Phytobacterium plantarum BL-17 for relieving atopic dermatitis and application thereof

Abstract

The invention provides a composition of probiotics and prebiotics with a function of relieving atopic dermatitis and application of the composition. Specifically, the invention provides a composition containing different proportions of breast milk oligosaccharide and plant lactobacillus BL-17, and an application of the composition in relieving atopic dermatitis. The preservation number of the lactobacillus plantarum BL-17 is CCTCC (China Center for Type Culture Collection) NO: M20221596, and the preservation number of the lactobacillus plantarum BL-17 is CCTCC NO: M20221596. The breast milk oligosaccharide comprises a fucosyl type oligosaccharide and a neutral non-fucosyl type oligosaccharide; the step of relieving the atopic dermatitis of the infants refers to the step of remarkably improving clinical pathological characteristics, and comprises the following steps: (1) remarkably improving the skin swelling degree of mice; (2) the total IgE level in serum and the levels of related cell factors IL-10 and the like are obviously regulated; and (3) the biased imbalance of the Th1 / Th2 cells is significantly adjusted. The composition can be used as a good functional food preparation for relieving atopic dermatitis, especially atopic dermatitis caused by food allergy. The invention provides a preparation method and application of the composition.

Description

TECHNICAL FIELD

[0001] The present application relates to a probiotic and prebiotic composition for alleviating atopic dermatitis and its application, in particular, a composition comprising Lactiplantibacillus plantarum BL-17 and human milk oligosaccharides, and related preparation methods and applications of the resulting composition. The composition can be applied in health foods and health care foods, belonging to the field of functional food technology. BACKGROUND

[0002] In infants, atopic dermatitis often occurs in the prodromal stage of food allergy. Many children with food allergy will first develop eczema in the first few months or years after birth, especially when exposed to certain food allergens (such as milk, eggs, nuts, etc.). These skin symptoms usually appear in the early stages of food allergy reactions, suggesting that the immune system may have already begun to react to certain foods. In recent years, the incidence of AD has gradually increased, affecting 10% to 20% of children, and persisting throughout adulthood in 1% to 3% of cases. The incidence in the infant population has shown an increasing trend year by year, with clinical manifestations of dry and itchy skin, erythema exudation, subacute, and chronic scaly skin.

[0003] The pathogenesis of AD is heterogeneous and can be triggered by immune dysregulation, impaired epidermal barrier, and interactions between genetic and environmental factors. Its main features include recurrent episodes, severe itching, immune system activation, TH2 cell expansion, early keratinocyte production of thymic stromal lymphopoietin (TSLP), and chronic keratinocyte proliferation and apoptosis. Most atopic dermatitis patients (about 80%) develop an immunoglobulin E (IgE)-related form of dermatitis with high serum IgE titers, previously known as "extrinsic" type of human atopic dermatitis.

[0004] In the late 20th century, researchers discovered the existence of helper T cell Th1 and Th2 subpopulations in humans. Both Th1 and Th2 are derived from precursor CD4+ T cells. Various cytokines play an important role in the differentiation of Th1 / Th2. Related studies have shown that the peripheral blood cells of atopic dermatitis patients are mainly of the Th2 type, and excessive IL-4 and IL-5 can be formed after stimulation by allergens, while the content of IFN-γ secreted by Th1 decreases sharply.

[0005] The primary event in the development of allergic disease is the generation of allergen-specific CD4+ T cells. Upon activation by APCs, naive T cells differentiate into Th2-type cells under the influence of IL-4, while Th1-type cells and IFN-γ are suppressed. Effector Th2 cells produce IL-4, IL-5 and IL-13, which drive B cells to produce allergen-specific IgE, promote eosinophil recruitment, mucus production, smooth muscle contraction and Th2 cell tissue homing.

