Preparation method of Cordyceps militaris fermentation filtrate, Cordyceps militaris fermentation filtrate and its application

Abstract

This application discloses a method for preparing Cordyceps militaris fermentation filtrate, the Cordyceps militaris fermentation filtrate, and its applications. The preparation method includes: mixing, dispersing, and culturing Cordyceps militaris mycelium with a liquid culture medium to obtain a high-density Cordyceps militaris seed liquid; inoculating the high-density Cordyceps militaris seed liquid into a fermentation culture medium for fermentation; adding Tween 80 during the mid-to-late logarithmic growth phase of the microorganisms during fermentation, and adding sophorolipids during the stable growth phase of the microorganisms; obtaining a mixed fermentation broth after fermentation; and subjecting the mixed fermentation broth to inactivation, centrifugation, freeze-thaw cycles, filtration, further inactivation, and preservation treatment to obtain the Cordyceps militaris fermentation filtrate. The Cordyceps militaris fermentation filtrate prepared according to the method of this application has a high cordycepin content, strong antioxidant capacity, and high stability, showing great application potential in cosmetics, health products, or pharmaceutical raw materials.

Description

Technical Field

[0001] This application belongs to the field of cosmetic technology, and in particular relates to a method for preparing Cordyceps militaris fermentation filtrate, the Cordyceps militaris fermentation filtrate and its application. Background Technology

[0002] Cordyceps militaris is a precious edible and medicinal fungus, not only delicious and nutritious but also possessing multiple effects such as immune regulation, anti-tumor activity, and anti-oxidation, making it highly popular among consumers. Cordyceps militaris extract contains various active substances, such as cordycepin, cordyceps polysaccharides, cordycepic acid, ergosterol, and adenosine, exhibiting strong biological activity. It is widely used in health foods and pharmaceuticals and also has broad development prospects in the cosmetics field. Among them, cordycepin (3'-deoxyadenosine) is one of the core active ingredients of Cordyceps militaris, possessing multi-dimensional biological activities such as anti-oxidation, anti-inflammation, moisturizing, and barrier repair, making it highly valuable for application in the cosmetics industry.

[0003] Existing Cordyceps militaris fermentation technologies generally suffer from low yields of cordycepin active ingredients and excessively long fermentation cycles, making them unsuitable for large-scale industrial production. Furthermore, if fermented Cordyceps militaris products are to be used in cosmetics, their impact on the sensory evaluation indicators and stability of the cosmetics must be further considered. Summary of the Invention

[0004] This application provides a Cordyceps militaris fermentation filtrate, its preparation method, and its application. The preparation process is low-cost and environmentally friendly. The resulting Cordyceps militaris fermentation filtrate has a high cordycepin content, strong antioxidant capacity, a clear and transparent appearance, and good stability, making it suitable for industrial production.

[0005] In a first aspect, this application provides a method for preparing Cordyceps militaris fermentation filtrate, comprising: mixing, dispersing, and culturing Cordyceps militaris mother culture with a liquid culture medium to obtain a high-density Cordyceps militaris seed solution, wherein the viable count of Cordyceps militaris in the high-density Cordyceps militaris seed solution is (7.0~7.5)×10⁻⁶. 7 CFU / mL; High-density Cordyceps militaris seed culture was inoculated into fermentation medium for fermentation. The inoculation volume of the high-density Cordyceps militaris seed culture was 4.5%~5.5% of the fermentation medium volume. During fermentation, surfactants were added in stages based on the microbial growth curve: Tween 80 was added in the middle and late logarithmic growth phase of the microorganisms, and sophorolipids were added in the stable growth phase of the microorganisms to obtain a mixed fermentation broth. The total amount of Tween 80 and sophorolipids added was 0.05 mg / mL~0.2 mg / mL based on the volume of the fermentation medium, and the mass ratio of Tween to sophorolipids was (1.5~2.5):1. The mixed fermentation broth was inactivated, centrifuged, freeze-thawed, filtered, inactivated again, and preserved to obtain Cordyceps militaris fermentation filtrate.

[0006] In any embodiment of this application, the mid-to-late logarithmic phase of microbial growth is 70-74 hours after the start of fermentation, the stable phase of microbial growth is 118-122 hours after the start of fermentation, and the total fermentation time is 6-8 days.

[0007] In any embodiment of this application, the fermentation medium includes a carbon source, a nitrogen source, a precursor, and inorganic salts.

[0008] In any embodiment of this application, the nitrogen source includes one or more of yeast extract, peptone, and glycine; the total content of the nitrogen source is less than 5 g / L based on the volume of the fermentation medium.

[0009] In any embodiment of this application, the total content of the nitrogen source is 2.3~3.7 g / L based on the volume of the fermentation medium.

[0010] In any embodiment of this application, the fermentation medium comprises: yeast extract 0.4~0.6 g / L, peptone 0.4~0.6 g / L, glycine 1.5~2.5 g / L, adenine 1.8~2.2 g / L, dipotassium hydrogen phosphate 0.4~0.6 g / L, magnesium sulfate heptahydrate 0.2~0.3 g / L, and glucose 30~50 g / L.

[0011] In any embodiment of this application, the step of mixing, dispersing and culturing Cordyceps militaris mother culture with liquid culture medium to obtain high-density Cordyceps militaris seed liquid includes at least one of crushing, grinding, shearing and homogenizing treatment.

[0012] In any embodiment of this application, the step of inactivating, centrifuging, freezing and thawing, filtering, re-inactivating and preservativeing ​​the mixed fermentation broth to obtain Cordyceps militaris fermentation filtrate includes freezing and thawing, with the freezing temperature being less than or equal to -20°C and the freezing time being greater than or equal to 24 hours.

[0013] In any embodiment of this application, the step of inoculating high-density Cordyceps militaris seed liquid into a fermentation medium for fermentation further includes the step of supplementing carbon source and / or precursor.

[0014] Secondly, this application provides a Cordyceps militaris fermentation filtrate, which is prepared according to the preparation method described in the first aspect.

[0015] Thirdly, this application provides the use of the Cordyceps militaris fermentation filtrate described in the second aspect in the preparation of cosmetics, health products or active pharmaceutical ingredients.

