TW202539716A - Probiotics composition and uses thereof - Google Patents

Abstract

The present invention discloses a probiotics composition for preparation of a composition to prevent, improve or relieve allergic syndromes. The probiotics composition comprises Bifidobacterium bifidum, Bifidobacterium longumor a combination thereof. After periodic administrations of the probiotics composition to a subject in need, serum levels of immune globulin and histamine are decreased, and dermal thickness, inflammation and mast cell density are also reduced. Allergic syndromes including dermatitis can be significantly improved.

Description

Probiotic compositions and their uses

This invention relates to the field of probiotics, particularly to probiotics of the genus Bifidobacterium ; this invention also relates to the use of probiotics, particularly to their use in regulating immunity and in relieving, preventing or treating allergic symptoms and inflammatory responses.

Probiotics generally refer to microorganisms that have positive benefits to the host (such as animals or humans) after being ingested. Common probiotics mainly include lactic acid bacteria and some yeasts. Lactic acid bacteria are a type of bacteria that can ferment carbohydrates to produce lactic acid. Currently, only a few strains have been proven to be beneficial to the host's health, and their application in promoting the host's intestinal health and regulating immunity has proven to have good effects.

The human gut contains up to 100 trillion bacteria, accounting for about 80% of the total human microbiome. 70% to 80% of human immune cells reside in the gut, thus providing a domain for interaction between the host's immune system and microorganisms. This makes probiotics play a crucial role in gut immune regulation. Common probiotics include lactic acid bacteria such as Enterococcus , Lactobacillus , and various species and strains of Bifidobacterium , while yeasts primarily include Saccharomyces boulardii . The efficacy of these probiotics in regulating immune function has been validated through numerous in vitro and clinical trials.

Generally speaking, probiotics regulate the host's immune function through various mechanisms. For example, regarding mucosal barrier function, probiotics can compete with invading organisms for attachment sites on mucosal cells, preventing bacteria from penetrating the mucosa and entering deeper tissues. On the other hand, probiotics have the ability to regulate the density of connections between intestinal epithelial cells, further maintaining the integrity of the gastrointestinal mucosa. Furthermore, probiotics can enhance innate immune function, such as increasing the activity of phagocytes and natural killer cells, and regulating the expression of related cytokines, thereby controlling the intensity of the inflammatory response. In addition, probiotics also have a positive effect on adaptive immunity, regulating the responsiveness of T and B lymphocytes to pathogens and inducing the secretion of various immunoglobulins.

Currently, due to environmental changes, the number of people seeking medical attention for allergies is increasing year by year. Asthma and atopic dermatitis (AD) continue to show significant incidence and prevalence globally, and these two diseases are more common in younger populations. Atopic dermatitis is a common and recurrent allergic skin disease, currently believed to be caused by Th1/Th2 dysbalance due to overactivation of Th2 cells. Th2 cytokines, such as interleukin-13, are usually observable. Extensive manifestations of IL-13; common symptoms of atopic dermatitis include itching, redness and swelling, desquamation, cracking, and crust formation; in infants and young children, the face is the most common site, while in children and adults, the flexor surfaces of joints such as the knees and elbows are more common; in addition, most patients also experience complications such as allergic rhinitis, asthma, and allergic conjunctivitis.

Clinically, the treatment of atopic dermatitis mainly relies on medications, such as oral antihistamines and antibiotics, or topical corticosteroids and immunosuppressive agents. However, drug treatment may inevitably cause side effects and other safety issues for patients. For example, JAK immunosuppressants have been found to affect blood cells, liver function, and blood lipids in clinical practice, and may also cause symptoms such as acne, headache, and nausea, and increase the risk of upper respiratory tract infections or herpes. Therefore, there is an urgent need to develop methods that can effectively treat, relieve, or prevent allergic diseases, including atopic dermatitis, without causing side effects.

Several applications of lactic acid bacteria strains have been developed in this field to improve atopic dermatitis or other allergic diseases. For example, Lactobacillus rhamnosus GG (LGG) or Bifidobacterium animalis subsp . lactis BB-12 has been added to hydrolyzed whey protein formulations and fed to breastfed infants who develop atopic dermatitis.

However, the effectiveness of probiotics still depends on their ability to successfully pass through the gastrointestinal tract and reach suitable implantation sites. Specifically, they need sufficient acid and bile salt tolerance to overcome defense mechanisms in the gastrointestinal tract, such as low-pH gastric acid and bile acids in the small intestine. Therefore, in order to improve and solve the problems encountered by previous technologies in the treatment, relief, or prevention of allergic diseases, the primary issue to be addressed is to select probiotic strains with high viability, acid and bile salt tolerance to ensure that they can pass through the gastrointestinal tract smoothly and exert their probiotic effects.

Therefore, one object of the present invention is to provide a probiotic composition comprising a long Bifidobacterium, a two Bifidobacterium, or a combination thereof.

