Buchnera aphidicola strain with stress resistance, fermentation product preparation and application thereof

Abstract

This disclosure relates to a stress-resistant strain of *Buchuriae*, its fermentation product formulation, and its applications, belonging to the field of microbial technology. The *Buchuriae* strain is classified and named... Buttiauxella ferragutiae The *Buchuria* strain BTN-HB-P48, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38093, was deposited on April 9, 2026. This *Buchuria* strain exhibits dual resistance to high temperatures and ultraviolet radiation. Its fermentation product filtrate, fermentation cell lysate, and PDRN-containing biological agents based on the fermentation cell lysate demonstrate in vitro DPPH free radical scavenging activity, in vitro cell barrier function, and soothing effects, making it suitable for use in daily chemical skincare products.

Description

Technical Field

[0001] This disclosure relates to the field of microbial technology, specifically to a stress-resistant strain of Buchuria, its fermentation product formulation, and its applications. Background Technology

[0002] With the diversification of skincare methods, environmental pollution, and changes in work and life pressures, people's demand for novel and effective skincare active ingredients is becoming increasingly strong. Compared with the extraction and enrichment of natural products and chemical synthesis, active ingredients obtained by microbial fermentation are more stable between batches and can be selectively modified to obtain target ingredients, which has certain advantages.

[0003] With the expanding market for microbial applications, the demand for microorganisms from different sources and with diverse functional characteristics is constantly increasing. Some microorganisms have attracted increasing attention due to their strong tolerance to extreme environments, i.e., stress resistance (high temperature, hyperosmolarity, ultraviolet radiation, etc.). Many studies have developed and applied some microorganisms with stress-resistant genes to prevent and control diseases in agriculture, medicine, and even skin care.

[0004] CN118556717A discloses an anti-ultraviolet microbial composition, its preparation method and application. The anti-ultraviolet microbial composition is not easily deactivated by ultraviolet radiation and can be used as a microbial insecticide, microbial growth regulator and other agents in agricultural production.

[0005] In the process of discovering microorganisms with stress-resistant genes, researchers have found that microorganisms originating from extreme environments, such as deserts and volcanic hot springs, possess stress-resistant properties. Furthermore, microbial preparations made from stress-resistant microorganisms have demonstrated certain anti-damage and skin-repairing effects. For example, the strain disclosed in CN118931973A is a thermophilic bacterium first discovered in hydrothermal vents on the seabed, capable of growing in high-temperature environments. The decolorized, deodorized, and sterilized fermentation products from this thermophilic bacterium have shown good effects in repairing photodamage to the skin.

[0006] While stable and highly effective active ingredients can be obtained through microorganisms, the requirements for microbial strains are relatively high. Current stress-resistant microorganisms are often obtained through screening under single stress conditions, such as heat-resistant strains screened from near natural hot springs, or strains obtained by screening for resistance to ultraviolet radiation. There is a lack of special stress-resistant strains screened under more comprehensive and complex environmental conditions, such as stress-resistant microorganisms screened from environments with large diurnal temperature differences, strong ultraviolet radiation, hypoxia, and low pressure.

[0007] Bacterium pumil ( ButtiauxellaSome strains of the genus *Brucella* naturally possess mild tolerance to high osmotic pressure. As Enterobacteriaceae microorganisms, their optimal temperature range is typically 20–40°C. However, no *Brucella* strains exhibiting multiple stress resistances have been reported. For example, the *Brucella* disclosed in CN118146979A only has the function of alleviating the growth stress of high-selenium environments on plants and promoting plant growth. Furthermore, stress-resistant *Brucella* strains have not been used in daily chemical skincare preparations, especially not in skincare preparations with skin barrier repair functions. Summary of the Invention

[0008] The purpose of this disclosure is to provide a bacterium with multiple stress resistance and to apply it to the repair of the skin barrier, so as to solve the above-mentioned technical problems.

[0009] Based on this, this disclosure provides a stress-resistant strain of *Buchuria*, wherein the *Buchuria* strain is classified and named as follows: Buttiauxella ferragutiae The B. tumefaciens strain BTN-HB-P48 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38093 and deposit date of April 9, 2026.

[0010] This disclosure also provides a fermentation product filtrate of a *Buchuria* strain, wherein the fermentation product filtrate is classified and named as described above. Buttiauxella ferragutiae The filtrate of the fermentation products of B. tumefaciens strain BTN-HB-P48 in the strain's liquid culture medium.

[0011] Preferably, the fermentation product filtrate is prepared in the following manner: S1. The aforementioned classifications are cultured in a culture medium and named accordingly. Buttiauxella ferragutiae The seed culture of B. tumefaciens strain BTN-HB-P48 was obtained. S2. The seed culture is inoculated into the strain liquid culture medium at an inoculation amount of 1% to 5% (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%) to obtain the fermentation product. The fermentation temperature is 25℃ to 37℃ (e.g., 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃), and the fermentation time is 24 h to 48 h (e.g., 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h). S3. Centrifuge the fermentation product, take the supernatant, and filter the supernatant to obtain the fermentation product filtrate.

[0012] This disclosure also provides a fermentation cell lysate of a Bacteroides strain, prepared in the following manner: S1. The aforementioned classifications are cultured in a culture medium and named accordingly. Buttiauxella ferragutiae The BTN-HB-P48 strain of Bucuna bacteria was used to obtain the seed culture of the Bucuna bacteria strain. S2. The seed culture is inoculated into the strain liquid culture medium at an inoculation amount of 1% to 5% (e.g., 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%) to obtain the fermentation product. The fermentation temperature is 25℃ to 37℃ (e.g., 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃), and the fermentation time is 24 h to 48 h (e.g., 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h). S3. Centrifuge the fermentation product, remove the supernatant, and collect the wet bacterial sludge; S4. The wet bacterial sludge is resuspended in ultrapure water to obtain a bacterial sludge resuspension, wherein the mass ratio of the wet bacterial sludge to the water is 1:130 to 1:25; S5. The bacterial sludge resuspension is homogenized under high pressure until no intact bacterial cells remain, to obtain the fermentation cell lysate of the Buchuria strain.

