Cordyceps sinensis fermentation filtrate as well as preparation method and application thereof

Abstract

The invention relates to cordyceps sinensis fermentation filtrate as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) mixing cordyceps sinensis powder with an alcoholic solution, carrying out ultrasonic extraction, centrifuging to take supernate, and drying to obtain a cordyceps sinensis matrix extract; (2) mixing the cordyceps sinensis powder, the cordyceps sinensis matrix extract and a basic fermentation culture medium to obtain a main fermentation culture medium, and inoculating the main fermentation culture medium with a zymophyte solution for fermentation culture to obtain a fermentation solution; and centrifuging the fermentation liquor, filtering, and collecting the filtrate to obtain the cordyceps sinensis fermentation filtrate. By optimizing the fermentation process, the cordyceps sinensis powder and the cordyceps sinensis matrix extract obtained by ultrasonic extraction of the cordyceps sinensis powder are jointly fermented, so that the bioavailability of the cordyceps sinensis can be fully improved, the functional components in the cordyceps sinensis fermentation filtrate are enriched, and the health-care effect of the cordyceps sinensis is improved. The prepared cordyceps sinensis fermentation filtrate has an excellent anti-aging effect.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and particularly relates to a cordyceps sinensis fermentation filtrate, a preparation method and an application thereof. Background Art

[0002] Cordyceps sinensis, a traditional and precious Chinese medicinal material, has a long history of use in medicine. Rich in bioactive ingredients such as nucleosides, polysaccharides, and sterols, it has long garnered significant attention for its immunomodulatory, antioxidant, and anti-fatigue benefits. With the advancement of modern biotechnology, research on Cordyceps sinensis continues to deepen, with the goal of more efficiently utilizing its active ingredients and expanding its application in health supplements, cosmetics, and other fields.

[0003] CN118697671A discloses a small molecule peptide composition obtained by fermenting Cordyceps sinensis. The small molecule peptide composition obtained by fermenting Cordyceps sinensis is a small molecule peptide composition obtained by fermenting Cordyceps sinensis containing polypeptides. The small molecule peptide composition obtained by fermenting Cordyceps sinensis containing polypeptides is obtained by mixing a fermentation product and a lysate obtained by fermenting Cordyceps sinensis with galactose yeast fermentation broth. The mass ratio of the galactose yeast fermentation broth to Cordyceps sinensis is 75-95:2-10. Through the above design, the invention obtains a small molecule peptide composition obtained by fermenting Cordyceps sinensis with good free radical scavenging, antioxidant enzyme activation, and cell repair promotion, and the antioxidant capacity of the composition is further enhanced by fermenting the raw materials with galactose yeast.

[0004] Microorganisms have powerful transformation capabilities. Their fermentation of plant materials can fully release the active ingredients in the plant, increasing their content and reducing their toxicity. This allows for further separation and extraction of these ingredients, making them more bioactive. However, there are few reports on further fermentation of Cordyceps sinensis using microorganisms.

[0005] Therefore, developing a method for preparing Cordyceps sinensis fermentation filtrate and improving the antioxidant and anti-aging effects of Cordyceps sinensis fermentation filtrate have become one of the technical problems that need to be solved urgently. Summary of the Invention

[0006] In response to the shortcomings of the prior art, the present invention aims to provide a Cordyceps sinensis fermentation filtrate, a preparation method, and applications thereof. The Cordyceps sinensis fermentation filtrate prepared by this method has multiple skin care benefits, including the ability to enhance free radical scavenging, maintain skin redox balance, and exhibit excellent anti-aging effects.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a Cordyceps sinensis fermentation filtrate, the preparation method comprising the following steps:

[0009] (1) mixing Cordyceps sinensis powder with an alcohol solution, performing ultrasonic extraction, centrifuging, collecting the supernatant, and drying to obtain a Cordyceps sinensis matrix extract;

[0010] (2) Cordyceps sinensis powder, Cordyceps sinensis matrix extract and basic fermentation medium are mixed to obtain a main fermentation medium, fermentation culture is inoculated into the main fermentation medium for fermentation culture to obtain a fermentation liquid; the fermentation liquid is centrifuged, filtered and the filtrate is collected to obtain a Cordyceps sinensis fermentation filtrate.

[0011] The present invention optimizes the fermentation process and co-fermentes cordyceps sinensis powder and a cordyceps sinensis matrix extract obtained by ultrasonic extraction of the cordyceps sinensis powder, thereby fully improving the bioavailability of cordyceps sinensis and enriching the effective ingredients in the cordyceps sinensis fermentation filtrate. The prepared cordyceps sinensis fermentation filtrate has an excellent anti-aging effect.

[0012] In addition, the present invention creatively discovered that the Cordyceps sinensis fermentation filtrate prepared by using Cordyceps sinensis powder and Cordyceps sinensis matrix extract together has better antioxidant and anti-aging effects than single Cordyceps sinensis powder or single Cordyceps sinensis matrix extract, indicating that Cordyceps sinensis powder and Cordyceps sinensis matrix extract have a significant synergistic effect in improving the anti-aging effect of the product.

[0013] Preferably, the material-liquid ratio of the Cordyceps sinensis powder to the alcohol solution in step (1) is 1:8-1:12 g / mL, for example, it can be 1:8 g / mL, 1:9 g / mL, 1:10 g / mL, 1:11 g / mL, 1:12 g / mL, etc.

[0014] Preferably, the alcohol solution comprises an aqueous solution of ethanol.

[0015] Preferably, the volume percentage of ethanol in the ethanol aqueous solution is 50-70%, for example, it can be 50%, 55%, 60%, 65%, 70%, etc.

[0016] Preferably, the temperature of the ultrasonic extraction in step (1) is 50-70° C., and the time of the ultrasonic extraction is 1-3 h.

[0017] Preferably, the power of the ultrasonic extraction in step (1) is 200-400W, and the frequency of the ultrasonic extraction is 30-50kHz.

[0018] Among them, the specific point values ​​in 50-70℃ can be selected from 50℃, 55℃, 60℃, 65℃, 70℃, etc., the specific point values ​​in 1-3h can be selected from 1h, 1.5h, 2h, 2.5h, 3h, etc., the specific point values ​​in 200-400W can be selected from 200W, 250W, 300W, 350W, 400W, etc., and the specific point values ​​in 30-50kHz can be selected from 30kHz, 35kHz, 40kHz, 45kHz, 50kHz, etc.

[0019] Preferably, the fermentation broth in step (2) comprises a combined fermentation broth of Lactobacillus plantarum and Bifidobacterium bifidum.

[0020] Lactobacillus plantarum is a Gram-positive facultative anaerobic bacillus, commonly found in plant fermented foods. It can degrade the structural polysaccharides and proteins in the cell wall of Cordyceps sinensis and release small molecular active substances; Bifidobacterium bifidum is a Gram-positive anaerobic bacillus that can produce short-chain fatty acids to regulate the microecology, secrete β-galactosidase, arabinosidase, etc., specifically hydrolyze the glycosidic bonds in Cordyceps sinensis polysaccharides, and promote the release of active ingredients such as cordycepic acid and Cordyceps polysaccharides. It can cooperate with facultative anaerobic bacteria (Lactobacillus plantarum) to maintain the anaerobic fermentation system.