[0006] Treg has anti-inflammatory and autoimmune tolerance effects, while Th17 has strong pro-inflammatory effects, and the balance of Treg / Th17 plays an important role in maintaining the body's autoimmune homeostasis, and the imbalance of Treg / Th17 is a key factor in the pathogenesis of many inflammatory and autoimmune diseases. Treg can induce B cell activation to produce isotype IgG4 by secreting IL-10 and TGF-β, and inhibit the production of IgE. In addition, Treg directly inhibits the activation of allergen-specific Th2 cells, thereby maximally reducing the production of key cytokines IL-13, IL-4, and IL-5 in the allergic effector phase. Treg can also inhibit immune responses by directly inhibiting the activation of Th2 and Th17 cells and the activity of eosinophils and basophils, and even prevent mast cell degranulation through cell-to-cell contact.

[0007] The early life is a critical period for the development of allergy, and the immune system and gut microbiota of infants develop significantly. The first 1000 days after birth is considered to be the key period in which external factors (including nutrition) have the greatest impact on long-term immunity. Emerging research shows that HMOs found in breast milk may have potential therapeutic effects on infant allergy. HMOs are composed of glucose, galactose, fucose, sialic acid and N-acetylglucosamine, and together constitute the third most abundant component in breast milk. So far, more than 200 structurally different HMOs have been identified. Related studies have shown that the supplementation of prebiotics in early life can significantly reduce the incidence of allergic diseases in infants and young children within two years.

[0008] Based on the types of HMOs in breast milk, the synergistic effect between breast milk oligosaccharides is particularly important, especially in the regulation of intestinal microorganisms, immune enhancement, anti-inflammatory, anti-infection and neural development. The interaction between different oligosaccharides enables breast milk to provide more comprehensive and long-lasting health protection for infants. For example, sialylated oligosaccharides have anti-viral and anti-bacterial effects, and can inhibit the invasion of pathogenic bacteria by binding to their receptors. The combined use of 3'-SL and 6'-SL can more comprehensively enhance the immune defense of infants and reduce the incidence of infection.

[0009] The allergy protection properties of HMOs may reflect a variety of direct and indirect mechanisms, including modification of the gut microbiome, maturation of the epithelial barrier, regulation of immune regulatory and inflammation-related genes.

[0010] In recent years, probiotics as a natural microecological regulator have gained increasing attention in the application of alleviating food allergies and atopic dermatitis. Food allergies are often accompanied by an overreaction of the immune system. Probiotics can help reduce the overreaction to foreign substances by regulating the intestinal microbiota and improving the intestinal barrier function, thereby alleviating allergic symptoms. Some studies have found that specific probiotic strains can reduce allergy-related inflammatory factors, alleviate skin symptoms, and even improve skin barrier function. In the treatment of atopic dermatitis, the anti-inflammatory effect of probiotics is particularly important. They can help reduce skin inflammation caused by immune disorders by enhancing intestinal immune regulation.

[0011] Therefore, in the field of health foods and functional foods, HMOs may have potential for treating atopic dermatitis, but the alleviation of related symptoms achieved by single breast milk oligosaccharides is poor. Therefore, further research is needed to find a combination of HMOs and Lactiplantibacillus plantarum BL-17 that can significantly alleviate infantile atopic dermatitis and optimize the ratio, providing a technical basis for future functional foods. SUMMARY

[0012] An object of the present application is to provide a composition that can alleviate atopic dermatitis in infants and young children.

[0013] The present inventors found in their research that the combination of 2'-fucosyllactose, lacto-N-neotetraose, lacto-N-tetraose, lactodifucopentaose II, 3'-fucosyllactose and Lactiplantibacillus plantarum BL-17 is beneficial for improving atopic dermatitis-related symptoms, specifically by regulating the levels of total IgE and related cytokines TSLP in serum, improving the proportion of immune cells, etc.

[0014] Furthermore, the present application combines 2'-fucosyllactose, lacto-N-neotetraose, lacto-N-tetraose, lactodifucopentaose II, lactodifucopentaose III, 3'-fucosyllactose and Lactiplantibacillus plantarum BL-17 to provide a new composition that can alleviate atopic dermatitis in infants and young children.