Another object of the present invention is to provide the use of the probiotic composition as described above for the preparation of a pharmaceutical composition for the prevention, improvement or relief of allergy symptoms, comprising administering an effective dose of the probiotic composition to a desired individual for a period of time.

As described above, the required individual includes a mammal and the allergic symptoms include rheumatoid arthritis, seborrheic dermatitis, contact dermatitis, atopic dermatitis, allergic dermatitis, or psoriasis.

As described above, the route of administration includes oral, intravenous, or gastrointestinal administration.

As described above, the pharmaceutical composition comprises a pharmaceutically and food-acceptable carrier, excipient, and/or additive.

As described above, the dosage form of the pharmaceutical composition includes powder, tablet, granule, suppository, microcapsule, ampoule, liquid spray or stopper.

As described above, the effective dose is (0.10 to 2.00) x 10 ¹¹ CFU per half-day or (0.20 to 4.00) x 10 ¹¹ CFU per day; the duration is at least 1 to 56 days, preferably at least 14 to 49 days, and more preferably 21 to 28 days.

Another object of the present invention is to provide the use of the probiotic composition as described above for the preparation of a nutritional composition for regulating cellular physiological activity, comprising administering an effective dose of the probiotic composition to a desired individual for a period of time to regulate its immune cells or immune cell populations.

As described above, the immune cells include natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, or dendritic cells, and the immune cell population includes any two or more of the following groups: natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells.

As described above, the effective dose is (0.10 to 2.00) x 10 ¹¹ CFU per half-day or (0.20 to 4.00) x 10 ¹¹ CFU per day; the duration is at least 1 to 56 days, preferably at least 14 to 49 days, and more preferably 21 to 28 days.

The term "probiotics" in this article refers to microorganisms that, when administered to a host in appropriate doses, can promote the host's health, such as Lactobacillus, Pediococcus, Bacillus, Bifidobacterium, and Lactobacillus rhamnosus. The term "prevention" refers to taking specific actions to stop or prevent a disease, condition, or its symptoms.

The term "treatment" refers to relieving, improving, or curing a disease, condition, or symptoms.

The term "mitigation" means reducing the scope, decreasing the quantity, or lessening the severity.

The term "effective dose" refers to a dosage sufficient to prevent, treat, or alleviate the disease, condition, or symptoms of the disease or condition.

The term "mammals" refers to all members of the mammal class, including humans, primates, poultry, and livestock, such as rabbits, pigs, sheep, and cattle, including but not limited to humans, rats, mice, guinea pigs, monkeys, pigs, sheep, cattle, horses, dogs, cats, birds, and fowl.

The term "medical or food acceptable excipient" refers to a compound that is compatible with other components in a pharmaceutical or food composition and will not cause harm to the desired individual when administered to that individual.

The term "immune cell" refers to any member of the innate immune cell population, including but not limited to natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells, and is not limited to specific subtypes of these cells that express special cell surface markers.

The first embodiment of the present invention provides a probiotic composition comprising one Bifidobacterium longum , one Bifidobacterium bifidum , or a combination thereof.

In some embodiments, the ratio of the number of the long-lived Bifidobacterium to one of the two Bifidobacteria is 2:1 to 1:2.

In several embodiments, the Bifidobacterium longum is Bifidobacterium longum S9, and biological storage has been completed at a designated storage facility in Japan.

In some embodiments, Bifidobacterium longum S9 exhibited acid and bile salt resistance, demonstrating good intestinal retention capacity. Cellular experiments confirmed that, compared to other strains of the same genus, Bifidobacterium longum S9 significantly reduced TH2 cell counts and increased TH1 cell and FoxP3 ⁺ cell counts. The number of Treg cells can reduce IL-4-induced inflammation and allergic reactions. Animal experiments have shown that, compared to other strains of the same genus, oral administration of Bifidobacterium longum S9 to animals with skin inflammation for 4 to 7 days can alleviate dermatitis reactions such as erythema, induration, or desquamation, and effectively increase skin moisture content and reduce the content of chemokines in the skin, such as thymus and activation-regulated chemokines. Chemokine (TARC) or CCL27, etc.; In addition, after oral administration of Bifidobacterium longum S9, the concentrations of immunoglobulins and histamine in the serum of animals also decreased significantly, and the dermatitis response, such as the thickness of the stratum corneum, the thickness of the epithelium, the degree of inflammation, and the density of fat cells, was significantly improved.

In other embodiments, the bifidobacterium is Bifidobacterium S2, and biodepositation has been completed at a designated storage facility in Japan.