[0021] The present invention creatively discovered that during the main fermentation process, the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum has better antioxidant and anti-aging effects than the single fermentation liquid of Lactobacillus plantarum or Bifidobacterium bifidum, indicating that Lactobacillus plantarum and Bifidobacterium bifidum can enhance the fermentation effect and have a significant synergistic effect in improving the anti-aging effect of Cordyceps sinensis fermentation filtrate.

[0022] Preferably, the total inoculation amount of the fermentation bacteria liquid is 10-12% by volume, for example, 10%, 10.5%, 11%, 11.5%, 12%, etc.

[0023] Preferably, the method for preparing the combined fermentation broth of Lactobacillus plantarum and Bifidobacterium bifidum comprises the following steps:

[0024] (i) Lactobacillus plantarum and Bifidobacterium bifidum were inoculated into MRS basal culture medium to obtain a first-level seed solution of Lactobacillus plantarum and a first-level seed solution of Bifidobacterium bifidum, respectively.

[0025] (ii) The first-level seed liquid of Lactobacillus plantarum and the first-level seed liquid of Bifidobacterium bifidum were inoculated into the MRS medium modified with Cordyceps sinensis powder to obtain the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum.

[0026] Preferably, the MRS medium modified with cordyceps powder comprises: 8-12 g / L peptone, 6-10 g / L beef extract powder, 3-5 g / L yeast extract, 18-22 g / L glucose, 50-80 g / L cordyceps powder, 2-4 mL / L olive oil, 0.8-1.2 g / L vitamin B1, 0.3-0.7 g / L L-cysteine, 3-7 g / L sodium acetate, and 1-5 g / L ammonium citrate.

[0027] Among them, the specific point values ​​in 8-12g / L can be selected from 8g / L, 9g / L, 10g / L, 11g / L, 12g / L, etc., the specific point values ​​in 6-10g / L can be selected from 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, etc., the specific point values ​​in 3-5g / L can be selected from 3g / L, 3.5g / L, 4g / L, 4.5g / L, 5g / L, etc., the specific point values ​​in 18-22g / L can be selected from 18g / L, 19g / L, 20g / L, 21g / L, 22g / L, etc., the specific point values ​​in 50-80g / L can be selected from 50g / L, 60g / L, 70g / L, 80g / L, etc., 2-4mL / L The specific point values ​​in 0.8-1.2g / L can be selected from 2mL / L, 2.5mL / L, 3mL / L, 3.5mL / L, 4mL / L, etc. The specific point values ​​in 0.8-1.2g / L can be selected from 0.8g / L, 0.9g / L, 1g / L, 1.1g / L, 1.2g / L, etc. The specific point values ​​in 0.3-0.7g / L can be selected from 0.3g / L, 0.4g / L, 0.5g / L, 0.6g / L, 0.7g / L, etc. The specific point values ​​in 3-7g / L can be selected from 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, etc. The specific point values ​​in 1-5g / L can be selected from 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, etc.

[0028] The invention can further enrich the effective components in the Cordyceps sinensis fermentation filtrate and improve the anti-aging effect of the Cordyceps sinensis fermentation filtrate by adding Cordyceps sinensis powder to the culture medium of the secondary fermentation.

[0029] In addition, by adding vitamin B1 and L-cysteine ​​to the culture medium at the same time, the anti-aging effect of the Cordyceps sinensis fermentation filtrate prepared was significantly higher than that of single vitamin B1 or single L-cysteine, indicating that vitamin B1 and L-cysteine ​​have a significant synergistic effect in improving the anti-aging effect of Cordyceps sinensis fermentation filtrate.

[0030] Preferably, the yeast extract is prepared by a preparation method comprising the following steps:

[0031] The baker's yeast is broken into pieces and mixed with water to form a slurry with a mass concentration of 15-20%, and the pH is adjusted to 7.0-7.5; complex enzyme I is added to enzymolyze the slurry, and then 5'-phosphodiesterase is added to enzymolyze the slurry; the enzymolysis product is centrifuged and filtered to obtain a yeast extract.

[0032] Preferably, the complex enzyme I comprises alkaline protease and flavor protease in a mass ratio of 1:0.5-1:1.

[0033] Preferably, the mass of the complex enzyme I is 0.2-0.5% of the mass of the slurry.

[0034] Preferably, the mass of the 5'-phosphodiester is 0.01-0.03% of the mass of the slurry.

[0035] Among them, the specific point values ​​in 1:0.5-1:1 can be selected from 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, etc., the specific point values ​​in 0.2-0.5% can be selected from 0.2%, 0.3%, 0.4%, 0.5%, etc., and the specific point values ​​in 0.01-0.03% can be selected from 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, etc.

[0036] Preferably, the temperature for enzymatic hydrolysis of the slurry by adding complex enzyme I is 50-60° C. and the time is 3-5 hours.

[0037] Preferably, the temperature for enzymatic hydrolysis of the slurry by adding 5'-phosphodiesterase is 50-70°C and the time is 1-3 hours.

[0038] Among them, the specific point values ​​in 50-60℃ can be selected from 50℃, 53℃, 56℃, 60℃, etc., the specific point values ​​in 3-5h can be selected from 3h, 3.5h, 4h, 4.5h, 5h, etc., the specific point values ​​in 50-70℃ can be selected from 50℃, 55℃, 60℃, 65℃, 70℃, etc., and the specific point values ​​in 1-3h can be selected from 1h, 1.5h, 2h, 2.5h, 3h, etc.

[0039] Preferably, the total inoculation amount of the primary seed solution of Lactobacillus plantarum and the primary seed solution of Bifidobacterium bifidum in step (ii) is 3-7%, for example, 3%, 4%, 5%, 6%, 7%, etc., in terms of volume percentage.

[0040] Preferably, when the primary seed liquid of Lactobacillus plantarum and the primary seed liquid of Bifidobacterium bifidum are inoculated into the MRS medium modified with Cordyceps sinensis powder, the ratio of the viable counts of Lactobacillus plantarum and Bifidobacterium bifidum is 1:3-3:1, for example, it can be 1:3, 2:3, 1:1, 2:1, 3:1, etc.

[0041] Preferably, the basic fermentation medium in step (2) comprises:

[0042] Glucose 25-35 g / L, soy peptone 10-20 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, MgSO4·7H2O 0.3-0.7 g / L, olive oil 1.5-2.5 mL / L.

