Avocado oil-splitting yeast fermentation filtrate, and preparation method and application thereof

Abstract

The application belongs to the technical field of cosmetic raw materials, and particularly relates to avocado oil split yeast fermentation filtrate and a preparation method and application thereof. The preparation method comprises the following steps: S1, performing squeezing treatment on dried avocado pulp to obtain avocado oil; S2, adding the avocado oil and a nitrogen source into a basic culture medium, inoculating split yeast for fermentation treatment, and filtering to obtain the avocado oil split yeast fermentation filtrate. The application takes avocado oil as a research object, combines split yeast for fermentation treatment, and adds a specific nitrogen source to optimize the fermentation process, so as to effectively improve the proliferation speed of the strain, and then ensure that macromolecular substances in the avocado oil are effectively converted into small molecular substances with higher activity. Through the conversion, the compound avocado oil split yeast fermentation filtrate not only maintains the original activity, but also has split yeast fermentation metabolic small molecular active products, so that the finally obtained fermentation filtrate has more excellent multiple effects such as in-vitro antioxidant effect.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic raw material technology, and in particular relates to an avocado oil Bifida ferment filtrate, its preparation method and application. Background Technology

[0002] Avocado, also known as butter fruit (Persea americana Mill.), is a plant belonging to the genus Persea in the family Lauraceae. Avocado oil, extracted from the pulp, is a natural plant oil rich in monounsaturated fatty acids, vitamin E, antioxidants, and other active ingredients, and is widely used in cosmetics and pharmaceuticals. Its high nutritional value makes it an important ingredient in anti-aging, moisturizing, and repairing skincare products, and it also has certain medicinal effects in promoting wound healing, anti-inflammation, and antibacterial properties. However, because the active ingredients in avocado oil are large molecules, their absorption and utilization efficiency by the human body is relatively low, especially in skin care and oral applications.

[0003] Most existing methods for preparing avocado oil employ cold pressing, solvent extraction, or supercritical fluid extraction. While these methods can extract the oil, they still face the challenge of efficient absorption of these active ingredients, even if they retain their properties. Furthermore, traditional technologies often fail to effectively convert large molecules into smaller active ingredients, thus reducing their effectiveness in cosmetics and pharmaceuticals. Current technologies also have significant limitations in improving the bioavailability and skin permeability of the ingredients.

[0004] Therefore, developing a preparation method that can convert macromolecules in avocado oil into small-molecule active ingredients, thereby significantly improving its absorption rate and bioactivity, has important practical application value. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an avocado oil Bifida ferment filtrate, its preparation method and application. By processing the pressed fruit oil with Bifida ferment filtrate, macromolecular substances are transformed into small, easily absorbed active ingredients. Combined with Bifida ferment filtrate metabolites as anti-wrinkle and firming active substances, the anti-wrinkle, firming, antioxidant, soothing, moisturizing and repairing effects of avocado oil ferment filtrate are effectively enhanced.

[0006] To achieve the above objectives, the technical solution adopted by the present invention includes:

[0007] In a first aspect, the present invention provides a method for preparing avocado oil Bifida ferment filtrate, comprising the following steps:

[0008] S1. Press the dried avocado pulp to obtain avocado oil;

[0009] S2. Add avocado oil and nitrogen source to the basic culture medium, inoculate with Bifida ferment lysate for fermentation, filter, and obtain the avocado oil Bifida ferment lysate.

[0010] The nitrogen source is at least one of hydrolyzed milk protein and yeast extract.

[0011] This invention uses physically pressed avocado oil as the research object, combined with Bifida ferment lysate fermentation, and optimizes the fermentation process by adding a specific nitrogen source to effectively increase the proliferation rate of the strain. This ensures that macromolecules in the avocado oil, such as unsaturated triglycerides, large-molecule proteins, and polysaccharides, are effectively converted into smaller-molecule triglycerides, small-molecule peptides, and small-molecule oligosaccharides—more active raw materials. Through this conversion, the compound avocado oil Bifida ferment lysate fermentation filtrate retains the activity of the original unsaturated fatty acids, polyphenols, tocopherols, etc., while also possessing the small-molecule active products of Bifida ferment lysate fermentation metabolism. This results in the final fermentation filtrate exhibiting superior in vitro antioxidant, anti-aging, firming, soothing, repairing, and moisturizing effects.

[0012] The innovative fermentation technology described in this invention transforms compound avocado oil and bifida ferment filtrate into a safe, non-irritating, and easily absorbed high-efficiency cosmetic ingredient. This ingredient not only effectively improves the skin's anti-aging effects and enhances its firmness and smoothness, but also provides soothing and repairing functions, offering a new direction for the development of high-efficiency skincare products.

