A method for extracting and separating rhodin from rhodotorula

Abstract

The present application relates to the field of microbial active ingredient extraction and separation, and particularly relates to a method for extracting and separating rhodovibrin, comprising the following steps: adding a first phase elution solution to total carotenoid extract containing rhodovibrin, then loading the total carotenoid extract into a solid phase extraction column, eluting the total carotenoid extract with the first phase elution solution, then eluting the total carotenoid extract with a second phase elution solution, and collecting the solution obtained by eluting the total carotenoid extract with the second phase elution solution; the first phase elution solution is a mixture of n-hexane and diethyl ether with a volume ratio of 7:1; the second phase elution solution is a mixture of methanol, acetone and n-hexane with a volume ratio of 2:2:1; the above method can obtain purified rhodovibrin, and the above method is simple, has few operation steps, has low reaction condition requirement, and can obtain rhodovibrin with high purity, thereby achieving the purpose of quickly and simply obtaining rhodovibrin with high purity.

Description

Technical Field

[0001] This invention relates to the field of extraction and separation of active ingredients from microorganisms, and specifically to a method for extracting and separating erythropoietin from red yeast. Background Technology

[0002] Carotenoids are a class of important pigments widely found in nature, generally appearing yellow, orange, or red. To date, nearly 750 types of carotenoids have been discovered, with β-carotene, lycopene, and lutein being the main representatives. Besides plant synthesis, some bacteria, yeasts, and molds are also natural sources of carotenoids. Yeasts of the genus *Rhodotorula* (*Rhodotorula spp.*) produce carotenoids, with *Rhodotorula mucilaginosa* capable of synthesizing β-carotene, γ-carotene, saccharin, and erythropoietin using different types of carbon sources. Humans and animals cannot synthesize carotenoids and must obtain them from food, where they must be digested and absorbed before utilization. Erythropoietin is an important carotenoid; its molecular structure contains a closed β-ionone ring and a long carbon chain skeleton rich in unsaturated double bonds. It is a precursor compound of vitamin A. Because its structure contains more unsaturated double bonds than lycopene, erythropoietin is considered to have stronger antioxidant properties than lycopene. Currently, this pigment has only been found in fungi, and studies have shown that it possesses various biological activities, including antioxidant, anti-inflammatory, antibacterial, and anticancer effects, making it a promising candidate for applications in food, feed, and biomedicine. Therefore, developing a low-cost and simple method for the production, extraction, and separation of erythropoietin from red yeast would be highly beneficial for its large-scale industrial production.

[0003] Red yeast erythrin is an intracellular product of red yeast, usually dispersed in lipid droplets within the cytoplasm. Therefore, its extraction, separation, and purification are quite difficult. A series of physical, chemical, and biological methods are usually required to break down the yeast cells and release the intracellular product. At the same time, due to its similar polarity and solubility to lipids and other carotenoids, the separation and purification of red yeast erythrin is usually difficult to achieve. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for extracting and separating red yeast erythrin.

[0005] Therefore, the present invention provides the following technical solution:

[0006] A method for extracting and separating erythropoietin from red yeast includes the following steps:

[0007] Add the first phase elution solution to the total carotenoid extract containing erythropoietin, then load the sample onto a solid phase extraction column, elute with the first phase elution solution, and then elute with the second phase elution solution. Collect the solution obtained by elution with the second phase elution solution.

[0008] The first phase elution solution is a mixture of n-hexane and diethyl ether in a volume ratio of 7:1;

[0009] The second phase elution solution is a mixture of methanol, acetone and n-hexane in a volume ratio of 2:2:1.

[0010] Optionally, the first phase elution solution is used to elute 5 column volumes;

[0011] And / or, the second phase elution solution elutes 5 column volumes.

[0012] Optionally, the ratio of the total carotenoid extract containing erythropoietin to the first phase elution solution is 1:10, with a ratio of mg:mL;

[0013] And / or, prior to sample loading, the solid-phase extraction column is equilibrated with 5 column volumes of n-hexane;

[0014] And / or, the solid phase extraction column is a silica solid phase extraction column with a capacity of 1 g / 6 mL adsorbent.

