A method for efficiently obtaining 7-dehydrocholesterol from a yeast fermentation broth

Abstract

The present application relates to the technical field of biological chemical industry, and particularly relates to a method for efficiently obtaining 7-dehydrocholesterol from yeast fermentation liquor. The method of the present application is to treat the yeast fermentation liquor with ionic liquid microemulsion, in which the ionic liquid dissolves polysaccharide on the surface of cell wall, and is used for assisting ultrasonic cell wall breaking, so as to shorten the ultrasonic cell wall breaking time, reduce the cell wall breaking power, and improve the cell wall breaking capacity. In addition, the ionic liquid microemulsion contains oil phase, which is a new process integrating the two-step traditional process of cell wall breaking and solvent extraction into one step, so as to shorten the overall process time, reduce the use of process equipment, simplify the process operation process, and can be completely applied to the environment-friendly industrial production, with low cost and wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, specifically to a method for efficiently obtaining 7-dehydrocholesterol from yeast fermentation broth. Background Technology

[0002] 7-Dehydrocholesterol (7-DHC) is a sterol compound and a precursor for the synthesis of vitamin D3. Under ultraviolet radiation, 7-Dehydrocholesterol is converted into vitamin D3 in human skin, thereby regulating calcium and phosphorus metabolism, immune responses, and cell differentiation. In recent years, 7-DHC has been widely used in the pharmaceutical industry for the synthesis of highly active vitamin D analogs (such as calcitriol) to treat osteoporosis; in the new materials industry for the synthesis of photoresponsive materials and liquid crystal compounds; and in the nutrition industry, primarily as a functional food additive and feed additive.

[0003] While traditional chemical methods for synthesizing 7-dehydrocholesterol are technologically mature and reliable with standardized reaction conditions, they suffer from drawbacks such as unstable raw materials, cumbersome processes, complex byproduct removal, high pollution risks, and low yields. Currently, microbial fermentation for 7-dehydrocholesterol preparation offers green sustainability and high selectivity. Patents such as Dalian Yinuo Biotechnology Co., Ltd.'s CN115044603B, Tianjin University's CN113151027B, and Jiangnan University's CN112813129B and CN114606147B primarily focus on strain screening and molecular modification for yeast fermentation of 7-dehydrocholesterol; research on cell wall disruption and extraction technologies for obtaining 7-dehydrocholesterol from fermentation broth is lacking. Furthermore, while patent CN117362376A discloses a method for extracting 7-dehydrocholesterol, it requires high temperature and strong alkali conditions, large amounts of organic solvents, and severe environmental pollution; patent CN115594727B involves cumbersome steps and has high operational requirements.

[0004] Currently, methods such as acid-base chemical disruption, high-pressure homogenization, and enzymatic disruption are commonly used to address the thick cell walls and difficulty in breaking down yeast cells. However, these methods suffer from drawbacks including high destructiveness, significant pollution, limited cell disruption rates, and high costs. Ionic liquids, as a novel "green" solvent and functional material, offer advantages such as strong dissolving power, low melting point, good thermal stability, and high design flexibility. However, common imidazole-based ionic liquids have poor biodegradability, posing environmental risks. Furthermore, the extraction of 7-dehydrocholesterol using a single ionic liquid relies on cell wall disruption, and extraction efficiency decreases when cell wall disruption capabilities are limited.

[0005] To address the shortcomings and deficiencies in existing research techniques for extracting 7-dehydrocholesterol from yeast, this invention proposes a type of choline-based ionic liquid microemulsion for cell wall disruption and extraction. This microemulsion possesses advantages such as biodegradability and being green and pollution-free, effectively improving extraction efficiency and product purity, and has broad prospects for industrial application. Summary of the Invention