[0013] This disclosure also provides a biological agent containing PDRN (polydeoxyribonucleotide), said biological agent being prepared in the following manner: S1. Take the aforementioned fermented cell lysate, add enzyme at a dosage of 1.25 mg proteinase K per gram of wet bacterial mud, and enzymatically hydrolyze in a water bath at 50–60℃ (50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃) for 2–4 h (e.g., 2h, 2.5h, 3h, 3.5h, 4h). S2. After enzymatic hydrolysis, the product is centrifuged to remove the precipitate and obtain a clear filtrate; S3. Precipitate the clarified filtrate with ethanol, wherein the volume ratio of ethanol to the clarified filtrate is 4 to 2:1, and collect the precipitate after precipitation. S4. The precipitate is washed and freeze-dried to obtain a biological agent containing PDRN.

[0014] Alternatively, the PDRN-containing biological agent may be prepared in the following manner: S1. Take the aforementioned fermented cell lysate, add enzyme at a dosage of 1.25 mg proteinase K per gram of wet bacterial mud, and enzymatically hydrolyze in a water bath at 50–60℃ (50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃) for 2–4 h (e.g., 2h, 2.5h, 3h, 3.5h, 4h). S2. After enzymatic hydrolysis, the product is centrifuged to remove the precipitate and obtain a clear filtrate; S3. Filter the clarified filtrate using an ultrafiltration membrane, and freeze-dry the filtrate to obtain a biological agent containing PDRN.

[0015] This disclosure also provides an application of the aforementioned fermentation cell lysate or the aforementioned biological agent in daily chemical products.

[0016] Preferably, the daily chemical products can be of various types, such as shampoo, hair care products, cosmetics, etc. The cosmetics can be toners, skin creams, lotions, serums, eye creams, face masks, makeup removers, facial cleansers, shower gels, shampoos, etc.

[0017] More preferably, the amount of the fermentation product filtrate added to the daily chemical product is 0.09% to 3% by mass percentage, and the amount of the PDRN-containing biological agent obtained by purification of the cell lysate added to the daily chemical product is 50 to 200 μg / mL.

[0018] The technical solution claimed in this disclosure achieves the following beneficial effects: 1. Through screening, a strain of B. tuberculosis BTN-HB-P48 (referred to as P48) from the extreme plateau environment (small red orchid growing in Shika Snow Mountain, Shangri-La, Yunnan) was obtained, enriching the resource of Yunnan-characteristic stress-resistant strains with dual stress resistance characteristics of high temperature (45℃) and UVC irradiation resistance.

[0019] 2. Provide the fermentation process and corresponding fermentation products of the aforementioned *Buchuria* strain. The fermentation products include *Buchuria* P48 fermentation filtrate and a PDRN-containing biological agent obtained by further refining the *Buchuria* P48 cell lysate. *Buchuria* P48 exhibits strong self-repair and related repair potential due to its vigorous vitality. The *Buchuria* P48 fermentation filtrate possesses certain in vitro DPPH free radical scavenging activity, and its PDRN-containing biological agent has in vitro cell barrier function and soothing effects, showing potential application prospects in the daily chemical skincare field and enriching the application areas of *Buchuria*. The specific efficacy of the obtained biological agent includes: 1) The DPPH scavenging rate of the blister bacterium fermentation filtrate prepared from the supernatant of blister bacterium P48 fermentation was 13.4% in vitro at a concentration of 25%; 2) A PDRN-containing biological agent obtained by further purification of Bucuna p48 cells, at an addition of 200 μg / mL, significantly increased the LOR of the HaCaT cell barrier gene by 14.2%. 3) The PDRN-containing biological agent obtained by further refining Bacillus p48 cells, at an addition of 200 μg / mL, significantly reduced the inflammatory factor NO in RAW264.7 cells induced by LPS stimulation by 57.23%.

[0020] 3. The biological raw material, Buchuria, used is safe, controllable, and environmentally friendly throughout the entire process from strain activation and cultivation to large-scale fermentation. It also exhibits good batch-to-batch stability, a relatively simple production process, and is easy to operate and replicate. It is suitable for large-scale industrial production and has great potential for developing skincare product raw materials, thus possessing good economic value. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 This disclosure includes a process flow diagram encompassing strain screening, stress resistance detection, and biological agent preparation.

[0023] Figure 2 This describes the growth of Bacteroides P48 at high temperatures.

[0024] Figure 3 Electrophoretic band images of PDRN-containing biopharmaceuticals based on Buchuria P48.

[0025] Figure 4 The results show the effects of PDRN-containing biopharmaceuticals based on Buchuria P48 on the regulation of HaCaT cell barrier genes.

[0026] Figure 5 This is an evaluation of the anti-inflammatory and soothing activity of a PDRN-containing biological agent based on Buchuria P48. Detailed Implementation

[0027] To make the objectives, technical solutions, and beneficial effects of the embodiments in this disclosure clearer, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this disclosure.

[0028] like Figure 1 As shown, this disclosure describes the isolation of a strain of *Bacillus pumilus* with dual resistance to ultraviolet C radiation and high temperature (45℃) from *Cymbidium goeringii* plants growing in Shika Snow Mountain, Shangri-La, Yunnan. Buttiauxella ferragutiae BTN-HB-P48 (P48 for short) exhibits vigorous vitality and inherent stress resistance and repair-promoting properties. Fermentation technology was used to develop related fermentation products of the stress-resistant strain *Buchuria pumil* P48, yielding fermentation filtrate of *Buchuria pumil* P48 and a PDRN-containing biological agent obtained from further lysis of *Buchuria pumil* cells. The biological agent obtained from the fermentation exhibited certain in vitro antioxidant, barrier repair, and anti-inflammatory soothing effects.

[0029] <Example 1> This embodiment provides Bacteroides ( Buttiauxella ferragutiae The screening and identification of BTN-HB-P48 (hereinafter referred to as P48) are carried out in the following steps: (1) Collect samples of small red orchid plants growing in Shika Snow Mountain, Shangri-La, Yunnan. After washing away the soil and dust, soak them in sterile water at 4°C for 18-24 h. Then, take an appropriate amount of soaking solution and spread it on the strain solid culture medium. After culturing at 37°C for 24-30 h, pick out colonies of different morphologies from the strain solid culture medium and streak them on the surface of the strain solid culture medium for purification.