[0015] Optionally, the preparation method of the total carotenoid extract containing erythropoietin includes the following steps:

[0016] (1) Fermenting carotenoid-producing red yeast (Rhodotorula) or Rhodosporidium to obtain fermentation broth, separating the fermentation broth to obtain wet mycelium containing red yeast erythrin;

[0017] (2) The wet mycelium is subjected to saponification treatment;

[0018] (3) Add deionized water and n-hexane to the saponification reaction solution obtained in step (2). The volume ratio of saponification reaction solution, deionized water and n-hexane is 1:1:1. Extract the solution, collect the upper liquid, and extract repeatedly until the bacterial cells are colorless. Combine the collected upper liquids and dry them in an inert protective atmosphere to obtain a total carotenoid extract containing red yeast erythrin.

[0019] Optionally, in step (1), the carotenoid-producing microorganism includes Rhodotorula spp.

[0020] And / or, in step (1), the seed culture of the erythropoietin-producing microorganism is inoculated into a synthetic culture medium with a carbon-nitrogen ratio of 50 to 200 and cultured at 30°C and 210 to 250 rpm for 72 to 120 hours.

[0021] Optionally, the carotenoid-producing microorganisms include Rhodotorula mucilaginosa, Rhodotorula toruloides, or Rhodotorula glutinis.

[0022] And / or, the synthetic culture medium with a carbon-to-nitrogen ratio of 50 to 200 comprises the following components: 50 g / L glucose, 1.7 g / L yeast basal medium free of amino acids and ammonium sulfate, and 0.55-2.2 g / L ammonium sulfate. The carbon-to-nitrogen ratio is calculated as the ratio of the number of available carbon moles in glucose to the number of available nitrogen moles in ammonium sulfate.

[0023] Optionally, in step (2), anhydrous ethanol containing 1-2M KOH is added to the wet bacterial cells to obtain a bacterial solution with a bacterial concentration of 5-10 g / L. Then, the solution is saponified at 45-65°C and 250-350 rpm in the dark for 0.5-3 hours.

[0024] Optionally, in step (3), the extraction conditions are to vortex for 1 to 3 minutes and centrifuge at 3800 to 4200 rpm for 5 to 10 minutes to separate the solution into layers.

[0025] Optionally, high-performance liquid chromatography (HPLC) can be used to analyze erythropoietin in yam. The chromatographic conditions are as follows: the column is packed with octadecylsilane-bonded silica gel; mobile phase A is a mixture of acetonitrile and water at a volume ratio of 9:1; mobile phase B is ethyl acetate; and the gradient elution program is as follows: 0–6 min, mobile phase B volume percentage 20% → 60%; 6–15 min, mobile phase B volume percentage 60%; 15–20 min, mobile phase B volume percentage 60% → 100%; 20–25 min, mobile phase B volume percentage 100% → 20%; 25–30 min, mobile phase B volume percentage 20%.

[0026] Optionally, the column temperature is 25°C in the chromatographic conditions.

[0027] And / or, under the chromatographic conditions, the DAD detection signal is 494 nm;

[0028] And / or, under the chromatographic conditions, the flow rate is 1 mL / min;

[0029] And / or, under the chromatographic conditions, the injection volume is 10–30 μL;

[0030] And / or, in the chromatographic conditions, the chromatographic column is an Agilent 5TC-C18(2), 250×4.6mm.

[0031] The technical solution of this invention has the following advantages:

[0032] 1. This invention provides a method for extracting and separating erythropoietin from erythropoietin, comprising the following steps: adding a first-phase elution solution to a total carotenoid extract containing erythropoietin, then loading the sample onto a solid-phase extraction column, eluting with the first-phase elution solution, followed by elution with a second-phase elution solution, and collecting the solution obtained by elution with the second-phase elution solution; the first-phase elution solution is a mixture of n-hexane and diethyl ether in a volume ratio of 7:1; the second-phase elution solution is a mixture of methanol, acetone, and n-hexane in a volume ratio of 2:2:1; this invention has found that, using solid-phase extraction technology, for a sample being a total carotenoid extract containing erythropoietin, firstly eluting with the aforementioned first-phase elution solution to remove non-polar carotenoids, and then eluting with the second-phase elution solution to remove erythropoietin, purified erythropoietin can be obtained. The above method is simple, has few operating steps, low reaction condition requirements, and yields high-purity erythropoietin, thereby achieving the goal of rapidly and easily obtaining high-purity erythropoietin;

[0033] Furthermore, the red yeast erythrin obtained by the above method is a pure natural product, free of artificial pigments or other chemical additives. The reagents and materials used are low in toxicity, readily available, and low in cost, and can be widely used in various fields such as food, cosmetics, and pharmaceuticals.

