture was inoculated into 100 mL of fresh secondary enrichment medium and enriched for another week. The enriched cultures were then serially diluted and spread onto the corresponding solid media for isolation and pure culture. The solid media were the corresponding enrichment media with an additional 2% agar. A total of 165 strains of bacteria, 35 strains of actinomycetes, 56 strains of yeast, and 67 strains of mold were isolated.

[0069] The isolated pure cultures were inoculated into the corresponding nitrogen-limiting media and cultured for 3-6 days. Bacteria were cultured on nitrogen-limiting M9 medium (formula: glucose 50 g / L, Na2HPO4·7H2O 12.8 g / L, KH2PO4 3 g / L, NaCl 0.5 g / L, NH4Cl 0.9 g / L, MgSO4 0.24 g / L, and CaCl2 0.011 g / L); actinomycetes were cultured on nitrogen-limiting Gao's No. 1 medium (formula: glucose 50 g / L, potassium nitrate 1.69 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.5 g / L, ferrous sulfate 0.01 g / L, sodium chloride 0.5 g / L, and potassium dichromate 0.1 g / L); and yeasts were cultured on nitrogen-limiting SD medium (formula: glucose 50 g / L, YNB 1.7 g / L, (NH4)2SO4). 1.1 g / L); the mold was cultured on nitrogen-limited PDB medium (formulation: glucose 50 g / L and potato extract powder 2 g / L, pH 5.6 ± 0.2). Cells were collected by centrifugation, freeze-dried, and directly derivatized with KOH methanol solution. GC-MS was used to qualitatively and quantitatively assess the ability of each strain to accumulate squalene glycerol. Yeast Y26 was found to produce the highest squalene glycerol, at 12 mg / L (calculated as squalene), with squalene accounting for 0.5% of the total lipids. Based on 18S rDNA, ITS, and morphological characteristics, yeast Y26 was identified as *Rhodotorula glutinis*, and named *Rhodotorula glutinis* Y26.

[0070] Example 2: EMS chemical mutagenesis of Rhodotorula glutinis Y26

[0071] Rhodotorula glutinis Y26 was inoculated into YPD medium and cultured until OD. 600 =2-3, centrifuge to collect bacterial cells, and resuspend in 100mM potassium phosphate buffer (pH 6.0) to OD. 600 =10, add EMS solution to a final concentration of 0.05 mol / L, shake in a shaker at 28℃ and 100 rpm for 30 minutes, then add 5% sodium thiosulfate solution to terminate the process. Inoculate the bacterial suspension at a ratio of 1:100 into YPD medium and incubate until OD... 600 =20, serially diluted and plated on YPD plates to isolate single colonies. Single colonies were picked and inoculated into SD nitrogen-limited medium for culture. The ability of each strain in the mutant library to produce oil and accumulate squalene was evaluated, and the optimal mutant was selected as the starting strain for the next round of mutagenesis. Among them, Rhodotorula glutinis M1 achieved a dry weight of 10.25 g / L in SD nitrogen-limited medium, with a stem cell oil content of 31.2%, an oil fermentation titer of 3.2 g / L, a squalene content of 2.1% in the oil, and a squalene fermentation titer of 67.2 mg / L, showing a significant improvement compared to the starting strain. Therefore, it was selected as the starting strain for the next round of mutagenesis.

[0072] Rhodotorula glutinis M1 was used, and the above mutagenesis process was repeated once to obtain the optimal mutant Rhodotorula glutinis M2. The Rhodotorula glutinis M2 had a dry weight of 10.11 g / L in SD nitrogen-limited medium, a stem cell oil content of 41.2%, an oil fermentation titer of 4.17 g / L, a squalene content of 8.9% in the oil, and a squalene fermentation titer of 370.7 mg / L, which was significantly improved compared to the starting strain. Rhodotorula glutinis M2 was used, and the same mutagenesis process was repeated once to obtain the optimal mutant Rhodotorula glutinis M3. The Rhodotorula glutinis M3 had a dry weight of 10.35 g / L in SD nitrogen-limited medium, a stem cell oil content of 45.5%, an oil fermentation titer of 4.7 g / L, a squalene content of 12.2% in the oil, and a squalene fermentation titer of 574.5 mg / L, which was also significantly improved compared to the starting strain. Further mutagenesis on this basis did not further improve the squalene production capacity of the strain, and the number of negative mutants increased significantly. Therefore, the optimal mutant Rhodotorula glutinis M3 obtained in the third round of mutagenesis was selected for subsequent culture medium formulation and fermentation parameter optimization experiments. Rhodotorula glutinis M3 was renamed Rhodotorula glutinis OMK-87 and was deposited at the China Center for Type Culture Collection (China, Wuhan, Wuhan University) on October 13, 2022, with accession number: CCTCC NO: M 20221564.

