A galactose yeast-like fungus purslane fermentation product and its preparation method and use

Abstract

The present invention belongs to the field of daily chemical new materials, and specifically relates to a method for preparing a galactose yeast-like bacteria purslane fermentation product, wherein the galactose yeast-like bacteria is used to ferment the fermentation culture liquid to prepare the galactose yeast-like bacteria purslane fermentation product; the purslane fermentation culture liquid comprises the following components: 0.01wt%-5wt% of purslane powder, 0.1wt%-10wt% of sugar, 0.1wt%-5wt% of yeast extract, and the balance is water. The purslane fermentation product prepared by the method is safe, mild and non-irritating, and has a good oil control effect. A second object of the present invention is to provide an application of the above-mentioned extract.

Description

Technical Field

[0001] The invention belongs to the field of daily chemical new materials, and particularly relates to a galactose yeast-like fungus purslane fermentation product and a preparation method and application thereof. Background Art

[0002] In recent years, with the development of the times and the growing needs of consumers, the innovation and transformation of the cosmetics industry has been continuously promoted. The cosmetics industry has experienced the direct application of natural plants and spices in skin care in the past primitive era, and then the widespread application of water-oil emulsification technology, which has promoted and given birth to a series of synthetic sugars, organic acids, ester alcohols and other fine chemical raw materials, which also constitute today's rich chemical system. Now with the rise of concepts such as pure beauty and functional cosmetics, it also reflects the increasing demand of the entire cosmetics industry for natural and pure functional raw materials. In order to meet consumers' pursuit of higher safety and efficacy of cosmetics, it has become an inevitable trend for the development of fermented cosmetics to make cosmetics without adding any chemical agents and only through fermentation. The preparation of plant fermented cosmetics is obtained by crushing plant raw materials or filtering and purifying plant juice after probiotic fermentation. Compared with traditional plant extraction technology, fermentation is carried out under milder conditions, while avoiding alcohol, acid and alkali and other components used in traditional plant extraction, which can retain the efficacy of plant active ingredients more comprehensively and to a higher degree.

[0003] The technical process of plant fermentation can be summarized as using abundant plant resources as raw materials, using specific microbial strains or probiotics, and combining modern biotechnology including enzyme engineering, separation and extraction, etc., to achieve effective extraction and transformation of plant ingredients by microorganisms. The advantages of plant fermentation technology in cosmetic research and development are mainly reflected in two aspects: on the one hand, the use of plant fermentation technology can break the barrier substances in the substrate, so that more active substances can be dissolved, and the effective extraction of plant active ingredients can be achieved; plant fermentation technology can also degrade and transform toxic substances in plants, making them safer raw materials than traditional plant extraction; in addition, in the process of microbial fermentation, the effective ingredients in plants can be made into small molecules, and new effective substances can be produced to obtain raw materials with better absorption; in the process of plant fermentation, the probiotics used in fermentation will also secrete a series of active ingredients that have the effect of enhancing the efficacy of the raw materials. On the other hand, plant fermentation cosmetics meet the four characteristics of cosmetics-high safety, relative stability, good usability and certain efficacy. After sterilization, separation and extraction of active ingredients to remove bacterial fragments and impurities, they can be used as terminal cosmetics or raw materials. The safety of plant fermented cosmetics is also in line with the concept of pure beauty. Since they use all-natural ingredients, they are safe for consumers' skin. To a large extent, due to the green and environmentally friendly production process, they are also clean and natural for the natural environment. In terms of efficacy, plant fermented cosmetics are achieved by gradually conditioning and improving the skin condition, rather than following the current concept of making single-site changes in whitening, anti-wrinkle, etc. Plant fermented cosmetics emphasize the overall concept.

[0004] Portulaca oleracea L., also known as Portulaca oleracea in Chinese medicine, is also known as Portulaca oleracea in Shenghuifang and Guazicai in Lingnan Caiyaolu. It is the whole herb of Portulaca oleracea, a plant of the genus Portulaca in the family Portulacaceae. Portulaca oleracea is widely produced and is distributed in many provinces in China, and is widely distributed in temperate and tropical regions around the world. There are many active ingredients in the whole herb of Portulaca oleracea, mainly alkaloids, flavonoids, terpenes and sterols, organic acids, coumarins, polysaccharides, alcohols and esters, aldehydes, glycosides, etc. Portulaca oleracea has multiple pharmacological effects, including regulating blood lipids, anti-atherosclerosis, anti-inflammatory, analgesic, antibacterial, anti-tumor, anti-oxidation, anti-aging, liver protection, enhancing the body's immunity, and effects on skeletal muscle and smooth muscle.

