WO2018188124A1 - Colonizing probiotic preparation, applications thereof, and medicament - Google Patents

Abstract

A colonizing probiotic preparation directly acting on the respiratory mucosa, comprising a surfactant and probiotics. The surfactant in the preparation not only increases the probability of and the rate at which the probiotics come into contact with respiratory mucosal cells, but also increases the adhesion rate of the probiotics on respiratory mucosal epithelial cells. The preparation is applicable in preparing a medicament for treating rhinitis, asthma, atopic dermatitis, cranial nerve disease, nasopharyngeal cancer, lung cancer, cold, or respiratory infection.

Description

Colonization probiotic preparation, application and medicament thereof

This application claims the priority of the Chinese patent application filed on April 11, 2017, the Chinese National Intellectual Property Office, application number CN201710232833.4, entitled "A colonized probiotic preparation and its application and drugs", the entire contents of which This is incorporated herein by reference.

Technical field

The present invention relates to the field of biotechnology, and in particular to a colonization probiotic preparation, an application thereof and a medicament.

Background technique

The respiratory tract of terrestrial vertebrates, such as humans, animals, and poultry, has long-term circulation of air containing microorganisms. The mucous membranes distributed on the inner surface of the respiratory tract constantly filter the microorganisms in the air. Therefore, a wide variety of microorganisms are inevitably present in the respiratory tract of terrestrial animals. These microbes interact with each other and also interact with the respiratory mucosa to form a sustainable micro-ecology.

Due to the connectivity of the respiratory tract, the population of microorganisms distributed in the respiratory tract of the same individual is substantially the same. However, due to the role of the Mucociliary Transport System (MCT), the number of microorganisms in the upper part of the respiratory tract (nasal cavity) is much larger than that in the lower part of the lower respiratory tract (bronchial).

Microorganisms in the respiratory tract, in accordance with the health effects of the host. Divided into three major categories of pathogenic bacteria, conditional pathogens and probiotics. By colonizing the mucosa, probiotics can continuously inhibit pathogenic bacteria, bind pathogenic bacteria, and balance host respiratory mucosal immunity.

Simple experiments have shown that sterile mice and sterile rabbits lacking probiotics, once exposed to the atmosphere, are not quickly killed by a few staphylococci or molds, or severe hypersensitivity reactions quickly die.

Clearly, probiotics that colonize the respiratory mucosa play an essential role in maintaining respiratory health. The existing research shows that the probiotics of the respiratory tract are mainly lactic acid bacteria, and some Bacillus species are not excluded. When the probiotic level of the respiratory tract (because of environmental air pollution, exposure to antibiotics and disinfectants, etc.) is reduced, the above effects are diminished, resulting in a variety of respiratory diseases. The above-mentioned series of events occur in the upper respiratory tract and manifest as various rhinitis (including allergic rhinitis). When it occurs in the lower respiratory tract, it is manifested as bronchitis, bronchitis and allergic asthma. Due to the interaction of probiotic mucosa, the reduction of probiotic membranes in the respiratory mucosa leads to an imbalance of mucosal immunity, especially the imbalance between Th1 type immunity and Th2 type immunity, resulting in IgE-mediated type I hypersensitivity. Due to the connectivity of the immune system, inflammatory factors act on the respiratory mucosa, skin, and intestines, leading to a variety of allergic diseases.

At present, the known method for increasing the level of respiratory probiotics is most effective in directly acting on the respiratory mucosa with an aqueous suspension containing probiotics. In the form of a liquid or an aerosol, it can be achieved by soaking the nose, or by washing the nose, or by dripping the nose, or by nasal spray. When using aerosols, the respiratory tract probiotics are often raised by inhalation of the mouth and nose or by direct inhalation into the lower respiratory tract.

Probiotics acting directly on the respiratory mucosa can adjust the respiratory micro-ecology and further adjust local or systemic immunity. Probiotics are not limited to live bacteria, and inactivated probiotics can produce the above adjustments. The probiotics are also not limited to respiratory origin. The lactic acid bacteria preparation carrying the vaccine gene can also be administered by directly acting on the respiratory tract to achieve vaccination.

However, the existing probiotic preparations acting on the respiratory mucosa can be transported away from the respiratory tract by the cilia-driven mucus blanket in just 10 minutes under the respiratory mucus transport. Therefore, it is difficult to prepare probiotic preparations directly acting on the respiratory mucosa. Adhesion is a problem of low adhesion rate, which in turn makes it difficult to fully function the corresponding probiotics.

In view of this, the present invention has been specifically proposed.

Summary of the invention

It is an object of the present invention to provide a colonized probiotic preparation for direct application to the respiratory mucosa, which has a higher adhesion rate of the probiotic bacteria in the respiratory mucosa than when administered alone.

Another object of the present invention is to provide a method for treating rhinitis or for treating asthma or for treating allergic dermatitis or for treating cranial nerve disease or anti-nasopharynx cancer or against lung cancer or anti-cold or preventing or treating respiratory infection The drug of the disease, after the above-mentioned drug directly acts on the respiratory mucosa, has a high adhesion rate in the respiratory mucosa, a long residence time of the active ingredient, and a long function of the drug effect, which helps to improve the healing effect of the corresponding treatment.

A further object of the present invention is to provide a colonization probiotic preparation as described above for use in the treatment of rhinitis or for the treatment of asthma or for the treatment of atopic dermatitis or for the treatment of cranial nerve diseases or against nasopharyngeal cancer or against lung cancer or anti-flu Or the use of drugs to prevent or treat respiratory infections.

Another object of the present invention is to provide a colonization probiotic preparation as described above for the treatment of rhinitis or for the treatment of asthma or for the treatment of atopic dermatitis or for the treatment of cranial nerve diseases or for anti-nasopharynx cancer or for anti-lung or anti-cold or for the prevention or treatment of respiratory infections.

Another object of the present invention is to provide a method for treating rhinitis or treating asthma or treating allergic dermatitis or treating cranial nerve diseases or against nasopharyngeal cancer or against lung cancer or anti-cold or preventing or treating respiratory infections.

The present invention is implemented as follows:

A colonized probiotic preparation for direct action on the respiratory mucosa, the colonization probiotic preparation comprising a surfactant and a probiotic.

A medicament for treating rhinitis or for treating asthma or for treating allergic dermatitis or for treating cranial nerve disease or for treating nasopharyngeal cancer or against lung cancer or anti-cold or preventing or treating respiratory infectious diseases, which comprises the above Colonization of probiotic preparations.

The above-mentioned colonized probiotic preparation is prepared for treating rhinitis or for treating asthma or for treating allergic dermatitis or for treating cranial nerve disease or anti-nasopharynx cancer or anti-lung cancer or anti-cold or preventing or treating respiratory infectious diseases. Application in medicine.

The above-mentioned colonized probiotic preparation is used for treating rhinitis or treating asthma or treating allergic dermatitis or treating cranial nerve disease or anti-nasopharynx cancer or anti-lung cancer or anti-cold or preventing or treating respiratory infection.

A method for treating rhinitis or treating asthma or treating allergic dermatitis or treating cranial nerve disease or anti-nasopharynx cancer or anti-lung cancer or anti-cold or preventing or treating respiratory infectious diseases, comprising: contacting an individual with the above-mentioned colonizing probiotic preparation The respiratory mucosa.

Optionally, the colonization probiotic preparation has a surface tension of less than 65 mN/m.

Optionally, the colonization probiotic preparation has a surface tension of 40-60 mN/m.

Optionally, the surfactant is selected from the group consisting of biosurfactants and vegetable surfactants.

Optionally, the biosurfactant is selected from the group consisting of glycolipids and lipopeptides selected from the group consisting of saponins and other surface active glycosides.

Optionally, the glycolipid is selected from the group consisting of rhamnolipid, trehalose lipids, sophorolipid, and mannose erythritol lipid (MEL) selected from the group consisting of a group consisting of surfactin, lichenysin, iturin, fengicin, plipastatin, spiroidesin, and tensin .

Optionally, the saponin is selected from the group consisting of ginsenoside, tea saponin, astragaloside, soy saponin, acacia saponin, zona saponin, saponin, diosgenin, and saponin.

Optionally, the colonizing probiotic preparation comprises a extract of Magnolia.

Optionally, the extract of Magnolia contains a surfactant.

Optionally, the colonizing probiotic preparation comprises a ginseng extract.

Optionally, the probiotic is a lactic acid bacterium, and the content of the lactic acid bacteria in the colonization probiotic preparation is 1×10 ⁴ -1× ^{10 12} /g.

Optionally, the probiotic is Lactobacillus.

Optionally, the Lactobacillus is Lactobacillus saliva, the colonization probiotic preparation further comprises a bacterial culture supernatant, the bacterial culture supernatant containing the surfactant, a surface tension of the colonized probiotic preparation It is 49-60 mN/m.

Optionally, the colonization probiotic preparation has a surface tension of 55 mN/m.

Optionally, the colonization probiotic preparation comprises NaCl, and the content of the NaCl in the colonization probiotic preparation is 2-60 g/Kg.

Optionally, the pH of the colonized probiotic preparation is 3.5-7.

Optionally, the probiotic is selected from the group consisting of inactivated probiotics and recombinant lactic acid bacteria.

Compared with the prior art, the beneficial effects of the present invention are:

The invention provides a colonization probiotic preparation for directly acting on a respiratory mucosa, which comprises a surfactant and a probiotic, and the surfactant modifies the sol layer and the gel layer of the respiratory mucus, thereby improving probiotic contact with the respiratory tract The probability and speed of mucosal cells. At the same time, the surfactant changes the cell surface hydrophobicity of probiotics, and improves the adhesion rate of these probiotics to the airway mucosal epithelial cells, thereby solving the problem that the probiotics in the prior art are difficult to colonize the respiratory mucosa under the removal of the ciliary transmission system. bottleneck. Therefore, the colonized probiotic preparation provided by the invention has higher adhesion rate on the respiratory mucosa, is easy to colonize the respiratory mucosa, has long-lasting function of the beneficial bacteria, and has broad application prospect, and can be applied to It is prepared in the fields of treating rhinitis or for treating asthma or for treating allergic dermatitis or for treating cranial nerve diseases or anti-nasopharynx cancer or anti-lung cancer or anti-cold or preventing or treating respiratory infectious diseases.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below in order to clarify the objects, the technical solutions and the advantages of the embodiments of the present invention. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained by commercially available purchase.

A colonized probiotic preparation, an application thereof and a medicament according to an embodiment of the present invention are specifically described below.

The ciliary transmission system on the respiratory mucosa consists of a sol layer and a gel layer of cilia and mucus that continuously transmit power. The cilia continuously brush the gel layer across the sol layer, and the gel layer carrying the foreign matter in the respiratory tract continuously moves toward the pharynx or nostrils and exits the respiratory tract. This process is not too long, usually around 10-30 minutes. It is easy to measure by means of saccharin experiments. This discharge of foreign matter is more rapid if sneezing or coughing occurs due to nerve reflexes.