[0030] The solid culture medium for the bacterial strains included: tryptone, yeast extract, sodium chloride, and agar; the solid culture medium for the seeds included: 10-30 g / L tryptone, 5-15 g / L yeast extract, 5-10 g / L sodium chloride, 15-20 g / L agar, with the remainder being water. The pH was calibrated to 7.0±0.2. The culture medium was autoclaved at 121℃ for 15 min, and 15-20 mL of agar was poured into plates before solidification.

[0031] (2) Observe the single colonies on different plates after purification under a microscope, select different strains with bacterial morphology for identification, and preserve the selected strains.

[0032] The 16S sequence of Bucuna p48 from *Pteris vittata* in Shangri-La, Yunnan, in this embodiment is as follows:

[0033] <Example 2> This embodiment provides a process for preparing seed culture of Bucuna p48 from *Cymbidium goeringii* plants in Lijiang, Yunnan. The specific method is as follows: (1) The strain of Bucula pumilus P48 from the Yunnan Lijiang Xiaohongmen orchid plant in Example 1 was streaked onto a solid culture medium plate and cultured at 37°C for 18-48 h to complete the strain recovery.

[0034] (2) Pick a single colony from the plate and inoculate it into the strain liquid culture medium. Incubate at 37°C for 24 h to complete one generation. Use the fermentation product obtained after two generations of culture and activation as the seed liquid.

[0035] The liquid culture medium for the strain may include: tryptone, yeast extract, and sodium chloride; the liquid culture medium for the seed includes: 10-30 g / L tryptone, 5-15 g / L yeast extract, 5-10 g / L sodium chloride, with the remainder being water, calibrated to pH 7.0±0.2, autoclaved at 121℃ for 15 min and cooled to room temperature.

[0036] <Example 3> This embodiment provides a Bacillus p48 fermentation filtrate, prepared using the following process: (1) The seed culture of Bucuna P48 obtained in Example 2 was inoculated into the strain liquid culture medium at an inoculation amount of 1% to 5%, the fermentation temperature was 37°C, the fermentation time was 24 to 48 h, and the shaking speed was set to 150 to 200 rpm.

[0037] (2) Centrifuge the fermentation mixture to remove insoluble matter such as bacterial cells. The centrifugation conditions are 5000~8000 g and the centrifugation time is 10~20 min. Then filter the supernatant through a 0.22 μm filter membrane to obtain the fermentation filtrate of Bacillus p48 prepared by microbial means.

[0038] <Example 4> This embodiment provides a lysate of Bacteroides P48 fermentation cells, prepared using the following process: (1) The seed culture of Bucuna P48 obtained in Example 2 was inoculated into the strain liquid culture medium at an inoculation amount of 2%, the fermentation temperature was 37°C, the fermentation time was 24 h, and the rotation speed was 200 rpm.

[0039] (2) After fermentation, centrifuge at 5000~8000 g for 10~20 min, collect the wet mycelium mud and weigh it.

[0040] (3) Prepare the mushroom mud resuspension solution according to the ratio of 2~8 g wet mushroom mud to 250 mL ultrapure water.

[0041] (4) The resuspension was treated with a high-pressure homogenizer at 1000~1200 bar for 3 cycles to obtain the fermentation cell lysate of Bucuna p48. When no intact cells were found during microscopic examination, the strain was confirmed to be completely lysed.

[0042] <Example 5> This embodiment provides a method for purifying and preparing a PDRN-containing biological agent based on Buchuria p48: (1) Prepare the fermentation cell lysate of Bucuna P48 according to the method in Example 4, wherein the preparation of the bacterial sludge resuspension is limited to step (3) of Example 4, with 4 g of wet bacterial sludge resuspended in 250 mL of ultrapure water, and the other conditions remain unchanged.

[0043] (2) Weigh the accurate volume of the high-pressure homogenized bacterial sludge resuspension, add NaCl until the final concentration of NaCl in the bacterial sludge resuspension is 3M, add enzyme at a dosage of 1.25 mg proteinase K per gram of wet bacterial sludge, and enzymatically hydrolyze in a water bath at 55℃ for 3 h. After enzymatic hydrolysis, centrifuge at 8000~12000 g for 10~15 min, discard the precipitate and take the supernatant to obtain a clear filtrate.

[0044] (3) Accurately weigh the volume of the clarified filtrate from step (2) and record it as V1. Add anhydrous ethanol of volume V2 to the clarified filtrate, V2 = 3 * V1. Mix well and place at 4℃ to precipitate overnight. After sufficient precipitation, centrifuge at 8000~12000 g for 10~15 min, discard the supernatant and take the precipitate.

[0045] (4) Wash the precipitate once with 1 / 3 to 1 volume of 70% ethanol, and then wash it a second time at a ratio of 10 mL of 70% ethanol for every 10 mg of wet precipitate.

[0046] (5) The precipitate was washed and dehydrated with an appropriate volume of anhydrous ethanol, centrifuged at 8000-12000 g for 10-15 min, the supernatant was discarded and the crude extract was collected. The crude extract was freeze-dried to obtain a white solid. After nanodrop analysis, it was found to contain more than 80% PDRN. The A268 / A280 ratio met the PDRN requirements (values ​​in the range of 1.8-2.0 and slightly higher, sharp, symmetrical, narrow peaks in the UV range with a very clear 260 nm peak, no shoulder, and no tail). The corresponding molecular weight could be determined by gel electrophoresis. The above-mentioned PDRN-containing biological agent product based on Buchuria P48 is denoted as S1.

[0047] <Example 6> This embodiment provides a method for purifying and preparing a PDRN-containing biological agent based on Buchuria p48: (1) Prepare the fermentation cell lysate of Bucuna P48 according to the method in Example 4, wherein the preparation of the bacterial sludge resuspension is limited to step (3) of Example 4, with 6 g of wet bacterial sludge resuspended in 250 mL of ultrapure water, and the other conditions remain unchanged.