[0073] Example 3: Optimization of culture medium composition and fermentation conditions for the production of squalene by Rhodotorula glutinis OMK-87

[0074] 3.1 Initial fermentation parameters

[0075] The culture medium composition and fermentation parameters of Rhodotorula glutinis OMK-87 were systematically optimized using a quadruple parallel bioreactor:

[0076] The initial culture medium formulation is as follows: ammonium sulfate 13 g / L, potassium dihydrogen phosphate 16 g / L, magnesium sulfate 3 g / L, calcium sulfate 0.8 g / L, sodium sulfate 2 g / L, trace element solution 1 ml / L, vitamin mixture 1 ml / L, and carbon source glycerol, along with water as the solvent. The carbon source glycerol is added as needed. The trace element solution consists of: boric acid 3.89 g / L, copper sulfate 205.9 mg / L, ferric chloride 3.22 g / L, manganese sulfate 0.98 g / L, sodium molybdate 1.86 g / L, and zinc sulfate 3.73 g / L. The vitamin mixture consists of: biotin 16.3 mg / L, calcium pantothenate 2.07 g / L, folic acid 16.3 mg / L, inositol 18.3 g / L, niacin 2.56 g / L, pyridoxine 2.56 g / L, riboflavin 1.23 g / L, and thiamine 1.36 g / L, and water as the solvent.

[0077] Fermentation conditions: initial fermentation pH was 4.8, initial fermentation temperature was 28℃, and fermentation time was 161 h.

[0078] Results: The dry weight of the cells obtained after fermentation under the above initial culture medium and fermentation conditions was 121.5 g / L, the oil fermentation titer was 45.6 g / L, and the squalene fermentation titer was 9.6 g / L.

[0079] 3.2 Screening of carbon sources

[0080] The optimal carbon source for the fermentation of Rhodotorula glutinis OMK-87 to produce squalene was determined.

[0081] Fermentation medium: Replace glycerol with glucose, sucrose, fructose or sorbitol as the carbon source in the above initial medium formula, while keeping the other components unchanged.

[0082] Fermentation conditions: pH 4.8, temperature 28℃, fermentation time 161h.

[0083] Result: See Figure 1 .

[0084] After fermentation under the above-mentioned fermentation medium and conditions, Rhodotorula glutinis OMK-87 showed good growth with glucose, sucrose, and fructose, except for its inability to effectively utilize sorbitol. Among these, glucose as the carbon source yielded the best fermentation results. After 161 hours of fermentation, the dry weight of the cells was 135.1 g / L, the oil fermentation titer was 58.7 g / L, and the squalene fermentation titer was 12.1 g / L, making it the best among all carbon sources. Furthermore, the oxygen demand of the cells was significantly lower with glucose as the carbon source than with other carbon sources, which is beneficial for industrial fermentation. Therefore, glucose was determined to be the optimal carbon source.

[0085] 3.3 Screening of Nitrogen Sources

[0086] The optimal nitrogen source for the fermentation of Rhodotorula glutinis OMK-87 to produce squalene was studied.

[0087] Fermentation medium: The nitrogen source in the initial medium formula was replaced by urea, yeast extract, and soybean peptone, respectively, instead of ammonium sulfate, while the other components remained unchanged.

[0088] Fermentation conditions: pH 4.8, temperature 28℃, fermentation time 161h.

[0089] Result: See Figure 2 .

[0090] After fermentation under the above-mentioned fermentation medium and conditions, except for ammonium sulfate, using organic nitrogen sources such as yeast extract and soybean peptone as nitrogen sources in the fermentation medium can effectively shorten the lag phase of the strain, but will severely inhibit the accumulation of oil and squalene by the strain. For example, when yeast extract is used as the nitrogen source, the dry weight of the cells after 161 hours of fermentation is 100.8 g / L, the oil fermentation titer is 23.7 g / L, and the squalene fermentation titer is 4.3 g / L. The squalene glycerol fermentation titer is much lower than that of the squalene glycerol fermentation titer of the medium using ammonium sulfate as the nitrogen source. Therefore, ammonium sulfate is determined to be the optimal nitrogen source.

