KR100274760B1 - Oral vaccine for helicobacter pyroli and method for preparing the same - Google Patents

Abstract

PURPOSE: An oral immunizing agent against Helicobacter pylori containing microspheres consisting of a biodegradable polymer and a Helicobacter pylori antigen as an effective component and a process for preparing the same are provided. Therefore, the agent can be effectively used for the prevention of infection of Helicobacter felis as well as Helicobacter pylori. CONSTITUTION: This oral immunizing agent contains microspheres having an average diameter of 0.1 to 100 micrometer as an effective component, wherein the microspheres comprise a Helicobacter pylori antigen prepared from one or more selected from the group consisting of one or more biodegradable polymers selected from the group consisting of a lactid polymer, a glycolide polymer and a copolymer of lactid and glycolide, Helicobacter pylori MBR12 and Helicobacter pylori18.

Description

Oral immunity against Helicobacter pylori and preparation method thereof

The present invention relates to oral immunity against Helicobacter pylori and a method for producing the same. More specifically, the present invention is a biodegradable polymer with Helicobacter pylori (Hilicobacter pylori) antigen comprising the microspheres consisting of the active ingredient, wherein in the blood and mucosal tissues - the same in the virulence, as well as to induce the generation of a Helicobacter pylori antibody Oral immunity against Helicobacter pylori that can protect even the infection of the microorganism Helicobacter felis and a method for producing the same.

In 1983, Australian microbiologists Marshall and Warren first discovered the same type of flexible bacteria in the gastric mucosal biopsy tissue of a patient with type B gastritis, followed by cultivation of an electric bacterium, resulting in the development of Campylobacter genus and Since identification of the new species involved , active research has been conducted on the link between Helicobacter pylori and diseases of the upper gastrointestinal tract, such as gastritis and peptic ulcer. Helicobacter pylori is a Gram-negative bacillus with a thin membrane-enclosed flagella and is known to propagate through the feces-oral route by releasing large amounts of urease. Currently, the pathogenesis of the disease caused by Helicobacter pylori first enters the gastric mucus layer using a flagella with a strong mobility of the bacteria entering the gastric lumen with food, and inhabits the upper part of the phase epithelial layer and the intercellular junction in the gastric mucus layer. By secreting a large amount of urease and changing the mucous layer to alkaline, it has been found to induce excessive gastric acid secretion promotion by gastrin, resulting in inflammation and ulceration. In addition, according to a recent study that found that Helicobacter pylori is an important risk factor for gastric adenocarcinoma, the International Health Organization has designated Helicobacter pylori as the first group carcinogen.

Infections with Helicobacter pylori have been reported in particular in developing countries, including Korea. According to Baek et al., It is estimated that more than 80% of normal Koreans are infected with Helicobacter pylori. Et al., Korean Journal of Microbiology, 25: 45-52, 1990). Therefore, the development of treatment and prevention methods for Helicobacter pylori infection is very urgent. The use of chemotherapy with antibiotics as a method for treating diseases caused by microbial infections may be effective in the short term, but it is not preferable because there is a risk of antibiotic resistant strains due to continuous use of antibiotics. In view of this, vaccination is the most desirable method for eradicating large-scale Helicobacter pylori for the entire population. As seen in the case of polio and diphtheria, which are now known as representative pathogenic microorganisms, the mass vaccine strategy has been very effective worldwide.

Regarding the development of a vaccine to prevent infection of Helicobacter pylori, Chin et al. Used mice and ferrets using Helicobacter pylori antigen and the cholera toxin (weak "CT"), a potent mucosal dajubvant. The oral immunity of (ferret) induced local immune responses to bacterial infections, particularly increasing the titer of intestinal IgA (see Cainn et al., Infect. Immun., 59: 5359-2363, 1991). This suggests that effective immunization against Helicobacter pylori is possible by using a vaccine. On the other hand, Helicobater felis, which is very similar to Helicobacter pylori, not only retains infection for 18 months in Valb / c mice, but also the low-grade ratio known to be caused by Helicobacter pylori in humans. By confirming the induction of low-graed B cell MALT lymphoma, it has been suggested that valve / medium mice can be used as an economical and convenient animal testing model essential for vaccine development studies (Lee et al. , Aliment Pharmacol. Ther. 10 (suppl. 1): 129-138, 1996). Chin et al. Also confirmed for the first time that mice were orally immunized with 1 mg of Helicobacter Felis antigen and 10 μg of cholera toxin, followed by oral administration of 10 ⁸ live Helicobacter Felis (Czinn). et al., Infect. Immun., 59: 2359-2363, 1991). In addition, many studies have been reported suggesting that the Helicobacter pylori vaccine can be developed by using the Helicobacter pylori antigen.

