Preparation method of yunling fungus ferment, product, preparation fungus and application thereof

Abstract

This invention provides a method for preparing a fermented product of *Saussurea involucrata*, the product itself, the strain used in its preparation, and its applications, belonging to the field of microbial fermentation. This invention also provides a novel strain isolated from *Saussurea involucrata* flora: *Bifidobacterium longum* subsp. *infantitidis* (…). Bifidobacterium longum subsp. infantis Xue-66 and Lactobacillus helveticus ( Lactobacillus helveticus Xue-123, the aforementioned Bifidobacterium longum subsp. infantileum Xue-66, Lactobacillus helveticus Xue-123 and Lactobacillus plantarum ( Lactiplantibacillus plantarum PQQ-1, when co-fermented with whey protein and collagen, yields a metabiotic called Yakult ferment. This Yakult ferment significantly inhibits tyrosinase activity and gene expression, exhibiting significant whitening effects and effectively increasing skin elasticity.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, specifically relating to a method for preparing Yakultek ferments, its products, the microorganisms used in preparation, and their applications. Background Technology

[0002] Snow lotus mushroom, also known as Tibetan kefir, is a milky white or light yellow gelatinous block-like substance. Its growth shape is irregular, mostly spherical or rice-grain-like. Its surface curls within the milky substance, allowing it to grow rapidly, forming cauliflower-like clumps that are elastic and have a sticky feel when squeezed, resembling the appearance of a snow lotus.

[0003] Yakult is a microbial mixture composed of lactic acid bacteria, acetic acid bacteria, and yeast. It has been reported that yakult, yakult fermented milk, and its metabolically active substances are effective in improving and preventing acute gastrointestinal diseases, preventing hyperlipidemia, regulating intestinal flora balance, and exhibiting anti-allergic, antibacterial, antioxidant, and immune-regulating effects.

[0004] Current research on *Saussurea involucrata* primarily focuses on analyzing its microbial community. Without understanding the composition of this community, it's impossible to delve into its fermentation process, metabolic mechanisms, or ensure the safety of its fermented products.

[0005] The existing technology CN113181086A provides the application of Yakult fermentation product in inhibiting tyrosinase activity and melanin production. This patent uses Yakult, which is composed of lactic acid bacteria, acetic acid bacteria and Bacillus, to ferment pure milk. However, the fermentation product only has a certain inhibitory effect on the production of melanin in cells when the amount is 2.5 mg / mL. This indicator still needs to be further improved.

[0006] He Binbin et al. evaluated the probiotic properties of lactic acid bacteria isolated from *Saussurea involucrata*. The DPPH free radical scavenging activities of the eight strains varied considerably. Strain RG exhibited the highest DPPH free radical scavenging activity at 72.93%, while BG-2, KF2-5, and NC-1 were at moderate levels of 34.24%, 32.13%, and 43.23%, respectively. BG-1, GG-1, GG-2, and CQ-2 were all below 30%. This study indicates that the lactic acid bacteria in *Saussurea involucrata* possess certain antioxidant capabilities, but most strains exhibit weak antioxidant capacity. Further research is needed to explore how to utilize *Saussurea involucrata* to prepare products with strong antioxidant properties. (He Shanshan, Wang Xiaorui, Peng Yuxi, et al. Probiotic properties of lactic acid bacteria in *Saussurea involucrata* [J]. Food Science, 2022, 43(02):210-216.)

[0007] Therefore, there is an urgent need to isolate and purify the microorganisms in *Saussurea involucrata*, and to explore its fermentation process and metabolic mechanisms, so as to obtain safe, controllable, and more effective *Saussurea involucrata* ferments. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a method for preparing Yakult-fermented bacteria, the product thereof, the bacteria used in the preparation, and its applications.

[0009] On the one hand, the present invention provides a method for preparing snow lotus ferment, which uses Bifidobacterium longum subsp. infantis Xue-66, Lactobacillus helveticus Xue-123 and Lactobacillus plantarum PQQ-1 to mix and ferment the substrate, and the post-biotic is separated from the fermentation product to obtain snow lotus ferment.