Such a moving speed, for direct action of the respiratory tract, probiotics released by the aqueous suspension and its droplets, contact the respiratory mucosal cells in a short period of time, and form adhesion with the adhesion receptor, which is a probiotic to colonize the respiratory tract and exert its biological activity. The premise of activity.

Based on this, in order to overcome the deficiencies of the existing probiotic preparations directly acting on the respiratory mucosa, it is difficult to adhere. On the one hand, the present invention provides a colonization probiotic preparation for directly acting on the respiratory mucosa, the above-mentioned colonization probiotic preparation Contains surfactants and probiotics.

Since the colonization probiotic preparation provided by the present invention contains a surfactant, it modifies the sol layer and the gel layer of the respiratory mucus, thereby increasing the probability and speed of probiotic contact with respiratory mucosal cells. At the same time, the surfactant changes the cell surface hydrophobicity (CSH) of the probiotics, and the adhesion rate of these probiotics to the respiratory mucosal epithelial cells is increased by more than 30%, thereby solving the problem that the probiotics in the prior art are difficult to be driven out under the ciliary transport system. A technical bottleneck that colonizes the respiratory mucosa. Therefore, the probiotics contained in the colonized probiotic preparation provided by the present invention have a higher adhesion rate in the respiratory mucosa and are more easily colonized in the respiratory mucosa, and are beneficial for long-lasting biological effects.

The surfactants in the colonized probiotic preparations provided by the invention play the role of promoting or improving the adhesion rate of the probiotics to facilitate colonization. The following methods are mainly used: (1) for the gel layer, the surfactant can make the gel The layer of mucopolysaccharide swells and pores are formed in the gel layer on the mucus surface of the respiratory tract. Thereby, the probiotics are more likely to enter the sol layer through the moving gel layer and come into contact with the respiratory mucosal cells. (2) For the sol layer of the mucus of the respiratory tract, the surfactant can change the viscosity of the sol, and the penetration-promoting effect can make the probiotics agglutinate more effectively and adhere to the mucosal cells of the respiratory tract to form a membrane. (3) The addition of the surfactant in the colonization probiotic preparation provided by the present invention can also change the cell surface hydrophobicity (CSH) of the probiotic bacteria and increase the adhesion rate of the probiotic bacteria to the respiratory mucosal epithelial cells.

Surfactants can occur individually or in combination by the above three modes of action. Thereby, the technical effect of promoting the adhesion of the probiotic bacteria to the respiratory mucosa and achieving the adhesion rate is achieved.

It should be noted that the colonization probiotic preparation provided by the present invention may be in the form of a powder, an aqueous suspension or a spray.

Optionally, in some embodiments of the invention, the above-described colonized probiotic formulation has a surface tension of less than 65 mN/m.

It should be noted that if the colonized probiotic preparation is a powder, the surface tension of the colonized probiotic preparation is tested as follows: the probiotic preparation is mixed with deionized water at a mass to volume ratio of 1:50, and then the precision is used. 0.1mN/m automatic interface tension meter measurement.

Controlling the surface tension of the colonized probiotics to a range of less than 65 mN/m helps to improve the adhesion rate of probiotics and mucosal cells in the colonized probiotic preparations, and also facilitates the colonization of probiotics to continue to exert biological effects.

Alternatively, in some embodiments of the invention, the above-described colonized probiotic formulation has a surface tension of from 40 to 60 mN/m. In the range of surface tension of 40-60 mN/m, the adhesion rate of probiotics to mucosal cells in the above-mentioned colonized probiotics preparation is further improved, which is more conducive to the colonization of probiotics, so that it continues to exert biological effects.

Still alternatively, in some embodiments of the invention, the colonization probiotic formulation has a surface tension of 55 mN/m.

Optionally, in some embodiments of the invention, the surfactant is selected from the group consisting of biosurfactants and vegetable surfactants.

Optionally, in some embodiments of the invention, the above biosurfactant is selected from the group consisting of glycolipids and lipopeptides; the above vegetable surfactants are selected from the group consisting of saponins and other surface active glycosides.

It will also be understood that in some embodiments of the invention, the surfactant is selected from the group consisting of glycolipids, lipopeptides, saponins, and other glycosides having surface-active functions.

Wherein the glycolipid is selected from the group consisting of rhamnolipid, trehalose lipids, sophorolipid, and mannose erythritol lipid (MEL);

The lipopeptide is selected from the group consisting of surfactant (surfactin), lichenysin, ituririn, fengicin, plipastatin, spiroidesin, and tensin (tensin). a group consisting of;

The saponin is selected from the group consisting of ginsenoside, tea saponin, astragaloside, soy saponin, acacia saponin, zona saponin, saponin, diosgenin, and saponin;

The glycoside is selected from the group consisting of glucosides and galactosides having surface-active functions.

It should be noted that the surfactant according to the present invention may be a single component having a high purity or a combination of a plurality of components, or may be a plant extract containing the above surfactant or a microorganism culture. These plant extracts or microbial cultures contain one or more ingredients of a surfactant.

Optionally, in some embodiments of the invention, the colonizing probiotic preparation comprises a extract of Magnolia, the extract of the above-mentioned Xinyi containing a surfactant, and the surfactant is a glycoside.

It is easy to understand that the composition of the extract of Magnolia Bark contains a variety of glycosides, so the extract of Magnolia Bark can be used as a source of surfactants, and the use of the extract of Magnolia Bark to prepare probiotics for probiotics with the probiotics is within the scope of the present invention.

By analyzing the composition of the extract of Magnolia biondii, the extract of Magnolia Biondii contains vanillic acid-4-O-β-D-glucoside, 3-methoxy-4-hydroxybenzene-1-O-β-D-glucoside, coffee Acid, 3,4,5-trimethoxybenzene-1-O-β-D-glucoside, benzyl-O-β-D-glucoside, benzyl-O-β-D-galactoside, vanilla Glycosidic substances such as acid glucose ester and 7-methoxycoumarin-6-O-β-D-glucoside.

Optionally, in some embodiments of the invention, the colonization probiotic preparation described above comprises a ginseng extract, the ginseng extract contains a surfactant, and the surfactant is saponin.

It is easy to understand that the main component of the ginseng extract is saponin, so the ginseng extract can be used as a source of the surfactant, namely saponin, and the ginseng extract can be used for formulating the probiotics with the probiotics, and also belongs to the protection of the present invention. range.

Ginsenoside is a sterol compound, triterpenoid saponin. It is mainly found in ginseng medicinal materials, including ginsenosides Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh, Ro, etc. It is regarded as a substance with extensive biological effects such as ginseng, notoginseng, and American ginseng. However, the molecular biological mechanism of ginsenosides specifically producing biological effects is unknown. The present invention is applied to a probiotic preparation to explain, in another aspect, a molecular biological mechanism for the biological effects of these traditional Chinese medicine ingredients.

Alternatively, in some embodiments of the present invention, the probiotic bacteria are lactic acid bacteria, and the content of the lactic acid bacteria in the colonization probiotic preparation is 1 × 10 ⁴ - 1 × 10 ¹² /g.

The probiotics preparations using lactic acid bacteria can act on the mucosa, adjust the mucosal micro-ecology, and interact with mucosal cells to produce biologically effective lactic acid bacteria. The following studies include the following lactic acid bacteria: Lactobacillus salivarius can reduce IgE for anti-allergy; Lactobacillus casei, Lactobacillus paracasei for improving the phagocytic ability of macrophages; Lactobacillus pentosus, Lactobacillus bulgaricus for adsorption of benzopyrene; Bifidobacterium infantis for self-transition inhibition of cancer cells; Cocci, can produce bacteriostats, used to inhibit harmful bacteria; most lactic acid bacteria can form a biofilm cloud barrier on the mucosal surface, occupying the colonization of harmful bacteria, reducing mucous membrane and antigen contact, is indispensable for mucous membrane biological.

Optionally, in some embodiments of the invention, the colonizing probiotic preparation comprises NaCl, and the content of the above NaCl in the colonization probiotic preparation is 2-60 g/Kg. NaCl helps to balance the osmotic pressure of the colonized probiotic preparation on the respiratory mucosa and prevents edema of the mucosa when the colonization probiotic preparation provided by the present invention is used. More optionally, in some embodiments of the invention, the amount of NaCl in the colonization probiotic formulation described above is 9 g/Kg or 9 g/L.

Alternatively, in some embodiments of the invention, the colonized probiotic formulation has a pH of 3.5-7. A low pH environment helps to increase the viability or duration of probiotics in colonized probiotic preparations. More optionally, in some embodiments of the invention, the pH of the colonized probiotic formulation is between 5.5 and 6.

It should be noted that the type of probiotic bacteria in the colonization probiotic preparation provided by the present invention is not limited to the above-mentioned types of lactic acid bacteria, and the designer may use the corresponding bacteria as probiotics according to the characteristics or use of the bacteria. For example, probiotics can be lactic acid bacteria Or yeast, Bacillus, etc., even other types of bacteria, even inactivated probiotics or inactivated conditional pathogens, and even DNA recombinant bacteria, all of which fall within the scope of the present invention.

Based on this, optionally, in some embodiments of the invention, the probiotic is a recombinant lactic acid bacterium.

Alternatively, in some embodiments of the invention, the recombinant lactic acid bacterium described above is a Bifidobacterium infantis containing foreign DNA.

Alternatively, in some embodiments of the invention, the probiotic described above is Lactobacillus casei.

Both lipoteichoic acid (LTA) and extracellular polysaccharide (EPS) of Lactobacillus casei promote the amount of macrophages phagocytizing neutral red. This means that Lactobacillus casei and its inactivated organisms can improve mucosal immunity, that is, the ability to kill viruses and germs, which is a powerful means of fighting colds and fighting diseases.

Alternatively, in some embodiments of the invention, the probiotic is a lactobacillus.

Optionally, in some embodiments of the present invention, the above-mentioned Lactobacillus is Lactobacillus saliva, and the above-mentioned colonization probiotic preparation further comprises a bacterial culture supernatant containing a surfactant, and the above-mentioned colonization probiotic preparation The surface tension is 60-49 mN/m. More optionally, the surface tension of the above-mentioned colonized probiotic preparation is 55 mN/m.

Optionally, in some embodiments of the present invention, the bacterial culture supernatant is one of a culture supernatant of Lactobacillus acidophilus, a culture supernatant of Lactobacillus plantarum, and a culture supernatant of Bacillus Or a combination of several.

High-adhesion lactic acid bacteria such as Lactobacillus acidophilus, Lactobacillus plantarum, and Bacillus, which can be metabolized by themselves to produce surfactants such as lipopeptides and/or glycolipids, which are Lactobacillus acidophilus, Lactobacillus plantarum, and The surface tension of the culture solution of Bacillus is reduced to 60 mN/m or less. In addition to promoting their own adhesion and colonization, this effect also has an effect of increasing adhesion rate and promoting colonization for other low adhesion Lactobacilli. However, in terms of specific biological effects, such as anti-allergic and promoting macrophage phagocytosis, these high-adhesion Lactobacilli are relatively weak, or even absent. Adhesion of highly bioactive strains to the mucous membranes through the culture of surfactant-producing strains is a revolutionary and creative approach to the greater role of probiotics.

