Method for improving anti-aging effect of eurotium cristatum tea compound fermentation liquor

Abstract

The invention provides a technical method for improving the anti-aging effect of eurotium cristatum tea compound fermentation liquor, and belongs to the technical field of microorganisms, the Latin name of eurotium cristatum is Eurotium cristatum, the eurotium cristatum is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC M 2024643. According to the method, the yeast seed solution is inoculated into the tea-sea buckthorn leaf culture solution fermented by the eurotium cristatum, so that the content of anti-aging active components such as procyanidine B2, quercitrin, cyanidin-3-glucoside, nicotinic acid, naringenin and rutin in the fermentation culture solution can be remarkably increased through co-fermentation, and the elastase inhibition rate can be remarkably increased; the anti-aging effect of a component system of the fermentation culture solution is enhanced. The prepared tea-hippophae rhamnoides leaf compound fermentation liquor is suitable for development of anti-aging cosmetics and has a wide market prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a method for improving the anti-aging effect of a composite fermentation liquid of Eurotium cristatum tea. Background Art

[0002] Elastin is a protein that is relatively abundant in human skin and makes the skin elastic and resilient. As we age, the skin's ability to synthesize elastin gradually decreases. At the same time, the elastin in the skin is constantly degraded by elastase, which is one of the main causes of skin aging.

[0003] Studies have shown that by inhibiting the activity of elastase, the decomposition of elastin in the skin can be reduced, thereby achieving the effect of preventing and delaying skin aging. As consumers' demand for anti-aging skin care continues to increase, more and more skin care brands have begun to focus on the research and application of inhibiting elastase. Many skin care products on the market currently contain ingredients that inhibit elastase, such as grape seed polyphenols, vitamin C, collagen, etc. However, these active ingredients are not very stable and are easily oxidized or denatured and inactivated by light, temperature, etc., so it is necessary to develop natural and stable substances that inhibit elastase.

[0004] Previous studies have shown that Eurotium cristatum K8824 was isolated and cultured from the strain of Shaanxi Fuzhuan tea. This Eurotium cristatum has antioxidant, anti-aging and whitening effects, among which the anti-aging effect is particularly prominent. According to the results of elastase inhibition rate, its elastase inhibition rate is significantly higher than that of Eurotium cristatum K8821. Summary of the invention

[0005] The present invention is based on Eurotium cristatum K8824, which has a fast growth rate and a fermentation broth with significant antioxidant, anti-aging and whitening effects. The strain has been deposited in the China Center for Type Culture Collection on April 7, 2024. The deposit number of Eurotium cristatum K8824 is: CCTCC M 2024643, and the Latin name is Eurotium cristatum. Address: Wuhan University, Wuhan City, Hubei Province, Postal Code: 430072, Telephone: 027-68754052.

[0006] A method for improving the inhibition rate of elastase in Eurotium cristatum K8824 fermentation broth, comprising the following steps:

[0007] (1) inoculating the seed liquid of Eurotium cristatum K8824 into the fermentation medium for primary fermentation, and culturing in a shaking incubator at 28±2° C. for 48 to 72 hours to obtain the tea-sea buckthorn leaf primary fermentation liquid;

[0008] (2) inoculating the yeast seed liquid into the tea-sea buckthorn leaf primary fermentation liquid, and fermenting the mixture in a shaking incubator at 28±2° C. for 24 to 72 hours;

[0009] (3) After the fermentation is completed, the fermentation culture liquid is centrifuged to obtain a supernatant;

[0010] (4) The obtained supernatant is filtered through a sterilizing filter to obtain a sterile tea-sea buckthorn leaf composite fermentation liquid.

[0011] Wherein, the preparation method of the fermentation medium is:

[0012] (1) Grinding tea and seabuckthorn leaves into fine powders of 40 to 100 mesh respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 7:3 to 5:4 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0013] (2) The tea-sea buckthorn leaf fermentation substrate was mixed with purified water in a ratio of 1:30 to 1:9 and stirred to obtain a fermentation medium, which was sterilized at 121° C. for 15 min.

[0014] Wherein, the yeast includes Angel Yeast and Saccharomyces cerevisiae.

[0015] The brewer's yeast is deposited in the China Center for Type Culture Collection, and the strain deposit number is CCTCC KY2008617.

[0016] Wherein, the inoculation amount of the seed solution of Eurotium cristatum K8824 is 3-8%.

[0017] Wherein, the inoculation amount of the yeast is 2-5%.

[0018] Wherein, the shaking table speed is 120-200 r / min.

[0019] Wherein, the centrifugal speed is 2800-3500r / min.

[0020] Wherein, the sterilizing filter is a sterilizing filter with a pore size of 0.15 to 0.30 μm.