[0015] The composition contains breast milk oligosaccharides 2'-FL 0-5370.2 mg / 10 g, 3-FL 1543.4-3333.4 mg / 10 g, LNnT, 1157.3-2500 mg / 10 g, LNT 1157.3-2500 / 10 g, LNFP-II 385.9-833.4 mg / 10 g, and LNFP-III 385.9-833.4 mg / 10 g.

[0016] Meanwhile, the application provides a preparation method of an active breast milk oligosaccharide preparation with the function of relieving atopic dermatitis, which comprises dissolving the breast milk oligosaccharides in a buffer solution to obtain the active breast milk oligosaccharide preparation.

[0017] Further, the buffer solution is a phosphate buffer solution.

[0018] In another aspect, the application also provides a food product comprising the composition of the application.

[0019] According to a specific embodiment of the application, in the composition of the application, the Lactiplantibacillus plantarum is Lactiplantibacillus plantarum BL-17 HN001 strain.

[0020] According to a specific embodiment of the application, in the composition of the application, the ratio of Lactiplantibacillus plantarum to breast milk oligosaccharides is 1 x 10 3 CFU: 0.1 g-10 g, preferably 1 x 10 12 CFU: 0.1 g-10 g, preferably 1 x 10 6 CFU: 0.1 g-10 g, preferably 1 x 10 10 CFU: 0.1 g-10 g, preferably 1 x 10

[0021] To solve the above technical problems, the application adopts the technical scheme of:

[0022] According to a specific embodiment of the application, the application provides a composition containing 2'-fucosyllactose, lacto-N-neotetraose, lacto-N-tetraose, lactodifucopentaose II, lactodifucopentaose III, 3'-fucosyllactose and Lactiplantibacillus plantarum BL-17, which helps to improve the health of infants and relieve the symptoms of atopic dermatitis.

[0023] The composition contains breast milk oligosaccharides 2'-FL 537 mg, 3-FL 154.3 mg, LNnT, 115.7 mg, LNT 115.7 mg, LNFP-II 38.6 mg, and LNFP-III 38.6 mg.

[0024] The above composition was dissolved in phosphate buffer solution of different dosages to obtain human milk oligosaccharide solutions of different concentrations (20 mg / mL, 100 mg / mL, and 200 mg / mL, respectively).

[0025] The *Lactobacillus plantarum* was cultured on MRS medium and incubated anaerobically at 37°C for 12–48 h. After obtaining the culture medium, the bacterial cells were obtained by centrifugation. The cells were washed 2–4 times with phosphate-buffered saline (pH 7.2) and resuspended with a cryoprotectant to a final concentration of 1 × 10⁻⁶. 8 CFU.

[0026] The present invention has the following beneficial effects:

[0027] (1) This invention investigated solutions containing *Lactobacillus plantarum* and different concentrations of human milk oligosaccharides. Animal experiments were conducted to verify the efficacy in alleviating food allergy symptoms based on clinical lesion severity, allergic symptoms, and immune response. Experimental studies revealed that, compared to the model group, the other intervention groups all alleviated atopic dermatitis symptoms in mice to some extent, reduced the impact of allergic reactions on mouse growth and development, and decreased antibody levels in the serum of allergic mice. Therefore, this invention demonstrates that the combination of human milk oligosaccharides and *Lactobacillus plantarum* has a significant alleviating effect on AD symptoms.

[0028] (2) This invention further explores the effect of solutions containing *Lactobacillus plantarum* and different concentrations of human milk oligosaccharide compositions on alleviating atopic dermatitis. The clinical pathological characteristics were further examined to investigate the alleviating effect of different concentrations of human milk oligosaccharide composition solutions combined with *Lactobacillus plantarum* on AD symptoms in mice. Attached Figure Description

[0029] Figure 1 The change in mouse ear thickness during modeling in Example 2;

[0030] Figure 2 The change in body weight of the mice in Example 2;

[0031] Figure 3 The concentration of TSLP in the mouse serum in Example 3;

