Sporobolomyces sp. SY-18 and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a strain of sporospora sp. SY-18 and application thereof. The Sporobolomyces sp.SY-18 is preserved in the China Center for Type Culture Collection on October 21, 2025, the preservation number is CCTCC NO: M 20252277, and the Sporobolomyces sp.SY-18 is classified and named as Sporobolomyces sp.SY-18. The Sporobolomyces sp.SY-18 has the advantages that the Sporobolomyces sp.SY-18 is named as Sporobolomyces After fermentation is carried out by using optimized culture medium components and fermentation conditions, the yield of the rhodotorula red pigment can be remarkably increased and reaches 1.6 + / -0.1 g / L, and efficient and reliable strain resources and process basis are provided for large-scale production of the rhodotorula red pigment.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of *Saccharomyces cerevisiae* SY-18 and its applications. Background Technology

[0002] Torularhodin is a lutein-like carotenoid with a molecular structure containing a β-ionone ring and a long-chain carbon skeleton with 13 conjugated double bonds, exhibiting provitamin A activity. Natural torichodin is primarily produced by a few yeast strains and has not been found in bacteria, algae, or plants. Torularhodin is characterized by its high polarity and vibrant color. Its antioxidant properties are superior to lycopene and β-carotene, and it is safe and non-toxic, thus attracting increasing attention. Recent studies have revealed that torichodin possesses broad-spectrum antioxidant, antibacterial, anti-inflammatory, anti-aging, and anti-tumor activities, thus showing significant potential applications in functional foods, animal feed, cosmetics, and pharmaceuticals.

[0003] The production of erythropoietin from erythropoietin can be achieved through chemical synthesis, but due to its large molecular weight, complex chemical structure, complicated synthesis process, easy generation of byproducts, and poor biological activity, the production cost is high. In contrast, fermentation using yeast offers advantages such as mild reaction conditions, simple nutritional requirements, fast production cycle, higher product stability and biological activity, no food safety risks, and greater consumer acceptance, making it the preferred method for large-scale production of erythropoietin from erythropoietin. Currently, the yield of erythropoietin from erythropoietin produced via microbial methods is extremely low, and the proportion of carotenoids is small, far from meeting the requirements of large-scale production. Therefore, it is necessary to discover a new strain of erythropoietin from erythropoietin that yields high levels of erythropoietin. Summary of the Invention

[0004] To discover a new strain of Rhodotorula rubigin-producing yeast, this invention provides a strain of Rhodotorula rubigin, SY-18, and its applications. The Rhodotorula rubigin-producing yeast SY-18 provided by this invention, when fermented using optimized culture medium components and fermentation conditions, can significantly increase the yield of Rhodotorula rubigin to 1.6 ± 0.1 g / L, providing an efficient and reliable strain resource and technological foundation for the large-scale production of Rhodotorula rubigin.

[0005] This invention provides a strain of *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Sporobolomyces sp)SY-18, the so-called sp. SY-18 was deposited at the China Center for Type Culture Collection on October 21, 2025, with accession number CCTCC NO: M 20252277.

[0006] The *Saccharomyces cerevisiae* SY-18 provided by this invention, when fermented using optimized culture medium components and fermentation conditions, can significantly increase the yield of erythromycin from *Saccharomyces cerevisiae* to 1.6 ± 0.1 g / L, providing an efficient and reliable strain resource and technological basis for the large-scale production of erythromycin from *Saccharomyces cerevisiae*.

[0007] This invention also provides the application of *Saccharomyces cerevisiae* SY-18 in the preparation of erythromycin, specifically including the following steps: Yeast SY-18 was fermented to obtain a fermentation broth. The cells were collected by centrifugation from the fermentation broth, and the cells were subjected to cell wall disruption to extract carotenoids. The carotenoids were then isolated and purified to obtain erythrozyme erythrin. The liquid fermentation medium used for fermentation contains a carbon source, a nitrogen source, and inorganic salts; the carbon source is sucrose, the nitrogen source is potato extract powder, and the inorganic salts are CuSO4.

[0008] Furthermore, the concentration of sucrose in the culture medium is 20 g / L to 50 g / L.

[0009] Furthermore, the concentration of the potato extract powder in the culture medium is 1 g / L to 16 g / L.

[0010] Furthermore, the concentration of CuSO4 in the culture medium is 0.1 g / L to 2 g / L.

