Fermentation filtrate with anti-wrinkle effect and preparation method thereof

Abstract

According to the fermentation filtrate with the anti-wrinkle effect and the preparation method, purely natural rice or brown rice is gelatinized to form a natural culture medium, so that the use of a synthetic culture medium and a chemical reagent is completely avoided, and a purely natural daily chemical care raw material without chemical residues can be obtained; rhizopus and fermentation brightening factors are subjected to mixed fermentation. The method has the technical advantages that the mixed bacteria are fermented at the same time, additional step-by-step fermentation culture is not needed, fermentation product filtrate without obvious ethanol can be obtained, the filtrate can remarkably improve the content of active ingredients such as exopolysaccharides, small molecule peptides and organic acids, and the obtained fermentation liquor has a remarkable improvement effect on skin inflammation repair.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a fermentation filtrate with anti-wrinkle effect and a preparation method thereof. Background Art

[0002] At present, people have conducted more in-depth research on the preparation method of the galactose yeast-like bacteria fermentation product filtrate, hoping to optimize the preparation process, make the quality more stable, and make the efficacy of the galactose yeast-like bacteria fermentation product filtrate equivalent to or even better than Pitera.

[0003] Chinese patent CN116869870A discloses a galactosaccharomyces-like fermentation product filtrate with moisturizing, firming and soothing effects, as well as a preparation method and application thereof. The method is to obtain a galactosaccharomyces-like fermentation product by fermenting the independently discovered and isolated Bethmus spores (preservation number is CCTCC NO:M 2023647) in a fermentation matrix containing grain mash, lactose and milk powder. The galactosaccharomyces-like fermentation product filtrate prepared by testing and comparison of the independently isolated Bethmus spores has good repair effect.

[0004] Chinese patent CN115125153A discloses the use of Trichosporon asahii in the preparation of galactosaccharomyces fermentation product filtrate. The galactosaccharomyces fermentation product filtrate prepared by fermenting Trichosporon asahii inhibits the oxidative stress of pro-inflammatory cytokines by activating the antioxidant system, has excellent antioxidant and anti-inflammatory properties, and can be used to produce antioxidant and anti-inflammatory products.

[0005] The existing patent can enrich the preparation method of the filtrate of the fermentation product of galactose yeast-like bacteria, and effectively improve the skin condition to a certain extent. However, the existing method requires additional enzymatic hydrolysis and conversion treatment of the substrate and then enzyme inactivation treatment. The fermentation needs to be carried out in steps. The ethanol content produced by fermentation is very high, and ethanol is highly irritating to the skin and can easily cause skin allergies. High-content ethanol cannot be directly used in skin care, and an additional ethanol removal process needs to be added. Therefore, the manufacturing process is relatively complex and has a great impact on the activity of the product. Summary of the invention

[0006] The purpose of the present invention is to provide a fermentation filtrate with anti-wrinkle effect and a preparation method thereof, so as to solve the technical problems that the filtrate of the traditional galactose yeast-like fermentation product contains ethanol, the process steps are complicated, and the activity of the product is reduced.

[0007] In order to solve the above technical problems, the specific technical solutions of the present invention are as follows:

[0008] In some embodiments of the present application, a fermentation filtrate with anti-wrinkle effect and a preparation method thereof are provided, comprising the following steps:

[0009] 1) preparing a base material: grinding one of rice or brown rice to obtain a powder, and steaming the powder;

[0010] 2) Dispersing the base material: adding rice flour or brown rice flour to the cooked powder, and dispersing and cooling;

[0011] 3) Sterilization treatment: After the cooled base material is heated to gelatinize, it is transferred to a fermentation tank for sterilization to obtain alcohol-free galactose yeast-like bacteria;

[0012] 4) Fermentation treatment: placing the non-alcoholic galactose yeast-like bacteria and the amplified Rhizopus seed solution in a culture medium for mixed fermentation;

[0013] 5) Post-treatment: The mixed solution after the fermentation treatment is subjected to high-pressure homogenization and bacteria-breaking treatment to obtain the alcohol-free galactose yeast-like bacteria fermentation product filtrate.