However, cholera toxin, which is used as a mucosal immune adjuvant by most researchers, is difficult to use in humans due to its toxicity, such as a very small amount of 5 µg, which can cause severe diarrhea in humans, and is also known as a mucosal immune adjuvant. LT (heat-labile enterotoxin of E. coli ) is also less dangerous to humans, but it is not desirable because it has a CT-like structure.

Recently, as an effective drug delivery system, there are microspheres prepared by capturing active substances such as proteins, peptides, hormones, etc. using high-rich compounds. By controlling the preparation method and the like, the decomposition rate and the release pattern of the active substance can be changed in various ways, and the immunity can be maintained for a long time with only one immunity. Among the polymer compounds that can be used for the production of microspheres, the most popular among them is a copolymer of lactide and glycide, which is a kind of biodegradable polymer (ploy (D, L-lactide-co-glycolide, hereinafter, " PLG has been used in humans as a suture for a long time and has the advantage of being harmless to humans because it is broken down and released in vivo. Also, chalacombe and others have PLG and ovalbumin. When oral immunization of valve / cid mice with microspheres was performed, it was confirmed that not only IgG in blood but also secretory IgA secreted from mucosal tissue were induced (see Challacombe et al., Immunology, 76: 164-168). , 1992), Moore et al., Patidos, et al., Have shown that immune responses involving cytotoxic T lymphocytes when immunized with microspheres containing proteins or peptides. Has reported that the bar (see: Moore et al, Vaccine, 13:. 1741-1749, 1995: Partidos et al, J. Immunol Methods, 20:.. 143-151, 1997).

Accordingly, the present inventors have made intensive studies to prepare oral immunity agents for Helicobacter pylori using biodegradable polymers. As a result, helicobacter using PLG (poly (D, L-lactide-co-glycolide)) is a kind of biodegradable polymer. Preparation of microspheres containing pylori antigens and oral facet of mice with electric microspheres resulted in the production of anti-Helicobacter pylori antibodies in blood and mucosal tissues, as well as the infection of Helicobacter pelis, a pathogenic microorganism of the same genus. It was confirmed that the defense can be completed, the present invention was completed.

After all, it is an object of the present invention to provide an oral immunity agent for Helicobacter pylori comprising a microsphere composed of a biodegradable polymer and Helicobacter pylori antigen as an active ingredient.

Another object of the present invention is to provide a method for the preparation of oral immunity against electric Helicobacter pylori.

FIG. 1A is a result of ELISA analysis of anti-helicobacter pylori IgG antibody titer in Val / C (Balb / c) mouse serum orally immunized with microspheres containing Helicobacter pylori antigen.

FIG. 1B is a result of ELISA analysis of anti-helicobacter pylori IgA antibody titer in valv / c mouse gastric lavage fluid orally immunized with microspheres containing Helicobacter pylori antigen.

FIG. 2 shows the results of immunofluorescence analysis of Helicobacter Felis infection in the gastric tissues of Balb / c mice treated with Helicobacter pylori, followed by oral immunization with microspheres containing Helicobacter pylori antigen.

The present inventors prepared microspheres containing Helicobacter pylori antigen by solvent evaporation or spray drying using poly (D, L-lactide-co-glycolide) (PLG), and then orally immunized mice with electric microspheres. The serum and gastric lavage of mice were collected and measured by anti-helicobacter pylori antibody titer by ELISA method. The serum and gastric lavage of immunized mice contained a large amount of anti-helicobacter pylori IgG antibody and IgA antibody, respectively. In addition, it was confirmed that when Helicobacter Felis, a pathogenic microorganism of the same genus as Helicobacter Pylori, was administered to mice immunized with electric microspheres, infection of Helicobacter Felis was effectively prevented.

Hereinafter, the present invention will be described in more detail.

