Paenibacillus sp. and application thereof in fermentation of antarctic raphidiopsis sp.

Abstract

This invention belongs to the field of microbial technology and provides a Bacillus strain and its application in the fermentation of *Trichoderma antarctica*. A Bacillus strain, named BTN-HB-D4, was obtained through microbial isolation and purification from *Trichoderma antarctica* plants obtained from Haba Snow Mountain in Yunnan Province. It was deposited at the Guangdong Microbial Culture Collection Center on September 20, 2024, with accession number GDMCC NO: 65160. A two-way fermentation of *Trichoderma antarctica* and *Trichoderma antarctica* yielded a fermentation product filtrate. This filtrate exhibits good antioxidant, anti-aging, and skin microecological regulation functions. Since the active ingredient is in the fermentation filtrate, the introduction of chemical reagents during purification or refining is avoided, ensuring safety and efficacy. The fermentation method is simple to operate and exhibits high batch stability, all of which indicate its great potential for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Bacillus species and its application in the fermentation of Antarctic spirochetes. Background Technology

[0002] Antarctic spirochetes is a high-level algae that grows in the deep sea near Antarctica. Its growth environment requires withstanding various extreme climatic conditions, exhibiting strong resistance to cold, pollution, and radiation. Antarctic spirochetes is widely used in the cosmetics industry, primarily for the extraction and refining of polysaccharides and other components, as well as the creation of various extract combinations. Current research mainly focuses on the extraction of polysaccharides from Antarctic spirochetes, the optimization of extraction methods, and the development of related compositions.

[0003] For example, Chinese patent CN117986396A discloses a method for preparing a water-soluble homogeneous polysaccharide from *Cladosporium antarctica*, along with its products and applications. By using subcritical water extraction and purification, a polysaccharide with lower molecular weight and greater homogeneity is obtained. This polysaccharide exhibits excellent biological activities, such as promoting the proliferation of skin fibroblasts, activating antimicrobial peptides and defensins, and accelerating wound healing. Furthermore, the patent also provides an application of this polysaccharide as an active ingredient in skincare products, offering researchers a more efficient and homogeneous polysaccharide resource.

[0004] Chinese patent CN113476485A discloses a method for preparing and applying seaweed extract. By using a combination of natural eutectic solvent and ultrasonic extraction, the seaweed extract can be prepared, which can significantly improve the antioxidant properties of the seaweed extract, mainly manifested in its ability to scavenge DPPH free radicals and ABTS free radicals.

[0005] While *Platycodon grandiflorus* from Antarctica is widely used in skincare products due to its excellent skincare effects, research on further developing its skincare efficacy through microbial fermentation is limited. Two-way fermentation of microorganisms promotes microbial metabolism, producing various active substances, and may also reduce toxicity and enhance efficacy through interactions with plants. Therefore, to address the shortcomings of current technology, it is necessary to screen a microorganism for two-way fermentation with *Platycodon grandiflorus*, verify the skincare efficacy of the fermentation broth, and develop a simpler preparation process to improve production and application efficiency, meeting the needs of industrialization and product development. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] Firstly, this invention obtains a strain of Bacillus by isolating and purifying microorganisms from *Prickly Pear* plants obtained from Haba Snow Mountain in Yunnan Province. The strain is classified as... BacillusThe sp., named BTN-HB-D4, was deposited on September 20, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 9, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO:65160.

[0008] Preferably, the 16S rDNA sequence of the above-mentioned Bacillus is shown in the sequence listing SEQ NO:1.

[0009] Secondly, the present invention provides a fermentation product of Antarctic spirochetes and Bacillus, using Bacillus with accession number GDMCC NO: 65160 as the fermentation bacteria to ferment Antarctic spirochetes and obtain the fermentation product of Antarctic spirochetes and Bacillus.

[0010] Thirdly, the present invention provides a fermentation product filtrate of Antarctic spirochetes and Bacillus, using Bacillus with accession number GDMCC NO: 65160 as the fermentation bacteria to ferment Antarctic spirochetes to obtain fermentation products, and the fermentation products are filtered to obtain a fermentation product filtrate of Antarctic spirochetes and Bacillus.

[0011] Fourthly, the present invention provides a method for preparing a filtrate of fermentation products from Antarctic spirochetes and Bacillus subtilis, comprising the following steps:

[0012] Step S1: Inoculate the Bacillus with accession number GDMCC NO: 65160 into LB liquid medium and culture with shaking to obtain seed culture;

[0013] Step S2: Inoculate the seed culture at an inoculum volume of 1-5% into the fermentation medium of Antarctic spirochetes and culture with shaking to obtain the fermentation product.

