CN116407569A - Antiallergic probiotic composition and application thereof - Google Patents

Abstract

The present invention provides the purposes of probiotics composition in the preparation prevention and/or treatment the medicine of the disease that is caused by allergic reaction, and described probiotics composition comprises the ratio of viable bacteria quantity is (50-150):(50-150 ):(5‑15):(0.5‑1.5) of Bifidobacteria, Lactobacilli, Cocci and Bacillus. The present invention also provides a pharmaceutical composition for preventing and/or treating diseases caused by allergic reactions and a kit containing the pharmaceutical composition, which includes a probiotic composition and an allergen preparation, such as an acarid pathogenic inhibitor, etc. . The probiotic composition of the invention can significantly treat allergic reactions, effectively resist the invasion of external allergens, and reduce the probability of allergic diseases in the body. At the same time, the probiotic microecological preparation contained in the composition is harmless and has no side effects on the body.

Description

Antiallergic probiotic composition and use thereof

technical field

The invention belongs to the field of medicine, and relates to an antiallergic probiotic composition and its use in the preparation of medicines for preventing and/or treating diseases caused by allergic reactions, as well as a pharmaceutical composition comprising a probiotic composition and an allergen preparation and A kit comprising the pharmaceutical composition.

Background technique

At present, allergic diseases have become a common public health problem. In 2005, the epidemiological survey of allergic diseases in 30 countries by the World Allergy Organization (WAO) showed that 22% of the total population in these countries suffered from allergic diseases, such as allergic rhinitis, asthma, conjunctival Inflammation, eczema, food allergies, drug allergies, etc. Moreover, the incidence of allergic diseases is increasing year by year, resulting in a huge medical burden. How to effectively prevent and treat allergic diseases has become a current research hotspot.

Allergy refers to the body's inappropriate immune response to the originally harmless substances in the environment. It is a systemic disease and has the following characteristics: multiple attacks in children, repeated attacks, multiple allergens, and multiple organ involvement. The high incidence of allergic disease in industrialized developed countries is partly due to smaller household size and reduced incidence of infection in children, reducing exposure to microorganisms that play an important role in the immune maturation of the infant host. Common allergens are pollen, house dust mites, mold, medications, food and animal hair and dander. The most common allergic diseases are rhinitis, asthma and atopic dermatitis. The side effects caused by common allergic reactions include: edema, increased vascular permeability, tracheal obstruction, local congestion, nasal mucosal edema, nasal mucosal irritation, bronchoconstriction, increased mucus secretion, airway inflammation, vomiting, diarrhea, itching, Urticaria, rash.

Allergic asthma is a chronic inflammatory disease. Symptomatic treatment of allergic diseases can be achieved through the use of antihistamines, β-agonists, and corticosteroids to improve allergic symptoms. The mainstay of treatment for many patients with uncontrolled asthma is frequent oral corticosteroids, which are associated with numerous adverse effects. In many countries in the world, the probability of children suffering from asthma is about 10%. Children suffering from asthma is caused by multiple factors, and the disease is prone to repeated attacks, which seriously affects children's health and normal learning. At present, inhaled glucocorticoids and long-acting β-receptor agonists are often used to treat such children clinically, but the effect is still unsatisfactory, and more effective treatment options are urgently needed. Immunotherapy methods for allergic diseases include: subcutaneous immunotherapy, such as the American TO-204 dust mite extract preparation used for subcutaneous immunotherapy in the United States and Japan, Danish Antuda-house dust mite allergen preparation, German Alog- Mite allergen injection; sublingual immunotherapy, such as Changdi-Farmentum farinae drops, American OdaCtra household dust mite inhibitor; oral immunotherapy, such as ragweed, birch pollen preparations; intranasal local specific immunotherapy; lymphatic Intratube immunotherapy, etc.

Mites are one of the most common allergens in children in our country, which can cause allergic rhinitis and asthma. The treatment of allergic rhinitis and asthma includes avoiding contact with allergens, drug control, AIT, patient education, etc. The use of inhaled glucocorticoid-based drug treatment can quickly and effectively relieve symptoms, but cannot change the natural course of rhinitis and asthma caused by allergens. Specific immunotherapy (SIT) is currently the only etiological treatment that can affect the natural course of allergy, but there are still reports of adverse reactions to this treatment. For example, local reactions manifest as local redness and itching around the injection site; systemic reactions manifest as asthma-related symptoms (cough, wheezing, chest tightness, decreased peak expiratory flow rate), upper respiratory tract symptoms (itchy throat), vomiting, abdominal pain, etc. Mite allergen preparations are mite allergen active substances prepared from inactivated pure-bred cultures of mites, such as mite bodies, body fragments, mite excreta, larvae, and eggs. In vivo diagnosis or desensitization therapy of allergic diseases caused by allergens. At present, the commercially available dust mite allergen preparation Antoda is a preparation containing mite allergen extract, and its indication is for mild to moderate allergic asthma and/or allergic rhinitis with a history of house dust mite sensitization Desensitization of patients. Its treatment is carried out in two stages, that is, the initial treatment stage and the maintenance treatment stage. The initial treatment stage is 1-15 weeks, and the maintenance treatment stage is 17-51 weeks. The treatment cycle is too long, which is not conducive to clinical application. bring inconvenience. In addition, the use of dust mite allergen preparation Antuda treatment may produce some adverse reactions, such as local allergic reactions, systemic allergic reactions and anaphylactic shock, etc., such as local allergic reactions mainly manifested as local swelling at the drug injection site, redness and Itching; systemic allergic reaction is mildly manifested as hay fever symptoms, moderately manifested as urticaria or asthma; anaphylactic shock manifested as dyspnea, generalized urticaria, angioedema, laryngeal edema with wheezing, asthma, hypotension, Nausea, vomiting, diarrhea, abdominal pain, loss of consciousness, convulsions and coma. There is no report in the prior art that can reduce adverse reactions while treating anti-allergic reactions.