[0011] Furthermore, the fermentation conditions include: an initial pH of 3 to 9 in the culture medium and a rotation speed of 120 rpm to 240 rpm.

[0012] Furthermore, the fermentation time is 70 h to 74 h.

[0013] Furthermore, the fermentation temperature is 20℃~37℃.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a high-yield erythromycin-producing yeast strain SY-18, with an erythromycin yield as high as 1.6±0.1 g / L. The fermentation method utilizes sucrose as the sole carbon source and potato extract as the sole nitrogen source, resulting in extremely low costs. Lower temperatures increase erythromycin yield, allowing for erythromycin production in cold seasons or environments to meet market demand.

[0015] Information on the preservation of biological materials: SY-18, referred to as *Saccharomyces cerevisiae* SY-18 in this invention, was deposited on October 21, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252277. The address of the depository is Wuhan University, Wuhan, China, postcode: 430072. It is classified and named as follows: Sporobolomyces sp. SY-18. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The colony morphology of *Saccharomyces cerevisiae* SY-18 is shown.

[0018] Figure 2 This is the standard curve for red yeast lycopene.

[0019] Figure 3 The figures show the changes in erythromycin production and cell biomass of various culture medium components at different concentrations. In the figures, A represents the changes in erythromycin production and cell biomass under different carbon sources; B represents the changes in erythromycin production and cell biomass under different carbon source concentrations; C represents the changes in erythromycin production and cell biomass under different nitrogen sources; D represents the changes in erythromycin production and cell biomass under different nitrogen source concentrations; E represents the changes in erythromycin production and cell biomass under different inorganic salts; and F represents the changes in erythromycin production and cell biomass under different inorganic salt concentrations.

[0020] Figure 4 The changes in erythrin production and cell biomass of red yeast in an orthogonal experiment of culture medium composition.

[0021] Figure 5 The figures show the changes in lycopene production and cell biomass of red yeast under different fermentation conditions; A shows the changes in lycopene production and cell biomass of red yeast at different pH levels; B shows the changes in lycopene production and cell biomass of red yeast at different fermentation speeds; C shows the changes in lycopene production and cell biomass of red yeast at different liquid volumes; and D shows the changes in lycopene production and cell biomass of red yeast at different temperatures. Detailed Implementation

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0023] Example 1: Isolation and identification of *Saccharomyces cerevisiae* strain SY-18.

[0024] 1. Strains Isolation and Purification Waxberries were collected from Daluo Mountain in Wenzhou City, Zhejiang Province. Under aseptic conditions, fresh waxberry pulp was cut using a sterile knife and placed into test tubes containing sterile water. The mixture was vortexed at high speed for 5 minutes, then allowed to stand for 1 minute. The supernatant was transferred to a new sterile centrifuge tube and diluted 10% with sterile water. 5 After dilution, 200 μL of the diluted solution was evenly spread onto YPD solid plates containing ampicillin and incubated at 28°C for 2 days. Based on the morphological characteristics of yeast colonies, single colonies with a redder color and larger shape were selected for shake-flask culture, and their colony cytological morphology was observed in the culture medium.

[0025] 2. Strain identification The strain was streaked in PDA medium (20 g / L glucose, 20 g / L agar, 8 g / L potato extract, balance distilled water, pH=6) and cultured at 28℃ for 6 days. The results are as follows: Figure 1 As shown, its colony morphology is characterized by being red, smooth, and glossy.

[0026] The genome of the strain was extracted using a biomolecular fungal genome extraction kit (B518227-0050). The ITS rDNA sample obtained after PCR amplification using universal primers ITS1 and ITS4 was sequenced. The sequencing results were then compared with existing data from *Saccharomyces koalaensis*. Sporobolomyces koalae The homology was 100%, and the strain was identified as *Saccharomyces cerevisiae*, named *Saccharomyces cerevisiae* SY-18.

[0027] Example 2: Pigment extraction and determination of *Saccharomyces cerevisiae* strain SY-18.

