Lactobacillus paracasei LPa-6 and application thereof in atopic dermatitis

Abstract

The invention belongs to the technical field of lactobacillus paracasei, and particularly relates to a lactobacillus paracasei LPa-6 strain and application thereof in atopic dermatitis. The lactobacillus paracasei LPa-6 strain not only has the performance of targeted inhibition of Enterobacter cloacae, Streptococcus sobrinus and Aloscardovia omnicola, but also can be used for increasing the concentration of anti-inflammatory factors, regulating Th1 / Th2 imbalance, increasing the abundance of effective microbial communities in intestinal tracts, and effectively relieving and improving the symptoms of atopic dermatitis, and can be used for preventing and treating enterobacter cloacae, Streptococcus sobrinus and Aloscardovia omnicola, and preventing and treating enterobacter cloacae, Streptococcus sobrinus and Aloscardovia omnicola.

Description

Technical Field

[0001] This application belongs to the technical field of Lactobacillus paracasei, specifically relating to a strain of Lactobacillus paracasei LPa-6 and its application in atopic dermatitis. Background Technology

[0002] Lactobacillus paracasei, as a recognized safe probiotic, has a broad application base in food fermentation, intestinal health maintenance, and mucosal protection. Its acid- and bile-salt-resistant physiological characteristics enable it to adapt to the complex microecological environment of the human gut and oral cavity. However, existing Lactobacillus paracasei strains mainly focus on single-function development, possessing only basic antibacterial activity or only assisting in the regulation of microecological balance. They lack the ability to target specific conditionally pathogenic bacteria, making it difficult to achieve synergistic control effects such as inhibiting pathogenic bacterial proliferation and blocking pathogenic bacterial colonization.

[0003] Furthermore, *Lactobacillus paracasei* can restore the balance of the skin microbiome, reduce inflammation and immune responses, and improve skin barrier function. Therefore, it is crucial to develop a strain of *Lactobacillus paracasei* that can target and inhibit opportunistic pathogens, enhance immunity, regulate gut microbiota balance, and improve allergies. Summary of the Invention

[0004] This application provides a strain of *Lactobacillus paracasei* LPa-6, with accession number CGMCC NO.37032. This strain not only exhibits targeted inhibition of *Enterobacter cloacae*, *Streptococcus sobrinus*, and *Alloscardovia omnicolens*, but also increases the concentration of anti-inflammatory factors, regulates Th1 / Th2 imbalance, and enhances the abundance of beneficial intestinal flora, effectively alleviating and improving symptoms of atopic dermatitis.

[0005] This application provides an antibacterial preparation comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of *Lactobacillus paracasei* LPa-6 as described in accession number CGMCC NO.37032.

[0006] This application provides a targeted adhesion copolymerizing agent comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of *Lactobacillus paracasei* LPa-6 as described in accession number CGMCC NO.37032.

[0007] This application provides a dried body prepared by freeze-drying Lactobacillus paracasei LPa-6 as described in accession number CGMCC NO.37032. The dried body has a water content of ≤5% and a viable count retention rate of ≥85% after rehydration.

[0008] This application provides a composition with antibacterial and copolymerization functions, characterized in that it comprises at least one of the following: Lactobacillus paracasei LPa-6 as described in preservation number CGMCC NO.37032, the antibacterial agent, the targeted adhesion copolymerization agent, and the dried body.

[0009] This application provides for the application of *Lactobacillus paracasei* LPa-6 as described in accession number CGMCC NO.37032, the application including the preparation of fermentation preparations of *Lactobacillus paracasei* LPa-6; the preparation of antibacterial agents; the preparation of targeted adhesion copolymers; and the preparation of at least one of the following: Attached Figure Description

[0010] Figure 1 The colony morphology of Lactobacillus paracasei LPa-6 on a plate is shown in the example.

[0011] Figure 2 Microscopic images of Lactobacillus paracasei LPa-6 provided for this example.

[0012] Figure 3 The inhibition zone diagram of Lactobacillus paracasei LPa-6 against rare pathogens provided for the test case.

[0013] Figure 4 A bar graph showing the secretion of interferon-gamma stimulated by L. paracasei LPa-6 in the test case.

[0014] Figure 5 A bar graph showing the effect of Lactobacillus paracasei LPa-6 on macrophage proliferation, provided as a test example.

[0015] Figure 6 Images of swelling of the right ear in each group of mice provided for the test cases.

[0016] Figure 7 A bar chart showing the thickness of the right ear of each group of mice provided for the test cases.

[0017] Figure 8 Physical images of skin lesions on the backs of mice in each group provided for the test cases.

[0018] Figure 9 Pathological sections of skin tissue from the backs of mice in each group were provided for the test cases.

[0019] Figure 10Toluidine blue staining images of mast cells in the dorsal skin lesions of mice in each group provided for the test cases.

[0020] Figure 11 A bar chart showing the results of mast cell count analysis in the dorsal skin lesions of mice in each group provided for the test cases.

[0021] Figure 12 A bar chart showing the results of serum IL-13 analysis in each group of mice provided for the test cases.

[0022] Figure 13 A bar chart showing the results of serum gamma-interferon analysis for each group of mice provided for the test cases.

[0023] Figure 14 A bar chart showing the species composition analysis results at the phylum level of the gut microbiota of each group of mice provided for the test cases.

[0024] Figure 15 A bar chart showing the results of the ratio analysis of Firmicutes and Bacteroidetes in the gut microbiota of each group of mice provided for the test cases.

[0025] In the above figure, different letters represent statistically significant differences between groups (p < 0.05). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0027] Current research mainly focuses on the inhibitory effect of Lactobacillus paracasei on Escherichia coli. This belongs to the classic mechanism by which probiotics inhibit the growth of pathogens by competing for living space and producing antibacterial substances (such as organic acids, hydrogen peroxide, and bacteriocins).

[0028] However, unlike its significant inhibition of *Escherichia coli*, *Lactobacillus paracasei* showed little inhibitory effect against other pathogenic *Enterobacter* species. For example, in in vitro antimicrobial assays against pathogens from different food sources (FrontVetSci.2022Dec15;9:1025677.doi:10.3389 / fvets.2022.1025677), *Lactobacillus paracasei* exhibited weak or absent antimicrobial activity against foodborne *Enterobacter cloacae* isolates. The study noted that compared to ATCC standard strains, foodborne pathogens (including *Enterobacter cloacae*) often exhibited smaller or absent inhibition zones (<6 mm).

