Bifidobacterium fermentation product as well as preparation method and application thereof

Abstract

The invention discloses a bifidobacterium fermentation product as well as a preparation method and application thereof. According to the invention, placenta protein polypeptide hydrolysate is adopted as a substrate, Bifidobacterium animalis Ba5 obtained by screening is fermented to obtain the bifidobacterium animalis Ba5, and the preservation number of the bifidobacterium animalis Ba5 is GDMCC No.66814. The invention further discloses a preparation method of the bifidobacterium animalis Ba5. Experiments prove that the obtained bifidobacterium fermentation product can significantly improve the activity of skin cells, also has a significant effect in the aspect of oxidation resistance, and has the potentials of light damage repair, moisturizing, whitening and barrier repair. The preparation method of the bifidobacterium fermentation product can realize double assurance of efficiency and efficacy stability, provides a more efficient and safer application scheme, has the advantages of differentiation, stability, high bioavailability and environmental protection, and has extremely high promotable value and economic value.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a Bifidobacterium fermentation product, its preparation method, and its application. Background Technology

[0002] In existing technologies, probiotics (such as Bifidobacteria and Lactobacillus) are defined as beneficial live microorganisms that primarily function by regulating the gut microbiota and are widely used in cosmetics, health products, and other fields. Bifidobacteria have attracted considerable attention due to their unique morphology and dominant role in skin health.

[0003] Protein hydrolysates are a class of protein molecules obtained through protease hydrolysis. Rich in peptides, amino acids, and a range of trace components, they are widely used in cosmetics, such as hydrolyzed egg white and hydrolyzed fish skin protein, which are listed in the cosmetic ingredient catalog. In recent years, based on safety and efficacy considerations, the cosmetic industry has increasingly favored ingredients like Bifida Ferment Lysate (Bifidobacterium) fermentation product filtrate. These metabolites produced by Bifidobacterium fermentation are rich in polysaccharides, proteins, and peptides, and have been proven to have skincare benefits such as moisturizing, antioxidant, and anti-aging effects. They have also been approved for use as cosmetic ingredients.

[0004] In the fermentation process of Bifidobacteria, bifidus factors are crucial for its proliferation. Among these, protein hydrolysate-based bifidus factors, such as hydrolysates of whey protein, lactoferrin, and casein, are the most widely used. Industrially, soybean trypsin hydrolysate and yeast extract are commonly used as fermentation medium components for Bifidobacteria, while the use of animal protein polypeptide hydrolysates is relatively limited. Meanwhile, existing technologies for preparing Bifidobacteria fermentation product filtrates also face several technical challenges, such as insufficient research on the compatibility of fermentation substrates with the metabolic characteristics of the strains, leading to low substrate conversion efficiency. Production efficiency is affected by factors such as operator skill, high production costs, and long production cycles. The core limiting factor is that Bifidobacteria strains cannot fully express their metabolic potential due to the lack of an optimized culture system; this technical bottleneck directly restricts the industrialization of high-value-added fermentation products.

[0005] Metabolomics, as a science that studies the metabolites of organisms (with molecular weights typically less than 1500 Da), can reflect the physiological state of an organism. Untargeted metabolomics, through comprehensive analysis of samples, seeks differentially expressed metabolites and can be used to screen biomarkers or reveal their biological functions and mechanisms of action. This technology has been widely applied in various research fields. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the purpose of this invention is to provide a Bifidobacterium fermentation product, its preparation method, and its application. The Bifidobacterium fermentation product of this invention specifically overcomes the problems of low efficiency and difficulty in enhancing activity in existing Bifidobacterium fermentation products. It uses placental protein polypeptide hydrolysate as a substrate and is obtained through fermentation with animal Bifidobacterium. Experiments have shown that the obtained Bifidobacterium fermentation product can significantly enhance skin cell vitality, and it also has significant antioxidant effects, as well as potential for photodamage repair, moisturizing, whitening, and barrier repair. The preparation method of the Bifidobacterium fermentation product of this invention achieves a dual guarantee of efficiency and efficacy stability, providing a more efficient and safer application solution, while also possessing advantages in differentiation, bioavailability, and environmental friendliness, exhibiting extremely high scalability and economic value.

[0007] In a first aspect, the present invention provides a Bifidobacterium animalis, namely Bifidobacterium animalis Ba5, taxonomically named Bifidobacterium animalis, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 6, 2025, with accession number GDMCC No. 66814.

[0008] In some embodiments of the present invention, the colony morphology of the animal Bifidobacterium Ba5 is milky white, round and convex, and has a smooth surface.

[0009] In some embodiments of the present invention, the animal Bifidobacterium Ba5, after Gram staining, appears blue-purple and irregularly rod-shaped under a microscope, and is a typical Gram-positive bacterium.

