Schizochytrium fermentum filtrate with moisturizing and repairing effects, and preparation method and application thereof

Abstract

The application belongs to the technical field of microbial fermentation, and specifically discloses a bifida yeast fermentation product filtrate with moisturizing and repairing effects, and a preparation method and application thereof. The preparation method of the bifida yeast fermentation product filtrate comprises the following steps: (1) strain activation: inoculating Bifidobacterium adolescentis into a first seed culture medium for anaerobic culture to obtain activated bacterial liquid; (2) seed liquid culture: inoculating the activated bacterial liquid into a second seed culture medium and continuously transferring for anaerobic culture twice to obtain a third-level seed liquid; (3) fermentation culture: inoculating the third-level seed liquid into a fermentation tank for culture to obtain a fermentation product; and (4) separation and purification: filtering and decolorizing the fermentation product to obtain the bifida yeast fermentation product filtrate. The bifida yeast fermentation product filtrate prepared by the application has high active ingredient content, good stability, and good moisturizing and repairing effects.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a Bifida ferment filtrate with moisturizing and repairing effects, its preparation method, and its application. Background Technology

[0002] Bifida ferment filtrate is the liquid filtered after Bifidobacterium fermentation. It contains no bacterial cells and mainly consists of metabolites such as short-chain fatty acids, extracellular polysaccharides, free amino acids, and peptides. Among these, polysaccharides form a protective film on the skin's surface, effectively locking in moisture and resisting free radical damage, thus providing excellent moisturizing and antioxidant effects. Amino acids and peptides not only participate in normal skin metabolism but also penetrate deep into the skin's basal layers, repairing damaged cells and stimulating collagen production, thereby enhancing skin elasticity. Therefore, Bifida ferment filtrate has enormous market application potential.

[0003] The currently disclosed Bifida ferment lysate fermentation processes have the following shortcomings: low fermentation efficiency, long cycle, and high energy consumption; low and unstable yield of target active ingredients, with large batch-to-batch differences. This paper aims to solve the problems of low fermentation efficiency and insufficient active ingredients in existing technologies. Summary of the Invention

[0004] In view of the above, it is necessary to provide a Bifida ferment filtrate with moisturizing and repairing effects, its preparation method, and its application. The Bifida ferment filtrate prepared by this invention has a high content of active ingredients, good stability, and good moisturizing and repairing effects.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a Bifida ferment filtrate includes the following steps:

[0007] (1) Activation of bacterial strain: Bifidobacterium adolescentis was inoculated into the first seed culture medium and cultured anaerobically to obtain activated bacterial solution;

[0008] (2) Seed culture: The activated bacterial solution was inoculated into the second seed culture medium and transferred to anaerobic culture twice to obtain the third seed culture;

[0009] (3) Fermentation culture: The three-stage seed liquid is inoculated into a fermenter containing fermentation culture medium and anaerobic fermentation culture is carried out to obtain fermented product; wherein, the anaerobic fermentation culture includes a first anaerobic fermentation, a second anaerobic fermentation and a third anaerobic fermentation;

[0010] The conditions for the first anaerobic fermentation were: temperature 36-37℃, pH 6.0-6.2, rotation speed 100-110 rpm, and fermentation time 8-10 h.

[0011] The conditions for the second anaerobic fermentation are: temperature 38.5-39℃, pH 5.8-6.0, rotation speed 110-115 rpm, and fermentation time 10-12 h.

[0012] The conditions for the third anaerobic fermentation are: temperature 35-35.5℃, pH 5.8-6.0, rotation speed 95-100 rpm, and fermentation time 2-4 hours.

[0013] (4) Separation and purification: After filtering and decolorizing the fermentation product, the Bifida ferment filtrate can be obtained.

[0014] Furthermore, in step (1), the Bifidobacterium adolescentis is Bifidobacterium adolescentis HH404-1, which is classified as Bifidobacterium adolescentis. It is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) at the address of Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 22, 2025, with the accession number GDMCC No: 67151.

[0015] Further, the first seed culture medium comprises the following raw material components by weight percentage: peptone 1.0-1.2%, beef extract 0.8-1.2%, yeast extract 0.4-0.7%, glucose 1.8-2.3%, dipotassium hydrogen phosphate 0.1-0.3%, triammonium citrate 0.1-0.3%, sodium acetate 0.4-0.6%, magnesium sulfate 0.01-0.04%, manganese sulfate 0.003-0.006%, and Tween-80 0.10-0.15%; the second seed culture medium comprises the following raw material components by weight percentage: glucose 2.3-2.7%, peptone 0.8-1.2%, yeast extract 0.3-0.7%, magnesium sulfate 0.01-0.02%, manganese sulfate 0.003-0.006%, sodium acetate 0.4-0.6%, and ammonium citrate 0.13-0.16%.

[0016] Furthermore, in step (3), during the first anaerobic fermentation, a first solution is added; during the second anaerobic fermentation, a second solution is added; and during the third anaerobic fermentation, a third solution is added; the first solution comprises glucose and yeast treatment, the second solution comprises glucose, yeast treatment and glutamine, and the third solution comprises trehalose and serine.

[0017] Further, in step (3), the first solution comprises the following components by mass percentage: 5-10% glucose and 0.5-1% yeast treatment; the second solution comprises the following components by mass percentage: 5-10% glucose, 1-2% yeast treatment and 0.1-0.3% glutamine; the third solution comprises the following components by mass percentage: 0.1-0.3% trehalose and 0.03-0.05% serine.

[0018] Furthermore, the first solution is added at a rate of 2-5% of the initial fermentation material mass, the second solution is added at a rate of 4-7% of the initial fermentation material mass, and the third solution is added at a rate of 1-2% of the initial fermentation material mass.

[0019] Furthermore, the preparation method of the yeast treatment product is as follows: the yeast cells are crushed at 30-40 MPa to obtain a bacterial solution, a complex enzyme is added to the bacterial solution, and the solution is enzymatically hydrolyzed at 45-50℃ and pH 5.0-5.5 for 2-3 hours to obtain the yeast treatment product; wherein the complex enzyme is prepared by mixing cellulase and β-glucanase in a mass ratio of 1:2.

[0020] Furthermore, the fermentation medium comprises the following raw material components in weight percentage: glucose 4.0-5.0%, yeast treatment 1.5-1.8%, peptone 0.8-1.2%, dipotassium hydrogen phosphate 0.4-0.7%, licorice extract 0.3-0.5%, manganese sulfate 0.01-0.02%, magnesium sulfate 0.01-0.03%, anhydrous sodium acetate 0.4-0.7%, ammonium citrate 0.12-0.18%, and cysteine ​​0.1-0.3%.