[0014] Step S3: After centrifuging the fermentation product, take the supernatant and filter the supernatant to obtain the fermentation product filtrate of Antarctic spirochetes and Bacillus.

[0015] Preferably, the powder of Antarctic spirulina is pulverized through a 30-mesh sieve to remove large pieces of plant material and then mixed with ultrapure water at a mass ratio of 0.1% to 10%, preferably 5%, to serve as the fermentation medium, i.e., Antarctic spirulina fermentation medium.

[0016] Preferably, the components of LB liquid culture medium are: 10g peptone, 5g yeast extract, 10g NaCl, and 1L ultrapure water.

[0017] Preferably, in step S1, Bacillus BTN-HB-D4 is inoculated into LB liquid medium and cultured at 25℃~37℃ and 0~200rpm for 16h~48h to obtain seed culture; more preferably, the conditions are cultured at 37℃ and 200rpm for 24h.

[0018] Preferably, in step S2, the obtained seed liquid is inoculated at 1-3% into the fermentation medium of *Clerodendrum anthocyanin* for fermentation culture, and the fermentation product is obtained after culturing at 25℃-37℃ and 0-200rpm for 16-48h; more preferably, the inoculation amount is 2%, and the culture is carried out at 30℃ and 200rpm for 48h.

[0019] Preferably, the obtained fermentation product is centrifuged at 8000 rpm for 15 min, the bacterial sludge is discarded and the supernatant is collected. After filtration through a 0.22 μm filter membrane, the fermentation product filtrate of Bacillus thuringiensis BTN-HB-D4 is obtained, which is also called the fermentation product filtrate of Antarctic spirochetes and Bacillus thuringiensis BTN-HB-D4, or simply fermentation filtrate.

[0020] Fifthly, the present invention provides an application of the above-obtained fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis in the preparation of cosmetics or skin care products with antioxidant, anti-aging, and skin microecological regulation properties.

[0021] In a sixth aspect, the present invention provides a cosmetic or skin care product that has antioxidant, anti-aging, and skin microecological regulation properties, containing the above-mentioned fermentation product filtrate of Antarctic spirochetes and Bacillus.

[0022] This invention obtained a strain of Bacillus by isolating and purifying microorganisms from *Pyracantha fortuneana* plants obtained from Haba Snow Mountain in Yunnan Province. Bacillus A fermentation product filtrate containing multiple active ingredients, named BTN-HB-D4, was obtained through bidirectional fermentation with Antarctic spirochetes. This filtrate is safe, easy to prepare, and contains various active ingredients. It exhibits multiple skincare benefits, including antioxidant and anti-aging properties, promotion of fibroblast proliferation and repair, and regulation of the skin's microecology. It can improve skin condition from multiple perspectives and has practical application value, making it suitable as a functional ingredient in the cosmetics industry.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] 1. The fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis BTN-HB-D4 at a concentration of 0.0625% exhibited excellent HAS inhibition ability, with an HAS inhibition rate of 83.96%;

[0025] 2. The filtrate of fermentation products of Antarctic spirochetes and Bacillus subtilis BTN-HB-D4 at a concentration of 3% can significantly inhibit the expression of matrix metalloproteinases 1 and 3 (MMP1, MMP3);

[0026] 3. A 3% concentration of the fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis BTN-HB-D4 significantly promoted the proliferation of fibroblasts;

[0027] 4. The fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis BTN-HB-D4 exhibited significant DPPH and ABTS free radical scavenging capabilities, with a DPPH scavenging rate of 13.03% and an ABTS scavenging rate of 31.30% at a 10% concentration.

[0028] 5. The fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis BTN-HB-D4 can strongly inhibit the growth of pathogenic Propionibacterium acnes ATCC 6919, while the inhibitory effect on non-pathogenic Propionibacterium acnes ATCC 11828 is weaker. Therefore, this fermentation filtrate has the effect of regulating the skin microecology.

[0029] 6. The bidirectional fermentation of Bacillus and Antarctic Cladosporium avoids the introduction of chemical reagents during refining or purification because the active ingredient is found in the fermentation filtrate. The resulting fermentation filtrate of Antarctic Cladosporium and Bacillus BTN-HB-D4 contains abundant natural active ingredients, and the absence of added chemical reagents ensures safety and efficacy. This fermentation method is simple to operate and exhibits high batch stability, all of which demonstrate its significant potential for large-scale industrial production. Attached Figure Description

[0030] Figure 1 This is a graph showing the cell proliferation promotion efficacy test results of Test Example 4 of the present invention;

[0031] Figure 2 This is a graph showing the test results of the matrix metalloproteinase (MMP) inhibition ability of Test Example 5 of the present invention; wherein, Figure 2 In the diagram, A represents the relative expression level of matrix metalloproteinase 1 (MMP1) mRNA. Figure 2 B in the diagram represents the relative expression level of matrix metalloproteinase 3 (MMP3) mRNA.