[0010] In some embodiments of the present invention, the 16S rRNA sequence of the animal Bifidobacterium Ba5 is shown in SEQ ID NO.1.

[0011] In a second aspect, the present invention provides a method for preparing a Bifidobacterium fermentation product, comprising the following steps: fermenting placental hydrolysate with Bifidobacterium.

[0012] In some embodiments of the present invention, the placental hydrolysate includes products obtained by enzymatic hydrolysis of animal placenta using proteases.

[0013] In some embodiments of the present invention, the protease includes at least one of alkaline protease, flavor protease, trypsin, bromelain, neutral protease, and papain.

[0014] In some embodiments of the present invention, the protease is a neutral protease.

[0015] In some embodiments of the present invention, the conditions for enzymatic hydrolysis are: enzymatic hydrolysis temperature of 40°C and enzymatic hydrolysis time of 1 hour.

[0016] In some embodiments of the present invention, the animal is a non-human animal.

[0017] In some embodiments of the present invention, the non-human animal includes any viviparous animal.

[0018] In some embodiments of the present invention, the Bifidobacterium is Bifidobacterium animalis.

[0019] In some embodiments of the present invention, the animal bifidobacterium is the animal bifidobacterium Ba5 described above.

[0020] In some embodiments of the present invention, the Bifidobacterium is a Bifidobacterium obtained by culturing in MRS medium.

[0021] In some embodiments of the present invention, the composition and preparation method of the MRS culture medium (solid) are as follows: Weigh and mix 10.0g of casein digest, 10.0g of beef extract powder, 4.0g of yeast extract powder, 2.0g of triammonium citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 20.0g of glucose, 1.08g of Tween-80, 0.5g of L-cysteine ​​hydrochloride, and 15.0g of agar. Dissolve in water, adjust the pH to 7.0, and bring the volume to 1.0L. Sterilize at 121°C for 15 minutes. When preparing MRS liquid culture medium, agar is not added.

[0022] In some embodiments of the present invention, the fermentation conditions are: anaerobic fermentation at 32-40℃ for 25-40 hours.

[0023] In some embodiments of the present invention, the fermentation temperature is 35-40°C.

[0024] In this invention, the term "anaerobic" refers to an environment with an oxygen content of less than 0.2%.

[0025] In some embodiments of the present invention, the preparation method specifically involves: picking a single colony of Bifidobacterium animalis and inoculating it into MRS liquid culture medium, anaerobically culturing it at 35-40℃ for 36-48 hours to obtain a seed culture, inoculating the seed culture into placental protein polypeptide hydrolysate, and anaerobically culturing it at 35-40℃ for 25-30 hours to obtain the Bifidobacterium fermentation product.

[0026] In some embodiments of the present invention, the inoculation amount of the seed solution is 1%-5%.

[0027] In some embodiments of the present invention, the inoculation concentration OD of the seed solution is...600 It ranges from 1.00 to 2.00.

[0028] A third aspect of the present invention provides a Bifidobacterium fermentation product prepared by the preparation method described above.

[0029] In some embodiments of the present invention, the fermentation products of the Bifidobacterium include, but are not limited to, substances such as cytosine, S-methyl-5'-thioadenosine, β-D-glucose, and N-acetyl-β-mannosamine.

[0030] In some embodiments of the present invention, the fermentation products of Bifidobacterium may also include, but are not limited to, substances such as pyruvic acid, nonanoic acid, cis-aconitine, mevalonic acid, and histidine.

[0031] In some embodiments of the present invention, the form of the Bifidobacterium fermentation product is not limited, and any pharmaceutical form in the art can be reasonably adopted according to the purpose of use, storage requirements, etc., including but not limited to: lyophilized powder, solution, etc.

[0032] In a fourth aspect, the present invention provides a Bifidobacterium fermentation product filtrate, wherein the Bifidobacterium fermentation product filtrate is the filtrate of the Bifidobacterium fermentation product described in the above aspect.

[0033] In some embodiments of the present invention, the filtration pore size of the Bifidobacterium fermentation product filtrate is 0.2-0.5 μm.

[0034] In some embodiments of the present invention, the Bifidobacterium fermentation product is centrifuged before filtration.

[0035] In some embodiments of the present invention, the centrifugation conditions are: 10000-14000 rpm, 10-30 min.

[0036] In some embodiments of the present invention, the form of the Bifidobacterium fermentation product filtrate is not limited, and any pharmaceutical form in the art can be reasonably adopted according to the purpose of use, storage requirements, etc., including but not limited to: lyophilized powder, solution, etc.

[0037] A fifth aspect of the present invention provides the use of the Bifidobacterium fermentation product or Bifidobacterium fermentation product filtrate described above in the preparation of pharmaceuticals or cosmetics.