[0016] Compared to existing technologies, the method for preparing Cordyceps militaris fermentation filtrate provided in this application employs high-density liquid microbial fermentation, effectively increasing the biomass and growth point density at the fermentation starting point. This allows the cells to rapidly overcome the lag phase, shorten the logarithmic growth phase, enter the stationary phase earlier, and efficiently synthesize active ingredients such as cordycepin and polysaccharides. Based on the characteristics of the cell growth and product synthesis cycle during Cordyceps militaris fermentation, a staged addition strategy is adopted to add Tween 80 and sophorolipids to the fermentation system. By controlling the timing and amount of addition, Tween 80 and sophorolipids synergistically disrupt cell walls and promote release. This significantly increases the release of intracellular substances such as cordycepin and polysaccharides, enhances the target efficacy of the fermentation broth, and prevents the fermentation broth from becoming too viscous or forming foam. It also improves dissolved oxygen efficiency and enhances the efficiency and effect of post-treatment purification, making the fermentation filtrate more suitable for cosmetic applications. After fermentation, multi-physics field coupling purification is used to efficiently remove impurities such as macromolecular polysaccharides and proteins from the fermentation broth without introducing any exogenous reagents, successfully transforming the fermentation broth into a clear and transparent yellow liquid. Furthermore, this application further reduces the conductivity of the Cordyceps militaris fermentation filtrate by controlling the total amount of nitrogen source added to the fermentation medium to below 5 g / L, significantly improving its stability in cosmetic applications. According to the method provided in this application, a Cordyceps militaris fermentation filtrate with high cordycepin content and excellent antioxidant capacity is prepared with a significantly shorter process cycle compared to existing technologies. This fermentation filtrate is clear and transparent in appearance and exhibits good stability, showing promising application prospects in cosmetics, health products, or pharmaceutical raw materials, especially suitable for applications as a cosmetic raw material. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments of this application will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram showing the changes in mycelium and cordycepin production during the fermentation process of Example 1 of this application.

[0019] Figure 2 This is the liquid chromatography spectrum of cordycepin standard.

[0020] Figure 3 This is a schematic diagram of the cordycepin standard curve.

[0021] Figure 4 This is a schematic diagram of the standard curve for DPPH free radical scavenging rate.

[0022] Figure 5 This is a schematic diagram of the standard curve of ABTS free radical scavenging rate. Detailed Implementation

[0023] To make the purpose, technical solution, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the implementation details described in this specification are merely for illustrative purposes and are not intended to limit the scope of this application.

[0024] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0025] It should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0026] Unless otherwise stated, the values ​​of the parameters mentioned in this application can be measured using various measurement methods commonly used in the art (e.g., they can be tested according to the methods given in the embodiments of this application). Unless otherwise stated, the test temperature for all parameters mentioned in this application is 25°C and the test pressure is standard atmospheric pressure.

[0027] The foregoing description of this application is not intended to describe every disclosed implementation or method. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments, which can be used in various combinations. The examples listed are representative only and should not be construed as exhaustive.

[0028] In view of the deficiencies of the prior art, this application provides a Cordyceps militaris fermentation filtrate, its preparation method and application. The preparation process is low-cost and environmentally friendly. The obtained Cordyceps militaris fermentation filtrate has high cordycepin content, strong antioxidant capacity and high stability, and is suitable for industrial production.

[0029] The first aspect of this application provides a method for preparing Cordyceps militaris fermentation filtrate, comprising: mixing, dispersing, and culturing a Cordyceps militaris mother culture with a liquid culture medium to obtain a high-density Cordyceps militaris seed solution, wherein the viable count of Cordyceps militaris in the high-density Cordyceps militaris seed solution is (7.0~7.5)×10⁻⁶. 7 The high-density Cordyceps militaris seed culture was inoculated into a fermentation medium for fermentation. The inoculation volume of the high-density Cordyceps militaris seed culture was 4.5% to 5.5% of the volume of the fermentation medium. During fermentation, Tween 80 and sophorolipids were added in stages based on the microbial growth curve: Tween 80 was added in the middle and late stages of the logarithmic growth phase of the microorganisms, and sophorolipids were added in the stationary phase of the microorganisms to obtain a mixed fermentation broth. The total amount of Tween 80 and sophorolipids added was 0.05 mg / mL to 0.2 mg / mL based on the volume of the fermentation medium, and the mass ratio of Tween 80 to sophorolipids was (1.5 to 2.5):1. The mixed fermentation broth was inactivated, centrifuged, freeze-thawed, filtered, inactivated again, and preserved to obtain Cordyceps militaris fermentation filtrate.

[0030] First, this application controls the viable count of Cordyceps militaris in high-density Cordyceps militaris seed solution to (7.0~7.5)×10⁻⁶. 7 Inoculated with cfu / mL at a ratio of 4.5%–5.5% of the fermentation medium volume, this provides a sufficient quantity and highly active starting microbial community for the fermentation process. This effectively increases the biomass and growth point density at the fermentation start point, enabling the cells to quickly overcome the lag phase and shorten the logarithmic growth phase, thus entering the stationary phase earlier and efficiently transitioning to the synthesis of active metabolites such as cordycepin. The high-density microbial community not only enhances the directionality and efficiency of the fermentation process, improving the yield of the target product and the antioxidant capacity of the fermentation broth from the source, but also further reduces overall production costs due to the shortened fermentation cycle and increased metabolic efficiency, enhancing the feasibility and economic viability of the entire green and environmentally friendly production process for industrial-scale application.

[0031] As an example, the viable count of Cordyceps militaris in a high-density Cordyceps militaris seed solution can be 7.0 × 10⁻⁶. 7 cfu / mL, 7.05×10 7 cfu / mL, 7.1×10 7 cfu / mL, 7.15×10 7 cfu / mL, 7.2×10 7 cfu / mL, 7.25×10 7 cfu / mL, 7.3×10 7 cfu / mL, 7.31×10 7 cfu / mL, 7.32×10 7 cfu / mL, 7.33×10 7cfu / mL, 7.34×10 7 cfu / mL, 7.35×10 7 cfu / mL, 7.36×10 7 cfu / mL, 7.37×10 7 cfu / mL, 7.38×10 7 cfu / mL, 7.39×10 7 cfu / mL, 7.40×10 7 cfu / mL, 7.45×10 7 cfu / mL, 7.5×10 7 cfu / mL, or any range of the above values.

[0032] As an example, the inoculation volume of high-density Cordyceps militaris seed liquid can be 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5% of the fermentation medium volume, or any range of the above values.

[0033] Furthermore, during the fermentation process of inoculating high-density Cordyceps militaris seed liquid into the fermentation medium, Tween 80 and sophorolipids were added to the fermentation system in stages based on the microbial growth curve.

[0034] As a biological term familiar to those skilled in the art, a microbial growth curve is a graph describing the change in the number of microbial communities in a culture medium over time. The vertical axis represents the number of viable cells or cell weight, and the horizontal axis represents the culture time. This curve consists of four phases: lag phase, logarithmic phase, stationary phase, and death phase. The number of microorganisms in the culture medium can be directly determined by plate counts, turbidimetry, or by OD... 600 Indirect methods such as optical density measurement and fluorescence detection are used for determination.

