Rhodotorula graminis lipid extract, preparation method thereof and application of rhodotorula graminis lipid extract in cosmetics

Abstract

The invention provides a novel Rhodotorula graminearum strain RS-Y-red and a preparation method thereof. The invention also provides a method for preparing a yeast lipid extract by fermenting the novel Rhodotorula graminis strain and the yeast lipid extract prepared by the method. The invention further provides application of the yeast lipid extract in preparation of cosmetics or medicines and cosmetics or medicines containing the yeast lipid extract.

Description

Technical Field

[0001] This invention relates to the fields of microbial fermentation technology, biological resource development, and cosmetic technology. Specifically, this invention relates to a novel *Heterobryonic acid* yeast (…). Rhodotorula graminis The invention relates to a yeast strain, a method for preparing a yeast lipid extract by fermentation using the novel *Rhodotorula granatum* strain, and the yeast lipid extract obtained by the method. The invention further relates to the use of the yeast lipid extract in the preparation of cosmetics or pharmaceuticals, and cosmetics or pharmaceuticals comprising the yeast lipid extract. Background Technology

[0002] With the increasing demand from consumers for natural and functional cosmetics, more and more research is focusing on the efficient development and utilization of renewable natural resources. Microbial fermentation, as a green and controllable method, has significant advantages in the acquisition of functional oils. In particular, oil-producing yeasts, due to their high lipid accumulation capacity, controllability, and sustainability, have gradually become an important means of developing high-value natural oils in recent years.

[0003] Currently, most cosmetic oil raw materials on the market are derived from plant extracts, such as jojoba oil and rosehip oil, or from animal sources, such as fish oil and beeswax. However, plant oils face challenges such as limitations imposed by climate and origin, and environmentally unfriendly extraction processes, while animal-derived raw materials are restricted due to ethical and safety concerns. In contrast, fermented oils derived from oil-producing yeasts offer advantages such as stable raw materials, high yield, good controllability, and environmental friendliness. Furthermore, their fatty acid composition can be controlled through cultivation conditions, thereby endowing them with specific biological activities.

[0004] Although some oil-producing yeasts (such as...) have already been found... Yarrowia lipolytica , Rhodosporidium toruloides While research has focused on the application of microorganisms (such as ginseng root symbiotic microorganisms) in biodiesel and food additives, studies on developing specific strains from naturally derived microorganisms, especially those from medicinal plants (such as ginseng root symbiotic microorganisms), into highly active functional oils for application in cosmetics are still relatively rare. Ginseng, as a traditional Chinese medicine, may harbor symbiotic microbial resources with unique metabolic potential in its root micro-ecosystem, providing new insights for discovering novel oil-producing yeasts with unique functional oils.

[0005] Therefore, the novel oil-producing yeast strain isolated from ginseng roots not only produces oils with excellent physicochemical properties but may also carry the unique metabolic capabilities of active ingredients from the ginseng root microbiota, possessing potential application value in anti-oxidation, anti-inflammation, and promoting skin barrier repair. Based on the above considerations, this invention provides a uniquely sourced and functionally prominent oil-producing yeast and its fermented oil, applying it to cosmetics to achieve the development and innovative application of a new source of natural, highly effective functional oils. Summary of the Invention

[0006] This invention has discovered a novel *Polygonum chinense* yeast derived from ginseng roots, which has been named *Polygonum chinense* RS-Y-red. Rhodotorula graminis The strain RS-Y-red is deposited at the China Center for Type Culture Collection (CCC) under accession number M20251301. This invention also provides a method for preparing a yeast lipid extract using the novel *Rhodotorula granatum* fermentation, and the yeast lipid extract obtained by the method. The yeast lipid extract of this invention possesses antioxidant, barrier repair, moisturizing, and anti-aging effects.

[0007] The first aspect of the present invention relates to a strain of Rhodotorula granatum, which is deposited at the China Center for Type Culture Collection with accession number M20251301.

[0008] A second aspect of the present invention relates to a method for preparing yeast lipid extracts by fermentation using a strain of Rhodotorula granatum, wherein the Rhodotorula granatum strain is a strain of Rhodotorula granatum deposited at the China Center for Type Culture Collection with accession number M20251301.

[0009] A third aspect of the invention relates to a yeast lipid extract prepared by the method described in the second aspect of the invention.

[0010] The fourth aspect of the invention relates to the use of the yeast lipid extract according to the third aspect of the invention in the preparation of cosmetics / pharmaceuticals.

[0011] The fifth aspect of the invention relates to a cosmetic or pharmaceutical product comprising a yeast lipid extract according to the third aspect of the invention, wherein the yeast lipid extract comprises 0.1% to 99% by weight of the cosmetic or pharmaceutical product. Attached Figure Description

[0012] Figure 1 The phylogenetic tree of yeast strains obtained in this invention, identified based on 16S / ITS whole-genome sequencing, is shown.

[0013] Figure 2 The GC-MS analysis results of the yeast lipid extract prepared according to the present invention are shown.

[0014] Figure 3 The results of DPPH free radical scavenging rate determination for yeast oil at different concentrations are shown.

[0015] Figure 4 The results of the assay of cytotoxicity of different concentrations of yeast oil on HFF-1 cells are shown (expressed as cell viability).

[0016] Figure 5The results show the fluorescence intensity of intracellular reactive oxygen species (ROS) after HFF-1 cells were treated with different concentrations of yeast oil.

[0017] Figure 6 The study showed the effect of different concentrations of yeast oil on the relative mRNA expression level of filaggrin in HaCaT cells.

[0018] Figure 7 The study showed the effect of different concentrations of yeast oil on the relative expression level of type I collagen mRNA in HDF cells.

[0019] Figure 8 The study showed the effect of different concentrations of yeast oil on the relative expression level of type III collagen mRNA in HDF cells.

[0020] Figure 9 The study showed the effect of different concentrations of yeast oil on the relative mRNA expression level of elastin in HDF cells. Invention Details

[0022] definition

[0023] In this article, the terms “s”, “min” and “h” stand for “second”, “minute” and “hour”, respectively.