[0048] (2) Weigh the accurate volume of the high-pressure homogenized bacterial sludge resuspension, add NaCl until the final concentration of NaCl in the bacterial sludge resuspension is 3M, add enzyme at a dosage of 1.25 mg proteinase K per gram of wet bacterial sludge, and enzymatically hydrolyze in a water bath at 55℃ for 3 h. After enzymatic hydrolysis, centrifuge at 8000~12000 g for 10~15 min, discard the precipitate and take the supernatant to obtain a clear filtrate, and record the volume of the clear filtrate as V1.

[0049] (3) The clarified filtrate obtained in step (2) was passed through a 100 kDa ultrafiltration membrane, the retentate was collected, and the retentate was lyophilized. The resulting white solid was analyzed by nanodrop and found to contain more than 80% PDRN. The A268 / A280 ratio met the PDRN requirements (values ​​in the range of 1.8 to 2.0 and slightly higher, sharp, symmetrical, narrow peaks in the UV spectrum, and a very clear 260 nm peak with no shoulder or tail). The corresponding molecular weight could be determined by gel electrophoresis. The above-mentioned PDRN-containing biopharmaceutical product based on Buchuria P48 is designated as S2.

[0050] <Comparative Example 1> This comparative example provides liquefied Serratia marcescens ( ) Serratia liquefaciens R34 seed solution preparation method: The Serratia liquefiedis R34 was also a bacterial strain obtained by isolating, purifying, and preserving a small red orchid plant sample grown in Shika Snow Mountain, Yunnan Province, according to the method in Example 1.

[0051] The seed culture was prepared according to the method in Example 2, except that Bucuna P48 in Example 2 was replaced with Serratia liquefiediformis R34, and all other conditions remained the same.

[0052] <Comparative Example 2> This comparative example provides *Pseudomonas* spp. ( Pseudomonas Q98 Seed Liquid Preparation Method: Pseudomonas spp. Q98 was also a bacterial strain obtained by isolating, purifying, and preserving a small red orchid plant sample growing in Shika Snow Mountain, Shangri-La, Yunnan, according to the method in Example 1.

[0053] The seed culture was prepared according to the method in Example 2, except that Bucuna P48 in Example 2 was replaced with Pseudomonas Q98, while the other conditions remained the same.

[0054] <Comparative Example 3> This comparative example provides Bacteroides ( Buttiauxella P47 Seed Liquid Preparation Method: Bucula p47 is a bacterial strain that was isolated, purified, and preserved from a sample of sea cucumber plant in Jidi Village, Shangri-La, Yunnan Province, according to the method described in Example 1.

[0055] The seed culture was prepared according to the method in Example 2, except that Bucuna P48 in Example 2 was replaced with Bucuna P47, and all other conditions remained the same.

[0056] <Comparative Example 4> This comparative example provides *Microbacterium brevicornu* from brine shrimp (… Exiguobacterium artemiae P75 Seed Liquid Preparation Method: Artemia microbacterium P75 is a bacterial strain that was isolated, purified, and preserved from a sample of Hydrangea macrophylla plant from Shika Snow Mountain in Shangri-La, Yunnan Province, according to the method described in Example 1.

[0057] The seed culture was prepared according to the method in Example 2, but the Bacterium pumil P48 in Example 2 was replaced with Microbacterium bacillus P75, and the other conditions remained the same.

[0058] <Comparative Example 5> This comparative example provides Pseudomonas aeruginosa ( Pseudomonas yamanorum Q94 Seed Liquid Preparation Method: The *Pseudomonas aeruginosa* Q94 is a bacterial strain isolated, purified, and preserved from a whole-plant sample of the large edible fungus *Boletus lucidum* from Shika Snow Mountain in Shangri-La, Yunnan Province, according to the method described in Example 1.

[0059] The seed culture was prepared according to the method in Example 2, but the Bucuna P48 in Example 2 was replaced with Pseudomonas aeruginosa Q94, and the other conditions remained the same.

[0060] <Comparative Example 6> This comparative example uses commercially available PDRN products derived from salmon, denoted as PDRN product S0; Dissolve an appropriate amount of the finished product powder in ultrapure water to a test sample concentration of 1 mg / mL for later use.

[0061] <Test Example 1> This test example examines the heat resistance (45℃) of Bacteroides p48. The specific method is as follows: Take 2 mL of seed liquid from each of Examples 2 and Comparative Examples 1-5, add 1 mL of each culture medium to the seed solid culture medium, and spread it evenly with a disposable spreader. Label the strain number corresponding to each plate. Thus, each of Examples 2 and Comparative Examples 1-5 has two seed solid plates with 1 mL of seed liquid added.

[0062] Two solid plates containing seeds from Examples 2 and Comparative Examples 1-5 (with added seed solution) were placed in incubators at 37°C and 45°C respectively, and cultured for 48 h. The plate cultured at 37°C was designated as ck. The colony growth of each plate was observed and recorded, and the results are shown in Table 1.

[0063] Table 1 Example 2 yes yes yes Comparative Example 1 yes no no Comparative Example 2 yes no no Comparative Example 3 yes no no Comparative Example 4 yes yes yes Comparative Example 5 yes no no As shown in Table 1, the bacteria *Bacillus pumilus* P48 from *Orchidonia spp.* in Example 2 and *Microbacterium bacillus* P75 from *Ilex chinensis* in Comparative Example 4 exhibit high-temperature (45℃) resistance. Both bacteria grew distinct colonies in incubators at 37℃ and 45℃, as shown in Table 1. Figure 2 As shown.

[0064] The bacteria *Serratia liquefaction* R34 from *Ormosia simonii* in Comparative Example 1 and *Pseudomonas* Q98 from *Ormosia simonii* in Comparative Example 2 do not exhibit high-temperature resistance and can only grow in an incubator at 37°C. Furthermore, the bacteria *Buchuria* P47 from *Stichopus japonicus* in Comparative Example 3 and *Pseudomonas aeruginosa* Q94 from the large edible fungus *Boletus luridus* in Comparative Example 5 also do not exhibit high-temperature resistance and can only grow in an incubator at 37°C.