[0021] The present invention also provides a filtrate of Bifida ferment lysate prepared by the above preparation method.

[0022] The present invention also provides an application of the above-mentioned Bifida ferment filtrate in the preparation of cosmetics.

[0023] Furthermore, the cosmetic is a cosmetic with moisturizing and / or repairing effects.

[0024] The present invention has the following beneficial effects:

[0025] 1. This invention employs a three-stage fermentation control strategy during the main fermentation process by adjusting fermentation parameters. During the rapid cell growth stage, controlling fermentation conditions at 36-37℃, pH 6.0-6.2, and 100-110 rpm effectively promotes cell growth and increases cell volume. When cell growth stabilizes, controlling fermentation conditions at 38.5-39℃, pH 5.8-6.0, and 110-115 rpm helps increase the metabolic rate and promotes the accumulation of target metabolites, thereby increasing the content of active ingredients. In the later stages of fermentation, reducing temperature and rotation speed reduces the synthesis of byproducts, stabilizes activity, and thus improves product purity and stability.

[0026] 2. Specific feedstocks are added to each of the three stages of the main fermentation process of this invention to match the nutritional needs of the microbial cells at different stages, avoiding substrate deficiency or inhibition, thereby improving the yield and stability of active ingredients. In the early stage of fermentation, the addition of glucose and yeast treatment products provides carbon and nitrogen sources, which promotes cell growth and increases cell mass. In the middle stage of fermentation, the addition of glucose, yeast treatment products, and glutamine effectively induces product synthesis and promotes the accumulation of active metabolites. In the later stage of fermentation, the addition of trehalose and serine maintains cell activity and promotes product release.

[0027] 3. The fermentation medium of the present invention is nutritionally complete, providing balanced nutrition to the cells and activating related metabolic pathways, improving fermentation initiation force, significantly increasing cell density and metabolic activity, thereby effectively improving fermentation efficiency and effect.

[0028] 4. The *Bifidobacterium adolescentis* isolated by this invention exhibits excellent anaerobic fermentation performance, high bacterial growth, and abundant metabolite synthesis. The fermentation filtrate obtained through anaerobic fermentation has a high content of active substances and good stability. When applied to cosmetics, it can effectively provide moisturizing, antioxidant, repairing, and brightening effects. Furthermore, by controlling the appropriate amount added, it will not damage the structure of the cosmetics, will not adversely affect the stability of the cosmetics, and will not cause quality problems such as discoloration or layering.

[0029] In summary, this invention, through optimization of the fermentation medium combined with staged fermentation condition control and dynamic feeding strategy, improves the synthesis efficiency of metabolites, resulting in high content of target active ingredients and good stability in the prepared Bifida ferment filtrate, which has excellent skin care effects, especially good repair and moisturizing effects, making it suitable for cosmetic development. On the other hand, it improves the controllability of the preparation process, thereby reducing batch differences, improving product consistency and reliability, and facilitating large-scale scaling and production automation. Attached Figure Description

[0030] Figure 1This is a colony diagram of the Bifidobacterium adolescentis strain HH404-1 of this invention.

[0031] Figure 2 This is a microscopic image of the Bifidobacterium adolescentis strain HH404-1 of this invention.

[0032] Figure 3 This is an ambient temperature diagram used in this invention to measure the efficacy of serums S1-S7.

[0033] Figure 4 This is an environmental humidity diagram used in this invention to measure the efficacy of serums S1-S7.

[0034] Information on the preservation of biological materials

[0035] The strain information deposited in this application is: Bifidobacterium adolescentis HH404-1, classified as Bifidobacterium adolescentis, deposited at Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 22, 2025, with accession number GDMCC No: 67151. Detailed Implementation

[0036] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0037] In this invention, unless otherwise specified, all reagents and consumables used are purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and techniques used are conventional methods and techniques in the field.

[0038] First, this invention provides a strain of Bifidobacterium adolescentis. This Bifidobacterium adolescentis was isolated and screened from the feces of centenarians in Bama Longevity Village, Guangxi Zhuang Autonomous Region, and underwent biochemical and molecular biological identification, the specific process of which is shown below.

[0039] Isolation and screening of strains

[0040] The Bifidobacterium adolescentis was isolated from the feces of centenarians in Bama Longevity Village, Guangxi Zhuang Autonomous Region. The specific isolation and screening methods are as follows:

[0041] Aseptically weigh 25g of feces from a long-lived elderly person, add it to 225mL of physiological saline, and then dilute the bacterial suspension to 10. -6 10 -7 10-8 Three gradients were used, and then 0.2 mL was transferred to Hengate anaerobic roll culture medium and incubated at 37°C for 48-72 h. Small to medium-sized colonies with smooth, convex, even edges, milky white or slightly yellowish color, and soft texture were selected and inoculated into Bifidobacterium BS liquid medium. After anaerobic incubation for 24-48 h, Gram staining and microscopic examination were performed. Colonies exhibiting Bifidobacterium morphology (with a clear zone around the colony, smooth and even edges, white or slightly yellowish color) were selected and inoculated into Bifidobacterium BS liquid medium. They were incubated at 37°C for 24 h under both aerobic and anaerobic conditions, while simultaneously performing KOH, catalase, indole, and glucose metabolism tests. Bacteria that grew under anaerobic conditions, did not grow under aerobic conditions, were negative for KOH, catalase, and indole tests, and could metabolize glucose but did not produce gas were preliminarily identified as the selected Bifidobacteria. The preliminarily screened Bifidobacteria were inoculated into MRS liquid medium and cultured. Colonies with good results were picked and repeatedly streaked and purified. After screening, a strain of Bifidobacterium adolescentis was obtained, numbered HH404-1, and stored in glycerol at -80℃.

[0042] 1. Biochemical identification of the strain

[0043] Observe the morphology, color, etc. of the colonies of the strain on the surface of MRS medium according to Bergey's Manual of Bacteriological Identification (9th Edition). The colony morphology diagram is shown in the figure below. Figure 1 As shown; young cultures were selected, smeared, Gram-stained, and observed under a microscope for bacterial morphology, size, Gram staining reaction, and the presence, morphology, and attachment position of spores, etc. The microscopic images are shown below. Figure 2 As shown.

[0044] 3. Molecular biological identification of the strain

[0045] Taxonomic identification of the target strain HH404-1 was performed using 16S rDNA sequence analysis.