[0013] Preferably, the avocado oil has a mass percentage content of 0.5-0.75% in the basal culture medium.

[0014] More preferably, the avocado oil has a mass percentage content of 0.75% in the basal culture medium.

[0015] This invention uses avocado oil as the carbon source for fermentation. Its concentration significantly affects the growth and proliferation of *Bifida flavours yeast* strains. Excessive avocado oil concentration leads to high lipid accumulation, inhibiting normal bacterial metabolism and affecting oxygen solubility in the culture medium, causing localized hypoxia and reducing the strain's proliferation rate. Conversely, insufficient avocado oil concentration results in inadequate carbon source supply, limiting strain growth and reproduction and affecting metabolic processes, thus reducing the production of small-molecule bioactive products. Experimental studies have shown that using the optimal concentration of avocado oil as the carbon source effectively increases the total bacterial count after fermentation, ensuring the superior efficacy of the final fermentation filtrate.

[0016] Preferably, the nitrogen source is hydrolyzed milk protein, and the mass percentage of the hydrolyzed milk protein in the basal culture medium is 0.5-1.5%.

[0017] Preferably, the hydrolyzed milk protein has a mass percentage of 0.75% in the basal culture medium.

[0018] Experimental studies revealed that the type and concentration of nitrogen source added during fermentation significantly impact the growth of Bifida Ferment Lysate. Using hydrolyzed milk protein as the specific nitrogen source at a concentration of 0.75% effectively increased the total number of Bifida Ferment Lysate colonies after fermentation, thereby enhancing the anti-wrinkle, firming, and antioxidant properties of the avocado oil fermentation filtrate. Conversely, using other nitrogen sources significantly inhibited the proliferation of Bifida Ferment Lysate strains; furthermore, concentrations of hydrolyzed milk protein exceeding the specified range also inhibited normal growth of Bifida Ferment Lysate, ultimately affecting the formation of active products in the fermentation filtrate.

[0019] Preferably, the fermentation conditions are: fermentation at 32-37℃ for 28-42 hours.

[0020] Preferably, the fermentation treatment conditions are: fermentation at 37°C for 30 hours.

[0021] Precise control of fermentation time and temperature is also crucial for ensuring the efficient proliferation of Bifida Ferment Lysate strains. Experimental studies have shown that fermentation at 37℃ for 30 hours maximizes the total number of Bifida Ferment Lysate colonies after fermentation, thereby optimizing the conversion of avocado oil and maximizing its anti-aging and soothing effects.

[0022] Preferably, during fermentation, an activated Bifida ferment lysate suspension is inoculated for fermentation, wherein the inoculation amount of the Bifida ferment lysate suspension is 3-7% (v / v).

[0023] More preferably, the inoculation amount of the Bifida Ferment Lysate suspension is 5% (v / v).

[0024] Preferably, the basal culture medium further comprises the following components in weight percentage: 0.1-1% sucrose, 0.1-0.4% dipotassium hydrogen phosphate, 0.2-0.8% sodium acetate, 0.02-0.08% magnesium chloride, 0.01-0.02% calcium chloride, 0.001-0.01% manganese sulfate, 0.1-0.3% L-histidine, 0.02-0.08% L-cysteine, and the balance being water.

[0025] Secondly, the present invention provides avocado oil Bifida ferment filtrate prepared by the aforementioned preparation method.

[0026] Thirdly, the present invention provides the application of the avocado oil bifida ferment filtrate in cosmetics.

[0027] Preferably, the cosmetics include antioxidant, anti-wrinkle and firming, soothing, moisturizing, and / or repairing cosmetics.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) This invention focuses on the physical pressing extraction of avocado oil and employs a fermentation process using Bifidobacterium longum (Bifida ferment lysate) to transform macromolecular unsaturated triglycerides, macromolecular proteins, and macromolecular polysaccharides in avocado oil into small-molecule triglycerides, small-molecule polypeptides, and small-molecule oligosaccharides, which are active raw materials. The preparation process described in this invention ensures that the compound avocado oil Bifida ferment lysate retains the original activity of unsaturated fatty acids, polyphenols, tocopherols, etc., while also possessing small-molecule active products from Bifida ferment lysate metabolism. This makes it a safe and non-irritating cosmetic raw material with in vitro antioxidant, anti-wrinkle, firming, soothing, repairing, and moisturizing effects.