[0032] Figure 3 The graph shows the results of the LOR (Large-Olefin Protein) expression level test in Test Example 6 of the present invention.

[0033] Figure 4 The figure shows the test results of microecological regulation in Test Example 7 of the present invention. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0035] Unless otherwise specified, the raw materials and materials used in the embodiments of the present invention were purchased through general commercial channels.

[0036] The source information of the raw materials, materials, and instruments involved in the following embodiments or comparative examples is as follows:

[0037] Bacillus ( Bacillus BTN-HB-D12, self-developed, see the example description;

[0038] Bacillus ( Bacillus BTN-HB-J1, self-developed, see the example description;

[0039] Weissella esculenta ( Weissella cibaria BTN-HB-F1, self-developed, see the example description;

[0040] yeast ( Saccharomyces BTN-HB-M9, self-developed, see the example description;

[0041] In this invention, the culture media used in the following embodiments, comparative examples, and test examples are as follows:

[0042] LB liquid medium: 10g peptone, 5g yeast extract, 10g NaCl, 1L ultrapure water.

[0043] LB solid medium: Add 2% agar powder to LB liquid medium.

[0044] Antarctic Clematis fermentation medium: 50g Antarctic Clematis powder, 1L ultrapure water.

[0045] MRS liquid culture medium: 10g peptone, 8g beef meal, 4g yeast powder, 20g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1g Tween, 1L ultrapure water.

[0046] MRS solid medium: Add 2% agar powder to MRS liquid medium.

[0047] YPD liquid culture medium: 10g peptone, 5g yeast extract, 20g glucose, 1L ultrapure water.

[0048] YPD solid medium: Add 2% agar powder to YPD liquid medium.

[0049] BHI solid culture medium: 10g peptone, 5g sodium chloride, 2g glucose, 12.5g dehydrated calf brain extract, 5g dehydrated calf heart extract, 2.5g disodium hydrogen phosphate, 1L ultrapure water.

[0050] BHI solid medium: Add 2% agar powder to BHI liquid medium.

[0051] All the culture media prepared above were sterilized at 121℃ for 15 minutes before use.

[0052] Example 1: Isolation, purification and identification of bacterial strains

[0053] The seeds of *Prickly ash* (a type of wild fruit) were collected from Haba Snow Mountain, placed in sterile bags, and brought back to the laboratory for bacterial isolation. The specific process for bacterial isolation is as follows:

[0054] The collected *Prickly pear* fruits were rinsed with running water, then rinsed three times with sterile water. They were then soaked in a 1% sodium hypochlorite solution for 20 minutes, rinsed with sterile water, and then soaked in 75% ethanol for 10 seconds. Residual ethanol was removed by rinsing with sterile water. The fruits were crushed in a sterile mortar and resuspended in sterile water. The suspension was serially diluted to 10, 100, and 1000 times, and 100 μL of each was spread onto LB agar plates, with three replicates per gradient. The plates were incubated at 37°C for 24 hours. Single colonies were picked for isolation and purification, yielding a *Bacillus* strain named BTN-HB-D4, which has been deposited at the Guangdong Microbial Culture Collection Center (GDMCC NO: 65160) on September 20, 2024. Its 16S rDNA sequence is shown in SEQ NO: 1.

[0055]

[0056] Example 2

[0057] This embodiment describes the preparation of fermentation product filtrate from Antarctic Cladosporium and Bacillus subtilis BTN-HB-D4. The specific steps are as follows:

[0058] Step S1, Seed culture preparation: Bacillus BTN-HB-D4 was inoculated into LB liquid medium and cultured at 37℃ and 200rpm for 24h to obtain seed culture.

[0059] Step S2, Fermentation broth preparation: Bacillus BTN-HB-D4 seed culture was inoculated at a rate of 1% into the fermentation medium of Antarctic Trichoderma that had been sterilized by high temperature and high pressure, and cultured at 25℃ and 150 rpm for 24 h to obtain the fermentation broth, which is the fermentation product of Antarctic Trichoderma and Bacillus.

[0060] Step S3, filtrate preparation: After centrifuging the fermentation broth at 8000 rpm for 15 min, the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane to obtain the fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis BTN-HB-D4, namely the fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis.