[0046] Strain HH404-1 was sent to the Sequencing and Identification Department of Shanghai Sangon Biotech for sequencing identification. The sequencing results are as follows:

[0047] GCGGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCTTCATGGAGTCGGGTTGCAGACTCCAATCCGAACTGAGACCGGTTTTAAGGGATCCGCTCCACCTCACAGTGTCGCATCCCGTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCCCCCGTGAGTTCCCACCACGACGTGCTGGCAACACAGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCCGCCCCGAAGGGAGACCGTATCTCTACGGCTGTCGGGAACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTGGCTCCGACACGGAGACCGTGGAATGGTCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCAAGCCCGCCCGTACCCGGCGCGGATCCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAATTCCGGATAACGCTTGCACCCTACGAATTACCGCGG

[0048] The sequencing results were compared and analyzed in the NCBI ribosome database. 16S rDNA sequence analysis showed that the strain of this invention shared 100% homology with *Bifidobacterium adolescentis*, indicating that this strain is *Bifidobacterium adolescentis*.

[0049] The strain was deposited as follows: Bifidobacterium adolescentis HH404-1, classified as Bifidobacterium adolescentis, deposited at Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 22, 2025, with accession number GDMCC No: 67151.

[0050] Secondly, the present invention provides a Bifida ferment filtrate.

[0051] The method for preparing the Bifida ferment filtrate includes the following steps:

[0052] (1) Activation of bacterial strain: Bifidobacterium adolescentis was inoculated into the first seed culture medium and cultured anaerobically to obtain activated bacterial solution;

[0053] (2) Seed culture: The activated seed culture was inoculated into the second seed culture medium and transferred to anaerobic culture twice to obtain the third seed culture;

[0054] (3) Fermentation culture: The three-stage seed liquid is inoculated into a fermenter containing fermentation culture medium and anaerobic fermentation culture is carried out to obtain fermented product; wherein, the fermentation culture includes a first anaerobic fermentation, a second anaerobic fermentation and a third anaerobic fermentation;

[0055] The conditions for the first anaerobic fermentation were: temperature 36-37℃, pH 6.0-6.2, rotation speed 100-110 rpm, and fermentation time 8-10 h.

[0056] The conditions for the second anaerobic fermentation are: temperature 38.5-39℃, pH 5.8-6.0, rotation speed 110-115 rpm, and fermentation time 10-12 h.

[0057] The conditions for the third anaerobic fermentation are: temperature 35-35.5℃, pH 5.8-6.0, rotation speed 95-100rpm, fermentation time 2-4h, at which time the residual sugar in the tank is <1g / L;

[0058] (4) Separation and purification: After filtering and decolorizing the fermentation product, the Bifida ferment filtrate can be obtained.

[0059] Preferably, in step (1), the Bifidobacterium adolescentis is Bifidobacterium adolescentis HH404-1, which is classified as Bifidobacterium adolescentis. It is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on October 22, 2025, with the accession number GDMCC No: 67151.

[0060] Preferably, in step (1), the first seed culture medium comprises the following raw material components in the following mass percentages: peptone 1.0-1.2%, beef extract 0.8-1.2%, yeast powder 0.4-0.7%, glucose 1.8-2.3%, dipotassium hydrogen phosphate 0.1-0.3%, triammonium citrate 0.1-0.3%, sodium acetate 0.4-0.6%, magnesium sulfate 0.01-0.04%, manganese sulfate 0.003-0.006%, and Tween-80 0.10-0.15%.

[0061] For example, in the first seed culture medium, the mass percentage of peptone can be 1.0%, 1.1%, or 1.2%, etc.; the mass percentage of beef extract can be 0.8%, 0.9%, 1.0%, or 1.2%, etc.; the mass percentage of yeast powder can be 0.4%, 0.6%, or 0.7%, etc.; the mass percentage of glucose can be 1.8%, 2.0%, 2.1%, or 2.3%, etc.; the mass percentage of dipotassium hydrogen phosphate can be 0.1%, 0.2%, or 0.3%, etc.; the mass percentage of triammonium citrate can be 0.1%, 0.2%, or 0.3%, etc.; the mass percentage of sodium acetate can be 0.4%, 0.5%, or 0.6%, etc.; the mass percentage of magnesium sulfate can be 0.003%, 0.004%, or 0.006%, etc.; and the mass percentage of Tween-80 can be 0.10%, 0.12%, or 0.15%, etc.

[0062] Preferably, in step (1), the anaerobic culture conditions are 37-38℃ for 16-20h. For example, the anaerobic culture temperature can be 37℃ or 38℃, and the culture time can be 16h, 18h, or 20h.

[0063] Preferably, in step (2), the two consecutive anaerobic cultures specifically involve: inoculating the activated bacterial solution into the second seed culture medium and anaerobically culturing it at 36-38℃ for 12-16 hours to obtain a primary seed culture; inoculating the primary seed culture into the first seed culture medium and anaerobically culturing it at 36-38℃, 95-110 rpm, and pH 6.0-6.2 for 10-12 hours to obtain a secondary seed culture; and inoculating the secondary seed culture into the second seed culture medium and anaerobically culturing it at 36-38℃, 100-110 rpm, and pH 6.0-6.2 for 10-13 hours to obtain a tertiary seed culture.

[0064] Preferably, in step (2), the second seed culture medium comprises the following raw material components in the following mass percentages: glucose 2.3-2.7%, peptone 0.8-1.2%, yeast extract 0.3-0.7%, magnesium sulfate 0.01-0.02%, manganese sulfate 0.003-0.006%, sodium acetate 0.4-0.6%, and ammonium citrate 0.13-0.16%.

[0065] For example, in the second seed culture medium, the mass percentage of glucose can be 2.3%, 2.5%, or 2.7%, etc.; the mass percentage of peptone can be 0.8%, 1.0%, or 1.2%, etc.; the mass percentage of yeast extract can be 0.3%, 0.5%, or 0.7%, etc.; the mass percentage of magnesium sulfate can be 0.01%, 0.015%, or 0.02%, etc.; the mass percentage of manganese sulfate can be 0.003%, 0.005%, or 0.006%, etc.; the mass percentage of sodium acetate can be 0.4%, 0.5%, or 0.6%, etc.; and the mass percentage of ammonium citrate can be 0.13%, 0.15%, or 0.16%, etc.