[0006] The purpose of this invention is to provide a method for efficiently obtaining 7-dehydrocholesterol from yeast fermentation broth, in order to solve the problems of traditional acid-base chemical cell disruption, high-pressure homogenization physical cell disruption, and enzymatic cell disruption, which are highly destructive, polluting, have limited cell disruption rates, and are costly. In addition, common imidazole ionic liquids have poor degradability and pose environmental hazards. Furthermore, the extraction of 7-dehydrocholesterol from a single ionic liquid depends on cell disruption treatment, and the extraction capacity decreases when the cell disruption capacity is limited.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a method for efficiently obtaining 7-dehydrocholesterol from yeast fermentation broth, comprising the following steps:

[0008] S1. Choline-based ionic liquid, composite surfactant, oil phase and aqueous phase are formulated and prepared according to the weight ratio and process to obtain ionic liquid microemulsion; S2. Centrifuge the yeast fermentation broth containing 7-dehydrocholesterol, collect the bacterial sludge and wash it several times to obtain pure bacterial sludge; S3. Mix the ionic liquid microemulsion with the pure bacterial mud, stir at medium temperature and treat with high temperature ultrasound to break the cell wall and extract the solution. S4. Centrifuge the cell wall-breaking extract and collect the lower solid phase bacterial mud and aqueous phase, the middle emulsion phase and the upper oil phase respectively. S5. The lower solid phase bacterial sludge is returned for multiple extractions; the middle emulsion layer collects ionic liquid and composite surfactants for reuse; the upper oil phase has a total extraction yield of approximately 95% for 7-dehydrocholesterol.

[0009] Furthermore, the choline-based ionic liquid uses choline as a cation donor; the anion donor includes, but is not limited to, amino acids such as tryptophan, phenylalanine, histidine, and alanine; and fatty acids such as arachidic acid, myristic acid, stearic acid, and lauric acid.

[0010] Furthermore, the composite surfactant is composed of a surfactant and a cosolvent; wherein the surfactant is a nonionic surfactant such as Tween-80 or Brij-35, and the cosolvent is one of n-butanol, ethanol, and isopropanol; the weight ratio of surfactant to cosolvent is 1:1 to 5:1, and the overall HLB value is 10 to 20.

[0011] Furthermore, the oil phase is one of ethyl oleate, medium-chain triglycerides, tocopheryl acetate, and isopropyl palmitate.

[0012] Furthermore, the aqueous phase is one of Tris-HCl buffer, phosphate buffer, and citrate buffer with a pH of 7.2 to 7.8.

[0013] Furthermore, the weight ratio formulation and process are as follows: 10%~25% choline-based ionic liquid and 20%~40% composite surfactant are mixed and stirred at 30~60°C until a homogeneous transparent colloid is formed.

[0014] Furthermore, the weight ratio formulation and process are as follows: 35-55% aqueous phase is added dropwise to the above transparent colloid at a uniform rate; the addition time is 5-15 minutes, and the stirring speed is 400-800 rpm to form a pre-emulsion.

[0015] Furthermore, the weight ratio formulation and process are as follows: 12%~20% of the oil phase is added to the above pre-emulsion; stirring time is 10~30 min, and the rotation speed is 600~1000 rpm; followed by ultrasonication time is 1~15 min, the power is 150W~300W, and the frequency is 20~40kHz; forming an ionic liquid microemulsion with a particle size of 100~400 micrometers.

[0016] Furthermore, the yeast fermentation broth is one of Yeast lipolytica fermentation broth, Saccharomyces cerevisiae fermentation broth, and Pichia pastoris fermentation broth.

[0017] Furthermore, the yeast fermentation broth is centrifuged at a speed of 6000~12000 rpm.

[0018] Furthermore, in the aforementioned bacterial mud washing, the detergent is deionized water, the washing frequency is 2 to 7 times, and the final conductivity is 50 to 500 μS / cm.

[0019] Furthermore, the pure bacterial sludge has a moisture content of 60-90%.

[0020] Furthermore, the weight ratio of the ionic liquid microemulsion to the bacterial sludge is 4:1 to 15:1.

[0021] Furthermore, the medium-temperature stirring is carried out at a temperature of 35~50℃, a stirring time of 30~100min, and a stirring speed of 100~600rpm.