[0035] During the mid-to-late logarithmic growth phase of Cordyceps militaris liquid fermentation, the cell biomass approaches its peak, and the synthesis of secondary metabolites such as cordycepin and polysaccharides begins to flourish. At this stage, adding the relatively mild nonionic surfactant Tween 80 to the fermentation system is crucial. Tween 80 can penetrate the lipid bilayer of the cell membrane, increasing cell membrane fluidity and gently promoting the release of early-synthesized intracellular products, thus mitigating feedback inhibition. During the stationary growth phase of Cordyceps militaris liquid fermentation, the cell biomass tends to stabilize due to nutrient consumption and metabolite accumulation, while product synthesis reaches its peak. At this stage, adding the more potent biobased surfactant sophorolipids to the fermentation system can deeply release the large amount of products accumulated intracellularly and specifically disperse extracellular polysaccharides. The above-mentioned phased addition strategy is based on the characteristics of cell growth and product synthesis cycle during Cordyceps militaris fermentation. It enables Tween 80 and sophorolipids to synergistically break cell walls and promote release. While significantly increasing the release of intracellular substances such as cordycepin and polysaccharides and enhancing the target efficacy of the fermentation broth, it can also prevent the fermentation filtrate from becoming too viscous or forming foam, improve dissolved oxygen efficiency, effectively reduce the viscosity of the fermentation filtrate, and improve the efficiency and effect of post-processing purification, making it more suitable for cosmetic applications.

[0036] Besides the timing of addition, the total amount and ratio of Tween 80 and sophorolipid are also key factors affecting the effect. If the total amount of Tween 80 and sophorolipid is too low, the effect of increasing cordycepin yield and enhancing the efficiency of the fermentation filtrate will be insignificant. If the total amount of Tween 80 and sophorolipid is too high, it can lead to premature cell lysis and death, thus affecting the continuous synthesis of the product. The most significant effect in improving cordycepin yield and the efficiency of the fermentation filtrate is achieved when the mass ratio of Tween 80 to sophorolipid is (1.5~2.5):1.

[0037] As an example, the total amount of Tween 80 and sophorolipid added can be 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.20 mg / mL, or any range of the above values.

[0038] As an example, the mass ratio of Tween to sophorolipid can be 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, or any range of the above values.

[0039] As an example, the amount of Tween added, based on the volume of the fermentation medium, is 0.03 mg / mL to 0.14 mg / mL, preferably 0.05 mg / mL to 0.12 mg / mL, more preferably 0.08 mg / mL to 0.10 mg / mL, and particularly preferably 0.10 mg / mL.

[0040] As an example, the amount of sophorolipid added, based on the volume of the fermentation medium, is 0.01 mg / mL to 0.08 mg / mL, preferably 0.02 mg / mL to 0.06 mg / mL, more preferably 0.03 mg / mL to 0.04 mg / mL, and particularly preferably 0.05 mg / mL.

[0041] Furthermore, after fermentation, this application employs a purely physical post-treatment process combining inactivation, centrifugation, freeze-thaw cycles, and filtration. Through multi-field coupling of thermal field, centrifugal force field, phase change field, and membrane sieving field, impurities such as macromolecular polysaccharides and proteins in the fermentation broth are efficiently removed without the introduction of any exogenous reagents. The originally viscous and turbid fermentation broth is transformed into a clear and transparent yellow liquid. The resulting Cordyceps militaris fermentation filtrate not only has strong antioxidant capacity and high stability, but also has a short process cycle, controllable raw material costs, and a green and environmentally friendly process, making it more suitable for industrial-scale production.

[0042] In some embodiments, during the fermentation process of inoculating high-density Cordyceps militaris seed liquid into a fermentation medium, the aeration rate is 0.8 vvm to 1.2 vvm (e.g., 0.8 vvm, 0.9 vvm, 1.0 vvm, 1.0 vvm, 1.2 vvm, or any range of the above values); the stirring speed is 200 rpm to 700 rpm; the temperature is 25℃ to 30℃ (e.g., 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, or any range of the above values); dissolved oxygen is ≥40%; and the pH is not lower than 5.0.

[0043] In some implementations, the mid-to-late logarithmic phase of microbial growth refers to 65-80 hours after the start of fermentation, the stationary phase of microbial growth refers to 120-140 hours after the start of fermentation, and the total fermentation time is 6-8 days.

[0044] As an example, Tween 80 is added to the system after 70-74 hours (e.g., 70h, 71h, 72h, 73h, 74h, or any range of the above values) of fermentation culture after inoculating high-density Cordyceps militaris seed culture into the fermentation medium; sophorolipid is added to the system after 118-122 hours (e.g., 118h, 119h, 120h, 121h, 122h, or any range of the above values) of fermentation culture after inoculating high-density Cordyceps militaris seed culture into the fermentation medium. As an example, the total fermentation time is 6-8 days (e.g., 6 days, 7 days, 8 days, or any range of the above values), which is significantly shorter than the existing Cordyceps militaris fermentation process and is more suitable for industrial production.

[0045] In some embodiments, the fermentation medium includes a carbon source, a nitrogen source, a precursor, and inorganic salts. The carbon source includes at least one selected from glucose, fructose, galactose, sucrose, maltose, lactose, and starch; the precursor includes at least one selected from adenine, guanine, and hypoxanthine; and the inorganic salts include phosphorus-based inorganic salts, potassium-based inorganic salts, magnesium-based inorganic salts, or inorganic salts providing trace elements such as iron, zinc, copper, and manganese.

[0046] In some embodiments, the nitrogen source in the fermentation medium includes one or more of yeast extract, peptone, and glycine; the total nitrogen source content, based on the volume of the fermentation medium, is less than 5 g / L. During Cordyceps militaris fermentation, the core role of the nitrogen source is to provide nitrogen for the growth of Cordyceps militaris mycelium, metabolic activities, and the synthesis of active ingredients. Generally, a higher nitrogen source content is beneficial for mycelial growth and metabolism. However, the inventors of this application have discovered that when the nitrogen source content in the Cordyceps militaris fermentation medium exceeds 5 g / L, it leads to the excessive generation of high-conductivity products, resulting in a conductivity of over 5000 μS / cm for the final Cordyceps militaris fermentation filtrate. When this filtrate is further applied to cosmetic compositions such as emulsions, precipitation and stratification easily occur, which is detrimental to the stability of the cosmetics. Therefore, controlling the total nitrogen source content in the fermentation medium to below 5 g / L can effectively reduce the conductivity of the Cordyceps militaris fermentation filtrate and improve its stability in cosmetic applications.

[0047] In some embodiments, the total nitrogen source content, based on the volume of the fermentation medium, is 2.3 to 3.7 g / L. As an example, the total nitrogen source content can be 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L, or any range of the above values.

[0048] In some embodiments, the fermentation medium, by its total volume, comprises the following components in the following amounts: yeast extract 0.4-0.6 g / L, peptone 0.4-0.6 g / L, glycine 1.5-2.5 g / L, adenine 1.8-2.2 g / L, dipotassium hydrogen phosphate 0.4-0.6 g / L, magnesium sulfate heptahydrate 0.2-0.3 g / L, glucose 30-50 g / L, with the balance being deionized water.