Lactobacillus belongs to the genus generally regarded as safe (G.R.A.S). It is currently known that Lactobacillus is a group of microorganisms that live in the body and are beneficial to the health of the host. They can maintain human health and regulate immune function. Specific strains of the Lactobacillus genus have been found to colonize the intestinal mucosa and assist in the maintenance of human and animal health, typically at concentrations above 1 billion live bacteria. In recent years, a variety of probiotic live bacterial preparations have been developed at home and abroad. The basic guiding principle is to use strains of normal physiological flora of humans or animals, after screening and cultivation, to make various dosage forms of live bacterial preparations through various channels. Then put it back into its original environment, so as to play its natural physiological role. The research on the use of probiotics in health foods and medicines started relatively late in my country, and Bifidobacteria began to be used in various health foods in the 1990s. The dosage forms of probiotics currently on the market are roughly divided into three categories, namely capsules, such as Meichangan, Juke, Zhengchangsheng, Beifeida, Livzon Changle, etc.; granules and powders, such as Mamiai, Bifikang, Changlekang, etc.; tablets, such as probiotics, Jinbifid, probiotic composition, lactobacillus, etc. These probiotic products are mainly used for intestinal-related digestive system diseases.

For some allergens, no drugs have been developed to treat the corresponding allergic diseases. The main treatment method is to use drugs to control allergic symptoms (that is, symptomatic medication); but drugs cannot change the process of allergic diseases, so Also can not fundamentally achieve the healing effect.

Contents of the invention

The purpose of the present invention is to provide a probiotic composition that is safe to use and has anti-allergic effects, which is used to resist the invasion of external allergens and reduce the probability of allergic diseases in the body. Allergen-induced specific IgE secreted by plasma cells is a prerequisite for type I hypersensitivity and is also an important evaluation index for the level of hypersensitivity. IgE binds to receptors on the surface of basophils or mast cells, leading to the release of hypersensitive reaction factors such as histamine, cytokines, chemokines, and lipid mediators, resulting in hypersensitivity reactions. We use ELISA and other immunological detection methods The IgE level in the test animals is detected to measure the allergic reaction state of the animals. IL-2 is a cytokine produced by T cells and mainly acts on T cells. Its main function is to promote the proliferation and differentiation of T cells, increase the synthesis of cytokines, and promote the development of regulatory T cells; it can lead to the proliferation and differentiation of NK cells. Activation, proliferation of B cells and antibody synthesis; can stimulate the activation of cytotoxic lymphocytes and macrophages, and is an important factor in mediating and maintaining the body's immune protection function, but it is not directly involved in type I hypersensitivity. We also studied the body level of IL-2 to investigate the effect of the drug on the body's protective immune function while treating allergic symptoms. After a large number of experimental screening and research, the inventors of the present invention unexpectedly found that the quadruple live bacteria preparation, which is currently mainly used for digestive system diseases such as diarrhea, constipation, and indigestion caused by intestinal flora imbalance, also has a significant anti-inflammatory effect. Allergic activity can be used to prevent and treat allergic diseases, and the use of such active probiotics without harmful ingredients will not produce side effects on the human body. In addition, the inventors of the present invention unexpectedly found that the combination of the quadruple live bacteria preparation and the allergen preparation can change the treatment process of allergic diseases, achieve better treatment of allergic reactions, and fundamentally achieve Healing effect; in addition, the anti-allergic treatment cycle of quadruple live bacteria preparation or allergen preparation can be greatly shortened by adopting the treatment means of the present invention; at the same time, the composition of the present invention can significantly reduce the occurrence of adverse reactions in the process of treating allergic reactions , provides great favorable conditions for anti-allergic treatment.

In one aspect, the present invention provides the use of a probiotic composition in the preparation of a medicament for preventing and/or treating diseases caused by allergic reactions, wherein the probiotic composition comprises Bifidobacterium, Lactobacillus, Coccus and Bacillus.

According to an embodiment of the use of the present invention, the probiotic composition may comprise bifidobacteria with a ratio of (50-150):(50-150):(5-15):(0.5-1.5) of the number of live bacteria. Bacilli, Lactobacilli, Cocci and Bacillus.

According to a preferred embodiment of the use of the present invention, the probiotic composition comprises bifidobacteria with a ratio of (80-120):(80-120):(8-12):(0.8-1.2) of the number of live bacteria. Bacilli, Lactobacilli, Cocci and Bacillus.

According to a preferred embodiment of the use of the present invention, the total number of viable bacteria contained in the probiotic composition is not less than 1×10 ⁵ cfu/g. More preferably, the viable count of Bifidobacteria in the probiotic composition is at least 1×10 ⁶ cfu/g; the viable count of Lactobacillus is at least 1×10 ⁶ cfu/g; the viable count of cocci is at least It is 1×10 ⁶ cfu/g; and the viable count of Bacillus is at least 1×10 ⁵ cfu/g. According to an embodiment of the use of the present invention, the bifidobacterium can be selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve One or more of Bifidobacterium breve, Bifidobacterium infantis or Bifidobacterium longum. According to a preferred embodiment of the use of the present invention, the bifidobacterium is Bifidobacterium infantis.

According to the embodiments of the purposes of the present invention, the lactobacillus can be selected from Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus casei (Lactobacillus casei), Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus delbrueckii subsp. Lactobacillus delbrueckii subsp. Bulgaricus (Lactobacillus bulgaricus)), Lactobacillus delbrueckii subsp. Lactis, Lactobacillus fermentium, Lactobacillus gasseri, Lactobacillus helveticus Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, salivary milk One or more of Lactobacillus salivarius, Lactobacillus sakei or Lactobacillus curvatus. According to a preferred embodiment of the use of the present invention, the lactobacillus is Lactobacillus acidophilus.

According to an embodiment of the use of the present invention, the coccus may be selected from Enterococcus faecalis; Streptococcus, such as Streptococcus thermophilus; Lactococcus, such as Lactococcus lactis Lactococcus Lactis subsp.lactis, Lactococcus Lactis subsp.cremoris, Lactococcus Lactis subsp.diacetylactis; Leuconostoc, e.g. Leuconostoc mesenteroides subsp. mesenteroides; Pediococcus, such as Pediococcus acidilactici, Pediococcus pentosaceus; or Staphylococcus, such as Staphylococcus bovis One or more of Staphylococcus vitulinus, Staphylococcus xylosus, Staphylococcus carnosus. According to a preferred embodiment of the use of the present invention, the coccus is Enterococcus faecalis.