[0032] Figure 4 The total IgE concentration in the mouse serum of Example 3;

[0033] Figure 5 The concentration of cytokines in the mouse serum of Example 3;

[0034] Figure 6 The concentration of cytokines in the mouse serum of Example 3;

[0035] Figure 7The concentration of mMCP-1 in mouse serum in Example 3;

[0036] Figure 8 The gene expression levels of mouse transcription factors Foxp3 and RORγt in Example 4;

[0037] Figure 9 The gene expression levels of mouse transcription factors T-bet and GATA-3 in Example 4; Detailed Implementation

[0038] The preservation information involved in this invention is as follows:

[0039] Preservation name: Lactobacillus plantarum BL-17;

[0040] Latin name Lactiplantibacillus plantarum BL-17;

[0041] Accession number: CCTCC NO: M 20221596;

[0042] Depository: China Center for Type Culture Collection;

[0043] Address of collection: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0044] Date of deposit: October 19, 2022.

[0045] The present invention will be further described below with reference to specific embodiments.

[0046] Example 1: A method for preparing a human milk oligosaccharide mixture formulation for relieving atopic dermatitis, comprising the following steps:

[0047] (1) Weigh 537 mg of 2'-fucosyl lactose, 154.3 mg of 3'-fucosyl lactose, 115.7 mg of lactose-N-neotetrasaccharide, 115.7 mg of lactose-N-tetrasaccharide, 38.6 mg of lactofucrose pentasaccharide II, and 38.6 mg of lactofucrose pentasaccharide III on a balance.

[0048] (2) Dissolve each of the human milk oligosaccharides in (1) above in 20 mL of phosphate buffer solution, mix well until each HMO is completely dissolved, and obtain a 50 mg / mL human milk oligosaccharide solution;

[0049] (3) Dissolve each of the human milk oligosaccharides in (1) above in 10 mL of phosphate buffer solution, mix well until each HMO is completely dissolved, and obtain a human milk oligosaccharide solution of 100 mg / mL.

[0050] (4) Dissolve each of the human milk oligosaccharides in (1) above in 5 mL of phosphate buffer solution, mix well until each HMO is completely dissolved, and obtain a human milk oligosaccharide solution of 200 mg / mL.

[0051] Example 2: Preparation of *Lactobacillus plantarum* bacterial culture, including the following steps:

[0052] (1) Take a small amount of bacterial culture from the preserved *Lactobacillus plantarum* strain (which can be cryopreserved, lyophilized, or sampled from an agar slant) and inoculate it into the prepared MRS liquid medium in a clean bench. The inoculation amount is generally 3%;

[0053] (2) After inoculation, the culture bottle is placed in a constant temperature incubator and cultured at 37°C for 24 hours. The turbidity of the culture medium is checked regularly to determine the growth of the bacteria.

[0054] (3) After the culture is completed, centrifuge the culture medium at 6000 rpm for 15 minutes to separate the bacterial cells. After centrifugation, discard the supernatant and retain the precipitate, which is the bacterial cell;

[0055] (4) Wash the bacterial cells 2-4 times with phosphate buffer at pH 7.2, then resuspend them with a protectant until the bacterial concentration is not less than 1×10⁻⁶. 8 CFU / ml was used to obtain a bacterial suspension.

[0056] Example 3:

[0057] Verification of the efficacy of combinations containing different concentrations of human milk oligosaccharide solutions and Lactiplantibacillus plantarum BL-17 in relieving atopic dermatitis:

[0058] Forty-eight SPF-grade female Balb / c juvenile mice were randomly divided into six groups of eight mice each: NC (blank control), MC (model), intervention group A (50 mg / mL human milk oligosaccharide + Lactiplantibacillus plantarum BL-17 culture), intervention group B (100 mg / mL human milk oligosaccharide + Lactiplantibacillus plantarum BL-17 culture), intervention group C (200 mg / mL human milk oligosaccharide + Lactiplantibacillus plantarum BL-17 culture), and PC (positive control). To confirm the synergistic effects among the various HMOs and between the HMO mixture and BL-17, the experiment also included a BL-17 group (BL-17 alone intervention group) and HMO mixtures of different concentrations alone intervention groups. AHMO B HMO C (Human milk oligosaccharide 50 mg / mL intervention group, human milk oligosaccharide 100 mg / mL intervention group, human milk oligosaccharide 200 mg / mL intervention group). HMO1, HMO2, and HMO3 (2'-fucosylvose + 3'-fucosylvose intervention group, lactose-N-neotetrasaccharide + lactose-N-tetrasaccharide intervention group, lactofucopentose II + lactofucopentose III intervention group) were kept in a natural light / dark cycle (12-hour light cycle) and provided with water and food at will. Mice were acclimatized for 1 week before the experiment.

[0059] In short, the experiment lasted for four weeks. After the adaptation period, on days 1, 4, 7, and 10, 200 μL of a 1.0% DNCB solution was applied to the backs of mice in both the model and treatment groups to sensitize and challenge their skin. Subsequently, on days 14, 17, 20, 23, 26, and 29, 20 μL of a 0.5% DNCB solution was applied to the backs of mice in both the model and treatment groups.

[0060] Mice were administered gavage from day 14 until the end of the experiment. All groups had free access to water and food.

[0061] After the final provocation, the dermatitis symptoms of the mice were scored.

[0062] Before the end of the experiment, the ear thickness of each group of mice was measured, and the results are as follows: Figure 1 As shown in the figure. Before modeling began, the weight of each mouse in each group was measured. During modeling and treatment, the mouse weight was measured every three days to observe weight changes. A weight change curve was plotted, and the results are shown in the figure. Figure 2 Observe the degree of skin damage in mice and score them according to the clinical symptoms of allergies in mice using the mouse allergy symptom scoring scale. After dissecting the mice, remove the thymus and spleen, clean the surrounding tissues, and weigh them using an electronic balance. The immune organ index is calculated using the following formula:

[0063]

[0064] The results obtained in this embodiment are analyzed as follows: Figure 1 , Figure 2 As shown:

[0065] Within 30 minutes after the final stimulation, the skin damage in mice was assessed. No allergic symptoms were observed in any mice in the control group; only a few mice in the positive control group showed mild damage, with 3 developing erythema; the model group showed more severe skin damage, with 3 exhibiting desquamation / dryness; the skin lesions in the remaining treatment groups were alleviated, with the HMO mixture and BL-17 combination group showing more significant relief compared to the HMO mixture and BL-17 group. The experiment demonstrates that the combined application of HMO mixture and Lactiplantibacillus plantarum BL-17 is more effective in alleviating skin lesions in mice with atopic dermatitis.

[0066] Changes in mouse body weight during modeling period, such as Figure 2 As shown: During the sensitization phase (0-14 days), the body weight of mice in the control group steadily increased, while the body weight of mice in the model group showed a decreasing trend. Mice in groups A, B, and C all showed some degree of body weight increase, although their growth rate was lower than that of the control group. During the challenge phase (14-29 days), the average body weight of mice in the model group continued to decrease. No significant decrease in body weight was observed in the daily gavage HMO mixture group, BL-17 group, and HMO mixture + BL-17 group, although their growth rate was lower than that of the control group. Among these, the body weight change trend of the HMO mixture + BL-17 group was closer to that of the control group than the other groups, indicating that both the HMO composition and BL-17 can alleviate the impact of allergic reactions on mouse growth and development to a certain extent. The combination of the two can minimize the impact of atopic dermatitis on mouse growth and development.