[0021] Beneficial Effects

[0022] First, through the previous research, it was found that the Eurotium cristatum K8824 preserved in the present invention can adapt well to the environment of tea and sea buckthorn leaves, and the fermentation speed is significantly higher than that of other Eurotium cristatum. When the fermentation substrate is a mixture of tea leaves and sea buckthorn leaves, and Eurotium cristatum K8824 and Saccharomyces cerevisiae CCTCC KY 2008617 are used for fermentation, the fermentation liquid has a strong anti-aging effect. When the fermentation liquid substrate is a single raw material of tea or sea buckthorn leaves, or the strain is a single strain of Eurotium cristatum K8824 or Saccharomyces cerevisiae CCTCC KY 2008617, the anti-aging effect in the fermentation liquid is weakened. After analysis, it was found that Eurotium cristatum K8824 and Saccharomyces cerevisiae CCTCC KY 2008617 have a synergistic effect, and a higher anti-aging component is produced in the fermentation liquid of tea and sea buckthorn leaves. The inventor analyzed the main components of the fermentation liquid.

[0023] The results showed that the main anti-aging active substances in the tea-sea buckthorn leaf composite fermentation liquid were: Procyanidin B2, Quercitrin, O-Succinyl-L-homoserine, Betaine, beta-D-Glucopyranuronic acid, Caproic acid, Cyanidin 3-glucoside, nicotinic acid, Vanillate, naringenin, rutin, 2-Ketobutyric acid and N-Acetylglutamic acid. After analysis, it was found that by initially inoculating Eurotium cristatum K8824 in the tea-sea buckthorn leaf fermentation medium for 48 hours and then inoculating yeast CCTCC KY 2008617 for 24 hours, the relative contents of active substances with anti-aging ability such as procyanidin B2, quercitrin, cyanidin 3-glucoside, nicotinic acid, naringenin, and rutin in the tea-sea buckthorn leaf composite fermentation liquid were increased by 183%, 159%, 187%, 155%, 132%, and 330%, respectively, compared with the fermentation filtrate to which Eurotium cristatum was added alone, resulting in enhanced anti-aging ability.

[0024] The invention adopts a mixed bacteria cascade co-fermentation technical method to prepare a fermentation liquid with anti-aging activity, and the elastase inhibition rate can reach 97.36%. After the fermentation liquid is processed into cosmetics, the anti-aging activity in the cosmetics is well maintained after being stored for about 6 to 12 months. According to the results of storage experiments, the initial elastase inhibition rate of the anti-aging cosmetics prepared by the invention is 82.76%, the elastase inhibition rate after being stored at 4°C for 6 months is 78.62%, and the elastase inhibition rate after being stored for 12 months is 74.48%; the elastase inhibition rate after being stored at room temperature for 6 months is 70.60%; the elastase inhibition rate after being stored for 12 months is 60.4%. The retention rate of the elastase inhibition rate after being stored at 4°C for 12 months can still reach 90% of the initial inhibition rate, and the stability is good. The technical method is green and simple in process, and the production cost is more controllable than that of the extraction method. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a substance and efficacy correlation analysis diagram of the tea-sea buckthorn leaf composite fermentation liquid of the present invention.

[0026] Figure 2 It is a material identification diagram of proanthocyanidin B2, quercetin, cyanidin-3-glucoside, nicotinic acid, naringenin and rutin in the tea-sea buckthorn leaf composite fermentation liquid of the present invention. DETAILED DESCRIPTION

[0027] The present invention is described in detail below with reference to examples. All methods and techniques, unless otherwise specified, are conventional methods and techniques.

[0028] Method for preparing fermentation broth

[0029] ① After grinding tea and seabuckthorn leaves into 40 mesh fine powder respectively, the tea and seabuckthorn leaves are mixed evenly in a mass ratio of 6:4 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0030] ② Mix the tea-sea buckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stir evenly to obtain a fermentation medium, and sterilize at 121°C for 15 minutes;

[0031] ③ The seed liquid of Eurotium cristatum was inoculated into the fermentation medium for primary fermentation, the inoculation amount was 3-8%, and the shaking culture was carried out at 28±2°C for 48-72h, and the shaking speed was 120-200r / min. For data unification, the inoculation amount of Eurotium cristatum in Example 1 was 5%, and the shaking culture was carried out at 28±2°C for 48h, and the shaking speed was 150r / min.

[0032] The preparation method of the activated seed liquid of the crown-like spores K8824 is as follows: streaking the preserved crown-like spores K8824 on a PDA plate culture medium, placing it under 28±1°C for activation for 48 to 72 hours, scraping the spores on the plate, washing them with sterile water, fully shaking and mixing the spore liquid to form a spore suspension, and adjusting the concentration of the spore suspension to 1.0×10 7 CFU / mL, and the activated seed solution of Eurotium cristatum K8824 was obtained.

[0033] The method for preparing the brewer's yeast seed liquid is as follows: the yeast is inoculated into a YPD liquid medium and cultured for 24 to 36 hours to obtain a yeast seed liquid, and then the spore suspension is adjusted to 1.0×10 8 CFU / mL, and obtain the Saccharomyces cerevisiae seed liquid.

[0034] The preparation method of Angel yeast seed liquid is as follows: open the bag of Angel high-activity dry yeast next to the flame of an alcohol lamp, take 2g of yeast powder and add it to the sterilized 5% glucose solution, place it on a shaker at 28±1℃ for shaking culture and activation for 3-6h to obtain Angel yeast seed liquid, and then adjust the concentration to 1.0×10 8 CFU / mL, and get Angel yeast seed liquid.