[0005] Fermented yeast itself contains many enzymes and trace elements, and during the fermentation process, rich nutritional products such as small molecule peptides, amino acids and small molecule polysaccharides are regenerated, making the efficacy more comprehensive. Yeast fermentation products are widely used in the field of cosmetics because they are rich in active ingredients (free amino acids, small molecule peptides, vitamins, etc.). Their main functions include whitening, moisturizing, delaying skin aging, regulating the pH of the skin and repairing damaged skin. Galactocystis is a special yeast called Saccharomyces cerevisiae. During the fermentation process, it can secrete small molecules such as vitamin B group, minerals, amino acids, organic acids, inorganic acids, etc. that are beneficial to skin care. When used in fermentation, the fermentation product has excellent skin care effects and is an excellent skin conditioner.

[0006] At present, galactose yeast-like bacteria are mainly used in the fermentation of sugars such as galactose and lactose. The products are mainly yeast-like bacteria fermentation products such as galactose yeast-like bacteria filtrate. However, its application in plant fermentation production is still blank. There is no report on the exploration of plant fermentation research and development of Purslane using galactose yeast-like bacteria and the efficacy testing and analysis of cosmetics prepared by it.

[0007] The process of purslane fermentation liquid is relatively mature. The difference between the existing processes lies in the difference in fermentation bacteria and the difference in fermentation process points. The fermentation bacteria are commonly used in combination of one or more of plant lactobacillus, yeast, and rice wine yeast.

[0008] The study found that the factors affecting the performance of Purslane fermentation broth are not limited to the above factors.

[0009] Prior art 1: CN104887563A discloses a snow chrysanthemum tea anti-allergic mask cream and a preparation method thereof, which uses probiotics Geotrichum candidum and Saccharomyces boulardii pure strain fermented licorice, oat kernels, purslane, and snow grass 5-10 parts each, and extracts of cherry leaves, comfrey leaves, neem leaves, knotweed roots, scutellaria roots, lotus, honeysuckle, peony roots, burdock leaves, elm inner bark, magnolia bark, arnica, mulberry leaves, and white willow bark;

[0010] Its main effect is anti-allergic.

[0011] After further research, it was found that when Galactomyces-like yeasts fermented plant ingredients or plant extracts to obtain good 5α-reductase inhibition, there was obvious antagonism between different plants. 5α-reductase inhibition is a very important aspect to achieve oil control.

[0012] Therefore, the technical problem solved in this case is: how to improve the oil control effect of purslane fermented liquid. Summary of the invention

[0013] In view of the deficiencies in the prior art, the first object of the present invention is to provide a galactosyl yeast-like fungus purslane fermentation product and a preparation method thereof. The purslane fermentation product prepared by the method is safe, mild and non-irritating, and has a good oil control effect.

[0014] The second object of the present invention is to provide the application of the above extract.

[0015] In order to achieve the first invention object, the present invention adopts the following technical solutions:

[0016] A method for preparing a galactosaccharomyces-like bacteria purslane fermentation product, wherein the galactosaccharomyces-like bacteria are used to ferment a purslane fermentation culture solution to obtain the galactosaccharomyces-like bacteria purslane fermentation product;

[0017] The purslane fermentation culture solution comprises the following components: 0.01wt%-5wt% purslane powder, 0.1wt%-10wt% sugar, 0.1wt%-5wt% yeast extract, and the balance is water;

[0018] The preservation number of the galactose yeast-like bacteria is: GDMCC No: 64235; preservation date: January 2, 2024; preservation unit: Guangdong Microbiological Culture Collection Center; preservation address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0019] In the above-mentioned method for preparing the galactose yeast-like bacteria purslane fermentation product, the galactose yeast-like bacteria are preliminarily activated to obtain a galactose yeast-like bacteria seed liquid, and the galactose yeast-like bacteria seed liquid is added to the purslane fermentation culture liquid for fermentation to obtain the galactose yeast-like bacteria purslane fermentation product;

[0020] The process of the activation treatment is:

[0021] The galactose yeast-like bacterial liquid is added to the sterilized activation culture medium, and the galactose yeast-like bacterial activation liquid is obtained by primary culture.