As a supplement to the above-described embodiment, it is preferred to remove the culture solution of the cells, and secondly to lyse the inactivated culture solution. The high-adhesive rate of Lactobacillus bacteria on the mucosa, will undoubtedly reduce the bioavailability (low adhesion rate) of the target lactic acid bacteria adhesion rate, will also reduce the program to improve the target probiotic adhesion rate technology effect. However, this method of not discarding the cells producing E-BS lactic acid bacteria will undoubtedly reduce the cost and process difficulty, and in some cases, it is also a secondary selection scheme.

In combination with the above aspects, in another aspect, the present invention also provides a method for treating rhinitis or for treating asthma or for treating allergic dermatitis or for treating cranial nerve disease or anti-nasopharynx cancer or anti-lung cancer or anti-cold Or a medicament for preventing or treating a respiratory infectious disease, which comprises the colonized probiotic preparation of any of the above.

Bacterial rhinitis is caused by excessive reproduction of harmful bacteria. After colonization on the mucosa, Lactobacillus can inhibit these harmful bacteria, relieve their colonization, and further eliminate infection.

Any of the above-described colonized probiotic preparations provided by the present invention can achieve this effect, especially a probiotic preparation containing a surfactant and Lactobacillus casei. Allergic rhinitis, allergic asthma and allergic dermatitis are IgE-mediated allergic diseases. After the probiotic preparation containing lichenin and Lactobacillus saliva acts on the mucosa, the Lactobacillus biofilm formed on the one hand can isolate the actual contact between the antigen and the mucosa, and on the other hand, it can stimulate the mucosal immune system to increase the secretion of INF-γ and reduce The secretion of IL-4 reduces the level of IgE in local and even serum, thereby eliminating the root cause of allergic diseases. Under the promotion of adhesion of surfactants, Lactobacillus infantis is colonized from the nasal cavity in the nasopharynx, and is very close to the nasopharyngeal cancerous anaerobic zone. It has a greater probability of migration and inhibition of nasopharyngeal carcinoma formation. If the recombinant DCN gene is carried, Lactobacillus infantarum that accumulates in the anaerobic region of nasopharyngeal carcinoma expresses a protein that inhibits tumors and promotes apoptosis, and such an anticancer effect is stronger. Due to the similarity of the basic structure of the terrestrial vertebrate respiratory tract and the brain, the probiotics may have different functional correspondences and strain performance differences for different terrestrial vertebrates, but based on the technology of overcoming the mucus ciliary transport system common to terrestrial vertebrates Problem, this technical solution has a common technical effect

In a further aspect, the present invention provides the preparation of any of the above-mentioned colonized probiotic preparations for the treatment of rhinitis or for the treatment of asthma or for the treatment of atopic dermatitis or for the treatment of cranial nerve diseases or for anti-nasopharynx or anti-lung cancer Or use in medicines that are resistant to colds or to prevent or treat respiratory infections.

In still another aspect, the present invention provides the above-described colonization probiotic preparation for treating rhinitis or treating asthma or treating allergic dermatitis or treating cranial nerve disease or anti-nasopharynx cancer or anti-lung cancer or anti-cold or preventing or treating respiratory infection .

In still another aspect, the present invention provides a method of treating rhinitis or treating asthma or treating atopic dermatitis or treating a cranial nerve disease or anti-nasopharynx cancer or against lung cancer or anti-cold or preventing or treating a respiratory infection, comprising: Contacting the respiratory mucosa of an individual with any of the above-described colonizing probiotic preparations.

Optionally, in some embodiments of the invention, the respiratory mucosa comprises one or more of a nasal mucosa, a tracheal mucosa, or a mucosa of the inner surface of the bronchus.

In summary, the colonization probiotic preparation provided by the invention can modify the sol layer and the gel layer of the respiratory mucus by the added surfactant, thereby improving the probability and speed of probiotics in the colonization probiotic preparation contacting the respiratory mucosa cells. . At the same time, the surfactant changes the cell surface hydrophobicity of probiotics, and improves the adhesion rate of these probiotics to the airway mucosal epithelial cells, thereby solving the problem that the probiotics in the prior art are difficult to colonize the respiratory mucosa under the removal of the mucociliary transport system. Technical bottlenecks. The colonized probiotic preparation provided by the invention has high adhesion rate on the respiratory mucosa, is easy to colonize the respiratory mucosa, and has long-lasting function of the beneficial bacteria, and has broad application prospects.

The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

Example 1

The colonized probiotic preparation provided by the embodiment is used for directly acting on the respiratory mucosa, and comprises a surfactant and a probiotic. Among them, the probiotic is Lactobacillus saliva, and the surfactant is lichenysin. The concentration of Lactobacillus saliva is 1×10 ⁸ CFU/ml, the content of lichenin is 5 mg/L, the surface tension is 55 mN/m, and the NaCl content is 9 g/kg, pH 5.5. The colonized probiotic preparation is an aqueous suspension.

1.1 Preparation method of the colonization probiotic preparation provided by the embodiment:

1.1.1 Lactobacillus salivarius strain (ATCC 11741) was selected for culture. Culture conditions: 28 ° C, inoculum amount of 5%, the medium was MRS, anaerobic culture, the initial pH of the MRS medium was 5.5, and cultured for 15 h.

1.1.2 After the end of the culture, centrifuge at 4500 rpm for 10 min to collect the Lactobacillus salivarius cells.

1.1.3 A strain of Bacillus licheniformis (ATCC 39307) was selected for culture to obtain a culture medium containing lipopeptides, particularly lichenin (a natural surfactant derived from microorganisms). Medium formula: soluble starch 20.0g / L, NH ₄ NO ₃ 4.0g / L, KH ₂ PO ₄ 3.0g / L, Na ₂ HPO ₄ 3.0g / L, yeast powder 0.5g / L, FeSO ₄ 6.8μmol / L, ZnSO ₄ 0.038 mmol / L, CaCl ₂ 0.5 mmol / L, MgSO ₄ 0.2 mmol / L, MnSO ₄ 0.02 mmol / L, EDTA 50 μmol / L, pH 7.4; 500 ml flask is filled with 200 ml, inoculum 5.0%, culture temperature 37 ° C, culture time 48 h or so.

1.1.4 After the culture is completed for a predetermined period of time and the surface tension of the culture solution is determined to fall below 50 mN/m, the culture solution is taken, centrifuged at 8000 rpm for 20 min, the bacteria are removed twice, and the supernatant is taken. The obtained supernatant was adjusted to pH 2.0 with concentrated HCl, and a flocculent precipitate appeared. The mixture was allowed to stand overnight at 4 ° C, and the precipitate was collected by centrifugation at 10,000 rpm for 30 min, and washed once with acid water of pH 2.0. The precipitate was then dissolved in a NaOH solution to give a pH of 7.0 and lyophilized to give a crude lichenysin. The crude product was extracted in CH ₂ Cl ₂ , evaporated to dryness under reduced pressure, and dissolved in dilute NaOH solution to form a multi-bubble liquid. The surface tension was determined to be about 35.0 mN/m using a JYN 2200A automatic interfacial tension meter. The above multi-bubble liquid was filtered through Whatman No. 4 filter paper, and the filtrate was again adjusted to pH 2.0 with HCl, and then centrifuged to obtain a precipitate. Purified lichenin was obtained by vacuum drying to remove residual moisture from the precipitate.

By analyzing the above purified lichenin component, it is determined that the surfactant is:

Ring-[L-Gln1→D-Leu2→L-Leu3→L-Val4→L-Asp5→D-Leu6→L-Ile7-β-OH fatty acid].

1.1.5 Dissolve 0.5 mg of the above pure lichenin in 100 ml of deionized water to prepare a solution containing 5 mg/L of lichenin. The surface tension of the above solution was measured using a JYN 2200A automatic interfacial tension meter at 25 ° C to be 55 mN/m.

1.1.6 Take 100ml of the above solution, add 0.9g of NaCl, add Lactobacillus salivarius and mix it to obtain a suspension of Lactobacillus salivarius, and determine the Lactobacillus salivarum content by using a spectrophotometer to be 1×10 ⁸ CFU/ml, and add lactic acid. The pH value was determined to be 5.5 with a pH meter (if the pH was higher than 5.5, lactic acid was added, and if the above suspension was added to Lactobacillus saliva), a colonized probiotic preparation which can act on the respiratory tract was obtained.

1.2 Cell surface hydrophobicity (CSH) detection of probiotics in colonized probiotic preparations provided in this example

1.2.1 The hydrophobicity of the above Lactobacillus salivarius bacteria is detected by the hydrocarbon compound adhesion technique (WATH):

The salivary lactic acid bacteria obtained in the above step 1.1.2 were washed 4 times with a sterile NaCl solution (the solution was prepared in deionized water to be 0.5 mol/L). After vacuum freeze-drying, 100 mg of Lactobacillus salivarius cells were weighed and shaken in a 50 ml solution containing 100 mmol/L of NaHCO ₃ at 30 ° C for 1 h. Add 1 ml of n-hexadecane to the above solution, shake at 37 ° C for 1 h, centrifuge at 4000 rpm for 6 min, freeze the precipitate in a vacuum and accurately weigh it to obtain a weight of C, and calculate the adhesion rate B = (100-C)%. This was repeated 3 times, and the results were averaged and expressed as (mean ± standard deviation) %.

As a result, the cell surface hydrophobicity of the above Lactobacillus salivarius cells was (22.62 ± 1.63)%.

The salivary lactic acid bacteria cells collected in the aforementioned step 1.1.2 were washed 4 times with a sterile NaCl solution (NaCl solution was prepared in double deionized water at a concentration of 0.5 mol/L). After vacuum freeze-drying, 100 mg of Lactobacillus salivarius cells were weighed and mixed in 50 ml of a sterile culture supernatant containing 100 mmol/L of NaHCO ₃ and lichenin 5 mg/L, and shaken at 30 ° C for 1 h. Add 1 ml of n-hexadecane to the above solution, shake at 37 ° C for 1 h, centrifuge at 4000 rpm for 6 min, freeze the precipitate in a vacuum and accurately weigh it to obtain a weight of C (mg), and calculate the adhesion rate B = (100-C)%. . The results were repeated 3 times, and the results were averaged, and the results were expressed as mean ± standard deviation. As a result, Lactobacillus salivarius treated with a solution containing 5 mg/L of lichenin was found to have a surface hydrophobicity of (71.61 ± 2.35)%.

The results showed that the surface hydrophobicity of Lactobacillus saliva treated with a solution containing 5 mg/L of lichenin was significantly higher than that of Lactobacillus saliva which was not treated with the lichenin solution. The difference was statistically significant (p < 0.05). An increase in the hydrophobicity of the surface of the probiotic means an increase in its adhesion rate.