[0043] Among them, the specific point values ​​in 25-35g / L can be selected from 25g / L, 27g / L, 29g / L, 31g / L, 33g / L, 35g / L, etc., the specific point values ​​in 10-20g / L can be selected from 10g / L, 12g / L, 14g / L, 16g / L, 18g / L, 20g / L, etc., the specific point values ​​in 0.5-1.5g / L can be selected from 0.5g / L, 0.7g / L, 0.9g / L, 1.1g / L, 1.3g / L, 1.5g / L, etc. The specific point values ​​in 0.3-0.7g / L can be selected as 0.3g / L, 0.4g / L, 0.5g / L, 0.6g / L, 0.7g / L, etc. The specific point values ​​in 1.5-2.5mL / L can be selected as 1.5mL / L, 1.7mL / L, 1.9mL / L, 2.1mL / L, 2.3mL / L, 2.5mL / L, etc.

[0044] Preferably, the mass ratio of the Cordyceps sinensis powder to the Cordyceps sinensis matrix extract in step (2) is 2.5:1-4:1, for example, it can be 2.5:1, 3:1, 3.5:1, 4:1, etc.

[0045] Preferably, the total amount of Cordyceps sinensis powder and Cordyceps sinensis matrix extract added to the main fermentation medium is 70-100 g / L, for example, 70 g / L, 80 g / L, 90 g / L, 100 g / L, etc.

[0046] Preferably, the fermentation culture in step (2) includes a primary fermentation and a secondary fermentation performed sequentially.

[0047] The conditions for the primary fermentation are: temperature of 27-29° C., pH value of 6.8-7.2, dissolved oxygen content of 30-50%, and fermentation time of 2-2.5 days.

[0048] The conditions for the secondary fermentation are: temperature of 24-26° C., pH value of 6.8-7.2, dissolved oxygen content of 30-50%, and fermentation time of 5-7 days.

[0049] Among them, the specific point values ​​in 27-29℃ can be selected from 27℃, 27.5℃, 28℃, 28.5℃, 29℃, etc., the specific point values ​​in 24-26℃ can be selected from 24℃, 24.5℃, 25℃, 25.5℃, 26℃, etc., the specific point values ​​in 6.8-7.2 can be selected from 6.8, 6.9, 7, 7.1, 7.2, etc., the specific point values ​​in 30-50% can be selected from 30%, 35%, 40%, 45%, 50%, etc., the specific point values ​​in 2-2.5 days can be selected from 2 days, 2.25 days, 2.5 days, etc., and the specific point values ​​in 5-7 days can be selected from 5 days, 5.5 days, 6 days, 6.5 days, 7 days, etc.

[0050] The role of the primary fermentation is mainly to promote bacterial proliferation and primary metabolism: in the primary fermentation stage (27-29 ℃, 2-2.5 days), the conditions close to the optimal growth temperature of Lactobacillus plantarum (28-30 ℃) can activate the expression of heat shock proteins (HSPs), promote rapid bacterial proliferation, and at the same time, this temperature range is also compatible with the adaptive growth of Bifidobacterium bifidum. In this stage, 30-50% high dissolved oxygen promotes aerobic metabolism, so that the biomass of Lactobacillus plantarum reaches 10 within 48 hours. 9 CFU / mL, a neutral pH environment (6.8-7.2) can avoid premature acidification and inhibit the growth of bifidobacteria, and the bacteria synthesize a large amount of proteases, glycoside hydrolases and energy metabolism-related coenzymes through primary metabolism. The role of the secondary fermentation is to enrich the secondary metabolites of the bacteria. After entering the secondary fermentation stage (24-26°C, 5-7 days), the temperature is lowered to induce a cold stress response, and the expression of cold shock proteins (CSPs) is upregulated to enhance enzyme stability. At the same time, the metabolic rate is reduced to prolong the synthesis period of secondary metabolites, thereby increasing the activity of cordycepin synthase by 30-40%, and inhibiting adenosine deaminase activity to reduce adenosine degradation. The present invention can significantly increase the content of effective ingredients in the Cordyceps sinensis fermentation filtrate by conducting a primary fermentation at 27-29°C and a secondary fermentation at 24-26°C.

[0051] Preferably, the fermentation culture further comprises adding a fermentation promoter I on the 1st to 2nd day of the secondary fermentation.

[0052] Among them, day 1-2 can be day 1, day 1.5, day 2, etc.

[0053] The fermentation promoting agent I comprises glucose and glutamine.

[0054] Glucose is a crucial carbon source in the fermentation system, easily absorbed and utilized by the microorganisms, rapidly promoting their proliferation. It also participates as a metabolic substrate in various biochemical reactions, providing a material basis for the synthesis of active ingredients and helping to increase the accumulation of active ingredients in the fermentation product. Glutamine is a key nitrogen source in the fermentation system, not only meeting the nitrogen requirement for bacterial growth and promoting its proliferation, but also further regulating bacterial metabolic pathways, enhancing microbial metabolic activity, promoting the release and conversion of active ingredients in Cordyceps sinensis, and further increasing the content of active ingredients in the fermentation filtrate.

[0055] The present invention creatively discovered that, during the secondary fermentation process, supplementing glucose and glutamine can further enrich the effective components in the Cordyceps sinensis fermentation filtrate and enhance the anti-wrinkle and anti-aging effects of the Cordyceps sinensis fermentation filtrate.

[0056] Preferably, the mass ratio of glucose to glutamine is 4:1-6:1, for example, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, etc.

[0057] Preferably, the addition amount of the fermentation promoting agent I is 0.5-0.8% of the mass of the main fermentation medium, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, etc.

[0058] Preferably, the fermentation culture further comprises adding a fermentation promoter II on the 3rd to 4th day of the secondary fermentation.

[0059] Among them, day 3-4 can be day 3, day 3.5, day 4, etc.

[0060] The fermentation promoting agent II includes adenylic acid, guanylic acid and arginine.

[0061] Adenosine is a key substance in energy metabolism, providing energy support for microbial proliferation and enhancing the stability of active ingredients in the fermentation system. Guanylate regulates microbial metabolic pathways, aiding the decomposition and conversion of active ingredients in Cordyceps sinensis and enhancing the activity of fermentation products. Arginine provides a nitrogen source for the bacteria, promoting protein synthesis and accelerating bacterial proliferation. Its metabolites also optimize the fermentation environment and enhance the system's antioxidant capacity.

[0062] The present invention creatively discovered that during the secondary fermentation process, the addition of adenylic acid, guanylic acid, adenylic acid and guanylic acid can further enrich the effective components in the Cordyceps sinensis fermentation filtrate, and the three have a significant synergistic effect in improving the antioxidant and anti-aging effects of the Cordyceps sinensis fermentation filtrate.

[0063] Preferably, the mass ratio of adenylate, guanylate and arginine is (0.5-1.5):(0.1-1):(0.1-1).

[0064] Among them, the specific point values ​​in 0.5-1.5 can be selected from 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, etc., and the specific point values ​​in 0.1-1 can be selected from 0.1, 0.3, 0.5, 0.7, 0.9, 0.1, etc.