[0022] Furthermore, the high-temperature ultrasound is characterized by a temperature of 40~70℃, an ultrasonic power of 100~400W, and an ultrasonic frequency of 20~60kHz; it is pulsed ultrasound, with an on / off cycle of 2~7s and a total number of cycles of 30~100, and a total ultrasound time of 2~25min.

[0023] Furthermore, the centrifugation separation is carried out at a speed of 4000~12000 rpm for a time of 2~30 min.

[0024] Furthermore, the lower solid phase bacterial sludge is extracted 1 to 6 times.

[0025] Furthermore, the ionic liquid in the intermediate emulsion layer is collected and then combined with a composite surfactant, an oil phase, and an aqueous phase to prepare an ionic liquid microemulsion, which is reused 2 to 15 times.

[0026] The beneficial effects of this invention are: 1. This invention designs an ionic liquid microemulsion system, characterized by the ionic liquid dissolving polysaccharides on the cell wall surface to assist in physical ultrasonic cell wall disruption, reducing ultrasonic disruption time and power, and improving the ability to fully disrupt the cell wall; it solves the problems of high cost of enzymatic cell wall disruption, strong destructiveness and high pollution of chemical cell wall disruption, and high energy consumption and incomplete cell wall disruption of single physical cell wall disruption. 2. This invention uses ionic liquid microemulsion as an extractant. Its composite surfactant can increase the contact between ionic liquid and lipid, enabling efficient extraction of 7-DHC from the oil phase. It has the advantages of reducing usage risks, improving extraction efficiency, reducing environmental pollution, and maintaining product stability, thus avoiding the shortcomings of traditional organic extractants. 3. This invention combines ionic liquid microemulsion and ultrasound to process fermented sludge and collect intracellular substances. It is a new process that integrates the two traditional steps of cell wall disruption and solvent extraction into one step, which shortens the overall process time, reduces the use of process equipment, and simplifies the process operation. 4. This invention can efficiently recover and reuse the previous batch of ionic liquids and composite surfactants, and prepare the next batch of ionic liquid microemulsions, achieving low-cost recycling. 5. The method of the present invention can be fully applied to environmentally friendly industrial production, and the cost is low. Attached Figure Description

[0027] Figure 1 This is a purity test chart of the product obtained by the method of this invention; Figure 2 This is a purity test chart of the product obtained from Comparative Example 1. Detailed Implementation

[0028] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention.

[0029] Example 1 In this embodiment, an ionic liquid microemulsion is prepared.

[0030] Take 10 portions of choline-based ionic liquid. In this embodiment, the cation donor of the choline-based ionic liquid is choline, and the anion donor is phenylalanine.

[0031] Take 20 parts of the composite surfactant. In this embodiment, the composite surfactant is Tween-80 and ethanol, and the mass ratio of Tween-80 to ethanol is 3:1.

[0032] Take 55 portions of the aqueous phase. In this embodiment, the aqueous phase is a Tris-HCl buffer solution with pH=7.5.

[0033] Take 15 parts of the oil phase. In this embodiment, the oil phase is ethyl oleate.

[0034] In this embodiment, a choline-based ionic liquid is mixed with a composite surfactant and stirred at 50°C until a homogeneous transparent colloid is formed. Then, an aqueous phase is added dropwise to the homogeneous transparent colloid at 600 rpm to obtain a pre-emulsion. An oil phase is added to the pre-emulsion, and the mixture is stirred at 800 rpm for 20 min, followed by sonication for 10 min (200 W, 24 kHz) to obtain the ionic liquid microemulsion of the present invention. The particle size of the obtained ionic liquid microemulsion is mainly distributed in the range of 125-180 micrometers (particle size refers to the size of cells in the ionic liquid microemulsion).

[0035] Example 2 In this embodiment, the ionic liquid microemulsion prepared in Example 1 was used to extract 7-DHC from the yeast fermentation broth.