[0014] In some embodiments of the present application, in step 1, one of the rice or the brown rice is crushed to 100-140 mesh, and 95 parts of deionized water are added and the temperature is raised to 60° C. for steaming.

[0015] In some embodiments of the present application, the fraction of rice flour or brown rice flour in step 2 is 8 portions.

[0016] In some embodiments of the present application, the base material in step 3 is heated to 80°C, gelatinized for 3 hours, and then the fermentation tank is sterilized and cooled to 35°C.

[0017] In some embodiments of the present application, the alcohol-free galactose yeast-like bacteria in step 4 is a yeast factor, which has been deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.31156.

[0018] In some embodiments of the present application, the amplified Rhizopus seed liquid in step 4 is Rhizopus oryzae.

[0019] In some embodiments of the present application, the ratio of the amplified Rhizopus seed liquid to the alcohol-free galactose yeast-like bacteria in step 4 is 2:1, and the culture medium is inoculated for fermentation. The fermentation culture is started at a temperature of 28°C and a rotation speed of 220r; after 24 hours, the temperature is increased to 35°C and the stirring speed is 180r; the total fermentation time is 72h.

[0020] In some embodiments of the present application, the high-pressure homogenization sterilization treatment in step 5 has a sterilization condition of 60 MPa, a single time of 30-45 minutes, and the sterilization is performed twice.

[0021] In some embodiments of the present application, the alcohol-free galactose yeast-like bacteria fermentation product filtrate prepared from the alcohol-free galactose yeast-like bacteria fermentation product filtrate has the functions of removing spots, repairing skin wrinkles and relieving inflammation.

[0022] In some embodiments of the present application, the alcohol-free galactose yeast-like fermentation product filtrate having the effects of removing spots, repairing skin wrinkles and relieving inflammation is used in the preparation of cosmetics.

[0023] Compared with the prior art, the beneficial effect of the present invention is that the natural culture medium is made of pure natural rice or brown rice after gelatinization, which completely avoids the use of synthetic culture medium and chemical reagents, and is conducive to obtaining pure natural daily care raw materials without chemical residues; by fermenting Rhizopus and fermentation factors with mixed bacteria. The technical advantage is that mixed bacteria are fermented simultaneously, and no additional step-by-step fermentation culture is required, and a fermentation product filtrate without obvious ethanol can be obtained. The filtrate can significantly increase the content of active ingredients such as exopolysaccharides, small molecule peptides, and organic acids, so that the obtained fermentation liquid has a significant improvement effect on skin repair and inflammation repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0025] Figure 1 A schematic diagram of the yeast strain provided by the embodiment of the present invention;

[0026] Figure 2 A schematic diagram of the test results of a DPPH free radical scavenging experiment provided in an embodiment of the present invention;

[0027] Figure 3 A schematic diagram of the ABTS free radical scavenging activity provided by an embodiment of the present invention;

[0028] Figure 4 A schematic diagram of amino acid composition analysis provided in an embodiment of the present invention.

[0029] Figure 5 Schematic diagram of the yeast bright factor strain under a microscope provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0030] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0031] In order to better understand the purpose, structure and function of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings.

[0032] See attached Figure 1-5 As shown, according to some embodiments of the present application, the following steps are included:

[0033] 1) preparing base material: grinding rice or brown rice powder to 100-140 mesh, and heating and steaming 95 parts of deionized water to 60° C.;

[0034] 2) Dispersing the base material: adding rice flour or brown rice flour to the cooked powder, and dispersing and cooling;

[0035] 3) Sterilization treatment: after the cooled base material is heated and gelatinized, it is transferred to a fermentation tank for sterilization to obtain alcohol-free galactose yeast-like bacteria, wherein the base material is heated to 80° C. for gelatinization for 3 hours, transferred to a fermentation tank for sterilization, and cooled to 35° C.; it should be noted that the galactose yeast-like bacteria is specifically the yeast factor, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, and the deposit number is CGMCC No.31156;