[0034] 2. The present invention provides a method for extracting and separating erythropoietin from Rhodotorula rubigin. The method for preparing the total carotenoid extract containing erythropoietin from Rhodotorula rubigin includes the following steps: (1) fermenting carotenoid-producing Rhodotorula rubigin or Rhodosporidium rubigin to obtain a fermentation broth, separating the fermentation broth to obtain wet mycelium containing erythropoietin from Rhodotorula rubigin; (2) saponifying the wet mycelium; (3) adding deionized water and n-hexane to the saponification reaction solution obtained in step (2). The saponification reaction solution, deionized water, and n-hexane were mixed in a volume ratio of 1:1:1 for extraction. The supernatant was collected, and extraction was repeated until the bacterial cells were colorless. The collected supernatants were combined and dried in an inert atmosphere to obtain a total carotenoid extract containing yeast erythrin. This invention found that prior to saponification, cell disruption not only failed to improve the recovery rate of carotenoids, especially yeast erythrin, but also led to the loss of carotenoids, particularly yeast erythrin, resulting in a low recovery rate. However, it was found that during the saponification process, the OH groups in the saponification system... -It can dissolve the protein components of yeast cell walls, assist ethanol solution in penetrating the cytoplasm and saponifying and dissolving the oils, thereby dissolving the pigment components and achieving rapid extraction and separation of the pigment components from the oil components. Therefore, this invention omits the steps of drying and breaking the cell walls of the cells, and instead selects to separate the fermentation broth, wash away the residual fermentation broth, retain the wet cells, and directly saponify the wet cells, which improves the recovery rate of red yeast erythrin and reduces the number of operation steps and production costs. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 The HPLC chromatogram of red yeast erythrin standard;

[0037] Figure 2 The HPLC chromatogram of the total carotenoid extract is shown below.

[0038] Figure 3 The HPLC chromatogram of the eluent from the first phase eluent;

[0039] Figure 4 HPLC chromatogram of the eluent from the second phase eluent;

[0040] Figure 5 The images show the separation effect of total carotenoid extract from red yeast on SPE columns; Figure A shows the first phase elution effect of total carotenoids; Figure B shows the first phase elution effect of total carotenoids. Detailed Implementation

[0041] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0042] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0043] The YPD agar medium in the following examples consists of: 10 g / L yeast extract, 20 g / L peptone, 20 g / L agar, and 20 g / L glucose; the YPD liquid medium consists of: 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose.

[0044] Example 1: Extraction and isolation of erythropoietin from Rhodotorula glutinis

[0045] The method for extracting and separating red yeast erythrin in this embodiment includes the following steps:

[0046] The strain used in this embodiment, Rhodotorula mucilaginosa CGMCC 2.4005, was purchased from the China General Microbiological Culture Collection Center.

[0047] (1) Preparation of Rhodotorula glutinis seed culture: The freeze-dried Rhodotorula glutinis was dissolved in sterile water and streaked onto yeast extract peptone glucose (YPD) agar medium. The medium was then inverted in a 30°C constant temperature incubator and cultured for 7 days. Single colonies were picked from the above plates and inoculated into 100-500 mL of YPD liquid medium. The medium was then pre-cultured in shake flasks at 30°C and 210 rpm for 72 h to obtain Rhodotorula glutinis seed culture.