[0029] Based on this, this application obtained a strain of *Lactobacillus paracasei* LPa-6 through screening. This strain, with accession number CGMCC NO.37032, not only exhibits significant antibacterial activity against *Enterobacter cloacae*, *Streptococcus distantly*, and *Scardovician glomerulosa*, but also targets and adheres to these three pathogenic bacteria, while simultaneously producing multiple active ingredient forms, including live bacteria, dead cells, inactivated cells, and metabiotics. This solves the technical problems of existing strains having single functions, limited formulation forms, and insufficient targeting. The specific screening and identification process of this strain is as follows:

[0030] (1) Isolation and screening of strains

[0031] The *Lactobacillus paracasei* LPa-6 provided in this application was isolated from infant feces. The collected samples were serially diluted with 0.85% physiological saline under aseptic conditions, and then plated on LBS agar plates at an appropriate gradient, incubated at 37°C for 48-72 hours. Colony morphology was observed visually, and suspected single colonies were picked for microscopic examination, followed by preliminary screening and purification. The purified strain was incubated in MRS broth at 37°C for 8-12 hours, centrifuged to remove the supernatant, and resuspended in sterile 25% glycerol aqueous solution, then stored at -80°C.

[0032] (2) Morphological characteristics and identification

[0033] like Figure 1 and Figure 2 As shown, *Lactobacillus paracasei* LPa-6 grew well on MRS solid plates. Single colonies were milky white, round, raised, medium-sized, with regular edges and smooth surfaces. Microscopic examination revealed that the bacteria were rod-shaped, arranged singly, in pairs, or in chains, consistent with the staining characteristics of Gram-positive bacteria.

[0034] The selected target strain was cultured in liquid medium, and bacterial cells were collected. Genomic DNA was extracted, and PCR amplification was performed using universal primers 27F / 1492R. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 35 cycles, followed by a final extension at 72℃ for 10 min. The content and purity of the PCR amplification products were then tested. After passing the tests, the products were sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. Based on the sequencing results, homology comparison was performed using the BLAST tool in the NCBI database, and the obtained strain was identified as *Lactobacillus paracasei*. This strain was named *Lactobacillus paracasei* LPa-6 and deposited for preservation. Its preservation information is as follows:

[0035] Accession number: CGMCC NO.37032

[0036] Classification and nomenclature: Lactobacillus paracasei LPa-6

[0037] Latin name: Lacticaseibacillus paracasei

[0038] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0039] Address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0040] Deposit date: December 11, 2025

[0041] The embodiment also provides an antibacterial preparation comprising at least one or more of the following: dead cells, live cells, and inactivated cells of *Lactobacillus paracasei* LPa-6 (accession number CGMCC NO.37032). The antibacterial effect against the target bacteria is achieved through different cell forms, adapting to different product processes and application requirements.

[0042] In some embodiments, the Lactobacillus paracasei LPa-6 strain according to this application may be an isolated bacterial strain or a pure culture colony.

[0043] In some of the formulations provided in the embodiments, the concentration of at least one or more of the dead bacterial cells, live bacterial cells, and inactivated bacterial cells is 10. 2 Up to 10 17 Within the range of colony-forming units per gram or per milliliter (CFU / g or CFU / mL), for example, in 10 5 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 6 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 7 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 8 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 9 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 10 -10 17 Within the range of CFU / g or CFU / mL, for example, in the range of 10 11 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 12 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 13 -10 16Within the range of CFU / g or CFU / mL, for example, in the range of 10 7 -10 16 Within the range of CFU / g or CFU / mL, for example, in the range of 10 8 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 9 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 10 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 11 -10 15 Within the range of CFU / g or CFU / mL, for example, in the range of 10 12 -10 15 Within the range of CFU / g or CFU / mL.

[0044] In the context of this application, *Lactobacillus paracasei* LPa-6, as defined herein, can be provided in the composition according to this application in the form of at least one or more of dead, live, and inactivated bacterial cells. Live bacterial cells refer to live *Lactobacillus paracasei* bacteria with intact cell structure, capable of normal metabolism and reproduction, for example, cultured in a culture medium (such as MRS medium), centrifuged, washed to retain viability, and typically preserved in lyophilized form (such as lyophilized bacterial powder). For example, live bacterial cells refer to the bacterial solid obtained by collecting the strain after fermentation in MRS medium by centrifugation and washing with physiological saline. Dead bacterial cells refer to bacterial cells that have died naturally or lost their activity through physical / chemical treatment (such as high temperature, ultraviolet light), and whose cell structure may be intact or partially destroyed. For example, dead bacterial cells are obtained by freeze-drying live bacterial cells, grinding and crushing them, and passing them through a 200-mesh sieve to obtain bacterial fragment powder (protein content ≥30%). Inactivated bacterial cells specifically refer to bacterial cells that have been killed by controlled methods (such as heat inactivation, formaldehyde treatment, high pressure treatment) but retain their cell surface structures (such as cell walls, capsules). Inactivated bacteria emphasize "structural preservation," while dead bacteria may suffer structural damage due to processing methods. For example, inactivated bacteria are produced by sterilizing live bacteria with moist heat at 60 °C for 30 min (or treating with 0.5% formaldehyde solution for 1 h) to ensure no live bacteria (live bacteria count <10 CFU / mL using plate counting method), thus preserving the bacterial structure and metabolites.

[0045] In some of the formulations provided, Lactobacillus paracasei LPa-6 may be used as an active ingredient in a mixture of at least one or more of dead, live and inactivated cells, with the active ingredient comprising 0.0001% (w / w) to 99% (w / w) of the total mass of the formulation.

[0046] In some embodiments, the preparation process of live Lactobacillus paracasei LPa-6 cells includes:

[0047] Lactobacillus paracasei LPa-6 strain was inoculated into seed culture medium and anaerobically cultured at 37°C for 18 h to obtain seed culture (viable count ≥1×10⁻⁶). 9 The seed culture medium consisted of: 5.0 g / L yeast extract, 10.0 g / L tryptone, 6.0 g / L potassium dihydrogen phosphate, 0.034 g / L ferrous sulfate, 0.575 g / L magnesium sulfate, 20.0 g / L glucose, 25.0 g / L sodium acetate, 2.0 g / L ammonium citrate, and 0.12 g / L manganese sulfate. The pH was adjusted to 5.5, and then 1.0 mL / L Tween-80 and 1.3 mL / L glacial acetic acid were added. The mixture was heated and stirred to dissolve in 1000 mL of distilled water, and then autoclaved at 121°C for 15 min. The solid culture medium contained 15.0 g / L agar.

[0048] Inoculate the seed culture at a 5% inoculation rate into the fermentation medium and anaerobic ferment at 37℃ for 24 hours. The viable cell count in the fermentation broth should be ≥1×10¹. 0 CFU / mL; the fermentation medium contained: glucose 20.0 g / L, yeast peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, anhydrous sodium acetate 5.0 g / L, Tween-80 1.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium sulfate heptahydrate 0.58 g / L, and manganese sulfate monohydrate 0.19 g / L, dissolved in 1000 mL of distilled water, adjusted to pH 6.8, and autoclaved at 115 °C for 20 min.

[0049] The fermentation broth was centrifuged at 8000 r / min for 3 min, the bacterial precipitate was collected, and washed twice with sterile physiological saline to obtain a live bacterial concentrate (live count ≥1×10¹¹ CFU / g).