[0022] Adding galactose yeast-like bacteria activation liquid to the sterilized seed culture medium, performing secondary culture to obtain galactose yeast-like bacteria seed liquid;

[0023] The activation culture medium and the seed culture medium both contain the following ingredients: 0.1wt%-10wt% sugar, 0.1wt%-5wt% yeast extract, and the balance is water.

[0024] In the above-mentioned method for preparing the galactose yeast-like fungus purslane fermentation product, the sugars in the purslane fermentation culture solution, activation culture medium and seed culture medium are independently one or more combinations of glucose, lactose and galactose.

[0025] In the above-mentioned method for preparing the galactose yeast-like bacteria purslane fermentation product, the mass ratio of the galactose yeast-like bacteria to the purslane fermentation culture solution is 0.01-5:100; the content of live bacteria per gram of galactose yeast-like bacteria is 1x10 6 ~1x10 7 cfu / g.

[0026] In the method for preparing the galactose yeast-like fungus purslane fermentation product, the fermentation temperature is 25° C.-45° C.; and the fermentation time is 6 h-72 h.

[0027] In the above-mentioned method for preparing the galactose yeast-like fungus purslane fermentation product, the fermentation temperature is 30° C. and the fermentation time is 24 hours.

[0028] In the above-mentioned preparation method of galactose yeast-like fungus purslane fermentation product, the fermentation operation is carried out in a fermentation tank, and the fermentation program is set as follows: when fermenting for 0-4 hours, the rotation speed is set to 60 rpm; when fermenting for 4-8 hours, the rotation speed is set to 80 rpm; when fermenting for 8-16 hours, the dissolved oxygen content value of the fermentation tank is set to 20%, the stirring speed is associated with the dissolved oxygen content, and the speed is automatically compensated and adjusted; when fermenting for 16-48 hours, the rotation speed is set to 80 rpm; stirring is stopped after 48 hours of fermentation.

[0029] In the above-mentioned method for preparing the galactose yeast-like fungus purslane fermentation product, the purslane fermentation culture solution includes the following components: 0.1wt%-0.5wt% purslane powder, 0.3wt%-5wt% sugar, 0.1wt%-2wt% yeast extract, and the balance is water.

[0030] Meanwhile, the present invention also discloses a purslane fermentation product, which is prepared by using any of the above methods for preparing a galactose yeast-like fungus purslane fermentation product.

[0031] Finally, the present invention also discloses the use of the galactose yeast-like fungus purslane fermentation product as described above in preparing cosmetics.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention uses galactose yeast-like bacteria to ferment purslane fermentation culture fluid to ferment purslane. Yeast extract is introduced into the fermentation culture fluid. The yeast extract is rich in soluble components in yeast cells such as amino acids, peptides, nucleotides, etc. The role of the yeast extract in the culture fluid is to supply nutrients required for bacterial growth, thereby achieving the purpose of expanding the culture of high-density galactose yeast-like bacteria cells on the one hand, and achieving the purpose of improving the oil control effect of the fermentation product on the other hand.

[0034] The present invention utilizes liquid high-density fermentation technology to achieve high-density galactose yeast-like bacteria cell expansion culture. During its own metabolism, the galactose yeast-like bacteria can efficiently extract the active ingredients of purslane and degrade the toxicity of natural plants. On the other hand, it can secrete its own yeast active ingredients, so that the fermentation product of the galactose yeast-like bacteria purslane has good safety, mildness and non-irritation, and has good oil control effect.

[0035] As a further optimization of the present invention, by performing secondary activation culture on the galactose yeast-like bacteria and using yeast extract as one of the active ingredients of the activation culture medium, the activity of the galactose yeast-like bacteria can be improved, making it more adaptable to the purslane fermentation culture fluid, and further achieving the purpose of expanding the culture of high-density galactose yeast-like bacteria cells.