According to an embodiment of the use of the present invention, the Bacillus is selected from one or more of Bacillus cereus or Bacillus coagulans. According to a preferred embodiment of the use of the present invention, the Bacillus is Bacillus cereus.

Preferably, the strain preservation number of the Bifidobacterium infantis is CGMCC No.0460.1; the strain preservation number of the Lactobacillus acidophilus is CGMCC No.0460.2; the strain preservation number of the Enterococcus faecalis is CGMCC No. .0460.3; the strain preservation number of the Bacillus cereus is CGMCC No.0460.4.

According to a specific embodiment of the use of the present invention, the probiotic composition comprises a baby with a ratio of (80-120):(80-120):(8-12):(0.8-1.2) of the number of viable bacteria Bifidobacterium, Lactobacillus acidophilus, Enterococcus faecalis and Bacillus cereus.

According to the embodiment of the use of the present invention, the disease caused by the allergic reaction may be selected from one or more of anaphylaxis, wheal, allergic rhinitis, allergic asthma or food allergy.

According to an embodiment of the use according to the invention, the allergic reaction may be caused by one or more allergens selected from the group consisting of pollen allergens, milk allergens, venom allergens, egg allergens, weed allergens, pasture allergens Allergens, tree allergens, shrub allergens, flower allergens, vegetable allergens, grain allergens, fungal allergens, fruit allergens, berry allergens, nut allergens, seed allergens, soybean allergens, fish allergens , shellfish allergens, seafood allergens, meat allergens, spice allergens, insect allergens, mite allergens (e.g. house dust mite), mold allergens, animal allergens, pigeon tick allergens, worm allergens, Soft coral allergens, animal dander allergens, nematode allergens, or rubber tree allergens.

According to an embodiment of the use of the present invention, the allergic reaction may be caused by one or more allergens selected from the group consisting of inhalant allergens, such as pollen particles, dust mite excreta, fungal hyphae and spores, Insect venom, animal fur, etc.; food allergens such as milk, eggs, fish, shrimp, crab shells and other food proteins or peptides; enzymes such as cysteine protein in dust mites, etc.

In a preferred embodiment of the use according to the invention, the allergic reaction is caused by a mite allergen.

According to an embodiment of the use of the present invention, the probiotic composition may also include Propionibacterium (Propionibacterium), such as Propionibacterium freudenreichii subsp. One or more of Propionibacterium acidipropionici), wherein the probiotic composition comprises a ratio of viable bacteria numbers of (50-150):(50-150):(2.5-7.5):(0.5-1.5): (2.5-7.5) Bifidobacteria, Lactobacilli, Cocci, Bacillus and Propionibacterium. According to a preferred embodiment of the purposes of the present invention, the probiotic composition comprises a ratio of the number of live bacteria of (80-120):(80-120):(8-12):(0.8-1.2):(4 -6) Bifidobacteria, Lactobacilli, Cocci, Bacillus and Propionibacterium.

According to the embodiment of the use of the present invention, the probiotic composition may further comprise Kluyveromyces (Kluyveromyces), such as one or more of Kluyveromyces marxianus (Kluyveromyces marxianus), wherein the probiotic The bacteria composition comprises bifidobacteria, lactobacilli, cocci, and spores with a ratio of (50-150):(50-150):(5-15):(0.25-0.75):(0.25-0.75) of the number of live bacteria Bacillus and Kluyveromyces. According to a preferred embodiment of the purposes of the present invention, the probiotic composition comprises a ratio of the number of live bacteria of (80-120):(80-120):(8-12):(0.8-1.2):(0.4 -0.6) of Bifidobacteria, Lactobacilli, Cocci, Bacillus and Kluyveromyces.

According to an embodiment of the use according to the invention, the medicament may also comprise an allergen preparation.

According to an embodiment of the use of the present invention, the ratio of the probiotic composition to the allergen preparation may be (1×10 ⁵ to 1×10 ⁹ cfu):(20 to 2×10 ⁵ SQ-U), Preferably (1×10 ⁶ to 1×10 ⁸ cfu): (1000 to 2×10 ⁵ SQ-U).

According to an embodiment of the use of the present invention, the allergen preparation may be selected from microbial allergen preparations, mite allergen preparations, parasite allergen preparations, pollen allergen preparations, xenogeneic animal serum allergens One or more of preparations, drug allergen preparations or chemical reagent allergen preparations. According to a preferred embodiment of the use according to the invention, the allergen preparation is a mite allergen preparation, eg a house dust mite allergen preparation or a dust mite allergen preparation.

According to a preferred embodiment of the use of the present invention, the house dust mite allergen preparation is a mite allergen extract, for example, the extraction of mite body, body fragments, mite excrement, larvae and/or eggs things.

According to a preferred embodiment of the use according to the invention, said probiotic composition is placed separately from the allergen preparation. Further preferably, the probiotic composition is an oral preparation, and the allergen preparation is an injection.

On the other hand, the present invention also provides a pharmaceutical composition for preventing and/or treating diseases caused by allergic reactions, which comprises a probiotic composition and an allergen preparation, wherein the probiotic composition comprises the number of live bacteria The ratio is (50-150):(50-150):(5-15):(0.5-1.5) of Bifidobacterium, Lactobacillus, Coccus and Bacillus; and said probiotic composition is combined with allergen The ratio of the preparation is (1×10 ⁵ to 1×10 ⁹ cfu):(20 to 2×10 ⁵ SQ-U).

According to a preferred embodiment of the pharmaceutical composition of the present invention, the probiotic composition comprises a ratio of the number of viable bacteria (80-120): (80-120): (8-12): (0.8-1.2) Bifidobacteria, Lactobacilli, Cocci and Bacillus.