1.3 Detection of adhesion rate of probiotics to human nasal mucosa cells (HNE) in colonized probiotic preparations provided in this example

1.3.1 Nasal polyps mucosa was taken during surgery for human nasal polyps. Rinse with PBS to remove mucus, blood, necrotic tissue and submucosal tissue, and remove the mucosal layer. Rinse and soak for 3 times with 4 ° C sterile PBS solution. After pipetting with 0.1% collagenase type I, it was digested at 37 ° C for 30 min. Gently blow the digested mucosal tissue to remove the epithelial cells and remove the mucosal mass. The cell suspension was centrifuged at 1000 rpm, 4 ° C for 5 min. Discard the supernatant. A medium containing DMEM/F12 (1:1, containing 1 × 10 ⁵ U/L penicillin, 100 mg/L streptomycin) containing 10% fetal calf serum was added to the pellet. After gently pipetting, the cell suspension was inoculated into the culture plate, and cultured in a 37 ° C saturated humidity CO ₂ cell incubator. The culture medium was changed every 2 days and cultured for 7 days.

1.3.2 The cultured human nasal mucosa cells (HNE) were digested with trypsin-EDTA digestion solution, and then adjusted to a cell concentration of 1.0×10 ⁵ /ml with DMEM complete nutrient solution, and mounted on a 6-well tissue culture plate. 2 ml of each well was incubated in a 5% CO ₂ , 95% air incubator at 37 ° C until the cells grew to a monolayer. The DMEM nutrient solution in each well of the tissue culture plate was discarded and washed twice with sterile PBS buffer.

1.3.3 One of the wells was digested with 0.5 ml of trypsin-EDTA, and 0.5 ml of PBS was added, shaken with a micro-oscillator and pipetted with a pipette, the cells were completely digested and mixed, and the cells were counted by a hemocytometer. concentration. Two additional wells, one well and 1 ml of DMEM stock solution and 1 ml of sterile FBS buffer adjusted suspension of Lactobacillus saliva (1 × 10 ⁸ CFU / ml) as a control; another well added 1ml DMEM stock solution and 1ml of this The colonized probiotic preparation (L. salivarius concentration of 1 x 10 ⁸ CFU/ml) provided in the examples. The two wells were mixed with a pipette and incubated in an anaerobic incubator at 37 ° C for 2 h.

1.3.4 After incubation, discard the mixture of each well in the tissue culture plate and wash it 5 times with sterile PBS buffer to remove unadhered probiotics. After adding 0.4 ml of trypsin-EDTA digestive juice for digestion, 0.6 ml of sterile PBS buffer was added, and the mixture was subjected to gradient dilution, methanol fixation for 15 min, Gram staining, and plate count to calculate the number of adhered bacteria. The adhesion rate of different bacterial suspensions was calculated according to the following formula: Adhesion rate (CFU/cel1) = number of adherent bacteria/cell number. The results are as follows.

The Lactobacillus salivarius in the colonized probiotic preparation provided in this example has a adhesion rate to human nasal mucosa cells (HNE) of (3.17±0.23) CFU/cell. In the control group, the Lactobacillus salivarius adjusted with FBS buffer containing no lignin had an adhesion rate of (1.78±0.17) CFU/cell.

Thus, the adhesion rate of probiotics (L. saliva) to human nasal cells in a suspension of Lactobacillus saliva suspension containing lipopeptide surfactant, Lichenysin (i.e., the colonized probiotic preparation of the present example), Higher than the bacterial suspension without the surfactant. The difference was statistically significant (p < 0.05).

The increase in the adhesion rate of probiotics to mucosal cells means that their biological effects are multiplied. At the same time, it means that probiotics have more opportunities to colonize the mucosa and play a role in the rapid removal of the mucociliary transport system.

Previous studies have shown that Lactobacillus salivarius can increase the expression of IFN-γ and lower serum IgE by acting on mucous membranes, and has better anti-allergic effects. However, the lower adhesion rate of Lactobacillus saliva has a major technical obstacle directly affecting the respiratory mucosa containing the nasal cavity. However, the present invention overcomes this technical obstacle by adding a surfactant such as a lipopeptide, such as lichenin, to increase the adhesion rate of Lactobacillus salivarius to nasal epithelial cells. Therefore, the probiotics in the colonized probiotic preparation provided by the present embodiment have a higher adhesion rate to the respiratory mucosa cells, especially the nasal mucosa cells, and are easily colonized in the respiratory mucosa, and the probiotics continue to function for a long time.

1.4 The blank saccharin test of the colonized probiotic preparation provided by the present embodiment

36 healthy subjects were selected. On the first day, all the blank saccharin test was performed to test the mucociliary transmission time. After 6 days of rest, on the 7th day, all the saline was immersed in the nasal cavity, and the saccharin test was performed at 5 min intervals to test the mucociliary transmission. Time; rest for 6 days, on the 14th day, all the colony probiotics provided in this example were used to soak the nasal cavity, and the saccharin test was performed at 5 min intervals to test the mucociliary transit time. On the 7th day and the 14th day, the liquid amount of the immersion nasal cavity was 200 ml, and the immersion time was not less than 15 minutes.

The mucociliary transmission rate of the saccharin test was compared three times. RESULTS: The mucosal cilia transmission rate after the nasal cavity was diluted with the colonized probiotics provided in this example was (13.97±2.15) mm/min, which was significantly greater than the blank saccharin test (10.64±3.25) mm/min and the nasal wash with saline. The mucociliary transmission rate of the posterior cavity was 11.02±5.13 mm/min. The differences were statistically significant (p < 0.05).

It is thus shown that the colonized probiotic preparation provided by the present embodiment directly acts on the respiratory tract by immersing the nasal mucosa, and the cilia toxicity is smaller than that of the physiological saline. Probiotic preparations containing biosurfactants can also continuously produce ATP to supply more energy to the ciliated cells and promote their activity. This experiment also shows that the colonization probiotic preparation provided by the present embodiment is applied to respiratory care, which can promote the nasal mucociliary transmission system to discharge harmful foreign matter from the respiratory tract.

1.5 The effect of colonized probiotics provided in this example on total IgE in serum of patients with allergic diseases

Thirty-six patients with allergic diseases with abnormal serum IgE levels were selected. 19 people were allergic rhinitis patients, 12 were allergic rhinitis with asthma, and 5 were allergic dermatitis patients. Inclusion conditions: The respective allergic symptoms were obvious, and no immunosuppressive agents such as glucocorticoids were used within 3 months. The serum IgE level by enzyme-linked immunosorbent assay (ELISA) was not detected to be less than 100 IU/ml within six months.

Patients with allergic disease were randomly divided into three groups (experimental group and saline control group, no surfactant control group), 12 people in each group. Serum IgE levels were determined by enzyme-linked immunosorbent assay (ELISA): 2 ml of venous blood was taken from a routine fasting, placed in a test tube without anticoagulant, allowed to stand for 10 min after hemagglutination, centrifuged at 3000 rpm for 10 min, serum was separated, and refrigerated at 20 ° C. For inspection. Detection was performed using an enzyme-linked immunosorbent assay. Data processing was performed using statistical software SPSS 13.0, and the results were expressed as average values. Using the above method, serum IgE levels of the three groups were measured on the first day. The test results were: (511 ± 93.54) IU/ml in the experimental group and (507 ± 95.32) IU/ml in the saline control group. The surfactant-free control group was (520 ± 89.44) IU/ml.

The saline control group was immersed in the lower part of each patient's nasal cavity with 200 ml of physiological saline once a day for 21 consecutive days. The dipping time is not less than 15 minutes. On day 22, serum IgE levels were measured. The test result was (493±90.18) IU/ml.

No surfactant control group for 21 consecutive days, once a day with a surfactant-free, Lactobacillus saliva content of 1 × 10 ⁸ CFU / ml, NaCl 0.9g / L, pH value of 5.5. Probiotic bacteria suspension 200 ml of liquid was dipped in the lower part of the nasal cavity of each patient. The dipping time is not less than 15 minutes. On day 22, serum IgE levels were measured. The test result was (302 ± 98.01) IU / ml.

The experimental group was immersed in the lower nasal cavity of each patient once a day for 21 consecutive days with 200 ml of the colonized probiotic preparation provided in this example. The dipping time is not less than 15 minutes. On day 22, serum IgE levels were measured and the results were (183 ± 56.18) IU/ml.

According to the above test results, the colonization probiotic preparation provided in the present example significantly reduced serum IgE levels in patients with IgE-mediated allergic diseases by acting on the nasal mucosa (p<0.05). The colonized probiotic preparations provided in this example are more effective than physiological saline in reducing serum IgE levels of IgE-mediated allergic diseases. The difference was statistically significant (p < 0.05). The colonized probiotic preparations provided in this example are also more effective than probiotic preparations without surfactants in reducing serum IgE levels of IgE-mediated allergic diseases. The difference was statistically significant (p < 0.05).

In the nasal cavity, Lactobacillus saliva does not face the killing of digestive juice. Its adhesion rate to mucosal cells, which is improved by the action of surfactants, can overcome the repelling of cilia mucus transport and reliably colonize the respiratory mucosa. This also indicates that the probiotic preparation provided by the embodiment of the present invention acts on the respiratory tract and can be applied to treat IgE-mediated allergic diseases, for example, diseases including allergic rhinitis, allergic asthma, allergic dermatitis and the like.

Example 2

The colonized probiotic preparation provided by the embodiment is used for directly acting on the respiratory mucosa, and comprises a surfactant and a probiotic. Among them, the probiotic is Lactobacillus casei, the surfactant is surfactant (surfactin), the surface tension of the colonization probiotic preparation is 55 mN/m, the pH is 6, the NaCl content is 9 g/L, and the surfactant (surfactin) The content was 14.5 mg/L and the probiotic content was 1 x 10 ⁸ CFU/ml.

2.1 The preparation method of the colonization probiotic preparation provided by the embodiment is as follows:

2.1.1 Lactobacillus casei (ATCC 393) was selected. The fresh bacterial culture cultured overnight was inoculated into MRS medium, the inoculum amount was 1%, the culture temperature was 28 ° C, and the anaerobic culture was allowed to stand in the incubator for about 15 hours. After the completion of the culture, the cells were centrifuged at 4500 rpm for 10 min, and the Lactobacillus casei bacteria were collected.