[0038] In some embodiments of the present invention, Bifidobacterium fermentation products or Bifidobacterium fermentation product filtrate, Bifidobacterium lysate and extracts, etc., have been included in the "Catalogue of Used Cosmetic Raw Materials (2021 Edition)" issued by the National Medical Products Administration.

[0039] In some embodiments of the present invention, the pharmaceutical or cosmetic product has at least one of the following functions (1)-(6):

[0040] (1) Improve cell viability or promote cell proliferation;

[0041] (2) Skin whitening;

[0042] (3) Antioxidant;

[0043] (4) Moisturizing;

[0044] (5) Promotes skin repair;

[0045] (6) Build or repair the skin barrier.

[0046] In some embodiments of the present invention, the pharmaceutical or cosmetic product also contains excipients.

[0047] In some embodiments of the present invention, the excipients include pharmaceutically and / or cosmetically acceptable excipients.

[0048] In this invention, the term "cosmetically acceptable adjuvant" refers to "cosmetically or pharmaceutically acceptable carrier," that is, any conventional cosmetic ingredient or pharmaceutical carrier that can be used in the methods disclosed in this invention. For specific selections, please refer to Remington's Pharmaceutical Sciences, Ewmartin Mack Publishing Co., Easton, Pa., 15th edition (1975).

[0049] In some embodiments of the present invention, the cosmetic-acceptable excipients and pharmaceutical-acceptable excipients include, but are not limited to, diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, crospovidone, etc.), lubricants (such as talc, hydrogenated vegetable oil, polyethylene glycol, etc.), colorants (such as titanium dioxide, methylene blue, etc.), coating materials, solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), and chelating agents (such as disodium EDTA).

[0050] The beneficial effects of this invention are:

[0051] This invention uses placental protein polypeptide hydrolysate as a fermentation substrate and obtains it through fermentation with Bifidobacterium animalis. Based on experimental findings, Bifidobacterium animalis is the probiotic that can ferment placental protein polypeptide hydrolysate and produce significant skin cell activity in this test. Further metabolomics analysis effectively identified key differential metabolites and potential biomarkers, elucidated their potential mechanisms of action, and demonstrated the compatibility between the substrate and microbial fermentation.

[0052] The Bifidobacterium fermentation product or Bifidobacterium fermentation product filtrate in this invention not only demonstrates outstanding effects in improving cell vitality, whitening, and anti-oxidation, but also possesses potential for photodamage repair, moisturizing, and barrier repair. It provides the cosmetics industry with a new high-quality raw material option and shows great potential in enhancing product efficacy, opening up new research and development directions and application prospects for the industry.

[0053] The method for preparing Bifidobacterium fermentation products or Bifidobacterium fermentation product filtrate in this invention is reliable and can consistently improve efficiency. The resulting product provides a more efficient and safer option for applications, and possesses multiple advantages such as differentiation, high bioavailability, and environmental friendliness. Attached Figure Description

[0054] Figure 1 This is a colony morphology diagram of Bifidobacterium animalis Ba5 in this invention.

[0055] Figure 2 This is a Gram staining image of Bifidobacterium animalis Ba5 in this invention.

[0056] Figure 3 This is an amplified image of the 16S rRNA gene sequence of Bifidobacterium animalis Ba5 in this invention.

[0057] Figure 4 A comparative diagram showing the effects of fermentation product filtrates A, B, C, and D on skin cell activity.

[0058] Figure 5 The total ion chromatograms of three batches of fermentation product filtrate B in positive ion mode (A) and negative ion mode (B) are overlaid spectra.

[0059] Figure 6 This is a graph showing the percentage of metabolites identified in fermentation product filtrate B across various chemical categories.

[0060] Figure 7 This is a differential metabolic volcano diagram of fermentation product filtrate B.

[0061] Figure 8 The top 10 metabolites of fermentation product filtrate B with the highest differences in positive ion mode are listed below.

[0062] Figure 9The top 10 metabolites of fermentation product filtrate B with the highest differences under negative ion mode are listed.

[0063] Figure 10 The results show the test results of HSF cell viability for fermentation product filtrate B.

[0064] Figure 11 The results are from the tyrosinase inhibition test of fermentation product filtrate B.

[0065] Figure 12 The results are from the free radical scavenging test of fermentation product filtrate B.

[0066] Figure 13 The results show the cell damage repair (A) and antioxidant (B) capabilities of fermentation product filtrate B.

[0067] Figure 14 The results are from the assay of moisturizing-related gene expression in fermentation product filtrate B.

[0068] Figure 15 The results show the expression of barrier-related genes in fermentation product filtrate B. Detailed Implementation

[0069] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0070] Experimental materials

[0071] In the following examples, the composition and preparation method of the MRS solid culture medium were as follows: 10.0 g of casein digest, 10.0 g of beef extract powder, 4.0 g of yeast extract powder, 2.0 g of triammonium citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0 g of dipotassium hydrogen phosphate, 20.0 g of glucose, 1.08 g of Tween-80, 0.5 g of L-cysteine ​​hydrochloride, and 15.0 g of agar were weighed and mixed, dissolved in water, the pH was adjusted to 7.0, and the volume was brought to 1.0 L. The medium was then sterilized at 121°C for 15 min. When preparing the MRS liquid culture medium, agar was not added.