In some embodiments, Bifidobacterium S2 exhibits acid and bile salt resistance, demonstrating its excellent intestinal retention capacity. Animal experiments have confirmed that, compared to other strains of the same genus, oral administration of Bifidobacterium S2 to animals suffering from skin inflammation for 4 to 7 days can alleviate dermatitis reactions such as erythema, induration, or desquamation, effectively increase skin moisture content, and reduce the content of chemokines in the skin, such as thymus and activation-regulated chemokines. Chemokine (TARC) or CCL27, etc.; In addition, after oral administration of Bifidobacterium S2, the concentrations of immunoglobulins and histamine in the serum of animals also decreased significantly, and the dermatitis response, such as the thickness of the stratum corneum, the thickness of the epithelium, the degree of inflammation, and the density of fat cells, was significantly improved.

A second embodiment of the invention provides the use of a probiotic composition for preparing a pharmaceutical composition for preventing, improving or alleviating allergy symptoms, comprising administering an effective dose of the probiotic composition to a desired individual for a period of time, wherein the probiotic composition is the probiotic composition provided as in the first embodiment.

In this embodiment, the required individual includes a mammal, which in some specific examples includes a mouse or a human.

The allergic symptoms include, but are not limited to, rheumatoid arthritis, seborrheic dermatitis, contact dermatitis, atopic dermatitis, allergic dermatitis, or psoriasis.

In this embodiment, the route of administration includes, but is not limited to, oral, intravenous, or gastrointestinal administration; the dosage form of the pharmaceutical composition includes powder, tablet, granule, suppository, microcapsule, ampoule, liquid spray, or plug; in some examples, an oral powder is provided. Specifically, for humans, it can be taken with a fluid (e.g., water), such as by dissolving the powder in water and then taking it, or by encapsulating the powder in a capsule and taking it with water, or by mixing it with various foods or beverages and consuming it, without any particular limitation.

In this embodiment, the pharmaceutical composition includes a pharmaceutically and food-acceptable carrier, excipient, and/or additive.

In some embodiments, the provided pharmaceutical composition is used to alter or reduce the expression of at least one of CCL20, CCL22, CCL27, VEGF, IL1RA, and CCL18 in skin lesions of a desired individual suffering from atopic dermatitis (AD). This alteration or reduction includes administering Bifidobacterium longum S9, Bifidobacterium bifidum S2, or a combination thereof to the desired individual suffering from AD, wherein, compared to the non-lesional skin of an individual without AD or the non-lesional skin of the desired individual, the expression of at least one of CCL20, CCL22, CCL27, VEGF, IL1RA, and CCL18 is reduced. In an individual's skin lesions, at least one of TARC, CCL20, CCL22, CCL27, VEGF, and CCL18 is upregulated, and administration of Bifidobacterium longum S9, Bifidobacterium bifidum S2, or a combination thereof reduces the expression of at least one of TARC, CCL20, CCL22, CCL27, and CCL18 in the skin lesions and/or increases the expression of TARC and CCL27 in the skin lesions.

In this embodiment, the time refers to the period required for an effective dose of the combination to achieve the aforementioned effect during a continuous period; in a specific embodiment, the time is at least 1 to 56 days, preferably at least 14 to 49 days, and more preferably 21 to 28 days; the effective dose refers to the amount of the combination administered at specific time intervals within the time period to achieve a sufficient quantity of the *Bifidobacterium longum*, the *Bifidobacterium bifidum*, or a combination thereof; for example, for an adult human, the effective dose is 0.10 to 2.00 x ^10¹¹ CFU/60 kg per half-day or (0.20 to 4.00) x ^10¹¹ CFU/60 kg per day, preferably 0.35 to 1.25 x 10¹¹ CFU per half-day ^. CFU/60kg or (0.70 to 2.50) x 10 ¹¹ CFU/60kg daily.

A third embodiment of the present invention provides the use of a probiotic composition for preparing a nutritional composition that modulates cellular physiological activity, comprising administering an effective dose of the probiotic composition to a desired individual for a period of time to modulate its immune cells or immune cell populations, wherein the probiotic composition is the probiotic composition provided as in the first embodiment.

In this embodiment, the immune cells include, but are not limited to, natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, or dendritic cells. The immune cell population includes, but is not limited to, any combination of two or more of the following groups: natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells. In some embodiments, the provided nutrient composition is used to alter, reduce, or increase the number of TH1 cells, TH2 cells, TH17 cells, Treg cells, and FoxP3 ⁺ cells in the desired individual immune cells. The change, decrease, or increase in the number of at least one of the Treg cells also includes administering Bifidobacterium longum S9, Bifidobacterium bifidum S2, or a combination thereof to the desired individuals; in some specific examples, compared to desired individuals without the administration of the strain, the number of at least one of TH1 cells and FoxP3 ⁺ Treg cells in the desired individuals is increased, and the number of at least one of TH2 cells, TH17 cells, and Treg cells in the desired individuals is decreased, wherein the administration of Bifidobacterium longum S9, Bifidobacterium bifidum S2, or a combination thereof decreases the number of TH2 cells in the desired individuals and increases the number of TH1 cells and FoxP3 ⁺ Treg cells.