According to a preferred embodiment of the pharmaceutical composition of the present invention, the total number of viable bacteria contained in the probiotic composition is not less than 1×10 ⁵ cfu/g. More preferably, the viable count of Bifidobacteria in the probiotic composition is at least 1×10 ⁶ cfu/g; the viable count of Lactobacillus is at least 1×10 ⁶ cfu/g; the viable count of cocci is at least It is 1×10 ⁶ cfu/g; and the viable count of Bacillus is at least 1×10 ⁵ cfu/g. According to the embodiments of the pharmaceutical composition of the present invention, the bifidobacterium can be selected from Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve One or more of Bifidobacterium breve, Bifidobacterium infantis or Bifidobacterium longum. According to a preferred embodiment of the pharmaceutical composition of the present invention, the bifidobacterium is Bifidobacterium infantis.

According to the embodiment of the pharmaceutical composition of the present invention, the lactobacillus can be selected from Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus casei (Lactobacillus casei), Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus delbrueckii Lactobacillus delbrueckii subsp. Bulgaricus (Lactobacillus bulgaricus)), Lactobacillus delbrueckii subsp. Lactis, Lactobacillus fermentium, Lactobacillus gasseri, Switzerland Lactobacillus (Lactobacillus shelveticus), Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, One or more of Lactobacillus salivarius, Lactobacillus sakei, or Lactobacillus curvatus. According to a preferred embodiment of the pharmaceutical composition of the present invention, the lactobacillus is Lactobacillus acidophilus.

According to an embodiment of the pharmaceutical composition of the present invention, the coccus may be selected from Enterococcus faecalis; Streptococcus, such as Streptococcus thermophilus; Lactococcus, such as lactic acid milk Lactococcus Lactis subsp.lactis, Lactococcus Lactis subsp.cremoris, Lactococcus Lactis subsp.diacetylactis; Leuconostoc, e.g. Leuconostoc mesenteroides subsp. mesenteroides; Pediococcus, such as Pediococcus acidilactici, Pediococcus pentosaceus; or Staphylococcus, such as Staphylococcus bovis One or more of Staphylococcus vitulinus, Staphylococcus xylosus, Staphylococcus carnosus. According to a preferred embodiment of the pharmaceutical composition of the present invention, the coccus is Enterococcus faecalis.

According to the embodiment of the pharmaceutical composition of the present invention, the Bacillus is selected from one or more of Bacillus cereus or Bacillus coagulans. According to a preferred embodiment of the pharmaceutical composition of the present invention, the Bacillus is Bacillus cereus.

According to a preferred embodiment of the pharmaceutical composition of the present invention, the probiotic composition comprises a ratio of the number of live bacteria of (80-120):(80-120):(8-12):(0.8-1.2) Bifidobacterium infantis, Lactobacillus acidophilus, Enterococcus faecalis and Bacillus cereus; and the ratio of the probiotic composition to the allergen preparation is 1×10 ⁶ to 1×10 ⁸ cfu): (1000 to 2×10 ⁵ SQ-U).

Preferably, the strain preservation number of the Bifidobacterium infantis is CGMCC No.0460.1; the strain preservation number of the Lactobacillus acidophilus is CGMCC No.0460.2; the strain preservation number of the Enterococcus faecalis is CGMCC No. .0460.3; the strain preservation number of the Bacillus cereus is CGMCC No.0460.4.

According to the embodiment of the pharmaceutical composition of the present invention, the disease caused by the allergic reaction may be selected from one or more of anaphylaxis, wheal, allergic rhinitis, allergic asthma or food allergy.

According to an embodiment of the pharmaceutical composition of the present invention, the allergic reaction may be caused by one or more allergens selected from the group consisting of pollen allergens, milk allergens, venom allergens, egg allergens, weed allergens , pasture allergens, tree allergens, shrub allergens, flower allergens, vegetable allergens, grain allergens, fungal allergens, fruit allergens, berry allergens, nut allergens, seed allergens, soybean allergens, fish Allergens, shellfish allergens, seafood allergens, meat allergens, spice allergens, insect allergens, mite allergens (eg house dust mite), mold allergens, animal allergens, pigeon tick allergens, worm allergies allergens, soft coral allergens, animal dander allergens, nematode allergens, or rubber tree allergens.

According to an embodiment of the pharmaceutical composition of the present invention, the probiotic composition may also include Propionibacterium (Propionibacterium), such as Propionibacterium freudenreichiisubsp. One or more of bacillus (Propionibacterium acidipropionici), wherein the probiotic composition comprises a ratio of the number of viable bacteria (50-150):(50-150):(2.5-7.5):(0.5-1.5 ): (2.5-7.5) of Bifidobacteria, Lactobacilli, Cocci, Bacillus and Propionibacterium. In a preferred embodiment of the pharmaceutical composition according to the present invention, the probiotic composition comprises a ratio of the number of live bacteria of (80-120):(80-120):(8-12):(0.8-1.2) : (4-6) Bifidobacteria, Lactobacilli, Cocci, Bacillus and Propionibacterium.

According to an embodiment of the pharmaceutical composition of the present invention, the probiotic composition may further comprise Kluyveromyces (Kluyveromyces), such as one or more of Kluyveromyces marxianus (Kluyveromyces marxianus), wherein the The probiotic composition comprises Bifidobacterium, Lactobacillus, Coccus with a ratio of (50-150):(50-150):(5-15):(0.25-0.75):(0.25-0.75) of the number of viable bacteria , Bacillus and Kluyveromyces. In a preferred embodiment of the pharmaceutical composition according to the present invention, the probiotic composition comprises a ratio of the number of live bacteria of (80-120):(80-120):(8-12):(0.8-1.2) : (0.4-0.6) of Bifidobacteria, Lactobacilli, Cocci, Bacillus and Kluyveromyces.

According to an embodiment of the pharmaceutical composition of the present invention, the allergen preparation may be selected from microbial allergen preparations, mite allergen preparations, parasite allergen preparations, pollen allergen preparations, xenobiotic serovar One or more of allergen preparations, drug allergen preparations or chemical reagent allergen preparations. According to a preferred embodiment of the pharmaceutical composition of the present invention, the allergen preparation is a mite allergen preparation, such as a house dust mite allergen preparation or a dust mite allergen preparation.

In a preferred embodiment of the pharmaceutical composition according to the present invention, the probiotic composition is placed separately from the allergen preparation. Further preferably, the probiotic composition is an oral preparation, and the allergen preparation is an injection.