[0049] Preferably, the fermentation substrate, by its total volume, comprises the following components in the following amounts: yeast extract 0.5 g / L, peptone 0.5 g / L, glycine 2 g / L, adenine 2 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.25 g / L, glucose 40 g / L, with the balance being deionized water.

[0050] In some embodiments, the step of mixing, dispersing and culturing Cordyceps militaris mother culture with liquid culture medium to obtain high-density Cordyceps militaris seed liquid includes at least one of crushing, grinding, shearing and homogenizing treatments.

[0051] In the seed culture preparation stage, the Cordyceps militaris mother culture is physically dispersed through methods such as crushing, grinding, shearing, or homogenization to thoroughly break down the mycelial clusters, thereby creating numerous uniform mycelial growth points in the liquid culture medium. This effectively increases the cell density and activity of the seed culture, providing a sufficient quantity and vigorous microbial community for subsequent fermentation. Once this high-quality seed culture is introduced into the fermenter, it can rapidly adapt to the environment, multiply quickly, effectively shorten the fermentation cycle, and promote earlier and more efficient shift of microbial metabolism towards the synthesis of active substances such as cordycepin. This physical dispersion strategy enhances fermentation efficiency from the source, laying a solid foundation for achieving high-yield, highly antioxidant products and a low-cost, green industrialization of the entire production process.

[0052] In some embodiments, the steps of inactivating, centrifuging, freezing and thawing, filtering, re-inactivating and preservativeing ​​the mixed fermentation broth to obtain Cordyceps militaris fermentation products include freezing and thawing, with the freezing temperature being less than or equal to -20°C and the freezing time being greater than or equal to 24 hours.

[0053] In the post-processing stage, a deep freeze-thaw process with a freezing temperature ≤-20℃ and a time ≥24 hours is adopted. By utilizing the phase transformation stress during the aqueous phase crystallization and melting process, the colloidal stability and spatial conformation of macromolecular polysaccharides and proteins are effectively destroyed, causing them to fully aggregate and precipitate. As a core link in the multi-physics purification technology, this process efficiently removes the main impurities that cause the fermentation broth to become viscous and turbid without introducing any organic solvents or chemical reagents. This effectively reduces the load on subsequent membrane filtration. This purely physical impurity removal method not only completely preserves the concentration and antioxidant capacity of small molecule active substances such as cordycepin, but also further reduces production costs and environmental pressure by avoiding the use of expensive or high-risk chemical reagents. This provides a reliable guarantee for achieving high clarity and high stability of the final product and the green industrialization of the entire production process.

[0054] As an example, inactivation includes high-temperature sterilization of the mixed fermentation broth at a temperature of 60°C to 115°C (e.g., 60°C, 95°C, 105°C, 115°C, or any range of the above values) and a sterilization time of 5 min to 60 min (e.g., 5 min, 10 min, 20 min, 30 min, 60 min, or any range of the above values).

[0055] As an example, centrifugation includes centrifuging the inactivated mixed fermentation broth to remove precipitate, with a centrifugation speed of 7500 rpm to 8000 rpm (e.g., 7500 rpm, 7600 rpm, 7700 rpm, 7800 rpm, 7900 rpm, 8000 rpm, or any range of the above values), and a centrifugation time of 8 min to 12 min (e.g., 8 min, 9 min, 10 min, 11 min, 12 min, or any range of the above values).

[0056] As an example, freeze-thaw involves fully freezing the centrifuged mixed fermentation broth at a temperature less than or equal to -20°C for a time greater than or equal to 24 hours; then thawing it at room temperature (20°C to 30°C, preferably 25°C) to precipitate large molecules with low solubility, which can be removed by further centrifugation (7500 rpm to 8000 rpm for 8 to 12 minutes; preferably 8000 rpm for 10 minutes).

[0057] As an example, filtration includes ceramic membrane filtration of the mixed fermentation broth after freeze-thaw treatment, with the aim of removing large molecular organic matter.

[0058] As an example, the filtration pore size of the ceramic membrane is 50nm~200nm (such as 50nm, 100nm, 200nm, or any range of the above values), preferably 50nm.

[0059] As an example, re-inactivation includes high-temperature sterilization of the filtered mixed fermentation broth at a temperature of 60°C to 115°C (e.g., 60°C, 95°C, 105°C, 115°C, or any range of the above values) and a sterilization time of 5 min to 60 min (e.g., 5 min, 10 min, 20 min, 30 min, 60 min, or any range of the above values).

[0060] In some embodiments, the step of inoculating high-density Cordyceps militaris seed culture into a fermentation medium for fermentation further includes the step of supplementing a carbon source and / or a precursor; the carbon source includes at least one of glucose, fructose, galactose, sucrose, maltose, lactose, and starch; the precursor includes at least one of adenine, guanine, and hypoxanthine.

[0061] In some embodiments, the volume of the precursor added is 5% to 10% of the volume of the fermentation medium (e.g., 5%, 6%, 7%, 8%, 9%, 10%, or any range of the above values), preferably 8%.

[0062] During fermentation, the addition of carbon sources such as glucose and precursors such as adenine provides ample nutrients for the efficient metabolism of Cordyceps militaris. The timely replenishment of carbon sources maintains the vigorous life activities and energy supply of the fungi, while the addition of precursors such as adenine directly provides the molecular framework for the biosynthesis of cordycepin, thereby significantly improving the yield of the target active product and the antioxidant capacity of the fermentation filtrate.

[0063] In some implementations, adding a carbon source refers to adjusting the concentration of glucose in the system to be no less than 5 g / L (such as 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L, or any range of the above values).

[0064] In some implementations, preservation treatment refers to adding preservatives to the fermentation filtrate.

[0065] As an example, the preservative may be at least one of 1,2-pentanediol, 1,2-hexanediol, and p-hydroxyacetophenone.

[0066] As an example, the preservative content in the Cordyceps militaris fermentation filtrate is 1% to 8% by mass (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or any range of the above values).

[0067] As an example, the mass content of 1,2-pentanediol in the Cordyceps militaris fermentation filtrate is 1% to 5% (e.g., 1%, 2%, 3%, 4%, 5%, or any range of the above values).

[0068] As an example, the mass content of 1,2-hexanediol in the Cordyceps militaris fermentation filtrate is 1% to 2% (e.g., 1%, 1.5%, 2%, or any range of the above values).

[0069] As an example, the mass content of p-hydroxyacetophenone in the Cordyceps militaris fermentation filtrate is 0.1% to 0.3% (e.g., 0.1%, 0.2%, 0.3%, or any range of the above values).

[0070] The second aspect of this application provides a Cordyceps militaris fermentation filtrate, which is prepared according to the preparation method described in the first aspect.