[0030] (2) The avocado bifida ferment filtrate obtained by the preparation method of the present invention has a large number of water-soluble and oil-soluble active substances, and after biological fermentation, it is more biocompatible and easily absorbed by the skin. Attached Figure Description

[0031] Figure 1 Fresh avocado flesh ( Figure 1 A) Dried avocado fruit (after drying) Figure 1 B) and avocado oil obtained through physical pressing ( Figure 1 C) Images;

[0032] Figure 2 Images of fermentation broth after fermentation treatment with different strains;

[0033] Figure 3 Images of fermentation broth after fermentation treatment with different concentrations of hydrolyzed milk protein;

[0034] Figure 4 Images of fermentation broth after fermentation treatment with different avocado oil concentrations;

[0035] Figure 5 Figure 5 shows the results of the keratinocyte scratch test for different test substances in Example 5;

[0036] Figure 6 The image shows the results of the chicken embryo chorioallantoic membrane test on the avocado oil bifida ferment filtrate described in this invention. Detailed Implementation

[0037] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0038] Unless otherwise specified, all raw materials used in the following examples and comparative examples are commercially available general-purpose materials.

[0039] Example 1

[0040] This embodiment uses seven different bacterial strains (Candida utilis, Pichia pastoris, Candida bumblebee, Bacillus subtilis, Lactobacillus plantarum, Bifidobacterium longum, and Aspergillus niger) to ferment a substrate containing a certain proportion of avocado oil. The growth and quantity of the bacteria were assessed by counting the bacteria in diluted plate cultures. The antioxidant activity of the fermentation products was then evaluated through an in vitro antioxidant test (DPPH free radical scavenging rate) to select the optimal fermentation strain. The specific experimental methods are as follows:

[0041] S1. Preparation of avocado oil: Fresh avocados purchased directly are peeled, pitted, and cut into small pieces. They are then dried at a low temperature (40-50℃) and pressed directly using physical pressing technology to obtain avocado oil. Figure 1 (AC) represent fresh avocado pulp, dried avocado, and avocado oil obtained through physical pressing, respectively.

[0042] S2. Preparation of avocado oil Bifida ferment filtrate:

[0043] (1) Activation of strains: Use an inoculation loop to pick up one loop of different bacterial suspensions from the cryopreservation tube and inoculate them into the corresponding slant culture medium. After culturing at an appropriate temperature for a certain period of time to the appropriate density, the culture is passaged again to the appropriate density. Then, the colonies on the slant are washed off with 10 mL of physiological saline to obtain 7 different fermentation bacterial suspensions.

[0044] (2) Experimental procedure: Eight appropriately numbered containers were used. 1% (w / w, the same below) avocado oil and 1% hydrolyzed milk protein were accurately weighed into the basal culture medium, and the remaining amount was supplemented with deionized water to 100%. After mixing thoroughly, the culture medium was sterilized by high-pressure steam at 121℃ for 15 minutes. After cooling to room temperature, the container surface was disinfected by spraying with alcohol and transferred to an anaerobic chamber. 5% (v / v) of the corresponding pre-activated bacterial suspension was added to each culture flask according to its number. An equal volume of deionized water was added to the blank control group. After mixing, samples were taken for initial viable cell counting. After incubation at 37℃ for 28 hours, the mixture was shaken and mixed, and samples were taken again for viable cell counting. The fermentation broth was then filtered to obtain the avocado oil Bifida ferment filtrate. The antioxidant properties of the seven fermentation filtrates were determined by a routine in vitro antioxidant test (DPPH free radical scavenging rate). The specific results are shown in Table 1.

[0045] The basal culture medium also includes the following components in weight percentage: 0.5% sucrose (w / w), 0.2% dipotassium hydrogen phosphate, 0.5% sodium acetate, 0.05% magnesium chloride, 0.015% calcium chloride, 0.005% manganese sulfate, 0.2% L-histidine and 0.05% L-cysteine.

[0046] Table 1

[0047]

[0048] As shown in Table 1, there are significant differences in the DPPH free radical scavenging rate of the fermentation filtrate obtained after fermentation treatment with different strains. The DPPH free radical scavenging rate of avocado oil without the addition of strains is only 17.29%, while the DPPH free radical scavenging rate of the filtrate obtained after fermentation treatment with Bifidobacterium longum (Bifida ferment lysate) reaches 79.42%, showing the best antioxidant properties and the fastest proliferation rate in the culture medium environment with avocado oil as the carbon source. Figure 2 Images of fermentation broth after fermentation treatment with different strains.