[0061] Example 3

[0062] This embodiment describes the preparation of filtrate from the fermentation products of *Trichoderma antarctica* and Bacillus subtilis BTN-HB-D4. The seed culture preparation method is the same as in Example 2. The fermentation broth preparation scheme is as follows: Bacillus subtilis BTN-HB-D4 seed culture is inoculated at a 2% inoculum into *Trichoderma antarctica* fermentation medium that has been sterilized by high temperature and high pressure, and cultured at 30℃ and 200 rpm for 48 h to obtain the fermentation broth; the fermentation broth is centrifuged at 8000 rpm for 15 min, and the supernatant is collected. The supernatant is filtered through a 0.22 μm filter membrane to obtain the filtrate from the fermentation products of *Trichoderma antarctica* and Bacillus subtilis BTN-HB-D4.

[0063] Example 4

[0064] This embodiment describes the preparation of filtrate from the fermentation products of *Trichoderma antarctica* and *Bacillus subtilis* BTN-HB-D4. The seed culture preparation method is the same as in Example 2. The fermentation broth preparation scheme is as follows: *Bacillus subtilis* BTN-HB-D4 seed culture is inoculated at a rate of 3% into *Trichoderma antarctica* fermentation medium that has been sterilized by high temperature and high pressure, and cultured at 37℃ and 200 rpm for 48 h to obtain the fermentation broth; the fermentation broth is centrifuged at 8000 rpm for 15 min, and the supernatant is collected. The supernatant is filtered through a 0.22 μm filter membrane to obtain the filtrate from the fermentation products of *Trichoderma antarctica* and *Bacillus subtilis* BTN-HB-D4.

[0065] Comparative Example 1

[0066] The fermentation broth preparation method is as follows: the fermentation medium of Antarctic spirochetes is not inoculated with microorganisms, and after autoclaving, it is placed at 30℃ and shaken at 200rpm for 48h. After centrifuging the fermentation broth at 8000rpm for 15min, the supernatant is collected. The supernatant is filtered through a 0.22μm filter membrane to obtain the Antarctic spirochetes extract.

[0067] Comparative Example 2

[0068] The preparation method is the same as in Example 3, except that the strain is replaced with Bacillus ( ). Bacillus sp.) BTN-HB-L10.

[0069] Comparative Example 3

[0070] The preparation method is the same as in Example 3, except that the strain is replaced with Bacillus ( ). Bacillus sp.) BTN-HB-L21.

[0071] Comparative Example 4

[0072] Seed culture preparation: *Westernella esculenta* (… Weissella cibaria BTN-HB-F1 was inoculated into MRS liquid medium and cultured at 37℃ and 200rpm for 24h to obtain seed culture.

[0073] Fermentation broth preparation: The seed culture of *Westernella antarcticis* BTN-HB-F1 was inoculated at a rate of 3% into the fermentation medium of *Trichoderma antarcticis* that had been sterilized by high temperature and high pressure. The medium was then incubated at 37℃ and 200 rpm for 48 h with shaking. After centrifugation of the fermentation broth at 8000 rpm for 15 min, the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane to obtain the fermentation product filtrate of *Trichoderma antarcticis* and *Westernella antarcticis* BTN-HB-F1.

[0074] Comparative Example 5

[0075] Seed culture preparation: Yeast ( Saccharomyces BTN-HB-M9 was inoculated into YPD liquid medium and cultured at 27℃ and 200rpm for 24h to obtain seed culture.

[0076] Fermentation broth preparation: The BTN-HB-M9 yeast seed culture was inoculated at a rate of 2% into the Antarctic Trichoderma fermentation medium that had been sterilized by high temperature and high pressure. The medium was then cultured at 30℃ and 200 rpm for 48 h with shaking. After centrifugation of the fermentation broth at 8000 rpm for 15 min, the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane to obtain the fermentation product filtrate of Antarctic Trichoderma and BTN-HB-M9 yeast.

[0077] Comparative Example 6

[0078] The seed culture preparation method is the same as in Example 2. The fermentation broth preparation scheme is as follows: Bacillus BTN-HB-D4 seed culture is inoculated into LB liquid medium at an inoculation amount of 3%, and cultured at 37°C and 200 rpm for 48 h with shaking. After centrifuging the fermentation broth at 8000 rpm for 15 min, the supernatant is collected. The supernatant is filtered through a 0.22 μm filter membrane to obtain the Bacillus BTN-HB-D4 fermentation product filtrate.

[0079] Test Example 1

[0080] DPPH free radical scavenging ability test:

[0081] A 0.1 mg / mL solution of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) was prepared with anhydrous ethanol and stored in the dark. Sample groups, sample blank control groups, control groups, and control blank control groups were set up, with two replicates for each group. In the sample group, 100 μL of the fermentation product filtrate from Examples 2-4 and Comparative Examples 1-6 was added to each well of a 96-well plate as the test sample. Then, an equal volume of 100 μL of DPPH solution was added, and the plate was shaken well.