[0071] In some embodiments, the cordycepin content in the Cordyceps militaris fermentation filtrate is greater than or equal to 0.7 g / L (e.g., 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, or any range of the above values).

[0072] The third aspect of the embodiments of this application provides the application of the Cordyceps militaris fermentation filtrate described in the second aspect in the preparation of cosmetics, health products or active pharmaceutical ingredients.

[0073] As an example, cosmetics can be formulated into various forms such as moisturizing toner (for gently hydrating the skin), nourishing toner (to replenish the skin's nutrients), nourishing cream (for deep nourishment), massage cream (for massage to promote absorption), serum (with high concentration of active ingredients), eye cream (for eye care), cleansing cream (for gentle cleansing), cleansing foam (for rich foam cleansing), cleansing water (for refreshing cleansing), face mask (for intensive care), spray (for convenient moisturizing), or powder (for easy carrying and use).

[0074] This diverse range of dosage forms allows the cosmetic compositions of the present invention to be better suited to different skin types and usage scenarios. For example, oily skin may prefer a refreshing lotion or spray, while dry skin may benefit from a nourishing cream or massage cream. Furthermore, the different dosage forms facilitate product packaging and use; for instance, powders are convenient for travel, while sprays can be used for large-area skin moisturizing.

[0075] Optionally, cosmetics may also include excipients, which include at least one of the following: fragrances, dyes, emulsifiers, stabilizers, lubricants, solvents, humectants, emollients, wetting agents, film-forming agents, UV absorbers, essential oils, vitamins, trace metals, anti-irritants, antimicrobial agents, antioxidants, chelating agents, preservatives, pH adjusters, skin conditioning agents, thickeners, and organosilicon compounds.

[0076] Example

[0077] The following embodiments describe the disclosure of this application in more detail. These embodiments are for illustrative purposes only, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0078] The Cordyceps militaris used in the embodiments of this application ( Cordyceps malitaris Purchased from Beijing Huayan Shijia Quality Inspection Technology Co., Ltd.; Product number: HYCC54871.

[0079] The sources of some of the reagents used in Examples 1-8 and Comparative Examples 1-12 are as follows:

[0080] 1. Tween-80 was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0081] 2. Sophorolipids were purchased from Shanghai Baihaobo Biotechnology Co., Ltd.

[0082] 3. Yeast extract powder was purchased from Angel Yeast Co., Ltd.

[0083] 4. Peptone was purchased from Angel Yeast Co., Ltd.

[0084] 5. Glycine was purchased from Shanghai Titan Technology Co., Ltd.

[0085] 6. Adenine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0086] 7. Dipotassium hydrogen phosphate was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0087] 8. Magnesium sulfate heptahydrate was purchased from Shanghai Titan Technology Co., Ltd.

[0088] 9. Glucose was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0089] 10. Sodium hydroxide was purchased from Shanghai Titan Technology Co., Ltd.

[0090] 11. The total antioxidant capacity assay kit (ABTS microplate method) was purchased from Shanghai Titan Technology Co., Ltd.

[0091] 12. The DPPH free radical scavenging rate assay kit (microplate method) was purchased from Shanghai Titan Technology Co., Ltd.

[0092] Example 1

[0093] (1) Preparation of high-density Cordyceps militaris seed liquid

[0094] Take a 3.0–3.5 cm diameter Cordyceps militaris agar plate culture and place it in a sterile immersion cup. Add 30 mL of liquid culture medium and mechanically break it up for 1 minute to ensure thorough mycelial dispersion. Then transfer the mixture to a 500 mL wide-mouth Erlenmeyer flask (200 mL volume) and incubate at 25°C and 230 rpm in the dark on a shaker for 3 days. Perform three replicates. After incubation, take 20 mL of sample from each group, centrifuge at 9000 rpm for 10 minutes, collect the mycelial precipitate, and dry it at 60°C to constant weight. Measure the dry weight of the mycelium. Simultaneously, serially dilute the sample with sterile water to 10⁻⁶. 7 times (specifically 10) 4 times, 10 5 times, 10 6 times, 10 7 (times), take 10 respectively 4 Up to 10 7 100 µL of the diluted solution was spread onto a PDA plate and incubated at 25°C for 3 days. The viable count was 7.33 × 10⁻⁶. 7 cfu / mL.

[0095] The liquid culture medium formula, based on the total volume of the liquid culture medium, is as follows: 20 g / L glucose, 2 g / L potato starch, 2 g / L yeast extract, 2 g / L peptone, 1 g / L dipotassium hydrogen phosphate, and 0.5 g / L magnesium sulfate heptahydrate.

[0096] (2) Cordycepin hyper-enrichment induced fermentation process

[0097] High-density Cordyceps militaris seed liquid (viable count 7.33 × 10⁻⁶) 7 Yeast extract (cfu / mL) was inoculated at a 5% inoculum into a 5 L fermenter (3.5 L of liquid volume). Based on the total volume of the fermentation medium, the composition of the fermentation medium was: 0.5 g / L yeast extract, 0.5 g / L peptone, 2 g / L glycine, 2 g / L adenine, 0.5 g / L dipotassium hydrogen phosphate, 0.25 g / L magnesium sulfate heptahydrate, and 40 g / L glucose. Fermentation conditions were: temperature 27℃, rotation speed 200-700 rpm, aeration rate 1 vvm, dissolved oxygen ≥40%, pH not lower than 5.0. Glucose was added in stages before 140 h of fermentation, and the glucose concentration was maintained at ≥5 g / L throughout the process. Fed-feed fermentation was carried out for 7 days. Tween 80 (350 mg) was added at 72 h of fermentation, and sophorolipid (175 mg) was added at 120 h. The total amount of Tween 80 and sophorolipid added was 0.15 mg / mL based on the volume of the fermentation medium. Daily sampling and monitoring of mycelial dry weight and cordycepin yield (see schematic diagram of changes in mycelial and cordycepin yield during fermentation). Figure 1), glucose residual amount, and supplement glucose appropriately (control the glucose concentration in the system ≥5 g / L).

[0098] (3) Multiphysics field coupling purification and post-processing

[0099] After fermentation, post-processing was performed, including sterilization at 105℃ for 20 min, centrifugation at 8000 rpm for 10 min to collect the supernatant, freezing at -20℃ for 24 h, thawing at room temperature, centrifugation again, and then filtration through a 50 nm ceramic membrane to obtain the filtered fermentation filtrate. The filtered fermentation filtrate was sterilized again at 105℃ for 20 min, cooled to room temperature, and then subjected to preservation treatment to obtain Cordyceps militaris fermentation filtrate. Based on the total mass of Cordyceps militaris fermentation filtrate, the preservation system included 4% 1,2-pentanediol, 1% 1,2-hexanediol, and 0.2% p-hydroxyacetophenone.