[0066] For example, in step (2), the anaerobic culture temperature of the activated bacterial solution can be 36℃, 37℃, or 38℃, and the culture time can be 12h, 14h, or 16h, etc.; the anaerobic culture temperature of the primary seed solution can be 36℃, 37℃, or 38℃, the rotation speed can be 95rpm, 100rpm, or 110rpm, etc., the pH can be 6.0, 6.1, or 6.2, etc., and the culture time can be 10h, 11h, or 12h, etc.; the anaerobic culture temperature of the secondary seed solution can be 36℃, 37℃, or 38℃, etc., the rotation speed can be 100rpm, 105rpm, or 110rpm, etc., the pH can be 6.0, 6.1, or 6.2, etc., and the culture time can be 10h, 12h, or 13h, etc.

[0067] Preferably, in step (3), the inoculation amount of the tertiary seed solution is 5-10%. For example, the inoculation amount of the tertiary seed solution can be 5%, 7%, 9% or 10%.

[0068] Preferably, in step (3), the fermentation medium comprises the following raw material components by mass percentage: glucose 4.0-5.0%, yeast treatment 1.5-1.8%, peptone 0.8-1.2%, dipotassium hydrogen phosphate 0.4-0.7%, licorice extract 0.3-0.5%, manganese sulfate 0.01-0.02%, magnesium sulfate 0.01-0.03%, anhydrous sodium acetate 0.4-0.7%, ammonium citrate 0.12-0.18%, and cysteine ​​0.1-0.3%.

[0069] For example, in the fermentation medium, the mass percentage of glucose can be 4.0%, 4.3%, 4.7%, 5.0%, etc.; the mass percentage of yeast treatment can be 1.5%, 1.7%, 1.8%, etc.; the mass percentage of peptone can be 1.5%, 1.7%, 1.8%, etc.; the mass percentage of dipotassium hydrogen phosphate can be 0.4%, 0.4%, 0.7%, etc.; the mass percentage of licorice extract can be 0.3%, 0.4%, 0.5%, etc.; the mass percentage of manganese sulfate can be 0.01%, 0.015%, 0.02%, etc.; the mass percentage of magnesium sulfate can be 0.01%, 0.02%, 0.03%, etc.; the mass percentage of anhydrous sodium acetate can be 0.4%, 0.6%, 0.7%, etc.; the mass percentage of ammonium citrate can be 0.12%, 0.16%, 0.18%, etc.; and the mass percentage of cysteine ​​can be 0.1%, 0.2%, 0.3%, etc.

[0070] For example, in step (3), the temperature of the first anaerobic fermentation can be 36℃, 36.5℃ or 37℃, the pH can be 6.0, 6.1 or 6.2, the rotation speed can be 100rpm, 105rpm or 110rpm, and the fermentation time can be 8h, 9h or 10h; the temperature of the second anaerobic fermentation can be 38.5℃, 38.7℃ or 39℃, the pH can be 5.8, 5.9 or 6.0, the rotation speed can be 110rpm, 112rpm or 115rpm, and the fermentation time can be 10h, 11h or 12h; the temperature of the third anaerobic fermentation can be 35℃, 35.2℃ or 35.5℃, the pH can be 5.8, 5.9 or 6.0, the rotation speed can be 95rpm, 98rpm or 100rpm, and the fermentation time can be 2h, 3h or 4h.

[0071] Preferably, in step (3), a first solution is added during the first fermentation; a second solution is added during the second fermentation; and a third solution is added during the third fermentation. The first solution comprises glucose and yeast extract, the second solution comprises glucose, yeast extract, and glutamine, and the third solution comprises trehalose and serine. Specifically, the feed rate is dynamically adjusted based on online dissolved oxygen (DO) and pH feedback. When DO rises sharply or pH fluctuates abnormally, the feed rate is reduced; when DO decreases steadily, the feed rate is increased.

[0072] Preferably, the first solution comprises the following components by mass percentage: 5-10% glucose and 0.5-1% yeast extract; the second solution comprises the following components by mass percentage: 5-10% glucose, 1-2% yeast extract and 0.1-0.3% glutamine; and the third solution comprises the following components by mass percentage: 0.1-0.3% trehalose and 0.03-0.05% serine.

[0073] For example, in the first solution, the mass percentage of glucose can be 5%, 7%, 9%, 10%, etc., and the mass percentage of yeast treatment can be 0.5%, 0.8%, 1%, etc.; in the second solution, the mass percentage of glucose can be 5%, 7%, 9%, 10%, etc., and the mass percentage of yeast treatment can be 1%, 1.5%, 2%, etc.; the mass percentage of glutamine can be 0.1%, 0.2%, or 0.3%, etc.; in the third solution, the mass percentage of trehalose can be 0.15%, 0.2%, 0.3%, etc., and the mass percentage of serine can be 0.03%, 0.04%, 0.05%, etc.

[0074] Preferably, the amount of the first solution added is 2-5% of the mass of the initial fermentation material, the amount of the second solution added is 4-7% of the mass of the initial fermentation material, and the amount of the third solution added is 1-2% of the mass of the initial fermentation material.

[0075] For example, the amount of the first solution added can be 2%, 4%, or 5% of the initial fermentation material mass, the amount of the second solution added can be 4%, 6%, or 7% of the initial fermentation material mass, and the amount of the third solution added can be 1%, 1.5%, or 2% of the initial fermentation material mass.

[0076] Preferably, in step (3), the yeast treatment is prepared by: crushing the yeast cells at 30-40 MPa to obtain a bacterial solution, adding a complex enzyme to the bacterial solution, and enzymatically hydrolyzing at 45-50℃ and pH 5.0-5.5 for 2-3 hours to obtain the yeast treatment; wherein the complex enzyme is prepared by mixing cellulase and β-glucanase in a mass ratio of 1:2.

[0077] For example, the crushing pressure of the brewing yeast cells can be 30MPa, 34MPa or 40MPa, the enzymatic hydrolysis temperature of the bacterial solution can be 45℃, 47℃ or 50℃, the enzymatic hydrolysis pH can be 5.0, 5.3 or 5.5, and the enzymatic hydrolysis time can be 2h, 2.5h or 3h.

[0078] Preferably, in step (4), the filtration is performed using a ceramic membrane; the decolorization can be performed using activated carbon or molecular sieves.

[0079] The present invention also provides an application of the above-mentioned Bifida ferment filtrate in the preparation of cosmetics.

[0080] Preferably, the cosmetic is a cosmetic with moisturizing and / or repairing effects.

[0081] For example, the dosage forms of the product include emulsions, creams, liquids, and gels.