[0024] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized in," and is inclusive or open-ended, not excluding additional, undescribed elements or method steps. However, in this document, each reference to "comprising" is intended to cover alternative embodiments of "consistently composed of" and "consisting of," wherein "consisting of" excludes any unspecified elements or steps, and "consisting of" allows the inclusion of other elements or steps that are not described and do not materially affect the essential or essential characteristics and novelty of the composition or method discussed.

[0025] As used herein, the term “about” means ±10% of the value modified by the term, more preferably ±5%, and most preferably ±2%. Therefore, those skilled in the art can clearly determine the range of the term “about” based on the modified value.

[0026] In this article, the terms "yeast oil," "yeast fat," and "yeast lipid extract" have the same meaning and are used interchangeably, all referring to yeast extracts derived from Rhodotorula glutinis (Yeast Oil). Rhodotorula graminisThis is an extract primarily composed of lipid components, obtained through physical cell disruption and extraction / separation processes (such as, but not limited to, supercritical CO2 fluid extraction) after fermentation and culture. Given its biological origin and processing characteristics, this extract may contain trace amounts of non-lipid components (such as cell wall polysaccharides, proteins, pigments, or culture medium residues) in addition to the main lipids (e.g., triglycerides, fatty acids, sterols, carotenoids, etc.). This does not affect its overall properties and function as a "lipid extract."

[0027] The term "feed-batch fermentation" as used in this paper refers to a fermentation method in which a portion of the culture medium is initially added, followed by the addition of one or more specific nutrients (such as carbon sources, nitrogen sources, etc.) to the fermentation system in batches or continuously, according to a pre-set strategy, fermentation process parameters, or metabolic requirements. This method differs from simple batch culture with a single feeding and aims to avoid substrate inhibition, optimize metabolic pathways, and improve the final yield of the target product.

[0028] The term "feedback culture based on carbon source concentration monitoring" used herein is a preferred and specific implementation of "batch fed fermentation culture." Specifically, "feedback culture based on carbon source concentration monitoring" refers to a process in which a portion of the culture medium is initially added during fermentation, followed by real-time or intermittent monitoring of the concentration of key carbon sources (such as glucose) in the fermentation broth, using this monitoring data as a feedback signal. When the carbon source concentration falls below a preset first concentration threshold, a feeding operation is initiated to restore and maintain the concentration at the threshold level. Furthermore, at a specific fermentation stage (such as the stationary phase of cell growth), the target concentration can be switched to a higher second concentration threshold, thereby achieving stepwise enhanced control over cell metabolism and product synthesis.

[0029] In this document, the term "fatty acid composition in fatty acids and triglycerides" refers to the fatty acid profile composed of free fatty acids and esterified fatty acids bound to triglyceride molecules in the extract.

[0030] The term "made from" is inclusive or open-ended, and does not exclude additional, undescribed elements. For example, "the composition is made from component a and component b" does not mean that the composition consists only of component a and component b; rather, this open-ended expression allows the composition to contain other ingredients besides components a and b, such as, but not limited to, excipients, carriers, and excipients.

[0031] Gramine red yeast strain

[0032] The first aspect of the present invention relates to a strain of Rhodotorula granatum, which is deposited at the China Center for Type Culture Collection with accession number M20251301.

[0033] In some implementations, the *Herbanemycin* strain is named *Herbanemycin RS-Y-red*.

[0034] Preparation method of yeast lipid extract

[0035] A second aspect of the present invention relates to a method for preparing yeast lipid extracts by fermentation using a strain of Rhodotorula granatum, wherein the Rhodotorula granatum strain is a strain of Rhodotorula granatum deposited at the China Center for Type Culture Collection with accession number M20251301.

[0036] In some implementations, the method includes the following steps:

[0037] (1) The *Rhodotorula granatum* strain was seed cultured to obtain *Rhodotorula granatum* seed liquid;

[0038] (2) The *Hemiberlesia javanica* seed culture was inoculated into a fermentation medium for fermentation culture; and

[0039] (3) After fermentation, yeast lipids are extracted from the culture.

[0040] In some implementations, the fermentation culture is a fed-batch fermentation culture, which is a feedback-based fed-batch culture based on carbon source concentration monitoring, and includes:

[0041] During the first fermentation stage, the carbon source concentration is maintained at a first predetermined level;

[0042] After reaching the predetermined fermentation node, the process transitions to the second fermentation stage, where the carbon source concentration is increased and maintained at a second predetermined level higher than the first predetermined level.

[0043] In some embodiments, the carbon source is glucose. The initial basal medium used in the fed-batch fermentation culture contains glucose, yeast extract, malt extract, ammonium sulfate, peptone, magnesium sulfate, and trace elements, and the pH of the initial basal medium is 5.5-6.5.

[0044] In some embodiments, the initial basal medium used in the fed-batch fermentation culture has a pH of 5.5-5.6, 5.6-5.7, 5.7-5.8, 5.8-5.9, 5.9-6.0, 6.0-6.1, 6.1-6.2, 6.2-6.3, 6.3-6.4, or 6.4-6.5.

[0045] In some embodiments, the initial basal medium used in the fed-batch fermentation culture contains 60-80 g / L of glucose. In some embodiments, the initial basal medium used in the fed-batch fermentation culture contains 60-62 g / L, 62-64 g / L, 64-66 g / L, 66-68 g / L, 68-70 g / L, 70-72 g / L, 72-74 g / L, 74-76 g / L, 76-78 g / L, or 78-80 g / L of glucose.

[0046] In some embodiments, the initial basal medium used in the fed-batch fermentation culture comprises 2-3 g / L of yeast extract. In some embodiments, the initial basal medium used in the fed-batch fermentation culture comprises 2-2.2 g / L, 2.2-2.4 g / L, 2.4-2.6 g / L, 2.6-2.8 g / L, 2.8-3 g / L, 3-3.2 g / L, 3.2-3.4 g / L, 3.4-3.6 g / L, 3.6-3.8 g / L, or 3.8-4 g / L of yeast extract.