[0035] Test Example 1: DPPH-free radical scavenging experiment

[0036] Prepare 0.5mmol / L DPPH-ethanol solution, dilute the fermentation sample solution 10 times, take 0.1mL sample and add 0.9mL DPPH solution to mix, the control group is 0.1mL distilled water and add 0.9mL DPPH solution to mix, react in the dark for 30min, measure its absorbance at a wavelength of 517nm, and the DPPH free radical scavenging rate is calculated by the formula:

[0037] DPPH free radical scavenging rate (%) = (1-A1 / A0) × 100%, A1 is the sample absorbance; A0 is the blank control absorbance. The higher the scavenging rate, the better the antioxidant effect of the fermentation filtrate.

[0038] Test Example 2: Hydroxyl free radical scavenging ability determination

[0039] Experimental method: A colorimetric kit (Nanjing Jiancheng Bioengineering Institute) was used to detect the hydroxyl radical scavenging ability. According to the instructions of the hydroxyl radical scavenging ability assay kit (A018-1-1), 0.2 mL of a 10-fold diluted fermentation sample solution was added to the Fenton system to generate hydroxyl radicals. The absorbance of each sample was measured at 550 nm using the Griess reagent.

[0040] Test Example 3: Determination of ABTS free radical scavenging ability

[0041] Experimental method: A colorimetric kit (Nanjing Jiancheng Bioengineering Institute) was used to detect the antioxidant capacity of ABTS free radicals. According to the kit instructions (A015-2), the concentration of the sample was diluted 10 times, and the antioxidant capacity of ABTS free radicals was determined. The absorbance was detected at 734 nm, and the value was expressed as the value equivalent to 1 mmol / L Trolox.

[0042] Test Example 4: FRAP Total Antioxidant Capacity Assay

[0043] Experimental method: A colorimetric kit (Nanjing Jiancheng Bioengineering Institute) was used to detect the FRAP free radical antioxidant capacity. According to the kit instructions (A015-3-1), a 10-fold diluted sample solution (5 μL) was added to 2,4,6-tripyridine-s-triazine (TPTZ) in an acidic medium to detect FRAP. The absorbance was measured at 593 nm, and the FRAP value was expressed as the equivalent of 1 mmol / L FeSO4-7H2O. The FRAP total antioxidant capacity results are shown in Table 1.

[0044] Test Example 5: Tyrosinase Inhibition Ability Determination

[0045] Experimental method: Refer to "T / GDCA 006-2021 Daily Chemical Raw Materials Tyrosinase Activity Inhibition Test Method (In Vitro Method)" to conduct tyrosinase activity inhibition test, and the fermentation filtrate sample was diluted 10 times for measurement.

[0046] Test Example 6: Determination of Elastase Activity Inhibition Rate

[0047] Experimental method: 85μL 50mmol / L Tris-HCL buffer (pH 8.0) was mixed with 15μL of 100-fold diluted sample solution, and then 25μL of elastase solution (60mU / mL) was added. The mixture was incubated at 25°C for 15min, followed by the addition of 25uL 1.015mmol / L N-succinyl-alanine-alanine-alanine-p-nitroaniline (AAAPAN) solution. After 15min, the absorbance was measured at 410nm. Both the elastase solution and the AAAPAN solution were prepared with 50mmol / L Tris-HCl buffer (pH 8.0), and the inhibition rate of the tested sample on the activity of elastase was calculated.

[0048] The elastase activity inhibition rate is calculated by the following formula: elastase activity inhibition rate (%) = [1-(CD) / (AB)] x 100%, A is the absorbance of the reaction solution without sample; B is the absorbance of the reaction solution without sample and enzyme; C is the absorbance of the reaction solution containing sample and enzyme; D is the absorbance of the reaction solution without enzyme. The higher the elastase inhibition rate, the better the anti-aging effect of the fermentation filtrate.

[0049] Example 1

[0050] A method for improving the inhibition rate of elastase activity in the fermentation liquid of Eurotium cristatum K8824. The inventors isolated a Eurotium cristatum from Shaanxi Fuzhuan tea, which grows fast in a seabuckthorn leaf-tea mixture and can significantly inhibit elastase. The seabuckthorn leaf-tea fermentation liquid has antioxidant, anti-aging and whitening effects. The strain was deposited in the China Center for Type Culture Collection on April 7, 2024. The Eurotium cristatum K8824 has a preservation number of CCTCC M 2024643, a Latin name of Eurotium cristatum, and an address of Wuhan University, Wuhan City, Hubei Province, with a zip code of 430072 and a telephone number of 027-68754052.

[0051] The morphological characteristics of Eurotium cristatum K8824 preserved in the present invention are as follows: the colony is milky white to light yellow, the surface is smooth and moist, the hyphae are slender, the branches are sparse, the edge of the colony is light yellow, the center is convex, and the color becomes darker to yellow and dark yellow as it approaches the center of the colony. The spores are oval, the cap is bowl-shaped, the inner wall is smooth, and the outer wall has a radial texture. This embodiment is based on the above-mentioned K8824 strain, and Angel Yeast is added to the traditional fermentation process for fermentation, and the specific method is as follows:

[0052] (1) Grinding tea and seabuckthorn leaves into 40 mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 6:4 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0053] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0054] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 5%, and cultured in a shaking incubator at 28±2° C. for 48 h to obtain tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed was 150 r / min.