[0049] Example 2

[0050] This embodiment uses soybean peptone, acid-hydrolyzed casein peptone, bacteriological peptone, yeast extract, skim milk powder, peptone (Maclean's), beef peptone, hydrolyzed milk protein, and peptone (Haibo Biotechnology) as different nitrogen sources. The growth and quantity of the bacterial strains were investigated by counting the bacteria on anaerobic plates after dilution, in order to select the optimal nitrogen source. The specific experimental methods are as follows:

[0051] S1. Preparation of avocado oil: Same as in Example 1;

[0052] S2. Preparation of avocado oil Bifida ferment filtrate:

[0053] (1) Activation of strain: Use an inoculation loop to pick up the Bifida ferment lysate from the cryopreservation tube, inoculate it into the corresponding slant culture medium, culture it at an appropriate temperature for a certain time until the appropriate density is reached, then subculture it again to the appropriate density, and then wash off the colonies on the slant with 10 mL of physiological saline to obtain the Bifida ferment lysate suspension.

[0054] (2) Experimental procedure: Ten appropriately numbered containers were used, and 1% (w / w, the same below) of the nine pre-prepared proteins was accurately weighed. The blank control group was replaced with 1% deionized water and 1% avocado oil. The composition of the remaining culture media was the same as in Example 1. After mixing, the containers were autoclaved at 121℃ for 15 min. After cooling to room temperature, the container surface was disinfected by spraying alcohol and then transferred to an anaerobic chamber. 5% (v / v) pre-activated Bifida ferment lysate suspension was added to each bottle, and the containers were cultured at 37℃ for 26 h. After shaking and mixing, samples were taken for viable cell counting. The specific results are shown in Table 2.

[0055] Table 2

[0056]

[0057]

[0058] As shown in Table 2, the absence of a nitrogen source during fermentation significantly inhibits the growth of Bifida Ferment Lysate, ultimately affecting the formation of active products in the fermentation filtrate and consequently its antioxidant properties. Furthermore, the use of different nitrogen sources also significantly affects the proliferation rate of Bifida Ferment Lysate. Using acid-hydrolyzed casein peptone and yeast extract as nitrogen sources better promotes the growth of Bifida Ferment Lysate, especially hydrolyzed milk protein, which exhibits the highest total colony count in plate culture and the highest DPPH free radical scavenging rate in the filtrate obtained after fermentation.

[0059] Example 3

[0060] In this embodiment, Bifida ferment lysate was fermented in culture media with different concentrations of hydrolyzed milk protein (0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, and 5%). The growth and quantity of the bacterial strain were examined by counting the cells in diluted agar plates to screen for the optimal nitrogen source concentration. The specific experimental methods are as follows:

[0061] S1. Preparation of avocado oil: Same as in Example 1;

[0062] S2. Preparation of avocado oil Bifida ferment filtrate:

[0063] (1) Strain activation: Same as in Example 2;

[0064] (2) Experimental procedure: Nine appropriately labeled containers were used. 0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, and 5% hydrolyzed milk protein and 1% avocado oil were accurately weighed into the basal culture medium (the remaining components were the same as in Example 1). The remaining amount was brought to 100% with deionized water. After mixing, the containers were autoclaved at 121°C for 15 minutes. After cooling to room temperature, the containers were disinfected by spraying alcohol on their surfaces and then transferred to an anaerobic chamber. 5% (V / V) pre-activated Bifida ferment lysate suspension was added to each container, and the mixture was incubated at 37°C for 28 hours. After shaking and mixing, samples were taken for viable cell counting. Specific results are shown in Table 3.

[0065] Table 3

[0066]

[0067]

[0068] As shown in Table 3, precise control of the concentration of hydrolyzed milk protein has a significant impact on the proliferation of Bifida ferment lysate. When the concentration of hydrolyzed milk protein is within the preferred range of 0.5-1.5%, the total number of Bifida ferment lysate colonies after fermentation reaches 4.2 × 10⁻⁶. 8 The above factors facilitate the conversion of macromolecular active substances in avocado oil and increase the content of metabolic active products of the strain in the fermentation filtrate, thereby effectively improving the multiple functions of the fermentation filtrate. In particular, the proliferation rate of Bifida Ferment Lysate is optimal when the concentration of hydrolyzed milk protein is 0.75%. Figure 3 Images of fermentation broth after fermentation treatment with different concentrations of hydrolyzed milk protein.