2.1.2 Bacillus subtilis (ATCC 2233), 50 ml of culture medium was placed in a 150 ml flask, sterilized, and seeded for 12 hours, inoculated in an amount of 2%, and cultured at 37 ° C. Among them, the slant seed culture medium (g/L) components: beef extract 3, peptone 10, NaCl 5, agar 20, pH 7.0; fermented seed medium formula (in g/L): glucose 10 g / L, NH ₄ NO ₃ 4g/L, KH ₂ PO ₄ 4g/L, Na ₂ HPO ₄ ·12H ₂ O 12g/L, Mg ₂ SO ₄ 0.2g/L, CaCl ₂ 1×10 ^-3 g/L, FeSO ₄ 6× 10 ^-2 g / L, MnSO ₄ 6 × 10 ^-1 g / L, yeast extract 1g / L, beef extract 1g / L; fermentation medium (g / L) components: glucose 40g / L, NH ₄ NO ₃ 4g /L, KH ₂ PO ₄ 4g / L, Na ₂ HPO ₄ · 12H ₂ O 12g / L, MgSO ₄ 0.2g / L, CaCl ₂ 1 × 10 ^-3 g / L, FeSO ₄ 6 × 10 ^-2 g / L, MnSO ₄ 6 g / L, yeast extract 1 g / L, beef extract 1 g / L. After culturing for 42 hours, the cells were removed by centrifugation at 4500 rpm for 10 min. The supernatant of the culture liquid containing the surfactant was obtained by a water bath at 80 ° C for 2 hours. The sterile culture supernatant supernatant having a surface tension of 55 mN/m was obtained by diluting the supernatant of the sterile culture solution with deionized water and determining by an automatic interfacial tension meter. Comparing the "concentration-surface tension" curve of surfactant (surfactin), it was confirmed that the corresponding concentration of surfactant (surfactin) in the culture supernatant dilution was 14.5 mg/L.

2.1.3 Take 100 ml of the supernatant of the above culture solution, add 0.9 g of NaCl, add Lactobacillus casei, and determine the content of Lactobacillus casei to 1×10 ⁸ CFU/ml by spectrophotometer, add lactic acid, and determine with pH meter. The pH is 5.5, resulting in a colonized probiotic preparation that acts on the respiratory tract.

2.2 Surface hydrophobicity detection of the colonized probiotic preparation provided in the present example: the detection method is the same as step 1.2.

Results: The cell surface hydrophobicity (CSH) of the surfactant-treated Lactobacillus casei was (59.01±3.12)%, which was significantly higher than that of the surfactant-free Lactobacillus casei cell surface hydrophobicity. (26.73 ± 2.37)%. The difference was statistically significant (p < 0.05).

2.3 Detection of adhesion rate of the colonized probiotic preparation provided in the present example: the method is the same as step 1.3.

Results: The adhesion rate of Lactobacillus casei in human colonic probiotics provided by this example to human nasal mucosa cells (HNE) was (5.13±0.21) CFU/cell, which was significantly higher than FBS without surfactant. Buffer-adjusted adhesion rate of Lactobacillus casei (2.11 ± 0.38) CFU/cell). The difference was statistically significant (p < 0.05).

2.4 Anti-respiratory disease infection experiment of colonized probiotics preparation provided by the present embodiment

In vitro cell experiments showed that both lipoteichoic acid (LTA) and extracellular polysaccharide (EPS) of Lactobacillus casei promoted the amount of macrophages phagocytizing neutral red. It is indicated that Lactobacillus casei has the ability to stimulate and enhance the body's ability to kill viruses and germs. The probiotic preparation of the present embodiment can be applied to the respiratory tract for use in the prevention of colds caused by viruses and/or germs. The colonized probiotic preparation of the present example was sprayed in the air during the initial stage of infectious influenza. After inhalation of terrestrial vertebrates, Lactobacillus casei in the droplets colonizes the respiratory tract, producing an effect that promotes macrophage phagocytosis. The colonized probiotic preparation of the present embodiment can protect inhalers, prevent respiratory infections, and improve the body's resistance to colds.

Nine primary school classes have been selected for two students (2) who have a cold. The number of students in each class is not less than 30, and the students are 7-8 years old. The six classes were divided into a control group, a control group and an experimental group, three each. In the 2 classes of the group, the students sprayed lactic acid-containing physiological saline before entering the classroom, and the pH was 5.5. In the two classes of the second group, the students were sprayed with physiological saline containing Lactobacillus casei 1×10 ⁸ CFU/ml and lactic acid before entering the classroom, and the pH was 5.5. In the two classes of the experimental group, the colonized probiotic preparation of the present example was sprayed before the students entered the classroom. One week later, the number of follow-up colds in the control group was (4.3 ± 1.2). In the control group, the number of subsequent infections was (3.1±1.6). In the experimental group, the number of subsequent infections averaged (1.3 ± 0.7). In the class sprayed with the colonized probiotic preparation of the present example, the number of subsequent colds was significantly smaller than the class using the suspension of Lactobacillus casei. The difference was statistically significant (p < 0.05). More significantly less than the class using lactic acid containing saline. The difference was statistically significant (p < 0.05).

The colonized probiotic preparation provided by the embodiment can be applied to the prevention and treatment of respiratory diseases, and can prevent the spread of the cold.

Example 3

The colonized probiotic preparation provided by the embodiment is used for directly acting on the respiratory mucosa, and comprises a surfactant and a probiotic. Among them, the probiotic is inactivated Lactobacillus casei, and the surfactant is surfactant. The surface tension of the colony probiotic preparation is 40 mN/m, the pH is 5.5, the NaCl content is 9 g/L, and the surfactant content is 16 mg/ L, the inactivated probiotic content is 1 × 10 ⁸ /ml.

3.1 Preparation method of the colonization probiotic preparation provided by the embodiment:

The preparation method of the colonization probiotic preparation provided in this embodiment is basically the same as the step 2.1 in the second embodiment, except that the Lactobacillus casei bacteria in this embodiment are inactivated at 80 ° C for 20 minutes, and then inactivated. The Lactobacillus casei and the sterile culture supernatant are used to prepare a colonization probiotic preparation.

3.2 Cell surface hydrophobicity detection of probiotics in the colonized probiotic preparation provided in the present example: the detection method is the same as step 1.2. Probiotics were selected to be inactivated Lactobacillus casei at a concentration of 1 × 10 ⁸ /ml.

Results: The cell surface hydrophobicity of the surfactant-treated inactivated L. casei was (57.38±6.32)%, which was significantly higher than that of the surfactant-treated inactivated Lactobacillus casei. (29.37±7.02)%. The difference was statistically significant (p < 0.05).

3.3 Detection of the adhesion rate of the colonized probiotic preparation and the respiratory tract cell-chicken tracheal epithelial cell (CTE) provided by the present example:

CTE medium: DMEM/F12 was added to a final concentration of 10 μg/ml INS, 0.1 μg/ml HC, 1 mmo1/L glutamine, 10 μg/m TF, 25 ng/ml human epidermal growth factor (EGF) bovine pituitary Extract (BP), 100 IU/ml penicillin, 100 μg/ml streptomycin, 50 IU/ml amphotericin B and 10 IU/ml gentamicin, 5% fetal bovine serum.

Isolation and culture of chicken trachea epithelium (CTE): 1 day old non-immune healthy chicks were selected. Sterilely take the gas tube to the hilum and carefully peel off the surface fascia of the trachea. Rinse repeatedly with ice-cold PBS, with the trachea pale and no mucus. Sterile cotton was ligated to the lower end of the bronchus, 0.1% prolase was injected into the trachea from the other end, the trachea was filled, then ligated and immersed in ice-cold DMEM/F12, digested at 4 °C for 10 h, then the trachea was removed and the end was collected. The suspension was stopped by adding 10% fetal bovine serum. The suspension was filtered through a 400 mesh sieve and a 40 μm filter, and the filtrate was collected, followed by centrifugation at 1000 rpm for 8 min. Aspirate the supernatant to collect the cell pellet. The cells were squirted into suspension with CTE medium, inoculated into a 90 mm cell culture dish, and placed at 37 ° C in a volume of 5% CO ₂ incubator for 72 h, and then changed every 2 to 3 d. 1 time. Cultivate for 7 days.

The cultured chicken tracheal epithelial cells (CTE) were digested with trypsin-EDTA digestion solution, and then the cell concentration was adjusted to 1×10 ⁵ cells/ml with CTE culture medium without fetal bovine serum, and mounted on a 6-well tissue culture plate. 2 ml of each well was incubated in a 5% CO ₂ , 95% air incubator at 37 ° C until the cells grew to a monolayer. The CTE medium in each well of the tissue culture plate was discarded, and the plate was washed twice with a sterile PBS buffer.

One of the wells was digested with 0.5 ml of trypsin-EDTA digestion solution, and 0.5 ml of PBS was added thereto, shaken with a micro-oscillator and pipetted with a pipette, the cells were completely digested and mixed, and the cell count was calculated by a blood cell counting plate.

Two additional wells, one well plus 1 ml of fetal bovine serum-free CTE medium, and 1 ml of sterile FBS buffer adjusted inactivated Lactobacillus casei suspension (1 × 10 ⁸ /ml); 1 ml of CTE culture medium without fetal bovine serum and 1 ml of the colonization probiotic preparation of this example.

The two wells were mixed with a pipette and incubated in an anaerobic incubator at 37 ° C for 2 h. After incubation, the mixture of each well in the tissue culture plate was discarded and washed 5 times with sterile PBS buffer to remove unadhered probiotics. Add 0.4ml trypsin-EDTA After digesting the chemical solution, 0.6 ml of sterile PBS buffer was added, and the mixture was subjected to gradient dilution, fixed by methanol for 15 min, Gram stain, and the number of adhering bacteria was counted by a plate count.

Calculate the adhesion rate of different bacterial suspensions according to the following formula: Adhesion rate (pieces / cel1) = number of adhering bacteria / number of cells

As a result, the adhesion rate to the chicken tracheal epithelial cells (CTE) by the inactivated Lactobacillus casei adjusted with the supernatant of the aseptic culture liquid containing the surfactant was (1.37±0.36) cells/cell.

Inactivated Lactobacillus casei adjusted with surfactant-free FBS buffer, adhesion rate:

(0.59 ± 0.15) / cell.

It can be seen that the probiotic preparation of the present embodiment contains surfactant (surfactin), and the inactivated Lactobacillus casei treated with a surface tension of 40 mN/m has a significantly higher adhesion rate to chicken tracheal epithelial cells (CTE) than does not. The adhesion rate of surfactant-inactivated Lactobacillus casei to CTE alone. The difference was statistically significant (p < 0.05).

3.4 Anti-respiratory disease infection experiment of colonized probiotic preparation provided in this embodiment

Randomly assigned 500 7-day-old healthy meat cocks to 9 chicken houses. 50 chickens per house. Three chicken houses were randomly selected as the control group, three chicken houses were used as the control two groups, and three chicken houses were used as the experimental group. A spray containing 500 ml/d of E. coli 1 × 10 ⁵ /ml was sprayed in all the houses. When two chickens in a house have symptoms of avian colibacillosis, stop spraying E. coli spray. In the control group, physiological saline containing lactic acid was sprayed at a pH of 5.5. In the control two groups, physiological saline containing lactic acid and containing inactivated L. casei (1 × 10 ⁸ /ml) was sprayed at a pH of 5.5. In three experimental groups, the colonized probiotic preparation of the present example was sprayed. All chicken houses were sprayed with their respective reagents three times a day, 500 ml each time.