[0100] Examples 2 to 8

[0101] The methods for preparing Cordyceps militaris fermentation filtrate in Examples 2-8 are basically the same as those in Example 1, except that the viable cell count in the Cordyceps militaris seed liquid, inoculation volume, nitrogen source weight in the culture medium, time and amount of Tween 80 and sophorolipid added, fermentation days, and freeze-thaw conditions have been adjusted within the reasonable range provided in this application. Table 1 shows the key parameters of Examples 2-8; parameters not shown in Table 1 are the same as those in Example 1.

[0102] Comparative Example 1

[0103] The only difference between Comparative Example 1 and Example 1 is that the viable Cordyceps militaris cell density in the Cordyceps militaris seed solution prepared in Comparative Example 1 is low, only 1.10 × 10⁻⁶. 6 cfu / mL.

[0104] Comparative Example 2

[0105] The only difference between Comparative Example 2 and Example 1 is that the inoculation volume of the high-density Cordyceps militaris seed liquid in Comparative Example 2 is only 2% of the fermentation medium volume.

[0106] Comparative Example 3

[0107] The only difference between Comparative Example 3 and Example 1 is that Tween 80 or sophorolipid was not added during the fermentation process.

[0108] Comparative Example 4

[0109] The only difference between Comparative Example 4 and Example 1 is that the total amount of Tween 80 and sophorolipid added during fermentation was 0.4 mg / mL (based on the total volume of the fermentation medium).

[0110] Comparative Example 5

[0111] The only difference between Comparative Example 5 and Example 1 is that Tween 80 and sophorolipid were not added at 72h and 120h respectively during the fermentation process, but all 0.15mg / mL Tween 80 (based on the total volume of the fermentation medium) was added at 72h.

[0112] Comparative Example 6

[0113] The only difference between Comparative Example 6 and Example 1 is that Tween 80 and sophorolipid were not added at 72h and 120h respectively during the fermentation process, but all 0.15mg / mL of sophorolipid (based on the total volume of the fermentation medium) was added at 120h.

[0114] Comparative Example 7

[0115] The only difference between Comparative Example 7 and Example 1 is that the mass ratio of Tween 80 and sophorolipid added during the fermentation process is 1:1.

[0116] Comparative Example 8

[0117] The only difference between Comparative Example 8 and Example 1 is that the mass ratio of Tween 80 and sophorolipid added during the fermentation process is 3:1.

[0118] Comparative Example 9

[0119] The only difference between Comparative Example 9 and Example 1 is that Tween 80 was added 36 hours after the start of fermentation, which is the early stage of the fermentation process, i.e. the initial stage of the logarithmic phase.

[0120] Comparative Example 10

[0121] The only difference between Comparative Example 10 and Example 1 is that Tween 80 and sophorolipid were added to the culture medium together 72 hours after the start of fermentation.

[0122] Comparative Example 11

[0123] The only difference between Comparative Example 11 and Example 1 is that the sophorolipid was added 108 hours after the start of fermentation, when the strain was nearing its death phase.

[0124] Comparative Example 12

[0125] The only difference between Comparative Example 12 and Example 1 is that the freeze-thaw step was omitted in the purification and post-treatment after fermentation.

[0126] Table 1 shows the key parameters of Comparative Examples 1 to 12. Parameters not shown in Table 1 are the same as those in Example 1.

[0127] Table 1 Key parameters of Examples 1-8 and Comparative Examples 1-12

[0128]

[0129] Performance testing

[0130] The performance of the Cordyceps militaris fermentation filtrates prepared in Examples 1-8 and Comparative Examples 1-12 was tested using the following methods:

[0131] (1) Appearance:

[0132] Observe the color and clarity of the prepared Cordyceps militaris fermentation filtrate.

[0133] (2) Viscosity:

[0134] Viscosity was measured using an AMETEK Brookfield DV2TLVTJ0 viscometer. Before testing, the rotor was removed, the instrument was leveled and zeroed, a suitable rotor was immersed below the surface of the sample liquid, the rotation speed was adjusted to an appropriate value, and the viscosity value was read and recorded.

[0135] (3) Turbidity (OD) 600 ):

[0136] Turbidity was measured using a Thermo Scientific Multiskan SkyHigh microplate reader, with OD values ​​as the metric. 600 The value represents the turbidity of the sample. The specific method is as follows: First, measure the absorbance of a blank 96-well plate at a wavelength of 600 nm and record it as A. 空白 Subsequently, 200 μL of the sample to be tested was added to the well, and the absorbance was measured again at a wavelength of 600 nm, recorded as A. 测定 The final sample turbidity is calculated according to formula A. 测定 -A 空白 calculate.

[0137] (4) Cordycepin content (g / L):

[0138] A Watershes Arc HPLC system equipped with a 2998 PDA detector and a Waters XBridge C18 column was used. Methanol (phase A) and water (phase B) were used as the mobile phase in a 15:85 ratio, and the flow rate was set to 1 mL / min. A cordycepin standard curve was prepared before detection using cordycepin standards (see the HPLC chromatogram of the cordycepin standards). Figure 2A series of standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 g / L were prepared with ultrapure water. After filtration through a 0.22 μm filter membrane, the peak areas were measured by HPLC, and the peak areas were recorded. A standard curve was plotted based on this (the standard curve for cordycepin is shown in Figure 3). The fermentation broth to be tested was filtered through a 0.22 μm filter membrane, and the peak areas were measured under the same HPLC conditions. The cordycepin content was calculated based on the standard curve.

[0139] (5) Antioxidant capacity:

[0140] (5-1) DPPH free radical scavenging ability: DPPH was dissolved in 95% ethanol to prepare a 0.2 mM DPPH working solution (prepared fresh for each use). Simultaneously, a 1 g / L vitamin C (Vc) stock solution was prepared with deionized water and further diluted to 10, 20, 30, and 40 mg / L standard solutions for plotting standard curves (see DPPH free radical scavenging rate standard curve). Figure 4 During the assay, 50 μL of the test solution and 150 μL of DPPH working solution were added to a 96-well plate for the experimental group; 50 μL of the test solution and 150 μL of 95% ethanol were added to the background color group; and the two control groups were 50 μL of deionized water and 150 μL of DPPH working solution, and 50 μL of deionized water and 150 μL of 95% ethanol, respectively. After reacting at room temperature in the dark for 10 min, the absorbance of each group was measured at 515 nm using a microplate reader and recorded as A1 (experimental group), A2 (background color group), A3 (DPPH control), and A4 (ethanol control), respectively. The DPPH free radical scavenging rate was calculated as follows: Scavenging rate (%) = [(A3-A4)-(A1-A2)] / (A3-A4)×100. A standard curve was plotted based on the scavenging rate of the Vc standard solution. The scavenging rate of the test sample was then converted into an equivalent Vc concentration (μg / mL) to represent its DPPH free radical scavenging ability.