[0069] Example 4

[0070] In this embodiment, Bifida ferment lysate was fermented in culture media with different avocado oil carbon source concentrations (0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, and 5%). The growth and quantity of the bacterial strain were examined by counting the cultures under anaerobic conditions after dilution, in order to select the optimal avocado oil concentration. The specific experimental method is as follows:

[0071] S1. Preparation of avocado oil: Same as in Example 1;

[0072] S2. Preparation of avocado oil Bifida ferment filtrate:

[0073] (1) Strain activation: Same as in Example 2;

[0074] (2) Experimental procedure: Nine appropriately labeled containers were used. 0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, and 5% avocado oil and 1% hydrolyzed milk protein were accurately weighed into the basal culture medium (the remaining components were the same as in Example 1). The remaining amount was made up to 100% with deionized water. After mixing, the containers were autoclaved at 121°C for 15 minutes. After cooling to room temperature, the containers were disinfected by spraying alcohol on the surface and then transferred to an anaerobic chamber. 5% (V / V) pre-activated Bifida ferment lysate suspension was added to each container, and the containers were incubated at 37°C for 24 hours. After shaking and mixing, samples were taken for viable cell counting. The specific results are shown in Table 4.

[0075] Table 4

[0076]

[0077]

[0078] As shown in Table 4, when the concentration of avocado oil is within the preferred range of 0.5-0.75%, it can more effectively promote the proliferation and growth of Bifida ferment lysate, especially when the concentration is 0.5%, the effect of promoting the growth of the strain is the best. Figure 4 Images of fermentation broth after fermentation treatment with different concentrations of avocado oil.

[0079] Example 5

[0080] In this embodiment, fermentation was carried out at different temperatures (23℃, 28℃, 32℃, 37℃, 42℃, 47℃, and 52℃). The growth and quantity of the microbial strain were examined based on plate counts under anaerobic conditions after dilution of the bacterial solution to select the optimal incubation temperature. The specific experimental methods are as follows:

[0081] S1. Preparation of avocado oil: Same as in Example 1;

[0082] S2. Preparation of avocado oil Bifida ferment filtrate:

[0083] (1) Strain activation: Same as in Example 2;

[0084] (2) Experimental procedure: Seven appropriate containers were selected and numbered. The culture medium composition and sterilization procedure were the same as in Example 1. After cooling to room temperature, the container surfaces were disinfected by spraying with alcohol and then transferred to an anaerobic chamber. 5% (V / V) pre-activated Bifida ferment lysate suspension was added to each bottle, and the containers were incubated at 23℃, 28℃, 32℃, 37℃, 42℃, 47℃, and 52℃ for 25 h, respectively. After incubation, the containers were shaken to mix and samples were taken for viable cell counting. The specific results are shown in Table 5.

[0085] Table 5

[0086]

[0087]

[0088] As shown in Table 5, when the fermentation temperature of Bifida Ferment Lysate was 32-37℃, the total number of bacterial colonies of the strain was the highest. In particular, the strain growth effect was the best at 37℃, and the DPPH free radical scavenging rate of the obtained fermentation filtrate also reached the highest level.

[0089] Example 6

[0090] This embodiment sets up different culture times (20h, 22h, 24h, 26h, 28h, 30h, 42h, 44h, 46h, 48h) for fermentation treatment. The growth and quantity of the bacterial strain are examined based on plate counts under anaerobic conditions after dilution of the bacterial solution to select the optimal culture time. The specific experimental methods are as follows:

[0091] S1. Preparation of avocado oil: Same as in Example 1;

[0092] S2. Preparation of avocado oil Bifida ferment filtrate:

[0093] (1) Strain activation: Same as in Example 2;

[0094] (2) Experimental procedure: Seven appropriate containers were selected and numbered. The culture medium composition and sterilization procedure were the same as in Example 1. After cooling to room temperature, the container surface was disinfected by spraying alcohol and then transferred to an anaerobic chamber. 5% (V / V) pre-activated Bifida ferment lysate suspension was added to each bottle, and the containers were incubated at 37℃ for 20h, 22h, 24h, 26h, 28h, 30h, 42h, 44h, 46h, and 48h, respectively. After incubation, the containers were shaken to mix and samples were taken for viable cell counting. The specific results are shown in Table 6.

[0095] Table 6

[0096]

[0097]

[0098] As shown in Table 6, when using the optimal fermentation time of 28-42h for Bifida ferment lysate fermentation, the total number of bacterial colonies of the strain is the highest. In particular, the strain has the best growth effect at 30h, and the DPPH free radical scavenging rate of the fermentation filtrate is the highest.

[0099] Example 7

[0100] This embodiment provides a method for preparing avocado oil Bifida ferment filtrate, including the following steps:

[0101] S1. Preparation of avocado oil: Fresh avocados purchased directly are peeled, pitted, and cut into small pieces. They are then dried at a low temperature (40-50℃) and pressed directly using physical pressing technology to obtain avocado oil.