The present inventors used Helicobacter pylori antigen purified from two kinds of Helicobacter pylori MBRI2 (KCTC 0216BP), Helicobacter pylori MBRI8 (KCTC 021BP) already isolated in Korea as Helicobacter pylori antigen (see Korean patent) Pub. 97-43025). First, PLG was dissolved in an organic solvent, homogenized by addition of an electric Helicobacter pylori antigen, and then the organic solvent was removed to prepare an average diameter of the microspheres in which the Helicobacter pylori antigen was collected to be 0.1 to 40 µm. The removal of the electric organic solvent was performed by adding and homogenizing a polyvinyl alcohol solution to the electric homogenate, and then volatilizing the organic solvent with stirring, or volatilizing the organic solvent by spraying the electric homogenate directly through a nozzle.

Subsequently, in order to confirm that the microspheres obtained in the past induce an effective immune response against Helicobacter pylori, the microspheres are suspended in sodium carbonate solution to oral immunization of 5-6 week old Val / c mice. And two more immunizations, one week apart. Serum and gastric lavage fluid were collected at 1 month intervals from 2 weeks from the last day of additional immunization, and anti-helicobacter pylori antibody titer was measured by ELISA method. As a result, when the mice were orally immunized with microspheres containing Helicobacter pylori antigen, high anti-Helicobacter pylori antibody titer was confirmed not only in serum but also in gastric lavage fluid. Especially, the ecology of Helicobacter pylori inhabiting gastric mucosal tissue was considered. The fact that high anti-Helicobacter pylori antibody titers are observed in gastric lavage suggests that immunization with microspheres containing Helicobacter pylori antigen is very effective in eradicating and reinfecting Helicobacter pylori.

In addition, the same method as described above to determine whether the antibody induced by the Helicobacter pylori antigen contained in the electrical microsphere can also protect against infection by Helicobacter Fellis inhabiting gastric tissue with pathogenic microorganisms of the same genus as Helicobacter pylori. Valve / sea mice were immunized. After another immunization once again at 2 months from the last day of additional immunization, 1 week later, live Helicobacter Felis was orally administered over 2 days, and after 2 weeks thereafter, the gastric tissues of the mice were harvested, and immunofluorescence assay was applied to Helicobacter Felis. Infection was confirmed. As a result, when immunized with microspheres in which the Helicobacter pylori antigen was collected, it was confirmed that the infection rate by Helicobacter Felis significantly decreased. These results suggest that anti-Helicobacter pylori antibodies, particularly secretory IgA antibodies, formed by immunization with electric microspheres can effectively protect against infection of Helicobacter felis.

On the other hand, oral immunity agent comprising a microsphere composed of the biodegradable polymer of the present invention and the Helicobacter pylori antigen as an active ingredient from the group consisting of a polymer of lactide, a polymer of glycolide and a copolymer of lactide and glycoride It may comprise one or more biodegradable polymers selected, the inherent viscosity of the electrically biodegradable polymer is preferably 0.1 to 1.2 dL / g, molecular weight is 10 to 250 kDa, preferably 10 to 150 kDa. In a preferred embodiment of the present invention, the content ratio of lactide and glycidide is 50:50 in weight ratio, and a copolymer of lactide and glycidide having a molecular weight of 20 kDa is used, but the lactide in the copolymer of lactide and glycolide is used. The content ratio of and glycoride is not particularly limited. In other words, the oral immunity agent comprising a microsphere composed of the biodegradable polymer of the present invention and the Helicobacter pylori antigen as an active ingredient, a variety of biodegradable polymers each having a different viscosity, molecular weight, and content ratio of lactide and glycidide May comprise microspheres simultaneously. On the other hand, the organic solvent used in the manufacture of the microsphere of the present invention may be used dichloromethane, ethyl acetate, benzyl alcohol, acetone, dimethyl sulfoxide, ethanol and / or dimethyl formamide.

Oral immunity comprising a microsphere composed of the biodegradable polymer of the present invention and the Helicobacter pylori antigen as an active ingredient can be prepared orally administered in a conventional oral dosage form such as liquids, tablets, capsules, granules, such Oral dosage forms contain a pharmaceutically acceptable buffer such as sodium bicarbonate, potassium bisulfate and sodium phosphate to increase or neutralize the pH of the gastric juice, thereby preventing the Helicobacter pylori antigen protein exposed during degradation of the biodegradable polymer. It can be stably protected and can be prepared by adding various pharmaceutically acceptable carriers such as stabilizers and sweeteners.