[0055] (4) After the fermentation of Eurotium cristatum K8824 for 48 h, the Angel yeast seed solution was inoculated into the tea-sea buckthorn leaf fermentation solution for co-fermentation. The inoculation amount was 3%, and the co-culture was carried out in a shaking incubator at 28±2°C for 24 h at a shaking speed of 150 r / min.

[0056] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 3000 r / min.

[0057] (6) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf composite fermentation liquid. The measured data are shown in Table 1.

[0058] Example 2

[0059] This example is based on the above-mentioned K8824 strain, and Saccharomyces cerevisiae is added to the traditional fermentation process for fermentation, wherein the original source of Saccharomyces cerevisiae is the China Center for Type Culture Collection, and the strain collection number is CCTCC KY 2008617. The specific method is as follows:

[0060] (1) Grinding tea and seabuckthorn leaves into 40 mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 6:4 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0061] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0062] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 5%, and cultured in a shaking incubator at 28±2° C. for 48 h to obtain a tea-sea buckthorn leaf fermentation filtrate, wherein the shaking speed was 150 r / min.

[0063] (4) After the fermentation of Eurotium cristatum K8824 for 48 h, the brewer's yeast seed solution was inoculated into the tea-sea buckthorn leaf fermentation solution for co-fermentation at an inoculation amount of 3%. The co-culture was carried out in a shaking incubator at 28±2°C for 24 h at a shaking speed of 150 r / min.

[0064] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 3000 r / min.

[0065] (6) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf composite fermentation liquid. The measured data are shown in Table 1.

[0066] Comparative Example 1

[0067] The comparative experiment was based on the above-mentioned K8824 strain. In the traditional fermentation process, no yeast was added to perform a single fermentation of Eurotium cristatum. The specific method is as follows:

[0068] (1) Grinding tea and seabuckthorn leaves into 40 mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 6:4 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0069] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0070] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for fermentation, with an inoculation amount of 5%, and cultured in a shaking incubator at 28±2° C. for 72 h to obtain tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed was 150 r / min.

[0071] (4) After fermentation, the tea-sea buckthorn leaf fermentation liquid was centrifuged to obtain a supernatant at a centrifugal speed of 3000 r / min.

[0072] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf fermentation filtrate. The measured data are shown in Table 1.

[0073] Comparative Example 2

[0074] The comparative experiment was based on the above-mentioned K8824 strain. No yeast was added during the traditional fermentation process, and pure green tea was used as the substrate for single fermentation with Eurotium cristatum. The specific method is as follows:

[0075] (1) Grinding tea into 40 mesh fine powder to obtain a tea fermentation substrate;

[0076] (2) mixing the tea fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0077] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for fermentation at an inoculation amount of 5%. The culture was shaken at 28±2° C. for 72 h to obtain tea fermentation liquid, wherein the shaking speed was 150 r / min.

[0078] (4) After fermentation, the tea fermentation liquid was centrifuged to obtain the supernatant at a centrifugal speed of 3000 r / min.

[0079] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea fermentation filtrate. The measured data are shown in Table 1.

[0080] Comparative Example 3

[0081] The comparative experiment was based on the above-mentioned K8824 strain. No yeast was added in the traditional fermentation process, and pure seabuckthorn leaves were used as substrates for single fermentation with Eurotium cristatum. The specific method is as follows:

[0082] (1) grinding tea into 40 mesh fine powder to obtain seabuckthorn leaf fermentation substrate;

[0083] (2) mixing the seabuckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0084] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for fermentation at an inoculation amount of 5%. The culture was shaken at 28±2° C. for 72 h to obtain the seabuckthorn leaf fermentation liquid, wherein the shaking speed was 150 r / min.

[0085] (4) After the fermentation is completed, the seabuckthorn leaf fermentation liquid is centrifuged to obtain a supernatant at a centrifugal speed of 3000 r / min.

[0086] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile seabuckthorn leaf fermentation filtrate. The measured data are shown in Table 1.

[0087] Comparative Example 4

[0088] This example is based on the above-mentioned K8824 strain, uses pure green tea as a substrate, and adds Angel Yeast to the traditional fermentation process for fermentation. The specific method is as follows:

[0089] (1) Grinding tea into 40 mesh fine powder to obtain tea fermentation substrate;

[0090] (2) mixing the tea fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0091] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 5%, and cultured in a shaking incubator at 28±2°C for 48 h to obtain tea fermentation liquid, wherein the shaking speed was 150 r / min.

[0092] (4) After the fermentation of Eurotium cristatum K8824 for 48 h, the Angel yeast seed solution was inoculated into the tea fermentation solution for co-fermentation at an inoculation amount of 3%. The co-culture was carried out in a shaking incubator at 28±2°C for 24 h at a shaking speed of 150 r / min.

[0093] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 3000 r / min.

[0094] (6) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea fermentation liquid. The measured data are shown in Table 1.