[0102] S2. Preparation of avocado oil Bifida ferment filtrate:

[0103] (1) Activation of strain: Use an inoculation loop to pick up the Bifida ferment lysate from the cryopreservation tube, inoculate it into the corresponding slant culture medium, culture it at an appropriate temperature for a certain time until the appropriate density is reached, then subculture it again to the appropriate density, and then wash off the colonies on the slant with 10 mL of physiological saline to obtain the Bifida ferment lysate suspension.

[0104] (2) Experimental procedure: Accurately weigh 0.75% hydrolyzed milk protein and 0.75% avocado oil; the remaining culture medium composition is the same as in Example 1. After mixing, autoclave at 121℃ for 15 min, cool to room temperature, disinfect the container surface with alcohol spray, and transfer to an anaerobic chamber. Add 5% (V / V) pre-activated Bifida ferment lysate suspension to each bottle, incubate at 37℃ for 30 h, and filter after fermentation to obtain the avocado oil Bifida ferment lysate fermentation filtrate. Example 1. In vitro antioxidant DPPH free radical scavenging rate test.

[0105] This example uses the avocado oil Bifida ferment filtrate prepared in Example 7 as the test sample to test its antioxidant enhancement effect before and after fermentation. The DPPH free radical scavenging rate was tested according to the national standard GB / T 39100-2020 "Determination of Antioxidant Activity of Peptides: DPPH and ABTS Methods". The specific process is as follows:

[0106] Take the prepared avocado oil Bifida ferment filtrate, dilute it 4 times, and pipette 1.25 mL into a 10 mL volumetric flask. Add distilled water to bring the volume to 10 mL and shake well. For the experimental group, pipette 200 μL of the test solution and 600 μL of 50 ppm DPPH solution into a 2 mL disposable centrifuge tube. For the blank group, pipette 200 μL of distilled water and 600 μL of anhydrous ethanol into a 2 mL disposable centrifuge tube. Mix them thoroughly with a vortex mixer and react at room temperature in the dark for 30 min. Then centrifuge for 5 min. Pipe 180 μL of the supernatant after centrifugation into a 96-well plate and measure the absorbance at 517 nm using a microplate reader.

[0107] Experimental results showed that the 25% avocado oil Bifida ferment filtrate prepared in Example 7 was used as the test sample, and the free radical scavenging rate at 50 ppm DPPH was 85.76%, indicating that the sample had strong in vitro antioxidant and free radical scavenging effects.

[0108] Example 2. Cytotoxicity test

[0109] This example uses the avocado oil Bifida ferment filtrate prepared in Example 7 as the test sample to test its cytotoxicity. The specific cytotoxicity test method is as follows:

[0110] Cell cultures were seeded at an appropriate density into 96-well plates, with 100 μL seeded per well, and incubated in a CO2 incubator for 24 h. After incubation, the culture medium in the 96-well plates was discarded, and different concentrations of the test solution were added. A blank control group and a positive control group were also set up, with 100 μL / well for each concentration, and six replicates were added. The plates were then incubated in a CO2 incubator for 24 h. After incubation, the cell state was observed and recorded. The culture medium was discarded, and the cells were washed 1-2 times with PBS. MTT working solution was added at 50 μL / well, and the plates were incubated in a CO2 incubator for 4 h. The culture medium was then discarded, and 150 μL of DMSO was added to each well. The plates were shaken for 10 min, and the absorbance was measured at 570 nm using a microplate reader, with 630 nm as the reference wavelength. Cell viability was calculated based on the measurement results. When the cell viability of the sample at the corresponding concentration in the 96-well plate was ≥90%, and there was no significant difference in morphology between the cells in the sample group and the blank group, it was considered that the sample had no significant cytotoxicity at that concentration.

[0111] Cell viability formula: Cell viability (%) = OD样品 / OD 空白 ×100%,

[0112] Where: OD 样品 —Mean OD value of the sample group; OD 空白 —Mean OD value of the blank control group.

[0113] Experimental results showed that, using the avocado oil Bifida ferment filtrate prepared in Example 7 as the test sample, the cytotoxicity test results showed that the non-toxic concentration for human skin fibroblasts HFF-1, mouse mononuclear macrophage leukemia cell line RAW264.7, mouse melanocytes B16, and HaCat keratinocytes was 5.0%.

[0114] Example 3. Anti-wrinkle and firming effect test

[0115] This study used the avocado oil Bifida ferment filtrate prepared in Example 7 as the test sample. The anti-wrinkle and firming effect of the sample was determined by testing its effect on enhancing type I collagen in human fibroblasts. The specific method is as follows:

[0116] 1) Cell seeding: Human skin fibroblasts HFF-1 were seeded into 96-well plates at an appropriate density (60% confluence was achieved 24 hours after seeding), 100 μL per well, and incubated in a CO2 incubator for 24 hours;

[0117] 2) Drug administration: Discard the culture medium in the 96-well plate, dilute the test substance to 2.5% with cell culture medium and add it to the test substance wells. Add 100 μL of culture medium containing 100 ng / mL TGFβ1 to the positive control wells and add cell culture medium to the blank control wells. After drug administration, place the 96-well plate in a CO2 incubator and incubate for 24 h.