[0036] The yeast bright factor strain, cells cultured under aerobic conditions or microaerobic conditions have an oblong shape, and have arthrospores but no conidia. It comes from naturally fermented millet juice, collected in Changshou County, Chongqing, and identified as a galactose yeast-like fungus by fungal 26srDNA. The strain yeast bright factor is round or oval under a microscope; it grows on a solid plate of malt juice and can form milky white, dull colonies with neat edges; such as Figure 1 As shown, the bacteria grow evenly in the malt wort liquid culture medium, and the bacteria present a white precipitate after long-term storage. The optimal growth temperature is 28°C.

[0037] Nucleic acid identification

[0038] Pick a single colony and place it in malt juice liquid medium. After culturing at 30℃ overnight, centrifuge at 10,000 rpm for 5 minutes to collect the bacteria, and operate according to the steps of the yeast genomic DNA extraction kit. The primers used are yeast universal primers NL1 and NL4, and the PCR amplification system is a 50μL system, 95℃ pre-denaturation for 5 minutes, 94℃ for 1 minute, 52℃ for 1 minute, 72℃ for 90 seconds, 36 cycles; 72℃ extension for 10 minutes.

[0039] After comparing the sequencing results of the PCR products with the standard sequences published in GenBank, it was concluded that the yeast strain was a galactosaccharomyces-like bacterium.

[0040] 4) Fermentation treatment: the amplified Rhizopus seed solution (preferably Rhizopus oryzae) and the fermentation factor seed solution are inoculated into the culture medium at a ratio of 2:1 for fermentation, the fermentation culture is started at a temperature of 28°C and a rotation speed of 220r; after 24 hours, the temperature is raised to 35°C and the stirring speed is 180r; the total fermentation time is 72h;

[0041] 5) Post-treatment: After fermentation, the fermented material is subjected to high-pressure homogenization to break the bacteria. The breaking condition is 60Mpa, the time is 30-45 minutes, and the bacteria are broken twice.

[0042] Physical and chemical indicators of the filtrate of the prepared alcohol-free galactose yeast-like fermentation product

[0043] 1. Test Method

[0044] (1) Appearance and smell:

[0045] 100 g of the filtrate of the fermentation product of the galactosidase-like bacteria obtained in the example and the comparative example and the SK-II finished product were placed in a clean beaker respectively, and the appearance was observed and the odor was smelled.

[0046] (2) Solid content test:

[0047] 2 g (m0) of the galactosaccharomyces fermentation product filtrate obtained in the embodiment and the comparative example and the SK-II finished product were respectively placed at 95°C for drying for 120 min, taken out and placed in a dryer, cooled to 25°C, and weighed m1; then the solid content was calculated according to the formula solid content = (m1 / m0)*100%.

[0048] (3) Irritation test:

[0049] The filtrate of the galactose yeast-like bacteria fermentation product obtained in the example was subjected to multiple skin irritation tests according to the method of Chapter 6.4 of the "Technical Specification for Safety of Cosmetics" (2015 edition), and the acute eye irritation test was conducted according to the method of Chapter 6.5 of the "Technical Specification for Safety of Cosmetics" (2015 edition).

[0050] (4) Detection of bacterial concentration and component content:

[0051] The filtrates of the galactose yeast-like bacteria fermentation products obtained in the example and the comparative example were respectively centrifuged at a speed of 3500 r / min for 30 min, the supernatant was discarded, the wet bacteria were collected, and the concentration of the wet bacteria in the filtrate, as well as the contents of small molecule polypeptides, total sugars, amino acids, and organic acids in the filtrate / essence were calculated.