[0010] Specifically, the mass ratio of Bifidobacterium longum subsp. infantis Xue-66, Lactobacillus helveticus Xue-123, and Lactobacillus plantarum PQQ-1 is 0.5-2:1-3:1-5.

[0011] More specifically, the mass ratio of Bifidobacterium longum subsp. infantis Xue-66, Lactobacillus helveticus Xue-123, and Lactobacillus plantarum PQQ-1 is 0.5-1:1-1.5:1-3.

[0012] More specifically, the mass ratio of Bifidobacterium longum subsp. infantis Xue-66, Lactobacillus helveticus Xue-123, and Lactobacillus plantarum PQQ-1 is 1-2:1.5-3:3-5.

[0013] Specifically, the substrates include collagen and whey protein.

[0014] More specifically, by weight, the amount of collagen used is 15-20%, and the amount of whey protein used is 10-15%.

[0015] Preferably, the amount of collagen used is 15-18% by weight, and the amount of whey protein used is 10-13%.

[0016] Preferably, the amount of collagen used is 18-20% by weight, and the amount of whey protein used is 13-15%.

[0017] More specifically, the substrate also includes lactose.

[0018] Preferably, the amount of lactose used is 3-10% by weight.

[0019] More preferably, the amount of lactose used is 7-9% by weight.

[0020] In some specific embodiments of the present invention, the amount of lactose used is 7-8% by weight.

[0021] In some specific embodiments of the present invention, the amount of lactose used is 8-9% by weight.

[0022] Specifically, the temperature control conditions for the mixed fermentation process are: 45℃-25℃, with a stepwise change of 4-6℃ / h.

[0023] More specifically, the culture conditions during the mixed fermentation process are: rotation speed 300-500 rpm, fermentation time 20-24 h.

[0024] Preferably, the rotation speed during the mixed fermentation process is 300-400 rpm.

[0025] Preferably, the rotation speed during the mixed fermentation process is 400-500 rpm.

[0026] Specifically, the fermentation products are separated into postbiotics through centrifugation and filtration.

[0027] More specifically, the centrifugation is ultra-high-speed centrifugation, and the conditions for high-speed centrifugation are: 2-8℃, 10000-12000 g, 10-20 min.

[0028] Preferably, the centrifugation is ultra-high speed centrifugation, and the conditions for high-speed centrifugation are: 2-8℃, 11000-12000 g, 15-20 min.

[0029] Preferably, the centrifugation is ultra-high-speed centrifugation, and the conditions for high-speed centrifugation are: 2-8℃, 10000-11000 g, 10-15 min.

[0030] More specifically, the filter membrane used in the filtration step has a mesh size of 100-400.

[0031] Preferably, the filter membrane used in the filtration step has a mesh size of 300-400.

[0032] Preferably, the filter membrane used in the filtration step has a mesh size of 100-300.

[0033] In another aspect, the present invention provides a snow lotus ferment obtained by the above preparation method.

[0034] In another aspect, the present invention provides the application of the above-mentioned snow lotus ferment in inhibiting tyrosinase activity, inhibiting tyrosinase expression, promoting collagen synthesis and / or anti-oxidation.

[0035] In another aspect, the present invention provides the application of the above-mentioned snow lotus ferment in the preparation of products that inhibit tyrosinase activity, inhibit tyrosinase expression, promote collagen synthesis and / or have antioxidant properties.

[0036] Specifically, the products include, but are not limited to, skincare or cosmetic products.

[0037] On another aspect, the present invention provides a new strain of Bifidobacterium longum subsp. infant, Xue-66, obtained by purifying the snow lotus bacterial community. The Bifidobacterium longum subsp. infant, Xue-66, is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 33981.

[0038] On another note, the present invention provides a new strain of Lactobacillus helveticus Xue-123 obtained by purifying the snow lotus bacterial community. Lactobacillus helveticus Xue-123 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 33982.