[0048] (2) Production of red yeast erythrin: The prepared red yeast seed culture was centrifuged at 3800 rpm for 5 min to collect cells. The cells were washed twice with sterile water, and the supernatant was discarded. The cells were then inoculated into a synthetic medium with a carbon-nitrogen ratio of 100 or 200 at a concentration of 1 g / L. The culture was carried out at 30℃ and 210 rpm for 96 h to obtain a fermentation broth containing red yeast erythrin. The synthetic medium with a carbon-nitrogen ratio of 100 or 200 consisted of 50 g / L glucose, 1.7 g / L yeast basal medium (Y1251, Sigma) without amino acids and ammonium sulfate, and 1.1 g / L or 0.55 g / L ammonium sulfate. The carbon-nitrogen ratio was calculated as the ratio of the number of available carbon moles in glucose to the number of available nitrogen moles in ammonium sulfate.

[0049] (3) Cell collection: Centrifuge the fermentation broth obtained from the expansion culture in step (2) at 3800 rpm for 5 min, wash twice with sterile water, discard the supernatant to obtain cells containing red yeast erythrin.

[0050] (4) Separation and extraction of oil and carotenoids: Add a certain volume of anhydrous ethanol containing 1.1M KOH to the bacterial cells obtained in step (3) to make the final concentration of bacterial cells about 6g / L. Saponify for 1 hour at 50℃ and 350rpm on a magnetic stirrer in the dark.

[0051] (5) Extraction of total carotenoids: After the saponification reaction in step (4) is completed and the reaction solution is cooled, add the same volume of deionized water and n-hexane as the reaction solution (i.e., saponification reaction solution: deionized water: n-hexane volume ratio = 1:1:1), vortex for 1-3 min, centrifuge at 3800 rpm for 5 min to separate the solution into layers, collect the upper layer, repeatedly add n-hexane to the precipitate, and repeatedly extract until the yeast cells are colorless; combine the collected upper liquid, blow dry under nitrogen, and the residual component is the total carotenoid extract of Rhodotorula glutinis;

[0052] (6) Separation of red yeast erythrin: The total carotenoid extract described in step (5) and the first phase elution solution were mixed at a ratio of 1:10 and then transferred to a solid phase extraction (SPE) column (silica solid phase extraction column, 1g / 6mL adsorbent) that had been equilibrated with 5 column volumes of n-hexane. The remaining carotenoid extract in the original container was rinsed with a certain volume of the first phase elution solution and added to the column. After the sample was loaded onto the SPE column, the first phase nonpolar carotenoids were eluted with 5 column volumes of the first phase elution solution. After all the liquid on the column had passed through, a new glass tube was replaced to collect the second phase. Red yeast erythrin was eluted with 5 column volumes of the second phase elution solution and the eluent of the second phase elution solution was collected. The solvent in the second phase was dried under nitrogen. The remaining component was red yeast erythrin. The first phase elution solution was a mixture of n-hexane and diethyl ether in a volume ratio of 7:1. The second phase elution solution was a mixture of methanol, acetone and n-hexane in a volume ratio of 2:2:1. See the diagram showing the separation effect of total carotenoid extract from red yeast on SPE column. Figure 5 (A: The first-phase elution effect of total carotenoids was achieved in three parallel experiments using synthetic medium with a carbon-to-nitrogen ratio of 100, as described in this example. This effect was also achieved in synthetic medium with a carbon-to-nitrogen ratio of 100, as well as in Examples 2-3, eluting pigments other than erythropoietin. B: The second-phase elution effect of total carotenoids was achieved in three parallel experiments using synthetic medium with a carbon-to-nitrogen ratio of 100, as described in this example. This effect was also achieved in synthetic medium with a carbon-to-nitrogen ratio of 100, as well as in Examples 2-3, eluting pure erythropoietin.)

[0053] (7) The total carotenoid extract obtained in step (5), the first phase elution solution eluent obtained in step (6), and the second phase elution solution eluent obtained in step (6) were analyzed by high performance liquid chromatography:

[0054] Test solution: Dissolve the total carotenoid extract, the eluent from the first phase elution solution, and the eluent from the second phase elution solution in 1.5 mL of acetone containing 0.2% (w / v) BHT (butylated hydroxytoluene). Filter the solutions through a 0.45 μm filter membrane to a brown liquid chromatography vial before loading.

[0055] Reference solution: Dissolve the carotene standard in 1.5 mL of acetone containing 0.2% (w / v) BHT (butylated hydroxytoluene), and filter through a 0.45 μm filter membrane to a brown liquid chromatography vial before loading.