[0036] At the same time, the present invention also selected a variety of plant ingredients recognized in the art as having excellent 5α-reductase inhibitory effects, such as Platycladus orientalis leaves (Research progress on the chemical composition, physiological activity and anti-hair loss function of Platycladus orientalis leaves, "China Wild Plant Resources", June 2010), Polygonum multiflorum (Research on the anti-hair loss efficacy of composite extracts of Platycladus orientalis leaves and Polygonum multiflorum, "China Wild Plant Resources", May 2021), and Ligustrum lucidum (Screening of type II 5α-reductase inhibitors from traditional Chinese medicine and research on the hair growth promoting effect of Ligustrum lucidum alcohol extract, May 2020, Guangdong Pharmaceutical University, master's thesis) and Portulaca oleracea for composite fermentation, which showed obvious antagonism.

[0037] The above verification process shows that the fermentation culture of purslane with galactose yeast-like bacteria excludes the co-fermentation of similar functional plant ingredients in terms of 5α-reductase inhibition, and the effect of single fermentation is significantly better than that of compound fermentation. This has proposed a new direction for the development of oil control function of purslane. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a graph showing the test results of the chicken embryo chorioallantoic membrane of the formula of Example 1 of the present invention (0min);

[0039] Figure 2 This is a graph showing the test results of the chicken embryo chorioallantoic membrane of the formula of Example 1 of the present invention (5 min);

[0040] Figure 3 This is the result of the chicken embryo chorioallantoic membrane test using normal saline (0min);

[0041] Figure 4 This is the result of the chicken embryo chorioallantoic membrane test using normal saline (5min);

[0042] Figure 5 This is the result of the chicken embryo chorioallantoic membrane test using sodium hydroxide solution (0min);

[0043] Figure 6 This is a graph showing the test results of chicken embryo chorioallantoic membrane using sodium hydroxide solution (5 minutes). DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0045] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0046] Supplier information and / or product information

[0047] Purslane powder: Haozhou Yuanshengtang Pharmaceutical Co., Ltd., primary agricultural product, 3000g / bag, packaging date 2024.3.23;

[0048] Lactose imported from the United States: Zhengzhou Jieshang Biotechnology Co., Ltd., 1000g / bag, production date 2022.12.24;

[0049] Yeast extract: Guangzhou Dingguo Biotechnology Co., Ltd. 500g / bottle, brand OXOID, model LP0021B, source: imported;

[0050] The deposit information of Galacto-Saccharomyces cerevisiae is as follows:

[0051] The taxonomic name of the galactose yeast-like fungus is: Geotrichum candidum; the Latin name of the galactose yeast-like fungus is: Geotrichum candidum; the galactose yeast-like fungus is deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), with the deposit number: GDMCC NO: 64235, the deposit date: January 2, 2024; the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0052] Example 1

[0053] Preparation of a galactosyl yeast-like fungus purslane fermentation product

[0054] Step 1: Prepare activation medium and seed medium by mixing 20g glucose, 5g yeast extract and the rest water per kg, adjust the pH value of the solution to 6.0, sterilize the medium at 121°C for 20 min, add 1g frozen galactose yeast sample solution (concentration: 1x106 cfu / g) were cultured in the activation medium. The constant temperature was set at 28°C and cultured for 24 hours to obtain the activated solution of galactosaccharomyces cerevisiae. After testing, the concentration of live bacteria in the activated solution of galactosaccharomyces cerevisiae was 0.9x10 8 cfu / g.

[0055] Step 2: After the primary culture, fresh galactosaccharomyces cerevisiae activated liquid was inoculated into the seed culture medium and cultured at 28°C for 24 hours to obtain galactosaccharomyces cerevisiae seed liquid. The concentration of live bacteria in the galactosaccharomyces cerevisiae seed liquid was 1.5x10 9 cfu / g.

[0056] The weight of the galactose yeast-like bacteria activation solution added to every 1 kg of seed culture medium is 2 g.

[0057] Step 3: Prepare a fermentation medium by mixing 20g of glucose, 10g of lactose, 2.5g of purslane powder, 10g of yeast extract, and the balance of water per kg. After the secondary culture is completed, fresh galactose yeast seed liquid is inoculated into the fermentation medium, 2g of galactose yeast-like bacteria seed liquid is inoculated into each kg of the fermentation medium, the temperature is set to a constant temperature of 30°C, the total fermentation time is 48h, and the fermentation program is set as follows: when fermenting for 0-4h, the speed is set to 60 rpm; when fermenting for 4-8h, the speed is set to 80 rpm; when fermenting for 8-16h, the dissolved oxygen content value of the fermentation tank is set to 20%, the stirring speed is associated with the dissolved oxygen content, and the speed is automatically compensated and adjusted; when fermenting for 16-48h, the speed is set to 80 rpm; stirring is stopped after 48h fermentation.