[0039] On the other hand, the present invention provides the application of the above-mentioned Bifidobacterium longum subsp. infantis Xue-66 and Lactobacillus helveticus Xue-123 in the preparation of Yakult ferments.

[0040] Compared with the prior art, the present invention has the following advantages:

[0041] (1) The present invention uses plant lactobacillus PQQ-1, helvetica lactobacillus Xue-66, and bifidobacterium longum subsp. infantis Xue-123 in combination with optimized fermentation conditions, which can significantly improve fermentation efficiency and reduce the fermentation time.

[0042] (2) The snow lotus ferment provided by the present invention has the ability to significantly inhibit tyrosinase activity and inhibit the gene expression of tyrosinase, and has significant whitening effect.

[0043] (3) At the same time, compared with unfermented collagen, fermented collagen can induce the synthesis of its own type I collagen-related genes at a lower concentration. col1a1a, col1a1b, eln1 and eln2 Its ability can effectively increase skin elasticity.

[0044] Preservation Instructions 1

[0045] Biomaterial name: Xue-66.

[0046] Taxonomous name: Bifidobacterium longum infantis subspecies Bifidobacterium longum subsp. infantis .

[0047] Date of preservation: March 26, 2025.

[0048] Accession number: CGMCC No.33981.

[0049] Preservation institution: China General Microbiological Culture Collection Center, China Microbial Culture Collection Committee.

[0050] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0051] Preservation Instructions 2

[0052] Biomaterial name: Xue-123.

[0053] Taxonomous name: Lactobacillus helveticus Lactobacillus helveticus

[0054] Date of preservation: March 26, 2025.

[0055] Accession number: CGMCC No.33982.

[0056] Preservation institution: China General Microbiological Culture Collection Center, China Microbial Culture Collection Committee.

[0057] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0058] Preservation Instructions 3

[0059] Biomaterial name: PQQ-1.

[0060] Taxonomous name: Lactobacillus plantarum Lactiplantibacillus plantarum .

[0061] Date of preservation: December 12, 2023.

[0062] Accession number: CGMCC No.29303.

[0063] Preservation institution: China General Microbiological Culture Collection Center, China Microbial Culture Collection Committee.

[0064] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0065] Figure 1 This is a typical image of the melanin signal intensity in the head of a zebrafish after sample treatment in Example 1.

[0066] Figure 2 The value represents the melanin signal intensity in the zebrafish head after sample treatment in Example 1. *** indicates a comparison with the model control group, p<0.001.

[0067] Figure 3 The OD of zebrafish tyrosinase activity after sample treatment in Example 1 is shown. 475nm Values, ** represent p < 0.01 compared with the model control group, *** represent p < 0.001 compared with the model control group.

[0068] Figure 4 The percentage of melanin in zebrafish after sample treatment in Example 1 is represented by **, which indicates a comparison with the model control group (p < 0.01), and ***, which indicates a comparison with the model control group (p < 0.001).

[0069] Figure 5 For example 1 tyr Relative gene expression levels, ** represents p < 0.01 compared with the model control group, *** represents p < 0.001 compared with the model control group.

[0070] Figure 6 For example 2 col1a1a Relative gene expression levels, ** represents p < 0.01 compared with the normal control group, *** represents p < 0.001 compared with the normal control group.

[0071] Figure 7 For example 2 col1a1b Relative gene expression levels, * indicates p < 0.05 compared with the normal control group, ** indicates p < 0.01 compared with the normal control group.

[0072] Figure 8 For example 2 eln1 Relative gene expression levels, * indicates p < 0.05 compared to the normal control group, ** indicates p < 0.01 compared to the normal control group, and *** indicates p < 0.001 compared to the normal control group.

[0073] Figure 9 For example 2 eln2 Relative gene expression levels, * indicates p < 0.05 compared to the normal control group, ** indicates p < 0.01 compared to the normal control group, and *** indicates p < 0.001 compared to the normal control group.