[0047] In some embodiments, the initial basal medium used in the fed-batch fermentation culture comprises 6-10 g / L of malt extract. In some embodiments, the initial basal medium used in the fed-batch fermentation culture comprises 6-6.5 g / L, 6.5-7 g / L, 7-7.5 g / L, 7.5-8 g / L, 8-8.5 g / L, 8.5-9 g / L, 9-9.5 g / L, or 9.5-10 g / L of malt extract.

[0048] In some embodiments, the initial basal medium used in the fed-batch fermentation culture contains 1-3 g / L of ammonium sulfate. In some embodiments, the initial basal medium used in the fed-batch fermentation culture contains 1-1.5 g / L, 1.5-2 g / L, 2-2.5 g / L, or 2.5-3 g / L of ammonium sulfate.

[0049] In some embodiments, the initial basal medium used in the fed-batch fermentation culture contains 2-3 g / L of peptone. In some embodiments, the initial basal medium used in the fed-batch fermentation culture contains 2-2.2 g / L, 2.2-2.4 g / L, 2.4-2.6 g / L, 2.6-2.8 g / L, or 2.8-3 g / L of peptone.

[0050] In some embodiments, the initial basal medium used in the fed-batch fermentation culture contains 0.1-2 g / L magnesium sulfate. In some embodiments, the initial basal medium used in the fed-batch fermentation culture contains 0.1-0.3 g / L, 0.3-0.5 g / L, 0.5-0.7 g / L, 0.7-0.9 g / L, 0.9-1.1 g / L, 1.1-1.3 g / L, 1.3-1.5 g / L, 1.5-1.7 g / L, or 1.7-2.0 g / L magnesium sulfate.

[0051] In some embodiments, the first predetermined level of carbon source concentration is 20-40 g / L. In some embodiments, the first predetermined level of carbon source concentration is 20-25 g / L, 25-30 g / L, 30-35 g / L, or 35-40 g / L.

[0052] In some embodiments, the second predetermined level of carbon source concentration is 40-60 g / L. In some embodiments, the second predetermined level of carbon source concentration is 40-45 g / L, 45-50 g / L, 50-55 g / L, or 55-60 g / L.

[0053] In some embodiments, the fermentation temperature for the first fermentation stage is 25-30 °C. In some embodiments, the fermentation temperature for the first fermentation stage is 25-26 °C, 26-27 °C, 27-28 °C, 28-29 °C, or 29-30 °C.

[0054] In some embodiments, the fermentation temperature for the second fermentation stage is 15-25 °C. In some embodiments, the fermentation temperature for the second fermentation stage is 15-16 °C, 16-17 °C, 17-18 °C, 18-19 °C, 19-20 °C, 20-21 °C, 21-22 °C, 22-23 °C, 23-24 °C, or 24-25 °C.

[0055] In some implementations, the fermentation time for the first fermentation stage is 50-70 hours. In other implementations, the fermentation time for the first fermentation stage is 50-51 hours, 51-52 hours, 52-53 hours, 53-54 hours, 54-55 hours, 55-56 hours, 56-57 hours, 57-58 hours, 58-59 hours, 59-60 hours, 60-61 hours, 61-62 hours, 62-63 hours, 63-64 hours, 64-65 hours, 65-66 hours, 66-67 hours, 67-68 hours, 68-69 hours, or 69-70 hours.

[0056] In some implementations, the fermentation time for the second fermentation stage is 30-40 hours. In some implementations, the fermentation time for the second fermentation stage is 30-31 hours, 31-32 hours, 32-33 hours, 33-34 hours, 34-35 hours, 35-36 hours, 36-37 hours, 37-38 hours, 38-39 hours, or 39-40 hours.

[0057] In some embodiments, the amount of seed culture inoculated in the fermentation culture step is 5-15% (v / v). In some embodiments, the amount of seed culture inoculated in the fermentation culture step is 5-6% (v / v), 6-7% (v / v), 7-8% (v / v), 8-9% (v / v), 9-10% (v / v), 10-11% (v / v), 11-12% (v / v), 12-13% (v / v), 13-14% (v / v), or 14-15% (v / v).

[0058] In some implementations, the aeration rate during the fermentation culture step is 0.5–1.5 vvm. In other implementations, the aeration rate during the fermentation culture step is 0.5–0.6 vvm, 0.6–0.7 vvm, 0.7–0.8 vvm, 0.8–0.9 vvm, 0.9–1.0 vvm, 1.0–1.1 vvm, 1.1–1.2 vvm, 1.2–1.3 vvm, 1.3–1.4 vvm, or 1.4–1.5 vvm.

[0059] In some implementation schemes, the cell concentration at the end of the fermentation culture can reach O ≈ 100-200. In some implementation schemes, the cell concentration at the end of the fermentation culture can reach O. ≈ 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190 or 190-200.

[0060] In some implementations, the dry weight of the cells at the end of the fermentation culture is 60-100 g / L. In other implementations, the dry weight of the cells at the end of the fermentation culture is 60-65 g / L, 65-70 g / L, 70-75 g / L, 75-80 g / L, 80-85 g / L, 85-90 g / L, 90-95 g / L, or 95-100 g / L.

[0061] In some implementations, the total lipid accumulation at the end of the fermentation culture accounts for 40-60% of the cell dry weight. In some implementations, the total lipid accumulation at the end of the fermentation culture accounts for 40-45%, 45-50%, 50-55%, or 55-60% of the cell dry weight.

[0062] In some implementations, the step of extracting yeast lipids from the culture includes:

[0063] (a) Collecting and crushing the yeast cells obtained from fermentation; and

[0064] (b) Using supercritical C Fluid extraction technology is used to extract yeast lipids from broken bacterial cells.