[0052]

[0053]

[0054] The performance of the alcohol-free galactose yeast-like bacteria fermentation broth in terms of solid content, appearance, odor, wet bacterial concentration, small molecule polypeptide content, total sugar content, amino acid content, organic acid content, etc. is significantly better than that of the existing patents and the comparison samples of SK-II Magic Water. It can be seen that the present invention uses the fermentation factor as the fermentation bacteria, adopts the oxygen-controlled fermentation culture mode, and combines the natural culture medium of rice, which not only improves the appearance and smell of the galactose yeast-like bacteria fermentation product filtrate, but also makes the galactose yeast-like bacteria fermentation product filtrate light yellow and transparent, and obtains the fermentation broth with only trace amounts of ethanol, and does not require a professional ethanol removal process. The galactose yeast-like bacteria fermentation broth prepared and fermented according to the Chinese patents CN 115125153 A and CN 116869870A technology has a high ethanol content, which requires special removal before use. Therefore, the preparation technology of the alcohol-free galactose yeast-like bacteria fermentation broth is simpler and more efficient than the existing patents and has obvious advantages. The use of the substrate is very simple, the physical and chemical indicators are excellent and stable, and the efficiency of bacterial fermentation and the content of secondary metabolites can be significantly improved.

[0055] Determination of DPPH clearance rate

[0056] DPPH (2-2diphenyl hydrazy) is a stable free radical with non-shared electrons (N·) inside the molecule. When it reacts with electrons and free radicals, it will be converted into a stable structure (NH). DPPH appears purple in aqueous solution, but when the free radicals are removed and become a covalent bond structure, the color will turn into light yellow. At this time, we use a spectrophotometer to measure in the range of 515-517nm to determine the DPPH free radical scavenging ability.

[0057] Methods: Prepare 0.2mmol DPPH solution with 95% ethanol, take 100μL DPPH solution into 96-well plate, then add 100μL sample solution of different concentrations (0.1-10mg / mL), shake and mix, incubate at room temperature in the dark for 30min, replace sample solution with ethanol in control group, and measure absorbance at 517nm with microplate reader. The calculation formula of DPPH free radical scavenging activity is as follows:

[0058] DPPH free radical scavenging rate (%) = (A control - A sample) / A control × 100; where A control and A sample are the absorbance values ​​of the control group and the experimental group, respectively.

[0059] DPPH free radical scavenging test results

[0060] like Figure 2As shown, all three exhibited DPPH radical scavenging activity with a dose effect (P<0.05), but the scavenging ability of the filtrate of the fermentation product of alcohol-free galactosyl yeast was stronger at the same dose. When its concentration increased to 5 mg / mL, the DPPH radical scavenging activity was 101%8±1.55%, respectively, indicating that the filtrate of the fermentation product of alcohol-free galactosyl yeast had a stronger DPPH radical scavenging activity.

[0061] Hydroxyl radical scavenging activity

[0062] Hydroxyl free radicals are the most active type of free radicals, which can react with substances such as amino acids, proteins, lipids, and nucleic acids, leading to peroxidation of amino acids, proteins, lipids, etc. and DNA mutation, thereby causing oxidative damage.

[0063] Take a 96-well plate, add 30 μL 1,10-phenanthroline (5.0 mM), 50 μL FeSO 4 (5.0 mM), 50 μL EDTA (15 mM) and 30 μL PBS buffer (0.2 M, pH 7.4) to 30 μL sample group solutions of different concentrations (1-50 mg / mL), mix well, add 30 μL H2O2 (1%) to start the Fenton reaction, incubate at 37 ° C for 1 h, and then measure the absorbance value at 536 nm. The sample solution is replaced by deionized water in the damaged group, and the sample group solution and H2O2 are replaced by deionized water in the non-damaged group. The hydroxyl radical scavenging activity is calculated as follows:

[0064] Hydroxyl radical scavenging rate (%) = (A sample - A damage) / (A non-damage - A damage) × 100; where A sample, A damage and A non-damage are the absorbance values ​​of the experimental group, the damaged group and the non-damaged group, respectively.