[0056] Chromatographic conditions: The column was an Agilent 5TC-C18(2), 250×4.6mm; mobile phase A was a mixture of acetonitrile and water in a volume ratio of 9:1; mobile phase B was ethyl acetate; the gradient elution program was as follows: 0–6 min, mobile phase B volume percentage 20%→60%; 6–15 min, mobile phase B volume percentage 60%; 15–20 min, mobile phase B volume percentage 60%→100%; 20–25 min, mobile phase B volume percentage 100%→20%; 25–30 min, mobile phase B volume percentage 20%; column temperature was 25℃; DAD detection signal was 494nm; flow rate was 1mL / min; injection volume was 20μL.

[0057] The quantification of erythromycin was calculated based on the absorption peak area of ​​the erythromycin standard. Standard curves were plotted for erythromycin at concentrations of 0.05, 0.1, 0.25, 0.5, 1, 2, 4, and 8 mg / L. The linear relationship of erythromycin was good within the concentration range of 0.05–8 mg / L, with a linear regression equation of y = 290.96859x - 26.23447 and a correlation coefficient R0. 2 =0.99962. HPLC analysis showed that the retention time of the red yeast erythrin standard under the conditions of this example was 10.057 min. (See...) Figure 1 After culturing in a synthetic medium with a carbon-to-nitrogen ratio of 100, the yield of erythromycin in *Rhodopsinus* under the conditions of this example was 0.034 mg / g of cells (cells in step (3)), with a recovery rate of 76%. The purity was 100% according to HPLC chromatogram analysis. The results are shown in [see attached table]. Figure 2-4 After being cultured in a synthetic medium with a carbon-to-nitrogen ratio of 200, under the experimental conditions, the yield of erythromycin in red yeast was 0.056 mg / g of cells (cells in step (3)), the recovery rate was 79%, and the purity was 100% according to HPLC chromatogram analysis.

[0058] Red yeast erythrin recovery rate = Red yeast erythrin content recovered in the eluent of the second phase elution solution / Total red yeast erythrin content in the total carotenoid extract × 100%.

[0059] Example 2: Extraction and isolation of erythropoietin from Rhodotorula rubrum

[0060] This embodiment provides a method for extracting and separating red yeast erythrin, including the following steps:

[0061] The strain used was one strain of Rhodotorula toruloides, CICC 32489, purchased from the China Industrial Microbial Culture Collection Center.

[0062] (1) Preparation of Rhodotorula rubra seed culture: The freeze-dried Rhodotorula rubra was dissolved in sterile water, streaked onto yeast extract peptone glucose (YPD) agar medium, and inverted in a 30℃ constant temperature incubator for 7 days; single colonies were picked from the above plate and inoculated into 100-500 mL of YPD liquid medium, and pre-cultured in a shake flask at 30℃ and 250 rpm for 72 h to obtain Rhodotorula rubra seed culture.

[0063] (2) Production of red yeast erythrin: The prepared Rhizopus cylindrica seed liquid was centrifuged at 4200 rpm for 5 min to collect cells. The cells were washed twice with sterile water, and the supernatant was discarded. The cells were then inoculated into a synthetic medium with a carbon-nitrogen ratio of 50 at a cell mass concentration of 1 g / L. The culture was carried out at 30℃ and 250 rpm for 96 h to obtain a fermentation broth containing red yeast erythrin.

[0064] (3) Cell collection: Centrifuge the fermentation broth obtained from the expansion culture in step (2) at 4200 rpm for 5 min, wash twice with sterile water, discard the supernatant to obtain cells containing red yeast erythrin.

[0065] (4) Separation and extraction of oil and carotenoids: Add a certain volume of anhydrous ethanol containing 2M KOH to the bacterial cells obtained in step (3) to make the bacterial cell concentration about 5g / L, and saponify for 0.5h at 65℃ and 300rpm in the dark on a magnetic stirrer.