In this embodiment, the definitions of the time and the effective dose can be found in the detailed definitions of the fourth embodiment, and will not be repeated here.

1. Strains Isolation and Identification

The inventors continuously tracked 338 healthy full-term infants for at least 52 weeks and diagnosed 36 infants with symptoms of atopic dermatitis (hereinafter referred to as the AD group; the other infants were referred to as the non-AD group). The AD group was further subdivided according to their usual food source into a breastfed group (n=12), a formula-fed group without added oligosaccharides (n=19), and a formula-fed group with added oligosaccharides (n=5). Fecal samples were collected from these infants at 0, 1, 4, 8, 16, 24, and 52 weeks of age, and analyzed using Shannon... The α-diversity of fecal microbiota was analyzed using indices such as index, species_richness, and Chao1, while the β-diversity of fecal microbiota was analyzed using community distance indices such as Bray_Curtis, uniFrac_unweighted, and uniFrac_weighted.

Analysis, as shown in Figure 1a, revealed no significant difference in α-diversity between the AD group and the non-AD group (P > 0.05); however, in week 16, as shown in Figure 1a, a significant difference in β-diversity was observed between the AD group and the non-AD group (unifrac_unweighted P < 0.001; unifrac_weighted P = 0.046).

Next, the significantly different bacterial flora were further analyzed using LefSe and LDA scores. As shown in Figure 1b, compared with the non-AD group, the AD group with atopic dermatitis before one year of age showed a significant decrease in the abundance of Bifidobacterium spp. in the gut at 16 weeks of age, especially Bifidobacterium longum . Therefore, at 16 weeks of age in infants and young children, the abundance of Bifidobacterium spp. was significantly reduced. At one week old, the abundance of Bifidobacterium longum in a baby's feces can serve as a biomarker for predicting the development of atopic dermatitis. It can also be inferred that Bifidobacteria in breast milk or the infant's gut play a protective role for infants, especially against immune dysregulation-related inflammatory responses, including atopic dermatitis.

The following further explains the methods for isolating and identifying the strains, and selects probiotic strains with acid- and bile-resistant properties for subsequent animal experiments to verify the immunomodulatory effects of these probiotics.

(1) Strain isolation: The feces provided by the donor were serially diluted and inoculated into MRS solid medium and anaerobic cultured at 37°C for 3 to 5 days. Single colonies on the medium were picked and isolated by streak to obtain pure colonies. The pure colonies were inoculated into LB liquid medium and anaerobic cultured at 37°C for 12 to 16 hours. Then, 20% glycerol was added and stored at -80°C.

(2) Strain identification: The colonies isolated by MALDI-TOF were identified as Bifidobacterium bifidum and Bifidobacterium longum , respectively.

After obtaining the above strains, the survival rate and environmental stress tolerance of these strains were further tested, as well as their physiological characteristics of acid resistance and bile salt resistance.

2. Growth test

The above-mentioned strains were cultured in MRS medium and incubated at 37°C for 16 to 18 hours. The absorbance at 600 nm was measured using a spectrophotometer, and the bacterial count (CFU/ml) was determined using the plate count method. The test results are shown in Table 1. Table 1 strain Growth activity (CFU/mL) Acid resistance Bile salts Bile salt hydrolase activity (cm) S2 0.22 x ^10⁹ + + 2.3 S9 6.4 x ^10⁹ + ++ 1.2 LGG

3. Acid resistance test

The bacterial culture obtained above was diluted or concentrated to ^10⁹ CFU per milliliter. One milliliter of the adjusted bacterial culture was taken from each bacterial culture, and 9 milliliters of pH 2.0 phosphate buffer solution were added to each. The cultures were incubated at 37°C for 3 hours. Another bacterial culture was added to a pH 7.2 phosphate buffer solution under the same conditions as a control group. After incubation, the cultures were sequentially diluted and cultured on MRS agar-agar using the tilting method. The cultures were incubated at 37°C for 48 hours, and the number of surviving probiotics was counted. The test results are shown in Table 1 above.

4. Bile salt tolerance test

In this test, the bacterial suspension adjusted to a concentration of ^10⁹ CFU/ml was used to analyze the tolerance of the test strains to bile in MRS-bile broth (0.3% ox gall bile). The bacterial suspension was transferred to 9 ml of MRS broth with or without 0.3% ox gall bile and incubated for 24 hours. After incubation, the absorbance at 600 nm was measured using a spectrophotometer to evaluate the tolerance of the test strains in the presence of bile. The test results are shown in Table 1.

The results showed that strains S2 and S9 both had the ability to withstand acid and bile salts, with S9 performing better.