[0074] Figure 10 The results of the ABTS clearance ability in Example 3 are shown. ** represents the comparison with Comparative Example 1, p<0.01, and *** represents the comparison with Comparative Example 1, p<0.001. Detailed Implementation

[0075] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0076] Example 1: Preparation method of Yakult fermentation product

[0077] 1. Substrate preparation: By weight, collagen: 18%, whey protein: 13%, lactose: 8%, balance: water. Sterilize at 95°C for 30 minutes.

[0078] 2. Inoculation: By weight, Xue-66:Xue-123:PQQ-1 = 1:1.5:3, and the inoculation dose is 5.0 × 10⁻⁶. 9 cfu / L.

[0079] 3. Fermentation: Initial temperature 45℃, fermentation for 16 hours, followed by a stepwise cooling rate of 5℃ / h, with the final temperature reduced to 25℃. The final fermentation time was 24 hours. Rotation speed: 300 rpm

[0080] 4. Postbiotic separation and purification: Temperature 4℃, 11000 g, 15 minutes, filter through a 300-mesh filter.

[0081] Example 2: Preparation method of Yakult fermentation product

[0082] 1. Substrate preparation: By weight, collagen: 15%, whey protein: 10%, lactose: 7%, balance: water. Sterilize at 95°C for 30 minutes.

[0083] 2. Inoculation: By weight, Xue-66:Xue-123:PQQ-1 = 0.5:1:1, the inoculation dose is 5.0 × 10⁻⁶. 9 cfu / L.

[0084] 3. Fermentation: Initial temperature 40℃, fermentation for 14 hours, followed by a stepwise cooling rate of 5℃ / h, with the final temperature reduced to 25℃. The final fermentation time was 24 hours. Rotation speed: 400 rpm

[0085] 4. Postbiotic separation and purification: Temperature 4℃, 12000 g, 20 minutes, filter through a 100-mesh filter.

[0086] Example 3

[0087] 1. Substrate preparation: By weight, collagen: 20%, whey protein: 15%, lactose: 9%, balance: water. Sterilize at 95°C for 30 minutes.

[0088] 2. Inoculation: By weight, Xue-66:Xue-123:PQQ-1 = 2:3:5, and the inoculation dose is 5.0 × 10⁻⁶. 9 cfu / L.

[0089] 3. Fermentation: Initial temperature 45℃, fermentation for 18 hours, followed by a stepwise cooling rate of 5℃ / h, with the final temperature reduced to 30℃. The final fermentation time was 20 hours. Rotation speed: 500 rpm

[0090] 4. Postbiotic separation and purification: Temperature 4℃, 10000 g, 10 minutes, filter through a 400-mesh filter.

[0091] Comparative Example 1: Preparation method of Yakult ferment.

[0092] 1. Substrate preparation: By weight, collagen: 18%, whey protein: 13%, lactose: 8%, balance: water. Sterilize at 95°C for 30 minutes.

[0093] 2. Inoculation: By weight, Xue-66:Xue-123 = 1:1.5, and the inoculation dose is 5.0 × 10⁻⁶. 9 cfu / L.

[0094] 3. Fermentation: Initial temperature 45℃, fermentation for 16 hours, followed by a stepwise cooling rate of 5℃ / h, with the final temperature reduced to 25℃. The final fermentation time was 24 hours. Rotation speed: 300 rpm

[0095] 4. Postgenetic separation and purification: Temperature 4℃, 12000 g, 15 minutes, filter through a 300-mesh filter.

[0096] Comparative Example 2

[0097] 1. Substrate preparation: By weight, collagen: 18%, whey protein: 13%, lactose: 8%, balance: water. Sterilize at 95°C for 30 minutes.

[0098] 2. Inoculation: By weight, Xue-123:PQQ-1 = 1.5:3, the inoculation dose is 5.0 × 10⁻⁶. 9 cfu / L.

[0099] 3. Fermentation: Initial temperature 45℃, fermentation for 16 hours, followed by a stepwise cooling rate of 5℃ / h, with the final temperature reduced to 25℃. The final fermentation time was 24 hours. Rotation speed: 300 rpm

[0100] 4. Postgenetic separation and purification: Temperature 4℃, 12000 g, 15 minutes, filter through a 300-mesh filter.