[0082] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0083] Example 1

[0084] This embodiment provides a method for preparing Bifida ferment filtrate, the preparation method comprising the following steps:

[0085] 1. Activation of microbial strains

[0086] (1) Preparation of the first seed culture medium

[0087] Weigh the following components by mass percentage: peptone 1.0%, beef extract 1.0%, yeast powder 0.5%, glucose 2.0%, dipotassium hydrogen phosphate 0.2%, triammonium citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.005%, and Tween-80 0.1%. Prepare the first seed culture medium according to the above formula and adjust its pH to 6.5. Dispense the prepared seed culture medium into screw-cap test tubes, sterilize at 115℃ for 30 minutes, and cool for later use.

[0088] (2) Activation culture

[0089] Bifidobacterium adolescentis HH404-1 was inoculated into the first seed culture medium and placed in an anaerobic workstation for anaerobic culture at 37°C for 18 hours to obtain activated bacterial solution.

[0090] 2. Seed scaling-up culture

[0091] (1) Preparation of the second seed culture medium

[0092] Weigh the following components by mass percentage: glucose 2.5%, peptone 1%, yeast extract 0.5%, magnesium sulfate 0.01%, manganese sulfate 0.005%, anhydrous sodium acetate 0.5%, and ammonium citrate 0.15%. Prepare the second seed culture medium according to the above formula and adjust its pH to 6.5. Dispense the prepared seed culture medium into screw-cap test tubes, sterilize at 115℃ for 30 minutes, and cool for later use.

[0093] (2) Primary seed culture

[0094] At an inoculation rate of 5%, the activated bacterial solution was transferred to a 250 mL filament bottle containing 200 mL of the second seed culture medium and placed in an anaerobic workstation for 14 h at 37 °C to obtain the primary seed culture.

[0095] (3) Secondary seed culture

[0096] At an inoculation rate of 5%, the primary seed culture was transferred to a 5L seed tank containing 4L of secondary seed culture medium. After inoculation, high-purity nitrogen (99.99%) was introduced into the seed tank for 10 minutes to completely replace the air in the tank. After the dissolved oxygen was reduced to 0, the tank was clamped and pressurized. The tank was then cultured at 37°C, 100 rpm, and pH 6.0 (controlled by adding ammonia) for 12 hours to obtain the secondary seed culture.

[0097] (3) Three-stage seed culture

[0098] At an inoculation rate of 5%, the secondary seed culture was transferred to a 150L seed tank containing 80L of the second seed culture medium. After inoculation, high-purity nitrogen (99.99%) was introduced for 30 minutes to completely replace the air in the tank. After the dissolved oxygen was reduced to 0, the tank was kept under pressure and cultured at 37℃, 100rpm, and pH 6.0 (controlled by adding ammonia) for 12 hours to obtain the tertiary seed culture.

[0099] 3. Fermentation culture

[0100] (1) Preparation of yeast-treated products

[0101] The yeast cells were crushed at 30 MPa to obtain a bacterial solution. A complex enzyme, prepared by mixing cellulase and β-glucanase at a mass ratio of 1:2, was added to the bacterial solution. The solution was then enzymatically hydrolyzed at 45°C and pH 5.0 for 3 hours to obtain the yeast-treated product for later use.

[0102] (2) Preparation of licorice extract

[0103] Licorice powder and ethanol were mixed at a ratio of 1:8 and extracted at 70°C for a total of 3 times, 30 minutes each time. The extracts were combined, concentrated, and dried to obtain the licorice extract for later use.

[0104] (3) Preparation of fermentation medium

[0105] Weigh the following components by weight percentage: glucose 4.5%, yeast treatment 1.5%, peptone 1.0%, dipotassium hydrogen phosphate 0.5%, licorice extract 0.4%, manganese sulfate 0.01%, magnesium sulfate 0.01%, anhydrous sodium acetate 0.5%, ammonium citrate 0.15%, and cysteine ​​0.2%. Sterilize the glucose separately at 115°C for 20 min. Sterilize the remaining components together at 121°C for 30 min. Mix thoroughly after sterilization to obtain the fermentation medium, ready for use.

[0106] (4) Primary fermentation

[0107] At an inoculation rate of 7%, the tertiary seed culture was inoculated into a 1.5-ton fermenter containing 1.05 tons of fermentation medium. After inoculation, high-purity nitrogen (99.99%) was introduced for 50 minutes to completely replace the air in the tank. After the dissolved oxygen level dropped to zero, the pressure was maintained, and then the fermentation was carried out in three anaerobic stages, wherein:

[0108] The conditions for the first anaerobic fermentation were: temperature 37℃, pH 6.2, rotation speed 110 rpm, and fermentation time 8 hours.

[0109] The conditions for the second anaerobic fermentation were: temperature 39℃, pH 6.0, rotation speed 115 rpm, and fermentation time 11 h.

[0110] The conditions for the third anaerobic fermentation are: temperature 35℃, pH 5.8, rotation speed 100rpm, fermentation time 4h. At this time, the residual sugar in the tank is <1g / L. After stopping the tank, the fermentation product is obtained.

[0111] 4. Separation and purification

[0112] The fermentation product is purified by removing the bacterial cells and insoluble matter from the fermentation material using a ceramic membrane to obtain a clear fermentation broth; the clarified fermentation broth is then decolorized using activated carbon to obtain the Bifida ferment filtrate.

[0113] Example 2

[0114] This embodiment provides a method for preparing Bifida ferment filtrate, the preparation method comprising the following steps:

[0115] 1. Activation of microbial strains

[0116] (1) Preparation of the first seed culture medium

[0117] Weigh the following components by mass percentage: peptone 1.0%, beef extract 1.0%, yeast powder 0.5%, glucose 2.0%, dipotassium hydrogen phosphate 0.2%, triammonium citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.005%, and Tween-80 0.1%. Prepare the first seed culture medium according to the above formula and adjust its pH to 6.5. Dispense the prepared seed culture medium into screw-cap test tubes, sterilize at 115℃ for 30 minutes, and cool for later use.

[0118] (2) Activation culture

[0119] Bifidobacterium adolescentis HH404-1 was inoculated into the first seed culture medium and placed in an anaerobic workstation for anaerobic culture at 37°C for 18 hours to obtain activated bacterial solution.

[0120] 2. Seed scaling-up culture

[0121] (1) Preparation of the second seed culture medium

[0122] Weigh the following components by mass percentage: glucose 2.5%, peptone 1%, yeast extract 0.5%, magnesium sulfate 0.01%, manganese sulfate 0.005%, anhydrous sodium acetate 0.5%, and ammonium citrate 0.15%. Prepare the second seed culture medium according to the above formula and adjust its pH to 6.5. Dispense the prepared seed culture medium into screw-cap test tubes, sterilize at 115℃ for 30 minutes, and cool for later use.