[0065] Preferably, the addition amount of the fermentation promoter II is 0.1-0.3% of the mass of the main fermentation medium, for example, it can be 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, etc.

[0066] In a second aspect, the present invention provides a Cordyceps sinensis fermentation filtrate prepared by the method for preparing the Cordyceps sinensis fermentation filtrate according to the first aspect.

[0067] In a third aspect, the present invention provides a use of the Cordyceps sinensis fermentation filtrate as described in the second aspect in cosmetics.

[0068] The numerical range described in the present invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] The present invention optimizes the fermentation process and co-fermentes cordyceps sinensis powder and a cordyceps sinensis matrix extract obtained by ultrasonic extraction of the cordyceps sinensis powder, thereby fully improving the bioavailability of cordyceps sinensis and enriching the effective ingredients in the cordyceps sinensis fermentation filtrate. The prepared cordyceps sinensis fermentation filtrate has an excellent anti-aging effect.

[0071] Furthermore, during the main fermentation process, the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum has better antioxidant and anti-aging effects than the single fermentation liquid of Lactobacillus plantarum or Bifidobacterium bifidum, indicating that Lactobacillus plantarum and Bifidobacterium bifidum can enhance the fermentation effect and have a significant synergistic effect in improving the anti-aging effect of Cordyceps sinensis fermentation filtrate.

[0072] Furthermore, by adding vitamin B1 and L-cysteine ​​simultaneously to the MRS culture medium modified with Cordyceps sinensis powder, the anti-aging effect of the Cordyceps sinensis fermentation filtrate prepared was significantly higher than that of single vitamin B1 or single L-cysteine, indicating that vitamin B1 and L-cysteine ​​have a significant synergistic effect in improving the anti-aging effect of Cordyceps sinensis fermentation filtrate.

[0073] Furthermore, during the main fermentation process, the first fermentation is carried out at 27-29° C. and the second fermentation is carried out at 24-26° C., which can significantly increase the content of the effective ingredients in the Cordyceps sinensis fermentation filtrate.

[0074] Furthermore, during the secondary fermentation process, glucose and glutamine are added to further enrich the effective components in the Cordyceps sinensis fermentation filtrate, thereby improving the anti-wrinkle and anti-aging effects of the Cordyceps sinensis fermentation filtrate.

[0075] Furthermore, during the secondary fermentation process, the addition of adenylic acid, guanylic acid, adenylic acid and guanylic acid can further enrich the effective components in the Cordyceps sinensis fermentation filtrate, and the three have a significant synergistic effect in improving the antioxidant and anti-aging effects of the Cordyceps sinensis fermentation filtrate. DETAILED DESCRIPTION

[0076] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0077] The sources of various raw materials in the following specific embodiments are shown in Table 1.

[0078] Table 1

[0079]

[0080]

[0081] The formula of the MRS basal medium involved in the following specific embodiment is: peptone 10g / L, beef extract powder 8g / L, yeast extract 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, triammonium citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.04g / L, Tween-80 1mL / L.

[0082] The Lactobacillus plantarum involved in the following specific embodiments is CICC 23114.

[0083] The Bifidobacterium bifidum involved in the following specific embodiments is CICC 10395.

[0084] The yeast extract involved in the following specific embodiments is prepared in the following manner:

[0085] First, baker's yeast cells are collected by centrifugation, washed 2-3 times with sterile saline or deionized water to remove culture medium residues, and then subjected to cell wall disruption. Water is added to prepare a slurry with a mass concentration of 18% and the pH is adjusted to 7.2. Subsequently, a multi-stage enzymatic hydrolysis process is performed, wherein alkaline protease and flavor protease are first enzymatically hydrolyzed at 55°C and pH 8.0 in a ratio of 1:0.5 (the total amount of enzyme added is 0.3% of the slurry mass), and then nucleic acid is converted with 5'-phosphodiesterase at a mass ratio of 0.02% at 60°C and pH 6.0 for 2 hours. The enzymatic hydrolyzate is inactivated at 90°C and then decolorized and debittered by centrifugation, ceramic membrane filtration, and activated carbon adsorption. Finally, the yeast extract is fractionally concentrated using ultrafiltration-nanofiltration membranes, evaporated at low temperature to a solid content of 40%, and then spray-dried.

[0086] Example 1

[0087] (1) Raw material preparation:

[0088] (1.1) Preparation of Cordyceps sinensis powder: Fresh Cordyceps sinensis was cleaned and dried at 40°C until the moisture content was ≤8%. The powder was obtained by using a jet mill to control the particle size to ≤50 μm.

[0089] (1.2) Preparation of Cordyceps Matrix Extract: Cordyceps powder was mixed with 60% (v / v) ethanol aqueous solution at a solid-liquid ratio of 1:10 g / mL and ultrasonically extracted (ultrasonic extraction temperature: 60°C, time: 2 h, power: 300 W, frequency: 40 kHz). The supernatant was centrifuged at 8000 rpm for 15 min, filtered through a 0.45 μm membrane, and concentrated under reduced pressure (50°C / -0.09 MPa) to remove ethanol. The supernatant was then spray-dried to obtain the Cordyceps Matrix Extract.

[0090] (2) Culture medium preparation:

[0091] The formula of MRS medium modified with Cordyceps sinensis powder is as follows: peptone 10 g / L, beef extract powder 8 g / L, yeast extract 4 g / L, glucose 20 g / L, Cordyceps sinensis powder 70 g / L, olive oil 3 mL / L, vitamin B1 1 g / L, L-cysteine ​​0.5 g / L, sodium acetate 5 g / L, and ammonium citrate 3 g / L.

[0092] The formula of the main fermentation medium is: Cordyceps sinensis powder 70 g / L, Cordyceps sinensis matrix extract 20 g / L, glucose 30 g / L, soy peptone 15 g / L, potassium dihydrogen phosphate 1 g / L, MgSO4·7H2O 0.5 g / L, and olive oil 2 mL / L.

[0093] (3) Preparation of the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum:

[0094] (i) inoculating Lactobacillus plantarum into MRS basal medium, culturing at 37° C. for 24 h, and obtaining a primary seed solution of Lactobacillus plantarum; inoculating Bifidobacterium bifidum into MRS basal medium, culturing at 37° C. for 24 h, and obtaining a primary seed solution of Bifidobacterium bifidum.

[0095] (ii) The viable bacterial count was set to 1×10 9 CFU / mL of the first-level seed solution of Lactobacillus plantarum and the number of viable bacteria was 1×10 9 The first-level seed liquid of Bifidobacterium bifidum with a volume ratio of 3:2 was inoculated into the MRS medium modified with Cordyceps sinensis powder (total inoculation amount was 5%) to obtain the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum.