[0101] Example 1: Oral whitening effect

[0102] 1. Testing materials:

[0103] 1.1 Sample:

[0104] The fermented product of *Syzygium spp.* was used directly from the stock solution prepared in the example.

[0105] Positive control: POLA Whitening Pills (hereinafter referred to as POLA Whitening Pills), grayish-white tablets, batch number B862, solvent is ultrapure water.

[0106] 1.2 Laboratory Animals

[0107] Zebrafish were all raised in aquarium water at 28℃ (water quality: 200mg of quick-dissolving sea salt added per 1L of reverse osmosis water, conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100mg / L CaCO3).

[0108] 1.3 Reagents

[0109] Melanocyte-stimulating hormone (batch number J13HS188447, Shanghai Yuanye Biotechnology Co., Ltd.); ChamQ universal real-time quantitative PCR premix (batch number 027E2201CD, Novizan); First-strand synthesis kit (genome-free) (batch number X1213, Tiangen Biotech (Beijing) Co., Ltd.); Universal RNA extraction kit (catalog number TL2204001643C, Foshan Aowei Biotechnology Co., Ltd.).

[0110] 2. Evaluation of oral skin whitening efficacy (phenotype)

[0111] Wild-type AB strain zebrafish, 3 days post-fertilization, were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Water-soluble samples (concentrations shown in Table 1) were administered, along with a positive control of POLA whitening pills at a concentration of 25.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all experimental groups received melanocyte-stimulating hormone injections into the yolk sac to establish a zebrafish melanin-increasing model. After treatment at 28℃ for 1 day, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were analyzed and collected using ImageJ advanced image processing software to analyze the melanin signal intensity in the zebrafish head. The statistical analysis results of this index were used to evaluate the oral whitening efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance. Experimental results are shown in Table 1. Figure 1-2 .

[0112] Table 1. Evaluation results of oral whitening efficacy (phenotype) of the samples (n=10)

[0113]

[0114] Compared with the model control group, ***p<0.001.

[0115] Under the experimental conditions, the fermented product of *Syzygium spp.* has an oral whitening effect, specifically by reducing the intensity of melanin signal in the head of zebrafish.

[0116] 3. Evaluation of oral skin whitening efficacy (tyrosinase activity and melanin percentage)

[0117] Wild-type AB strain zebrafish, 3 days post-fertilization, were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Water-soluble samples (concentrations shown in Table 2) were administered, along with a positive control of POLA whitening pills at a concentration of 25.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all experimental groups received melanocyte-stimulating hormone injection into the yolk sac to establish a zebrafish melanin-increasing model. The experiment was conducted in triplicate. After treatment at 28℃ for 1 day, zebrafish samples were collected. Data were collected using a multi-functional microplate reader (SPARK, TECAN) according to the instructions for measuring tyrosinase activity and melanin percentage. The tyrosinase activity and melanin percentage in zebrafish were analyzed, and the oral whitening efficacy of the samples was evaluated based on the statistical analysis results of these indicators. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. p < 0.05 indicated statistical significance. Experimental results are shown in Table 2. Figures 3-4 .

[0118] Table 2. Evaluation of the oral whitening efficacy of the samples (tyrosinase activity and melanin percentage) Experimental results (n=3)

[0119]

[0120] Compared with the model control group, **p<0.01, ***p<0.001.

[0121] Under the experimental conditions, the fermented product of *Syzygium squarrosum* has oral whitening effects, specifically by reducing tyrosinase activity and melanin content.

[0122] 3. Evaluation of oral whitening efficacy ( tyr Gene)

[0123] zebrafish tyr The gene is a member of the tyrosinase gene family, and is related to human... tyr Genetic homology. Its encoded TYR (tyrosinase) is the main rate-limiting enzyme in melanin formation, directly mediating melanin production through the oxidation of tyrosine, the substrate for melanin synthesis. The synthesis of melanin primarily involves the microphthalmia transcription factor (MTBF). mitf oculocutaneous albinism type II ( oca2 ), tyrosinase ( tyr ), dopatin tautomerase ( dct ), tyrosinase-associated protein 1 ( tyrp1 (and other genes.) Sakeh's research indicates that decreased melanin content and tyrosinase activity are associated with... tyr There is an indirect relationship between gene downregulation and gene downregulation.