After 7 days, the number of chickens that continued to develop E. coli in the control group was (13.2 ± 3.6). The number of chickens infected with E. coli continued to be (8.4 ± 4.2) in the control group. The number of chickens in the experimental group continued to be (1.4 ± 0.8).

The above results show that the number of chickens that sprayed the colonized probiotic preparation of the present example continued to be significantly lower than the two control groups in which the lactic acid physiological saline and the inactivated cheese lactic acid bacteria suspension were sprayed. The difference was statistically significant (p < 0.05). This indicates that the colonized probiotic preparation provided in the present example acts on the respiratory tract, and the inactivated lipoteichoic acid (LTA) and extracellular polysaccharide (EPS) of L. casei still promote the phagocytic ability of macrophages. The infection of chicken E. coli disease is effectively prevented, which also indicates that the colonized probiotic preparation of the present embodiment can be applied to the prevention and treatment of livestock and poultry such as chicken respiratory diseases.

Example 4

The colonization probiotic preparation provided by the embodiment is used for directly acting on the respiratory mucosa, and comprises a surfactant, a probiotic, and NaCl, citric acid. Among them, the probiotic is inactivated Lactobacillus casei 1x10 ⁸ / ml, and the surfactant is chlorhexidine acetate and surfactant. The chlorine containing acetic acid has been determined to be 5 mg/L, and the concentration of surfactant is 14.5 mg/L. The surface tension of the probiotic preparation is 40 mN/m, the pH is 6, and the NaCl concentration is 60 g/L.

4.1 The preparation method of the colonization probiotic preparation provided by the embodiment: taking the supernatant of the culture supernatant containing the surfactant concentration of 14.5 mg/L in step 2.1, adding 6 g of NaCl, adding chlorhexidine acetate 5 mg/L, The surface tension was determined by an automatic interfacial tension meter. The surface tension was lower than 40 mN/m and diluted with deionized water. When the surface tension was higher than 40 mN/m, the supernatant of the culture solution was adjusted to obtain a compound surface with a surface tension of 40 mN/m. Active agent solution.

Add the inactivated Lactobacillus casei to obtain the suspension of Lactobacillus casei, and determine the Lactobacillus casei content by 1x10 ⁹ /ml by spectrophotometer, add citric acid to adjust the pH (if the pH is higher than 6, add citric acid, below 6) The above Lactobacillus casei suspension was added, and the pH value was determined by a pH meter to obtain a colonized probiotic preparation which can act on the respiratory tract.

4.2 Surface hydrophobicity test of the colonized probiotic preparation provided in the present example: the detection method is the same as step 3.2, as follows.

Results: The cell surface hydrophobicity of the inactivated Lactobacillus casei treated with the compound surfactant of this example was: (52.47±5.54)%, which was significantly higher than that of the surfactant-treated inactivated Lactobacillus casei cells. Surface hydrophobicity (29.37 ± 7.02)%. The difference was statistically significant (p < 0.05).

4.3 Detection of adhesion rate of the colonized probiotic preparation provided in the present example: the method is the same as step 3.3.

As a result, the inactivated Lactobacillus casei in the colonized probiotic preparation provided in the present example had an adhesion rate to chicken tracheal epithelial cells (CTE) of (1.53 ± 0.32) / cell. Significantly higher than the surfactant-free FBS buffer adjusted inactivated Lactobacillus casei (0.59 ± 0.15) / cell. The difference was statistically significant (p < 0.05).

4.4 The effect of this example on the treatment of swine hyperthermia syndrome

Pig hyperthermia syndrome is a disease with high morbidity and mortality. The main clinical manifestations of sick pigs are elevated body temperature, depression, loss of appetite or abolition, difficulty breathing, gasping, and some pigs with redness of the skin. Symptoms such as purple, a few pores have bleeding points. Pneumonia symptoms can occur before the pigs die.

Forty pigs with pig hyperthermia syndrome were randomly divided into three groups. A control group of 16 rats was used for cephalosporin within 1 day of high fever, and intramuscularly, and injected once per 0.1 kg of KG body weight. Change to a normal pig house for normal feeding, daily use 20ml of normal saline, spray into the nostrils three times; control two groups of 16, in the pigs with high fever within 1 day, using cephalosporin, intramuscular injection, per KG weight of 0.15ml injection 1 Next, change into a clean pig house, use 20ml of inactivated bacterial suspension of Lactobacillus casei at 1x10 ⁸ /ml per day, spray into the nostrils three times; 16 rats in the experimental group, use 1 day in the high fever of the piglets Cephalosporin, intramuscularly injected, once per KG body weight was injected 0.15 ml, and was exchanged into a clean pig house. 20 ml of the colonized probiotic preparation of the present example was sprayed into the nostrils three times a day.

After 7 days, the progress of the piglets with hyperthermia syndrome and the proportion of pneumonia occurred. RESULTS: The proportion of pneumonia in the control group was 56.3%. The proportion of pneumonia in the control group was 31.3%. The experimental group was 12.5%. It can be seen that the incidence of cerebral pneumonia in the hyperthermia syndrome of cephalosporin injection combined with the colonization of probiotics preparation of the present embodiment is significantly lower than that of the control group in which the cephalosporin and cephalosporin combined with the inactivated suspension of L. casei suspension is sprayed. Incidence.

This indicates that the colonized probiotic preparation provided by the present embodiment acts on the respiratory tract, effectively preventing the progression of the porcine hyperthermia syndrome, which also indicates that the colonized probiotic preparation of the present embodiment can be applied to the prevention and treatment of respiratory diseases related to livestock and poultry such as pigs. .

Example 5

The colonization probiotic preparation provided by the embodiment is used for directly acting on the respiratory mucosa, and comprises a surfactant, a probiotic and NaCl. Among them, the probiotic is Lactobacillus saliva, and the surfactant is a combination of probiotic surfactant (E-BS) and rhamnolipid (RL) produced by Lactobacillus acidophilus and Lactobacillus plantarum. The colonization probiotic preparation had a surface tension of 55 mN/m, a probiotic concentration of 1×10 ⁷ CFU/ml, a pH of 5.5, and a NaCl concentration of 7.6 g/L.

5.1 Preparation method of colonization probiotic preparation provided by the embodiment:

Lactobacillus acidophilus ATCC4356 and Lactobacillus plantarum ATCC8014 were separately inoculated into MRS medium, cultured at 37 ° C for 24 h, and activated 2 to 3 times.

Plant medium: Magnolia 10g / L, leek 10g / L, pine mushroom 10g / L, ginseng 100g / L, radish 100g / L, bitter gourd 10g / L; plant raw materials chopped and added deionized water; ozone disinfection for 20 minutes, After standing for 30 min, oligo-isomaltose 80 g/L and NaCl 38 g/L were added to become a plant medium.

1% of the activated Lactobacillus acidophilus strain and 1% of the activated Lactobacillus plantarum strain were inoculated in the plant medium, and anaerobic culture was carried out at 32 ° C for 24 hours. Transfer to 25 ° C anaerobic culture for more than 12d.

The culture was filtered through a three-layer filter cloth, and the filtrate was centrifuged at 4500 rpm for 20 min to remove the cells. After the culture supernatant of Lactobacillus acidophilus was obtained, the supernatant was obtained. Add 4 times deionized water to dilute.

The surface tension of the culture diluent was tested using a JYN 2200A automatic interfacial tension meter. The surface tension of the culture dilution was adjusted to 55 mN/m with deionized water and rhamnolipid. When the surface tension is higher than 55 mN/m, rhamnolipid is added, and below 55 mN/m, a NaCl deionized aqueous solution having a final concentration of 7.6 g/L of NaCl is added.

The Lactobacillus salivarius in Example 1 was added to the culture dilution to obtain a suspension of Lactobacillus salivarius, and the content of Lactobacillus salivarius was determined by spectrophotometer to be 1×10 ⁷ CFU/ml, and pH was adjusted by adding lactic acid (pH was determined by pH meter). The value is 5.5. If it is higher than 5.5, the lactic acid is continuously added, and if it is less than 5.5, the above-mentioned bacterial suspension is added, thereby obtaining a probiotic preparation which can act on the respiratory tract.

Lactobacillus salivarius has a good anti-allergic effect, but the adhesion rate is low. Lactobacillus acidophilus and Lactobacillus plantarum have a high adhesion rate, but the anti-allergic biological effect is low. Probiotic surfactants produced by Lactobacillus acidophilus and Lactobacillus plantarum can promote adhesion of almost all strains of the genus Lactobacillus. The types and ratios of probiotic surfactants are complex due to changes in the conditions of the culture process. After dilution, it is usually not necessarily the best surface tension of 55mN/m to promote the adhesion of the target cells. The adjustment of rhamnolipid or deionized water can overcome this process obstacle.

It should be noted that rhamnolipids can also be replaced by one or more of the following surfactants: trehalose lipids, sophorolipid, mannose erythritol lipid (MEL) ), Surfactin, lichenysin, ituririn, fengicin, plipastatin, spiroidesin, Bacircines ), Liposan, Tensin, Halobacillin, Isohalobacillin, Daitocidin, Pumilacidin, Ginsenoside, Tea Seed saponin, astragalus saponin, sapindus saponin, yellow flower saponin.

5.2 Cell surface hydrophobicity detection of probiotics in the colonized probiotic preparation provided in this example: the detection method is the same as step 1.2. A suspension of Lactobacillus salivarius at a concentration of 1 x 10 ⁷ CFU/ml was used.

Results: The surface hydrophobicity of the Lactobacillus salivarius treated with the compound surfactant in this example was (93.31±4.24)%. Significantly higher than the surface hydrophobicity (27.35 ± 1.48)% of Lactobacillus saliva without probiotic surfactant. The difference was statistically significant (p < 0.05).

5.3 Detection of Adhesion Rate of Colonized Probiotic Preparation Provided by the Present Example: The method is the same as step 1.3. The bacterial suspension adjusted with PBS was selected to be 1 × 10 ⁷ CFU/ml.

Results: The adhesion rate of probiotics to human nasal mucosa cells (HNE) in the colonized probiotics provided in this example was (8.94±0.51) CFU/cell, which was higher than that of surfactant-free bacterial suspension on human nasal mucosa. Cell (HNE) adhesion rate (1.13 ± 0.28) CFU / cell. The difference was statistically significant (p < 0.05).

5.4 Effect of colonizing probiotics preparation of this example on allergic rhinitis

According to the diagnostic criteria of allergic rhinitis of the Chinese Medical Association Otolaryngology Branch, 120 patients with allergic rhinitis were selected, including 67 males and 53 females; the youngest was 5 years old and the largest was 56 years old. 35 years old; the longest course of disease is 10 years and the shortest is 2 years. Excretion conditions: antibiotics were used within 3 months, glucocorticoids were used within 3 months, and probiotic preparations were used.

The patients were randomly divided into the experimental group (probiotics preparation dipping group) 40 cases, the control group (normal saline dipping group) 40 cases, the control two groups (probiotic bacteria containing saline dipping group) 40 cases. There were no significant differences in gender, age, and duration of the three groups of patients, which were comparable.