[0050] In some embodiments, the preparation process of dead Lactobacillus paracasei LPa-6 cells includes:

[0051] Take the above-mentioned live bacterial concentrate and sterilize it by moist heat (121℃, 0.1MPa for 20 min) or dry heat (160℃ for 2 h). If no live bacteria are detected after sterilization (plate count method), it is considered dead bacterial.

[0052] In some embodiments, the preparation process of inactivated Lactobacillus paracasei LPa-6 cells includes:

[0053] Take the live bacterial cell concentrate and inactivate it using low-temperature plasma inactivation (100W power, 5min) or ultraviolet inactivation (254nm, 30min). After inactivation, ensure that there are no live bacteria and retain the adhesion sites and antibacterial components on the bacterial surface.

[0054] In some embodiments, the preparation process of Lactobacillus paracasei LPa-6 mixed bacterial cells includes: mixing live cells, dead cells, and inactivated cells in any mass ratio, such as live cells:dead cells = 1:1, live cells:inactivated cells = 2:1, live cells:dead cells:inactivated cells = 1:1:1, etc.

[0055] In some embodiments, the antibacterial preparation further includes excipients such as stabilizers, carriers, and dispersants. The stabilizers are selected from trehalose, skim milk powder, or xanthan gum; the carriers are selected from maltodextrin, lactose, or corn starch; and the dispersants are selected from polyethylene glycol or Tween 80.

[0056] In some embodiments, the antibacterial preparation is a powder, tablet, ointment, emulsion, oil, suspension, lotion, gel, paste, foam, dairy product, gel, mist, or spray.

[0057] In some embodiments, the antibacterial preparation is a powder containing live *Lactobacillus paracasei* LPa-6 cells. Specifically, it contains 1×10¹¹ CFU / g of *Lactobacillus paracasei* LPa-6 live cell concentrate, maltodextrin, and trehalose. For example, the antibacterial preparation contains 10g of 1×10¹¹ CFU / g *Lactobacillus paracasei* LPa-6 live cell concentrate, 80g of maltodextrin, and 10g of trehalose. The live cell concentrate of *Lactobacillus paracasei* LPa-6 can be mixed evenly with maltodextrin and trehalose, and then aseptically packaged (1g / bag) to obtain the powdered antibacterial preparation.

[0058] In some embodiments, the antibacterial agent is a liquid formulation containing dead *Lactobacillus paracasei* LPa-6 cells. Specifically, it contains 1 × 10¹¹ CFU / g of dead *Lactobacillus paracasei* LPa-6 cells, xanthan gum, and sterile physiological saline. For example, the antibacterial agent contains 5 g of 1 × 10¹¹ CFU / g dead *Lactobacillus paracasei* LPa-6 cells, 0.5 g of xanthan gum, and 94.5 mL of sterile physiological saline. The dead cells and xanthan gum can be added to sterile physiological saline, stirred to dissolve, and then homogenized at 10,000 r / min for 10 min to obtain the liquid antibacterial agent.

[0059] In some embodiments, the antibacterial agent is a granule containing live and inactivated Lactobacillus paracasei LPa-6. Specifically, it contains 5g of not less than 5 × 10⁻⁶ cells. 8 CFU / g of live Lactobacillus paracasei LPa-6 cells, 5g 5×10 8 Inactivated Lactobacillus paracasei LPa-6 cells (CFU / g), 70g lactose, and 20g corn starch. 5g of at least 5×10⁻⁶ CFU / g of these can be added. 8 CFU / g of live Lactobacillus paracasei LPa-6 cells, 5g 5×10 8Inactivated Lactobacillus paracasei LPa-6 cells (CFU / g), 70g lactose, and 20g corn starch were mixed, and 5% povidone K30 aqueous solution was added as a binder. The mixture was granulated, dried at 60 ℃ for 1 hour, and then packaged to obtain granular antibacterial preparations.

[0060] These antibacterial agents inhibit the proliferation of Enterobacter cloacae, Streptococcus distantis, and Skadoviridae through the antibacterial activity of live, dead, or inactivated bacteria.

[0061] Live bacteria can colonize the intestines or oral cavity, continuously proliferate, and produce antibacterial substances (such as lactic acid and bacteriocins), achieving long-lasting antibacterial effects. Dead bacteria retain their cell wall components (such as peptidoglycan) and residual antibacterial substances, exhibiting antibacterial activity without colonization, high stability, and tolerance to high temperatures, acids, and alkalis. Inactivated bacteria maintain structural integrity, possessing both the stability of dead bacteria and the ability to assist in blocking target bacterial colonization through surface adhesion sites, thus combining antibacterial and copolymerization-aiding functions. Mixed bacteria can synergistically leverage the advantages of different bacterial forms, such as the long-lasting antibacterial effect of live bacteria and the immediate antibacterial effect of dead bacteria, enhancing the overall antibacterial efficacy of the formulation.

[0062] The embodiments also provide an antibacterial preparation comprising a metabiotic and / or synbiotic of Lactobacillus paracasei LPa-6.

[0063] In this article, "metapiogenics" refers to the collective term for physiologically active bacterial components and metabolites produced by probiotics (such as *Lactobacillus paracasei* in this article) after specific processing. Its core characteristic is that it can exert its functions without relying on the live state of the bacteria. Specifically, "metapiogenics" can include at least one of the following: inactivated bacterial cells, solutions containing inactivated bacterial cells, fermentation broth containing inactivated bacterial cells, or inactivated fermentation solutions; or the "beneficial legacy" left after the death of probiotics or their metabolites.

[0064] In some embodiments, the formulation may be provided in solid, liquid, viscous, emulsion, or dry form. The formulations provided in some examples are preferably formulated as pastes, soft gelatin capsules, hard gelatin capsules, powders, talc, granules, beads, lozenges, effervescent tablets, lozenges, chewable tablets, sublingual tablets, oils, liquids, solutions, tinctures, emulsions, fruit juices, concentrates, syrups, sprays, mists, drinking ampoules, gels, gums, tablets, or coated pills.

[0065] Some of the embodiments provide formulations that are powders, tablets, ointments, emulsions, oils, suspensions, lotions, gels, pastes, foams, dairy products, gels, mists, sprays, or fermented preparations.

[0066] In some embodiments, the metabiotic is a solution obtained by passing the fermentation broth of Lactobacillus paracasei LPa-6 through a water bath at 70 °C for 30 min.

[0067] In some embodiments, the antibacterial preparation comprises a metabiotic of *Lactobacillus paracasei* LPa-6 and excipients. The excipients include a diluent, a preservative, and a thickener. The diluent is sterile saline or maltodextrin. The preservative is sodium benzoate or potassium sorbate. The thickener is sodium carboxymethyl cellulose or gelatin.

[0068] In some embodiments, the antibacterial agent is a liquid formulation containing Lpa-6 postbiotic from *Lactobacillus paracasei*. For example, the *Lactobacillus paracasei* fermentation broth is prepared to a viable count of not less than 1 × 10⁻⁶. 10 CFU / mL, incubate in a 70 ℃ water bath for 30 min, take 100 mL of the supernatant, add 0.1 g of potassium sorbate, stir to dissolve, sterilize, and dispense into 10 mL / bottles to obtain a liquid antibacterial preparation.