In another aspect, the present invention also provides a kit for preventing and/or treating diseases caused by allergic reactions, which comprises the pharmaceutical composition described in the present invention.

According to a preferred embodiment of the kit of the present invention, the probiotic composition and the allergen preparation are placed separately. Further preferably, the probiotic composition is an oral preparation, and the allergen preparation is an injection.

The invention successfully expands the quadruple or more probiotic composition containing bifidobacterium, lactobacillus, coccus or bacillus from the field of digestive system to a new field of anti-allergy treatment, which has important clinical application significance and value.

The anti-allergic probiotic composition of the present invention has low content of quadruple live bacteria, good therapeutic effect, can significantly treat allergic reactions, and reduce the probability of occurrence of allergic diseases in a body. At the same time, the components of the probiotic microecological preparation contained in the probiotic composition are harmless and have no side effects on the body. More importantly, the present invention also uses the quadruple live bacteria preparation in combination with the allergen preparation, which can change the course of allergic diseases, significantly improve the efficacy of immunotherapy, and realize the best treatment for allergic reactions, which can fundamentally In addition, the treatment method of the present invention can greatly shorten the anti-allergic treatment period of the quadruple live bacteria preparation or allergen preparation; meanwhile, the composition of the present invention can greatly alleviate the existing Allergic symptoms under the treatment plan of allergen preparations, thereby reducing the occurrence of adverse reactions. In addition, the composition of the present invention can significantly increase the level of IL-2 while treating allergic reactions, thereby enhancing the immune protection ability of the body, eliminating the side effects of lowering immunity that are often accompanied by existing allergy treatment schemes, and providing clinical anti-allergic diseases. It provides a valuable composition for the treatment of and immunity enhancement.

Description of drawings

Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Figure 1 shows the changes in Derp-specific IgE in mice after modeling;

Figure 2 shows the change of Derp-specific IgE in mice before/after 4 weeks of treatment;

Figure 3 shows the change of Derp-specific IgE in mice before/after 12 weeks of treatment;

Figure 4 shows the changes of Derp-specific IgE in mice before and after challenge in different treatment cycles;

Figure 5 shows the allergic symptom score of mice treated for 12 weeks;

Figure 6 shows the changes of Derp-specific cytokines secreted by spleen lymphocytes of mice treated for 12 weeks.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention.

The reagents and preparation methods used in the following examples are described below:

1. House dust mite allergen preparation (AIT): Antuda, manufacturer: ALK-Abello A/S. Ingredients: mite allergen extract (Mites Allergen Product), excipients: aluminum hydroxide, sodium chloride, sodium bicarbonate, phenol 5mg/ml, water for injection.

Dosage:

The dose of the house dust mite allergen preparation per mouse is 10000 SQ-U (specification 100000 SQ-U/ml), which is approximately equal to one-tenth of the clinical human dose.

2. Mite allergen (Derp), manufacturer Zhejiang Wowu Biotechnology Co., Ltd.

3. The preparation method of single or multiple live bacteria preparations:

(1) Dosage of quadruple probiotic suspension in the experimental group:

The strain preservation number of Bifidobacterium infantis is CGMCC No.0460.1; the strain preservation number of Lactobacillus acidophilus is CGMCC No.0460.2; the strain preservation number of Enterococcus faecalis is CGMCC No.0460.3; the strain preservation number of Bacillus cereus The species deposit number is CGMCC No.0460.4.

The administration concentration of Bifidobacterium infantis (Y) was 1.36×10 ⁶ cfu per mouse; the administration concentration of Lactobacillus acidophilus (S) was 1.36×10 ⁶ cfu per mouse; The administration concentration of cocci (F) was 1.36×10 ⁵ cfu per mouse; the administration concentration of Bacillus cereus (L) was 1.36×10 ⁴ cfu per mouse (the dosage was approximately equal to One-tenth of the clinical human dose).

Embodiment 1 establishes mouse allergy model

SPF grade Balb/c mice, female, aged 5-8 weeks were selected for the experiment. Modeling was performed using mite allergens. After the mice were adaptively fed, they were sensitized with Derp (20 μg/ml, 0.1 ml/mouse) by subcutaneous injection. The sensitization cycle was sensitization every other day, a total of 3 times. After the mice were sensitized, the interval was 1 week. The mice were challenged with Derp (80 μg/ml, 40 μl/mouse) in the form of nasal drops. The stimulation cycle was 1 time/day for 7 consecutive days.

After the modeling was completed, blood was collected from the face of the mice, centrifuged, and the serum was separated. The ELISA method was used to detect the changes of Derp-specific IgE in the serum. The results are shown in Table 1 and Figure 1.

Table 1 Changes of Derp-specific IgE in mice after modeling (MEAN±SD)

group MEAN SD no normal control group 0.08 0.00 10 Model control group 0.64 0.16 10

The results showed that after the mice were sensitized and challenged by Derp, compared with the normal control group, the serum Derp IgE level of the model control group was significantly increased (P<0.001), indicating that the mite allergen-induced mouse allergy model was successfully established .

Embodiment 2 4 weeks treatment experiment

1. Establish a mouse allergy model according to the method in Example 1.

2. The model animals were randomly divided into the model control group, the AIT group (Antidal house dust mite allergen preparation), the SLK group (quadruple probiotic powder suspension), and the SLK+AIT group (combined treatment group: four Probiotics powder suspension + antidermatitis house dust mite allergen preparation), 10 mice in each group, and another 10 normal mice were selected as the normal control group, and were treated as follows:

a. Subcutaneous injection: normal saline (control group) or house dust mite allergen preparation (experimental group), 0.1ml/monkey (specification 100000SQ-U/ml), once a week, continuous treatment for 4 weeks;

b. Gastrointestinal administration: normal saline (control group) or probiotic suspension (experimental group), 0.2ml/rat, once a day, for 4 weeks of continuous treatment.

After 4 weeks of desensitization treatment, ELISA method was used to detect the changes of Derp-specific IgE in serum of mice in each group, and the results are shown in Table 2 and Figure 2.