[0065] In some embodiments, the bacterial culture is centrifuged to collect bacterial cells before step (a). In some embodiments, the centrifugation is performed at a speed of 6000-10000 rpm. In some embodiments, the centrifugation is performed at a speed of 6000-7000 rpm, 7000-8000 rpm, 8000-9000 rpm, or 9000-10000 rpm. In some embodiments, the centrifugation is performed for 5-20 minutes. In some embodiments, the centrifugation is performed for 5-10, 10-15, or 15-20 minutes.

[0066] In some embodiments, in step (a), the yeast cells are broken by mechanical grinding. In some embodiments, the mechanical grinding method is bead grinding.

[0067] In some implementations, in step (b), at supercritical C Anhydrous ethanol is added as an entrainer during extraction.

[0068] In the following implementation scheme, when conducting supercritical C Before extraction, the broken yeast cells obtained in step (a) are dried until the moisture content is less than 10%.

[0069] In some implementation schemes, supercritical C The temperature during the extraction step is 40-60°C. In some embodiments, supercritical C... The temperature during the extraction step is 40-45°C, 45-50°C, 50-55°C, or 55-60°C.

[0070] In some implementation schemes, supercritical C The pressure during the extraction step is 20-40 MPa. In some embodiments, supercritical C... The pressure during the extraction step is 20-25 MPa, 25-30 MPa, 30-35 MPa, or 35-40 MPa.

[0071] In some implementation schemes, supercritical C The proportion of entrainer added in the extraction step is C. 1-15% of the flow rate. In some implementations, supercritical C The proportion of entrainer added in the extraction step is C. 1-5%, 5-10%, or 10-15% of the flow rate.

[0072] In some implementation schemes, supercritical C The extraction time is 0.5-5 h. In some implementations, supercritical C... The extraction time is 0.5-1 h, 1-1.5 h, 1.5-2 h, 2-2.5 h, 2.5-3 h, 3-3.5 h, 3.5-4 h, 4-4.5 h or 4.5-5 h.

[0073] In some implementations, in supercritical C Following extraction, yeast lipids are precipitated by fractional depressurization, and residual solvent is removed under vacuum. In some embodiments, fractional depressurization is performed at 35–45 °C and 5–8 MPa.

[0074] In some embodiments, the seed culture is carried out in a liquid culture medium containing at least one stage of pre-culture, the liquid culture medium containing a carbon source and a nitrogen source; the carbon source includes glucose, and the nitrogen source includes at least one of yeast extract, malt extract, and peptone.

[0075] In some embodiments, the liquid culture medium for seed culture contains 15-25 g / L of glucose. In some embodiments, the liquid culture medium for seed culture contains 15-16 g / L, 16-17 g / L, 17-18 g / L, 18-19 g / L, 19-20 g / L, 20-21 g / L, 21-22 g / L, 22-23 g / L, 23-24 g / L, or 24-25 g / L of glucose.

[0076] In some embodiments, the liquid culture medium for seed culture comprises 2-4 g / L of yeast extract. In some embodiments, the liquid culture medium for seed culture comprises 2-2.2 g / L, 2.2-2.4 g / L, 2.4-2.6 g / L, 2.6-2.8 g / L, 2.8-3.0 g / L, 3.0-3.2 g / L, 3.2-3.4 g / L, 3.4-3.6 g / L, 3.6-3.8 g / L, or 3.8-4.0 g / L of yeast extract.

[0077] In some embodiments, the liquid culture medium for seed culture comprises 2-4 g / L of malt extract. In some embodiments, the liquid culture medium for seed culture comprises 2-2.2 g / L, 2.2-2.4 g / L, 2.4-2.6 g / L, 2.6-2.8 g / L, 2.8-3.0 g / L, 3.0-3.2 g / L, 3.2-3.4 g / L, 3.4-3.6 g / L, 3.6-3.8 g / L, or 3.8-4.0 g / L of malt extract.

[0078] In some embodiments, the liquid culture medium for seed culture contains 4-6 g / L of peptone. In some embodiments, the liquid culture medium for seed culture contains 4-4.2 g / L, 4.2-4.4 g / L, 4.4-4.6 g / L, 4.6-4.8 g / L, 4.8-5.0 g / L, 5.0-5.2 g / L, 5.2-5.4 g / L, 5.4-5.6 g / L, 5.6-5.8 g / L, or 5.8-6.0 g / L of peptone.

[0079] In some embodiments, the pH of the liquid culture medium used for seed culture is 5.5-6.5. In some embodiments, the pH of the liquid culture medium used for seed culture is 5.5-5.6, 5.6-5.7, 5.7-5.8, 5.8-5.9, 5.9-6.0, 6.0-6.1, 6.1-6.2, 6.2-6.3, 6.3-6.4, or 6.4-6.5.

[0080] In some embodiments, the seed culture temperature is 25-30 °C. In some embodiments, the seed culture temperature is 25-26 °C, 26-27 °C, 27-28 °C, 28-29 °C, or 29-30 °C.

[0081] In some embodiments, the seed culture time is 48-96 h. In some embodiments, the seed culture time is 48-60 h, 60-72 h, 72-84 h, or 84-96 h.

[0082] Yeast lipid extract

[0083] A third aspect of the invention relates to a yeast lipid extract prepared by the method described in the second aspect of the invention.

[0084] In some embodiments, the yeast lipid extract comprises fatty acids, triglycerides, sterols, and carotenoids.

[0085] In some embodiments, the fatty acid composition of the fatty acids and triglycerides includes oleic acid, octadecenoic acid, palmitic acid, linoleic acid, and linolenic acid.

[0086] In some embodiments, the octadecenoic acid includes, but is not limited to, 9-octadecenoic acid and its cis-trans isomers, 11-octadecenoic acid and its cis-trans isomers, 13-octadecenoic acid and its cis-trans isomers, and 9-cis,11-trans-octadecadienoic acid.

[0087] In some embodiments, the yeast lipid extract, directly analyzed by GC-MS, contains the following characteristic spectral peaks: methyl trans-9-octadecenoate, ethyl palmitate, and palmitic acid.