5. Bile salt hydrolases (Bile salt hydrolase , BSH) Activity testing

Further screening of the above-mentioned strains was conducted using the bile salt hydrolase (BSH) screening plate method. The method is briefly described as follows: Sterilized paper tablets were placed flat with sterile tweezers in solutions containing 0.5% taurodeoxycholic acid sodium salt (TDCA) and 0.37 g/L CaCl₂. On _MRS agar plates or MRS agar plates without any bile salts, bacterial suspensions that had been incubated overnight were dropped onto paper tablets. After anaerobic incubation at 37°C for 48-72 hours, the diameter of the precipitate ring was measured. The test results are shown in Table 1. The results show that strains S2 and S9 exhibited varying degrees of bile salt hydrolase activity, with S2 showing better performance.

6. Cell culture

Mouse macrophages RAW264.7 (registration number: BCRC 60001, hereinafter referred to as RAW264.7 cells) were purchased from the Bioresource Preservation and Research Center of the Food Industrial Research Institute in Hsinchu, Taiwan. The culture medium used was DMEM medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 1.85g sodium bicarbonate.

7. Immunomodulation test

RAW264.7 cells (5 × ^10⁵ cells/well) were seeded in 24-well plates. When the cells reached 80% confluence, they were washed with phosphate buffer, and then 900 μl of fresh cell culture medium was added. Strains S2 and S9 were then added separately, with *Lactobacillus rhamnosus* GG (hereinafter referred to as strain LGG) used as a control strain. The viable cell count in all cultures was 5 × ^10⁵. CFU, wherein the bacterial culture was first rinsed with phosphate buffer, the supernatant was removed, and then reconstituted with cell culture medium; after culturing in an anaerobic incubator for 24 hours in 24-well plates, the cell supernatant was collected, and the contents of IL-6, TNF-α and IL-1β in the cell supernatant were analyzed by an ELISA kit (R&D Systems).

As shown in Figures 1c to 1e, strain S2 promoted the secretion of IL-6 and TNF-α more than strain LGG; strain S9 promoted the secretion of IL-1β more than strain LGG.

8. Anti-inflammatory trial

RAW264.7 cells (5 × ^10⁵ cells/well) were seeded in 24-well plates. When the cells reached 80% confluence, they were washed with phosphate buffer, and 800 μl of fresh cell culture medium was added. Then, bacterial cultures of strains S2, S9, and LGG (5 × ^10⁵ CFU) were added, along with endotoxin lipopolysaccharide (1 μg/mL) to induce cellular inflammation. These bacterial cultures were prepared by first washing with phosphate buffer, removing the supernatant, and then rehydrating with cell culture medium. The 24-well plates were incubated in an anaerobic incubator for 24 hours. The cell supernatant was then collected and analyzed using an ELISA kit (R&D). Systems) analyzed the levels of TNF-α and IL-6 in cell supernatant.

As shown in Figures 1f to 1g, strain S2 showed a better inhibitory effect on TNF-α secretion compared to strain LGG; strain S9 showed a better inhibitory effect on IL-6 secretion compared to strain LGG.

9. Strain identification

Following the aforementioned immunomodulatory and anti-inflammatory tests, strains S2 and S9, possessing both immunomodulatory and anti-inflammatory functions, were sent to the Bioresource Preservation and Research Center of the Food Industry Development and Research Institute for identification of their scientific names. The identification results are as follows:

Strain S2 is a Gram-positive bacterium. 16S rDNA sequence analysis and Biolog identification system analysis showed that strain S2 has 100% similarity to Bifidobacterium bifidum , with a partial sequence shown in SEQ ID NO: 1. The comprehensive identification results indicate that strain S2 is a Bifidobacterium bifidum strain, and it is named Bifidobacterium S2 in this invention.

Strain S9 is a Gram-positive bacterium. Analysis of its 16S rDNA partial sequence and API identification system showed that strain S9 shared over 99% similarity with * Bifidobacterium longum *, as shown in SEQ ID NO: 2. The comprehensive identification results indicate that strain S9 is a * Bifidobacterium longum* subsp . *infancier *, and it is named *Bifidobacterium longum* S9 in this invention.

16S rDNA identification revealed that the 16S rDNA sequence of Bifidobacterium longum S9 is shown in SEQ ID NO.1, and the 16S rDNA sequence of Bifidobacterium bifidum S2 is shown in SEQ ID NO.2. Based on sequence alignment using the BLAST (Basic Local Alignment Search Tool) database, the species names of the two strains were determined to be Bifidobacterium longum and Bifidobacterium bifidum.

10. test animals

The animal experiments described in this article used male HR-1 hairless mice (female HR-1 mice) (3 weeks old, weighing approximately 20 g), purchased from the National Laboratory Animal Center (R.O.C.). All experimental animals were individually housed in animal rooms with 12-hour light and 12-hour dark cycles, maintained at a temperature of 21-24°C and a relative humidity of 45-70%, with adequate hydration and food supplies. All experimental procedures involving the animals were approved by the Institutional Animal Care and Use Committee of National Chung Kung University and in accordance with the Guide for the Care and Use of Laboratory Animals (NIH) of the National Institutes of Health (NIH). Animals) will be used to carry out this.