[0072] In the following embodiments, the placental protein polypeptide hydrolysate was prepared by the following method:

[0073] (1) Enzymatic hydrolysis: The placenta is thoroughly cleaned and then enzymatically hydrolyzed with a protease. The protease can be alkaline protease, flavor protease, trypsin, neutral protease, etc. In this example, a neutral protease was used. The hydrolysis temperature was 40°C and the hydrolysis time was 1 hour to obtain the hydrolysis product.

[0074] (2) Filtration: After the enzymatic hydrolysis is completed, coarse filtration and fine filtration are performed in sequence to obtain placental protein polypeptide hydrolysate.

[0075] In the following examples, the Bifidobacterium used is Bifidobacterium animalis Ba5.

[0076] Example 1

[0077] This embodiment provides a method for isolating and purifying a strain of Bifidobacterium animalis, specifically as follows:

[0078] (1) Separation, purification and morphological identification

[0079] Fresh breast milk samples from healthy volunteers were mixed thoroughly, and 1 mL was taken as the stock solution. The stock solution was serially diluted with sterile water. The appropriate dilution was then spread on MRS solid medium containing mupirocin and anaerobically incubated at 37°C for 48 h. Typical single colonies of Bifidobacterium were picked and streaked again on MRS solid medium containing mupirocin for purification. After further incubation, single colonies were picked and transferred to MRS liquid medium for enrichment. The culture was then stored at -80°C with 25% glycerol to obtain strain Ba5.

[0080] The morphological results of strain Ba5 are as follows Figure 1 As shown, the colonies of strain Ba5 are milky white, round and raised, with a smooth surface.

[0081] Activate strain Ba5, pick a single colony and transfer it to MRS liquid medium. Incubate anaerobically at 37°C for 48 h. Take 5 μL of bacterial suspension and drop it onto the center of a glass slide. Fix the slide by intermittent baking with an alcohol lamp. Add crystal violet staining solution and stain for 1 min, then wash with water. Add Gram's iodine solution and act for 1 min, then wash with water. Add decolorizing alcohol for about 30 s, then add safranin counterstaining solution and counterstain for 1 min. Wash with water, dry, and examine under a microscope.

[0082] The results are as follows Figure 2 As shown, strain Ba5 appears blue-purple and irregularly rod-shaped under a microscope, typical of Gram-positive bacteria.

[0083] (2) Identification of 16S rRNA

[0084] The genome of strain Ba5 was extracted using a commercially available kit, and its 16S rRNA sequence was amplified using universal bacterial primers 27F / 1492R. The results were detected by agarose gel electrophoresis. Figure 3 As shown.

[0085] Based on the electrophoresis results, an amplification product with a size of approximately 1500 bp was selected for sequencing identification.

[0086] The nucleotide sequence of the strain's 16S rRNA, as shown in SEQ ID NO: 1, was obtained through sequencing.

[0087]

[0088] Homology sequence alignment and analysis of its 16S rRNA were performed on NCBI, and the results showed that the closest genus was Bifidobacterium animalis, with a similarity of 99.86%. Therefore, the strain was named Bifidobacterium animalis Ba5.

[0089] The obtained Bifidobacterium animalis Ba5 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 6, 2025. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66814. The taxonomic name is Bifidobacterium animalis.

[0090] Example 2

[0091] This embodiment provides a method for preparing Bifidobacterium fermentation product filtrate, specifically as follows:

[0092] The frozen Bifidobacterium animalis Ba5 was activated, and single colonies with good growth were picked and inoculated into MRS liquid medium. The culture was then incubated anaerobicly at 37°C for 36-48 hours to obtain a seed culture. The OD value was adjusted. 600 The inoculum concentration was 1.00-2.00. Then, the seed culture was inoculated into placental protein polypeptide hydrolysate at a 2% inoculum concentration and anaerobically cultured at 37°C for 28 hours to obtain the fermentation product. The fermentation broth was centrifuged at 12000 rpm for 10-30 minutes. After precipitation, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the Bifidobacterium fermentation product filtrate (hereinafter referred to as fermentation product filtrate B).

[0093] Example 3

[0094] This embodiment provides a method for preparing Bifidobacterium fermentation product filtrate. The specific preparation steps are the same as in Embodiment 1, except that the basal culture medium MRS is used as the fermentation substrate.

[0095] Example 4

[0096] This embodiment provides a method for preparing Bifidobacterium fermentation product filtrate. The specific preparation steps are the same as in Embodiment 1, except that Sigma-Aldrich (a hydrolysate of lactalbumin) is used as the fermentation substrate.