[0123] (2) Primary seed culture

[0124] At an inoculation rate of 5%, the activated bacterial solution was transferred to a 250 mL filament bottle containing 200 mL of the second seed culture medium and placed in an anaerobic workstation for 14 h at 37 °C to obtain the primary seed culture.

[0125] (3) Secondary seed culture

[0126] At an inoculation rate of 5%, the primary seed culture was transferred to a 5L seed tank containing 4L of secondary seed culture medium. After inoculation, high-purity nitrogen (99.99%) was introduced into the seed tank for 10 minutes to completely replace the air in the tank. After the dissolved oxygen was reduced to 0, the tank was clamped and pressurized. The tank was then cultured at 37°C, 100 rpm, and pH 6.0 (controlled by adding ammonia) for 12 hours to obtain the secondary seed culture.

[0127] (3) Three-stage seed culture

[0128] At an inoculation rate of 5%, the secondary seed culture was transferred to a 150L seed tank containing 80L of the second seed culture medium. After inoculation, high-purity nitrogen (99.99%) was introduced for 30 minutes to completely replace the air in the tank. After the dissolved oxygen was reduced to 0, the tank was kept under pressure and cultured at 37℃, 100rpm, and pH 6.0 (controlled by adding ammonia) for 12 hours to obtain the tertiary seed culture.

[0129] 3. Fermentation culture

[0130] (1) Preparation of yeast-treated products

[0131] The yeast cells were crushed at 30 MPa to obtain a bacterial solution. A complex enzyme, prepared by mixing cellulase and β-glucanase at a mass ratio of 1:2, was added to the bacterial solution. The solution was then enzymatically hydrolyzed at 45°C and pH 5.0 for 3 hours to obtain the yeast-treated product for later use.

[0132] (2) Preparation of licorice extract

[0133] Licorice powder and ethanol were mixed at a ratio of 1:8 and extracted at 70°C for a total of 3 times, 30 minutes each time. The extracts were combined, concentrated, and dried to obtain the licorice extract for later use.

[0134] (3) Preparation of fed-batch solution

[0135] Preparation of the first solution: Mix 8% glucose and 0.5% yeast treatment material evenly by mass percentage to obtain the first solution, and set aside for later use;

[0136] Preparation of the second solution: Mix 10% glucose, 1.5% yeast treatment and 0.02% glutamine by mass percentage to obtain the second solution, and set aside for later use;

[0137] Preparation of the third solution: Mix 0.2% trehalose and 0.03% serine evenly by mass percentage to obtain the third solution, which is then set aside.

[0138] (4) Preparation of fermentation medium

[0139] Weigh the following components by weight percentage: glucose 4.5%, yeast treatment 1.5%, peptone 1.0%, dipotassium hydrogen phosphate 0.5%, licorice extract 0.4%, manganese sulfate 0.01%, magnesium sulfate 0.01%, anhydrous sodium acetate 0.5%, ammonium citrate 0.15%, and cysteine ​​0.2%. Sterilize the glucose separately at 115°C for 20 min. Sterilize the remaining components together at 121°C for 30 min. Mix thoroughly after sterilization to obtain the fermentation medium, ready for use.

[0140] (5) Primary fermentation

[0141] At an inoculation rate of 7%, the tertiary seed culture was inoculated into a 1.5-ton fermenter containing 1.05 tons of fermentation medium. After inoculation, high-purity nitrogen (99.99%) was introduced for 50 minutes to completely replace the air in the tank. After the dissolved oxygen level dropped to zero, the pressure was maintained, and then the fermentation was carried out in three anaerobic stages, wherein:

[0142] The conditions for the first anaerobic fermentation were: temperature 37℃, pH 6.2, rotation speed 110 rpm, and fermentation time 8 hours; and during the fermentation process, the first solution was added in a flow-through at a rate of 3% of the initial fermentation material mass.

[0143] The conditions for the second anaerobic fermentation were: temperature 39℃, pH 6.0, rotation speed 115 rpm, and fermentation time 11 h; and during the fermentation process, the second solution was added in a flow-through manner at a rate of 5% of the initial fermentation material mass.

[0144] The conditions for the third anaerobic fermentation are: temperature 35℃, pH 5.8, rotation speed 100rpm, fermentation time 4h. At this time, the residual sugar in the tank is <1g / L. During the fermentation process, the third solution is added, and the amount added is 1.5% of the initial fermentation material mass. After the tank is stopped, the fermentation product is obtained.

[0145] 4. Separation and purification

[0146] The fermentation product is purified by removing the bacterial cells and insoluble matter from the fermentation material using a ceramic membrane to obtain a clear fermentation broth; the clarified fermentation broth is then decolorized using activated carbon to obtain the Bifida ferment filtrate.

[0147] Comparative Example 1

[0148] Comparative Example 1 provides a method for preparing Bifida ferment filtrate. The preparation method differs from the preparation method of Example 1 only in step 3 (4). Specifically, step 3 (4) of Comparative Example 1 involves inoculating the tertiary seed liquid into a 1.5-ton fermenter containing 1.05 tons of fermentation medium at an inoculation rate of 7%. After inoculation, high-purity nitrogen (99.99%) is introduced for 50 minutes to completely replace the air in the tank. After the dissolved oxygen is reduced to 0, the pressure is maintained, and fermentation is carried out at 37°C, 100 rpm, and pH 6.0 for 24 hours. At this time, the residual sugar in the tank is <1 g / L. After stopping the fermentation, the fermentation product is obtained.

[0149] Comparative Example 2

[0150] Comparative Example 2 provides a method for preparing Bifida ferment filtrate. The preparation method differs from the preparation method of Example 2 only in step 3 (5) of the main fermentation. Specifically, step 3 (5) of Comparative Example 2 involves inoculating the tertiary seed liquid into a 1.5-ton fermenter containing 1.05 tons of fermentation medium at an inoculation rate of 7%. After inoculation, high-purity nitrogen (99.99%) is introduced for 50 minutes to completely replace the air in the tank. After the dissolved oxygen is reduced to 0, the pressure is maintained, and the fermenter is heated at 37°C for 10 minutes. Fermentation was carried out at 0 rpm and pH 6.0 for 23 hours. At this time, the residual sugar in the tank was <1 g / L. After the fermentation was stopped, the fermentation product was obtained. During the first 1-8 hours of fermentation, the first solution was added at a rate of 3% of the initial fermentation product mass. During the second 8-19 hours of fermentation, the second solution was added at a rate of 5% of the initial fermentation product mass. During the third 19-24 hours of fermentation, the third solution was added at a rate of 1.5% of the initial fermentation product mass. After the fermentation was stopped, the fermentation product was obtained.