In addition, an oral immune agent comprising a microsphere composed of the biodegradable polymer of the present invention and a helicobacter pylori antigen as an active ingredient may be added to antibiotics to facilitate eradication and infection prevention of Helicobacter pylori, and various anti-ulcer agents Adding them may shorten the treatment period for gastritis or gastric or duodenal ulcer.

The dosage of the oral immunity agent comprising a microsphere composed of the biodegradable polymer of the present invention and the Helicobacter pylori antigen as an active ingredient is generally once a day based on an adult having a weight of 60 kg based on the active ingredient. Administration from 15 mg to 1.5 g is preferred, and may be determined by the experience of one of ordinary skill in the art.

On the other hand, if necessary, re-administration at intervals of one week or two weeks can induce an additional antigen stimulation reaction, and the amount of additional antigen stimulation may be administered at or below the initial dose. have.

Oral immunity agent comprising a microsphere composed of a biodegradable polymer of the present invention and a Helicobacter pylori antigen as an active ingredient, from 0.625g to 100 times the effective amount of 5 to 6 weeks old valve / seed mice having a weight of 20 to 25g As a result of oral administration of 62.5 mg, all 10 animals survived, and it was found that the oral immunity agent containing the microsphere composed of the biodegradable polymer of the present invention and the Helicobacter pylori antigen as an active ingredient in an effective range was a safe drug.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Preparation of Microphone Sphere

Example 1-1 Preparation of Microspheres by a Solvent Evaporation Method

A Helicobacter pylori antigen for microsphere fabrication using the H. pylori antigen purified from the inventors Helicobacter pylori MBRI2 of two previously separate (Helicobacter pylori MBRI2, KCTC 0216BP), and Helicobacter pylori MBRI8 (Helicobacter pylori MBRI8, KCTC 0218BP ) It was. In order to prepare microspheres, first, PLG (poly (D, L-lactide-co-glycolide), Wakao Chemical Inc., Japan) having a weight ratio of lactide and glycorides of 50:50 and a molecular weight of 20 kDa was obtained from dichloromethane. Or dissolved in ethyl acetate at a concentration of about 1-20% (w / v). Then, 1 to 10% (w / v) antigen solution diluted with sterile water was added to the electric PLG solution so that the volume ratio was 1: 5 to 1:20, and 5,000 to 10,000 using a homogenizer. Homogenize at rpm for 5-10 minutes. Subsequently, the electric sample and the 0.5-20% polyvinyl alcohol solution were mixed, homogenized using a homogenizer at 1,000 to 10,000 rpm for 10 to 20 minutes, and then the organic solvent was evaporated with stirring at 300 to 500 rpm for 24 hours. , A solid sample was obtained. The solid sample was washed three times with distilled water, resuspended in an appropriate volume of distilled water, lyophilized and stored in a vacuum desiccator. As a result of measuring the average diameter of the microspheres prepared in the same manner as above, it was confirmed that it is about 0.5 to 20㎛.

Example 1-2 Preparation of Microspheres by a Spray Drying Method

In the same manner as in Example 1-1, the PLC was dissolved in an organic solvent, the antigen solution was added to the electric PLC solution, and then homogenized using a homogenizer for 5 to 10 minutes. 450, Branson, USA) was further homogenized for about 2 to 5 minutes under conditions of output 3-5. Subsequently, the electric homogenate was sprayed and dried using a mini sprayer (Buchi Mini-sprayer model B-191, Buchi, Swizerland) at a spray temperature of 30 to 70 ° C. and a spray rate of 400 to 700 Nl / h. Then, to remove traces of residual moisture and organic solvent, vacuum dried at room temperature for 24 hours, and stored in a vacuum desiccator. As a result of measuring the average diameter of the microspheres prepared in the same manner as above, it was confirmed that it is about 1 to 80㎛.