[0096] (4) Preparation of Cordyceps sinensis fermentation filtrate:

[0097] The main fermentation medium was inoculated with a combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum (inoculation amount was 12%), and a primary fermentation culture was carried out: the fermentation temperature was controlled at 28° C., the pH value was adjusted to 7, the dissolved oxygen content was maintained at 40%, and the fermentation was carried out for 2 days;

[0098] Subsequently, a secondary fermentation was carried out, the fermentation temperature was controlled at 25°C, the pH value was adjusted to 7, the dissolved oxygen content was maintained at 40%, and the secondary fermentation was carried out for 6 days; and on the first day of the secondary fermentation, 0.6% of the mass of the main fermentation medium of fermentation promoter I (glucose and glutamine in a mass ratio of 5:1) was added. On the third day of the secondary fermentation, 0.2% of the mass of the main fermentation medium of fermentation promoter II (adenylate, guanylate and arginine in a mass ratio of 1:0.5:0.5) was added to obtain a fermentation liquid.

[0099] The fermentation broth was cooled to 20°C to terminate the reaction, and cross-flow filtration was performed using a 0.1μm α-Al2O3 membrane assembly under a transmembrane pressure difference of 0.3MPa. The operating temperature was maintained at 25°C and reverse pulse cleaning was performed at 0.6MPa every 30 minutes. The broth was then passed through a 10kDa hollow fiber ultrafiltration membrane (0.4MPa) and then a 1kDa spiral nanofiltration membrane (1.2MPa) for graded concentration. The final filtrate was concentrated under vacuum conditions of 45°C and 0.095MPa. The concentrate was freeze-dried and reconstituted with water to obtain a Cordyceps sinensis fermentation filtrate with a solid content of 30%.

[0100] Example 2

[0101] (1) Raw material preparation:

[0102] (1.1) Preparation of Cordyceps sinensis powder: Fresh Cordyceps sinensis was cleaned and dried at 40°C until the moisture content was ≤8%. The powder was obtained by using a jet mill to control the particle size to ≤50 μm.

[0103] (1.2) Preparation of Cordyceps Matrix Extract: Cordyceps powder was mixed with 50% (v / v) ethanol aqueous solution at a solid-liquid ratio of 1:8 g / mL and ultrasonically extracted (ultrasonic extraction temperature: 50°C, time: 3 h, power: 200 W, frequency: 30 kHz). The supernatant was centrifuged at 7000 rpm for 20 min, filtered through a 0.45 μm membrane, and concentrated under reduced pressure (50°C / -0.09 MPa) to remove ethanol. The supernatant was then spray-dried to obtain the Cordyceps Matrix Extract.

[0104] (2) Culture medium preparation:

[0105] The formula of MRS medium modified with Cordyceps sinensis powder is as follows: peptone 8 g / L, beef extract 6 g / L, yeast extract 3 g / L, glucose 18 g / L, Cordyceps sinensis powder 50 g / L, olive oil 2 mL / L, vitamin B1 0.8 g / L, L-cysteine ​​0.3 g / L, sodium acetate 3 g / L, and ammonium citrate 5 g / L.

[0106] The formula of the main fermentation medium is: Cordyceps sinensis powder 50 g / L, Cordyceps sinensis matrix extract 20 g / L, glucose 25 g / L, soy peptone 10 g / L, potassium dihydrogen phosphate 0.5 g / L, MgSO4·7H2O 0.3 g / L, and olive oil 1.5 mL / L.

[0107] (3) Preparation of the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum:

[0108] (i) inoculating Lactobacillus plantarum into MRS basal medium, culturing at 37° C. for 24 h, and obtaining a primary seed solution of Lactobacillus plantarum; inoculating Bifidobacterium bifidum into MRS basal medium, culturing at 37° C. for 24 h, and obtaining a primary seed solution of Bifidobacterium bifidum.

[0109] (ii) The viable bacterial count was set to 1×10 9 CFU / mL of the first-level seed solution of Lactobacillus plantarum and the number of viable bacteria was 1×10 9 The first-level seed liquid of Bifidobacterium bifidum with a CFU / mL volume ratio of 1:3 was inoculated into the MRS medium modified with Cordyceps sinensis powder (total inoculation amount was 3%) to obtain the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum.

[0110] (4) Preparation of Cordyceps sinensis fermentation filtrate:

[0111] The main fermentation medium was inoculated with a combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum (inoculation amount was 10%), and a primary fermentation culture was performed: the fermentation temperature was controlled at 27° C., the pH value was adjusted to 6.8, the dissolved oxygen content was maintained at 30%, and the fermentation was carried out for 2 days;

[0112] Subsequently, a secondary fermentation was carried out, the fermentation temperature was controlled at 24°C, the pH value was adjusted to 6.8, the dissolved oxygen content was maintained at 30%, and the secondary fermentation was carried out for 5 days; and on the first day of the secondary fermentation, 0.5% of the mass of the main fermentation medium of fermentation promoter I (glucose and glutamine in a mass ratio of 4:1) was added, and on the third day of the secondary fermentation, 0.1% of the mass of the main fermentation medium of fermentation promoter II (adenylate, guanylate and arginine in a mass ratio of 0.5:1:0.1) was added to obtain a fermentation liquid.

[0113] The fermentation broth was cooled to 15°C to terminate the reaction, and cross-flow filtration was performed using a 0.1μm α-Al2O3 membrane assembly under a transmembrane pressure difference of 0.2MPa. The operating temperature was maintained at 25°C and reverse pulse cleaning was performed every 30 minutes at 0.6MPa. The broth was then passed through a 10kDa hollow fiber ultrafiltration membrane (0.3MPa) and then a 1kDa spiral nanofiltration membrane (1MPa) for graded concentration. The final filtrate was concentrated under vacuum conditions of 45°C and 0.095MPa. The concentrate was freeze-dried and reconstituted with water to obtain a Cordyceps sinensis fermentation filtrate with a solid content of 30%.

[0114] Example 3

[0115] (1) Raw material preparation:

[0116] (1.1) Preparation of Cordyceps sinensis powder: Fresh Cordyceps sinensis was cleaned and dried at 40°C until the moisture content was ≤8%. The powder was obtained by using a jet mill to control the particle size to ≤50 μm.

[0117] (1.2) Preparation of Cordyceps Matrix Extract: Cordyceps powder was mixed with 70% (v / v) ethanol aqueous solution at a solid-liquid ratio of 1:12 g / mL and ultrasonically extracted (ultrasonic extraction temperature: 70°C, time: 1 h, power: 400 W, frequency: 50 kHz). The supernatant was centrifuged at 9000 rpm for 12 min, filtered through a 0.45 μm membrane, and concentrated under reduced pressure (50°C / -0.09 MPa) to remove ethanol. The supernatant was then spray-dried to obtain the Cordyceps Matrix Extract.

[0118] (2) Culture medium preparation:

[0119] The formula of MRS medium modified with Cordyceps sinensis powder is as follows: peptone 12 g / L, beef extract powder 10 g / L, yeast extract 5 g / L, glucose 22 g / L, Cordyceps sinensis powder 80 g / L, olive oil 4 mL / L, vitamin B1 1.2 g / L, L-cysteine ​​0.7 g / L, sodium acetate 7 g / L, and ammonium citrate 1 g / L.