[0065] The above results confirm that the high-temperature (45℃) resistance characteristic has certain uniqueness and is not necessarily related to the source or species of the strain. In order to screen for strains with multiple resistance characteristics, a further test example 2 was designed to test the resistance to ultraviolet (UVC irradiation).

[0066] <Test Example 2> This test examines the UV (UVC irradiation) resistance of Bacteroides p48. The specific method is as follows: (1) The turbidity meter was zeroed and calibrated using the liquid culture medium of the strain in Example 2.

[0067] (2) Take the seed liquid from Example 2 and Comparative Examples 1-5 and dilute it with the strain liquid culture medium. After dilution, the turbidity of the seed liquid diluted in Example 2 and Comparative Examples 1-5 was measured by a turbidity meter and was 0.50±0.3.

[0068] (3) Add 1 mL of the seed diluent adjusted in step (2) to the 24-well plate. The seed diluents of Example 2 and Comparative Examples 1 to 5 each need to be inoculated in 8 wells in parallel.

[0069] (4) Place the UVC lamp 15 cm vertically above the 24-well plate, cover 4 wells in the plate with aluminum foil, and then turn on the UVC lamp for 25 s to ensure that 4 out of the 8 wells in each strain in Example 2 and Comparative Examples 1-5 are evenly irradiated with UVC for 25 s and 4 are in an untreated state.

[0070] (5) Use an ELISA reader to detect and record the absorbance A at 600 nm for each well at 0 h after UV treatment. 处理0h The absorbance of the seed dilution solution without UV treatment after shading was measured at 600 nm as A. 对照0h .

[0071] (6) The 24-well plate was incubated at 37°C for 24 h, and the absorbance A at 600 nm of each well was measured 24 h after UV treatment. 处理24h The absorbance of the seed dilution solution without UV treatment after shading was measured at 600 nm as A. 对照24h .

[0072] (7) Based on the results of the two tests, the growth inhibition rate of each strain after UVC irradiation was calculated as follows:

[0073] A positive growth inhibition rate after UVC irradiation indicates that the growth of the strain is inhibited by UVC irradiation. The larger the value, the more obvious the inhibition, and the strain has no or weak resistance to UV irradiation. A negative growth inhibition rate after UVC irradiation indicates that the growth of the strain is not inhibited by UVC irradiation, the strain still has strong vitality under UVC irradiation, and the strain has strong resistance to UV irradiation.

[0074] The growth inhibition rate of strains in Example 2 and Comparative Examples 1-5 after UVC irradiation was calculated, and the results are shown in Table 2.

[0075] Table 2 Example 2 -33.59% yes Comparative Example 1 5.22% no Comparative Example 2 12.55% no Comparative Example 3 -2.34% yes Comparative Example 4 18.07% no Comparative Example 5 0.81% no As shown in Table 2, the bacteria Bacillus p48 from *Ormosia simonii* in Example 2 and the bacteria Bacillus p47 from *Stichopus japonicus* in Comparative Example 3 have resistance to ultraviolet (UVC) irradiation. Their growth was not significantly inhibited after 24 h of UVC irradiation.

[0076] The bacteria *Serratia liquefaction* R34 from *Lysimachia christinae* (comparative example 1), *Pseudomonas* spp. Q98 from *Lysimachia christinae* (comparative example 2), *Microbacterium bacillus* P75 from *Hydrangea spp.* (comparative example 4), and *Pseudomonas aeruginosa* Q94 from the large edible fungus *Boletus luridus* (comparative example 5) all lacked resistance to ultraviolet (UVC) radiation, and their growth was inhibited to some extent after 24 h of UVC irradiation.

[0077] The above results confirm that the resistance to ultraviolet (UVC) radiation has certain unique characteristics and is not necessarily related to the source or species of the strain.

[0078] Based on the results of Test Examples 1 and 2, among the six strains tested, only *Bacteroides buchuriae* P48, derived from *Ormosia simsii* in Example 2, possesses both high-temperature (45°C) and UVC irradiation resistance. Therefore, *Bacteroides buchuriae* P48 from Example 2 was subsequently selected for the preparation and efficacy verification of related bio-fermentation agents.

[0079] <Test Example 3> Using the "Cosmetic Free Radical (DPPH) Scavenging Test Method (T / SHRH006-2018)" as the reference standard, the DPPH free radical scavenging rate (%) of the Bacteroides p48 fermentation filtrate prepared under the process conditions of Example 3 and the blank control group were tested: (1) Preparation of the reagent: Weigh 10 mg of DPPH reagent accurately, dissolve it in anhydrous ethanol by sonication, and after the reagent is completely dissolved, make up to 100 mL to prepare a DPPH solution with a concentration of 0.1 mg / mL. Store in a dark environment for later use.

[0080] (2) Experiment: Sample group, control group and control group were set up in 96-well plate, with 3 replicates in each group; blank control was set up in both sample group and control group. The amount added in each well and the distribution in 96-well plate are as follows: The sample group consisted of 150 μL of fermentation filtrate of Bacillus p48 from Example 3 at different concentrations, with 150 μL of DPPH alcohol solution added. The blank sample group consisted of 150 μL of fermentation filtrate sample solution of Bacteroides p48 from Example 3 at different concentrations, with 150 μL of anhydrous ethanol added. The control group consisted of 150 μL of DPPH solution with 150 μL of sample solvent (ultrapure water). The control blank group consisted of 150 μL of anhydrous ethanol and 150 μL of sample solvent (ultrapure water).

[0081] After the sample was added, the mixture was allowed to react at room temperature in the dark for 30 min, then shaken to mix, and the absorbance was measured at 517 nm.

[0082] (3) DPPH removal rate calculation: Clearance rate (%) = [1 - (As - As0) / (Ac - Ac0)] × 100%.

[0083] In the formula, As represents the absorbance measured in the sample group; As0 represents the absorbance measured in the blank sample group; Ac represents the absorbance measured in the control group; and Ac0 represents the absorbance measured in the control group.