[0082] Clearance rate = [1 - (Asample - Asampleblank) / (Acontrol - Acontrolblank)] × 100%,

[0083] In the above formula:

[0084] Asample: The absorbance of the sample group is measured.

[0085] Asampleblank: Absorbance measured in the blank control group;

[0086] Acontrol: The control group measures absorbance.

[0087] Acontrolblank: Used to control the absorbance of the blank control group.

[0088] The results are shown in Table 1, in which the DPPH removal rate of the 10% diluted fermentation filtrate in Example 3 reached 13.03%.

[0089] Test Example 2

[0090] ABTS free radical scavenging ability test:

[0091] Accurately weigh 28.4 mg of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and dissolve it in 10 mL of ultrapure water. Weigh 6.7 mg of potassium persulfate and dissolve it in 10 mL of ultrapure water. Mix the ABTS solution and potassium persulfate solution at a volume ratio of 1:1 and let it stand overnight at room temperature in the dark to obtain the ABTS stock solution. Dilute the ABTS stock solution to make its absorbance at a wavelength of 734 nm 0.7 ± 0.1 to obtain the ABTS working solution. The ABTS working solution should be prepared fresh before use.

[0092] Fermentation product filtrates from Examples 2-4 and Comparative Examples 1-6 were used as test samples. Sample groups and control groups were set up. In the sample group, 180 μL of ABTS working solution and 20 μL of test sample were added to a 96-well plate. After reacting for 180 min, the absorbance of the reaction system at a wavelength of 734 nm was measured. In the control group, ultrapure water was used instead of fermentation product filtrate, and other conditions were the same as those in the sample group.

[0093] Clearance rate = [1 - (Asample / Acontrol)] × 100%,

[0094] In the formula:

[0095] Asample: The absorbance of the sample group is measured.

[0096] Acontrol: The control group measures absorbance.

[0097] The results are shown in Table 1. In Example 3, the ABTS removal rate of the 10% diluted fermentation filtrate reached 31.30%.

[0098] Test Example 3

[0099] Hyaluronidase (HAS) inhibition ability test:

[0100] (1) Preparation of medicines:

[0101] Prepare a 0.25 mg / mL hyaluronidase solution and a 1 mg / mL sodium hyaluronate solution by dissolving in 0.1 mM acetate buffer; prepare a 12.5 mM calcium chloride solution and a 0.4 M sodium hydroxide solution by using ultrapure water; prepare a 4 M potassium borate solution by using ultrapure water and let it stand overnight before use; take 0.8 g of p-dimethylaminobenzaldehyde, add it to 20 ml of acetic acid, and then add 5 ml of concentrated hydrochloric acid (store away from light) as a colorimetric reagent.

[0102] (2) Set up groups:

[0103] Sample group (Group A): The test samples are the fermentation product filtrates prepared in Examples 2-4 and Comparative Examples 1-6; the test samples, hyaluronidase and sodium hyaluronate will be added to this group at the same time.

[0104] Sample blank group (Group B): The test sample is the fermentation product filtrate prepared by Examples 2-4 and Comparative Examples 1-6; only the test sample is added to this group, without adding hyaluronidase and sodium hyaluronate.

[0105] In the control group (Group C), the sample to be tested is the sample solvent (ultrapure water). Hyaluronidase and sodium hyaluronate will be added to this group at the same time.

[0106] The control group (Group D) used ultrapure water as the sample solvent and did not add hyaluronidase or sodium hyaluronate.

[0107] (3) Experimental steps:

[0108] Step (a): According to the grouping requirements, add 0.5 mL of the sample to be tested to groups A, B, C and D respectively; then add 0.5 mL of acetate buffer to groups A, B and C, and add 0.5 mL of sample solvent to group D; incubate at 37℃ for 20 min.

[0109] After steps (b) and (a) are completed, 100 μL of calcium chloride is added to each group and incubated at 37°C for 20 min.

[0110] After steps (c) and (b) are completed, add 0.5 mL of sodium hyaluronate to group A and group B respectively, and add 0.5 mL of acetate buffer to group C and group D respectively; incubate at 37°C for 40 min.

[0111] After steps (d) and (c) are completed, 100 μL of sodium chloride and 100 μL of potassium iodate are added to each group respectively. Then, the mixture is placed in a water bath at 85°C for 5 min, followed by an ice bath for 2 min, and then placed at room temperature for 5 min to obtain the reaction solution.

[0112] Step (e): After shaking the reaction solution obtained after step (d) evenly, add 100 μL of each solution to a 96-well plate and add 100 μL of colorimetric reagent to each well.