[0058] After the fermentation culture is completed, the fermentation broth is sterilized at 121°C for 20 minutes under high temperature and high pressure, and then the bacteria and the fermentation broth are separated at 15,000 rpm, and the fermentation broth supernatant is collected after centrifugation. The sterilized fermentation broth supernatant is clarified and refined to obtain the galactose yeast-like fungus purslane fermentation product.

[0059] Example 2

[0060] Preparation of a galactosyl yeast-like fungus purslane fermentation product

[0061] The preparation method is the same as that of Example 1, except that the added amount of purslane powder in the fermentation medium is 2.5 g, glucose is 25 g, lactose is 12.5 g, and yeast extract is 20 g.

[0062] 1 kg of activation medium and seed medium contains 25 g of glucose, 12.5 g of lactose and 20 g of yeast extract.

[0063] Example 3

[0064] The preparation method is the same as that of Example 1, except that the added amount of purslane powder in the fermentation medium is 0.25 g, glucose is 10 g, lactose is 10 g, and yeast extract is 2 g.

[0065] 1 kg of activation medium and seed medium contains 10 g of glucose, 10 g of lactose and 2 g of yeast extract.

[0066] Example 4

[0067] The preparation method is the same as that of Example 1, except that the added amount of purslane powder in the fermentation medium is 0.125 g, glucose is 40 g, lactose is 5 g, and yeast extract is 15 g.

[0068] 1 kg of activation medium and seed medium contains 40 g of glucose, 5 g of lactose and 15 g of yeast extract.

[0069] Example 5

[0070] The preparation method is the same as that of Example 1, except that 5 g of galactosidase-like bacteria seed liquid is inoculated into each kg of fermentation medium.

[0071] Example 6

[0072] The preparation method is the same as that of Example 1, except that 10 g of galactosidase-like bacteria seed liquid is inoculated into each kg of fermentation medium.

[0073] Example 7

[0074] Preparation of a galactosyl yeast-like fungus purslane fermentation product

[0075] The preparation method is the same as that of Example 1, except that the fermentation medium contains 20 g of Tremella fuciformis extract, 20 g of glucose, 10 g of lactose, 2.5 g of purslane powder, and 10 g of yeast extract.

[0076] 1 kg of activation culture medium and seed culture medium contains 10 g of tremella extract, 25 g of glucose, 12.5 g of lactose and 20 g of yeast extract.

[0077] Example 8

[0078] Preparation of a fermentation product of galactosyl yeast-like bacteria, purslane and Polygonum multiflorum

[0079] The preparation method is the same as that of Example 1, except that the fermentation medium is prepared by mixing 20 g glucose, 10 g lactose, 2.5 g purslane powder, 2.5 g Polygonum multiflorum powder, 10 g yeast extract, and the balance water per 1 kg.

[0080] 1 kg of activation culture medium and seed culture medium contains 10 g of tremella extract, 25 g of glucose, 12.5 g of lactose and 20 g of yeast extract.

[0081] Example 9

[0082] Preparation of a fermentation product of galactosyl yeast-like fungus, purslane and oriental arborvitae

[0083] The preparation method is the same as that of Example 1, except that the fermentation medium is prepared by mixing 20 g glucose, 10 g lactose, 2.5 g purslane powder, 2.5 g orientalis powder, 10 g yeast extract, and the balance water per kg.

[0084] 1 kg of activation culture medium and seed culture medium contains 10 g of tremella extract, 25 g of glucose, 12.5 g of lactose and 20 g of yeast extract.

[0085] Example 10

[0086] Preparation of a fermentation product of galactosyl yeast-like bacteria, purslane and Ligustrum lucidum

[0087] The preparation method is the same as that of Example 1, except that the fermentation medium is prepared by mixing 20 g of glucose, 10 g of lactose, 2.5 g of purslane powder, 2.5 g of Ligustrum lucidum powder, 10 g of yeast extract, and the balance of water per 1 kg.