[0124] Wild-type AB strain zebrafish, 3 days post-fertilization, were randomly selected and placed in 6-well plates, with 30 zebrafish per well. Water-soluble samples (concentrations shown in Table 4) were administered, along with a positive control of POLA whitening pills at a concentration of 25.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all experimental groups received melanocyte-stimulating hormone injection into the yolk sac to establish a zebrafish melanin-increasing model. The experiment was conducted in triplicate. After treatment at 28℃ for 1 day, total RNA was extracted from each group of zebrafish using an automated nucleic acid extractor. The concentration and purity of total RNA were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from the zebrafish sample was used to synthesize 20.0 μL of cDNA according to the cDNA first-strand synthesis kit instructions, and the cDNA was detected by q-PCR. β-actin and tyr The primers used for gene expression are shown in Table 3. β-actin As an internal reference for gene expression, calculation tyr The relative RNA expression levels of the gene. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. p < 0.05 indicated statistical significance. Experimental results are shown in Table 4 and... Figure 5 .

[0125] Table 3 Primer sequence information

[0126]

[0127] Table 4 Evaluation of the oral whitening efficacy of the samples ( tyr Genetic experimental results (n=3)

[0128]

[0129] Compared with the model control group, **p<0.01, ***p<0.001.

[0130] Under the conditions of this experiment, the fermented product of *Syzygium squarrosum* has an oral whitening effect, specifically manifested in its ability to downregulate skin. tyr Relative gene expression levels.

[0131] Example 2: Increases skin elasticity

[0132] 1. Testing materials

[0133] 1.1 Sample

[0134] The fermented product of *Syzygium spp.* was used directly from the stock solution prepared in the example.

[0135] Positive control: Verisol collagen, white powder, batch number 893660, Jialida (Pingyang) Gelatin Co., Ltd., solvent is standard dilution water.

[0136] 1.2 Laboratory Animals

[0137] Zebrafish were all raised in aquarium water at 28℃ (water quality: 200mg of instant sea salt added per 1L of reverse osmosis water; conductivity 450-550μS / cm; pH 6.5-8.5; hardness 50-100mg / L CaCO3).

[0138] 1.3 Reagents

[0139] ChamQ Universal Real-Time Quantitative PCR Premix (Batch No. 027E2201CD, Novizan); cDNA First Strand Synthesis Kit (Genomic De-generated) (Batch No. X1213, Tiangen Biotech (Beijing) Co., Ltd.); Universal RNA Extraction Kit C (Catalog No. TL2204001643C, Foshan Aowei Biotechnology Co., Ltd.).

[0140] 2. Evaluation of efficacy in increasing skin elasticity

[0141] Skin growth, repair, nutrition, elasticity, and tension are all related to collagen. Its loss leads to decreased skin smoothness and wrinkles. In tetrapods, type I collagen is a trimer, mainly composed of two α1 chains and one α2 chain, which are... col1a1a and colla2 Genes encode collagen-related biological functions in connective tissue and bone. Three type I collagen genes exist in zebrafish, encoding Col1a1a, Col1a1b, and Col1a2 of the α1(I), α2(I), and α3(I) chains, respectively. Elastin is the main component of elastic fibers in skin tissue, providing structural support and protecting the skin from aging and sagging. Elastin is composed of two types of alternating short peptide segments. eln1 , eln2 These two genes, responsible for encoding different peptide segments of elastin, work together to regulate the expression level of elastin, making the skin firm and elastic.