[0028] 1. Extraction of carotenoids Prepare 3 L of PDA liquid medium (PDA medium without agar) and place it in a fermenter. Autoclave at 121℃ for 20 min. After cooling to room temperature, inoculate the SY-18 strain into the PDA liquid medium and pre-culture to the logarithmic growth phase. Inoculate the pre-cultured strain at a 4% inoculum (v / v). Maintain the temperature at 28℃, stirring speed at 300 rpm, dissolved oxygen (DO) at 40%, and aeration rate at 4 L / min. After culturing for 3 days, terminate the fermentation. Centrifuge the fermentation product at 8000 rpm for 5 min and collect the SY-18 cells. Since carotenoids are present within yeast cells and can only be extracted by cell disruption, the SY-18 cells were freeze-dried at -60℃ and 0.1 MPa for 2 days. Take 50 mg of dried bacterial cell sample, add 100 mL of DMSO, then place it in a water bath at 65°C for 30 min, add 3 mL of acetone and shake for 20 min, centrifuge at 10000 rpm for 10 min and take the supernatant to obtain carotenoids.

[0029] 2. Purification of red yeast erythrin Weigh 100 g of 300-mesh silica gel (30 mm × 300 mm); pour the weighed silica gel into the chromatographic column all at once, vibrate the tube wall to make it uniform and ensure the surface is flat. Then, evaporate the carotenoids together with the 200-mesh silica gel using a rotary evaporator at 60℃ and 180 rpm until dry, pour the evaporated silica gel into the column, and smooth the surface using the same method. Use a vacuum pump at 0.1 Pa for 1 h to purge the column, making the packing more dense and facilitating sample separation. After loading the sample, add 5 g of anhydrous sodium sulfate on top of the sample layer to mitigate the impact of the mobile phase during pouring, thus ensuring better and more uniform sample separation.

[0030] Using petroleum ether:chloroform at a volume ratio of 10:1 as the mobile phase, carotenoids were collected by utilizing color differences. Each gradient band was collected gradually until only the top red band remained. Ethyl acetate (1 L) was added to the elution column, yielding the crude extract of erythropoietin. This crude extract was concentrated using a rotary evaporator at 60°C and 180 rpm to obtain purified erythropoietin, which was stored at -20°C for further analysis. The entire experiment was conducted in the dark to prevent pigment denaturation.

[0031] 3. Pigment determination The purified yam erythrin was used to prepare concentration gradients (1 mg / mL, 2 mg / mL, 4 mg / mL, 5 mg / mL, 15 mg / mL, 20 mg / mL), and a standard curve was plotted using high-performance liquid chromatography (y=193239x-34314, R). 2 =0.9991), such as Figure 2The conditions for high performance liquid chromatography (HPLC) were as follows: mobile phase A was acetonitrile:water = 9:1 (V:V), mobile phase B was ethyl acetate, column temperature was 30℃, wavelength was 507 nm, flow rate was 1.0 mL / min, and injection volume was 20 μL.

[0032] Example 3: Application of Saccharomyces SY-18 strain in high-yield red yeast erythrin.

[0033] 1. Single-factor experiment on culture medium composition A small amount of bacterial cells was activated on PDA medium. The activated cells, after 48 h of activation, were inoculated into 100 mL / 250 mL (100 mL of medium in a 250 mL Erlenmeyer flask, 40% full) of seed culture fermentation medium (20 g / L glucose, 8 g / L potato extract, pH 6) for fermentation to obtain seed culture. The seed culture was then inoculated into 100 mL of liquid fermentation medium at a 10% (v / v) inoculation rate. Three parallel experiments were conducted. The basic fermentation method was: 20 g / L glucose, 8 g / L potato extract, pH 6, 28℃, 200 rpm for 72 h. Under this method, the yield of erythromycin obtained from red yeast was 1.2 ± 0.1 g / L. Different liquid culture media were used in the single-factor experiments as follows:

[0034] (1) With other factors of the basic fermentation method remaining unchanged, glucose was replaced with glycerol, xylose, fructose, and sucrose. The results are as follows: Figure 3 As shown in A, the production of red yeast lycopene is the largest when the carbon source is sucrose. (2) With other factors of the basic fermentation method remaining unchanged, the sucrose concentration was changed from 20 g / L to 10 g / L, 30 g / L, 40 g / L, and 50 g / L, and the results were as follows: Figure 3 As shown in B, the yield of red yeast lycopene is the highest when the sucrose concentration is 50 g / L. (3) With other factors of the basic fermentation method unchanged, potato extract powder was replaced with peptone, yeast extract, ammonium sulfate, and beef extract. The results were as follows: Figure 3 As shown in C, the yield of erythropoietin was highest when the nitrogen source was potato extract and peptone. (4) With other factors of the basic fermentation method remaining unchanged, the concentration of potato extract powder was changed from 8 g / L to 1 g / L, 4 g / L, 12 g / L, and 16 g / L. The results are as follows: Figure 3 As shown in D, the yield of red yeast lycopene is the highest when the concentration of potato extract powder is 12 g / L. (5) With other factors of the basic fermentation method remaining unchanged, 0.5 g / L of NaCl, Al2(NO)3, CaCl2, CuSO4, Fe2(SO)3 and KCl were added respectively, and the results are as follows. Figure 3 As shown in E, the red yeast lycopene production is highest when the inorganic salt is CuSO4; (6) With other factors of the basic fermentation method remaining unchanged, the CuSO4 concentration was changed from 0.5 g / L to 0.1 g / L, 0.25 g / L, 1 g / L, and 2 g / L, and the results are as follows: Figure 3 As shown in F, the yield of erythropoietin in erythropoietin was highest when the CuSO4 concentration was 0.25 g / L.