[0088] 1 kg of activation culture medium and seed culture medium contains 10 g of tremella extract, 25 g of glucose, 12.5 g of lactose and 20 g of yeast extract.

[0089] Embodiment 11

[0090] The method is substantially the same as Example 1, except that: in step 3, the fermentation procedure is as follows: the dissolved oxygen content in the fermentation tank is set to 30%, the stirring speed is related to the dissolved oxygen content, the speed is automatically compensated and adjusted, and the fermentation time is 24 hours at 30°C.

[0091] Example 12

[0092] The method is substantially the same as Example 1, except that: in step 3, the fermentation procedure is as follows: the dissolved oxygen content in the fermentation tank is set to 30%, the stirring speed is related to the dissolved oxygen content, the speed is automatically compensated and adjusted, and the fermentation time is 48 hours at 30°C.

[0093] Performance Testing

[0094] Test Example 1 Safety test of galactosyl yeast-like fungus purslane fermentation product

[0095] Test Method

[0096] Chicken chorioallantoic membrane test

[0097] Control and test sample treatment

[0098] Sample: The fermentation product of galactosyl yeast and purslane from Example 1.

[0099] Negative control: normal saline.

[0100] Positive control: 0.1 mol / L NaOH solution.

[0101] Test operation steps

[0102] In this test, 6 chicken embryos were selected for each group, and the conditions of the chorioallantoic membrane were recorded with a camera. A polytetrafluoroethylene resin ring was placed on the chorioallantoic membrane of the chicken embryo and photographed. The sample to be tested was added to the polytetrafluoroethylene resin ring, the time of adding the sample was recorded, and the air chamber was covered with a moistened plastic wrap. The chicken embryo was moved to a constant temperature and humidity chamber for culture, and the degree of change of each toxic effect was observed.

[0103] Calculation results:

[0104] The endpoint evaluation method was used to perform the test, and the endpoint evaluation (ES) was calculated, with the result retained to two decimal places; the score of each chicken embryo = the sum of the bleeding, coagulation and vascular melting observed in each chicken embryo; the average value of the mathematical sum obtained for ES-6 chicken embryos. The average ES score was calculated according to the following formula.

[0105]

[0106] Result judgment criteria:

[0107] ES≤4, non-irritating; 4<ES≤12, mildly irritating; 12<ES<16, moderately irritating; ES≥16, strongly irritating / corrosive.

[0108] The test results of the chicken embryo chorioallantoic membrane test are shown in Table 1.

[0109] Table 1

[0110] Points result Negative control 0.00 Non-irritating Example 1 3.67 Non-irritating Positive Control 18.00 Strong irritation

[0111] As shown in Table 2, the purslane fermentation product prepared in Example 1 is non-irritating to the chicken embryo chorioallantoic membrane. After simultaneous verification, the purslane fermentation products of Saccharomyces cerevisiae prepared in Examples 2 to 12 are non-irritating to the chicken embryo chorioallantoic membrane.

[0112] Test Example 2 Efficacy test of purslane fermentation product

[0113] 1. Test Method

[0114] (1) 5α-reductase inhibition test

[0115] The experiment set up 4 groups, namely: sample group, positive control group, negative control group and blank group;

[0116] Sample group: Add 0.25mL PBS, 0.7mL enzyme extract, 0.6mL sample solution, 0.1mL testosterone solution and 0.35mL NADPH, react the test tube at 37℃ for 30min, add 2mL dichloromethane to stop the reaction after the reaction, shake for 1min and centrifuge at 3000r / min for 10min. Remove the upper aqueous phase and take 1mL of the lower organic phase (in the "dichloromethane-water" system, the white gel-like layer will separate the two phases in the middle). Evaporate the organic phase to dryness. The white powdery residue is dissolved in 1mL methanol.

[0117] Positive control group: add 0.25mL PBS, 0.7mL enzyme extract, 0.6mL finasteride solution, 0.1mL testosterone solution and 0.35mL NADPH, react the test tube at 37℃ for 30min, add 2mL dichloromethane to stop the reaction after the reaction, shake for 1min and centrifuge at 3000r / min for 10min. Remove the upper aqueous phase, take 1mL of the lower organic phase, evaporate the organic phase to dryness, and dissolve the white powdery residue in 1mL methanol.