[0036] In this embodiment, the yeast fermentation broth is Yeast lipolytica fermentation broth.

[0037] In this embodiment, 10 parts by weight of the ionic liquid microemulsion prepared in Example 1 are used.

[0038] Take 0.5L of Yeast lipophila fermentation broth (7-DHC content is 3810ppm), centrifuge at 10000rpm for 15min, collect the bacterial sludge, wash the bacterial sludge 5 times with deionized water, the endpoint conductivity is 268.0us / cm, and about 125g of pure bacterial sludge is obtained, with a water content of 70.65%; in this embodiment, the weight part of this pure bacterial sludge is set as 1 part.

[0039] Take 500g of the above ionic liquid microemulsion and mix it with 125g of pure bacterial mud. Stir at 40℃ and 400rpm for 80min, then raise the temperature to 60℃ and continuously stir with pulsed ultrasound (3s on / 5s off) for about 10min. The ultrasound intensity is 240W and the frequency is 40kHz; thus, the cell wall-breaking extract is obtained.

[0040] Centrifuge the cell-wall-breaking extract at 10,000 rpm for 10 min, and collect the lower solid phase bacterial mud and aqueous phase, the middle emulsion phase and the upper oil phase (referred to as one extraction).

[0041] Add 125g of ethyl oleate to the solid phase bacterial sludge, stir for 10min, centrifuge at 10000rpm for 10min, and collect the upper oil tank and the lower bacterial sludge phase; add ethyl oleate to the lower bacterial sludge phase and repeat the above extraction, for a total of 4 extractions. Figure 1 This is a graph showing the purity of the extracted product.

[0042] Table 1. Product purity and yield after each extraction cycle in this embodiment.

[0043] The middle emulsion layer was collected, mainly consisting of approximately 10 parts of choline-phenylalanine ionic liquid. An additional 20 parts of a composite surfactant (Tween-80 and ethanol in a 3:1 mass ratio) were also collected. 55 parts of the aqueous phase (Tris-HCl buffer, pH 7.5) and 15 parts of the oil phase (ethyl oleate) were also collected. The emulsion was prepared according to the ionic liquid microemulsion preparation process described in this invention. The change in cell wall disruption extraction rate with the number of recycling cycles is shown in the table below. Table 2. Variation of cell wall disruption extraction rate with the number of recycling cycles

[0044] It can be seen that the cell wall breaking and extraction ability decreases with the increase of the number of times it is reused; it is recommended to control the number of reuses to within 5 times.

[0045] Comparative Example 1 In this comparative example, the conventional cell wall disruption extraction method is compared with the method of the present invention.

[0046] Take 0.5L of Yeast lipolyticis fermentation broth from Example 2, centrifuge, wash and adjust the wet biomass to 20-30%, then homogenize and disrupt the cell walls three times using a high-pressure homogenizer (110-120 MPa). Microscopic examination shows that the cells are more than 90% broken. Add ethyl oleate to the above-mentioned cell wall disruption solution and extract four times.

[0047] Table 3. Product purity and yield after each extraction in this comparative example.

[0048] The purity of the products in this comparative example was tested as follows: Figure 2 As shown, it is evident that the purity of the product obtained by the conventional cell wall disruption extraction method in this comparative example is lower than that of the product obtained by the method of this invention.

[0049] This invention employs an ionic liquid microemulsion coupled with ultrasound. Choline-based ionic liquids can specifically dissolve yeast cell wall polysaccharides, and ultrasound, in conjunction with ionic liquids, achieves gentle yet thorough cell wall disruption. The composite surfactants in the ionic liquid microemulsion system significantly enhance the affinity between the ionic liquid and sterol lipids, promoting targeted extraction of 7-dehydrocholesterol from the oil phase. This achieves cell wall disruption and extraction in one step, reducing the dissolution of large amounts of intracellular proteins, polysaccharides, and other impurities while ensuring efficient enrichment of the target product. In contrast, Comparative Example 1 relies solely on high-pressure homogenization and mechanical physical cell wall disruption. This disruption depends on high-intensity shearing and the release of intracellular substances is non-selective. After disruption, extraction is performed solely with ethyl oleate, without the solubilizing and targeted extraction effects of ionic liquids. This results in incomplete release of 7-dehydrocholesterol and the dissolution of a large amount of intracellular impurities into the oil phase, ultimately leading to a product purity far lower than that of this invention. Furthermore, the stepwise process also causes product loss and a decrease in yield.