[0066] (5) Extraction of total carotenoids: After the saponification reaction in step (4) is completed and the reaction solution is cooled, add the same volume of deionized water and n-hexane as the reaction solution (i.e., the volume ratio of saponification reaction solution, deionized water and n-hexane is 1:1:1), vortex for 1-3 min, centrifuge at 4200 rpm for 10 min to separate the solution into layers, collect the upper layer, repeatedly add n-hexane to the precipitate, and repeatedly extract until the yeast cells are colorless; combine the collected upper liquids, blow dry under nitrogen, and the residual component is the total carotenoid extract of Rhodotorula glutinis;

[0067] (6) Separation of red yeast erythrin: The total carotenoid extract described in step (5) and the first phase elution solution were mixed at a ratio of 1:10 and then transferred to a solid phase extraction (SPE) column (silica solid phase extraction column, 1g / 6mL adsorbent) that had been equilibrated with 5 column volumes of n-hexane. The remaining carotenoid extract in the original container was rinsed with a certain volume of the first phase elution solution and added to the column. After the sample was loaded onto the SPE column, the first phase nonpolar carotenoids were eluted with 5 column volumes of the first phase elution solution. After all the liquid on the column had passed through, a new glass tube was replaced to collect the second phase. Red yeast erythrin was eluted with 5 column volumes of the second phase elution solution and the eluent of the second phase elution solution was collected. The solvent in the second phase was dried under nitrogen. The remaining component was red yeast erythrin. The first phase elution solution was a mixture of n-hexane and diethyl ether in a volume ratio of 7:1. The second phase elution solution was a mixture of methanol, acetone and n-hexane in a volume ratio of 2:2:1.

[0068] (7) The total carotenoid extract obtained in (5), the first phase elution solution eluent obtained in step (6), and the second phase elution solution eluent obtained in step (6) were detected by high performance liquid chromatography: the procedure was carried out according to step (7) in Example 1.

[0069] HPLC analysis showed that the yield of red yeast erythrin under the experimental conditions was 0.041 mg / g cell (cells in step (3)), with a recovery rate of 74% and a purity of 100% as determined by HPLC chromatogram analysis.

[0070] Example 3: Extraction and isolation of erythropoietin from Rhodotorula glutinis

[0071] This embodiment provides a method for extracting and separating red yeast erythrin, including the following steps:

[0072] The strain used was Rhodotorula glutinis CICC 31229, purchased from the China Industrial Microbial Culture Collection Center.

[0073] (1) Preparation of Rhodotorula glutinis seed culture: The freeze-dried Rhodotorula glutinis was dissolved in sterile water and streaked onto yeast extract peptone glucose (YPD) agar medium. The medium was inverted in a 30°C constant temperature incubator and cultured for 7 days. Single colonies were picked from the above plate and inoculated into 100-500 mL of YPD liquid medium. The medium was pre-cultured in a shake flask at 30°C and 210 rpm for 72 h to obtain Rhodotorula glutinis seed culture.

[0074] (2) Production of red yeast erythrin: The prepared red yeast seed liquid was centrifuged at 3800 rpm for 10 min to collect cells, washed twice with sterile water, and the supernatant was discarded. The cells were then inoculated into a synthetic medium with a carbon-nitrogen ratio of 100 at a cell mass concentration of 1 g / L and cultured at 30℃ and 230 rpm for 120 h to obtain a fermentation broth containing red yeast erythrin.

[0075] (3) Cell collection: Centrifuge the fermentation broth obtained from the expansion culture in step (2) at 3800 rpm for 10 min, wash twice with sterile water, discard the supernatant to obtain cells containing red yeast erythrin.

[0076] (4) Separation and extraction of oil and carotenoids: Add a certain volume of 1.1M KOH ethanol solution to the bacterial cells obtained in step (3) to make the bacterial cell concentration about 10g / L, and saponify for 3h at 45℃ and 250rpm in the dark on a magnetic stirrer.