[0069] In some embodiments, the antibacterial agent is a powder containing Lpa-6 postbiotic from *Lactobacillus paracasei*. For example, the *Lactobacillus paracasei* fermentation broth is prepared to a viable count of not less than 1 × 10⁻⁶. 10 After being heated to CFU / mL in a water bath at 70℃ for 30 minutes, the powder was freeze-dried. 1g of the powder was mixed evenly with 99g of maltodextrin and aseptically packaged (0.5g / bag) to obtain a powdered antibacterial preparation (postbiotic content 10μg / bag).

[0070] In some embodiments, the antibacterial agent is a gel formulation containing L. paracasei LPa-6 postbiotic. For example, the L. paracasei fermentation broth is prepared to a viable count of not less than 1 × 10⁻⁶. 10 After mixing CFU / mL with 20 mL of the solution in a 70 °C water bath for 30 min, and then homogenizing it with 2 g sodium carboxymethyl cellulose, 5 g glycerol, and 73 mL sterile water, the mixture was sterilized at 60 °C for 30 min and then cooled to obtain the gel antibacterial preparation.

[0071] These antibacterial agents utilize active ingredients in the metabiotics (e.g., bacteriocins can disrupt the cell membrane of target bacteria, and lactic acid can lower the environmental pH and inhibit the proliferation of target bacteria) to specifically bind to receptors on the cell membrane of target bacteria, forming pores that cause the leakage of intracellular substances and death of target bacteria; they also lower the pH of the application environment, disrupting the acid-base balance of target bacteria and inhibiting their metabolism and proliferation; and they compete with target bacteria for adhesion sites, indirectly assisting in antibacterial action.

[0072] More specifically, the antibacterial agent is selected from at least one of the following: antibacterial agents of Enterobacter cloacae, antibacterial agents of Streptococcus sobrinus, and antibacterial agents of Alloscardoviaomnicolens.

[0073] In some test cases, Lactobacillus paracasei LPa-6 fermentation broth was used as the test bacteria and Oxford cup tests were conducted with Enterobacter cloacae, Streptococcus distantis, and Skadoviridae fasciculata.

[0074] Enterobacter cloacae (CCTCC AB 2010162), Streptococcus sobrinus (ATCC 33478), and Alloscardoviaomnicolens (BNCC 382766) were inoculated into BHI liquid medium for activation and cultured at 37°C for 24 h. Bacterial colonies were then picked and dissolved in physiological saline to prepare a bacterial suspension, and the bacterial concentration was adjusted to 10⁻⁶. 8 CFU / mL. The fermentation broth of *Lactobacillus paracasei* LPa-6 was prepared using the method described above.

[0075] BHI medium containing 1.5% agar was cooled to approximately 55°C and then mixed thoroughly with suspensions of Enterobacter cloacae, Streptococcus distantly, and Scardovella fasciculata, ensuring that the viable counts of these bacteria were 10⁻⁶. 6 CFU / mL, then quickly pour into plates pre-placed with Oxford cups. After the culture medium cools and solidifies, remove the Oxford cups and inject 200 μL of viable bacteria (10⁶ CFU / mL) into each well. 8 The diameter of the inhibition zone was measured after fermentation broth of Lactobacillus paracasei LPa-6 at CFU / mL was incubated overnight at 37 °C for 24 h.

[0076] Depend on Figure 3 It can be seen that the inhibition zones of L. paracasei LPa-6 fermentation broth against Enterobacter cloacae, Streptococcus distantly, and Skadoviridae fasciculata were 12.50±0.50 mm, 12.17±0.76 mm, and 18.50±1.32 mm, respectively.

[0077] These test cases demonstrate that the *Lactobacillus paracasei* LPa-6 provided in this application can precisely inhibit specific target bacteria, adapting to the needs of target bacteria infection control in different scenarios. *Lactobacillus paracasei* LPa-6 avoids the impact of broad-spectrum antibacterial agents on beneficial bacteria; for example, oral preparations target only distant streptococci without disrupting the normal oral flora. Furthermore, *Lactobacillus paracasei* LPa-6 also solves the problem of existing antibacterial agents being "broad-spectrum but lacking specificity," improving antibacterial efficiency.

[0078] The embodiments also provide a targeted adhesion copolymerization agent, which comprises at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of *Lactobacillus paracasei* LPa-6. This agent blocks the colonization of the target bacteria on mucosal surfaces (such as the intestines and oral cavity) through targeted adhesion copolymerization between the bacterial strain and the target bacteria, achieving dual control by inhibiting proliferation and blocking colonization through synergistic antibacterial function.

[0079] In some embodiments, the targeted adhesion copolymerizing agent is a powder containing a live cell concentrate of *Lactobacillus paracasei* LPa-6. For example, the targeted adhesion copolymerizing agent contains 10g of a live cell concentrate of *Lactobacillus paracasei* LPa-6 at a concentration of 1×10¹¹ CFU / g, 0.3g of chitosan, 5g of galactooligosaccharides, and 84.7g of maltodextrin. After the components are mixed evenly, they are aseptically packaged (1g / bag) with a live cell count ≥1×10¹¹. 9 CFU / g.

[0080] In some embodiments, the targeted adhesion copolymerizing agent is a gel formulation containing inactivated Lactobacillus paracasei LPa-6 cells. For example, it contains 5g of inactivated Lactobacillus paracasei LPa-6 cells, 0.5g of sodium alginate, 8g of xylitol, 1g of sodium carboxymethyl cellulose, and 85.5 mL of sterile water. After mixing and homogenizing, the mixture is sterilized at 60 °C for 30 min and then cooled to obtain the gel.

[0081] In some embodiments, the targeted adhesion copolymerizing agent is a spray formulation containing live and inactivated *Lactobacillus paracasei* LPa-6 cells. For example, it comprises a 6 g mixture of live and inactivated cells (total concentration 1 × 10¹¹ CFU / g), 0.2 g chitosan, 5 g glycerol, and 88.8 mL sterile water. After stirring to dissolve, the mixture is filled into a spray bottle, with each 0.1 mL spray containing 6 × 10¹¹ CFU / g of the active ingredient. 8 CFU.

[0082] In some embodiments, the targeted adhesion copolymerizing agent is a targeted adhesion copolymerization of at least one of Enterobacter cloacae, Streptococcus sobrinus, and Alloscardoviaomnicolens.

[0083] This targeted adhesion copolymerizing microbial agent blocks the colonization of Enterobacter cloacae, Streptococcus distantis, and Skadoviridae maxima on the mucosal surface through targeted adhesion copolymerization. Simultaneously, it synergistically enhances the antibacterial activity of the bacteria, achieving dual control through inhibition and blocking. This targeted adhesion copolymerizing microbial agent overcomes the technical shortcomings of single antibacterial agents that "only inhibit proliferation but cannot block colonization," resulting in a more durable control effect. Furthermore, its strong targeting—copolymerizing only with the three target bacteria without affecting the colonization of beneficial bacteria—and its formulation form suitable for mucosal applications (such as oral gels and mucosal sprays) allow for the formation of localized high concentrations at the target bacteria colonization sites, enhancing the copolymerization effect.