[0088] In some embodiments, the yeast lipid extract, directly analyzed by GC-MS, contains characteristic spectral peaks with the following peak area ratios: 60-70% methyl trans-9-octadecenoate, 20-25% ethyl palmitate, and 1-5% palmitic acid.

[0089] In some embodiments, the yeast lipid extract, after methyl esterification, contains the following characteristic spectral peaks as analyzed by GC-MS: ethyl oleate, methyl 11-octadecenoate, ethyl palmitate, and methyl palmitate.

[0090] In some embodiments, the yeast lipid extract, after methylation, contains characteristic spectral peaks in the following peak area ratios as analyzed by GC-MS: 40-50% ethyl oleate, 10-20% methyl 11-octadecenoate, 10-15% ethyl palmitate, and 8-15% methyl palmitate.

[0091] In some embodiments, the fatty acid composition of the fatty acids and triglycerides also includes other saturated and unsaturated fatty acids, such as 2,2,4-trimethyl-1,3-pentanediol diisobutyric acid, cis-9-hexadecenoic acid, trans-9-octadecenoic acid, 11-octadecenoic acid, cis-13-octadecenoic acid, stearic acid, octadecanoic acid, myristic acid, 2-hexadecenoic acid, 3-hydroxyoctadecanoic acid, 9-cis,11-trans-octadecadienoic acid, 9-hexadecenoic acid, cis-11-octadecenoic acid, tetradecanoic acid, pentadecanoic acid, etc.

[0092] Used in the preparation of cosmetics or pharmaceuticals

[0093] The fourth aspect of the invention relates to the use of the yeast lipid extract according to the third aspect of the invention in the preparation of cosmetics / pharmaceuticals.

[0094] In some embodiments, the cosmetic or pharmaceutical product has antioxidant, moisturizing, anti-wrinkle, anti-aging, and / or barrier repair effects.

[0095] cosmetics or drugs

[0096] The fifth aspect of the invention relates to a cosmetic or pharmaceutical product comprising a yeast lipid extract according to the third aspect of the invention, wherein the yeast lipid extract comprises 0.1% to 99% by weight of the cosmetic or pharmaceutical product.

[0097] In some embodiments, the yeast lipid extract according to the third aspect of the invention accounts for 0.01%-20% by weight of the cosmetic or pharmaceutical product. In some embodiments, the yeast lipid extract according to the third aspect of the invention accounts for 0.01%-0.02%, 0.02%-0.03%, 0.03%-0.04%, 0.04%-0.05%, 0.05%-0.06%, 0.06%-0.07%, 0.07%-0.08%, 0.08%-0.09%, 0.09%-0.1%, 0.1%-1%, 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-6%, 6%-7%, 7%-8%, 8%-9%, 9%-10%, 10%-11%, 11%-12%, 12%-13%, 13%-14%, 14%-15%, 15%-16%, 16%-17%, 17%-18%, 18%-19%, or 19%-20% of the weight of the drug or cosmetic.

[0098] In some embodiments, the medicine or cosmetic is used for skin care. In some embodiments, the medicine and cosmetic are used for skin anti-oxidation, moisturizing, anti-wrinkle, anti-aging, and / or barrier repair. In some embodiments, the medicine and cosmetic comprise a yeast lipid extract according to the third aspect of the invention, and one or more pharmaceutically or cosmetically acceptable carriers, diluents, or excipients. In some embodiments, the medicine and cosmetic are creams, lotions, pastes, ointments, masks, gels, lotions, or serums. In some embodiments, the medicine and cosmetic of the present invention may also contain one or more other ingredients, such as plant extracts, nutritional additives, surfactants, fragrances and flavors, pigments, preservatives, antioxidants, moisturizers, UV absorbers, astringents, penetration enhancers, pH adjusters, etc. Those skilled in the art can make selections based on their common sense and specific needs.

[0099] In some embodiments, the drug or cosmetic may also contain one or more other cosmetic active ingredients, such as, but not limited to, moisturizers, keratin repair agents, oil controllers, anti-oil agents, hydrating agents, anti-allergens, anti-aging and wrinkle-reducing agents, etc. Detailed Implementation

[0100] The present invention can be implemented through the following embodiments, but the present invention is not limited thereto.

[0101] The instruments and equipment used in the embodiments of the present invention are all conventional instruments and equipment in the field and can be replaced by instruments and equipment that conform to the corresponding standards.

[0102] Unless otherwise specified, all Chinese herbal medicine raw materials used in the embodiments of the present invention are commercially available, and all reagents used in the embodiments of the present invention are commercially available analytical chemical reagents unless otherwise specified.

[0103] The raw materials and instruments mentioned in this invention are all commonly used in the art and are merely examples, not intended to limit the scope of protection of this invention. Those skilled in the art can select equivalent raw materials and related instruments based on the disclosure of this invention.

[0104] Experimental materials, equipment and statistical analysis methods

[0105] Experimental materials: Wheat germ extract solid culture medium and potato extract solid culture medium were purchased from Qingdao Haibo Biotechnology Co., Ltd.

[0106] Experimental reagents: glucose (Titan), yeast extract (Oxoid), malt extract (Qingdao Haibo Biotechnology), ammonium sulfate, peptone (Oxoid), magnesium sulfate (Titan), and trace elements (Titan).

[0107] Experimental equipment: 5L bioreactor, Shanghai Baoxing BIOTECH-5JGY.

[0108] Example

[0109] Example 1: Isolation, Identification and Preservation of New Microbial Strains

[0110] Colonies were isolated and purified from freshly collected ginseng root samples using a dilution plating method on wheat germ extract solid medium and potato extract solid medium, respectively, and incubated at 28°C for 72 hours. Pure single colonies were obtained after multiple rounds of subculturing. A yeast strain with an orange-red color, moist colonies, and color-producing ability was selected for further research.