[0067] The thymus index and spleen index of mice are shown in Table 3. There were no significant differences in the thymus index among the blank group, positive control group, model group, and groups A, B, and C (P<0.05). Compared with the blank group, the spleen index of the model group mice was significantly increased, with the highest spleen index of 6.64. This may be due to the severe allergic reaction in the model group mice, which, as the largest lymphoid organ in the body, triggered an immune response, leading to a significant increase in the spleen index (P<0.05). Compared with the model group, the spleen index of mice in groups A, B, and C decreased by 19.9%, 18.0%, and 23.2%, respectively. This indicates that compositions containing different human milk oligosaccharides and compositions containing *Lactobacillus plantarum* BL-17 can effectively reduce the immune organ response caused by allergies. Furthermore, comparisons between the intervention groups also show that the combined application of human milk oligosaccharides is more effective than single application; the combination of HMO and BL-17 can exert a more significant effect in alleviating AD. Based on the results, the combined administration of 100 mg / mL HMO mixture and BL-17 showed better relief of allergic symptoms in mice with atopic dermatitis.

[0068] Table 3 Effects of different interventions on rat immune organ indices

[0069]

[0070] Example 4

[0071] Effects of different concentrations of human milk oligosaccharide solutions combined with Lactiplantibacillus plantarum BL-17 on immune regulation in mice:

[0072] Mice were euthanized by cervical dislocation, and tissues and blood were collected for the following biomarker detection. A portion of the blood sample was centrifuged at 2500×g for 10 min, and serum was collected. Another portion of the blood sample was added to a heparin-containing centrifuge tube, mixed well, and centrifuged at 2500×g for 10 min, and plasma was collected. Serum and plasma were stored at -20℃ until the experiment. ELISA kits were used to determine the TSLP content and total IgE level in mouse serum, and the concentrations of related cytokines in mouse serum were detected.

[0073] All data are expressed as mean ± standard deviation. SPSS 22.0 software was used to analyze the experimental data, employing one-way analysis of variance (ANOVA) and Duncan's multiple comparison test. Graphs were plotted using Origin 2018 software. In all results, P < 0.05 was considered statistically significant, and P > 0.05 was considered not statistically significant.

[0074] The results obtained in this embodiment are analyzed as follows: Figure 3 , Figure 4 , Figures 5-7 As shown:

[0075] The level of TSLP in the serum of mice in each group was detected by sandwich ELISA, and the results are as follows: Figure 3As shown in the figure, compared with the blank group, the serum histamine level in the model group mice was significantly increased (P<0.05). TSLP is a major inflammatory mediator that can increase the permeability of capillary walls, leading to allergic symptoms such as tissue edema. Compared with the model group, the TSLP levels in the other intervention groups were significantly decreased (P<0.05), with the most significant decreases in TSLP levels in groups A, B, and C, and the lowest TSLP level in group B. This indicates that the combination of 100 mg / mL human milk oligosaccharide solution and Lactiplantibacillus plantarum BL-17 can effectively inhibit TSLP secretion. Moreover, the comparison between the intervention groups also shows that the combined application of human milk oligosaccharides is more effective than that of single application; the combination of HMO and BL-17 can exert a more significant effect in alleviating AD.

[0076] The levels of IgE in the serum of mice in each group were detected using a sandwich ELISA method. The results are as follows: Figure 4 As shown, compared with the control group, the serum IgE level in the PC group mice did not change significantly, while the serum IgE level in the model mice was significantly increased (P<0.05). IgE is an important mediator of food allergy reactions, and its increased level indicates that the mice have developed a severe allergic reaction. Compared with the model group, the IgE levels in the other intervention groups were significantly decreased (P<0.05), with the most significant decreases in IgE levels in groups A, B, and C, and the lowest IgE level in group B mice.