[0084] (4) Test Result Analysis The DPPH scavenging rate test results of the samples corresponding to the fermentation filtrate of Buchuria P48 in Example 3 with different concentrations are shown in Table 3.

[0085] Table 3

[0086] As shown in Table 3, the fermentation filtrate of Buchuria P48 has certain in vitro DPPH free radical scavenging activity and has the potential to be used as a raw material for biological antioxidants in daily chemical products.

[0087] <Test Example 4> This test example provides a method for testing PDRN concentration and purity. Using a Nanodrop nucleic acid detector, the concentration of PDRN in the product and the A260 / A280 ratio are accurately determined to verify the purity of PDRN in the product. (1) Instrument startup and calibration: Turn on the Nanodrop nucleic acid detector, preheat for 5 minutes, and enter the DNA detection mode; spot 1 μL of enzyme-free sterile water onto the detection probe, click "Blank" to perform blank calibration, after calibration, gently wipe the probe with a lint-free paper, repeat blank calibration twice to ensure that the blank value is stable (A260 value ≤ 0.01).

[0088] (2) Sample detection: Take 1 μL of the sample to be tested (the samples from Example 5, Example 6 and Comparative Example 6 are respectively prepared as 1 mg / mL test liquid), spot the sample onto the detection probe, click "Measure" to detect, and record the concentration (ng / μL) and A260 / A280 ratio of each sample in real time; each sample is tested 3 times, and after each test, gently wipe the probe with lint-free paper to avoid sample residue contamination.

[0089] (3) Post-test processing: After all samples have been tested, wipe the test probe repeatedly with enzyme-free sterile water 3-5 times, turn off the instrument, organize the test equipment, store the test samples and reagents according to the specifications, and record the test time.

[0090] (4) Result judgment criteria: a. Repeatability determination: If the RSD of each sample group is ≤5% in three repeated tests, and the RSD of the PDRN standard test is ≤5%, the test results are considered to have good repeatability. If RSD > 5%, the test result is deemed invalid, and the instrument status, sample mixing, and operational procedures must be checked before retesting.

[0091] b. Concentration determination: Since the sample to be tested was prepared at a concentration of 1 mg / mL, 1 μL was added to the Nanodrop nucleic acid detector for measurement. When the measurement result was ≥600 ng / μL, it was confirmed that the PDRN content in the sample was greater than 60%.

[0092] c. Purity determination: The A260 / A280 ratio is the core indicator for evaluating nucleic acid purity. It is used to determine whether there are impurities such as proteins and phenols in the PDRN product (proteins have characteristic absorption at 280nm, which will cause the ratio to decrease).

[0093] If the A260 / A280 ratio is between 1.8 and 2.0, it indicates that the PDRN product has high purity and no obvious protein or phenolic impurities.

[0094] (5) Analysis of the above sample concentration and purity test results The results of the above measurements are shown in Table 4.

[0095] Table 4

[0096] As shown in Table 4, the PDRN purity of the products obtained in Examples 5 and 6 was not significantly different from that of the commercially available salmon PDRN product in Comparative Example 6, where the PDRN content of the commercially available salmon in Comparative Example 6 was above 90%. Both Examples 5 and 6 yielded biological agents containing more than 60% PDRN.

[0097] Subsequently, product S1 from Example 5 was selected as a representative of the PDRN-containing biological agent of the stress-resistant strain Buchuria p48 for efficacy testing.

[0098] <Test Example 5> This test example uses agarose gel electrophoresis with a 250-10000 bp nucleic acid marker to accurately detect the molecular weight of PDRN in the PDRN-containing products of Example 5. The specific steps are as follows: (1) Preparation of agarose gel: Weigh 1.0 g of agarose using an electronic balance, put it into an Erlenmeyer flask, add 100 mL of 1×TAE electrophoresis buffer, and vortex to mix. Place the Erlenmeyer flask in a microwave oven and heat on medium-high heat until the agarose is completely melted (no bubbles and the solution is transparent). Remove and cool to 50~60℃, pour into a gel casting plate, insert a comb, and let stand at room temperature for 30 min until the gel is completely solidified.

[0099] (2) Instrument debugging: Place the solidified agarose gel into the electrophoresis tank, add 1×TAE electrophoresis buffer to the electrophoresis tank until the buffer covers the gel surface by 1~2 mm; pull out the comb and check whether the sample well is intact and leak-free; connect the power supply of the electrophoresis instrument, turn on the power switch, adjust to constant voltage 120 V mode, and preheat for 5 min.

[0100] (3) Sample and marker loading: Take 2 μL of diluted 250~10000 bp marker and slowly add it to the loading well (numbered as Marker well); take 2 μL of each of the mixed PDRN-containing samples (product S1 of Example 5 and product S0 of Comparative Example 6) and add them to the adjacent loading wells in sequence, mark the sample numbers, and avoid touching the edge of the loading well when loading the sample.

[0101] (4) Electrophoresis operation: After loading the sample, close the electrophoresis tank lid and confirm that the positive and negative terminals of the power supply are connected correctly (the sample end is connected to the negative terminal and the imaging end is connected to the positive terminal); turn on the electrophoresis instrument switch, start the electrophoresis program, set the electrophoresis time to 40 min, and observe the migration of the bromophenol blue band in the loading buffer during the period to prevent the band from running out of the gel.

[0102] (5) Staining and Imaging: After electrophoresis, turn off the power, disconnect the power supply, and remove the agarose gel; place the gel in EB staining solution and stain in the dark for 10 min, gently shaking the staining solution during this time to ensure uniform staining; after staining, remove the gel and rinse the surface with enzyme-free sterile water twice for 1 min each time to remove any residual EB staining solution; place the rinsed gel into a gel imaging system and image it under ultraviolet light to capture clear band images, and save the image file, such as... Figure 3 As shown.

[0103] Depend on Figure 3 It can be seen that the electrophoretic bands of product S1 (a representative of the anti-stress strain Buchuria p48 containing PDRN) in Example 5 and product S0 (PDRN from commercially available salmon) in Comparative Example 6 are clear and without obvious tailing, and their molecular weights are similar; in addition, according to the Maker, the molecular weight of product S1 in Example 5 is in the range of 250~500 bp.