[0141] (5-2) ABTS free radical scavenging ability: An equal volume of ABTS solution and oxidant was mixed to prepare an ABTS stock solution, which was then allowed to stand at room temperature in the dark for 12-16 h. Before use, the solution was diluted 30 times with deionized water to obtain the ABTS working solution. A 10 mM Trolox standard solution (6-hydroxy-2,5,7,8-tetramethyltryptane-2-carboxylic acid, a water-soluble vitamin E derivative) was diluted with deionized water to a series of concentrations of 0.05, 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM. 5 μL of each concentration of Trolox solution was placed in a 96-well plate, and 200 μL of ABTS working solution was added. The reaction was carried out at room temperature in the dark for 2 min, and the absorbance was measured at 734 nm using a microplate reader. This absorbance was recorded as A. XSeparately, take 5 μL of deionized water and 200 μL of ABTS working solution, and perform the same operation, measuring the absorbance as A0. The formula for calculating the ABTS free radical scavenging rate is: Scavenging rate (%) = (A0 - A X ) / A0×100. A standard curve of Trolox's ABTS radical scavenging rate was plotted based on the concentrations and scavenging rates of various Trolox products (see [link to Trolox standard curve]). Figure 5 The scavenging rate of the sample is converted into the equivalent Trolox concentration (mM) to represent its ABTS free radical scavenging ability.

[0142] (6) Electrical conductivity:

[0143] The pH conductivity was measured using a Mettler Toledo FE38 pH conductivity meter.

[0144] The performance test results of the Cordyceps militaris fermentation filtrate prepared in Examples 1-8 and Comparative Examples 1-12 are shown in Table 2:

[0145] Table 2 Performance Test Results

[0146]

[0147] Stability test

[0148] (1) Raw material stability:

[0149] The Cordyceps militaris fermentation filtrates prepared in Examples 1-8 (the Cordyceps militaris fermentation filtrate prepared in Comparative Examples 1-11 had low cordycepin content and little application value; the Cordyceps militaris fermentation filtrate prepared in Comparative Example 12 showed precipitation after being left at room temperature for one week, indicating obvious stability problems, so no raw material stability test was conducted) were separately packaged into transparent plastic bottles and placed under light, room temperature (about 25°C), 4°C, -20°C, 48°C and high and low temperature cycling (-20°C to 48°C, cycled once every 24 hours) conditions for 3 months to evaluate their stability indicators such as appearance, odor and clarity.

[0150] The results of the raw material stability study are shown in Table 3:

[0151] Table 3 Results of raw material stability study

[0152]

[0153] (2) Formulation stability:

[0154] The Cordyceps militaris fermentation filtrate prepared in Examples 1-8 was used as the active ingredient and added to the base emulsion formulation at a mass percentage of 3%. After thorough mixing, a test sample with an initial white emulsion state was obtained. This sample was then aliquoted and subjected to light, room temperature (approximately 25°C), 4°C, -20°C, 48°C, and high-low temperature cycling (-20°C to 48°C, cycled once every 24 hours) for two months to evaluate its stability indicators such as appearance, odor, and color, and to assess its stability as a component of a cosmetic formulation.

[0155] Based on the total mass of the base emulsion system, the formulations of each component in the base emulsion are shown in Table 4.

[0156] Table 4. Composition of Basic Emulsion Formulation

[0157]

[0158] The results of the formulation stability study are shown in Table 5:

[0159] Table 5 Results of Formulation Stability Study

[0160]

[0161] Based on the performance testing and stability evaluation data, we can conclude that:

[0162] Examples 1-8 describe the preparation of Cordyceps militaris fermentation filtrate using the method provided in this application. High-density liquid microbial inoculum is used for fermentation, effectively increasing the biomass and growth point density at the fermentation start point, enabling the cells to quickly overcome the lag phase, shorten the logarithmic growth phase, enter the stationary phase earlier, and efficiently synthesize active ingredients such as cordycepin and polysaccharides. Based on the characteristics of the cell growth and product synthesis cycle during Cordyceps militaris fermentation, a staged addition strategy is adopted. Tween 80 is added in the middle and late stages of the logarithmic growth phase, and sophorolipid is added during the stationary phase, with the addition ratio controlled within a reasonable range. Tween 80 and sophorolipid synergistically disrupt cell walls and promote release, significantly increasing the release of intracellular cordycepin and polysaccharides, enhancing the target efficacy of the fermentation filtrate, while also preventing the filtrate from becoming too viscous or forming foam, and improving dissolved oxygen efficiency. After fermentation, multi-physics field coupling purification is used to prepare a clear, transparent, yellow Cordyceps militaris fermentation filtrate without introducing any exogenous reagents. Testing showed that the viscosity and turbidity of the Cordyceps militaris fermentation filtrate prepared in Examples 1 to 8 were within an optimal range, and the appearance was clear and transparent, making it suitable as a raw material for use in cosmetics. The fermentation cycle was only 6 to 8 days, and the cordycepin content reached 0.85 to 0.90 g / L, exhibiting significant antioxidant effects. Furthermore, the raw material stability was investigated for 3 months under light, room temperature, 4℃, -20℃, 48℃ and high and low temperature cycling conditions, and the appearance, odor, and clarity remained stable.

[0163] In Examples 1-6, the total nitrogen source in the fermentation medium was controlled to be below 5 g / L, resulting in Cordyceps militaris fermentation filtrate with a conductivity ranging from 1000 to 3000 μS / cm. Stability tests showed that these filtrates exhibited good stability when applied to cosmetics. However, in Examples 7-8, the total nitrogen source in the fermentation medium exceeded 5 g / L, leading to an abnormally high conductivity in the resulting Cordyceps militaris fermentation filtrate. This suggests that the high nitrogen concentration induced the production of highly conductive metabolites during fermentation. However, the use of highly conductive raw materials in cosmetics can easily disrupt the ion balance of the formulation, potentially causing instability in the emulsion system. Stability testing results showed that under accelerated conditions such as light exposure or high / low temperatures, cosmetic compositions containing Cordyceps militaris fermentation filtrate from Examples 7-8 exhibited precipitation or stratification, while those containing Cordyceps militaris fermentation filtrate from Examples 1-6 showed good stability. This requires that the Cordyceps militaris fermentation filtrate from Examples 7 and 8 be further optimized for cosmetic formulation applications by improving the emulsification system or adding anti-ionic stabilizers. Therefore, from the perspective of formulation application, controlling the total amount of nitrogen source added to the fermentation medium to below 5 g / L is a preferred option, and more preferably 2.3 to 3.7 g / L.