[0065] About ABTS free radical scavenging activity

[0066] The ABTS free radical scavenging activity evaluation system is applicable to both fat-soluble and water-soluble compounds, and is particularly suitable for free radical scavenging activity evaluation of peptide substances such as protein hydrolysates. In the ABTS system, the antioxidant components in the filtrate of the fermentation product of non-alcoholic galactose yeast can react with free radicals as electron donors or proton donors.

[0067] ABTS reagent with a concentration of 7mM and potassium persulfate with a volume ratio of 1:1 were mixed and incubated at room temperature in the dark for 12 to 16 hours to prepare ABTS+ solution. Before use, it was diluted with 5mM PBS buffer to an absorbance of 0.70±0.02 at 734nm. 150μL of the diluted ABTS+ solution was added to 50μL of peptide solutions of different concentrations (0.01 to 1mg / mL) in a 96-well plate, incubated at room temperature for 10min, and the absorbance of the reaction system at 734nm was measured. The control group used PBS buffer instead of peptide solution.

[0068] The calculation formula of ABTS free radical scavenging activity is as follows:

[0069] ABTS free radical scavenging rate (%) = (A control - A sample) / A control × 100, where A control and A sample are the absorbance values ​​of the control group and the experimental group, respectively. Finally, the ABTS free radical scavenging activity is expressed by Trolox equivalents (TEAC, μM TE / gpeptide).

[0070] Amino acid composition analysis

[0071] The amino acid composition was determined according to the national standards GB / T 18246-2000

[178] and GB / T15400-1994

[179] .

[0072]

[0073] The antioxidant capacity of the filtrate of the fermentation product of non-alcoholic galactose yeast-like bacteria may be related to its special amino acid composition. Therefore, its amino acid composition (18 common amino acids) was determined. The results showed that the total amino acid content of the filtrate of the fermentation product of non-alcoholic galactose yeast-like bacteria was 86.74 mg / g. The liquid was prepared using the Chinese patent CN 115125153 A technology.

[0074] The total amino acid content is 51.79%, the total amino acid content of the liquid prepared by the Chinese patent CN 116869870 A technology is 54.18%, and the total amino acid content of the filtrate of the alcohol-free galactose yeast-like bacteria fermentation product is significantly improved compared with the existing patented technology.

[0075] Studies have found that some special amino acids such as His, Tyr, Met, Leu, Trp, Lys and Cys have strong antioxidant activity; His, Tyr, Met, Leu, Trp, Lys and Cys

[0076] Antioxidant amino acids account for 40.23% of the total amino acids. The liquid is prepared using Chinese patent CN 115125153 A technology.

[0077] His, Tyr, Met, Leu, Trp, Lys and Cys antioxidant amino acids account for 24.39% of the total amino acids, and the Chinese patent CN 116869870 A technology preparation liquid His, Tyr, Met, Leu, Trp, Lys and Cys antioxidant amino acids account for 24.27% of the total amino acids.

[0078] Safety Testing

[0079] The prepared alcohol-free galactose yeast-like fermentation product filtrate was commissioned to Guangzhou Kangzheng Testing Service Co., Ltd. to test acute skin irritation, skin allergy test, skin phototoxicity test, skin photoallergy test, and acute percutaneous toxicity test. The test results proved that the concentrate was completely non-toxic to human skin. In addition, the results of the acute eye irritation test proved that the concentrate was not irritating at all.

[0080] Inflammatory factor inhibition experiment

[0081] RAW 264.7 macrophages were used as the research object. The optimal concentration of the galactose yeast-like fermentation product filtrate obtained in the examples and comparative examples was first determined to be 3.0% by MTT cell proliferation experiment. Then, the cells were stimulated with lipopolysaccharide LPS (bacterial endotoxin) to establish a cell inflammation model. The galactose yeast-like fermentation product filtrate obtained in the examples and comparative examples and 0.5% quercetin (the group with quercetin added was used as a positive control) were added respectively, and the cells were cultured for 24 hours. The cell supernatant was collected, and finally the release level of the pro-inflammatory inflammatory factor TNF-α of RAW264.7 macrophages was analyzed by ELISA kit.