[0095] Comparative Example 5

[0096] This example is based on the above-mentioned K8824 strain, uses pure seabuckthorn leaves as a substrate, and adds Angel yeast to the traditional fermentation process for fermentation. The specific method is as follows:

[0097] (1) grinding seabuckthorn leaves into 40 mesh fine powder to obtain seabuckthorn leaf fermentation substrate;

[0098] (2) mixing the seabuckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0099] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 5%, and cultured in a shaking incubator at 28±2° C. for 48 h to obtain the seabuckthorn leaf fermentation liquid, wherein the shaking speed was 150 r / min.

[0100] (4) After the fermentation of Eurotium cristatum K8824 for 48 h, the Angel yeast seed solution was inoculated into the seabuckthorn leaf fermentation solution for co-fermentation at an inoculation amount of 3%. The co-culture was carried out in a shaking incubator at 28±2°C for 24 h at a shaking speed of 150 r / min.

[0101] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 3000 r / min.

[0102] (6) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile seabuckthorn leaf fermentation liquid. The measured data are shown in Table 1.

[0103] Comparative Example 6

[0104] This example is based on the above-mentioned K8824 strain, uses pure green tea as a substrate, and adds brewer's yeast to the traditional fermentation process for fermentation. The original source of brewer's yeast is the China Center for Type Culture Collection, and the strain collection number is CCTCC KY2008617. The specific method is as follows:

[0105] (1) Grinding tea into 40 mesh fine powder to obtain a fermentation substrate;

[0106] (2) mixing the tea fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0107] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 5%, and cultured in a shaking incubator at 28±2°C for 48 h to obtain tea fermentation liquid, wherein the shaking speed was 150 r / min.

[0108] (4) After the fermentation of Eurotium cristatum K8824 for 48 h, the brewer's yeast seed solution was inoculated into the tea fermentation solution for co-fermentation at an inoculation amount of 3%. The co-culture was carried out in a shaking incubator at 28±2°C for 24 h at a shaking speed of 150 r / min.

[0109] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 3000 r / min.

[0110] (6) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea fermentation liquid. The measured data are shown in Table 1.

[0111] Comparative Example 7

[0112] This example is based on the above-mentioned K8824 strain, uses pure seabuckthorn leaves as a substrate, and adds saccharomyces cerevisiae to ferment during the traditional fermentation process, wherein the original source of saccharomyces cerevisiae is the China Center for Type Culture Collection, and the strain collection number is CCTCCKY 2008617. The specific method is as follows:

[0113] (1) Grinding seabuckthorn leaves into 40-mesh fine powder to obtain a fermentation substrate;

[0114] (2) mixing the seabuckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0115] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 5%, and cultured in a shaking incubator at 28±2° C. for 48 h to obtain the seabuckthorn leaf fermentation liquid, wherein the shaking speed was 150 r / min.

[0116] (4) After the fermentation of Eurotium cristatum K8824 for 48 h, the cerevisiae seed solution was inoculated into the seabuckthorn leaf fermentation solution for co-fermentation at an inoculation amount of 3%. The co-culture was carried out in a shaking incubator at 28±2°C for 24 h at a shaking speed of 150 r / min.

[0117] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 3000 r / min.

[0118] (6) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile seabuckthorn leaf fermentation liquid. The measured data are shown in Table 1.

[0119] Comparative Example 8

[0120] This example is based on the above-mentioned K8824 strain. Angel yeast is added to the traditional fermentation process for simultaneous inoculation and fermentation. The specific method is as follows:

[0121] (1) Grinding tea and seabuckthorn leaves into 100-mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 6:4 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0122] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0123] (3) The seed liquid of Eurotium cristatum K8824 and the seed liquid of Angel Yeast were simultaneously inoculated into the fermentation medium, with the inoculation amount of Eurotium cristatum being 5% and the inoculation amount of yeast being 3%. The culture was carried out in a shaking incubator at 28±2°C for 72 h to obtain tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed was 150 r / min.

[0124] (4) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 3000 r / min.

[0125] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf composite fermentation liquid. The measured data are shown in Table 1.

[0126] Comparative Example 9

[0127] The comparative experiment was based on the above-mentioned K8824 strain. During the traditional fermentation process, brewer's yeast was added for simultaneous inoculation and fermentation. The original source of brewer's yeast was the China Center for Type Culture Collection, and the strain collection number was CCTCC KY2008617. The specific method is as follows:

[0128] (1) Grinding tea and seabuckthorn leaves into 40 mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 6:4 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0129] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0130] (3) The seed liquid of Eurotium cristatum K8824 and the seed liquid of Saccharomyces cerevisiae were simultaneously inoculated into the fermentation medium, the inoculation amount of Eurotium cristatum was 5%, and the inoculation amount of yeast was 3%, and the culture was carried out on a shaking table at 28±2°C for 72 hours to obtain tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed was 150 r / min.

[0131] (4) After fermentation, the tea-sea buckthorn leaf fermentation liquid was centrifuged to obtain a supernatant at a centrifugal speed of 3000 r / min.

[0132] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf fermentation filtrate. The measured data are shown in Table 1.