[0113] Step (f): Quickly place the 96-well plate into the microplate reader and shake at 37°C for 1 minute. Measure the absorbance at 585 nm after 10 minutes.

[0114] (4) Calculation method of hyaluronidase inhibition rate:

[0115] HAS inhibition rate (%) = [(CD) - (AB)] / (CD) × 100%,

[0116] In the formula: A, B, C, and D are the absorbance of groups A, B, C, and D measured at 585 nm, respectively.

[0117] The HAS inhibition rate test results are shown in Table 1. In Example 3, the HAS inhibition rate of the 0.0625% diluted fermentation filtrate reached 83.96%.

[0118] Table 1. Scavenging rates of DPPH, ABTS, and HAS by different fermentation supernatants

[0119]

[0120] As shown in Table 1, compared with the comparative examples of no microorganism inoculation (Comparative Example 1), no Antarctic spirochetes as fermentation substrate (Comparative Example 6), or fermentation with other microorganisms (Comparative Examples 2, 3, 4, 5), the fermentation product filtrate obtained by all the examples was better. Among the examples, the fermentation product filtrate obtained by the fermentation scheme in Example 3 was the best. Therefore, the comprehensive comparison found that the fermentation product filtrate obtained by bidirectional fermentation of Antarctic spirochetes and Bacillus subtilis BTN-HB-D4 (2% inoculation amount, 30℃, 200rpm shaking culture for 48h) was the best.

[0121] Test Example 4

[0122] Cell proliferation promotion efficacy test:

[0123] (1) Selection of cells and culture medium: Human foreskin fibroblasts (HFF cells) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; the culture medium was DMEM complete medium (containing 10% FBS fetal bovine serum).

[0124] (2) Experimental steps: Selected HFF cells with good morphology and in the logarithmic growth phase were seeded into 96-well plates and incubated in an incubator for 24 hours; sample groups, control groups, and zeroing groups were set up. In the sample group, the "fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis BTN-HB-D4" from Example 3 was taken respectively. Figure 1 The extracts were labeled "bidirectional fermentation - 3%" (as in Comparative Example 1) and "Antarctic Cladosporium extract" (represented as "bidirectional fermentation - 3%"). Figure 1 The test sample (represented as "unfermented -3%)" was added to the wells of HFF cells, with 3% of the test sample added to each well, in triplicate. The cells were incubated for 24 hours. After removing the supernatant from the DMEM complete medium, MTT test solution was added to each well, and the cells were incubated for another 2 hours. The absorbance at 570 nm was measured using a microplate reader. In the control group, the test sample was replaced with DMEM complete medium, and all other conditions were the same as in the sample group. In the zeroing group, no cells were seeded; only DMEM complete medium was added, and all other conditions were the same as in the sample group. Finally, the relative cell viability was calculated.

[0125] Relative cell viability (%) = (Asample - Ablank) / (Acontrol - Ablank) × 100%,

[0126] In the above formula:

[0127] Asample: Absorbance of the sample group

[0128] Acontrol: Absorbance of the control group

[0129] Ablank: Absorbance of the zeroing group.

[0130] The results are as follows Figure 1 As shown. Figure 1 In the figures, ** indicates a significant difference compared to the control group at p<0.01, and *** indicates a significant difference compared to the control group at p<0.001. “Unfermented -3%” represents “Antarctic Cladosporium extract” from Comparative Example 1, and “Dual fermentation -3%” represents “Antarctic Cladosporium and Bacillus BTN-HB-D4 fermentation product filtrate” from Example 3.

[0131] like Figure 1 As shown, compared with the unfermented extract of Antarctic spirochetes, the fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis BTN-HB-D4 had a more significant effect on promoting fibroblast proliferation at a concentration of 3%.

[0132] Test Example 5

[0133] Assay for inhibitory capacity of matrix metalloproteinases (MMPs):

[0134] (1) Selection of cells and culture medium: Human foreskin fibroblasts (HFF cells) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; the culture medium was DMEM complete medium (containing 10% FBS fetal bovine serum).

[0135] (2) Experimental procedure: Select HFF cells in the logarithmic growth phase with good morphology and seed them in 6-well plates at a density of 2.5 × 10⁻⁶. 5 Cells / wells were incubated in an incubator for 24 h; sample group, control group, and blank group were set up, with 3 replicate wells in each group; in the sample group, DMEM complete medium containing 3% fermentation product filtrate of Example 3 was added, and after treating the cells for 4 h, 400 μM H2O2 solution was added for induction treatment for 2 h, and DMEM complete medium containing 3% fermentation product filtrate of Example 3 was added again, and the cells were incubated in a 37°C, 5% CO2 incubator for another 24 h; no fermentation product filtrate was added to the control group, and the other conditions were the same as the sample group; no fermentation product filtrate was added to the blank group, and H2O2 solution was not used for induction, and the other conditions were the same as the sample group.