[0120] The formula of the main fermentation medium is: Cordyceps sinensis powder 60 g / L, Cordyceps sinensis matrix extract 15 g / L, glucose 35 g / L, soy peptone 20 g / L, potassium dihydrogen phosphate 1.5 g / L, MgSO4·7H2O 0.7 g / L, and olive oil 2.5 mL / L.

[0121] (3) Preparation of the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum:

[0122] (i) inoculating Lactobacillus plantarum into MRS basal medium, culturing at 37° C. for 24 h, and obtaining a primary seed solution of Lactobacillus plantarum; inoculating Bifidobacterium bifidum into MRS basal medium, culturing at 37° C. for 24 h, and obtaining a primary seed solution of Bifidobacterium bifidum.

[0123] (ii) The viable bacterial count was set to 1×10 9 CFU / mL of the first-level seed solution of Lactobacillus plantarum and the number of viable bacteria was 1×10 9 The first-level seed liquid of Bifidobacterium bifidum with a volume ratio of 3:1 was inoculated into the MRS medium modified with Cordyceps sinensis powder (total inoculation amount was 7%) to obtain the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum.

[0124] (4) Preparation of Cordyceps sinensis fermentation filtrate:

[0125] A combined fermentation culture of Lactobacillus plantarum and Bifidobacterium bifidum (inoculation amount of 12%) was inoculated into the main fermentation medium, and a primary fermentation culture was performed: the fermentation temperature was controlled at 29° C., the pH value was adjusted to 7.2, the dissolved oxygen content was maintained at 50%, and the fermentation was carried out for 2.5 days;

[0126] Subsequently, a secondary fermentation was carried out, the fermentation temperature was controlled at 26°C, the pH value was adjusted to 7.2, the dissolved oxygen content was maintained at 50%, and the secondary fermentation was carried out for 7 days; and on the second day of the secondary fermentation, 0.8% of the mass of the main fermentation medium of fermentation promoter I (glucose and glutamine in a mass ratio of 6:1) was added. On the fourth day of the secondary fermentation, 0.3% of the mass of the main fermentation medium of fermentation promoter II (adenylate, guanylate and arginine in a mass ratio of 1:0.1:1) was added to obtain a fermentation liquid.

[0127] The fermentation broth was cooled to 20°C to terminate the reaction, and cross-flow filtration was performed using a 0.1μm α-Al2O3 membrane assembly under a transmembrane pressure difference of 0.4MPa. The operating temperature was maintained at 25°C and reverse pulse cleaning was performed every 30 minutes at 0.6MPa. The broth was then passed through a 10kDa hollow fiber ultrafiltration membrane (0.5MPa) and then a 1kDa spiral nanofiltration membrane (1.5MPa) for graded concentration. The final filtrate was concentrated under vacuum conditions of 45°C and 0.095MPa. The concentrate was freeze-dried and reconstituted with water to obtain a Cordyceps sinensis fermentation filtrate with a solid content of 30%.

[0128] Example 4

[0129] This embodiment provides a method for preparing a Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, in step (2), Cordyceps sinensis powder is not added to the formula of the MRS medium modified with Cordyceps sinensis powder, and the remaining steps are consistent with Example 1.

[0130] Example 5

[0131] This embodiment provides a method for preparing a Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, in step (2), vitamin B1 is not added to the formula of the MRS medium modified with Cordyceps sinensis powder, and accordingly, the amount of L-cysteine ​​added is adjusted to 1.5 g / L. The remaining steps remain the same as in Example 1.

[0132] Example 6

[0133] This embodiment provides a method for preparing a Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, in step (2), L-cysteine ​​is not added to the formula of the MRS medium modified with Cordyceps sinensis powder, and accordingly, the amount of vitamin B1 added is adjusted to 1.5 g / L. The remaining steps remain the same as in Example 1.

[0134] Example 7

[0135] This embodiment provides a method for preparing a Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that step (3) is adjusted to prepare a single fermentation broth of Lactobacillus plantarum:

[0136] (i) Lactobacillus plantarum was inoculated into MRS basal medium and cultured at 37° C. for 24 h to obtain a primary seed solution of Lactobacillus plantarum.

[0137] (ii) The viable bacterial count was set to 1×10 9 The first-level seed liquid of Lactobacillus plantarum with a CFU / mL was inoculated into the MRS culture medium improved with Cordyceps sinensis powder (the total inoculation amount was 5%) to obtain a single fermentation liquid of Lactobacillus plantarum.

[0138] Accordingly, step (4) was adjusted to inoculating a single fermentation culture medium with Lactobacillus plantarum, and the remaining steps remained consistent with Example 1.

[0139] Example 8

[0140] This embodiment provides a method for preparing a Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that step (3) is adjusted to prepare a single fermentation broth of Bifidobacterium bifidum:

[0141] (i) Bifidobacterium bifidum was inoculated into MRS basal medium and cultured at 37°C for 24 hours to obtain a primary seed solution of Bifidobacterium bifidum.

[0142] (ii) The viable bacterial count was set to 1×10 9 The primary seed liquid of Bifidobacterium bifidum with a CFU / mL was inoculated into the MRS medium modified with Cordyceps sinensis powder (the total inoculation amount was 5%) to obtain a single fermentation liquid of Bifidobacterium bifidum.

[0143] Accordingly, step (4) was adjusted to inoculating a single fermentation culture medium with Bifidobacterium bifidum, and the remaining steps remained the same as in Example 1.

[0144] Example 9

[0145] This embodiment provides a method for preparing Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, during the secondary fermentation in step (4), the total mass of the fermentation agent I is kept unchanged, and the fermentation agent I is adjusted to a single glucose. The remaining steps are consistent with Example 1.

[0146] Example 10

[0147] This embodiment provides a method for preparing Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, during the secondary fermentation in step (4), the total mass of the fermentation agent I is kept unchanged, and the fermentation agent I is adjusted to a single glutamine. The remaining steps are consistent with Example 1.

[0148] Example 11

[0149] This embodiment provides a method for preparing Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, during the secondary fermentation in step (4), the total mass of the fermentation promoter II is kept unchanged, and the fermentation promoter II is adjusted to adenylate and guanylate with a mass ratio of 1:0.5. The remaining steps are consistent with Example 1.

[0150] Example 12

[0151] This embodiment provides a method for preparing Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, during the secondary fermentation in step (4), the total mass of the fermentation agent II is kept unchanged, and the fermentation agent II is adjusted to adenylate and arginine in a mass ratio of 1:0.5. The remaining steps are consistent with Example 1.

[0152] Example 13

[0153] This embodiment provides a method for preparing Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, during the secondary fermentation in step (4), the total mass of the fermentation promoter II is kept unchanged, and the fermentation promoter II is adjusted to a mass ratio of guanylate and arginine of 0.5:0.5. The remaining steps are consistent with Example 1.