[0077] (5) Extraction of total carotenoids: After the saponification reaction in step (4) is completed and the reaction solution is cooled, add the same volume of deionized water and n-hexane as the reaction solution (i.e., the volume ratio of saponification reaction solution, deionized water and n-hexane is 1:1:1), vortex for 1-3 min, centrifuge at 3800 rpm for 10 min to separate the solution into layers, collect the upper layer, repeatedly add n-hexane to the precipitate, and repeatedly extract until the yeast cells are colorless; combine the collected upper liquid, blow dry under nitrogen, and the residual component is the total carotenoid extract of Rhodotorula glutinis;

[0078] (6) Separation of red yeast erythrin: The total carotenoid extract described in step (5) and the first phase elution solution were mixed at a ratio of 1:10 and then transferred to a solid phase extraction (SPE) column (silica solid phase extraction column, 1g / 6mL adsorbent) that had been equilibrated with 5 column volumes of n-hexane. The remaining carotenoid extract in the original container was rinsed with a certain volume of the first phase elution solution and added to the column. After the sample was loaded onto the SPE column, the first phase nonpolar carotenoids were eluted with 5 column volumes of the first phase elution solution. After all the liquid on the column had passed through, a new glass tube was replaced to collect the second phase. Red yeast erythrin was eluted with 5 column volumes of the second phase elution solution and the eluent of the second phase elution solution was collected. The solvent in the second phase was dried under nitrogen. The remaining component was red yeast erythrin. The first phase elution solution was a mixture of n-hexane and diethyl ether in a volume ratio of 7:1. The second phase elution solution was a mixture of methanol, acetone and n-hexane in a volume ratio of 2:2:1.

[0079] (7) The total carotenoid extract obtained in (5), the first phase elution solution eluent obtained in step (6), and the second phase elution solution eluent obtained in step (6) were detected by high performance liquid chromatography, and the procedure was carried out in step (7) of Example 1.

[0080] HPLC analysis showed that the yield of red yeast erythrin was 0.056 mg / g cell (cells in step (3)), the recovery rate was 81%, and the purity was 100% according to HPLC chromatogram analysis.

[0081] Comparative Example 1

[0082] The difference between this comparative example and Example 3 is that the wet bacterial cells collected in step (3) were broken before saponification. Specifically, the bacterial cells obtained in step (3) were resuspended in deionized water and the cell concentration was adjusted to 10 g / L. The cells were then broken using a bead mill cell disruptor filled with 65% (v / v) 0.4 mm zirconia beads at a speed of 2039 rpm. The cell disruption suspension was taken every 2 minutes, centrifuged, and the supernatant was removed. A certain volume of 1.1 M KOH ethanol solution was added to make the bacterial cell concentration reach 10 g / L after disruption. The cells were then saponified for 2 hours at 45°C and 250 rpm in the dark on a magnetic stirrer.

[0083] Total carotenoid recovery = Total carotenoid content obtained by extracting cell fragments or intact cells with hexane after saponification / Dry weight of bacterial cells in step (3);

[0084] Red yeast erythrin recovery amount = Red yeast erythrin content obtained by extraction and separation in step (6) after cell disruption or saponification of intact cells / Dry weight of cell cells in step (3).

[0085] The calculated results of the total carotenoid recovery and red yeast lycopene recovery in this comparative example and Example 3 are shown in the table below:

[0086] Table 1. Recovery of total carotenoids and red yeast erythrin from cell disruption and direct saponification

[0087]

[0088] As shown in the table above, the bead mill cell disruptor can effectively disrupt yeast cells and release carotenoids in a relatively short time. However, its mechanical shearing force can also cause loss of carotenoids. Furthermore, after cell disruption, yeast erythropoietin is directly exposed to the alkaline saponification solution, resulting in a significant loss due to alkali neutralization. Yeast cells that are directly saponified without cell disruption can recover more total carotenoids and yeast erythropoietin.

[0089] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for extracting and separating erythropoietin from red yeast, characterized in that, Includes the following steps: Add the first phase elution solution to the total carotenoid extract containing erythropoietin, then load the sample onto a solid phase extraction column, elute with the first phase elution solution, and then elute with the second phase elution solution. Collect the solution obtained by elution with the second phase elution solution. The first phase elution solution is a mixture of n-hexane and diethyl ether in a volume ratio of 7:1; The second phase elution solution is a mixture of methanol, acetone and n-hexane in a volume ratio of 2:2:

1.

2. The method for extracting and separating erythropoietin from erythropoietin according to claim 1, characterized in that, The first phase elution solution was used to elute 5 column volumes. And / or, the second phase elution solution elutes 5 column volumes.