[0091] 3.4 Screening of Trace Elements

[0092] Investigating the effects of different trace elements on the cell growth, glucose metabolism, and squalene production of Rhodotorula glutinis OMK-87:

[0093] Fermentation medium: Based on the initial medium formula, a certain trace element was removed or added at 10 times the amount to investigate the effects of different trace elements on cell growth, glucose metabolism, and the ability to produce squalene.

[0094] Fermentation conditions: pH 4.8, temperature 28℃, fermentation time 161h.

[0095] result:

[0096] Screening and investigation revealed that after fermentation under the above-mentioned fermentation medium and conditions, removing iron from the initial medium formula resulted in a cell dry weight of 56.4 g / L after 161 hours of fermentation, an oil fermentation titer of 17.8 g / L, and a squalene fermentation titer of 2.3 g / L. The removal of other elements had no significant impact on cell growth, glucose metabolism, and the ability to produce squalene glycerides.

[0097] Screening and investigation revealed that after fermentation under the aforementioned fermentation medium and conditions, adding 10 times the amount of iron to the initial medium formula resulted in a slight increase in cell dry weight, a significant improvement in the cell's ability to metabolize glucose, and a substantial increase in the fermentation potency of squalene. After 161 hours of fermentation, the cell dry weight was 136.4 g / L, the oil fermentation potency was 61.2 g / L, and the squalene fermentation potency was 12.4 g / L. However, adding 10 times the amount of other trace elements had no significant effect on cell growth, glucose metabolism, or the ability to produce squalene.

[0098] Conclusion: Iron is beneficial for improving the cell growth, glucose metabolism, and squalene production of Rhodotorula glutinis OMK-87.

[0099] 3.5 Vitamin Screening

[0100] The effects of vitamins on the cell growth, glucose metabolism, and squalene production of Rhodotorula glutinis OMK-87 were investigated.

[0101] Fermentation medium: Based on the initial medium formula, a certain vitamin was removed or a certain vitamin was added at 10 times the amount to investigate the effects of different vitamins on cell growth, glucose metabolism, and the ability to produce squalene.

[0102] Fermentation conditions: pH 4.8, temperature 28℃, fermentation time 161h.

[0103] result:

[0104] Screening and investigation revealed that removing pantothenic acid or its salts from the initial culture medium formulation significantly affected the cell growth, glucose metabolism, and squalene production. For example, after removing pantothenic acid or its salts, the cell dry weight after 161 hours of fermentation was 54.6 g / L, the oil fermentation titer was 9.7 g / L, and the squalene fermentation titer was 1.4 g / L. However, after adding 10 times the amount of calcium pantothenic acid, the cell dry weight after 161 hours of fermentation was 145.3 g / L, the oil fermentation titer was 62.2 g / L, and the squalene fermentation titer was 16.8 g / L. This demonstrates that pantothenic acid or its salts have a significant promoting effect on the squalene production of Rhodotorula glutinis OMK-87.

[0105] 3.6 Investigation of Fermentation pH

[0106] Investigating the effect of fermentation pH on the production of squalene by Rhodotorula glutinis OMK-87:

[0107] Fermentation medium: initial culture medium.

[0108] Fermentation conditions: Fermentation temperature was 28℃, fermentation time was 161h, and fermentation pH was investigated at 2.5, 3.5, 4.8, 6.0, 7.0 and 8.0 respectively.

[0109] Result: See Figure 4 .

[0110] pH 6.0 is the optimal fermentation pH for the production of squalene by Rhodotorula glutinis OMK-87. A pH that is too low or too high will significantly affect the growth, metabolism, and ability of the strain to produce squalene.

[0111] 3.7 Investigation of Fermentation Temperature

[0112] Investigating the effect of fermentation temperature on the production of squalene by Rhodotorula glutinis OMK-87:

[0113] Fermentation medium: initial culture medium.

[0114] Fermentation conditions: Fermentation pH was 4.8, fermentation time was 161 h, and fermentation temperatures were investigated at 18℃, 23℃, 28℃, 32℃, and 37℃.