[0097] Example 5

[0098] This embodiment provides a method for preparing Bifidobacterium fermentation product filtrate. The specific preparation steps are the same as in Embodiment 1, except that Bifidobacterium longum is used to ferment placental protein polypeptide hydrolysate.

[0099] Comparative Example 1

[0100] This comparative example provides a method for preparing a filtrate of Bifidobacterium fermentation products, specifically as follows:

[0101] Bifidobacterium animalis Ba5 was activated and cultured according to the method in Example 2 to obtain a seed culture. Then, the seed culture was inoculated into fresh MRS liquid medium at an inoculation rate of 2%, and anaerobic culture was carried out at 37°C for 28 hours to obtain the fermentation broth. The broth was then centrifuged and filtered according to the method in Example 1 to obtain the Bifidobacterium fermentation product filtrate (hereinafter referred to as fermentation product filtrate A).

[0102] Comparative Example 2

[0103] This comparative example provides a method for preparing a filtrate of Bifidobacterium fermentation products, specifically as follows:

[0104] Bifidobacterium animalis Ba5 was activated and cultured according to the method in Example 2 to obtain a seed culture. Then, the seed culture was inoculated into lactalbumin hydrolysate at an inoculum volume of 2%, and anaerobic culture was carried out at 37°C for 28 hours to obtain the fermentation broth. The broth was then centrifuged and filtered according to the method in Example 1 to obtain the Bifidobacterium fermentation product filtrate (hereinafter referred to as fermentation product filtrate C).

[0105] Comparative Example 3

[0106] This comparative example provides a method for preparing a filtrate of Bifidobacterium fermentation products, specifically as follows:

[0107] Bifidobacterium longum was activated and cultured according to the method in Example 2 to obtain a seed culture. Then, the seed culture was inoculated into the placental protein polypeptide hydrolysate from Example 1 at an inoculation rate of 2%, and anaerobically cultured at 37°C for 28 hours to obtain the fermentation broth. The broth was then centrifuged and filtered according to the method in Example 1 to obtain the Bifidobacterium fermentation product filtrate (hereinafter referred to as fermentation product filtrate D).

[0108] Test Example 1

[0109] In this test example, skin cell viability was tested on the fermentation product filtrates (fermentation product filtrates A, B, C, and D) from the above embodiments and comparative examples. In this test example, the test subject was HaCaT cells, and the specific test steps were as follows:

[0110] HaCaT cells in the logarithmic growth phase were injected with 1×10 4Cells were seeded at a density of 1 cell per well in 96-well plates and cultured for 24 hours. Then, fermentation product filtrate A, B, C, or D was added at volume percentages of 0.3125%, 0.625%, 1.25%, 2.5%, 5%, and 10%, respectively, and cultured for another 24 hours. Cell viability was then assessed using the CCK8 assay.

[0111] The results are as follows Figure 4 As shown.

[0112] It was found that, compared with other comparative examples (P < 0.0001), fermentation product filtrate B obtained by specific fermentation of Bifidobacterium animalis Ba5 using placental protein polypeptide hydrolysate as a substrate significantly enhanced HaCaT cell viability at different concentrations, and its effect was superior to other fermentation product filtrates, showing statistically significant differences. Particularly noteworthy is that at a concentration of 2.5%, the cell viability-promoting effect of fermentation product filtrate B increased by more than 200%, initially revealing its potential to promote skin cell proliferation. Moreover, this proliferative activity showed a clear concentration dependence, that is, it increased with increasing concentration of fermentation product filtrate B. These results collectively confirm the rationality and unique advantages of the fermentation product filtrate B obtained in the embodiments of the present invention, based on the synergistic fermentation strategy of placental protein hydrolysate and Bifidobacterium animalis Ba5.

[0113] Test Example 2

[0114] Metabolites are intermediate or final products generated during metabolism. They participate in various biochemical reactions and maintain cellular life activities. Changes in metabolite levels can reflect changes brought about by the fermentation process and the physiological state that may result. In this test example, non-targeted metabolomics studies were conducted on fermentation product filtrate B from Example 2 above using liquid chromatography-mass spectrometry (LC-MS / MS) technology.

[0115] The specific steps are as follows:

[0116] Following the method described in the above embodiments, three batches of fermentation product filtrate B were prepared. Pre-cooled methanol / acetonitrile / water solution (mixing ratio 2:2:1, v / v / v) was added, and the mixture was vortexed and sonicated at low temperature for 30 min, then allowed to stand at -20℃ for 10 min. The mixture was then centrifuged at 14000g for 20 min at 4℃, and the supernatant was vacuum dried. For mass spectrometry analysis, 100 μL of acetonitrile / water solution (acetonitrile:water = 1:1, v / v) was added to reconstitute the solution. After vortexing, the mixture was centrifuged at 14000g for 15 min at 4℃, and the supernatant was injected for analysis.