[0133] Comparative Example 10

[0134] The comparative experiment was based on the above Angel yeast strains. In the traditional fermentation process, Angel yeast fermentation was performed alone without adding Eurotium cristatum. The specific method is as follows:

[0135] (1) Grinding tea and seabuckthorn leaves into 40 mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 6:4 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0136] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0137] (3) Inoculating Angel yeast seed liquid into fermentation medium with an inoculation amount of 3% for fermentation, and culturing in a shaking incubator at 28±2° C. for 72 h to obtain tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed is 150 r / min.

[0138] (4) After fermentation, the tea-sea buckthorn leaf fermentation liquid was centrifuged to obtain a supernatant at a centrifugal speed of 3000 r / min.

[0139] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf fermentation filtrate. The measured data are shown in Table 1.

[0140] Comparative Example 11

[0141] The comparative experiment was based on the above Angel yeast strains, using pure green tea as the substrate, and performing Angel yeast fermentation alone without adding Eurotium cristatum in the traditional fermentation process. The specific method is as follows:

[0142] (1) Grinding tea into 40 mesh fine powder to obtain tea fermentation substrate;

[0143] (2) mixing the tea fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0144] (3) Inoculating Angel Yeast seed liquid into fermentation medium for fermentation at an inoculation amount of 3%, and culturing on a shaking table at 28±2° C. for 72 h to obtain tea fermentation liquid at a shaking speed of 150 r / min.

[0145] (4) After fermentation, the tea fermentation liquid was centrifuged to obtain the supernatant at a centrifugal speed of 3000 r / min.

[0146] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea fermentation filtrate. The measured data are shown in Table 1.

[0147] Comparative Example 12

[0148] The comparative experiment was based on the above Angel yeast strain, using pure seabuckthorn leaves as the substrate, and performing Angel yeast fermentation alone without adding Eurotium cristatum in the traditional fermentation process. The specific method is as follows:

[0149] (1) grinding seabuckthorn leaves into 40 mesh fine powder to obtain seabuckthorn leaf fermentation substrate;

[0150] (2) mixing the seabuckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0151] (3) Inoculating Angel Yeast seed liquid into fermentation medium for fermentation at an inoculation amount of 3%, culturing on a shaking table at 28±2° C. for 72 h, and obtaining seabuckthorn leaf fermentation liquid at a shaking speed of 150 r / min.

[0152] (4) After the fermentation is completed, the seabuckthorn leaf fermentation liquid is centrifuged to obtain a supernatant at a centrifugal speed of 3000 r / min.

[0153] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile seabuckthorn leaf fermentation filtrate. The measured data are shown in Table 1.

[0154] Comparative Example 13

[0155] The comparative experiment was based on the above-mentioned Saccharomyces cerevisiae strains. In the traditional fermentation process, the Saccharomyces cerevisiae was fermented alone without adding Eurotium cristatum. The original source of Saccharomyces cerevisiae was the China Center for Type Culture Collection, and the strain collection number was CCTCCKY 2008617. The specific method is as follows:

[0156] (1) Grinding tea and seabuckthorn leaves into 40 mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 6:4 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0157] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0158] (3) The brewer's yeast seed liquid was inoculated into the fermentation medium for fermentation at an inoculation amount of 3%, and the culture was carried out on a shaking incubator at 28±2° C. for 72 h to obtain a tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed was 150 r / min.

[0159] (4) After fermentation, the tea-sea buckthorn leaf fermentation liquid was centrifuged to obtain a supernatant at a centrifugal speed of 3000 r / min.

[0160] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf fermentation filtrate. The measured data are shown in Table 1.

[0161] Comparative Example 14

[0162] The comparative experiment was based on the above-mentioned Saccharomyces cerevisiae strain, using pure green tea as the substrate, and fermenting with Saccharomyces cerevisiae alone without adding Eurotium coronatum in the traditional fermentation process. The original source of Saccharomyces cerevisiae was the China Center for Type Culture Collection, with the strain collection number CCTCC KY 2008617. The specific method is as follows:

[0163] (1) Grinding tea leaves into 40 mesh fine powder to obtain tea fermentation substrate;

[0164] (2) mixing the tea fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0165] (3) The brewer's yeast seed liquid was inoculated into the fermentation medium for fermentation at an inoculation amount of 3%, and the culture was carried out on a shaking incubator at 28±2° C. for 72 h to obtain a tea fermentation liquid, wherein the shaking speed was 150 r / min.

[0166] (4) After fermentation, the tea fermentation liquid was centrifuged to obtain the supernatant at a centrifugal speed of 3000 r / min.

[0167] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile tea fermentation liquid. The measured data are shown in Table 1.

[0168] Comparative Example 15

[0169] The comparative experiment was based on the above-mentioned Saccharomyces cerevisiae strain, with pure seabuckthorn leaves as the substrate, and the Saccharomyces cerevisiae fermentation was performed alone without adding Eurotium cristatum in the traditional fermentation process. The original source of Saccharomyces cerevisiae was the China Center for Type Culture Collection, with the strain collection number CCTCC KY 2008617. The specific method is as follows:

[0170] (1) grinding seabuckthorn leaves into 40 mesh fine powder to obtain seabuckthorn leaf fermentation substrate;

[0171] (2) mixing the seabuckthorn leaf fermentation substrate with purified water at a ratio of 1:30 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0172] (3) The saccharomyces cerevisiae seed liquid was inoculated into the fermentation medium for fermentation at an inoculation amount of 3%, and the culture was carried out in a shaking incubator at 28±2° C. for 72 h to obtain the seabuckthorn leaf fermentation liquid, wherein the shaking speed was 150 r / min.