[0136] (3) Gene expression level test: RNA was extracted from cells of the sample group, control group and blank group and the RNA content was measured. The qualified RNA was reverse transcribed into cDNA and the gene expression level was measured by real-time PCR. The calculation method was 2-ΔΔCt method and the internal reference gene was β-actin.

[0137] (4) Data Processing: Experimental data were processed using GraphPad and statistical analysis was performed using One-way ANDNA (and nonparametric) Turkey: Compare all pairs of columns. Each experiment was repeated at least three times. Data are expressed as Mean ± SEM. ### represents a significant difference between the control group and the blank group at p < 0.001, and *** represents a significant difference between the sample group and the control group at p < 0.001. Results are as follows: Figure 2 As shown, where Figure 2 In this context, A represents the relative mRNA expression level of matrix metalloproteinase 1 (MMP1). Figure 2 In this context, B represents the relative mRNA expression level of matrix metalloproteinase 3 (MMP3).

[0138] from Figure 2 It can be seen that, compared with the control group (H2O2), the fermentation product filtrate of *Trichoderma antarctica* and *Bacillus subtilis* BTN-HB-D4 obtained in Experiment 3 (two-way fermentation - 3%) can significantly inhibit MMP1 at a concentration of 3%. Figure 2 A) and MMP3 ( Figure 2 The expression B in the formula has anti-aging effects.

[0139] Test Example 6

[0140] LOR (Legume Integrin) expression level assay:

[0141] (1) Cell seeding: Cell and culture medium selection: Keratinocytes (HaCaT cells) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences; the culture medium was DMEM complete medium (containing 10% FBS fetal bovine serum). HaCaT cells in the logarithmic growth phase and with good morphology were seeded in 24-well plates at 2.5 × 10⁶ cells / well. 5 Cells / well were incubated in an incubator for 24 hours.

[0142] (2) Experimental grouping and administration: The experiment set up a control group and a sample group, with 3 replicates in each group. The sample group was given DMEM complete medium containing 3% fermentation product filtrate of Example 3, while the control group was not given fermentation product filtrate. The other conditions were the same as the sample group. After the cells were treated with the drug, they were incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0143] (3) Gene expression level test: RNA was extracted from cells of the sample group and the control group, and the RNA content was measured. The qualified RNA was processed according to PrimeScript. TM The RT Master Mix was reverse transcribed into cDNA according to the instructions, and gene expression levels were determined by quantitative real-time PCR. The 2-ΔΔCt method was used for calculation, with β-actin as the internal reference gene.

[0144] The results are as follows Figure 3 As shown, the filtrate of the bidirectional fermentation product of Antarctic spirochetes and Bacillus BTN-HB-D4 (bidirectional fermentation-3%) can effectively increase the expression level of LOR at a concentration of 3%, with an increase rate of 61.9%, and has the effect of skin barrier repair.

[0145] Test Example 7

[0146] Microecological regulation test:

[0147] The inhibitory ability of the samples against Propionibacterium acnes (ATCC 6919 and ATCC 11828) was tested to assess the impact of the samples on the skin microecology.

[0148] The following explanation uses Propionibacterium acnes ATCC 6919 as an example.

[0149] (1) Solution preparation:

[0150] Propionibacterium acnes ATCC 6919 was cultured on BHI solid medium and incubated in an anaerobic incubator at 37°C for 72 h. The bacterial cells were then resuspended in BHI liquid medium and the turbidity was adjusted to 0.5 to obtain the test solution for the model strain.

[0151] (2) Set up groups:

[0152] Sample group (Group A): The test sample was the original filtrate of fermentation product of Antarctic spirochetes and Bacillus BTN-HB-D4 prepared in Example 3. 1 mL of the model strain test solution and 1 mL of the test sample were added to a 96-well plate, respectively. After anaerobic culture at 37℃ for 48 h, 200 μL was taken into a 96-well plate and the absorbance value at 600 nm was measured.

[0153] Sample blank group (Group B): The sample to be tested was the original filtrate of fermentation product of Antarctic spirochetes and Bacillus BTN-HB-D4 prepared in Example 3. 1 mL of model strain test solution and 1 mL of sample to be tested were added to 96-well plates respectively. 200 μL was immediately taken into 96-well plates and the absorbance value at 600 nm was measured.