In the experimental group, 100 ml of the colonized probiotic preparation provided in the present example was used daily to dip the lower part and the back of the nasal cavity. The liquid temperature was 35 ° C, and the two nostrils were alternately lavaged until the preparation reached the mouth or another nostril, and the probiotic preparation was allowed to flow through the back of the nasal cavity (nasopharynx). After washing, bring a mask and incubate for half an hour. The body was examined by continuous dipping for 7 days and on the 9th day.

For the control group, 100 ml of physiological saline was used, and the cells were immersed in the above manner for 7 days, and the physical examination was performed on the 9th day.

For the control two groups, 100 ml of physiological saline containing Lactobacillus saliva 1×10 ⁷ CFU/ml was used, and the cells were immersed in the above manner for 7 days, and the physical examination was performed on the 9th day.

According to the evaluation criteria of allergic rhinitis, the symptoms were classified according to the physical signs: the inferior turbinate was in close contact with the nasal fundus and the nasal sinus, and the middle turbinate, or the middle turbinate mucosa polypoid, polyp formation, and 3 points were recorded; the inferior turbinate and There is a small gap between the lower nose and the bottom of the nose (or the nose), recorded as 2 points; the lower turbinate is slightly swollen, the nose is in the middle, the middle turb is still visible, record It is 1 point.

According to the sum of symptoms and signs before and after treatment, the percentage of improvement was evaluated according to the following formula: percentage = [(pre-treatment total score after treatment total score) / total score before treatment] x 100. If the percentage ≥ 51 is markedly effective, the 21≦ percentage is valid between ≦50 and the percentage ≤20 is invalid.

From the observation on the 9th day, the treatment group: 30 cases were markedly effective, 8 cases were effective, 2 cases were ineffective, the effective rate was 75%, and the total effective rate was 95%; the control group was markedly effective in 2 cases, effective in 4 cases, ineffective 34 For example, the apparent efficiency is 5% and the total effective rate is 15%. In the control group, 6 cases were markedly effective, 15 cases were effective, 19 cases were ineffective, the effective rate was 15%, and the total effective rate was 52.5%. The effective rate of the treatment group was greater than that of the two control groups. The difference was statistically significant (p < 0.05).

The probiotic preparation of the present embodiment, wherein the Lactobacillus salivarius has the effect of improving the expression of IFN-γ in the nasal mucosa-associated immune tissue and reducing the biological efficacy of IgE. In addition, its probiotic bacteria membrane formed on the nasal mucosa reconstitutes the microbial barrier of the nasal cavity. The nasal mucosa does not directly face the stimulus and antigen, which can increase the allergen tolerance threshold. The colonized probiotic preparation of the present embodiment is used for the treatment of allergic rhinitis, and has the fundamental effect of direct attack.

Among them, the most typical woman is a patient with old rhinitis for many years. First, a turbinate surgery was performed. After 1 year, he relapsed and glucocorticoids were sprayed. He did not improve, and then changed to oral administration. When her doctor advised her to inject glucocorticoids and do another nasal surgery, she was rejected by the woman. The female patient participated in the comparative trial and was assigned to the experimental group. After the experiment, all the symptoms of the woman disappeared completely. The patient claimed that the probiotic preparation saved her nose.

Many patients who participated in the treatment group, some had asthma or sinusitis, but still improved. In patients with marked improvement, the longest period of continuous improvement is 3 years without recurrence, and the shortest non-recurrence time is also more than 3 months. It can generally be done without recurrence for 1 year. (After observation and follow-up, the cause of recurrence may be related to the increase of smog and the use of antibiotics, resulting in the destruction of nasal probiotics again. Relapsed patients were re-dipped with the probiotic preparation of this example, and soon improved in less than 7 days) .

The colonized probiotic preparation of the present embodiment is used for treating allergic rhinitis and has special effects.

Example 6

The colonization probiotic preparation provided by the embodiment is used for directly acting on the respiratory mucosa, and comprises a surfactant, a probiotic and NaCl. Among them, the probiotic is a recombinant BCN of DCN gene, the bacterial content is 1×10 ⁷ CFU/ml, and the surfactant is ginsenoside. The colonization probiotic preparation had a surface tension of 60 mN/m, a pH of 7, and a NaCl concentration of 9 g/L.

6.1 Preparation method of colonization probiotic preparation provided by the embodiment:

30 μl of decorin (DCN) gene and PGEX-4T-1 plasmid were separately digested with EcoR I and Not I endonuclease in a 10×buffer reaction system for 2 h, and the digested product was subjected to agarose digestion. Identification by gel electrophoresis. A DNA gel fragment of 1.1 kbp and 5.0 kbp in length was cut using a blade according to the DNA Marker logo. The two gels were placed in an EP tube, and the DNA genes contained therein were recovered by using a gel extraction purification kit. The recovered PGEX-4T-1 destination vector and the DCN gene of interest were respectively taken as 10 μl and 30 μl, and added to a reaction system containing 2 μl of LT4 DNA ligase, 5 μl of 10×buffer, 3 μl of PEG 4000, and connected at 22° C., and left at 4° C. in a water bath overnight. The plasmid PGEX-4T-1-DCN was obtained.

Overnight cultured Bifidobacterium infantis ATCC 15697 was inoculated into PYG medium. The cells were anaerobic cultured at 37 ° C to an OD ₆₀₀ = 0.6, and the cells were collected by cryogenic centrifugation. Pre-cooled deionized water was washed 3 times and washed once with 10% glycerol. Then, 7 μl of plasmid PGEX-4T-1-DCN was added, and 10% glycerol was added to 100 μl. The mixture was placed in a pre-cooled electric shock cup and placed on ice for 5 min. The electroporator was adjusted to electric field strength: 20 kV/cm, capacitance: 25 μF, resistance: 200 Ω, and the recombinant plasmid was transformed into Bifidobacterium infantis after electric shock. After the electric shock is completed, immediately add 1 ml of PYG liquid medium to the electric shock cup, mix well, then transfer the liquid to 1.5 ml EP tube, and anaerobic culture for 1 h at 37 ° C. After the target carrier is fully expressed, the remaining bacterial liquid is tiled. The mixture was cultured in an anaerobic environment at 37 ° C in a Bs solid medium containing ampicillin. After 72 h, a well-grown single colony was selected from the culture medium, inoculated into a test tube containing ampicillin-containing PYG liquid medium, and anaerobic culture was carried out overnight at 37 ° C, and the test tube was taken out the next day. Obtained DCN gene recombinant Bifidobacterium infantis.

The above Bifidobacterium infantis was inoculated at a 5% inoculation amount, and anaerobic culture was carried out for 12 hours at 37 ° C in the following medium.

Bifidobacterium infantry fermentation medium (CPT) is: soybean protein 胨1.67%, casein 胨0.83%, lactose 0.5%, yeast leaching powder 0.5%, oligosaccharide 0.7%, carrot juice 5%.

After the culture was incubated for a predetermined period of time, it was centrifuged at 4500 rpm for 10 min, and the recombinant Bifidobacterium infantis was collected.

A salt solution was prepared using 0.9 g of NaCl and 100 ml of deionized water. The above Bifidobacterium infantis cells were added, and the content of Lactobacillus salivarius was determined to be 1×10 ⁷ CFU/ml by spectrophotometer, and 40 mg of ginsenoside was added to obtain a colonization probiotic preparation which can act on anticancer.

6.2 Hydrophobicity detection of the colonized probiotic preparation provided in the present embodiment: the detection method is the same as step 1.2.

Results: The surface hydrophobicity of the recombinant Bifidobacterium infantis (1×10 ⁷ CFU/ml) containing ginsenoside 0.4g/L used in this example was (65.11±6.43)%, which was greater than that of the recombinant infant without surfactant ginsenoside. The surface hydrophobicity of the Bifidobacterium suspension (1 × 10 ⁷ CFU/ml) was (53.72 ± 5.94)%.

6.3 Detection of Adhesion Rate of Colonized Probiotic Preparations Provided in the Examples:

Human nasopharyngeal carcinoma cell line CNE-2 was cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, penicillin 100 IU/ml, streptomycin 100 ng/ml. Culture conditions: 5% CO ₂ , 37 ° C. A 20 ml glass culture vial was inoculated with 200,000 cell lines CNE-2, and after 24 hours, it reached the logarithmic growth phase, and the old solution was removed, and 2.5 g/L of trypsin was digested into a single cell suspension.

The concentration of CNE-2 cells was adjusted to 1.0×10 ⁵ /ml in RPMI1640 medium plus 10% fetal bovine serum. The cells were packed in 3 wells of 6-well tissue culture plates at 2 ml per well at 37 °C. Incubate for 24 hours with 5% CO ₂ . Incubate until the cells grow to a single layer. The nutrient solution in each well of the tissue culture plate was discarded, and the plate was washed twice with sterile PBS buffer.

One of the wells was digested with 0.5 ml of trypsin-EDTA digestion solution, and 0.5 ml of PBS was added thereto, shaken with a micro-oscillator and pipetted with a pipette, the cells were completely digested and mixed, and the cell count was calculated by a blood cell counting plate.

Two additional wells, one well plus 1 ml of RPMI1640 medium, and 1 ml of sterile FBS buffer adjusted recombinant Bifidobacterium infantis suspension (1 × 10 ⁷ CFU/ml); one well plus 1 ml of RPMI1640 medium and 1 ml The colonized probiotic preparation of this example.

The two wells were mixed with a pipette and incubated in an anaerobic incubator at 37 ° C for 2 h. After incubation, the mixture of each well in the tissue culture plate was discarded and washed 5 times with sterile PBS buffer to remove unadhered probiotics. After adding 0.4 ml of trypsin-EDTA digestive juice for digestion, 0.6 ml of sterile PBS buffer was added, and the mixture was subjected to gradient dilution, methanol fixation for 15 min, Gram staining, and plate count to calculate the number of adhered bacteria.

The adhesion rate of different bacterial suspensions was calculated according to the following formula: Adhesion rate (CFU/cell) = number of adherent bacteria/cell number.

As a result, the adhesion rate of the recombinant Bifidobacterium infantis in the colonized probiotic preparation of the present example to the nasopharyngeal carcinoma cells was (6.58±0.32) CFU/cell, which was greater than that of the recombinant infant bifidobacteria without the surfactant. Adhesion rate of pharyngeal cancer cells (4.87±0.44) CFU/cell. The difference was statistically significant (p < 0.05).

6.4 Experimental study on the targeted therapy of colonized probiotics in the treatment of nasopharyngeal carcinoma

Sixteen patients with nasopharyngeal carcinoma in southern China were selected. Inclusion conditions: CT scan lesion size was 5mm-8mm, no spread, no radiotherapy.

Randomly divided into three groups. (Recombinant Bifidobacterium infantis) A control group of 8 was sprayed daily to the nasal cavity using a surfactant-free recombinant Bifidobacterium infantis 1 x 10 ⁷ CFU/ml bacterial suspension 10 ml twice a day. (Ginsenoside) 8 rats in the control group were sprayed daily with 10 ml of a solution containing ginsenoside 0.4 g/L twice a day. In the experimental group, 10 ml of the probiotic preparation of the present example was sprayed into the nasal cavity twice a day.