[0117] The results are as follows Figure 5-9 As shown.

[0118] By comparing the total ion chromatograms (TIC) of fermentation product filtrate samples, Figure 5It can be observed that the chromatographic peak response intensity and retention time of each replicate sample are highly consistent, indicating that the instrument error is small and the analysis results are reliable.

[0119] Further chemical classification and statistical analysis were performed on all identified metabolites (combining positive and negative ion patterns). Figure 6 It can be found that the filtrates of these fermentation products mainly contain organic acids, lipids, cyclic organic compounds, benzene rings, organic oxides, phenylpropanes / polyketides, and nitrogen-containing organic compounds.

[0120] Further analysis using variable weights (VIP) and multidimensional analysis revealed that the fermentation process significantly altered the metabolite profile. Combining univariate analysis (fold change, FC) and setting stringent screening criteria (OPLS-DA VIP > 1.0, P < 0.05, FC > 1.0 or < 1.0), significantly differentially expressed metabolites were identified. Figure 7 The results showed that 129 metabolites were upregulated and 59 were downregulated in the positive ion mode, while 93 metabolites were upregulated and 74 were downregulated in the negative ion mode, totaling 355 differentially regulated metabolites. This further demonstrates that the fermentation process significantly alters the metabolite profile. Significant upregulation or downregulation of a large number of metabolites was observed in both positive and negative ion modes. Figure 8 and Figure 9 The top 10 differentially expressed metabolites were shown. Further comprehensive KEGG pathway analysis revealed that these changes indicate that the fermentation process generates many metabolites related to antioxidant, whitening, moisturizing, and repair functions.

[0121] The fermentation system consists of microorganisms and substrate. Placental protein polypeptide hydrolysate is rich in D-ribose, adenine, and other substances. After fermentation with Bifidobacterium animalis, the levels of these substances decreased, indicating that they may have promoted ATP synthesis, providing more energy for fermentation. Simultaneously, adenosine was significantly reduced, while its derivative, S-methyl-5'-thioadenosine, significantly increased, reflecting the active glycosyl release and demethylation reactions during fermentation.

[0122] It is noteworthy that the content of the potent antioxidant NDGA (Nordihydroguaiaretic acid) also decreased significantly during fermentation, possibly due to degradation by microbial enzymes or structural modification. This may mean that NDGA is converted into milder, more skin-tolerant derivatives, such as N-acetyl-β-d-mannosamine detected in the above examples. This allows it to retain its antioxidant activity while reducing irritation and enhancing its brightening and soothing effects. These changes demonstrate the advantages of fermentation technology in optimizing molecular structure through biocatalysis to obtain more ideal cosmetic ingredients, meeting the current market demand for mild and highly effective products.

[0123] Furthermore, under negative ion mode, the upregulation of metabolites such as pyruvate and cis-aconitate confirms the activity of energy metabolism pathways such as glycolysis and the TCA cycle under anaerobic fermentation, and the production of various organic acids, also demonstrating the compatibility of placental protein hydrolysate with Bifidobacterium animalis. Histidine has antioxidant and moisturizing effects, helping to enhance the skin barrier function. The upregulation of histidine and the reduction of histidine-lysine dipeptide reveal that microbial fermentation promotes protein breakdown and amino acid release. The significant increase in mevalonic acid is also noteworthy. Its precursor, mevalonolactone, is an important cosmetic ingredient that rapidly converts to mevalonic acid upon contact with water. Mevalonic acid not only has excellent moisturizing and firming effects, but also effectively promotes the skin's own cholesterol synthesis, repairing the skin from the root. As we age, the skin's own synthesis capacity declines, making exogenous supplementation of mevalonic acid crucial for maintaining healthy and youthful skin.

[0124] Test Example 3

[0125] In this test example, the viability of HSF cells was tested on the fermentation product filtrate B from the above examples.

[0126] The specific testing method is as follows: HSF cells are used as the test subject, and the specific testing steps are as follows: HSF cells in the logarithmic growth phase are injected with 1.5 × 10⁻⁶ cells. 4 Cells were seeded at a density of cells / well in 96-well plates. After cell adhesion, the culture medium was aspirated, and the sample diluted with complete culture medium was added. After culturing for 48 hours, the results were analyzed using the CCK8 assay. Specifically, after adding CCK8, the cells were incubated at 37°C for 1.5 hours, and the OD450 nm absorbance was measured. The fermentation substrate was used as a blank control, and the cell viability of HSF after sample addition was calculated.

[0127] The results are as follows Figure 10 As shown.