The results showed that after 4 weeks, compared with the normal control group, the serum level of Derp IgE in the model control group remained high (P<0.001) (see Figure 2).

After 4 weeks of treatment, mice in each group were challenged with Derp nasal drops. The results showed that after stimulation, except for the normal control group, the serum Derp-specific IgE of mice in other groups all increased significantly (P<0.001). The IgE ratio after challenge changed 2.43 times compared with that before challenge, the IgE ratio changed 3.28 times after challenge in SLK group, and the IgE ratio changed 2.22 times after challenge in SLK+AIT group. Compared with the model control group, the serum Derp IgE of the mice in the AIT group (P<0.01), SLK group (P<0.05), and SLK+AIT group (P<0.01) all decreased significantly (see Figure 2). It can be seen from Table 2 that the stimulation was carried out after 4 weeks of desensitization treatment. It can be seen from the change levels of IgE in each experimental group that the AIT group, SLK group, and SLK+AIT group all had certain desensitization effects, and the SLK+AIT group The effect is better than that of AIT group and SLK group.

Table 2 Changes of Derp-specific IgE in mice after 4 weeks of treatment (MEAN ± SD)

Note: (1) Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001; (2) Compared with before stimulation, #P<0.05, ##P<0.01 , ###P<0.001.

Embodiment 3 12 weeks treatment experiment

1. Establish a mouse allergy model according to the method in Example 1.

2. The model animals were randomly divided into model control group, AIT group, SLK, SLK+AIT group, 10 in each group, and another 10 normal mice were selected as the normal control group, and treated as follows:

a. Subcutaneous injection: normal saline (control group) or house dust mite allergen preparation (experimental group), 0.1ml/monkey (specification 100000SQ-U/ml), once a week, continuous treatment for 12 weeks;

b. Gastrointestinal administration: normal saline (control group) or probiotic suspension (experimental group), 0.2ml/rat, once a day, for 12 consecutive weeks.

3. Treatment outcome

(1) Changes of Derp-specific IgE in mice after 12 weeks of treatment

After 12 weeks of desensitization treatment, ELISA method was used to detect the changes of Derp-specific IgE in serum of mice in each group, and the results are shown in Table 3 and Figure 3.

After 12 weeks of treatment, mice in each group were challenged with Derp nasal drops. The results showed that after challenge, except for the normal control group and the SLK+AIT group, the serum Derp-specific IgE of the mice in the other groups all increased significantly (P<0.001) (Figure 3). Among them, the IgE ratio of the model control group changed by 3.71 times compared with that before the challenge, the IgE ratio of the AIT group changed by 2.04 times after the challenge, the IgE ratio of the SLK group changed by 1.83 times after the challenge, and the SLK+AIT group changed by 1.83 times after the challenge. The IgE ratio changed by 1.57 times. Compared with the model control group, the serum Derp-specific IgE of mice in SLK group and SLK+AIT group was significantly decreased (SLK group: P<0.01; SLK+AIT group: P<0.001) (Figure 3). It can be seen from the stimulation effects of each group after 12 weeks of treatment (Table 3), that the AIT group, SLK group, and SLK+AIT group all have different degrees of desensitization effects, and the SLK+AIT group can be seen from the specific IgE level. It can be seen that the desensitization effect has been achieved; the desensitization effect of the SLK group is slightly better than that of the AIT group.

Table 3 Changes of Derp-specific IgE in mice after 12 weeks of treatment (MEAN ± SD)

Note: (1) Compared with the model control group, *P<0.05, **P<0.01, ***P<0.001; (2) Compared with before stimulation, #P<0.05, ##P<0.01 , ###P<0.001.

Figure 4 shows the changes of Derp-specific IgE in mice before and after challenge in different treatment cycles of 4 weeks and 12 weeks of desensitization treatment. It can be seen from the upper graph of Figure 4 (A: before stimulation) that with the continuous extension of the treatment cycle, the Derp-specific IgE in the AIT group and the SLK group showed an upward trend before the stimulation, indicating that the therapeutic effect was not achieved; IgE remained stable during treatment, indicating that the therapeutic effect was achieved. As can be seen from the lower panel of Figure 4 (B: after stimulation), after stimulation, the IgE in the mice of the AIT group still showed a rising trend during the treatment; while the IgE in the mice of the SLK group and SLK+AIT group appeared during the treatment. reduce. After 12 weeks of treatment, compared with the model control group, the serum Derp-specific IgE of the mice in the SLK+AIT group decreased significantly (P<0.01). It shows that the combined treatment of SLK and AIT can help alleviate the increase of IgE.

(2) Scores of allergic symptoms in mice after 12 weeks of treatment

After 12 weeks of treatment, the mice in each group were challenged with nasal drops for 7 consecutive days, and the allergic symptoms of the mice were counted on the 7th day. Figure 5 shows the allergic symptom scores of mice treated for 12 weeks. It can be seen from Figure 5 that compared with the model control group, the allergic symptom score of the SLK+AIT group was significantly reduced (P<0.01), and the scratching and wheezing of the mice were significantly reduced; while the allergic reaction symptoms of the allergen preparation AIT group were significantly lower than those of the four The combined live bacteria preparation SLK group and SLK+AIT group were serious, so the quadruple live bacteria preparation SLK group and SLK+AIT group can significantly reduce the occurrence of allergic symptoms, thereby reducing the incidence of adverse reactions.

(3) After 12 weeks of treatment, mouse spleen lymphocytes secrete Derp-specific IL-2

After 12 weeks of treatment, the mice were plucked from the eyeballs to collect blood, and sacrificed by neck dislocation to obtain splenic lymphocytes. The secretion of Derp-specific IL-2 by mouse spleen lymphocytes was detected by enzyme-linked immunospot (ELISPOT), and the results are shown in FIG. 6 . It can be seen from (A) of Figure 6 that after the combined treatment of SLK and AIT, the level of Derp-specific IL-2 secreted by mouse spleen lymphocytes can be significantly increased. The results showed that the SLK group and SLK+AIT group could achieve desensitization therapeutic effect by regulating the balance of Th1/Th2.