[0077] This study used sandwich ELISA to detect the serum levels of Th17 cytokines (IL-17) and Treg cytokines (TGF-β, IL-10) in mice of different groups. The results are as follows: Figure 5As shown. TGF-β and IL-10 are cytokines secreted by Treg cells that can inhibit T cell effects and are important factors in conditional immune responses. They are also anti-inflammatory and regulatory factors that induce tolerance. Compared with the control group, the serum levels of TGF-β and IL-10 in the model group mice were significantly increased (P<0.05), possibly due to the activation of the overall immune response in the model group, resulting in higher serum TGF-β and IL-10 levels. Compared with the model group, the TSLP levels in the other intervention groups were increased, with more significant increases in TGF-β and IL-10 levels in groups A, B, and C (P<0.05). IL-17, as a characteristic cytokine secreted by Th17 cells, plays a pro-inflammatory role in immune regulation. Compared with the control group and the positive control group, the serum IL-17 levels in the model group mice were significantly increased (P<0.05). Compared with the model group, the IL-17 levels in groups A, B, and C were significantly increased (P<0.05). The experimental results showed that the combination of human milk oligosaccharide solution with different concentrations of Lactiplantibacillus plantarum BL-17 could effectively upregulate the secretion of cytokines (TGF-β and IL-10) by Treg cells and downregulate the secretion of Th17 cytokine (IL-17), thus having a certain regulatory effect on the Th17 / Treg imbalance caused by food allergy.

[0078] The serum levels of Th1 cytokines (IFN-γ) and Th2 cytokines (IL-4 and IL-5) in each group of mice were detected using a sandwich ELISA method. The results are as follows: Figure 6As shown. IL-4 and IL-5 are important pro-inflammatory factors secreted by Th2 cells, and their levels are usually elevated during allergic reactions, making them important indicators of allergic responses. Compared with the control group, the serum levels of IL-4 and IL-5 in the model group mice were significantly increased (P<0.05). Compared with the model group, the IL-4 and IL-5 levels in the other intervention groups were decreased, with the decreases being more significant in groups A, B, and C (P<0.05). Interferon-γ (IFN-γ) is a cytokine for Th1 cell differentiation. It prevents the promoting effect of IL-4 on MHC class II molecules in B cells, thereby inhibiting Th2 cell-mediated humoral immunity and regulating the shift of immune direction from humoral immunity to cellular immunity. Compared with the control group, the serum levels of IFN-γ in the model group mice were significantly increased (P<0.05), which may be due to the overall activation of Th0 cell differentiation caused by the inflammatory response. Compared with the MC group, the IFN-γ levels in mice in groups A, B, and C were significantly increased (P<0.05). Among them, the IFN-γ level in group B mice was the highest. The experimental results indicate that gavage administration of a combination of human milk oligosaccharide solution with different concentrations of Lactiplantibacillus plantarum BL-17 can effectively upregulate the secretion of Th1 cytokines (IFN-γ) and downregulate the secretion of Th2 cytokines (IL-4 and IL-5), thereby playing a certain regulatory role in the Th1 / Th2 imbalance caused by food allergies.

[0079] The level of mMCP-1 in the serum of mice in each group was detected by sandwich ELISA, and the results are as follows: Figure 7 As shown in the figure, mast cell degranulation can induce an inflammatory response, and the serum mMCP-1 level can reflect the degree of mast cell degranulation. Compared with the control group, the serum mMCP-1 level of mice in the model group was significantly increased (P<0.05), indicating that food allergy significantly induces mast cell activation in mice. Compared with the model group, the mMCP-1 level of mice fed with a combination of different concentrations of human milk oligosaccharide solution and Lactiplantibacillus plantarum BL-17 was significantly decreased (P<0.05). Among them, the mMCP-1 level of mice in group B was the lowest, which was most similar to the result of the positive control group, indicating that it can effectively inhibit mast cell degranulation and thus alleviate allergy symptoms.

[0080] Example 5

[0081] Effects of different concentrations of human milk oligosaccharide solutions combined with Lactiplantibacillus plantarum BL-17 on gene expression in mice:

[0082] The expression levels of Th1 / Th2 and Th17 / Treg-related cytokines and transcription factors in the small intestine of mice were detected by real-time quantitative polymerase chain reaction (RT-PCR).

[0083] Take 10-20 mg of fresh mouse small intestine tissue and add it to Buffer RL1 in the kit. Use an electric homogenizer to thoroughly homogenize the tissue. Then, extract RNA according to the kit instructions. Detect the RNA concentration using a nucleic acid analyzer; an A260 / A280 value between 1.8 and 2.0 is acceptable for subsequent experiments. Perform cDNA synthesis according to the kit, preparing the reaction system on ice, gently mixing each component as it is added. The reverse transcription reaction conditions are shown in Table 4.