[0115] Result: See Figure 5 .

[0116] 28℃ is the optimal fermentation temperature for Rhodotorula glutinis OMK-87 to produce squalene. Fermentation temperatures that are too low or too high will significantly affect the growth, metabolism, and ability of the strain to produce squalene.

[0117] 3.8 Determination of the optimal fermentation medium

[0118] Optimal fermentation medium: Based on the results of single-factor experiments, response surface methodology was used to further optimize the medium components. The optimal fermentation medium composition was obtained as follows: ammonium sulfate 16 g / L, initial glucose 30 g / L, potassium dihydrogen phosphate 14 g / L, magnesium sulfate 4 g / L, ferrous sulfate 20 mg / L, calcium sulfate 0.5 g / L, sodium sulfate 0.8 g / L, trace element solution 1 ml / L, and vitamin mixture 1 ml / L, with the remainder being water. The carbon source glucose was added as needed. The trace element solution composition was: boric acid 3.89 g / L, copper sulfate 205.9 mg / L, ferric chloride 3.22 g / L, manganese sulfate 0.98 g / L, sodium molybdate 1.86 g / L, and zinc sulfate 3.73 g / L, with the remainder being water. The vitamin mixture consists of: biotin 16.3 mg / L, calcium pantothenate 20.66 g / L, folic acid 16.3 mg / L, inositol 18.3 g / L, niacin 2.56 g / L, pyridoxine 2.56 g / L, riboflavin 1.23 g / L, and thiamine 1.36 g / L, with the remainder being water.

[0119] Example 4: Production of low erucic acid and high squalene glycerides (fermentation in a 30L fermenter)

[0120] The ability of Rhodotorula glutinis OMK-87 to produce squalene was verified in a 30L fermenter. Rhodotorula glutinis OMK-87 was cultured sequentially on solid slant culture medium and seed culture medium to obtain a seed culture. This seed culture was inoculated into the optimal fermentation medium obtained in Example 3 and cultured at a pH of 6.0, a temperature of 28°C, a stirring speed of 100-600 rpm, an aeration rate of 1-1.5 VVM, and a dissolved oxygen content >40%. Fermentation was carried out after the glucose in the substrate was depleted and the dissolved oxygen content rebounded, with a 70% glucose solution added, controlling the residual glucose concentration at 1-10 g / L, a stirring speed of 400-600 rpm, and maintaining a dissolved oxygen content >15%. During the early stage of fermentation, ammonia was used to control the pH to provide sufficient nitrogen for cell growth. Once the cell growth reached OD... 600When the pH reached approximately 300, a 30% NaOH solution was used to control the pH and limit the nitrogen source to stimulate the accumulation of squalene glycerol in the cells. After a final fermentation time of 132 hours, the cell dry weight reached 186.6 g / L, the cell oil content was 57.8%, and the oil fermentation potency was 107.9 g / L. The total oil contained 26.1% squalene and 1.6% erucic acid, equivalent to a squalene fermentation potency of 28.2 g / L.

[0121] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A type of round red yeast ( Rhodotorula toruloides It is deposited at the China Center for Type Culture Collection, accession number: CCTCC NO: M 20221564.

2. The use of the Rhodotorula glutinis of claim 1 in the preparation of squalene or squalene glycerides.

3. A method for preparing squalene, comprising: The round red yeast of claim 1 is placed in a fermentation medium and fermented to obtain squalene.

4. The method according to claim 3, comprising: After being cultured in an expanded manner, the Rhodotorula glutinis described in claim 1 was fermented in a fermentation medium to obtain squalene.

5. The method according to claim 3, wherein the fermentation medium comprises a carbon source, a nitrogen source, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, calcium sulfate, sodium sulfate, trace elements, and vitamins.

6. The method according to claim 5, wherein the carbon source comprises at least one selected from glucose, sucrose, fructose, sorbitol, and glycerol.

7. The method according to claim 5, wherein the carbon source is glucose.

8. The method according to claim 5, wherein the nitrogen source comprises at least one of ammonium sulfate, urea, yeast extract, and soybean peptone.

9. The method according to claim 5, wherein the nitrogen source is ammonium sulfate.

10. The method according to claim 5, wherein the trace element includes at least one selected from iron, boron, copper, manganese, molybdenum, and zinc.