[0173] (4) After the fermentation is completed, the seabuckthorn leaf fermentation liquid is centrifuged to obtain a supernatant at a centrifugal speed of 3000 r / min.

[0174] (5) The obtained supernatant was filtered through a 0.22 μm sterilizing filter to obtain a sterile seabuckthorn leaf fermentation filtrate. The measured data are shown in Table 1.

[0175] Table 1 The results of the determination of various data of different strains and different culture media

[0176]

[0177] Example 3

[0178] This example is based on the above-mentioned K8824 strain, and Saccharomyces cerevisiae is added to the traditional fermentation process for fermentation, wherein the original source of Saccharomyces cerevisiae is the China Center for Type Culture Collection, and the strain collection number is CCTCC KY 2008617. The specific method is as follows:

[0179] (1) Grinding tea and seabuckthorn leaves into 100-mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 6:4 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0180] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water at a ratio of 1:20 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 20 min;

[0181] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 3%, and cultured in a shaking incubator at 28±2° C. for 24 h to obtain tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed was 120 r / min.

[0182] (4) After the fermentation of Eurotium cristatum K8824 for 72 h, the brewer's yeast seed solution was inoculated into the tea-sea buckthorn leaf fermentation solution for co-fermentation. The inoculation amount was 3%, and the culture was shaken for 24 h at 28±2°C and the shaking speed was 120 r / min.

[0183] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 3500 r / min.

[0184] (6) The obtained supernatant was filtered through a 0.15 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf composite fermentation liquid.

[0185] Example 4

[0186] This example is based on the above-mentioned K8824 strain, and Saccharomyces cerevisiae is added to the traditional fermentation process for fermentation, wherein the original source of Saccharomyces cerevisiae is the China Center for Type Culture Collection, and the strain collection number is CCTCC KY 2008617. The specific method is as follows:

[0187] (1) Grinding tea and seabuckthorn leaves into 60 mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 7:3 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0188] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water at a ratio of 5:90 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0189] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 8%, and cultured in a shaking incubator at 28±2° C. for 60 h to obtain tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed was 200 r / min.

[0190] (4) After the fermentation of Eurotium cristatum K8824 for 60 h, the brewer's yeast seed solution was inoculated into the tea-sea buckthorn leaf fermentation solution for co-fermentation. The inoculation amount was 2%, and the culture was shaken for 24 h at 28±2°C and the shaking speed was 200 r / min.

[0191] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 2800 r / min.

[0192] (6) The obtained supernatant is filtered through a 0.30 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf composite fermentation liquid.

[0193] Example 5

[0194] This example is based on the above-mentioned K8824 strain, and commercially available Angel yeast is added to the traditional fermentation process for fermentation. The specific method is as follows:

[0195] (1) Grinding tea and seabuckthorn leaves into 60 mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 6:5 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0196] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water in a ratio of 1:9 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 15 min;

[0197] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 5%, and cultured in a shaking incubator at 28±2° C. for 72 h to obtain tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed was 180 r / min.

[0198] (4) After the fermentation of Eurotium cristatum K8824 for 72 h, the Angel yeast seed solution was inoculated into the tea-sea buckthorn leaf fermentation solution for co-fermentation. The inoculation amount was 5%, and the culture was shaken for 36 h at 28±2°C and the shaking speed was 180 r / min.

[0199] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 3200 r / min.

[0200] (6) The obtained supernatant is filtered through a 0.25 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf composite fermentation liquid.

[0201] Example 6

[0202] This example is based on the above-mentioned K8824 strain, and Saccharomyces cerevisiae is added to the traditional fermentation process for fermentation, wherein the original source of Saccharomyces cerevisiae is the China Center for Type Culture Collection, and the strain collection number is CCTCC KY 2008617. The specific method is as follows:

[0203] (1) Grinding tea and seabuckthorn leaves into 100-mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 6:5 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0204] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water in a ratio of 1:9 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 20 min;

[0205] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 3%, and cultured in a shaking incubator at 28±2° C. for 24 h to obtain tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed was 120 r / min.

[0206] (4) After the fermentation of Eurotium cristatum K8824 for 24 h, the brewer's yeast seed solution was inoculated into the tea-sea buckthorn leaf fermentation solution for co-fermentation. The inoculation amount was 3%, and the culture was shaken for 24 h at 28±2°C and the shaking speed was 120 r / min.

[0207] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 3300 r / min.

[0208] (6) The obtained supernatant is filtered through a 0.15 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf composite fermentation liquid.