[0084] Table 4 Reaction conditions for reverse transcription

[0085]

[0086] The design and synthesis of primers are shown in Table 5.

[0087] Table 5 Primer Design and Synthesis

[0088]

[0089] Use 2 -ΔΔCt The relative expression levels of Foxp3, RORγt, T-bet, and GATA-3 mRNA in each group were analyzed using a method with GAPDH as an internal reference gene.

[0090] The results obtained in this embodiment are analyzed as follows: Figures 8-9 As shown

[0091] When antigens induce an allergic reaction, the jejunum exhibits more significant structural damage; therefore, we used the jejunum to measure transcription factors. T-bet and GATA-3 genes are specific transcription factors in Th1 and Th2 cells, respectively. Compared to the control group, the relative mRNA expression of the T-bet gene in the jejunum of the model group mice was significantly decreased (P<0.05), while the relative expression of the GATA-3 gene was significantly increased (P<0.05). Compared to the model group, mice administered a combination of different concentrations of human milk oligosaccharide solution and Lactiplantibacillus plantarum BL-17 via gavage showed a significant increase in the relative expression of the T-bet gene (P<0.05). Foxp3 and RORγt genes are key transcription factors in Treg and Th17 cells, respectively. Compared to the control group, the relative expression of the Foxp3 gene in the jejunum of the model group mice was significantly decreased (P<0.05), while the relative expression of the RORγt gene was significantly increased (P<0.05). Compared with the model, the relative expression level of Foxp3 gene was significantly increased in mice in groups A, B and C (P<0.05), and the relative expression level of RORγt gene was significantly decreased (P<0.05).

[0092] The transcriptional levels of different transcription factors determine the Treg / Th17 balance. RORγt is a transcription factor specific to Th17 cells, which can directly regulate their differentiation and function. Foxp3 is a characteristic regulator of Treg cells. The expression level of Foxp3 plays a crucial role in maintaining intestinal homeostasis, combating allergic reactions, and producing antigen-specific Tregs. T-bet and GATA-3 genes are specific transcription factors in Th1 and Th2 cells, respectively. Group B upregulated the expression levels of Foxp3 and T-bet genes, and downregulated the expression levels of RORγt and GATA-3 genes. The Foxp3 / RORγt and T-bet / GATA-3 ratios in Group B were significantly increased, indicating from the perspective of transcription factor expression levels that the composition described in this patent can enhance Th1-type immunity, achieve Th1 / Th2 immune balance in cell differentiation, promote Treg cell expression, and regulate the Treg / Th17 balance.

Claims

1. A composition comprising Lactobacillus plantarum and human milk oligosaccharides.

2. The composition of *Lactobacillus plantarum* and human milk oligosaccharides as described in claim 1, characterized in that, The *Lactiplantibacillus plantarum* BL-17 was mentioned, and the ratio of *Lactiplantibacillus plantarum* to human milk oligosaccharides was 1 × 10⁻⁶. 6 CFU ~ 1×10 10 CFU: 0.1g~10g.

3. The human milk oligosaccharide in the composition of claim 1 or 2 includes one or more of 2'-fucosylated lactose, lactose-N-neotetrasaccharide, lactose-N-tetrasaccharide, lactofucrose pentasaccharide II, and 3'-fucosylated lactose.

4. The use of the composition according to claim 1 or 2 in the preparation of food, medicine or health products having the ability to effectively relieve atopic dermatitis and its corresponding allergic symptoms.

5. A product for the prevention and / or treatment of atopic dermatitis, characterized in that, The product contains Lactobacillus plantarum BL-17.

6. The product for the prevention and / or treatment of atopic dermatitis as described in claim 5, characterized in that, In the product, the viable count of *Lactobacillus plantarum* BL-17 is not less than 1 × 10⁻⁶. 6 CFU / mL.

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