[0154] Comparative Example 1

[0155] This comparative example provides a method for preparing a Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, in step (2), the Cordyceps sinensis matrix extract is not added to the formula of the main fermentation medium. Accordingly, the amount of Cordyceps sinensis powder added is adjusted to 90 g / L. The remaining steps remain the same as in Example 1.

[0156] Comparative Example 2

[0157] This comparative example provides a method for preparing a Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, in step (2), Cordyceps sinensis powder is not added to the formula of the main fermentation medium. Accordingly, the addition amount of Cordyceps sinensis matrix extract is adjusted to 90 g / L. The remaining steps remain the same as in Example 1.

[0158] Comparative Example 3

[0159] This comparative example provides a method for preparing a Cordyceps sinensis fermentation filtrate, which differs from Example 1 only in that, in step (2), Cordyceps sinensis powder and Cordyceps sinensis matrix extract are not added to the formula of the main fermentation medium, and the remaining steps are consistent with Example 1.

[0160] Application Example 1

[0161] This application example provides an essence water, which includes, by weight percentage, 5% of the Cordyceps sinensis fermentation filtrate prepared in Example 1, 3% of polydimethylsiloxane, 5% of panthenol, 8% of glycerin, 3% of propylene glycol, 3% of sodium hyaluronate, and the balance being water.

[0162] Application Example 2-13

[0163] Application Examples 2-13 each provide an essence water, which differs from Application Example 1 only in that the Cordyceps sinensis fermentation filtrate prepared in Example 1 is replaced by the Cordyceps sinensis fermentation filtrate prepared in Examples 2-13 in equal mass, respectively, and the rest of the formula remains the same as Application Example 1.

[0164] Comparative Application Examples 1-3

[0165] Comparative Application Examples 1-3 each provide an essence water, which differs from Application Example 1 only in that the Cordyceps sinensis fermentation filtrate prepared in Example 1 is replaced by the Cordyceps sinensis fermentation filtrate prepared in Comparative Examples 1-3 in equal mass, respectively, and the rest of the formula remains the same as Application Example 1.

[0166] Test Example 1

[0167] Antioxidant capacity test.

[0168] (1) Test principle:

[0169] Excessive free radical production is the primary cause of both natural and photoaging skin aging. Therefore, scavenging free radicals can slow skin aging and reduce wrinkles. 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable, long-lived free radical. In the presence of free radical scavengers, DPPH ethanol solutions weaken their light absorption due to pairing with their single electrons. The degree of discoloration of DPPH ethanol solutions is linearly correlated with the number of electrons accepted, allowing evaluation of the free radical scavenging ability of the test sample, i.e., its antioxidant activity.

[0170] (2) Test method:

[0171] 1.0 mL of the Cordyceps sinensis fermentation filtrate prepared in each Example and Comparative Example (diluted 5-fold) was placed in a 10 mL centrifuge tube. 3.0 mL of DPPH solution (0.008 mmol / L) was added. The mixture was incubated at room temperature in the dark for 30 minutes. The absorbance was measured at 517 nm using anhydrous ethanol as a blank. The DPPH radical scavenging rate was calculated according to the following formula.

[0172] DPPH free radical scavenging rate = A0-(As-Ac) / A0×100%;

[0173] Where A0 is the absorbance of 1.0 mL distilled water + 3.0 mL DPPH solution, As is the absorbance of 1.0 mL sample solution + 3.0 mL DPPH solution, and Ac is the absorbance of 1.0 mL sample solution + 3.0 mL anhydrous ethanol.

[0174] The free radical scavenging abilities of the Cordyceps sinensis fermentation filtrates prepared in the Examples and Comparative Examples are shown in Table 2.

[0175] Table 2

[0176] Group DPPH free radical scavenging rate (%) Example 1 70.2 Example 2 68.5 Example 3 69.1 Example 4 58.3 Example 5 55.7 Example 6 62.4 Example 7 53.9 Example 8 57.1 Example 9 59.8 Example 10 54.6 Example 11 56.2 Example 12 51.3 Example 13 52.7 Comparative Example 1 39.5 Comparative Example 2 41.2 Comparative Example 3 35.8

[0177] Test Example 2

[0178] Evaluation of anti-aging efficacy.

[0179] 160 subjects aged 20-45 (80 males and 80 females) were randomly selected. The subjects presented with noticeable facial lines, wrinkles, and sagging. The subjects were divided into 16 groups. After cleansing their skin, each group of subjects took an appropriate amount of the essence water product from Application Examples 1-13 and Comparative Application Examples 1-3 and evenly applied it to the entire face and eye area, gently massaging until completely absorbed. This was done once daily, morning and evening, for two months. Skin images were captured using the VISIA-CR facial image analyzer, and the wrinkle percentage was measured. The skin images were captured, and the crow's feet and wrinkle reduction rates were calculated.

[0180] Crow's feet wrinkle reduction rate = (percentage of crow's feet wrinkles before using the product - percentage of crow's feet wrinkles after using the product) / percentage of crow's feet wrinkles before using the product × 100%.

[0181] Nasal fold reduction rate = (nasolabial fold ratio before using the product - nasolabial fold ratio after using the product) / nasolabial fold ratio before using the product × 100%.

[0182] The test results are shown in Table 3.

[0183] Table 3

[0184]

[0185]

[0186] From the test data in Table 2 and Table 3, it can be seen that the Cordyceps sinensis fermentation filtrate involved in the present invention has excellent DPPH free radical scavenging activity and shows good anti-aging potential.

[0187] A comparison of the data from Example 1 / Application Example 1 and Comparative Examples 1-3 / Comparative Application Examples 1-3 shows that adding Cordyceps sinensis powder and Cordyceps sinensis matrix extract to the fermentation medium and fermenting the fermentation broth can improve the bioavailability of Cordyceps sinensis, enrich the effective ingredients in the Cordyceps sinensis fermentation filtrate, and enhance the anti-aging effect of the Cordyceps sinensis fermentation filtrate. In addition, Cordyceps sinensis powder and Cordyceps sinensis matrix extract have a significant synergistic effect in enhancing the anti-aging effect of the Cordyceps sinensis fermentation filtrate.

[0188] From the data of Example 1 / Application Example 1 and Example 4 / Application Example 4, it can be seen that during the preparation of the combined fermentation broth of Lactobacillus plantarum and Bifidobacterium bifidum, adding Cordyceps sinensis powder to the culture medium can further enrich the effective ingredients in the Cordyceps sinensis fermentation filtrate and improve the anti-aging effect of the Cordyceps sinensis fermentation filtrate.