[0118] 3) ELISA detection: After the culture is completed, collect the cell culture supernatant from each well and perform the detection according to the instructions of the Human Type I Collagen Enzyme-Linked Immunosorbent Assay Kit.

[0119] Type I collagen content calculation: Using professional curve generation software, the concentration of the standard in the kit is compared with the OD value. 450 The regression equation for the standard curve is calculated using the values, and the OD of the sample is used to calculate the standard curve. 450 Substitute the values ​​into the equation to calculate the type I collagen content of the samples. The final average of the three replicates for each group is taken as the final type I collagen result.

[0120] Formula for calculating the upregulation rate of type I collagen: Upregulation rate (%) = (T / C-1) × 100%, where: T—average content of type I collagen in the test substance; C—average content of type I collagen in the blank control.

[0121] Experimental results showed that using the 2.0% avocado oil Bifida ferment filtrate prepared in Example 7 as the test sample, the upregulation rate of human fibroblast type I collagen was 30.80%, indicating that the sample has a strong anti-wrinkle and firming effect.

[0122] Example 4. Soothing effect test

[0123] The avocado oil Bifida ferment filtrate prepared in Example 7 was used as the test sample. The soothing efficacy of the sample was determined by the macrophage NO inhibition rate test. The specific method is as follows:

[0124] 1) Cell seeding: Mouse mononuclear macrophage leukemia cell line RAW264.7 was seeded into 96-well plates at an appropriate density (90% confluence was achieved 24 hours after seeding), 100 μL per well, and incubated in a CO2 incubator for 24 hours.

[0125] 2) Drug administration: Discard the culture medium in the 96-well plate. Set up a positive control group, a negative control group, and a blank control group, with 6 replicates in each group. Add 50 μL of the test solution and 50 μL of LPS working solution to each well. Add 100 μL of dexamethasone sodium phosphate working solution to each well in the positive control group, 50 μL of test culture medium and 50 μL of LPS working solution to each well in the negative control group, and 100 μL of test culture medium to each well in the blank control group. After sample addition, incubate the 96-well plate in a CO2 incubator for 24 h.

[0126] 3) Gliese assay: After the culture is completed, collect 50 μL of cell culture supernatant from each well and put it into a new 96-well plate. Add 50 μL of Gliese solution to each well, mix well, and react in the dark for 10 min. Detect the OD value at 540 nm using a microplate reader.

[0127] NO relative content (%) = OD sample / OD negative control group × 100%;

[0128] Experimental results showed that using the 2.5% avocado oil Bifida ferment filtrate prepared in Example 7 as the test sample, the NO inhibition rate of RAW264.7 macrophages was 19.61%, and the NO inhibition rate of the 5.0% test sample was 41.58%, indicating that the sample has good soothing effects.

[0129] Example 5. Cell Repair Effect Test

[0130] This study used the avocado oil Bifida ferment filtrate prepared in Example 7 as the test sample. The repair effect of the sample was determined by a keratinocyte scratch assay. The specific method is as follows:

[0131] 1) Inoculation: Using a marker, draw horizontal lines evenly on the back of the cell culture plate with a ruler, passing through at least 5 lines in each well. Inoculate the cells into 12-well plates and incubate at 37°C in a 5% CO2 incubator for 24 h ± 2 h.

[0132] 2) Solution preparation: Prepare the test substance and positive control according to Table 7.

[0133] Table 7 Experimental Grouping and Concentration Settings

[0134]

[0135] 3) Sample Administration: After incubation, use a pipette tip, aligned with a ruler, to make a vertical cut along the horizontal line on the back of the well plate, ensuring the cut intersects the marking line. Discard the culture medium in each well. Slowly add sterile PBS / test culture medium to wash the adherent cells three times, removing the cut cells. Perform the drug administration procedure. Add test culture medium to the blank group, add culture medium containing 15% FBS to the positive group, and add test culture medium containing the test substance to the sample group. Incubate in a CO2 incubator for 24±2 h. According to the experimental grouping and concentration settings in Table 7, after the cells have grown in the 12-well plate for 24±2 h, administer samples to each group, with 3 replicates per group, 1 mL per well. Continue incubation at 37℃ in a 5% CO2 incubator for another 24±2 h.