[0128] It was found that, compared with the blank control group, fermentation product filtrate B significantly enhanced the viability of HSF cells (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001), with statistically significant differences. Particularly at a 10% concentration, fermentation product filtrate B promoted cell viability by over 150%, demonstrating potential for promoting skin cell proliferation. Furthermore, this proliferative effect increased with increasing concentration, exhibiting a clear concentration-dependent relationship.

[0129] Test Example 4

[0130] In this test example, the tyrosinase activity inhibition test was performed on fermentation product filtrate B from the above examples.

[0131] The specific test method is as follows: using kojic acid (KA) as a standard, and each sample is reacted with tyrosinase reagent, the tyrosinase inhibition rate of KA is calculated, and the standard curve is plotted.

[0132] like Figure 11 As shown in A, the standard curve equation for the tyrosinase inhibition rate of KA is:

[0133] y = -0.0007782x 2 +0.5060x+3.394, R 2 =0.9871.

[0134] The tyrosinase inhibition rate of the samples was calculated based on the standard curve of KA, and the results are as follows: Figure 11 As shown in B in the diagram.

[0135] It was found that the inhibitory effect of fermentation product filtrate B on tyrosinase was concentration-dependent, with an inhibition rate of nearly 40% at higher concentrations. This indicates that fermentation product filtrate B has potential skin-whitening effects.

[0136] Test Example 5

[0137] In this test example, the antioxidant potential of fermentation product filtrate B from the above examples was evaluated.

[0138] The specific testing method is as follows:

[0139] (1) Prepare ABTs working solution, add Trolox (V) E ) and fermentation product filtrate B, with Trolox (V E Using 1 as the standard, draw a standard curve.

[0140] like Figure 12 As shown in A, Trolox (V EThe standard curve for ABTs is y = 0.4832x + 0.2696, R0 2 =0.9983.

[0141] Then according to Trolox (V E The ABTs free radical scavenging rate of fermentation product filtrate B was calculated using the ABTs standard curve (e.g., ...). Figure 12 (B in the middle).

[0142] (2) Using Trolox (V E Using ) as the standard product, Trolox (V) is manufactured. E The relative fluorescence decay curve of ).

[0143] like Figure 12 In the C-value, it was found that at a concentration of 25 μg / mL, the fluorescence intensity decreased to near zero at 120 min. Generally speaking, the higher the oxidative free radical absorption capacity (ORAC) value of a sample, the stronger its antioxidant capacity.

[0144] like Figure 12 As shown in D, it can be observed that at the concentration used, fermentation product filtrate B achieves an ABTS scavenging rate of nearly 150% at a 10% volume ratio, while even at lower concentrations, fermentation product filtrate B still achieves an ORAC free radical scavenging rate of nearly 100%. This strongly demonstrates that fermentation product filtrate B not only possesses the potential whitening effects previously confirmed in experiments, but also exhibits significant potential antioxidant effects.

[0145] Test Example 6

[0146] In this test example, the cell-level damage repair and antioxidant capacity of fermentation product filtrate B from the above examples were tested.

[0147] The specific testing method for cell damage repair is as follows: using 2×10 4 HaCaT cells in the logarithmic cell phase were seeded into 96-well plates and irradiated with UVB (300 mJ / cm²) 24 h later. 2 The cells were added to the test samples (prepared using basal culture medium) according to the group, and cell viability was detected by CCK8 method after culturing for 24 hours. Untreated cells were used as the blank control group (CON), UVB-irradiated cells were used as the blank model group (MODEL), and serum FBS was used as the positive control.

[0148] Simultaneously, antioxidant capacity was tested using an H2O2 model. The specific testing method was as follows: a H2O2 (0.6 mM) oxidative damage model was established using HaCaT cells to detect cellular antioxidant capacity. After treating the cells with H2O2 and various test samples, cell viability was measured. The prerequisite for determining whether a sample possesses antioxidant capacity was that the sample could significantly improve cell viability in the antioxidant model. Untreated cells served as the blank control group (CON), H2O2-irradiated cells served as the blank model group (MODEL), and glutathione (L-GSH) served as the positive control.

[0149] The results are as follows Figure 13 As shown.

[0150] It was found that, compared with the treated blank model group (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001), fermentation product filtrate B not only effectively reduced UVB irradiation-induced cell damage but also significantly improved cell survival rate under H2O2 stimulation, and this improvement was significantly higher than that of the positive control group (*P<0.05, ***P<0.001), showing a statistically significant difference. This indicates that fermentation product filtrate B possesses strong cell damage repair and antioxidant capabilities.

[0151] Test Example 7

[0152] In this test case, the expression of moisturizing-related genes in fermentation product filtrate B from the above examples was tested. The specific test method was as follows: using HaCaT cells as a model, the effect of filtrate B on the expression of hydration-related genes was detected. AQP3, FLG, and CD44 are all genes related to moisturizing efficacy. After 24 hours of adherent culture, the experimental group cells were aspirated and treated with filtrate B for 48 hours. A blank control group (BC) was also established. After treatment, cells were digested with trypsin and collected. Total RNA was extracted from the cells (using a Magen kit), reverse transcribed into cDNA, and then the relative expression levels of AQP3, FLG, and CD44 genes were analyzed by quantitative real-time PCR (qPCR).