[0084] In some test cases, *Lactobacillus paracasei* LPa-6 or its cells were copolymerized with *Enterobacter cloacae*, *Streptococcus distantis*, and *Scardovician glomerulosa*, respectively.

[0085] The fermentation broth of Lactobacillus paracasei LPa-6 was prepared according to the above method. After centrifugation at 8000 r / min for 3 min, the bacterial precipitate was collected and washed twice with sterile physiological saline to obtain a live bacterial concentrate (live count ≥1×10¹¹ CFU / g).

[0086] The preparation process of live Enterobacter cloacae includes: Enterobacter cloacae is inoculated into BHI liquid medium for activation, cultured at 37℃ for 24 h, centrifuged at 8000 r / min for 3 min, the bacterial pellet is collected, and washed twice with sterile physiological saline to obtain live bacterial concentrate (live count ≥1×10¹¹ CFU / g).

[0087] The preparation process of viable Streptococcus distantly related cells includes: activation of Streptococcus distantly related cells by inoculation in BHI liquid medium, incubation at 37℃ for 24 h, centrifugation at 8000 r / min for 3 min, collection of cell pellet, washing twice with sterile physiological saline to obtain viable cell concentrate (viable cell count ≥ 1×10¹¹ CFU / g).

[0088] Preparation process of live cells of *Scardoviciana guangxi*: *Scardoviciana guangxi* was inoculated into BHI liquid medium for activation, cultured at 37℃ for 24 h, centrifuged at 8000 r / min for 3 min, the bacterial pellet was collected, and washed twice with sterile physiological saline to obtain live cell concentrate (live count ≥ 1 × 10¹¹ CFU / g).

[0089] Lactobacillus paracasei LPa-6 cells, live Enterobacter cloacae cells, live Streptococcus distantly related cells, and live Scaradoves fasciculata cells were resuspended in PBS to obtain solutions containing Lactobacillus paracasei LPa-6 with an OD600 value of 0.6, and solutions containing live Enterobacter cloacae cells, live Streptococcus distantly related cells, and live Scaradoves fasciculata cells with an OD600 value of 0.4. The Lactobacillus paracasei LPa-6 solution was mixed with equal volumes of the three pathogenic bacteria solutions and incubated at 37 °C. Samples were taken at 2 h, 5 h, 21 h, and 24 h of incubation to measure the OD600 value and calculate the cohesive force between LPa-6 and the pathogens. Cohesive strength (%) = [(Ax+Ay) / 2-A(x+y)] / [Ax+Ay / 2]×100%, where x and y represent the initial OD600 values ​​before mixing, and (x+y) represents the OD600 value of the mixture.

[0090] The results showed that the coagulation power of live Lactobacillus paracasei LPa-6 against the three rare pathogens was 33.73%, 37.07%, and 35.24%, respectively. This indicates that LPa-6 has a good inhibitory effect on the growth of the three rare pathogens and removes the pathogens through coagulation, which is beneficial to exerting a probiotic effect and promoting the body's health.

[0091] Atopic dermatitis (AD) is an inflammatory disease closely related to gut microbiota imbalance. Studies have confirmed that the ratio of Firmicutes to Bacteroidetes in the gut microbiota of AD model animals is significantly abnormal (usually manifested as decreased Firmicutes abundance and increased Bacteroidetes abundance). This microbiota imbalance further exacerbates intestinal mucosal barrier damage and induces systemic inflammatory responses. Therefore, regulating the composition of Firmicutes and Bacteroidetes in the gut and restoring microbiota balance has become a key direction for improving AD-related gut microbiota dysbiosis.

[0092] Lactobacillus paracasei, as a safe-grade probiotic, possesses acid resistance, bile salt resistance, and intestinal colonization capabilities. However, existing related formulations mostly focus on single antibacterial or broad-spectrum microbial regulation functions, lacking targeted regulatory effects on the specific microbial combination of Firmicutes and Bacteroidetes in the intestines of AD mice.

[0093] Based on the above-mentioned technical deficiencies, the embodiment also provides a preparation that alters the composition of Firmicutes and Bacteroidetes in the intestine of atopic dermatitis mice, comprising at least one or more of the following: dead cells, live cells, inactivated cells, postbiotics, and fermentation broth of Lactobacillus paracasei LPa-6.

[0094] The formulations provided in these embodiments that alter the composition of Firmicutes and Bacteroidetes in the intestines of mice with atopic dermatitis can use at least one or more of the following as active ingredients: dead, live, inactivated, or metabiotic cells of Lactobacillus paracasei LPa-6, and fermentation broth. Specifically, the formulation method is the same as that used in the above-mentioned antibacterial agents or targeted adhesion copolymers. The excipients are also formulated in the same way as those used in the above-mentioned antibacterial agents or targeted adhesion copolymers. The feasible forms or formulations of these formulations that alter the composition of Firmicutes and Bacteroidetes in the intestines of mice with atopic dermatitis are also the same as those used in the above-mentioned antibacterial agents or targeted adhesion copolymers, and will not be described in detail here.

[0095] In some test cases, the following tests were conducted:

[0096] 1. Lactobacillus paracasei LPa-6 stimulates the secretion of gamma-interferon.

[0097] Mice were euthanized, and their spleens were harvested. The spleen tissue was mechanically crushed to create free spleen cells, which were then preserved in isotonic saline to form a spleen cell suspension. The spleen cells were then subjected to a 4 × 10⁻⁶ ppm... 6 Add cells / mL to a 96-well plate, then add either Lactobacillus rhamnosus LGG culture or the LPa-6 culture of this application at a concentration of 4 × 10⁻⁶ cells / mL. 7 CFU / mL was added to a 96-well plate. An equal volume of culture medium was used as a blank control (CK), and 4 μg / mL concanavalin A (ConA) was used as a positive control. The treated spleen cells were cultured for 48 h, centrifuged, and the supernatant was collected. The concentration of IFN-γ in the supernatant was determined according to the ELISA kit manufacturer's instructions.

[0098] The concentrations of IFN-γ in different treatment groups are as follows: Figure 4 As shown in the figure, compared with the negative control CK, strain LPa-6 can promote the secretion of IFN-γ, and the concentration of IFN-γ in the LPa-6 group is higher than that in the control strain LGG group. This indicates that Lactobacillus paracasei LPa-6 can stimulate the secretion of IFN-γ to a certain extent, promote the Th1 immune response, regulate the Th1 / Th2 balance, and has the potential to improve allergies.