[0035] 2. Orthogonal experiment on culture medium composition An orthogonal experiment was conducted according to Table 1 to determine the composition of the culture medium, and the results are as follows: Figure 4 The highest yield of erythromycin was observed when the sucrose concentration was 30 g / L, the potato extract concentration was 8 g / L, and the inorganic salt concentration was 0.25 g / L.

[0036] Table 1 Orthogonal Experimental Design Table 3. The cultivation conditions were investigated using single-factor experiments. (1) With other factors of the basic fermentation method remaining unchanged, the pH was adjusted from 6 to 3, 4, 5, 7, and 9, and the results were as follows: Figure 5 As shown in A, the red yeast erythrin production is highest at pH 6. (2) With other factors of the basic fermentation method remaining unchanged, the rotation speed was adjusted from 200 rpm to 120 rpm, 150 rpm, 210 rpm, and 240 rpm, and the results are as follows. Figure 5 As shown in B, the red yeast lycopene production is highest when the rotation speed is 210 rpm; (3) With other factors of the basic fermentation method remaining unchanged, the liquid volume was adjusted from 40% to 5%, 10%, 20%, 30%, and 50%, and the results were as follows: Figure 5 As shown in C, the yield of red yeast erythrin is the highest when the liquid volume is 10%. (4) With other factors of the basic fermentation method remaining unchanged, the temperature was adjusted from 28℃ to 20℃, 25℃, and 37℃, and the results were as follows. Figure 5 As shown in D, the red yeast lycopene production is highest at a temperature of 20℃.

[0037] In summary, compared to the basic fermentation method, this strain SY-18, through single-factor experiments and orthogonal experiments, increased the yield of erythromycin from 1.2 g / L to 1.6 g / L, an increase of 33.33%. Investigations showed that the yield of erythromycin from this strain SY-18 was significantly higher than that of other strains (ranging from 0.46 mg / L to 286 mg / L). Verification has shown that this strain has certain practical value, and the optimal preparation conditions for erythromycin obtained are highly reliable.

[0038] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0039] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of *Saccharomyces cerevisiae* ( Sporobolomyces sp)SY-18, characterized in that, The *Saccharomyces cerevisiae* SY-18 was deposited at the China Center for Type Culture Collection on October 21, 2025, with accession number CCTCC NO: M 20252277.

2. The application of the *Saccharomyces cerevisiae* SY-18 according to claim 1 in the preparation of erythromycin, characterized in that, Specifically, the following steps are included: Yeast SY-18 was fermented to obtain a fermentation broth. The cells were collected by centrifugation from the fermentation broth, and the cells were subjected to cell wall disruption to extract carotenoids. The carotenoids were then isolated and purified to obtain erythrozyme erythrin. The liquid fermentation medium used for fermentation contains a carbon source, a nitrogen source, and inorganic salts; the carbon source is sucrose, the nitrogen source is potato extract powder, and the inorganic salt is CuSO4.

3. The application according to claim 2, characterized in that, The concentration of sucrose in the culture medium is 20 g / L to 50 g / L.

4. The application according to claim 2, characterized in that, The concentration of the potato extract powder in the culture medium is 1 g / L to 16 g / L.

5. The application according to claim 2, characterized in that, The concentration of CuSO4 in the culture medium is 0.1 g / L to 2 g / L.

6. The application according to claim 2, characterized in that, The fermentation conditions include: an initial pH of 3-9 in the culture medium and a rotation speed of 120-240 rpm.

7. The application according to claim 2, characterized in that, The fermentation time is 70 h to 74 h.

8. The application according to claim 2, characterized in that, The fermentation temperature is 20℃~37℃.

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