Example 2 Anti-Helicobacter Pylori Antitega Measurement

In order to confirm that the microspheres containing the Helicobacter pylori antigens obtained from Examples 1-1 and 1-2 induce an effective immune response against Helicobacter pylori, the concentration of the electric microspheres is 0.5 to 5 mg / ml. Suspended in a 7.5% NaHCO ₃ solution, orally immunized 0.5 ml per 5-6 week old Balb / c mice weighing 20 to 25 g, 2 at weekly intervals. More times immunized. Diagnosis kit for detecting antibodies containing Helicobacter pylori antigen isolated from Korea, which the inventors have already patented, by collecting serum and gastric lavage fluid every month from the second week after the last additional immunization date (see Korean Patent Publication No. 97 -43067), the anti-Helicobacter pylori antibody titer was measured by ELISA method. At this time, the control mice were administered with the phosphate buffer according to the same immunization schedule, and the serum was diluted 1:50 times with the sample diluent included in the diagnostic kit, the antibody was used for the measurement, the gastric lavage was not diluted Used directly. As a result of the measurement of the antibody, as shown in FIGS. 1A and 1B, when the mice were orally immunized with microspheres containing the Helicobacter pylori antigen, the antigen was higher in the gastric lavage (FIG. 1B) as well as in the serum (FIG. 1B) than in the control group. Helicobacter pylori antibody titer was seen, particularly at week 8 (the second serum and gastric lavage, ie 10 weeks from the last immunization), rather than at 4 weeks (the first serum and gastric lavage days, ie, 6 weeks from the last immunization date). Much higher antibodies were seen. Considering the ecology of Helicobacter pylori inhabiting gastric mucosal tissues, the fact that high IgA antibody titer is observed in gastric lavage fluid suggests that microspheres-immunized immunization with Helicobacter pylori antigen is very effective in eradicating and reinfecting Helicobacter pylori. Presented.

Example 3 Measurement of Defense Efficiency Against Helicobacter Felice Infection

To determine whether the antibodies produced when immunized with microspheres in which the antigens of Helicobacter pylori are captured can be protected against infection by Helicobacter felis, which is a pathogenic microorganism in the same genus as Helicobacter pylori, and lives in the stomach tissue. In the same manner as in Example 2, Val / C (Balb / c) mice were orally immunized. After another immunization two months after the last day of additional immunization, one week later, Helicobacter Felis (ATCC 49179), obtained from the United States spawn association, was diluted with phosphate buffer and ¹ × 10 ⁷ to 5 × 10 ⁷ dogs were injected orally over two days. Two weeks after the administration, the stomach tissues of the mice were collected, and immunofluorescence and hygiene urease tests were performed in the following manner.

Example 3-1 Immunofluorescence Analysis

In order to perform immunofluorescent stone, the stomach tissue of the mouse was taken and crushed using a wire mesh having a pore size of 40 to 80 µm, and the upper layer was centrifuged at 2,000 to 4,000 rpm for 5 to 10 minutes to remove the residue. The solution was recovered, and then centrifuged at 10,000 to 15,000 rpm for 20 to 30 minutes to obtain a fungal cell precipitate. The precipitate was suspended in an appropriate volume of phosphate buffer and added to each well of a 12 well slide glass, dried and fixed with acetone. Serum prepared from rats intraperitoneally immunized with Helicobacter Felis homogenate, which the inventors had previously isolated, was added to the electrically fixed sample, diluted 1:20 to 1: 100, and reacted at 37 ° C for 1 hour. Subsequently, the mixture was washed sequentially with distilled water and phosphate buffer, and diluted with 1: 500 to 1: 2000 goat anti-rat IgG antibody (Kirkegaard & Perry Labs, USA) to which the fluorescent material, fluorescein isothiocyanate (FITC) was bound, was diluted. After the reaction, the infection was confirmed by Helicobacter Felis under a fluorescence microscope. At this time, the control group was used as the gastric tissue of the mouse administered with phosphate buffer according to the same immunization schedule. As shown in Figure 2, when the immunized with microspheres trapped Helicobacter pylori antigen, it was confirmed that the infection rate by Helicobacter Felis significantly reduced compared to the control.

Example 3-2 Urease Test

Stomach tissues of mice administered Helicobacter felis were collected, washed with phosphate buffer, immersed in the urease test solution, and reacted at room temperature for 3 to 12 hours. Then, the color change of the test solution was observed, and the color change was quantified by spectroscopic method. As a result, when immunized with microspheres in which the Helicobacter pylori antigen was collected, it was confirmed that the color change, that is, urease activity was significantly lower than that of the control group. These results, together with the immunofluorescence results, suggest that anti-Helicobacter pylori antibodies, particularly secretory IgA antibodies, formed by immunization can effectively protect against infection of Helicobacter felis.