[0082]

[0083]

[0084] Promoting HaCaT cell repair experiments

[0085] Inoculate HaCaT cells (5×104 cells / well) into a 96-well plate and culture overnight until the cells adhere. Prepare 50 μg / ml SDS, add 100 μl to each well, and culture at 37°C in a 5% carbon dioxide incubator for 8 hours. Add 5% Luddite fermentation product filtrate Example 1-5 and Comparative Example 1-4 to each well, and replace the control group with an equal volume of PBS for 24 hours. Add 10 μl of CCK-8 solution to each well, culture for 4 hours, and detect the absorbance A at 450 nm.

[0086] The cell proliferation rate was calculated as follows: cell proliferation rate = (experimental group A-control group A) / control group A*100%. The calculation results are shown in the table below.

[0087]

[0088] This experiment shows that the galactose yeast-like fermentation product filtrate prepared by adopting an oxygen-controlled fermentation culture mode in combination with a specific natural culture medium can significantly promote the repair of HaCaT keratinocyte damage caused by SDS and increase the cell proliferation rate. The proliferation rate promoted by the alcohol-free galactose yeast fermentation product filtrate is 18.01%.

[0089] Skin external use test

[0090] 1. Skin spot remedy test

[0091] Women with facial skin spots were selected, excluding patients with liver disease or female diseases, and pregnant women who went to a dermatology hospital for the treatment of skin spots. After washing the face, the skin pores were opened by ozone and steam, and gauze impregnated with 5% (without antibacterial agent) filtrate of alcohol-free galactosidase-like bacteria fermentation product filtrate was applied to the spotted area. The coated gauze was covered with Saran-Wrap tape and infrared irradiated for 15 minutes to allow the filtrate to penetrate into the skin. The treatment was performed once a week and continued for 3 months. It was found that 28 women (78.2%) had a great improvement in their condition, 7 women (22.8%) had a relative improvement in their condition, and no case of contact dermatitis occurred. The proportion of patients with a high or relative improvement in their condition was as high as 100%.

[0092] 2. Repair of skin folds and fine wrinkles

[0093] Generally, women over 25 are nervous about skin folds and fine lines, especially women between 35 and 40 years old, this trend is obvious, but women over 40 years old are also somewhat nervous about skin folds and fine lines.

[0094] In this test, 59 women who went to a dermatology hospital were selected (8 women aged 25 to 30, 28 women aged 30 to 35, 11 women aged 35 to 40, and 12 women aged 40 to 45). After washing the facial skin, the skin pores were opened by ozone and steam, and guaze impregnated with the concentrate of the cell-free supernatant of the present invention (concentration of 2) was applied to the facial skin, and gauze was covered-ed with Saran-Wrap tape. Infrared irradiation was performed for 18 minutes to allow the test liquid to penetrate into the skin. It was found that after one treatment, 52 women (88%) had a great improvement in skin cracking, 7 women (12%) had a relative improvement in skin cracking, and there was no other deterioration in the condition of any of the tested women. In view of the fact that cracked skin develops into fine wrinkles, it is believed that the present test liquid can effectively treat cracked skin and has a preventive effect on the formation of fine wrinkles.

[0095] 3. Observation of the therapeutic effect of contact dermatitis

[0096] Three patients diagnosed with contact dermatitis were selected.

[0097] Gauze impregnated with 5% (without antibacterial agent) filtrate of non-alcoholic galactosyl fermentation product was applied to the affected area by a dermatologist. No steroids or anti-inflammatory drugs were used. About 20 ml of the test filtrate was given to the patient, and the test filtrate was applied twice a day, i.e., in the morning and evening for about 25 minutes. 12-24 hours after the start of treatment, the itching disappeared, and 72-96 hours after the start of treatment, the rash basically disappeared; after 1 week, no rash was observed.