[0111] The 16S / ITS whole genome sequencing of this yeast strain was completed by Beijing New Era Zhonghe Technology Co., Ltd. The resulting species identification tree after analyzing the genome sequencing results is shown below. Figure 1 As shown. This strain was identified as *Rhodotorula granatum* RS-Y-red by morphological observation and 16S / ITS whole-genome sequencing. Rhodotorula graminisstrain RS-Y-red). This strain has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, 430072, China. The relevant deposit information is as follows:

[0112]

[0113] Example 2: Preparation of yeast lipid extract

[0114] 1. Microbial culture

[0115] The Rhodotorula granatum RS-Y-red strain was pre-cultured in YM2 liquid medium in shake flasks for 72 hours at 28°C and 200 rpm. The bacterial culture was collected as seed culture for inoculation in the fermenter.

[0116] The YM2 liquid culture medium consisted of 20 g / L glucose, 3 g / L yeast extract, 3 g / L malt extract, and 5 g / L peptone, with a pH of 6.0 ± 0.2.

[0117] 2.5L tank batch feeding fermentation

[0118] Fed-batch fermentation was conducted using a 5L bioreactor (Shanghai Baoxing BIOTECH-5JGY) with an initial working volume of 2.5 L and a seed culture inoculum size of 8% (v / v). The initial basal culture medium consisted of: 70 g / L glucose, 2.4 g / L yeast extract, 8 g / L malt extract, 2 g / L ammonium sulfate, 2.4 g / L peptone, 0.5 g / L magnesium sulfate, and appropriate amounts of trace elements. The initial pH was set to 6.0 ± 0.2. The fermentation temperature was set at 28°C, the stirring speed at 400 rpm, and the aeration rate at 0.8 vvm.

[0119] During fermentation, dissolved oxygen and glucose levels were monitored. When the glucose concentration fell below 30 g / L, a 600 g / L glucose solution was continuously fed to maintain a carbon source concentration of at least 30 g / L. After 60 hours, the fermentation temperature was set to 20°C, and carbon source feeding was maintained to ensure a glucose concentration of at least 50 g / L. The entire fermentation cycle lasted approximately 96 hours, with the final cell concentration reaching O2. ≈ 150, corresponding to a dry weight of approximately 80 g / L, with total oil accumulation accounting for up to 50% of the bacterial dry weight.

[0120] 3. Oil extraction

[0121] After fermentation, the bacterial culture was centrifuged (8000 rpm, 10 min) to collect the bacterial cells, and then washed twice with distilled water. The collected bacterial cells were then broken up using a bead milling method.

[0122] The specific steps of the bead grinding method are as follows: the centrifuged bacterial sludge is diluted with 30% water and mixed well, then poured into a grinding cup. 40% (v / v) of 0.5-1.0 mm zirconium beads are added to each grinding cup. After sealing, the cup is placed in a planetary ball mill and continuously ground at a speed of 300-500 rpm for 10-20 minutes as one cycle. After each cycle, the grinding cup is placed in ice water to cool for 10 minutes to reduce the temperature rise of the material. The grinding is repeated 4 times, and the yeast cell breakage rate reaches more than 90%.

[0123] The cell lysate was dried to a moisture content of less than 10%, then loaded into a supercritical fluid extraction vessel and purged with high-purity C. The temperature was raised to 50 °C and the pressure increased to 30 MPa to bring it to a supercritical state; at C During continuous flow, it can be done according to C Anhydrous ethanol was added at a flow rate of 5–10% via an entrainer pump to enhance the dissolution of polar lipids, and the extraction time was controlled at approximately 2 hours. The extract was then subjected to a stepwise depressurization process at 35–45 °C and 5–8 MPa in a separator to precipitate and collect the lipids. Finally, residual ethanol was removed under vacuum. The resulting yeast lipids are referred to hereafter as Rs.

[0124] Example 3: Component Detection of Yeast Lipid Extract

[0125] 1. Experimental Objective

[0126] The fatty acid content of oils was determined using GB5009.168—2016, the standard for determination of fatty acids in food.

[0127] Experiment 1: Direct GC-MS detection.

[0128] Experiment 2: Transesterification (applicable to oils with a free fatty acid content of no more than 2%): Dissolve the oil in isooctane, add potassium hydroxide methanol solution and transesterify to form methyl ester. After the reaction is complete, neutralize the remaining potassium hydroxide with sodium bisulfate to avoid saponification of methyl ester.

[0129] 2. Experimental Instruments and Materials

[0130] 2.1 Experimental Apparatus

[0131] Heated magnetic stirrer RCT BasicIKA, vortex mixer, gas chromatograph 7890A, adjustable pipette.

[0132] 2.2 Experimental Materials

[0133] The yeast lipid extract prepared in Example 2.

[0134] 2.3 Experimental Reagents

[0135] Methanol; isooctane; sodium bisulfate; potassium hydroxide

[0136] Potassium hydroxide methanol solution (2 mol / L): Dissolve 1.31 g of potassium hydroxide in 10 mL of anhydrous methanol. The solution can be heated slightly, dried with anhydrous sodium sulfate, and filtered to obtain a clear solution.

[0137] 3. Experimental Methods

[0138] 3.1 Sample Pretreatment

[0139] Direct detection: Dissolve 10 mg of the oil sample in 2 mL of methanol and then analyze directly by GC-MS.

[0140] Methyl esterification: Dissolve 30 mg of the oil sample in 2 mL of isooctane. If necessary, gently heat the sample to dissolve it. Add 100 μL of potassium hydroxide methanol solution, stopper the container, shake vigorously for 30 seconds, and let stand until clear. Add approximately 500 mg of sodium bisulfate, shake vigorously, and neutralize the potassium hydroxide. After the salt precipitates, filter the supernatant for analysis.

[0141] 3.2 GC-MS Analysis

[0142] 3.2.1 Direct Analysis of GC Conditions

[0143] Capillary column: J&W 122-5532, column length 30 m, inner diameter 250 μm, film thickness 0.25 μm.

[0144] Programmed temperature rise: Initial temperature 60℃, hold for 1 minute;

[0145] 60℃~120℃, heating rate 10℃ / min;

[0146] 120℃~240℃, heating rate 8℃ / min, hold for 10 min.