[0142] Wild-type AB strain zebrafish, 4 days post-fertilization, were randomly selected and placed in 6-well plates, with 30 zebrafish per well. Water-soluble samples (concentrations shown in Tables 6 and 7) were administered, along with a positive control of 250 μg / mL verisol collagen. A normal control group was also included. Each well contained 3 mL of sample. Trial experiments were performed. After treatment at 28℃ for 1 day, total RNA was extracted from each group of zebrafish using an automated nucleic acid extractor. The concentration and purity of total RNA were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from the zebrafish sample was used to synthesize 20.0 μL of cDNA according to the cDNA first-strand synthesis kit instructions, and the cDNA was detected by q-PCR. β- actin, col1a1a, col1a1b, eln1 and eln2The primer sequences used for gene expression are shown in Table 5. 。 use β-actin As an internal reference for gene expression, calculation col1a1a, col1a1b, eln1 and eln2 The relative RNA expression levels of the genes. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. p < 0.05 indicated statistical significance. Experimental results are shown in Tables 6-7. Figure 6-9 .

[0143] Table 5 Primer sequence information

[0144]

[0145] Table 6. Results of the experiment evaluating the skin elasticity-enhancing effects of the samples 1 (n=3)

[0146]

[0147] Compared with the normal control group, *p<0.05, **p<0.01, ***p<0.001.

[0148] Table 7. Results of the experiment evaluating the skin elasticity-enhancing effects of the samples 2 (n=3)

[0149]

[0150] Compared with the normal control group, *p<0.05, **p<0.01, ***p<0.001.

[0151] Under the conditions of this experiment, the fermented product of *Syzygium squarrosum* has the effect of increasing skin elasticity, specifically manifested in its ability to upregulate... col1a1a, col1a1b, eln1 and eln2 Relative gene expression levels.

[0152] Example 3: ABTS clearing ability

[0153] 1. Testing materials

[0154] ABTS Free Radical Scavenging Ability Test Kit (Catalog No. BC4770, Beijing Solarbio Science & Technology Co., Ltd.)

[0155] 2. ABTS removal capability test

[0156] The snow lotus ferment prepared in Example 1 was tested according to the instructions of the ABTS free radical scavenging ability test kit. The experimental results are shown in [Figure 1]. Figure 10 ,Depend on Figure 10 It can be seen that the snow lotus ferment prepared in Example 1 can effectively resist oxidation.

[0157] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a fermented product of *Saussurea involucrata*, characterized in that, The substrate was fermented using a mixture of *Bifidobacterium longum* subsp. *infant* Xue-66, *Lactobacillus helveticus* Xue-123, and *Lactobacillus plantarum* PQQ-1. The postbiotics were isolated from the fermentation product to obtain the *Snow Lotus* ferment broth. *Bifidobacterium longum* subsp. *infant* Xue-66 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33981; *Lactobacillus helveticus* Xue-123 is deposited at the CGMCC with accession number CGMCC No. 33982; and *Lactobacillus plantarum* PQQ-1 is deposited at the CGMCC with accession number CGMCC No. 29303. The mass ratio of Bifidobacterium longum subsp. infantileum Xue-66, Lactobacillus helveticus Xue-123, and Lactobacillus plantarum PQQ-1 is 0.5-2:1-3:1-5; The substrates include collagen and whey protein; the amount of collagen used is 15-20% by weight, and the amount of whey protein is 10-15% by weight. The temperature control conditions for the mixed fermentation process are as follows: fermentation at 40-45℃ for 14-18 hours, followed by a step-down cooling process at 4-6℃ / h; the culture conditions for the mixed fermentation process include: rotation speed of 300-500 rpm and fermentation time of 20-24 hours.

2. The preparation method according to claim 1, characterized in that, The fermentation products were separated into postbiotics by centrifugation and filtration.

3. The preparation method according to claim 2, characterized in that, The centrifugation is ultra-high speed centrifugation, and the conditions for ultra-high speed centrifugation are: 2-8℃, 10000-12000 g, centrifugation for 10-20 minutes.

4. The preparation method according to claim 2, characterized in that, The filtration step uses a 100-400 mesh filter.

5. The snow lotus ferment obtained by the preparation method according to any one of claims 1-4.

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