[0153] The results are as follows Figure 14 As shown.

[0154] It was found that the expression levels of FLG, AQP3, and CD44 were closely related to skin moisturizing function. Compared with the blank control (P < 0.0001), the fermentation product filtrate B in the above examples significantly upregulated the expression levels of these three genes, with statistical significance. This indicates that the fermentation product filtrate B in the above examples may enhance the skin's hydration capacity by promoting the expression of these key genes, thereby possessing potential moisturizing effects.

[0155] Test Example 8

[0156] In this test example, the expression of skin barrier-related genes was tested in fermentation product filtrate B from the above examples.

[0157] The specific testing method was as follows: Using HaCaT cells as a model, the effect of filtrate B on the expression of skin barrier-related genes was detected. ZO-1 and IVL are barrier repair-related genes. After 24 hours of adherent culture, the experimental group cells were aspirated and treated with filtrate B for 48 hours. A blank control group (BC) was also set up. After treatment, the cells were digested with trypsin and collected. Total RNA was extracted from the cells (using a Magen kit), reverse transcribed into cDNA, and then the relative expression levels of ZO-1 and IVL genes were analyzed by quantitative real-time PCR (qPCR).

[0158] The results are as follows Figure 15 As shown.

[0159] It was found that ZO-1 and IVL are both known key genes involved in the maintenance and repair of skin barrier structure. Compared with the blank control (P < 0.0001), the fermentation product filtrate B in the above examples significantly upregulated the expression of these two genes, with statistical significance. This indicates that the fermentation product filtrate B in the above examples can enhance the integrity of the skin barrier by promoting the expression of these barrier-related genes, thereby possessing potential barrier repair efficacy.

[0160] In summary, this invention successfully prepared a Bifidobacterium fermentation product filtrate with significant efficacy (especially in promoting cell viability or proliferation) by fermenting placental protein polypeptide hydrolysate with Bifidobacterium animalis Ba5. This not only demonstrates its enormous potential in improving product performance but also ensures consistency for future large-scale production, fully reflecting the core technological advantages of this invention.

[0161] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A type of animal Bifidobacterium, characterized in that, The Bifidobacterium animalis mentioned is Bifidobacterium animalis Ba5, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 6, 2025, with accession number GDMCC No. 66814.

2. A method for preparing a Bifidobacterium fermentation product, comprising the following steps: It is obtained by fermenting placental hydrolysate with Bifidobacterium; in, The placental hydrolysate includes products obtained by enzymatic hydrolysis of animal placenta using proteases; Preferably, the bifidobacteria include animal bifidobacteria; Preferably, the animal bifidobacterium is the animal bifidobacterium as described in claim 1.

3. The preparation method according to claim 2, characterized in that, The Bifidobacterium is a Bifidobacterium obtained by culturing in MRS medium.

4. The preparation method according to claim 2, characterized in that, The fermentation conditions are: anaerobic fermentation at 32-40℃ for 25-40 hours; preferably, the fermentation temperature is 35-40℃.

5. The Bifidobacterium fermentation product prepared by the method according to any one of claims 2-4; Preferably, the Bifidobacterium fermentation product contains at least one of cytosine, S-methyl-5'-thioadenosine, β-D-glucose, N-acetyl-β-mannosamine, and vitamins.

6. The Bifidobacterium fermentation product according to claim 5, characterized in that, The fermentation product of Bifidobacterium also contains at least one of pyruvate, nonanoic acid, cis-aconitine, mevalonic acid and histidine.

7. A filtrate of Bifidobacterium fermentation products, characterized in that, The filtrate of the Bifidobacterium fermentation product is the filtrate of the Bifidobacterium fermentation product as described in claim 5 or 6. Preferably, the filtration pore size of the Bifidobacterium fermentation product filtrate is 0.2-0.5 μm.

8. The Bifidobacterium fermentation product filtrate according to claim 7, characterized in that, The Bifidobacterium fermentation product was centrifuged before filtration. Preferably, the centrifugation conditions are: 10000-14000 rpm, 10-30 min.

9. Use of the Bifidobacterium fermentation product according to any one of claims 5-6 or the filtrate of the Bifidobacterium fermentation product according to any one of claims 7-8 in the preparation of pharmaceuticals or cosmetics.

10. The use according to claim 9, characterized in that, The medicine or cosmetic has at least one of the following functions (1)-(6): (1) Improve cell viability or promote cell proliferation; (2) Skin whitening; (3) Antioxidant; (4)Moisturizing; (5) Promotes skin repair; (6) Build or repair the skin barrier.

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