[0147] 3.2.2 GC conditions after methyl esterification

[0148] Capillary column: J&W 122-7062, column length 60m, inner diameter 250 μm, film thickness 0.25μm.

[0149] Programmed temperature rise: Initial temperature 80℃;

[0150] 80℃~160℃, heating rate 20℃ / min;

[0151] 160℃~220℃, heating rate 10℃ / min;

[0152] Set the temperature to 220℃~250℃, with a heating rate of 3℃ / min, and hold for 10min.

[0153] 3.2.3 MS conditions:

[0154] Ionization method: EI; Electron energy: 70 eV; Transfer line temperature: 290°C; Ion source temperature: 230°C; Quadrupole temperature: 150°C; Mass range: 50–600. Database: NIST11.L.

[0155] 4. Experimental Results

[0156] The results of direct GC-MS and GC-MS analysis after methylation are as follows: Figure 2 As shown, the main fatty acid composition of the lipid extract (Rs) of *Rhodotorula glutinis*, as detected by direct injection analysis by GC-MS, was methyl trans-9-octadecenoate, ethyl palmitate, and palmitic acid. GC-MS analysis after methylation treatment with potassium hydroxide-methanol solution showed that the main fatty acid composition was ethyl oleate, methyl 11-octadecenoate, ethyl palmitate, and methyl palmitate.

[0157] Example 4: Antioxidant efficacy test

[0158] 1. DPPH free radical scavenging experiment

[0159] Dissolve an appropriate amount of DPPH solid powder in anhydrous ethanol until the absorbance at 517 nm is 1.6. Dilute the yeast oil (Rs) serially using DMSO as the solvent. Add 100 μL of the analyte / DMSO mixture to a 96-well plate, setting up three replicates. Add 100 μL of the prepared DPPH solution to each well to obtain the sample group / blank group. Add 100 μL of the analyte / DMSO mixture to a 96-well plate, setting up one well, and add 100 μL of anhydrous ethanol to obtain the sample control group / blank control group. Incubate at room temperature in the dark for 30 min. Then measure the absorbance at 517 nm.

[0160] Inhibition rate % = [1 - (Sample group - Sample control group) / (Blank group - Blank control group)] 100%

[0161] Conclusion: Figure 3 As shown, yeast lipids (Rs) exhibit strong DPPH free radical scavenging ability, showing a gradient-dependent effect. The DPPH scavenging rate at 10% is close to 100%, and its half-inhibitory concentration (IC50) is very low. 50 The value was 1.91%. Therefore, Rs has a strong free radical scavenging ability and plays a role in resisting oxidative stress.

[0162] 2. Cytotoxicity and reactive oxygen species inhibition rate

[0163] The cultured HFF-1 cells were diluted with DMEM medium (containing 10% FBS and 1% penicillin antibiotics) to a final concentration of 2.5 × 10⁻⁶. 5 Cell suspension was added at a rate of 100 µL per well to each 96-well plate and incubated in a CO2 incubator for 12 h. The cultured cells were then replaced with DMEM medium (containing 1% penicillin antibiotics) diluted with the sample and incubated for another 12 h. After incubation, the sample was discarded and washed, and DCFH-DA and HOECHST were added to each well for 20 min of incubation. After incubation, the probe was discarded and washed, and 200 µmol / L H2O2 was added to each well for 30 min of treatment. ROS fluorescence intensity was measured at 485 nm (excitation) and 525 nm (emission); HOECHST fluorescence was measured at 350 nm (excitation) and 461 nm (emission) to reflect cell viability.

[0164] Reactive oxygen species inhibition rate % = [1 - (sample group - blank control group) / (H2O2 model group - blank control group)] 100%

[0165] Conclusion: The fluorescence intensity results of the HOECHST probe show that ( Figure 5 HFF-1 cells did not exhibit cytotoxicity when incubated with yeast oil Rs, indicating that Rs is safe and non-toxic within the detection range. Furthermore, it exhibited strong antioxidant activity against ROS, such as... Figure 5 As shown in Table 1, when the Rs concentration is 0.025%, the fluorescence intensity level of ROS is the same as that of EGCG at 20 µg / mL. Its antioxidant effect exhibits a concentration-dependent characteristic. At 0.025%, the ROS inhibition rate reaches 72.66%, and even at the lowest detection concentration of 0.0005%, a relatively high ROS inhibition level of 39.45% is still observed. This demonstrates that Rs, as an active oil, possesses strong antioxidant properties and has significant application value in skin care.

[0166] Table 1. ROS inhibition rate

[0167]

[0168] Example 5: Barrier Repair and Moisturizing Efficacy Testing

[0169] 2×10 5 HaCaT cells were seeded at a density of 10 cells / mL in 24-well plates. After cell adhesion the following day, the cells were incubated with 0.2 J / cm² water. 2Cells were treated with UVB irradiation for 1 min, and then incubated with samples prepared in DMEM. The control and model groups (UVB groups) received no sample treatment. Cells were cultured for 24 h. After 24 h of continuous culture, cells were lysed and total RNA was collected. RNA was extracted and purified using the SteadyPure Universal RNA Extraction Kit. Reverse transcription and probe modification were performed using HiScript® III All-in-one RT SuperMix Perfect for qPCR and AceQ® qPCR SYBR Green Master Mix (Vazyme). The mRNA expression level of FLG was then detected.

[0170] Conclusion: Filamentin (FLG) is a key protein in maintaining skin barrier function and moisturizing ability. It plays a role in the stratum corneum, helping the skin retain moisture, maintain elasticity, and resist external stimuli and water loss. The experimental results for the mRNA expression level of filaggrin (FLG) are as follows: Figure 6 As shown, incubation of skin cells with 0.025% and 0.05% Roil significantly enhanced the gene expression of filaggrin (FLG) in skin cells, with increases of 2.03-fold and 3.62-fold, respectively, compared to the UVB model group. Therefore, Roil plays an important role in skin barrier repair and moisturizing.