[0099] 2. Gastrointestinal tolerance test of LPa-6

[0100] (1) Preparation of artificial gastric juice and bovine bile powder: Prepare 0.5% physiological saline, adjust the pH to 3 with dilute hydrochloric acid (HCl), then add pepsin with a final concentration of 0.3%, dissolve it completely, filter it with a 0.22 μm microporous membrane for sterilization, and set it aside; add 0.2% (w / v) sodium thioglycolate to MRS medium, then add bovine bile powder with a final concentration of 0.3%, filter it for sterilization, and set it aside for use;

[0101] (2) Experimental procedure: Take 1 mL of the cultured third-generation bacterial solution, centrifuge, discard the supernatant, add 1 mL of artificial gastric juice, mix well, and incubate at 37 ℃. Perform gastric juice tolerance tests for 0 h and 3 h respectively. Take 1 mL of the cultured third-generation bacterial solution, centrifuge, discard the supernatant, add 1 mL of artificial bile, mix well, and incubate at 37 ℃. Perform tolerance tests for 0 h and 3 h respectively. The tolerance rate of the strain (%) = Nt / N0 × 100%, where N0 represents the number of viable bacteria (CFU / mL) of the strain at 0 h, and Nt represents the number of viable bacteria (CFU / mL) of the strain at 3 h.

[0102] The tolerance results of *Lactobacillus paracasei* LPa-6 in gastric juice and bile salts are shown in Table 1. As shown in Table 1, the tolerance rates of this strain after 3 h of treatment in gastric juice and bile acids were 97.38 ± 8.75% and 68.23 ± 5.04%, respectively, indicating that this strain has the potential to successfully enter the human gastrointestinal tract.

[0103] Table 1. Tolerance of Lactobacillus paracasei LPa-6 to gastric juice and bile salts.

[0104]

[0105] 3. Determination of the hydrophobicity of the cell surface of Lactobacillus paracasei LPa-6

[0106] Centrifuge the activated LPa-6 bacterial suspension at 10000g for 1 min. Wash the bacterial cells twice with PBS solution (pH 7.4) and resuspend them in PBS. Adjust the bacterial suspension concentration to 1×10⁻⁶. 8 The absorbance of the bacterial suspension at 600 nm was measured simultaneously using CFU / mL and recorded as A0. 12 mL of the bacterial suspension was mixed with 4 mL of xylene, vortexed at 25°C for 1 min, and allowed to stand for 10 min. The mixture was then vortexed again for 1 min, allowed to stand for 20 min to separate into layers, and the absorbance of the clear lower aqueous phase at 600 nm was measured and recorded as A1. PBS buffer was used as a blank control. Surface hydrophobicity was expressed as the percentage of organic solvent adhered to by the bacteria, and the results are shown in Table 2.

[0107] The statistical results in the table show that LPa-6 has a surface hydrophobicity of up to 85.85%, indicating that LPa-6 can interact with other microorganisms in the gut and host cells, effectively exerting its probiotic function.

[0108] Table 2. Hydrophobicity of the cell surface of Lactobacillus paracasei LPa-6

[0109]

[0110] 4. Effects of LPa-6 on macrophage proliferation

[0111] The concentration of RAW264.7 macrophages was adjusted to 4 × 10⁻⁶ cells. 5 Cells were seeded at 100 μL / well in 96-well plates and cultured for 24 h. The supernatant was then aspirated, and the cells were washed twice with PBS. Two experimental groups were set up: a control group (100 μL of LDM-1 was added to each well) and a probiotic group (100 μL of bacterial suspension was added to each well, with a bacterial concentration:cell concentration ratio of 100:1). After cell culture, the supernatant was aspirated (and the cells were washed once with PBS), 10 μL of CCK-8 reagent was added, and the cells were incubated at 37 ℃ in the dark for 1 h. The OD value at 450 nm was measured using a microplate reader, and the cell proliferation rate of RAW264.7 macrophages was calculated.

[0112] Cell proliferation rate (CV) = OD1 / OD2 × 100%

[0113] Note: CV - cell proliferation rate (%); OD1 - OD value of each treatment group at 450 nm wavelength; OD2 - OD value of the blank group at 450 nm wavelength.

[0114] The results of the LPa-6 assay on the proliferation of RAW264.7 cells were analyzed. Figure 5 As shown, the LPa-6 strain had a significantly higher ability to promote macrophage proliferation than Lactobacillus rhamnosus LGG (p < 0.05), indicating that LPa-6 can activate macrophages and thus improve the body's immunity.

[0115] 5. Application of LPa-6 and its post-biotics in alleviating atopic dermatitis in mice

[0116] (1) Experimental animals

[0117] Female BALB / c mice (18-20 g) free of specific pathogens were purchased from Beijing Spefol Biotechnology Co., Ltd., and housed in the animal room of the Hubei Provincial Center for Food and Drug Safety Evaluation. The temperature was maintained at 22 ± 2 ℃, the humidity at 50 ± 5%, and the light-dark cycle was 12 hours. Free access to water and food was provided. All methods used in this animal experiment were reviewed and approved by the Ethics Committee of the Hubei Provincial Center for Disease Control and Prevention (Ethics Evaluation Center Animal (Fujian) No. 202510331).

[0118] (2) Grouping

[0119] Before the experiment, the mice were acclimatized for one week, and then randomly divided into a normal control group (NC), a dermatitis model group (MC), a ketotifen drug group (Keto, 0.01g of Keto was weighed and dissolved in 100 mL of physiological saline, and dissolved by sonication for later use), a live Lactobacillus paracasei group (LPa-6), and a post-biotic group (LPa-6_P).

[0120] Before modeling, the fur on the backs of mice was shaved off using a shaver. A base solution was prepared using acetone and olive oil in a 4:1 ratio. Then, 0.5% and 0.2% DNFB sensitization drugs were prepared using 2,4-dinitrofluorobenzene (DNFB) and the base solution. Modeling began in the second week. On the first day of modeling, 0.5% DNFB was applied to the back skin of mice to sensitize them. On days 5, 8, 11, and 14, 0.2% DNFB solution was applied to the back skin and right ear of mice to induce atopic dermatitis. The control group mice were treated with the same amount of base solution.

[0121] Throughout the experiment, mice in the NC and MC groups were administered 0.2 mL of sterile saline by gavage daily, while mice in the Keto group were administered the same amount of drug solution (1 mg / kg body weight) by gavage daily.

[0122] The LPa-6 group mice were administered 0.2 mL of the solution via gavage daily, and the number of live bacteria was 10. 9 LPa-6 live bacterial suspension (CFU / animal) (Preparation method: collect overnight cultured LPa-6 bacterial sludge, wash twice with sterile physiological saline, and then concentrate to the target concentration).

[0123] LPa-6_P group mice were administered 0.2 mL of 10 by gavage daily. 9 CFU / animal LPa-6 dead bacterial suspension (preparation method: incubate LPa-6 live bacterial suspension in a 70 ℃ water bath for 30 min to obtain inactivated bacterial cells), until the end of the experiment, for a total of 3 weeks.

[0124] During the experiment, the thickness of the mouse's right ear was measured using a digital micrometer. After the experiment, the mice were euthanized, and samples were collected for subsequent analysis.