As described and demonstrated in detail above, the present invention provides an oral immunity agent for Helicobacter pylori comprising a microsphere consisting of a biodegradable polymer and Helicobacter pylori antigen as an active ingredient and a method of manufacturing the same. Oral immunity against Helicobacter pylori of the present invention induces humoral and mucosal immune responses, resulting in an increase in anti-helicobacter pylori antibodies in blood and mucus, thereby eradication of Helicobacter pylori inhabiting gastric mucosal tissue and In addition to being very effective in reinfection protection, it can be useful for the prevention of infection of Helicobacter Felice living in gastric tissue with pathogenic microorganisms of the same genus as H. pylori.

Claims (10)

A lactide polymer, a glyco lead polymers and lactide, and at least one selected from the group consisting of a copolymer of glycolic lead biodegradable polymer and Helicobacter pylori MBRI2 (Helicobacter pylori MBRI2, KCTC 0216BP), and Helicobacter pylori MBRI8 (Helicobacter pylori MBRI8 , KCTC 0218BP) oral immunity to Helicobacter pylori comprising a microsphere having an average diameter of 0.1 to 100 ㎛ composed of Helcobacter pylori antigen prepared from one or more selected from the group consisting of as an active ingredient. The method of claim 1, Biodegradable polymers have a molecular weight of 10 to 250 kDa, Intrinsic viscosity is characterized in that 0.1 to 1.2dL / g Oral immunity against Helicobacter pylori. The method of claim 1, Lactide is L-lactide, D-lactide or D, L-lactide Characterized Oral immunity against Helicobacter pylori. Helicobacter pylori MBRI2 ( Heicobacter pylori MBRI2, KCTC 0216BP) and at least one biodegradable polymer selected from the group consisting of the polymer of lactide, the polymer of glycide and the copolymer of lactide and glycoride Helicobacter pylori MBRI8 KCTC 0218BP) oral immunity against Helicobacter pylori further comprising a pharmaceutically acceptable buffer in a microsphere composed of Helicobacter pylori antigens prepared from one or more selected from the group consisting of: My. Helicobacter pylori MBRI2 ( Heicobacter pylori MBRI2, KCTC 0216BP) and at least one biodegradable polymer selected from the group consisting of the polymer of lactide, the polymer of glycide and the copolymer of lactide and glycoride a Helicobacter pylori (microspheres consisting of Helicobacter pylori) antigen prepared from at least one member selected from the group consisting of Helicobacter pylori MBRI8 KCTC 0218BP), oral immunization for pharmaceutical Helicobacter pylori, further comprising a carrier, which may be acceptable My. The group consisting of Helicobacter pylori MBRI2 (Heicobacter pylori MBRI2, KCTC 0216BP ) , and Helicobacter pylori MBRI8 (Helicobacter pylori MBRI8 KCTC 0218BP) of the organic yongmaen average diameter of 0.1 to 100㎛ a biodegradable polymer from 1 to 20% (w / v) Helicobacter pylori antigen solution prepared from at least one member selected from 1: 5 (v / v) to 1:20 (v / v) was added, homogenized at 5,000 to 10,000 rpm for 5 to 10 minutes, A method for preparing an oral immunity against Helicobacter pylori comprising the step of removing the organic solvent to obtain a microsphere. The method of claim 6, Homogenization is achieved by using an ultrasonic crusher Further homogenization for 2 to 5 minutes under conditions Characterized Method for preparing oral immunity against Helicobacter pylori. The method of claim 6, The organic solvent is 0.5-20% polyvinyl alcohol in the homogeneous liquid obtained from the former Mix the solution and 10 to 20 minutes at 1,000 to 10,000 rpm Homogenized, then stirred at 100-500 rpm for 24 hours While volatilizing the organic solvent to obtain a solid sample, The solid sample is washed three times with distilled water and washed in a suitable volume of distilled water. Removed by resuspension and lyophilization Characterized Method for preparing oral immunity against Helicobacter pylori. The method of claim 6, The organic solvent is a homogeneous liquid obtained from the previous injection temperature 30 ~ 70 ℃, Spray and dry under the conditions of 400 ~ 700N1 / h Obtaining a solid sample, the electric solid sample was stored at room temperature for 24 hours Characterized in that it is removed by vacuum drying Method for preparing oral immunity against Helicobacter pylori. The method of claim 6, Organic solvents include dichloromethane, ethyl acetate, benzyl alcohol, acetone, Consisting of dimethylsulfoxide, ethanol and dimethylformamide At least one selected from the group Method for preparing oral immunity against Helicobacter pylori.