[0104] <Test Example 6> This test utilizes in vitro HaCat cells to evaluate the skin barrier efficacy of PDRN-containing biologics based on Buchuria p48, verifying the effects of different PDRN-containing samples on HaCaT cell activity and their regulatory role in the relative expression levels of barrier gene (LOR) mRNA, thus validating the barrier repair effect of the test substance. The specific steps are as follows: (1) Cells and culture medium: Cell source: HaCaT cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences); Culture conditions: DMEM medium, 10% FBS fetal bovine serum; Test substances: Product S1 of Example 5 and Product S0 of Comparative Example 6.

[0105] (2) Effect of the test substance on HaCaT cell activity (CCK8 assay) a. HaCaT cells in the logarithmic growth phase and with good morphology were seeded into 96-well plates and incubated in an incubator for 24 h; b. The test substance (product S1 of Example 5 and product S0 of Comparative Example 6) was diluted with complete culture medium to 200 μg / mL, with 3 replicates for each concentration, and co-incubated with cells for 24 h. c. Remove the original supernatant from each well, add CCK8 test solution to each well, incubate for 2 h, and then measure the absorbance at 450 nm using a microplate reader to calculate the cell viability. Cell viability ≥80% P A concentration ≥0.05 was considered to indicate no significant toxicity. The results are shown in Table 5.

[0106] (3) Effects of the test substance on the regulation of HaCaT cell barrier genes a. HaCaT cells in the logarithmic growth phase and with good morphology were... Cells were seeded in 24-well plates and incubated for 24 h. b. Set up a control group (DMEM culture medium only) and an experimental group (product S1 of Example 5, product S0 of Comparative Example 6), with 3 replicates based on the highest safe concentration obtained in step (2). After drug administration, continue to maintain the concentration at 37°C and 5%... Incubate in an incubator for 24 hours; c. RNA extraction, RNA content determination, and reverse transcription to synthesize cDNA were performed sequentially, followed by quantitative real-time PCR to detect the expression of the barrier gene LOR.

[0107] (4) Result calculation and data analysis a. Cell viability assessment: If the cell viability rate of the test substance treatment group is ≥80% and there is no significant difference compared with the blank control group, the test substance HaCaT cells are determined to have no obvious toxicity, and subsequent barrier efficacy assessment can be performed; if the viability rate is <80%, the test substance is determined to have cytotoxicity, and its barrier efficacy assessment needs to be combined with the toxicity effect.

[0108] b. Calculation of quantitative real-time PCR results: The Comparative Ct (ΔΔCt) method was used, with β-actin as the internal reference gene; the calculation formula is RQ = Where ΔCt = ΔCt experimental group - ΔCt control group, ΔCt = CT target gene - CT β-actin, and the RQ value is the relative expression level of the target gene mRNA.

[0109] c. Data Analysis: Data was processed using GraphPad software, and statistical analysis was performed using one-way ANOVA (and non-parametric tests) and Turkey's method. Each experiment was repeated at least three times. P <0.05 is statistically significant and is marked with "*".

[0110] The results of the effect of the test substance on the regulation of HaCaT cell barrier genes are as follows: Figure 4 As shown in the figure. The control group is denoted as BC.

[0111] Table 5. Effects of PDRN-containing biopharmaceuticals based on Buchuria p48 on HaCaT cell viability

[0112] From Table 5 and Figure 4 The results showed that, at the detection concentration, the test substance S1 (Example 5 - a biological agent containing PDRN from the stress-resistant strain Bucuna p48) could significantly enhance the expression of the barrier gene LOR, demonstrating a superior barrier repair effect.

[0113] <Test Example 7> This test evaluated the anti-inflammatory and soothing effects of a PDRN-containing biologic agent based on Buchuria p48 in vitro using RAW264.7 mouse macrophages. It verified the influence of different PDRN-containing samples on RAW264.7 cell viability and their inhibitory effect on LPS-induced secretion of the inflammatory cytokine NO by RAW264.7 cells. The specific steps are as follows: (1) Cells and culture medium: Cells: RAW264.7 mouse macrophages; Culture conditions: DMEM medium, 10% FBS fetal bovine serum; Test substances: Product S1 of Example 5 and Product S0 of Comparative Example 6.

[0114] (2) Effect of the test substance on the viability of RAW264.7 cells (CCK8 assay) a. Log-phase, well-morphologically healthy RAW264.7 cells were seeded into 96-well plates and incubated in an incubator for 24 hours; b. The test substance (product S1 of Example 5 and product S0 of Comparative Example 6) was diluted with complete culture medium to 200 μg / mL, with 3 replicates for each concentration, and co-incubated with cells for 24 h. c. Remove the original supernatant from each well, add CCK8 test solution to each well, incubate for 2 h, and then measure the absorbance at 450 nm using a microplate reader to calculate the cell viability. Cell viability ≥80% P ≥0.05 μL indicates no significant toxicity. Results are recorded in Table 6.

[0115] (3) Detection of the anti-inflammatory and soothing effects of the test substance a. Take RAW264.7 cells in the logarithmic growth phase and with good morphology, seed them in 24-well plates, and incubate them in an incubator for 24 h; b. Experimental grouping: A blank control group, an LPS-induced stimulation group, a positive control group (dexamethasone), and experimental groups for each test sample (product S1 of Example 5 and product S0 of Comparative Example 6) were set up, with corresponding replicates for each group; c. Each group was subjected to 37℃ and 5% After incubation in the incubator for 2 hours, except for the blank control group, LPS was added to each well to induce stimulation, and incubation continued for 24 hours. d. Collect cell supernatants from each group and strictly follow the instructions of the nitric oxide assay kit to detect the NO content in the supernatant; e. The experimental results were analyzed using data processing software, and the results are as follows: Figure 5 As shown in the figure. The blank control group is labeled NC, the LPS-induced stimulation group is labeled LPS, the positive control group (dexamethasone) is labeled DEX, and the products in Example 5 and Comparative Example 6, which are experimental groups, are labeled S1 and S0, respectively.