[0125] (3) Mouse ear swelling test

[0126] Repeated topical application of DNFB induced clinical symptoms of atopic dermatitis in mice, and changes in ear thickness reflected the degree of ear swelling caused by local inflammatory infiltration. Figure 6 As shown, at the end of the experiment, the ear thickness of mice in different treatment groups was measured.

[0127] like Figure 7As shown, the ear thickness of mice in the MC group was significantly higher than that in the NC group (p < 0.01), indicating successful establishment of the atopic dermatitis mouse model. The ear thickness of mice in the Keto, LPa-6, and LPa-6_P groups was significantly lower than that in the MC group, and the ear thickness of mice in the LPa-6 and LPa-6_P groups was significantly lower than that in the Keto group. This demonstrates that intervention with live and inactivated LPa-6 strains provided in this application significantly improves ear thickness in atopic dermatitis mice compared to ketotifen.

[0128] (4) Evaluation of skin lesions and pathological symptoms on the back of mice

[0129] DNFB disrupts the skin barrier in mice, leading to significant skin lesions on the backs of the mice. After modeling, the skin lesions on the backs of the mice are as follows: Figure 8 As shown in the figure. Observation revealed that the MC group mice had typical dermatitis symptoms on their backs, accompanied by varying degrees of scratches and crusting scales, while the NC mice had smooth, rosy skin without scratches. The positive drug (Keto group) significantly alleviated the dermatitis symptoms on the backs of the mice, and they basically returned to normal. Compared with the model group, the pathological symptoms of the epidermal tissue on the backs of the mice in the LPa-6 and LPa-6_P groups were reduced.

[0130] Further staining of back skin samples from different treatment groups using hematoxylin-eosin staining was performed, such as... Figure 9 As shown in the figure, compared with the NC group, DNFB (MC group) induced skin inflammation, resulting in a significant increase in the thickness of the skin on the back of mice, accompanied by extensive infiltration of inflammatory cells, such as spongiosis and acanthosis. Similar to the alleviating effect of the positive control drug Keto, the intervention of live and dead LPa-6 bacteria significantly reduced the thickness of the skin on the back of mice, indicating that the ingestion of this strain significantly inhibited the immune inflammation at the skin lesions on the back of mice and had an alleviating effect on skin inflammation.

[0131] (5) Assessment of mast cell inflammatory infiltration in the skin of the back

[0132] Mast cells, mainly distributed in the skin and mucous membranes, are important effector cells in various allergic diseases, playing a crucial role, especially in atopic dermatitis. The various inflammatory mediators released after mast cell activation can further promote the development of skin lesions and the onset of pruritus symptoms in atopic dermatitis. Therefore, the effect of LPa-6 on mast cell inflammatory infiltration in the dorsal skin of mice with atopic dermatitis was evaluated.

[0133] Mast cell inflammatory infiltration in the dorsal skin of mice in different treatment groups is as follows: Figure 10 As shown in the figure. By toluidine blue staining, compared with the NC group, the MC group mice showed significant pathological symptoms of mast cell inflammatory infiltration in the skin lesions on their backs, and the number of mast cells was significantly increased.

[0134] Figure 11 The statistical results of the number of mast cells in the skin of mice in each group are shown. Figure 11 It was found that the number of mast cells in the skin of mice in the MC group was significantly higher than that in the NC group. Conversely, the number of mast cells in the skin of mice in the Keto, LPa-6, and LPa-6_P groups was significantly lower than that in the MC group. This indicates that the live and inactivated LPa-6 strain provided in this application, after intervention in mice with atopic dermatitis, can reduce the increase in the number of mast cells in the skin caused by this lesion, thereby improving and alleviating the clinical skin lesions of atopic dermatitis on the back of the mice.

[0135] (6) Detection of cellular inflammatory factors

[0136] After the experiment, orbital blood was collected from mice. The blood was allowed to stand at room temperature for 30 minutes, then centrifuged (3000 r / min, 10 min). The supernatant serum was collected, and the content of inflammatory factors in the serum of each group of mice was determined according to the detection method in the ELISA kit manufacturer's instructions.

[0137] The core immune mechanism of atopic dermatitis is an excessive Th2 immune response. Th2 immune cells release cytokines such as IL-4, IL-5, and IL-13, thereby disrupting the normal skin barrier and leading to severe skin lesions. Therefore, the concentration of the pro-inflammatory factor IL-13 in serum was measured. Figure 12 It can be seen that after local DNFB sensitization, the serum IL-13 level of mice in the model group increased significantly, while the serum IL-13 level of mice was significantly reduced after LPa-6 live bacteria intervention (p<0.05), and there was no significant difference in serum IL-13 level with the healthy group, indicating that LPa-6 live bacteria intake can inhibit Th2 immune response.

[0138] In inflammatory responses, regulatory T lymphocytes can play an important role in immunomodulation by producing cytokines, especially IFN-γ, which can suppress Th2 cells and effector cells in allergic inflammation. Figure 13 The statistical results of serum IFN-γ levels in each group of mice are shown.

[0139] like Figure 13As shown, the serum IFN-γ level in the MC group was significantly lower than that in the NC group, indicating that atopic dermatitis inhibits the Th1 immune response in mice, thereby reducing serum IFN-γ levels and exacerbating inflammatory symptoms. In contrast, the serum IFN-γ levels in the Keto, LPa-6, and LPa-6_P groups were significantly higher than those in the MC group, and the serum IFN-γ levels in the LPa-6 and LPa-6_P groups were significantly higher than those in the Keto group. This demonstrates that intervention with live and inactivated LPa-6 strains provided in this application in mice with atopic dermatitis has a better effect on promoting serum IFN-γ secretion than ketotifen, further indicating that both live and inactivated LPa-6 strains provided in this application help restore the balance of immune responses between Th1 and Th2, alleviating the pathological symptoms of atopic dermatitis.

[0140] (7) Changes in gut microbiota

[0141] After the experiment, the mice were euthanized and dissected, and their cecal contents were collected. The cecal contents were then sequenced and analyzed using 16S rRNA.

[0142] LPa-6 intake altered the composition of the gut microbiota in mice with atopic dermatitis. Figure 14 At the phylum level, the mouse gut microbiota is mainly composed of Firmicutes (Bacillota) and Bacteroidetes (Bacteroidota). The Bacillota / Bacteroidetes (B / B ratio) ratio has the potential to serve as a biomarker of gut microbiota in pathological conditions. Compared with the NC group, the MC group mice showed an increased relative abundance of Bacteroidetes and a decreased relative abundance of Firmicutes in their gut microbiota, thus resulting in a decreased B / B ratio. Compared with the decreased B / B ratio in the MC group, LPa-6 live bacteria intake significantly improved the B / B ratio. Figure 15 LPa-6 dead bacteria intake has no effect on the B / B ratio.

[0143] The above results indicate that ingestion of live Lactobacillus paracasei LPa-6 helps improve the intestinal flora imbalance induced by DNFB in mice and alleviates the clinical symptoms of atopic dermatitis.