[0151] Comparative Example 3

[0152] Comparative Example 3 provides a method for preparing Bifida ferment filtrate. The preparation method differs from the preparation method in Example 2 only in step 3 (5) of the main fermentation. Specifically, step 3 (5) of the main fermentation in Comparative Example 2 is as follows:

[0153] At an inoculation rate of 7%, the tertiary seed culture was inoculated into a 1.5-ton fermenter containing 1.05 tons of fermentation medium. After inoculation, high-purity nitrogen (99.99%) was introduced for 50 minutes to completely replace the air in the tank. After the dissolved oxygen level dropped to zero, the pressure was maintained, and then the fermentation was carried out in three anaerobic stages, wherein:

[0154] The conditions for the first anaerobic fermentation were: temperature 37℃, pH 6.2, rotation speed 110 rpm, and fermentation time 8 hours; and during the fermentation process, the first solution was added in a flow-through at a rate of 3% of the initial fermentation material mass.

[0155] The conditions for the second anaerobic fermentation were: temperature 39℃, pH 6.0, rotation speed 115 rpm, and fermentation time 11 h; and during the fermentation process, the first solution was added in a flow-through at a rate of 5% of the initial fermentation material mass.

[0156] The conditions for the third anaerobic fermentation are: temperature 35℃, pH 5.8, rotation speed 100rpm, fermentation time 4h. At this time, the residual sugar in the tank is <1g / L. During the fermentation process, the first solution is added, and the amount added is 1.5% of the initial fermentation material mass. After the tank is stopped, the fermentation product is obtained.

[0157] Comparative Example 4

[0158] Comparative Example 2 provides a method for preparing a Bifida ferment filtrate. The method differs from the method in Example 2 only in step 3(4) of preparing the fermentation medium. Specifically, step 3(4) of Comparative Example 2 involves weighing the following components by mass percentage: glucose 4.5%, yeast extract 1.5%, peptone 1.0%, dipotassium hydrogen phosphate 0.5%, licorice extract 0.4%, manganese sulfate 0.01%, magnesium sulfate 0.01%, anhydrous sodium acetate 0.5%, ammonium citrate 0.15%, and cysteine ​​0.2%. Glucose is sterilized separately at 115°C for 20 minutes. The remaining components are mixed and sterilized at 121°C for 30 minutes. After sterilization, the mixture is thoroughly mixed to obtain the fermentation medium, which is then ready for use.

[0159] Comparative Example 5

[0160] Comparative Example 2 provides a method for preparing a Bifida ferment filtrate. The method differs from the method in Example 2 only in step 3(4) of preparing the fermentation medium. Specifically, step 3(4) of Comparative Example 2 involves weighing the following components by mass percentage: glucose 4.5%, yeast extract 1.5%, peptone 1.0%, dipotassium hydrogen phosphate 0.5%, manganese sulfate 0.01%, magnesium sulfate 0.01%, anhydrous sodium acetate 0.5%, ammonium citrate 0.15%, and cysteine ​​0.2%. Glucose is sterilized separately at 115°C for 20 minutes. The remaining components are mixed and sterilized at 121°C for 30 minutes. After sterilization, the mixture is thoroughly mixed to obtain the fermentation medium, which is then ready for use.

[0161] The Bifida ferment filtrates prepared in Examples 1-2 and Comparative Examples 1-5 were applied to serums to obtain serums S1-S7, respectively. The specific serum formulations are shown in Table 1 below:

[0162] Table 1. Serum Formula (%, w / w)

[0163]

[0164] The preparation method of the above-mentioned essences S1-S7 is as follows: First, add phases B, C, and D to phase A in sequence, stir and disperse, heat to 80°C, soak and swell, stir and homogenize for 2-3 minutes to obtain a mixed phase; Second, mix the three raw materials of phase E and heat to 60°C, stir until the solid is completely dissolved; Finally, stir and cool the mixed phase to below 60°C, add the pre-dissolved phase E, stir evenly, and the corresponding essence can be obtained.

[0165] The moisturizing and repairing effects of the above-mentioned serums S1-S7 were measured using the following methods:

[0166] 1. Subjects

[0167] Subjects were screened based on their facial condition (those who reported having one of the following symptoms of impaired skin barrier function: dryness, peeling, redness, etc.) and other individual health conditions. A total of 84 subjects were selected and completed the study, with 0 dropouts. Among them, 70 were female and 14 were male, with an average age of 28.7 ± 5.6 years, meeting the subject voluntary inclusion criteria.

[0168] 2. Experimental Design

[0169] The participants were randomly divided into 7 groups of 12 people each (10 women + 2 men), designated as groups S1-S7 (corresponding to serums S1-S7). After cleansing, participants applied an appropriate amount of the corresponding product evenly to their facial skin and gently massaged until absorbed. The treatment was administered twice daily for 28 days. The testing period was from June 25, 2025 to July 23, 2025.

[0170] 3. Testing

[0171] The effectiveness was evaluated using a self-comparison principle before and after the event.

[0172] (1) Testing basis

[0173] "China Industry-University-Research Collaboration Promotion Association Group Standard: Test Methods for Seven Efficacy Items in Cosmetics (Anti-wrinkle, Firming, Moisturizing, Oil Control, Repairing, Nourishing, and Soothing)" T / CAB 0152-2022

[0174] (2) Instruments and equipment

[0175] The Corneometer CM825 skin moisture test probe, the CK Tewameter transdermal moisture loss tester, and the VISIA facial image analyzer all meet the performance requirements of the testing specifications.

[0176] (3) Testing environment

[0177] The instrument testing was conducted in an environment with a temperature of 21±1 °C and a relative humidity of 50±10% RH. Subjects were required to acclimatize to these conditions for 30 minutes before evaluation and testing. The ambient temperature and humidity during this test are shown in Figure 3 and Figure 4, respectively. Figure 4 As shown.

[0178] (4) Testing process

[0179] ① Cleaning: Before the test, cleanse your face with a cleansing product (Cetaphil facial cleanser) and dry with a lint-free, fluorescent agent-free tissue; repeat the cleaning process for each follow-up visit.