[0116] (4) Result judgment criteria a. Cell viability assessment: If the cell viability rate of the test substance treatment group is ≥80% and there is no significant difference compared with the blank control group, the test substance is determined to have no obvious toxicity to RAW264.7 cells and can be used for subsequent anti-inflammatory efficacy evaluation; if the viability rate is <80%, the test substance is determined to have cytotoxicity, and its anti-inflammatory efficacy evaluation needs to be combined with the toxicity effect. b. Determination of anti-inflammatory efficacy: Compared with the LPS-induced stimulation group, if the NO content in the supernatant of the test substance sample group is significantly reduced, the test substance is determined to have anti-inflammatory effect; the greater the reduction, the stronger the anti-inflammatory activity. If there is no significant reduction, the test substance is determined to have no obvious anti-inflammatory effect. c. Overall assessment: The test substance showed no significant toxicity to RAW264.7 cells and significantly inhibited LPS-induced NO secretion. Therefore, the test substance was deemed to have passed the test and possessed anti-inflammatory efficacy.

[0117] The significance of the positive control group (dexamethasone) and the experimental groups of each test substance sample compared with the LPS modeling group is indicated by "*". P <0.05 is statistically significant, indicating a certain inhibitory effect, and is marked with "*"; P <0.01 indicates significant inhibitory effect, marked as "**"; P<0.001 indicates extremely significant inhibitory effect, marked as "***". Furthermore, the significance of the LPS model group compared to the control group (NC) is represented by "#", with the specific number and meaning corresponding one-to-one with "*".

[0118] Table 6. Effects of the test substances on the viability of RAW264.7 cells

[0119] Depend on Figure 5 As shown in Table 6, under the experimental conditions of this test case, both sample S1 (Example 5 - biological agent containing PDRN from stress-resistant strain Bucuna p48) and S0 (Comparative Example 6 - from commercially available salmon) inhibited NO and had certain anti-inflammatory and soothing effects, with comparable efficacy.

[0120] The embodiments and application examples described above are merely illustrative descriptions of this disclosure and are not intended to limit the scope of this disclosure. Any modifications and improvements made by those skilled in the art to the technical solutions of this disclosure without departing from the spirit of this disclosure should fall within the protection scope defined by this disclosure.

Claims

1. A stress-resistant strain of *Buchuriae*, characterized in that, The *Buchuria* strain is classified and named as follows: Buttiauxella ferragutiae The B. tumefaciens strain BTN-HB-P48 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38093 and deposit date of April 9, 2026.

2. A fermentation product filtrate of a stress-resistant *Buchuriae* strain, characterized in that, Centrifuge the fermentation product of the *Buchuria* strain according to claim 1 in the strain's liquid culture medium, take the supernatant, and filter the supernatant through a 0.22 μm filter membrane to obtain the fermentation product filtrate.

3. The fermentation product filtrate according to claim 2, characterized in that, Prepared in the following manner: S1. Cultivate the *Buchuria* strain according to claim 1 in a culture medium to obtain a seed culture of the *Buchuria* strain; S2. The seed liquid is inoculated into the liquid culture medium of the strain at an inoculation amount of 1% to 5% and fermented to obtain the fermentation product. The fermentation temperature is 25℃ to 37℃ and the fermentation time is 24 h to 48 h. S3. Centrifuge the fermentation product, take the supernatant, and filter the supernatant through a 0.22 μm filter membrane to obtain the fermentation product filtrate.

4. A fermentation cell lysate of a stress-resistant Bacteroides strain, characterized in that, Prepared in the following manner: S1. Culture the *Buchuria* strain according to claim 1 in a culture medium to obtain a seed culture of the *Buchuria* strain; S2. The seed liquid is inoculated into the strain liquid culture medium at an inoculation amount of 1% to 5% and fermented to obtain the fermentation product. The fermentation temperature is 25℃ to 37℃ and the fermentation time is 24 h to 48 h. S3. Centrifuge the fermentation product, remove the supernatant, and collect the wet bacterial sludge; S4. Resuspend the wet bacterial sludge in ultrapure water to obtain a bacterial sludge resuspension; S5. The bacterial sludge resuspension is homogenized under high pressure until no intact bacterial cells remain, to obtain the fermentation cell lysate of the Buchuria strain.

5. A biological agent containing PDRN, characterized in that, Prepared in the following manner: S1. Take the fermented cell lysate according to claim 4, add enzyme at a dosage of 1.25 mg proteinase K per gram of wet bacterial mud, and enzymatically hydrolyze in a water bath at 50-60°C for 2-4 hours; S2. After enzymatic hydrolysis, the product is centrifuged to remove the precipitate and obtain a clear filtrate; S3. Precipitate the clarified filtrate with anhydrous ethanol, and collect the precipitate after precipitation; S4. The precipitate is washed and freeze-dried to obtain a biological agent containing PDRN.

6. A biological agent containing PDRN, characterized in that, Prepared in the following manner: S1. Take the fermented cell lysate according to claim 4, add enzyme at a dosage of 1.25 mg proteinase K per gram of wet bacterial mud, and enzymatically hydrolyze in a water bath at 50-60°C for 2-4 hours; S2. After enzymatic hydrolysis, the product is centrifuged to remove the precipitate and obtain a clear filtrate; S3. Filter the clarified filtrate using a 100 kDa ultrafiltration membrane, collect the retentate, and freeze-dry the retentate to obtain a biological agent containing PDRN.

7. The application of a fermentation product filtrate according to claim 2 or 3, or a fermentation cell lysate according to claim 4, or a biological agent according to claim 5 or 6 in the preparation of daily chemical products.

8. A daily chemical product, characterized in that, The daily chemical product comprises the fermentation product filtrate according to claim 2 or 3, or the fermentation cell lysate according to claim 4, or the biological agent according to claim 5 or 6.

9. The daily chemical product according to claim 8, characterized in that, The amount of the fermentation product filtrate according to claim 2 or 3 added to the daily chemical product is 0.09% to 3% by mass percentage.

10. The daily chemical product according to claim 8, characterized in that, The amount of the biological agent according to claim 5 or 6 added to the daily chemical product is 50 to 200 μg / mL.

Images