11. Statistical analysis

The values presented in this paper are expressed as mean ± standard deviation (mean ± SD). Analysis was performed using one-way ANOVA and commercially available software. A p-value < 0.05 was considered statistically significant. An asterisk (**) indicates p < 0.05, and an asterisk (***) indicates p < 0.005.

12. Immune cell population analysis

Bifidobacterium longum S9 isolated from PBMCs was co-cultured to analyze its cell-stimulating effect, particularly assessing the increase and decrease of T cell subpopulations, specifically by measuring the expression levels of TH1, TH2, TH17, Treg, and FoxP3 ⁺ Treg cells; in short, after co-culturing, the number of PBMC cells was adjusted to 5 x 10⁵ ^. /tube, and centrifuged at 400g for 5 minutes at 4°C, then the supernatant was removed; according to the required concentrations of IL-4, IL17, IFN-γ, IL-10 and FOXP3 antibodies, 100μl/tube was added respectively, incubated on ice in the dark for 30 minutes, then reconstituted with culture medium, centrifuged at low speed at 4°C, and then treated with fixation and permeabilization buffer. Finally, the cells were analyzed by FACSCalibur flow cytometry, and the results are shown in Figure 2; compared with the control group without probiotic stimulation, after treatment with Bifidobacterium longum S9, the proportion of TH2 cell population decreased significantly (from 52.994% to 45.720%), while the proportion of TH1 cell population (from 10.401% to 14.544%) and FoxP3 ⁺ Treg cells increased significantly. The percentage of cells that increased significantly (from 1.013% to 1.455%) suggests that Bifidobacterium longum S9 should have an anti-allergic effect.

13. HR-1 Prevention of atopic dermatitis in a hairless mouse model

Atopic dermatitis model was established by feeding HR-1 hairless mice with a diet containing 50% less zinc and 50% less magnesium (hereinafter referred to as AD-induced diet) for 8 weeks. During the experimental period (weeks 1 to 8), the control group (n=5) was fed a normal diet, while the atopic dermatitis group (AD group; n=5) was fed the AD-induced diet. In addition to being fed the AD-induced diet, the experimental groups were also given two strains of Bifidobacterium S2 (strain S2 group; n=5) and Bifidobacterium longum S9 (strain S group; n=5) in their drinking water (1x10⁸ ^). (CFU/ml, changed every two days); After the start of the experiment, the body weight, water intake and food intake of HR-1 mice were recorded weekly, and the preventive effect of probiotics on atopic dermatitis was evaluated by indicators such as the area and severity index of tinea (PASI), back water content (%), TEWL (transient water loss per hour), skin biomarker content, blood immune marker content and skin histology.

13.1. HR-1 Assessment of average body weight, water intake, and food intake in mice

As shown in Figures 3a to 3c, there were no statistically significant differences in average body weight, water intake (ml/week/mice), and food intake (g/day/mice) among the four groups of HR-1 hairless mice (control group, AD group, strain S2 group, and strain S9 group) until the end of the 8th week of the experiment.

13.2. Area and severity index of tinea psoriasis (PASI) evaluate

Starting from week 4 of the experiment, the appearance of HR-1 mice was photographed weekly to record the area of psoriasis and the Psoriasis Area Severity Index (PASI), and the recording continued until week 7. PASI measures the severity of psoriasis lesions based on area coverage and patch appearance, specifically considering the characteristics and percentage of the area of the patches covering the body. The characteristics of the patches were judged based on the degree of erythema, induration, and desquamation. The comprehensive judgment results are shown in Figures 4a to 4d.

In the AD group, mice showed significantly higher scores than the control group in terms of total PASI score, erythema, induration, and desquamation during weeks 4 to 7; the peak scores for each indicator also showed the same trend. In contrast, the S2 and S9 groups, which had probiotics added to their drinking water, showed significant improvements in total PASI score, erythema, induration, and desquamation.

13.3. Back water content (%) With dehydration per hour (TEWL) evaluate

At week 8 of the experiment, the moisture level and transepidermal water loss (TEWL) of the back skin of mice were measured using a Multiprobe MPA5 corneometer. As shown in Figure 5a, compared with the control group, the moisture level of the back skin of mice in the AD group decreased significantly to about 20% (p < 0.001). Compared with the AD group, the moisture level of the back skin of the experimental groups that continued to consume strain S2 or strain S9 was significantly improved (about 50%, p < 0.001).