[0118] Blank group: add 0.25mL PBS, 0.7mL enzyme extract, 0.6mL 75% ethanol solution, 0.1mL testosterone solution and 0.35mL NADPH, react the test tube at 37℃ for 30min, add 2mL dichloromethane to stop the reaction after the reaction, shake for 1min and centrifuge at 3000r / min for 10min. Remove the upper aqueous phase, take 1mL of the lower organic phase, evaporate the organic phase to dryness, and dissolve the white powdery residue in 1mL methanol.

[0119] Negative control group: add 0.25mL PBS, 0.7mL enzyme extract, 0.6mL PBS solution, 0.1mL testosterone solution and 0.35mL NADPH, directly add 2mL dichloromethane to stop the reaction, shake for 1min and centrifuge at 3000r / min for 10min. Remove the upper aqueous phase, take 1mL of the lower organic phase, evaporate the organic phase to dryness, and dissolve the white powdery residue in 1mL methanol.

[0120] Each group was subjected to HPLC analysis under the following analysis conditions: C18 (250 mm × 4.6 mm, 5 μm), column temperature 40 ° C, mobile phase methanol: water = 65:65 (v / v), flow rate 1 mL / min, UV detector, detection wavelength 242 nm.

[0121] Result calculation

[0122] Testosterone standard curve: Testosterone standard solutions of different concentrations were analyzed by HPLC, and a standard curve was prepared with concentration (X) as the horizontal axis and peak area (Y) as the vertical axis.

[0123] The testosterone content in the reaction solution was determined under the above chromatographic conditions, and the strength of the inhibitory activity was characterized by the inhibition rate, as shown below:

[0124]

[0125] Result judgment criteria

[0126] The 5α-reductase inhibition rate of the sample is higher than that of the negative control and has a significant difference (P < 0.05), which means that the test sample can be considered to have a certain oil control effect.

[0127] 2. Test results:

[0128] (1) The test results of 5α-reductase inhibition rate are shown in Table 3.

[0129] Table 3

[0130]

[0131]

[0132] Result analysis:

[0133] 1. As shown in Examples 1 to 4 in Table 3, the 5α-reductase inhibition rate decreased significantly with a significant decrease in the amount of purslane powder.

[0134] 2. It can be seen from Example 1, Example 5 and Example 6 in Table 3 that as the amount of seed liquid inoculated increases, the 5α-reductase inhibition rate increases accordingly. From an economic point of view, it is recommended to inoculate 2-5 g of galactosidase-like bacteria seed liquid per kg of fermentation medium.

[0135] 3. It can be seen from Example 1 and Example 7 in Table 3 that Tremella extract cannot improve the 5α-reductase inhibition rate, and has a certain antagonistic effect on the 5α-reductase inhibition rate of the fermentation broth.

[0136] 4. It can be seen from Examples 1, 8, 9 and 10 in Table 3 that the use of various Chinese herbal ingredients with potential 5α-reductase inhibition affects the efficacy of Portulaca oleracea powder, especially the antagonism of Ligustrum lucidum is more significant.

[0137] 5α-reductase is an important evaluation item for anti-hair loss. The results of Examples 8, 9 and 10 above prove from another perspective that not all the addition of other anti-hair loss ingredients is beneficial to the anti-hair loss effect of the purslane fermentation product. An optional implementation method is to blend the purslane fermentation product with other important extracts and sell and promote them as anti-hair loss products.

[0138] 5. As can be seen from Example 1, Example 11 and Example 12 in Table 3, the optimization of the fermentation procedure of the present invention is conducive to the improvement of the 5α-reductase inhibition rate.

[0139] Application example: An essence consists of phase A, phase B, phase C and phase D.

[0140] Phase A contains the following components in percentage by mass: 72.79 g water, 5 g glycerin, 0.25 g carbomer, 0.1 g sodium hyaluronate, and 0.05 g EDTA-disodium;

[0141] Phase B contains the following components in percentage by mass: 2 g water, 0.25 g arginine;

[0142] Phase C contains the following components in percentage by mass: 0.05 g of caprylhydroxamic acid, 0.5 g of 1,2-hexanediol, 1 g of butanediol, and 0.05 g of ethylhexylglycerol;

[0143] Phase D contained the following components in percentage by mass: 17.96 g of Galactomyces purslane fermentation filtrate;

[0144] A method for preparing an essence liquid comprises the following steps: dissolving phase B completely in advance, heating phase C in advance and dissolving completely for later use, adding phase A into an emulsifying pot, stirring and heating to 85°C, homogenizing for 3-5 minutes, keeping warm and stirring for 20 minutes, cooling to 75°C, adding the pre-dissolved phase B, continuing to cool to 40°C, adding the pre-dissolved phase C, stirring evenly, finally adding phase D, stirring at 30r / min for 10 minutes, and obtaining an essence liquid.