Three groups were treated continuously for 1 year, and the lesion size was again scanned with CT. The size of the lesion is constant or reduced, and the ratio of the number of people who do not spread to the number of groups can be regarded as the apparent efficiency of one-year conservative treatment. The effective efficiency of the colonized probiotic preparation provided by the present embodiment is 87.5%, which is significantly greater than the apparent efficiency of the control group of the recombinant Bifidobacterium infantis 50%, and the significant efficiency of the ginsenoside control group of 37.5%.

The colonized probiotic preparation of this example can be used for targeted treatment of nasopharyngeal carcinoma. Administration to the pharynx via the nasal cavity is safer and more effective than by injection.

Example 7

The colonization probiotic preparation provided by the embodiment is used for directly acting on the respiratory mucosa, and comprises a surfactant, a probiotic and NaCl. Among them, the probiotic is Lactococcus lactis, the concentration is 1×10 ⁵ CFU/ml, and the surfactant is Xinyi extract. The surface tension of the colonized probiotic preparation was 65 mN/m or less, the concentration of the extract of Magnolia biondii was 6.0 g/L, the pH was 7, and the NaCl concentration was 2 g/L.

7.1 Preparation method of colonizing probiotic preparation provided by the embodiment:

The content of 1 × 10 ⁷ CFU / g of Lactococcus lactis (Lactococcus lactis EU147310) embedded freeze - dried powder 1g, NaCl0.2g, deionized water was added 100ml double planetary mixer, magnolia extract 0.6g, thereby obtaining the present embodiment Examples of colonized probiotic preparations.

Vanillic acid 4-O-β-D-glucoside, 3-methoxy-4-hydroxybenzene-1-O-β-D-glucoside, vanillyl vanillyl ester, benzyl-O -β-D-galactoside, 3,4,5-trimethoxybenzene-1-O-β-D-glucoside, benzyl-O-β-D-glucoside is a vegetable surfactant material.

7.2 Cell surface hydrophobicity detection of probiotics in the colonized probiotic preparation provided in the present embodiment: the detection method is the same as step 1.2.

Results: The cell surface hydrophobicity of L. lactis treated by the extract of Magnolia biondii of this example was (32.51±5.21)%, which was significantly higher than that of the surfactant-free Lactococcus lactis (1x10 ⁵ CFU/ml). Sex (22.31 ± 1.67)%. The difference was statistically significant (p < 0.05).

7.3 Detection of adhesion rate of the colonized probiotic preparation provided in the present embodiment: the method is the same as step 1.3.

Results: The adhesion rate of Lactococcus lactis to human nasal mucosa in the colonized probiotic preparation of this example was (2.21±0.07) CFU/cell, which was higher than that of Lactococcus lactis (1×10 ⁵ CFU/ml without surfactant treatment). Adhesion rate (1.07 ± 0.14) CFU / cell.

7.4 Experiment of the effect of colonizing probiotics provided in this embodiment

Twenty healthy guinea pigs were selected, half male and half female, weighing 350-450 g. Guinea pigs were randomly divided into experimental and control groups by weight, with 10 rats in each group. Modeling of nasal hypersensitivity: firstly add 10 μl of 10% di-octyl isocyanate (TDI) olive oil solution, and add 2 sets of guinea pig bilateral anterior nares with a pipette, 5 μl per side, once a day, for 5 consecutive times. After ~7d, it was changed to once every other day.

After successful modeling, 10 μl of the probiotic preparation of the present example was administered to the observation group of guinea pigs, and the anterior nostrils of the guinea pigs were instilled into the anterior nares of the guinea pigs by a pipette, 5 μl per side, 4 times a day for 10 days. The control group was dripped into the nostrils with physiological saline in the same manner as above, and the administration time was synchronized.

Nasal symptoms were observed from the first day of administration. The time and severity of nasal symptoms such as nasal itching, sneezing, and salivation were observed, and the scores were recorded according to the following criteria: 1 nasal itching mild: lightly rubbed the nose several times. 1 point; nasal itching moderate: licking the nose is not limited. Count 2 points; nasal itching severe: nasal itching and scrubbing 3 points. 2 sneeze, 1 to 3 counts 1 point, 4 to 10 counts 2 points, more than 10 counts 3 points. 3 drooling, flow to the nose before the hole count 1 point, more than 2 points beyond the nose hole, turbulent full face count 3 points. The total score is calculated by the superposition method when recording, and the total of more than 5 points is the model success. At the time of administration, 2 hours after administration, each observation was carried out for 30 min.

The results showed that after 10 days, the animal score was >5, the experimental group was 2, and the control group was 7; the score was ≤5, the experimental group was 8 and the control group was 3. The difference was significant (P<0.01).

The colonized probiotic preparation of the present embodiment has a therapeutic effect on allergic rhinitis.

Example 8

The colonization probiotic preparation provided by the embodiment is used for directly acting on the respiratory mucosa, and comprises a surfactant, a probiotic, and a NaCl extract, and a water extract.

1 g of lyophilized powder of 1 × 10 ⁷ CFU/g of Lactococcus lactis EU147310 was embedded, 0.2 g of NaCl, 0.5 g of extract of Magnolia sinensis, and uniformly mixed together to obtain powdery colonization of the present example. Probiotic preparations.

When sprayed directly into the nasal cavity, or in a ratio of mass to volume ratio of 1:50 (ratio of colonized probiotic preparation to deionized water), the powdered colonized probiotic preparation is dispersed in deionized and then sprayed into the nasal cavity.

The dry powder of the colonized probiotic preparation provided by the present embodiment is obtained by dispersing the powdered colonization probiotic preparation in deionized water according to the ratio of the mass to volume ratio of 1:50 (the ratio of the colonized probiotic preparation to the deionized water). The same liquid probiotic formulation of Example 7 has the same efficacy as its properties.

In summary, the present invention provides a colonizing probiotic preparation for directly acting on a respiratory mucosa, which comprises a surfactant and a probiotic, and the surfactant modifies the sol layer and the gel layer of the respiratory mucus, thereby improving probiotics. The probability and rate of exposure of bacteria to respiratory mucosal cells. At the same time, the surfactant changes the cell surface hydrophobicity of probiotics, and improves the adhesion rate of these probiotics to the airway mucosal epithelial cells, thereby solving the problem that the probiotics in the prior art are difficult to colonize the respiratory mucosa under the removal of the mucociliary transport system. Technical bottlenecks.

Therefore, the probiotics contained in the colonization probiotic preparation provided by the invention have higher adhesion rate to the respiratory mucosa, and are more easily colonized in the respiratory mucosa, which is beneficial to the probiotics to continue to function for a long time. The colonization probiotic preparation provided by the invention has broad application prospects, and can be applied to preparation for treating rhinitis or for treating asthma or for treating allergic dermatitis or for treating cranial nerve disease or anti-nasopharynx cancer or anti-lung cancer Or in the field of anti-cold or drugs for preventing or treating respiratory infections.

The above description is only the preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.

Claims (20)

1. A colonization probiotic preparation characterized in that it is used for direct action on a respiratory mucosa, and the colonization probiotic preparation comprises a surfactant and a probiotic.
2. The colonizing probiotic preparation according to claim 1, wherein the colonization probiotic preparation has a surface tension of less than 65 mN/m.
3. The colonizing probiotic preparation according to claim 1 or 2, wherein the colonization probiotic preparation has a surface tension of 40 to 60 mN/m.
4. The colonizing probiotic preparation according to claim 2 or 3, wherein the surfactant is selected from the group consisting of a biosurfactant and a vegetable surfactant.
5. The colonizing probiotic preparation according to claim 4, wherein the biosurfactant is selected from the group consisting of glycolipids and lipopeptides selected from the group consisting of saponins and other surface-active glycosides. The group consisting of.
6. The colonizing probiotic preparation according to claim 5, wherein the glycolipid is selected from the group consisting of rhamnolipid, trehalose, sophorolipid, and mannose erythritol lipid, the lipopeptide A group consisting of a surfactant, a lichenin, a lutein, a fentanin, a large arbutin, a spirulin, and a tensin is selected.
7. The colonization probiotic preparation according to claim 5, wherein the saponin is selected from the group consisting of ginsenoside, tea saponin, astragaloside, soy saponin, acacia saponin, zona saponin, saponin, diosgenin, and yellow flower A group consisting of saponins.
8. The colonizing probiotic preparation according to any one of claims 1 to 7, wherein the colonization probiotic preparation comprises a Magnolia extract.
9. The colonizing probiotic preparation according to any one of claims 8 to 4, wherein the extract of Magnolia has a surfactant.
10. The colonizing probiotic preparation according to any one of claims 1 to 9, wherein the colonization probiotic preparation comprises a ginseng extract.
11. The colonization probiotic preparation according to any one of claims 1 to 10, wherein the probiotic is a lactic acid bacterium, and the content of the lactic acid bacteria in the colonization probiotic preparation is 1 x 10 ^{4 -} 1 x 10 ¹² / g .
12. The colonizing probiotic preparation according to any one of claims 1 to 10, wherein the probiotic is Lactobacillus.
13. The colonization probiotic preparation according to claim 12, wherein the lactobacillus is Lactobacillus saliva, the colonization probiotic preparation further comprises a bacterial culture supernatant, and the bacterial culture supernatant contains the The surfactant, the surface tension of the colonized probiotic preparation is 49-60 mN/m.
14. The colonizing probiotic preparation according to claim 13, wherein the colonization probiotic preparation has a surface tension of 55 mN/m.
15. The colonization probiotic preparation according to any one of claims 1 to 10, wherein the colonization probiotic preparation comprises NaCl, and the content of the NaCl in the colonization probiotic preparation is 2-60 g/Kg. .
16. The colonizing probiotic preparation according to any one of claims 1 to 10, wherein the colonizing probiotic preparation has a pH of 3.5-7.
17. The colonizing probiotic preparation according to any one of claims 1 to 10, wherein the probiotic is selected from the group consisting of inactivated probiotics and recombinant lactic acid bacteria.
18. The colonizing probiotic preparation according to claim 17, wherein the recombinant lactic acid bacterium is Bifidobacterium infantis containing foreign DNA.
19. A medicament for treating rhinitis or for treating asthma or for treating allergic dermatitis or for treating cranial nerve diseases or for treating nasopharyngeal cancer or against lung cancer or anti-cold or preventing or treating respiratory infectious diseases, characterized in that It comprises the colonized probiotic preparation of any one of claims 1-18.
20. The colonized probiotic preparation of any one of claims 1 to 19 for use in the treatment of rhinitis or for the treatment of asthma or for the treatment of atopic dermatitis or for the treatment of cranial nerve disease or anti-nasopharynx or anti-lung cancer or Use in medicines that are resistant to colds or to prevent or treat respiratory infections.