[0189] It can be seen from the data of Example 1 / Application Example 1 and Example 5-6 / Application Example 5-6 that in the preparation process of the combined fermentation broth of Lactobacillus plantarum and Bifidobacterium bifidum, vitamin B1 and L-cysteine ​​are simultaneously added to the culture medium. The anti-aging effect of the Cordyceps sinensis fermentation filtrate prepared is significantly higher than that of single vitamin B1 or single L-cysteine, indicating that vitamin B1 and L-cysteine ​​have a significant synergistic effect in improving the anti-aging effect of the Cordyceps sinensis fermentation filtrate.

[0190] From the comparison of the data of Example 1 / Application Example 1 and Example 7-8 / Application Examples 7-8, it can be seen that during the main fermentation process, the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum has better antioxidant and anti-aging effects than the single fermentation liquid of Lactobacillus plantarum or Bifidobacterium bifidum, indicating that Lactobacillus plantarum and Bifidobacterium bifidum have a significant synergistic effect in improving the anti-aging effect of Cordyceps sinensis fermentation filtrate.

[0191] Comparison of the data between Example 1 / Application Example 1 and Examples 9-10 / Application Examples 9-10 shows that the addition of glucose and glutamine during the secondary fermentation process can further enrich the effective components in the Cordyceps sinensis fermentation filtrate and enhance the anti-wrinkle and anti-aging effects of the Cordyceps sinensis fermentation filtrate.

[0192] From the comparison of the data of Example 1 / Application Example 1 and Examples 11-13 / Application Examples 11-13, it can be seen that the addition of adenylic acid, guanylic acid, adenylic acid, and guanylic acid during the secondary fermentation process can further enrich the effective components in the Cordyceps sinensis fermentation filtrate, and the three have a significant synergistic effect in improving the antioxidant and anti-aging effects of the Cordyceps sinensis fermentation filtrate.

[0193] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0194] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0195] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing Cordyceps sinensis fermentation filtrate, characterized in that: The preparation method comprises the following steps: (1) mixing Cordyceps sinensis powder with an alcohol solution, performing ultrasonic extraction, centrifuging, collecting the supernatant, and drying to obtain a Cordyceps sinensis matrix extract; (2) Cordyceps sinensis powder, Cordyceps sinensis matrix extract and basic fermentation medium are mixed to obtain a main fermentation medium, fermentation culture is inoculated into the main fermentation medium for fermentation culture to obtain a fermentation liquid; the fermentation liquid is centrifuged, filtered and the filtrate is collected to obtain a Cordyceps sinensis fermentation filtrate.

2. The preparation method according to claim 1, characterized in that The material-liquid ratio of the Cordyceps sinensis powder to the alcohol solution in step (1) is 1:8-1:12 g / mL; Preferably, the alcohol solution comprises an aqueous solution of ethanol; Preferably, the volume percentage of ethanol in the ethanol aqueous solution is 50-70%; Preferably, the temperature of the ultrasonic extraction in step (1) is 50-70° C., and the time of the ultrasonic extraction is 1-3 h; Preferably, the power of the ultrasonic extraction in step (1) is 200-400W, and the frequency of the ultrasonic extraction is 30-50kHz.

3. The preparation method according to claim 1 or 2, characterized in that The fermentation liquid in step (2) comprises a combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum; Preferably, the total inoculation amount of the fermentation broth is 10-12% by volume.

4. The preparation method according to claim 3, characterized in that The preparation method of the combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum comprises the following steps: (i) inoculating Lactobacillus plantarum and Bifidobacterium bifidum into MRS basal medium to obtain a first-level seed solution of Lactobacillus plantarum and a first-level seed solution of Bifidobacterium bifidum; (ii) inoculating the first-grade seed liquid of Lactobacillus plantarum and the first-grade seed liquid of Bifidobacterium bifidum into MRS medium modified with Cordyceps sinensis powder to obtain a combined fermentation liquid of Lactobacillus plantarum and Bifidobacterium bifidum; Preferably, the MRS medium modified with cordyceps sinensis powder comprises: 8-12 g / L peptone, 6-10 g / L beef extract powder, 3-5 g / L yeast extract, 18-22 g / L glucose, 50-80 g / L cordyceps sinensis powder, 2-4 mL / L olive oil, 0.8-1.2 g / L vitamin B1, 0.3-0.7 g / L L-cysteine, 3-7 g / L sodium acetate, and 1-5 g / L ammonium citrate. Preferably, the total inoculation amount of the first-stage seed solution of Lactobacillus plantarum and the first-stage seed solution of Bifidobacterium bifidum in step (ii) is 3-7% by volume; Preferably, when the first-level seed liquid of Lactobacillus plantarum and the first-level seed liquid of Bifidobacterium bifidum are inoculated into the MRS culture medium modified with Cordyceps sinensis powder, the ratio of the number of viable bacteria of Lactobacillus plantarum to that of Bifidobacterium bifidum is 1:3-3:

1.

5. The preparation method according to any one of claims 1 to 4, characterized in that The basic fermentation medium in step (2) comprises: Glucose 25-35 g / L, soy peptone 10-20 g / L, potassium dihydrogen phosphate 0.5-1.5 g / L, MgSO4·7H2O 0.3-0.7 g / L, olive oil 1.5-2.5 mL / L; Preferably, the mass ratio of the Cordyceps sinensis powder to the Cordyceps sinensis matrix extract in step (2) is 2.5:1-4:1; Preferably, the total amount of Cordyceps sinensis powder and Cordyceps sinensis matrix extract added to the main fermentation medium is 70-100 g / L.

6. The preparation method according to any one of claims 1 to 5, characterized in that The fermentation culture in step (2) includes a primary fermentation and a secondary fermentation carried out sequentially; The conditions of the primary fermentation are: temperature of 27-29° C., pH of 6.8-7.2, dissolved oxygen of 30-50%, and fermentation time of 2-2.5 days; The conditions for the secondary fermentation are: temperature of 24-26° C., pH value of 6.8-7.2, dissolved oxygen content of 30-50%, and fermentation time of 5-7 days.

7. The preparation method according to claim 6, characterized in that The fermentation culture further comprises adding a fermentation promoter I on the 1st to 2nd day of the secondary fermentation; The fermentation promoting agent I includes glucose and glutamine; Preferably, the mass ratio of glucose to glutamine is 4:1-6:1; Preferably, the addition amount of the fermentation promoting agent I is 0.5-0.8% of the mass of the main fermentation medium.

8. The preparation method according to claim 6 or 7, characterized in that The fermentation culture further comprises adding a fermentation promoter II on the 3rd to 4th day of the secondary fermentation; The fermentation promoting agent II includes adenylate, guanylate and arginine; Preferably, the mass ratio of adenylate, guanylate and arginine is (0.5-1.5):(0.1-1):(0.1-1); Preferably, the addition amount of the fermentation promoter II is 0.1-0.3% of the mass of the main fermentation medium.

9. A Cordyceps sinensis fermentation filtrate prepared by the method for preparing the Cordyceps sinensis fermentation filtrate according to any one of claims 1 to 8.

10. Use of the Cordyceps sinensis fermentation filtrate according to claim 9 in cosmetics.

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