The above test results show that, in terms of therapeutic effect, using the probiotic composition of the present invention or the probiotic composition of the present invention in combination with mite allergen preparations can effectively treat or alleviate allergic reactions. At the same time, the combination of probiotic composition of the present invention Compared with mite allergen preparation alone, the mite allergen preparation achieves the therapeutic effect faster and the treatment is more convenient. In addition, in terms of allergic symptom scores, the use of the probiotic combination of the present invention or the combination of the probiotic composition of the present invention in combination with mite allergen preparations can significantly reduce the occurrence of allergic symptoms and thus reduce the incidence of adverse reactions. The probiotic composition of the present invention or its combination with the mite allergen preparation can achieve better and faster therapeutic effect than the mite allergen preparation alone, and is expected to reduce the treatment expenses of patients.

Claims (15)

1. Use of a probiotic composition comprising bifidobacteria, lactobacilli, cocci and bacilli for the manufacture of a medicament for the prevention and/or treatment of diseases caused by allergic reactions.

2. Use according to claim 1, wherein the probiotic composition comprises live bacteriaThe total number is not less than 1×10 ⁵ cfu/g; preferably, the viable count of bifidobacteria in the probiotic composition is at least 1 x 10 ⁶ cfu/g; the viable count of Lactobacillus is at least 1×10 ⁶ cfu/g; the number of living bacteria of the coccus is at least 1×10 ⁶ cfu/g; and the viable count of the bacillus is at least 1 multiplied by 10 ⁵ cfu/g。

3. The use according to claim 1, wherein the bifidobacterium is selected from one or more of bifidobacterium adolescentis, bifidobacterium animalis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium infantis or bifidobacterium longum; preferably, the bifidobacterium is bifidobacterium infantis;

the lactobacillus is selected from one or more of lactobacillus acidophilus, lactobacillus casei, lactobacillus crispatus, lactobacillus bulgaricus, lactobacillus delbrueckii subspecies lactis, lactobacillus fermentum, lactobacillus grignard, lactobacillus helveticus, lactobacillus johnsonii, lactobacillus paracasei, lactobacillus plantarum, lactobacillus reuteri, lactobacillus rhamnosus, lactobacillus salivarius, lactobacillus sake or lactobacillus curvatus; preferably, the lactobacillus is lactobacillus acidophilus;

The coccus is selected from enterococcus faecalis; streptococci, such as streptococcus thermophilus; lactococcus, such as lactococcus lactis subspecies lactis, lactococcus lactis subspecies cremoris, lactococcus lactis diacetyl subspecies lactis; leuconostoc, such as Leuconostoc mesenteroides subspecies; pediococcus such as Pediococcus acidilactici, pediococcus pentosaceus; staphylococci such as one or more of staphylococcus calf, staphylococcus xylosus, staphylococcus sarcodactylis; preferably, the coccus is enterococcus faecalis;

the bacillus is selected from one or more of bacillus cereus and bacillus coagulans; preferably, the bacillus is bacillus cereus;

preferably, the bifidobacterium infantis has a collection number of CGMCC No.0460.1; the lactobacillus acidophilus has a strain preservation number of CGMCC No.0460.2; the preservation number of the enterococcus faecalis is CGMCC No.0460.3; the bacillus cereus has a strain preservation number of CGMCC No.0460.4.

4. The use according to any one of claims 1 to 3, wherein the probiotic composition comprises bifidobacteria, lactobacilli, cocci and bacilli in a ratio of viable bacteria number of (50-150): (50-150): (5-15): (0.5-1.5);

preferably, the probiotic composition comprises bifidobacteria, lactobacilli, cocci and bacilli in a ratio of the number of viable bacteria of (80-120): (80-120): (8-12): (0.8-1.2);

More preferably, the probiotic composition comprises bifidobacterium infantis, lactobacillus acidophilus, enterococcus faecalis and bacillus cereus in a ratio of the number of viable bacteria of (80-120): (80-120): (8-12): (0.8-1.2).

5. The use according to any one of claims 1 to 4, wherein the probiotic composition further comprises propionibacteria, such as one or more of the sub-species propionibacterium freudenreichii, propionibacterium propionicum, wherein the probiotic composition comprises bifidobacterium, lactobacillus, cocci, bacillus and propionibacteria in a ratio of viable bacteria count of (50-150): 2.5-7.5: 0.5-1.5: 2.5-7.5; preferably, the probiotic composition comprises bifidobacteria, lactobacilli, cocci, bacilli and propionibacteria in a ratio of the number of viable bacteria of (80-120): (80-120): (8-12): (0.8-1.2): (4-6); and/or

The probiotic composition further comprises kluyveromyces, such as one or more of kluyveromyces marxianus, wherein the probiotic composition comprises bifidobacteria, lactobacilli, cocci, bacilli and kluyveromyces in a ratio of viable count of (50-150): (50-150): (5-15): (0.25-0.75): (0.25-0.75); preferably, the probiotic composition comprises bifidobacteria, lactobacilli, cocci, bacilli and kluyveromyces in a ratio of the number of viable bacteria of (80-120): (80-120): (8-12): (0.8-1.2): (0.4-0.6).

6. The use according to any one of claims 1 to 5, wherein the medicament further comprises an allergen preparation selected from one or more of a microbial allergen preparation, a mite allergen preparation, a parasite allergen preparation, a pollen allergen preparation, a xenogeneic animal serum allergen preparation, a pharmaceutical allergen preparation or a chemical agent allergen preparation; most preferably, the allergen preparation is a mite allergen preparation, such as a house dust mite allergen preparation or a dust mite allergen preparation;

preferably, the ratio of the probiotic composition to allergen preparation is (1×10) ⁵ Up to 1X 10 ⁹ cfu) (20 to 2X 10 ⁵ SQ-U), preferably (1X 10) ⁶ Up to 1X 10 ⁸ cfu) (1000 to 2X 10 ⁵ SQ-U)。

7. The use according to any one of claims 1 to 6, wherein the disease caused by an allergic reaction is selected from one or more of systemic allergic disease, wind mass, allergic rhinitis, allergic asthma or food allergy;

preferably, the allergic reaction is caused by one or more allergens selected from the group consisting of: pollen allergens, milk allergens, venom allergens, egg allergens, weed allergens, pasture allergens, tree allergens, shrub allergens, flower allergens, vegetable allergens, cereal allergens, fungal allergens, fruit allergens, berry allergens, nut allergens, seed allergens, soybean allergens, fish allergens, shellfish allergens, seafood allergens, meat allergens, spice allergens, insect allergens, mite allergens, mould allergens, animal allergens, pigeon ticks allergens, helminth allergens, soft coral allergens, animal dander allergens, nematode allergens or rubber tree allergens; preferably, the allergic reaction is caused by a mite allergen.