[0180] ② Technicians sequentially measured baseline skin parameters, including VISIA (visual information over surface area), stratum corneum moisture content, and transepidermal water loss (TEWL), through facial image acquisition. During data acquisition, the operator ensured the subject was in a comfortable position to measure skin parameters in the test area. Each test probe was in complete contact with the skin and kept perpendicular during testing, and data were recorded. The same tests were repeated 4 weeks later (D28), and all values ​​were averaged.

[0181] (5) Statistical methods

[0182] SPSS software was used for statistical analysis of the data. Quantitative data were expressed as mean ± standard deviation and subjected to a normality test. If the data met the normality requirement, a paired t-test was used for comparisons of the data before and after the same data point; otherwise, a rank-sum test of two related samples was used.

[0183] 4. Test Results

[0184] (1) The results of the test of the moisture content of the stratum corneum of the subject's skin are shown in Table 2 below.

[0185] Table 2. Skin stratum corneum moisture content of subjects in each group

[0186]

[0187] (2) The results of the transepidermal water loss (TEWL) test are shown in Table 3 below.

[0188] Table 3 Transdermal water loss (TEWL) of subjects in each group

[0189]

[0190] (3) Skin redness index The results of the value detection are shown in Table 4 below.

[0191] Table 4 Skin redness index of subjects in each group value

[0192]

[0193] As shown in Tables 2-4, after using the corresponding serum for 28 days, the average skin stratum corneum moisture content of subjects in groups S1 and S2 increased by 71.07% and 77.78% respectively compared to before use (D0). This indicates that the Bifida Ferment Lysate Filtrate prepared in this invention has good moisturizing effects, and the transepidermal water loss (TEWL) and skin redness index are also significantly reduced. The values ​​decreased by at least 8.88% and 6.22% respectively, showing a significant improvement, indicating that the Bifida ferment filtrate prepared by this invention has good repair effects. Specifically, the moisturizing and repairing effects of groups S3 and S4 were significantly lower than those of groups S1 and S2, indicating that the three-stage fermentation control of this invention helps to increase the metabolic rate, promote the accumulation of target metabolites, and thus increase the content of active ingredients, thereby improving its moisturizing and repairing effects. The moisturizing and repairing effects of group S5 were significantly lower than those of group S4, indicating that the addition of specific solutions to the three-stage fermentation process of this invention can match the nutritional and metabolic needs of the cells at different stages, thereby increasing the yield and stability of active ingredients in the fermentation broth, and thus improving the moisturizing and repairing effects. The moisturizing and repairing effects of groups S6 and S7 were also reduced to varying degrees compared to group S2, indicating that the use of a specially treated yeast extract instead of yeast powder in the fermentation medium of this invention not only effectively improves nitrogen source utilization, but also, combined with licorice extract, better meets the growth needs of the cells, induces metabolite synthesis, and thus increases the content of effective active ingredients.

Claims

1. A method for preparing a Bifida ferment filtrate, characterized in that, Includes the following steps: (1) Activation of bacterial strain: Bifidobacterium adolescentis was inoculated into the first seed culture medium and cultured anaerobically to obtain activated bacterial solution; the Bifidobacterium adolescentis was Bifidobacterium adolescentis HH404-1, and the preservation number of Bifidobacterium adolescentis HH404-1 was GDMCC No: 67151; (2) Seed culture: The activated bacterial solution was inoculated into the second seed culture medium and transferred to anaerobic culture twice to obtain the third seed culture; (3) Fermentation culture: The three-stage seed liquid is inoculated into a fermenter containing fermentation culture medium and anaerobic fermentation culture is carried out to obtain fermented product; wherein, the anaerobic fermentation culture includes a first anaerobic fermentation, a second anaerobic fermentation and a third anaerobic fermentation; The conditions for the first anaerobic fermentation were: temperature 36-37℃, pH 6.0-6.2, rotation speed 100-110 rpm, and fermentation time 8-10 h. The conditions for the second anaerobic fermentation are: temperature 38.5-39℃, pH 5.8-6.0, rotation speed 110-115 rpm, and fermentation time 10-12 h. The conditions for the third anaerobic fermentation are: temperature 35-35.5℃, pH 5.8-6.0, rotation speed 95-100 rpm, and fermentation time 2-4 hours. During the first anaerobic fermentation, a first solution is added; during the second anaerobic fermentation, a second solution is added; and during the third anaerobic fermentation, a third solution is added. The first solution is composed of the following components by mass percentage: 5-10% glucose and 0.5-1% yeast treatment; the second solution is composed of the following components by mass percentage: 5-10% glucose, 1-2% yeast treatment, and 0.1-0.3% glutamine; and the third solution is composed of the following components by mass percentage: 0.1-0.3% trehalose and 0.03-0.05% serine. The fermentation medium is composed of the following raw material components in the indicated weight percentages: glucose 4.0-5.0%, yeast treatment 1.5-1.8%, peptone 0.8-1.2%, dipotassium hydrogen phosphate 0.4-0.7%, licorice extract 0.3-0.5%, manganese sulfate 0.01-0.02%, magnesium sulfate 0.01-0.03%, anhydrous sodium acetate 0.4-0.7%, ammonium citrate 0.12-0.18%, and cysteine ​​0.1-0.3%. The method for preparing the yeast treatment product is as follows: the yeast cells are crushed at 30-40 MPa to obtain a bacterial solution, a complex enzyme is added to the bacterial solution, and the solution is enzymatically hydrolyzed at 45-50℃ and pH 5.0-5.5 for 2-3 hours to obtain the yeast treatment product; wherein the complex enzyme is prepared by mixing cellulase and β-glucanase in a mass ratio of 1:

2. (4) Separation and purification: After filtering and decolorizing the fermentation product, the Bifida ferment filtrate can be obtained.

2. The method for preparing Bifida ferment filtrate according to claim 1, characterized in that, The first seed culture medium comprises the following raw material components in weight percentage: peptone 1.0-1.2%, beef extract 0.8-1.2%, yeast extract 0.4-0.7%, glucose 1.8-2.3%, dipotassium hydrogen phosphate 0.1-0.3%, triammonium citrate 0.1-0.3%, sodium acetate 0.4-0.6%, magnesium sulfate 0.01-0.04%, manganese sulfate 0.003-0.006%, and Tween-80 0.10-0.15%; the second seed culture medium comprises the following raw material components in weight percentage: glucose 2.3-2.7%, peptone 0.8-1.2%, yeast extract 0.3-0.7%, magnesium sulfate 0.01-0.02%, manganese sulfate 0.003-0.006%, sodium acetate 0.4-0.6%, and ammonium citrate 0.13-0.16%.

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