On the other hand, as shown in Figure 5b, the results of trans-epidermal water loss (TEWL) measured by the Tewameter TM210 instrument showed that, compared with the control group, the TEWL value of the back skin of the AD group mice was significantly increased to about 40 g/ ^m2 (p < 0.01). The TEWL value of the back skin of the experimental groups that continuously consumed strain S2 or strain S9 was also significantly improved, decreasing to about 20 g/ ^m2 (p < 0.001) and 30 g/ ^m2 (p < 0.05), respectively. The smaller the TEWL value, the less water loss from the skin surface, which means that the condition of the epidermal protective layer is better. The larger the TEWL value, the greater the water loss, which also means that the damage to the epidermal protective layer is more serious.

13.4. Assessment of skin biomarker content

At week 8 of the experiment, HR-1 hairless mice were sacrificed to collect skin samples from their backs. ELISA was used to determine whether the expression levels of intercytokines in the AD group, strain S2 group, and strain S9 group were significantly upregulated compared to the non-pathological skin of the control group. The evaluation results are shown in Figures 5c and 5d, which represent the protein content ratio (ng/mL) of intercytokines TARC and CCL27 identified in the skin samples, respectively.

As shown in Figure 4c, compared with the control group, the expression level of TARC protein in the skin samples of the AD group was significantly increased. In the experimental groups continuously ingested with strains S2 and S9, the expression level of TARC was significantly reduced (p < 0.01), similar to that of the control group. Similarly, as shown in Figure 4d, compared with the control group, the expression level of CCL27 protein in the skin samples of the AD group was also significantly increased. In the experimental groups continuously ingested with strains S2 and S9, the expression level of CCL27 was also significantly reduced (strain S2 group: p < 0.01; strain S9 group: p < 0.001).

13.5. Blood immune marker content assessment

Blood samples were collected from HR-1 hairless mice, and the levels of histamine and immunoglobulin E (IgE) in the serum were measured by ELISA to determine whether their levels were significantly increased in the AD group, strain S2 group, and strain S9 group compared with the levels in the non-pathological skin of the control group. The evaluation results are shown in Figures 6a and 6b, which represent the ratio of histamine and IgE levels (ng/mL) measured in the blood samples, respectively.

As shown in Figure 6a, compared with the control group, the histamine content in the blood samples of the AD group was significantly increased, reaching approximately 200 ng/mL. However, in the experimental groups continuously ingested with strains S2 and S9, although the histamine content also increased, it was significantly controlled. Similarly, as shown in Figure 6b, compared with the control group, the IgE content in the blood samples of the AD group was also significantly increased, reaching approximately 1000 ng/mL. In the experimental groups continuously ingested with strains S2 and S9, although the IgE content also increased, it was also significantly controlled.

13.6. Histological evaluation of skin sections

On the other hand, back skin tissue collected from HR-1 hairless mice at week 8 was further histologically stained with H&E and toluidine blue (TB), and pathologists evaluated the thickness of the stratum corneum, epidermal thickness, inflammation status, and mast cell density without knowing the grouping. In short, after sacrificing the mice, back skin was obtained as a skin sample (0.5 x 0.5 cm), and the muscle layer and fat layer in the skin sample were separated. The skin sample was fixed with a neutral buffer solution of 10% formalin, dried, and embedded in paraffin.

As shown in Figures 7a to 7d, H&E staining results indicate that the stratum corneum, epidermis, and dermis of the skin samples in the AD group were significantly thickened compared to the control group, which means an increase in the number of inflammatory cell infiltrations. As shown in Figures 7c to 7d, compared with the AD group, the thickening of the stratum corneum, epidermis, and dermis in the strain S2 and strain S9 groups was significantly controlled, showing that the intake of strain S2 or strain S9 has the effect of reducing inflammatory cell infiltration.

On the other hand, by evaluating the number of fat cells (mast) in the epidermal layer The density of fat cells was used as an assessment criterion for whether these strains effectively controlled skin inflammation. The density of fat cells and the corresponding inflammatory index are shown in Figure 7e. The density of fat cells and the inflammatory index of each group were determined by image analysis, and the statistical results are shown in Figures 7f to 7g. Compared with the control group, the inflammatory index and the density of fat cells in the AD group were significantly increased, indicating that fat cells accumulated in large numbers in the epidermal layer under OVA induction, causing infiltration. Compared with the AD group, the S2 and S9 groups showed significant improvement in both the inflammatory index and the density of fat cells, indicating that the intake of strain S2 or strain S9 has the effect of reducing the large number of fat cells accumulating in the epidermal layer, which helps to reduce skin inflammation.

The probiotics provided by this invention have demonstrated an immunomodulatory effect by regulating the number of peripheral blood mononuclear cells (PBMCs) in the consumer. In a model animal with atopic dermatitis, the consumption of probiotics effectively reduced dermatitis indices such as erythema, induration, and peeling on the consumer's skin, and significantly improved the water content and hourly water loss on the consumer's back, thus maintaining the consumer's skin health.