[0050] This invention is not limited to the preferred embodiments described above. Anyone can derive other forms of products under the guidance of this invention. However, regardless of any changes made in their shape or structure, any technical solution that is the same as or similar to this application falls within the protection scope of this invention.

Claims

1. An ionic liquid microemulsion, characterized in that, It comprises the following components in parts by weight: 10-25 parts of choline-based ionic liquid, 20-40 parts of composite surfactant, 12-20 parts of oil phase, and 35-55 parts of aqueous phase, for a total of 100 parts.

2. The ionic liquid microemulsion according to claim 1, characterized in that: The choline-based ionic liquid uses choline as a cation donor and amino acids and fatty acids as anion donors; the amino acids include tryptophan, phenylalanine, histidine and alanine; the fatty acids include arachidic acid, myristic acid, stearic acid and lauric acid.

3. The ionic liquid microemulsion according to claim 1, characterized in that: The composite surfactant includes a surfactant and a cosolvent; the surfactant is a nonionic surfactant, including Tween-80 and Brij-35; the cosolvent includes n-butanol, ethanol and isopropanol; the weight ratio of surfactant to cosolvent is 1~5:1, and the overall HLB value of the composite surfactant is 10~20.

4. The ionic liquid microemulsion according to claim 1, characterized in that: The oil phase includes ethyl oleate, medium-chain triglycerides, tocopheryl acetate, and isopropyl palmitate.

5. The ionic liquid microemulsion according to claim 1, characterized in that: The aqueous phase includes Tris-HCl buffer, phosphate buffer, and citrate buffer.

6. The method for preparing the ionic liquid microemulsion according to any one of claims 1-5, characterized in that: Choline-based ionic liquids were mixed with composite surfactants and stirred until a homogeneous transparent colloid was formed. Then, the aqueous phase was added dropwise to the homogeneous transparent colloid under stirring to form a pre-emulsion. Finally, the oil phase was added to the pre-emulsion under stirring and ultrasonication to form an ionic liquid microemulsion.

7. A method for efficiently obtaining 7-dehydrocholesterol from yeast fermentation broth, characterized in that: The process involves using the ionic liquid microemulsion described or prepared according to any one of claims 1-6, and includes the following steps: S1. Centrifuge the yeast fermentation broth containing 7-dehydrocholesterol and collect the bacterial sludge; S2. Mix the ionic liquid microemulsion with the bacterial sludge, stir and sonicate to obtain the extract; S3. Centrifuge the extract and collect the upper oil phase. 7-Dehydrocholesterol is enriched in the upper oil phase.

8. A method for efficiently obtaining 7-dehydrocholesterol from yeast fermentation broth according to claim 7, characterized in that: The weight ratio of the ionic liquid microemulsion to the bacterial sludge is 4:1 to 15:

1.

9. A method for efficiently obtaining 7-dehydrocholesterol from yeast fermentation broth according to claim 7, characterized in that, The stirring and ultrasonic treatment is as follows: first, stirring is performed at 35~50℃; then, ultrasonic treatment is performed at 40~70℃.

10. A method for efficiently obtaining 7-dehydrocholesterol from yeast fermentation broth according to claim 7, characterized in that: While collecting the upper oil phase, the lower solid phase bacterial sludge and aqueous phase, as well as the middle emulsion phase, are collected separately; the lower solid phase bacterial sludge is extracted multiple times; and the ionic liquid in the middle emulsion layer is recovered for recycling.

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