Claims

1. A method for preparing a galactosyl yeast-like fungus purslane fermentation product, characterized in that: The galactose yeast-like bacteria purslane fermentation product is prepared by fermenting the purslane fermentation culture liquid with galactose yeast-like bacteria; The purslane fermentation culture solution comprises the following components: 0.01wt%-5wt% purslane powder, 0.1wt%-10wt% sugar, 0.1wt%-5wt% yeast extract, and the balance is water; The deposit number of the galactosyl yeast-like bacteria is GDMCC No: 64235; the deposit date is January 2, 2024; Depository: Guangdong Microbiological Culture Collection Center; Deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The method for preparing the galactosyl yeast-like fungus purslane fermentation product according to claim 1, characterized in that: The galactose yeast-like bacteria are pre-activated to obtain a galactose yeast-like bacteria seed liquid, and the galactose yeast-like bacteria seed liquid is added to a purslane fermentation culture liquid for fermentation to obtain a galactose yeast-like bacteria purslane fermentation product; The process of the activation treatment is: Adding galactose yeast-like bacterial liquid to the sterilized activation medium, and performing primary culture to obtain galactose yeast-like bacterial activation liquid; Adding galactose yeast-like bacteria activation liquid to the sterilized seed culture medium, performing secondary culture to obtain galactose yeast-like bacteria seed liquid; The activation culture medium and the seed culture medium both contain the following ingredients: 0.1 wt%-10 wt% of sugar, 0.1 wt%-5 wt% of yeast extract, and the balance is water.

3. The method for preparing the galactosaccharomyces-like fungus purslane fermentation product according to claim 2, characterized in that: The sugars in the purslane fermentation culture solution, activation culture medium and seed culture medium are independently one or more combinations of glucose, lactose and galactose.

4. The method for preparing the galactosaccharomyces-like fungus purslane fermentation product according to claim 1, characterized in that: The mass ratio of the galactose yeast-like bacteria to the purslane fermentation culture liquid is 0.01-5:100; the content of live bacteria per gram of galactose yeast-like bacteria is 1×10 6 ~1x10 7 cfu / g.

5. The method for preparing the galactosaccharomyces-like fungus purslane fermentation product according to claim 1, characterized in that: The fermentation temperature is 25°C-45°C; the fermentation time is 6h-72h.

6. The method for preparing the galactosaccharomyces-like fungus purslane fermentation product according to claim 5, characterized in that: The fermentation temperature is 30°C; the fermentation time is 48h.

7. The method for preparing the galactosaccharomyces-like fungus purslane fermentation product according to claim 6, characterized in that: The fermentation operation is carried out in a fermentation tank, and the fermentation program is set as follows: when fermenting for 0-4 hours, the rotation speed is set to 60 rpm; when fermenting for 4-8 hours, the rotation speed is set to 80 rpm; when fermenting for 8-16 hours, the dissolved oxygen content value of the fermentation tank is set to 20%, the stirring speed is associated with the dissolved oxygen content, and the speed is automatically compensated and adjusted; when fermenting for 16-48 hours, the rotation speed is set to 80 rpm; stirring is stopped after 48 hours of fermentation.

8. The method for preparing the galactosyl yeast-like fungus purslane fermentation product according to claim 1, characterized in that: The purslane fermentation culture solution comprises the following components: 0.01wt%-5wt% purslane powder, 0.3wt%-5wt% sugar, 0.1wt%-2wt% yeast extract, and the balance is water.

9. A purslane fermentation product, characterized in that: The product is prepared by the method for preparing a galactosaccharomyces-like fungus purslane fermentation product as described in any one of claims 1 to 8.

10. Use of the purslane fermentation product according to claim 9 in preparing cosmetics.

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