3. The method for extracting and separating erythropoietin from erythropoietin according to claim 1 or 2, characterized in that, The ratio of the total carotenoid extract containing erythropoietin to the first phase elution solution is 1:10, with a ratio of mg:mL; And / or, prior to sample loading, the solid-phase extraction column is equilibrated with 5 column volumes of n-hexane; And / or, the solid phase extraction column is a silica solid phase extraction column with a capacity of 1 g / 6 mL adsorbent.

4. The method for extracting and separating erythropoietin from erythropoietin according to claim 1 or 2, characterized in that, The preparation method of the total carotenoid extract containing erythropoietin includes the following steps: (1) Red yeast that produces carotenoids ( Rhodotorula ) or Rhodotorula buergerianum ( Rhodotorula toruloides Fermentation is carried out to obtain a fermentation broth, and the fermentation broth is separated to obtain wet cells containing red yeast erythrin; (2) The wet mycelium is subjected to saponification treatment; (3) Add deionized water and n-hexane to the saponification reaction solution obtained in step (2). The volume ratio of saponification reaction solution, deionized water and n-hexane is 1:1:

1. Extract the solution, collect the upper liquid, and extract repeatedly until the bacterial cells are colorless. Combine the collected upper liquids and dry them in an inert protective atmosphere to obtain a total carotenoid extract containing red yeast erythrin.

5. The method for extracting and separating erythropoietin from erythropoietin according to claim 4, characterized in that, In step (1), the seed culture of the carotenoid-producing microorganism is inoculated into a synthetic culture medium with a carbon-nitrogen ratio of 50-200 and cultured at 30 °C and 210-250 rpm for 72-120 h.

6. The method for extracting and separating erythropoietin from erythropoietin according to claim 5, characterized in that, Carotenoid-producing microorganisms include Rhodotorula glutinis ( Rhodotorula mucilaginosa ), Rhodotorula buergerianum ( Rhodotorula toruloides ) or sticky red yeast ( Rhodotorula glutinis ); And / or, the synthetic culture medium with a carbon-to-nitrogen ratio of 50 to 200 comprises the following components: 50 g / L glucose, 1.7 g / L yeast basal medium free of amino acids and ammonium sulfate, and 0.55-2.2 g / L ammonium sulfate.

7. The method for extracting and separating erythropoietin from erythropoietin according to claim 4, characterized in that, In step (2), anhydrous ethanol containing 1-2 M KOH is added to the wet bacterial cells to obtain a bacterial solution with a bacterial concentration of 5-10 g / L. The solution is then saponified at 45-65 °C and 250-350 rpm in the dark for 0.5-3 h.

8. The method for extracting and separating erythropoietin from erythropoietin according to claim 4, characterized in that, In step (3), the extraction conditions are vortexing for 1-3 min and centrifuging at 3800-4200 rpm for 5-10 min to separate the solution into layers.

9. The method for extracting and separating erythropoietin from red yeast according to any one of claims 1-2 and 5-8, characterized in that, The study included the analysis of red yeast erythrin using high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: the column was packed with octadecylsilane-bonded silica gel; mobile phase A was a mixture of acetonitrile and water at a volume ratio of 9:1; mobile phase B was ethyl acetate; and the gradient elution program was as follows: 0–6 min, mobile phase B volume percentage 20%–60%; 6–15 min, mobile phase B volume percentage 60%; 15–20 min, mobile phase B volume percentage 60%–100%; 20–25 min, mobile phase B volume percentage 100%–20%; 25–30 min, mobile phase B volume percentage 20%.

10. The method for extracting and separating erythropoietin from erythropoietin according to claim 9, characterized in that, Under the specified chromatographic conditions, the column temperature is 25 °C; And / or, under the chromatographic conditions, the DAD detection signal is 494 nm; And / or, under the chromatographic conditions, the flow rate is 1 mL / min; And / or, under the chromatographic conditions, the injection volume is 10~30 μL; And / or, in the chromatographic conditions, the chromatographic column is an Agilent 5 TC-C18 (2), 250 × 4.6 mm.

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