[0209] Example 7

[0210] This example is based on the above-mentioned K8824 strain, and Saccharomyces cerevisiae is added to the traditional fermentation process for fermentation, wherein the original source of Saccharomyces cerevisiae is the China Center for Type Culture Collection, and the strain collection number is CCTCC KY 2008617. The specific method is as follows:

[0211] (1) Grinding tea and seabuckthorn leaves into 100-mesh fine powder respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 5.5:4.5 to obtain a tea-seabuckthorn leaf fermentation substrate;

[0212] (2) mixing the tea-sea buckthorn leaf fermentation substrate with purified water in a ratio of 8:92 and stirring evenly to obtain a fermentation medium, and sterilizing at 121° C. for 20 min;

[0213] (3) The seed liquid of Eurotium cristatum K8824 was inoculated into the fermentation medium for primary fermentation, with an inoculation amount of 3%, and cultured in a shaking incubator at 28±2° C. for 24 h to obtain tea-sea buckthorn leaf fermentation liquid, wherein the shaking speed was 120 r / min.

[0214] (4) After the fermentation of Eurotium cristatum K8824 for 48 h, the brewer's yeast seed solution was inoculated into the tea-sea buckthorn leaf fermentation solution for co-fermentation. The inoculation amount was 3%, and the culture was shaken for 24 h at 28±2°C and the shaking speed was 120 r / min.

[0215] (5) After the fermentation is completed, the fermentation medium is centrifuged to obtain the supernatant at a centrifugal speed of 2500 r / min.

[0216] (6) The obtained supernatant is filtered through a 0.15 μm sterilizing filter to obtain a sterile tea-sea buckthorn leaf composite fermentation liquid.

Claims

1. A method for improving the anti-aging effect of the composite fermentation liquid of Eurotium cristatum tea, characterized in that: The steps include: (1) The activated seed liquid of Eurotium cristatum K8824 was inoculated into the tea-sea buckthorn leaf fermentation medium for primary fermentation, and cultured in a shaking incubator at 28±2℃ for 24-72 h to obtain the tea-sea buckthorn leaf fermentation medium; (2) inoculating the yeast seed liquid into the tea-sea buckthorn leaf fermentation culture medium, and fermenting them in a shaking incubator at 28±2°C for 24-36 hours; (3) After the fermentation is completed, the co-fermentation culture liquid is centrifuged to obtain a supernatant; (4) The obtained supernatant is filtered through a sterilizing filter to obtain a sterile tea-sea buckthorn leaf composite fermentation liquid.

2. The method for improving the anti-aging effect of the composite fermentation liquid of Eurotium cristatum tea according to claim 1, characterized in that: The method for preparing the activated seed solution of Eurotium cristatum K8824 is as follows: The preserved Eurotium cristatum K8824 was streaked on a PDA plate medium and activated at 28±1℃ for 48-72h. The spores on the plate were scraped and washed with sterile water. The spore liquid was fully shaken and mixed to form a spore suspension, and the concentration of the spore suspension was adjusted to 1.0×10 7 CFU / mL, and the activated seed solution of Eurotium cristatum K8824 was obtained.

3. The method for improving the anti-aging effect of the composite fermented liquid of Eurotium cristatum tea according to claim 1, characterized in that: The preparation method of the fermentation medium is: (1) Grinding tea and seabuckthorn leaves into fine powders of 40-100 mesh respectively, and then mixing the tea and seabuckthorn leaves in a mass ratio of 7:3-5:5 to obtain a tea-seabuckthorn leaf fermentation substrate; (2) Mix the tea-sea buckthorn leaf fermentation substrate with purified water in a ratio of 1:30 to 1:9 and stir evenly to obtain a tea-sea buckthorn leaf fermentation medium, which is then sterilized.

4. The method for improving the anti-aging effect of the composite fermented liquid of Eurotium cristatum tea according to claim 1, characterized in that: The yeast is Angel Yeast or Saccharomyces cerevisiae; wherein, Saccharomyces cerevisiae is deposited in China Center for Type Culture Collection, and the strain deposit number is CCTCC KY 2008617.

5. The method for improving the anti-aging effect of the composite fermented liquid of Eurotium cristatum tea according to claim 4, characterized in that: The method for preparing the brewer's yeast seed liquid is as follows: the yeast is inoculated into a YPD liquid medium and cultured for 24 to 36 hours to obtain a yeast seed liquid, and then the spore suspension is adjusted to 1.0×10 8 CFU / mL, and obtain the cerevisiae seed liquid.

6. The method for improving the anti-aging effect of the composite fermentation liquid of Eurotium cristatum tea according to claim 1, characterized in that: The inoculation amount of the seed solution of Eurotium cristatum K8824 is 3-8%.

7. The method for improving the anti-aging effect of the composite fermented liquid of Eurotium cristatum tea according to claim 1, characterized in that: The inoculation amount of the yeast seed liquid is 2-5%.

8. The method for improving the anti-aging effect of the composite fermented liquid of Eurotium cristatum tea according to claim 1, characterized in that: The shaking table speed is 120~200 r / min.

9. The method for improving the anti-aging effect of the composite fermented liquid of Eurotium cristatum tea according to claim 1, characterized in that: The centrifugal speed is 2800~3500 r / min.

10. The method for improving the anti-aging effect of the composite fermented liquid of Eurotium cristatum tea according to claim 1, characterized in that: The sterilizing filter is a sterilizing filter with a pore size of 0.15-0.30 μm.

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