11. The method according to claim 5, wherein the trace element comprises at least one selected from ferric chloride, boric acid, copper sulfate, manganese sulfate, sodium molybdate, and zinc sulfate.

12. The method according to claim 5, wherein the vitamin comprises at least one of biotin, pantothenic acid or its salt, folic acid, inositol, niacin, pyridoxine, riboflavin, and thiamine.

13. The method according to claim 3, wherein the pH of the fermentation medium is 4.5-7.

0.

14. The method according to claim 3, wherein the pH of the fermentation medium is 6.

0.

15. The method according to claim 3, wherein the fermentation temperature is 25℃-30℃.

16. The method according to claim 3, wherein the fermentation temperature is 28°C.

17. The method according to claim 3, wherein the fermentation medium comprises, based on the total volume of the fermentation medium, 15 g / L-25 g / L ammonium sulfate, 30 g / L-600 g / L glucose, 13 g / L-22 g / L potassium dihydrogen phosphate, 3 g / L-8 g / L magnesium sulfate, 10 mg / L-40 mg / L ferrous sulfate, 0.2 g / L-2 g / L calcium sulfate, 0.2 g / L-2 g / L sodium sulfate, 0.5 ml / L-3 ml / L trace element solution, and 0.5 ml / L-3 ml / L vitamin mixture, and a solvent, wherein the solvent is water; The trace element solution comprises boric acid 2.23 g / L-5.67 g / L, copper sulfate 102.2 mg / L-309.6 mg / L, ferric chloride 1.22 g / L-4.55 g / L, manganese sulfate 0.32 g / L-1.53 ​​g / L, sodium molybdate 0.95 g / L-2.31 g / L, and zinc sulfate 1.23 g / L-4.87 g / L, as well as a solvent, which is water. The vitamin mixture contains biotin 2.3 mg / L-21.2 mg / L, calcium pantothenate 1.00 g / L-40.56 g / L, folic acid 2.6 mg / L-23.4 mg / L, inositol 4.3 g / L-23.1 g / L, niacin 1.02 g / L-3.56 g / L, pyridoxine 1.26 g / L-3.78 g / L, riboflavin 0.54 g / L-3.45 g / L, and thiamine 0.66 g / L-3.89 g / L, as well as a solvent, which is water.

18. The method according to claim 3, wherein the fermentation medium comprises, based on the total volume of the fermentation medium, 16 g / L ammonium sulfate, 300 g / L glucose, 14 g / L potassium dihydrogen phosphate, 4 g / L magnesium sulfate, 20 mg / L ferrous sulfate, 0.5 g / L calcium sulfate, 0.8 g / L sodium sulfate, 1 ml / L trace element solution, and 1 ml / L vitamin mixture, and a solvent, wherein the solvent is water; The trace element solution contains 3.89 g / L boric acid, 205.9 mg / L copper sulfate, 3.22 g / L ferric chloride, 0.98 g / L manganese sulfate, 1.86 g / L sodium molybdate, and 3.73 g / L zinc sulfate, as well as a solvent, which is water. The vitamin mixture contains 16.3 mg / L biotin, 20.66 g / L calcium pantothenate, 16.3 mg / L folic acid, 18.3 g / L inositol, 2.56 g / L niacin, 2.56 g / L pyridoxine, 1.23 g / L riboflavin, and 1.36 g / L thiamine, as well as a solvent, which is water.

19. The method according to claim 4, wherein the expanded culture comprises culture using solid slant culture medium and seed culture medium.

20. The method according to claim 3, wherein the fermentation pH is 2.0-8.

0.

21. The method according to claim 3, wherein the fermentation temperature is 18℃-37℃.

22. The method according to any one of claims 3-21, wherein the fermentation is carried out at a dissolved oxygen content of 5%-60%.

23. The method according to any one of claims 3-21, wherein the fermentation is carried out at a dissolved oxygen content of 15%-60%.

24. The method according to any one of claims 3-21, wherein the fermentation further comprises introducing air.

25. The method according to any one of claims 3-21, wherein the fermentation aeration ratio is 1.0 VVM - 1.5 VVM.

26. The method according to any one of claims 3-21, further comprising adding glucose solution after the dissolved oxygen content rebounds during fermentation.

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