[0171] Example 6: Anti-aging efficacy test

[0172] According to 2×10 per hole 5 Human dermal fibroblasts (HDF) were seeded into 24-well plates at a cell density of 1 cell / mL, with 3 replicates per group. Cells were shaken well and incubated overnight at 37°C with 5% CO2 for 16–24 h. After 24 h, the cells were washed twice with PBS to completely remove etoposide (ETO) residues. 500 μL of culture medium containing the analyte (i.e., yeast lipid extract) was added, and the cells were incubated for 16–24 h. After incubation, the cells were lysed, and total RNA was collected. RNA was extracted and purified using the SteadyPure Universal RNA Extraction Kit. Reverse transcription and probe modification were performed using HiScript® III All-in-one RT SuperMix Perfect for qPCR and AceQ® qPCR SYBRGreen Master Mix (Vazyme). The mRNA expression levels of COL-I, COL-III, and ELN were detected.

[0173] Conclusion: COL I (type I collagen) provides structural support and resilience to the skin, COL III (type III collagen) helps maintain skin elasticity and softness, and ELN (elastin) endows the skin with elasticity and retraction ability. These three components work together to maintain the normal shape and function of the skin. Their reduction in the skin is directly related to the aging phenotype of the skin, manifesting as facial wrinkles, sagging, and loss of elasticity. Low concentrations of yeast oil have a significant effect on promoting the increase of COL I, COL III, and ELN. Figures 7-9 As shown, yeast oil at a concentration of 0.005% can increase the relative mRNA expression levels of type I collagen, type III collagen, and elastin by 54.49%, 45.68%, and 97.71%, respectively, indicating that the yeast oil obtained in this invention can play a good anti-aging role.

Claims

1. A strain of *Rhodotorula granatum*, deposited at the China Center for Type Culture Collection (CCC) under accession number M20251301.

2. A method for preparing yeast lipid extract by fermentation using a strain of Rhodotorula granatum, wherein the Rhodotorula granatum strain is a strain of Rhodotorula granatum deposited at the China Center for Type Culture Collection with accession number M20251301.

3. The method according to claim 2, wherein the method comprises the following steps: (1) The *Rhodotorula granatum* strain was seed cultured to obtain *Rhodotorula granatum* seed liquid; (2) The *Hemiberlesia javanica* seed culture was inoculated into a fermentation medium for fermentation culture; and (3) After fermentation, yeast lipids are extracted from the culture.

4. The method according to claim 3, wherein the fermentation culture is a fed-batch fermentation culture, and the fed-batch fermentation culture is a feedback-based fed-batch culture based on carbon source concentration monitoring, comprising: During the first fermentation stage, the carbon source concentration is maintained at a first predetermined level; After reaching the predetermined fermentation node, the process transitions to the second fermentation stage, where the carbon source concentration is increased and maintained at a second predetermined level higher than the first predetermined level.

5. The method according to claim 4, wherein the carbon source is glucose, the initial basal culture medium used in the fed-batch fermentation culture comprises glucose, yeast extract, malt extract, ammonium sulfate, peptone, magnesium sulfate and trace elements, and the pH of the initial basal culture medium is 5.5-6.

5.

6. The method according to claim 5, wherein the initial basal culture medium comprises 60-80 g / L glucose, 2-3 g / L yeast extract, 6-10 g / L malt extract, 1-3 g / L ammonium sulfate, 2-3 g / L peptone, and 0.1-2 g / L magnesium sulfate; wherein the first predetermined level of carbon source concentration is 20-40 g / L, and the second predetermined level of carbon source concentration is 40-60 g / L; wherein the fermentation temperature of the first fermentation stage is 25-30 °C, and the fermentation time is 50-70 h; wherein the fermentation temperature of the second fermentation stage is 15-25 °C, and the fermentation time is 30-40 h.

7. The method according to any one of claims 3-6, wherein the step of extracting yeast lipids from the culture comprises: (a) Collect and break up the yeast cells obtained from fermentation; and (b) Using supercritical C Fluid extraction technology is used to extract yeast lipids from broken bacterial cells.

8. The method according to claim 7, wherein in step (a), yeast cells are broken by mechanical grinding; and in step (b), the mixture is subjected to supercritical C444. Anhydrous ethanol is added as an entrainer during extraction.

9. The method according to claim 7 or 8, wherein in supercritical C After extraction, yeast lipids were precipitated by stepwise depressurization, and residual solvent was removed under vacuum conditions.

10. The method according to any one of claims 3-9, wherein the seed culture is carried out in a liquid culture medium for at least one stage of pre-culture, the liquid culture medium comprising a carbon source and a nitrogen source; the carbon source comprising glucose, and the nitrogen source comprising at least one of yeast extract, malt extract and peptone.

11. The method according to claim 10, wherein the liquid culture medium comprises 15-25 g / L glucose, 2-4 g / L yeast extract, 2-4 g / L malt extract, 4-6 g / L peptone, and a pH of 5.5-6.

5.

12. The method according to claim 10 or 11, wherein the pre-culture conditions are: temperature 25-30 °C, culture time 48-96 h.

13. A yeast lipid extract prepared by any one of claims 2-12.

14. The yeast lipid extract according to claim 13, wherein the yeast extract comprises fatty acids, triglycerides, sterols, and carotenoids.

15. The yeast lipid extract according to claim 14, wherein the fatty acid composition of the fatty acids and triglycerides comprises oleic acid, octadecenoic acid, palmitic acid, linoleic acid, and linolenic acid.

16. Use of the yeast lipid extract according to any one of claims 13-15 in the preparation of cosmetics / pharmaceuticals.

17. The use according to claim 16, wherein the cosmetic / medication has antioxidant, moisturizing, anti-wrinkle, anti-aging and / or barrier repair effects.

18. A cosmetic or pharmaceutical product comprising a yeast lipid extract according to any one of claims 13-15, wherein the yeast lipid extract comprises 0.1%-99% by weight of the cosmetic or pharmaceutical product.

Images