[0098] Through the above technical solution, the technical effects produced in the embodiments of the present application are:

[0099] Pure natural rice or brown rice gelatinized as a natural culture medium completely avoids the use of synthetic culture media and chemical reagents, which is conducive to obtaining pure natural daily chemical care raw materials without chemical residues. Rhizopus and fermentation factors are mixed and fermented. The technical advantage is that the mixed bacteria are fermented simultaneously without the need for additional step-by-step fermentation culture, and a fermentation product filtrate without obvious ethanol can be obtained. The filtrate can significantly increase the content of active ingredients such as exopolysaccharides, small molecule peptides, and organic acids, so that the obtained fermentation liquid has a significant improvement effect on skin repair and inflammation repair.

[0100] In the description of the present application, it should be understood that the terms "center", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0101] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, "plurality" means two or more.

[0102] In the description of this application, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0103] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0104] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fermentation filtrate with anti-wrinkle effect and a preparation method thereof, characterized in that: The following steps are involved: 1) preparing a base material: grinding one of rice or brown rice to obtain a powder, and steaming the powder; 2) Dispersing the base material: adding rice flour or brown rice flour to the cooked powder, and dispersing and cooling; 3) Sterilization treatment: After the cooled base material is heated to gelatinize, it is transferred to a fermentation tank for sterilization to obtain alcohol-free galactose yeast-like bacteria; 4) Fermentation treatment: placing the non-alcoholic galactose yeast-like bacteria and the amplified Rhizopus seed solution in a culture medium for mixed fermentation; 5) Post-treatment: The mixed solution after the fermentation treatment is subjected to high-pressure homogenization and bacteria-breaking treatment to obtain the alcohol-free galactose yeast-like bacteria fermentation product filtrate.

2. The fermentation filtrate with anti-wrinkle effect and preparation method according to claim 1, characterized in that: In the step 1, one of the rice or the brown rice is crushed to 100-140 meshes, and 95 parts of deionized water are added and then heated to 60° C. for steaming.

3. The fermentation filtrate with anti-wrinkle effect and the preparation method according to claim 1, characterized in that: The fraction of the rice flour or brown rice flour in the step 2 is 8 parts.

4. The fermentation filtrate with anti-wrinkle effect and the preparation method according to claim 1, characterized in that: The base material in step 3 is heated to 80° C., gelatinized for 3 hours, and then the fermentation tank is sterilized and cooled to 35° C.

5. The fermentation filtrate with anti-wrinkle effect and the preparation method according to claim 1, characterized in that: The non-alcoholic galactose yeast-like bacteria in step 4 is a yeast factor, which has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.31156.

6. The fermentation filtrate with anti-wrinkle effect and the preparation method according to claim 1, characterized in that: The amplified Rhizopus seed liquid in step 4 is Rhizopus oryzae.

7. The fermentation filtrate with anti-wrinkle effect and the preparation method according to claim 1, characterized in that: The ratio of the amplified Rhizopus seed liquid to the alcohol-free galactose yeast-like bacteria in step 4 is 2:1, and the solution is inoculated into the culture medium for fermentation. The fermentation culture is started at a temperature of 28° C. and a rotation speed of 220 r. After 24 hours, the temperature is raised to 35° C. and the stirring speed is 180 r. The total fermentation time is 72 h.

8. The fermentation filtrate with anti-wrinkle effect and the preparation method according to claim 1, characterized in that: The high-pressure homogenization sterilization treatment in step 5 is carried out at a sterilization condition of 60 MPa for a single time of 30-45 minutes, and the sterilization is carried out twice.

9. The fermentation filtrate of any one of claims 1 to 8 having anti-wrinkle efficacy and the method for preparing the fermentation filtrate having the effects of removing spots, repairing skin wrinkles and relieving inflammation.

10. Use of the alcohol-free galactose yeast-like fermentation product filtrate having the effects of removing spots, repairing skin wrinkles and relieving inflammation according to claim 9 in the preparation of cosmetics.

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