[0164] The comparison between Comparative Examples 1-2 and Example 1 illustrates the impact of high-density Cordyceps militaris liquid inoculum on the technical effects of this application. The Cordyceps militaris seed liquid prepared in Comparative Example 1 had a low density of viable Cordyceps militaris cells, and the inoculum amount in Comparative Example 2 was insufficient. Therefore, the high-density liquid inoculum fermentation effect was not achieved, and the biomass and growth point density at the fermentation starting point could not be increased. Simultaneously, due to the low density of viable Cordyceps militaris cells in the fermentation system, the addition of Tween 80 and sophorolipids was relatively excessive, leading to premature lysis of some cells, which in turn affected the continuous synthesis of the product. Therefore, the cordycepin content and antioxidant efficacy in the Cordyceps militaris fermentation filtrate prepared in Comparative Examples 1 and 2 were significantly lower than those in Examples 1-8.

[0165] The comparison between Comparative Examples 3-11 and Example 1 illustrates the effect of staged addition of Tween 80 and sophorolipids during fermentation on the technical effects of this application. Comparative Example 3, without the addition of Tween 80 or sophorolipids, showed significantly lower cordycepin yield and antioxidant capacity in the fermentation filtrate. Comparative Example 4, with its excessively high total amount of Tween 80 and sophorolipids, may have caused premature cell lysis and death, negatively impacting the continuous synthesis of the product and thus adversely affecting cordycepin yield and the antioxidant efficacy of the fermentation filtrate. Comparative Examples 5-8, while maintaining the same total amount, varied the ratio of Tween 80 to sophorolipids: Comparative Example 5 only added Tween 80, Comparative Example 6 only added sophorolipids, and the ratios in Comparative Examples 7 and 8 were unreasonable. However, excessive addition of Tween 80 can cause excessive damage to the cell membrane, and excessive addition of sophorolipids can trigger partial cell lysis, thus terminating the metabolism of some cells. Neither can effectively increase cordycepin content and antioxidant efficacy, nor is it conducive to the properties of the prepared Cordyceps militaris fermentation filtrate. Comparative Examples 9-11 varied the timing of Tween 80 and sophorolipid addition: Comparative Example 9 added Tween 80 at an earlier stage (early logarithmic phase), when the cells had not yet accumulated sufficient product, resulting in a weak effect of Tween 80 in promoting product release and causing significant foaming in the fermentation system, which was detrimental to cell growth and metabolism. Comparative Example 10 added Tween 80 and sophorolipid simultaneously in the middle to late logarithmic phase of cell growth. Adding too much surfactant at once not only failed to produce any synergistic effect but also triggered cell lysis, leading to the cessation of metabolism in some cells and having a negative impact on cordycepin content and antioxidant efficacy. Comparative Example 11 added sophorolipid not during the stable growth phase but late into the near-decay phase; even with the addition of sophorolipid, it was impossible to significantly improve cordycepin content and antioxidant efficacy. Therefore, based on the characteristics of the cell growth and product synthesis cycle during Cordyceps militaris fermentation, the phased addition of Tween 80 and sophorolipid is crucial to achieving the technical effects of this application.

[0166] The comparison between Comparative Example 12 and Example 1 illustrates the impact of the purification step after fermentation on the technical effect of this application. Comparative Example 12 omitted the freeze-thaw step, and the prepared Cordyceps militaris fermentation filtrate showed obvious flocculent precipitate after one week of standing, making it unsuitable for cosmetic applications.

[0167] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing Cordyceps militaris fermentation filtrate, characterized in that, include: The mother culture of Cordyceps militaris was mixed, dispersed, and cultured with liquid culture medium to obtain a high-density Cordyceps militaris seed solution. The viable count of Cordyceps militaris in the high-density Cordyceps militaris seed solution was (7.0~7.5)×10⁻¹⁰. 7 cfu / mL; The high-density Cordyceps militaris seed culture was inoculated into a fermentation medium for fermentation. The inoculation volume of the high-density Cordyceps militaris seed culture was 4.5% to 5.5% of the fermentation medium volume. During fermentation, Tween 80 and sophorolipids were added to the fermentation medium in stages based on the microbial growth curve: Tween 80 was added in the middle and late stages of the logarithmic growth phase, which was 70 to 74 hours after the start of fermentation; sophorolipids were added in the stable growth phase, which was 118 to 122 hours after the start of fermentation. After fermentation, a mixed fermentation broth was obtained. The total amount of Tween 80 and sophorolipids added was 0.05 mg / mL to 0.2 mg / mL based on the volume of the fermentation medium, and the mass ratio of Tween 80 to sophorolipids was (1.5 to 2.5):

1. The mixed fermentation broth was subjected to inactivation, centrifugation, freeze-thaw, filtration, further inactivation, and preservation treatment to obtain Cordyceps militaris fermentation filtrate. The fermentation medium includes a carbon source, a nitrogen source, a precursor, and inorganic salts; the total nitrogen source content is less than 5 g / L based on the volume of the fermentation medium.

2. The method for preparing Cordyceps militaris fermentation filtrate according to claim 1, characterized in that, The total fermentation time is 6 to 8 days.

3. The method for preparing Cordyceps militaris fermentation filtrate according to claim 1, characterized in that, The nitrogen source includes one or more of yeast extract, peptone, and glycine.

4. The method for preparing Cordyceps militaris fermentation filtrate according to claim 3, characterized in that, The total nitrogen source content is 2.3~3.7 g / L based on the volume of the fermentation medium.

5. The method for preparing Cordyceps militaris fermentation filtrate according to claim 4, characterized in that, The fermentation medium comprises: yeast extract 0.4~0.6 g / L, peptone 0.4~0.6 g / L, glycine 1.5~2.5 g / L, adenine 1.8~2.2 g / L, dipotassium hydrogen phosphate 0.4~0.6 g / L, magnesium sulfate heptahydrate 0.2~0.3 g / L, and glucose 30~50 g / L.

6. The method for preparing Cordyceps militaris fermentation filtrate according to claim 1, characterized in that, In the step of mixing, dispersing and culturing Cordyceps militaris mother culture with liquid culture medium to obtain high-density Cordyceps militaris seed liquid, the dispersion includes at least one of crushing, grinding, shearing and homogenization treatment.

7. The method for preparing Cordyceps militaris fermentation filtrate according to claim 1, characterized in that, In the step of inactivating, centrifuging, freezing and thawing, filtering, re-inactivating and preservativeing ​​the mixed fermentation broth to obtain Cordyceps militaris fermentation filtrate, the freezing and thawing includes freezing and thawing, the freezing temperature is less than or equal to -20°C, and the freezing time is greater than or equal to 24 hours.

8. The method for preparing Cordyceps militaris fermentation filtrate according to claim 1, characterized in that, The step of inoculating the high-density Cordyceps militaris seed liquid into a fermentation medium for fermentation also includes the step of supplementing a carbon source and / or a precursor.

9. A Cordyceps militaris fermentation filtrate, characterized in that, The Cordyceps militaris fermentation filtrate is prepared by the method according to any one of claims 1-8.

10. The use of the Cordyceps militaris fermentation filtrate according to claim 9 in the preparation of cosmetics, health products or active pharmaceutical ingredients.

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