Biochemical analysis shows that the probiotics provided by this invention can reduce the concentration of chemotherapeutic factors, including TARC and CCL27, in the skin of consumers, and can simultaneously reduce serum IgE and histamine levels, thus alleviating inflammatory responses in consumers. Furthermore, histological examination reveals significant improvements in the thickness of the stratum corneum, epidermis, and dermis. As the inflammatory response decreases, the density of fat cells aggregated in the skin also decreases significantly.

In summary, the probiotics provided by this invention can reduce the inflammatory response, thereby reducing allergic symptoms and inflammation in the individual, and can effectively prevent, alleviate and treat inflammation caused by allergic reactions, such as rheumatoid arthritis, seborrheic dermatitis, contact dermatitis, atopic dermatitis, allergic dermatitis or psoriasis and other common skin allergy diseases.

(without)

Figure 1a shows the box-and-fiber diagram and community distance analysis to illustrate the diversity of probiotics in the gut of human infants and young children; Figure 1b is a bar chart illustrating the results of linear discriminant analysis (LDA) of various probiotics in the gut of 16-week-old human infants and young children; Figures 1c to 1e are bar charts illustrating the immunomodulatory effects of the selected strains on cells; Figures 1f to 1g are bar charts illustrating the anti-inflammatory effects of the selected strains on cells; Figure 2 shows the flow cytometry analysis results of the immune cell populations of experimental animals after ingestion of the selected strains; Figures 3a to 3c are line graphs used to assess the average body weight, food intake, and water consumption of animals with atopic dermatitis after feeding on selected strains. Figures 4a to 4d are bar graphs used to assess the skin health of animals with atopic dermatitis after feeding on selected strains, showing scores for PASI, erythema, induration, and peeling. Figures 5a to 5b are bar graphs showing the back skin water content and hourly water loss of animals with atopic dermatitis after feeding on selected strains. Figures 5c to 5d are bar graphs showing the TARC and CCL27 protein content of the back skin of animals with atopic dermatitis after feeding on selected strains. Figures 6a and 6b are bar graphs showing the levels of histamine and immunoglobulin IgE in the serum of atopic dermatitis model animals after ingestion of the selected bacterial strains; Figure 7a is a representative HE-stained skin section from atopic dermatitis model animals, used to present the baseline for measuring the thickness of the cuticle, epidermis, and dermis; Figures 7b and 7d are bar graphs illustrating the statistical results of the maximum thickness of the cuticle, epidermis, and dermis in skin sections from atopic dermatitis model animals; Figure 7e is a representative H&E histological staining map of skin sections from a model animal of atopic dermatitis, used to illustrate the density of different mast cells in the dermis and their corresponding assessment scores; Figures 7f and 7g are bar graphs used to illustrate the statistical results of inflammatory index and mast cell density in skin sections from a model animal of atopic dermatitis, respectively.

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Claims (10)

A probiotic composition comprising a long Bifidobacterium, a two Bifidobacterium, or a combination thereof. The use of the probiotic composition as described in claim 1 for the preparation of a pharmaceutical composition for the prevention, improvement or relief of allergy symptoms, comprising administering an effective dose of the probiotic composition to a desired individual for a period of time. As described in claim 2, the desired individual includes a mammal and the allergic symptoms include rheumatoid arthritis, seborrheic dermatitis, contact dermatitis, atopic dermatitis, allergic dermatitis, or psoriasis. The use as described in claim 2, wherein the route of administration includes oral, intravenous, or gastrointestinal administration. As described in claim 2, the pharmaceutical composition comprises a pharmaceutically and food-acceptable carrier, excipient, and/or additive. As described in claim 5, the dosage form of the pharmaceutical composition includes powder, tablet, granule, suppository, microcapsule, ampoule, liquid spray or stopper. As described in claim 2, the effective dose is (0.10 to 2.00) x 10 ¹¹ CFU per half-day or (0.20 to 4.00) x 10 ¹¹ CFU per day; the duration is at least 1 to 56 days, preferably at least 14 to 49 days, and more preferably 21 to 28 days. The use of the probiotic composition as described in claim 1 for preparing a nutritional composition that modulates cellular physiological activity includes administering an effective dose of the probiotic composition to a desired individual for a period of time to modulate its immune cells or immune cell populations. As described in claim 8, the immune cells include natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, or dendritic cells, and the immune cell population includes any two or more of the following groups: natural killer cells, mast cells, eosinophils, basophils, macrophages, neutrophils, and dendritic cells. For the purpose described in claim 8, the effective dose is (0.10 to 2.00) x 10 ¹¹ CFU per half-day or (0.20 to 4.00) x 10 ¹¹ CFU per day; the duration is at least 1 to 56 days, preferably at least 14 to 49 days, and more preferably 21 to 28 days.