[0154] Control group (Group C): The test sample was sterile PBS solution (phosphate buffered saline solution). 1 mL of the model strain test solution and 1 mL of sterile PBS were added to a 96-well plate, respectively. After incubation at 37℃ for 48 h, 200 μL was taken into a 96-well plate and the absorbance at 600 nm was measured.

[0155] Control group (D group): The test sample was sterile PBS solution. 1 mL of model strain test solution and 1 mL of sterile PBS were added to a 96-well plate, and 200 μL was immediately taken into the 96-well plate and the absorbance value at 600 nm was measured.

[0156] Microbial inhibition rate (%) = [(CD) - (AB)] / (CD) × 100%,

[0157] In the formula: A, B, C, and D are the absorbances of groups A, B, C, and D measured at 600 nm, respectively.

[0158] The culture time for the test solution of Propionibacterium acnes ATCC 11828 model strain was changed to 168 h, and the test culture time was changed to 72 h; all other aspects remained the same. The inhibition rate test results for these two Propionibacterium acnes strains are as follows: Figure 4 As shown.

[0159] Figure 4 The results showed that the fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis BTN-HB-D4 obtained in Example 3 could strongly inhibit the growth of pathogenic Propionibacterium acnes ATCC 6919, while the inhibitory effect on non-pathogenic Propionibacterium acnes ATCC11828 was weaker. Therefore, the fermentation filtrate has the effect of regulating the skin microecology.

[0160] The applicant declares that the above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the scope of protection and disclosure of the present invention.

Claims

1. A fermentation product of Antarctic spirochetes and Bacillus subtilis, characterized in that, The Bacillus species is classified as follows: Bacillus The Bacillus sp. strain BTN-HB-D4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 20, 2024, with accession number GDMCC NO: 65160. Using Bacillus with accession number GDMCC NO: 65160 as the fermentation strain, Antarctic spirochetes were fermented to obtain Antarctic spirochetes containing the aforementioned Bacillus and Bacillus fermentation products.

2. A filtrate of fermentation products from Antarctic spirochetes and Bacillus subtilis, characterized in that, The Bacillus species is classified as follows: Bacillus The Bacillus sp. strain BTN-HB-D4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 20, 2024, with accession number GDMCC NO: 65160. Bacillus with accession number GDMCC NO: 65160 was used as the fermentation bacteria to ferment Antarctic spirochetes to obtain fermentation products. The fermentation products were centrifuged and the supernatant was collected and filtered to obtain the filtrate of Antarctic spirochetes and Bacillus fermentation products.

3. A method for preparing the filtrate of fermentation products of Antarctic spirochetes and Bacillus as described in claim 2, characterized in that, Includes the following steps: Step S1: Inoculate the Bacillus with accession number GDMCC NO: 65160 into LB liquid medium and culture with shaking to obtain seed culture; Step S2: The seed liquid is inoculated into the fermentation medium of Antarctic spirochetes at an inoculation rate of 1% to 3%, and the fermentation product is obtained by shaking culture. Step S3: After centrifuging the fermentation product, take the supernatant and filter the supernatant to obtain the fermentation product filtrate of Antarctic spirochetes and Bacillus subtilis; In step S2, the preparation method of the Antarctic Clematis fermentation medium is as follows: Antarctic Clematis is selected as raw material and pulverized; then, Antarctic Clematis powder is added to ultrapure water at a mass percentage of 0.1% to 10%, and sterilized by steam at 121°C for 20 minutes.

4. The preparation method according to claim 3, characterized in that: In step S1, the seed culture is obtained by culturing at 25℃~37℃ and 150~200rpm for 16h~48h; in step S2, the fermentation product is obtained by culturing at 25℃~37℃ and 150~200rpm for 16h~48h; in step S3, the fermentation product is centrifuged at 8000rpm for 15min and the supernatant is collected. The supernatant is filtered through a 0.22μm filter membrane to obtain the filtrate of the fermentation product of Antarctic spirochetes and Bacillus.

5. The preparation method according to claim 3, characterized in that, The components of the LB liquid culture medium are: 10g peptone, 5g yeast extract, 10g NaCl, and 1L ultrapure water; the fermentation medium of Antarctic spirochetes is prepared by grinding Antarctic spirochetes into powder and mixing it with ultrapure water at a mass percentage of 5%.

6. The application of the fermentation product filtrate of Antarctic spirochetes and Bacillus as described in claim 2 in the preparation of cosmetics with antioxidant, anti-aging or skin microecological regulation properties.

7. A cosmetic product that has antioxidant, anti-aging, or skin microecological regulating properties, characterized in that, The filtrate contains the fermentation product of Antarctic spirochetes and Bacillus as described in claim 2.

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