8. A pharmaceutical composition for preventing and/or treating a disease caused by an allergic reaction, comprising a probiotic composition and an allergen preparation, wherein the probiotic composition comprises bifidobacteria, lactobacilli, cocci and bacilli in a ratio of viable bacteria count of (50-150): 5-15): 0.5-1.5.

9. The pharmaceutical composition of claim 8, wherein the allergen formulation is selected from one or more of a microbial allergen formulation, a mite allergen formulation, a parasite allergen formulation, a pollen allergen formulation, a xenogeneic animal serum allergen formulation, a pharmaceutical allergen formulation, or a chemical agent allergen formulation; most preferably, the allergen preparation is a mite allergen preparation, such as a house dust mite allergen preparation or a dust mite allergen preparation;

preferably, the ratio of the probiotic composition to the allergen preparation is (1×10) ⁵ Up to 1X 10 ⁹ cfu) (20 to 2X 10 ⁵ SQ-U), more preferably the ratio of the probiotic composition to the allergen preparation is (1X 10) ⁶ Up to 1X 10 ⁸ cfu) (1000 to 2X 10 ⁵ SQ-U)。

10. The pharmaceutical composition of claim 8, wherein the probiotic composition comprises a total number of viable bacteria of not less than 1 x 10 ⁵ cfu/g; preferably, the viable count of bifidobacteria in the probiotic composition is at least 1 x 10 ⁶ cfu/g; the viable count of Lactobacillus is at least 1×10 ⁶ cfu/g; the number of living bacteria of the coccus is at least 1×10 ⁶ cfu/g; and the viable count of the bacillus is at least 1 multiplied by 10 ⁵ cfu/g。

11. The pharmaceutical composition of claim 8, wherein the bifidobacterium is selected from one or more of bifidobacterium adolescentis, bifidobacterium animalis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium infantis, or bifidobacterium longum; preferably, the bifidobacterium is bifidobacterium infantis;

the lactobacillus is selected from one or more of lactobacillus acidophilus, lactobacillus casei, lactobacillus crispatus, lactobacillus bulgaricus, lactobacillus delbrueckii subspecies lactis, lactobacillus fermentum, lactobacillus grignard, lactobacillus helveticus, lactobacillus johnsonii, lactobacillus paracasei, lactobacillus plantarum, lactobacillus reuteri, lactobacillus rhamnosus, lactobacillus salivarius, lactobacillus sake or lactobacillus curvatus; preferably, the lactobacillus is lactobacillus acidophilus;

the coccus is selected from enterococcus faecalis; streptococci, such as streptococcus thermophilus; lactococcus, such as lactococcus lactis subspecies lactis, lactococcus lactis subspecies cremoris, lactococcus lactis diacetyl subspecies lactis; leuconostoc, such as Leuconostoc mesenteroides subspecies; pediococcus such as Pediococcus acidilactici, pediococcus pentosaceus; staphylococci such as one or more of staphylococcus calf, staphylococcus xylosus, staphylococcus sarcodactylis; preferably, the coccus is enterococcus faecalis;

The bacillus is selected from one or more of bacillus cereus and bacillus coagulans; preferably, the bacillus is bacillus cereus;

preferably, the bifidobacterium infantis has a collection number of CGMCC No.0460.1; the lactobacillus acidophilus has a strain preservation number of CGMCC No.0460.2; the preservation number of the enterococcus faecalis is CGMCC No.0460.3; the bacillus cereus has a strain preservation number of CGMCC No.0460.4.

12. The pharmaceutical composition according to any one of claims 8 to 11, wherein the probiotic composition comprises bifidobacteria, lactobacilli, cocci and bacilli in a ratio of viable bacteria number of (80-120): (80-120): (8-12): (0.8-1.2); preferably, the probiotic composition comprises bifidobacterium infantis, lactobacillus acidophilus, enterococcus faecalis and bacillus cereus in a ratio of the number of viable bacteria of (80-120): (80-120): (8-12): (0.8-1.2).

13. The pharmaceutical composition according to any one of claims 8 to 12, wherein the probiotic composition further comprises propionibacteria, such as one or more of the sub-species propionibacterium freudenreichii, propionibacterium propionicum, wherein the probiotic composition comprises bifidobacterium, lactobacillus, cocci, bacillus and propionibacteria in a ratio of viable bacteria count of (50-150): 2.5-7.5: 0.5-1.5: 2.5-7.5; preferably, the probiotic composition comprises bifidobacteria, lactobacilli, cocci, bacilli and propionibacteria in a ratio of the number of viable bacteria of (80-120): (80-120): (8-12): (0.8-1.2): (4-6); and/or

The probiotic composition further comprises kluyveromyces, such as one or more of kluyveromyces marxianus, wherein the probiotic composition comprises bifidobacteria, lactobacilli, cocci, bacilli and kluyveromyces in a ratio of viable count of (50-150): (50-150): (5-15): (0.25-0.75): (0.25-0.75); preferably, the probiotic composition comprises bifidobacteria, lactobacilli, cocci, bacilli and kluyveromyces in a ratio of the number of viable bacteria of (80-120): (80-120): (8-12): (0.8-1.2): (0.4-0.6).

14. A kit for preventing and/or treating a disease caused by an allergic reaction, comprising the pharmaceutical composition according to any one of claims 8 to 13.

15. Kit according to claim 14, wherein the probiotic composition and the allergen preparation are placed separately, preferably the probiotic composition is an oral preparation and the allergen preparation is an injection.

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