[0144] This application develops a formulation capable of altering the Firmicutes and Bacteroidetes phyla composition of the gut in atopic dermatitis mice using single or combined forms of live, dead, and inactivated *Lactobacillus paracasei* cells, metabiotics, and fermentation broth. The formulation offers comprehensive coverage of active ingredients, adapting to varying needs in terms of stability, onset speed, and duration of action. Its flexible combination options, including single, binary, ternary, and multi-component combinations, cater to diverse gut microbiota imbalances ranging from simple to complex. Furthermore, the formulation demonstrates high precision in regulation, targeting the specific Firmicutes and Bacteroidetes phyla combination in the gut of AD mice for significant therapeutic effects.

[0145] In addition, the embodiments also provide a dried body prepared by freeze-drying the aforementioned *Lactobacillus paracasei* LPa-6. The dried body has a water content of ≤5% and a viable bacterial count retention rate of ≥85% after rehydration. The dried body can be used as an intermediate or final product, facilitating storage, transportation, and subsequent formulation preparation.

[0146] In some embodiments, the preparation process of the dried body includes:

[0147] The fermentation broth of *Lactobacillus paracasei* LPa-6 (anaerobic fermentation at 37℃ for 24 h, viable cell count ≥1×10¹) was used to ferment the bacteria. 0 Centrifuge at 8000 r / min for 15 min (CFU / mL), collect the bacterial pellet, wash twice with sterile physiological saline, and resuspend in a solution containing a protectant (bacterial concentration 1×10¹¹ CFU / mL); wherein the protectant is at least one of skim milk powder, trehalose, sucrose, and mannitol; for example, add 10%~15% skim milk powder as protectant, or 5%~10% trehalose as protectant, or 8%~12% sucrose as protectant, or a total addition of 15%~20% skim milk powder and trehalose in a 2:1 mass ratio as protectant, or a total addition of 12%~18% sucrose and mannitol in a 3:1 mass ratio as protectant;

[0148] Aliquot the resuspension into lyophilization bottles and pre-freeze at -40℃ to -60℃ for 2 to 4 hours to ensure that the sample is completely frozen.

[0149] Vacuum degree 10~30Pa, temperature -30℃~-20℃, dry for 8~12h to remove free water from the sample;

[0150] Vacuum degree 5~10Pa, temperature 20℃~30℃, dry for 2~4h to remove bound water from the sample;

[0151] After drying, seal and store with a moisture content of ≤5%. After rehydration, the viable bacteria retention rate is ≥85%.

[0152] The resulting dried product is a loose white powder that is easily rehydrated (completely dissolved within 30 seconds after adding sterile saline); after sealing, it can be stored at -20℃ to 4℃ with a shelf life of 6 to 12 months (longer shelf life when stored at -20℃); it can be directly packaged as freeze-dried bacterial powder (end product) or used as an intermediate product for the preparation of bacterial agents and compositions.

[0153] The dried form provided in these embodiments, as a stabilized form of the strain, retains the dual functions of antibacterial and targeted adhesion copolymerization, facilitating storage, transportation, and subsequent processing. It exhibits high stability; a moisture content of ≤5% significantly reduces the metabolic activity of the strain, extending shelf life (6 months at 4°C, with a viable count still ≥1×10⁻⁶). 9 (CFU / g) It is easy to process, the dried body is in powder form, and can be directly mixed with excipients to prepare various formulations such as powders, granules, and capsules. The processing technology is simple. The transportation cost is low. The dried body is small in volume and light in weight, and does not require cold chain transportation (it can withstand short-term room temperature transportation), thus reducing logistics costs.

[0154] Furthermore, these dried products solve the technical problems of short storage period and high transportation cost of live bacterial preparations, improving the market circulation of the products; the live bacterial count retention rate after rehydration is ≥85%, ensuring that the functional activity of the strains is not affected by the drying process; they can be used as intermediate or end products, flexibly adapting to different industrialization needs, such as being sold directly as freeze-dried bacterial powder, or used to prepare compound preparations.

[0155] Based on the above embodiments, the examples also provide examples of combining at least one or more of these Lactobacillus paracasei LPa-6, antibacterial agents, targeted adhesion copolymerizing agents, formulations that alter the composition of Firmicutes and Bacteroidetes in the intestines of atopic dermatitis mice, and dried products. Through the synergistic effect of different components, multiple enhanced synergistic effects of antibacterial activity, copolymerization, and alteration of the composition of Firmicutes and Bacteroidetes in the intestines are achieved, adapting to complex application scenarios.

[0156] Based on the above embodiments, the examples also provide applications of *Lactobacillus paracasei* LPa-6, including: preparing fermentation preparations of *Lactobacillus paracasei* LPa-6; preparing antibacterial preparations; preparing targeted adhesion copolymerizing agents; preparing preparations that alter the composition of Firmicutes and Bacteroidetes in the intestines of atopic dermatitis mice; and preparing at least one of the following: dried body.

[0157] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application.

Claims

1. A strain of Lacticaseibacillus paracasei LPa-6, which is Lacticaseibacillus paracasei LPa-6 with accession number CGMCC NO.37032.

2. An antibacterial agent comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of *Lactobacillus paracasei* LPa-6 as described in claim 1.

3. The antibacterial agent according to claim 2 is selected from at least one of the following: antibacterial agents of Enterobacter cloacae, antibacterial agents of Streptococcus sobrinus, and antibacterial agents of Alloscardovia omnicolens.

4. A targeted adhesion copolymerizing agent comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of *Lactobacillus paracasei* LPa-6 as described in claim 1.

5. The targeted adhesion copolymerization agent according to claim 4 is used for targeted adhesion copolymerization of at least one of Enterobacter cloacae, Streptococcus sobrinus, and Alloscardoviaomnicolens.

6. A formulation for altering the composition of Firmicutes and Bacteroidetes in the intestine of mice with atopic dermatitis, comprising at least one or more of the following: dead cells, live cells, inactivated cells, metabiotics, and fermentation broth of Lactobacillus paracasei LPa-6 as described in claim 1.

7. The targeted adhesion copolymerizing agent according to claim 6 increases the relative abundance ratio of Firmicutes to Bacteroidetes in the intestine of mice with atopic dermatitis.

8. A dried body, prepared by freeze-drying of Lactobacillus paracasei LPa-6 as described in claim 1, wherein the dried body has a water content of ≤5% and a viable count retention rate of ≥85% after rehydration.

9. A composition, characterized in that, It comprises at least one of the following: Lactobacillus paracasei LPa-6 as described in claim 1, the antibacterial agent as described in claims 2-3, the targeted adhesion copolymerizing agent as described in claims 4-5, the formulation as described in claims 6-7, and the dried body as described in claim 8.

10. The application of Lactobacillus paracasei LPa-6 according to claim 1, wherein the application includes: Preparation of a fermentation formulation of Lactobacillus paracasei LPa-6; Preparation of antibacterial agents; Preparation of targeted adhesion copolymerizing bacterial agents; Preparation of formulations that alter the composition of Firmicutes and Bacteroidetes in the intestines of mice with atopic dermatitis; and Prepare at least one of the dried bodies.

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