[0136] 4) Detection: The cells were observed and photographed under a microscope at 0h and 24h after the scratch.

[0137] 5) Data processing: ImageJ software was used to analyze the scratch area.

[0138]

[0139] The experimental results showed that the keratinocyte migration rate was 21.91% when the 2.5% avocado oil Bifida ferment filtrate prepared in Example 7 was used as the test sample, indicating that the sample has good repair effects.

[0140] Example 6. Moisturizing effect test

[0141] This study used the avocado oil Bifida ferment filtrate prepared in Example 7 as the test sample. The moisturizing effect of the sample was determined by physicochemical analysis using a gravimetric method, as follows:

[0142] Three groups (A, B, and C) were set up, with three replicates in each group. 5g of test sample, deionized water, and a positive control were added to each container, and the total weight (M1) of the bottle (M0) and water was measured. The containers were placed in a constant temperature and humidity chamber at 20℃ and 65% humidity for a certain period, and then weighed again (M2). The humidity rate was calculated using the following formula:

[0143]

[0144] The experimental results showed that the avocado oil Bifida ferment filtrate was used as the test sample, and the 8-hour moisturizing rate was 93.47%, which was higher than the moisturizing rate of the 10% glycerol positive control (92.46%), indicating that the sample has good moisturizing effect.

[0145] Example 7. Chicken embryo safety and irritation test

[0146] This example uses the avocado oil Bifida ferment filtrate prepared in Example 7 as the test sample. The chicken embryo safety and irritation of the sample were tested using a chicken embryo experiment, referring to the chicken embryo chorioallantoic membrane test for eye irritation / corrosiveness in cosmetics (SN / T 2329-2009). The specific method is as follows:

[0147] 1) CAM Preparation: Candling of 9-day-old chicken embryos was performed. The air cell portion of the shell was removed using dental serrated forceps to expose the white membrane, handling it carefully to avoid damaging its integrity. A drop of 0.9% sodium chloride (NaCl) solution was added with a pipette to moisten the membrane, and the inner membrane was carefully removed with forceps, ensuring the vascular membrane remained undamaged. The structure of the vascular system was then observed again, and its integrity and suitability for experimental use were assessed.

[0148] 2) Pre-experiment test: Take 2 chicken embryos for the experiment to check the reactivity of this batch of chicken embryos and determine whether the reaction time method should be used in the formal experiment. The reaction time is limited to 5 minutes.

[0149] 3) Preliminary test: Using 6 chicken embryos, take 0.3 mL of the test substance, ensuring that at least 50% of the CAM surface is covered by the test substance. Immediately after the test substance is applied, observe the CAM reaction for 5 minutes and record the results. Determine whether the test substance is suitable for this method and determine whether the reaction time method or the endpoint evaluation method should be used in the formal test.

[0150] Figure 6 The results showed that the 100% avocado oil bifida ferment filtrate stock solution of the present invention was non-irritating.

[0151] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing avocado oil Bifida ferment filtrate, characterized in that, Includes the following steps: S1. Press the dried avocado pulp to obtain avocado oil; S2. Add avocado oil and nitrogen source to the basic culture medium, inoculate with Bifida ferment lysate for fermentation, filter, and obtain the avocado oil Bifida ferment lysate. The nitrogen source is hydrolyzed milk protein, and the mass percentage of the hydrolyzed milk protein in the basal culture medium is 0.5-1.5%. The avocado oil content in the basal culture medium is 0.5-0.75% by mass; The fermentation conditions are as follows: fermentation at 32-37℃ for 28-42 hours; The basal culture medium also includes the following components by weight percentage: 0.1-1% sucrose, 0.1-0.4% dipotassium hydrogen phosphate, 0.2-0.8% sodium acetate, 0.02-0.08% magnesium chloride, 0.01-0.05% calcium chloride, 0.001-0.01% manganese sulfate, 0.1-0.3% L-histidine, 0.02-0.08% L-cysteine, and the balance being water.

2. The method for preparing avocado oil Bifida ferment filtrate as described in claim 1, characterized in that, The hydrolyzed milk protein has a mass percentage of 0.75% in the basal culture medium.

3. The method for preparing avocado oil Bifida ferment filtrate as described in claim 1, characterized in that, The fermentation conditions were: fermentation at 37°C for 30 hours.

4. Avocado oil Bifida ferment filtrate prepared by the preparation method according to any one of claims 1-3.

5. The application of the avocado oil bifida ferment filtrate as described in claim 4 in cosmetics.

6. The application as described in claim 5, characterized in that, The cosmetics mentioned are antioxidant, anti-wrinkle and firming, soothing, moisturizing, and / or repairing cosmetics.

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