KR20020028547A - Composition of enteric-coated granules of oral typhoid vaccine and process for preparing the same - Google Patents

Abstract

PURPOSE: A process of preparing of an oral typhoid vaccine in granule form remarkably improved in viability by using a stabilizing agent during freeze drying after cultivation of ATCC-33459 and maximized in an arrival rate in the intestine is provided, thereby remarkably increasing a vaccine effect. The vaccine can be taken for children. CONSTITUTION: ATCC-33459 as a variant of attenuated typhoid bacillus is cultured, added with 0.1 to 10.0(w/v)% of sodium alginate and then freeze-dried by addition of 0.1 to 5.0M stabilizer alone or mixtures of two or more and 0.1 to 50.0%(w/v) of natural or synthetic polymer additives alone or mixtures of two or more, 0.01 to 20.0%(w/v) of an antioxidant alone or mixtures of two or more. The mixture is sprayed or dropped into 0.05 to 5.0M polyvalent cation solution to produce beads, which are left in place at 0 to 4deg.C for 1 to 5 hr, frozen at -20 to -70deg.C for 1 to 24 hr and freeze-dried for 1 to 48 hr.

Description

Enteric-coated granules of oral typhoid vaccine and process for preparing the same

Typhoid fever, a type of intestinal bacterial infection, is a major statutory communicable disease caused by diarrhea and fever caused by typhoid fever, which affects more than one billion people and kills millions of people annually in developing countries. Therefore, the development of vaccine against these diseases over the last few decades has made great progress, which is largely classified into parenteral and live oral vaccines. Since the bacteriophage has been killed by the pathogen, the effect of producing the antibody is much inferior to that of the viable vaccine, and the use of the live oral vaccine is gradually increasing due to the difficulty of applying it by injection. In particular, because typhoid bacteria are transmitted orally through the digestive system orally through water or food contaminated with the feces of the patient, the live vaccine is orally administered to the small intestine by oral administration of the live vaccine rather than causing the production of antibodies in the blood. Among the mucosal tissues, the antibody produced in the Peyer's patch, one of the lymphoid tissues, is concentrated, so the vaccine is effective in oral administration. However, the biggest problem when administering oral live vaccines is that most of them are killed by gastric acid when they pass through the stomach, so the intestinal delivery rate is low and thus hardly shows the effectiveness of the vaccine. Therefore, in order to solve this problem, conventional methods of administering sodium bicarbonate (NaHCO ₃ ) to neutralize gastric acid and then taking a vaccine and filling the capsule with the vaccine and then enteric coating the capsule surface Although it is used, the neutralization method of sodium bicarbonate of gastric acid is not only inconvenient to take, but also has a disadvantage that the degree of neutralization or neutralization time of gastric acid does not have a certain effect so as not to affect the live vaccine, The enteric coating capsule forming method has a high tendency for the capsule to break down due to the drying of about 12% of the moisture contained in the capsule itself due to the death of live bacteria and hot air when the capsule is coated with an enteric polymer material for a long time. Stability is shortened during preservation and the capsule is broken by the exercise of the stomach in the stomach when taken, By being coated on the fine when the engagement cavity occurs Frequent road during gastrointestinal passage penetrates the micro-balls acid has the disadvantage of a live vaccine of intestinal Rate of low.

In the present invention, in order to compensate for the disadvantages of the conventional method for the protection of live vaccine against gastric acid, the present invention was completed by adding a new stabilizer and a new enteric-coated granule manufacturing method during lyophilization.

Ty21a, which is used as an oral typhoid live vaccine, is attenuated as a mutant lacking Vi polysaccharide and lacking UDP-glucose-epimerase unlike wild bacteria, so it is weakly resistant to environmental changes. It is important to select a stabilizer so as not to cause cold damage since death occurs severely due to rapid freezing during freeze drying. The use of a stabilizer during lyophilization reduces the loss of activity by maintaining the original structure and increases the activity recovery upon restoration. In the present invention, the cells obtained by incubating Ty21a after centrifugation are used as primary stabilizers. Suspended in white sugar solution, and added polyhydric alcohols as a secondary stabilizer, and then added to the enteric coating material sodium alginate to dissolve evenly, and then stabilized during lyophilization by dropping or spraying into polycationic solution to prepare fine granules or beads It was. This suggests that hydrogen bonding between polyhydric alcohols and cell proteins increases the distance between proteins in the dehydration process, thus preventing lysis by intercellular aggregation.

By utilizing a new enteric coating technique that completes enteric coating at room temperature in a short time, a new production method for direct enteric coating of the enteric coating which hardly kills typhoid bacteria (Ty21a) during the enteric coating manufacturing process was established.

In more detail, as a strain for producing oral typhoid fever vaccine, Salmonella typhi Ty21a, an attenuated strain of typhoid fever, was used as an ATCC-33459 strain deposited with an international organization, and a single colony was used for brain heart infusion (BHI). The cells were inoculated in 100 ml of medium and shaken at 30 ° C. for 18 hours to collect the cells by centrifugation at 6000 g for 10 minutes, and suspended in 50 ml of a primary stabilizer (8% solution per bag). 0.1 ml of this suspension is placed in an ampoule, lyophilized, vacuum sealed, and used as a seed. The spawn ampoule is kept in the refrigerator and opened as necessary to use as spawn for the live vaccine production. Open the spawn ampoule and inoculate it on the inoculation medium (BHI), shake incubate at 30 ℃ for 18 hours, inoculate a certain amount of the culture medium into the production medium according to the production amount, incubate in large quantities, and centrifuge at 6000g for 10 minutes to collect the cells. After suspending in a tea stabilizer (8% solution of white sugar), add sucrose and mannitol as a secondary stabilizer at a concentration of 0.1 to 2.0 M, respectively, based on the typhoid suspension, and add sodium alginate to 0.1 to 5.0 (w / v)% and dissolved in 0.1-2.0 M calcium chloride (CaCl ₂ ) solution (pH 7.0) containing 0.1 g / m and mannitol, respectively, in the same volume as the typhoid suspension. Spraying results in ion exchange of sodium ions (Na ⁺ ) and calcium ions (Ca ²⁺ ) to form a calcium alginate film or coating that is poorly soluble in water. The curing time, i.e., soaking time in the calcium chloride solution, is 5-30 minutes, and the resulting film is stable for more than 2 hours in artificial gastric juice, is an enteric coating that disintegrates slowly in artificial intestinal fluid and completely disintegrates within 4 hours. The resulting beads or granules were collected by filtration and washed with sterile purified water, then adapted at low temperature at 0-4 ° C. for 1-2 hours, thinly spread on a freeze drying pan and frozen at -20-70 ° C. for 1-4 hours. Next, the lyophilizer was dried in vacuo for 12 to 24 hours. After completion of drying, measure the number of viable cells according to the conventional method and check whether there are 2 × 10 ⁹ cells per 1g, and fill the capsules or aluminum foil with a certain amount to make the final product.

As the primary stabilizer, an 8% solution of lactose may be used instead of an 8% solution of white sugar.

As the second stabilizer, glucose, fructose, glycerin, lactose, galactose, maltose, sorbitol, xylitol, trehalose, and adonitol are used instead of glucose and mannitol. ) May be used alone or in combination of two or more, and is used at a concentration of 0.1 to 2.0M based on the typhoid bacteria suspension, preferably at a concentration of 0.2 to 0.5M.

The sodium alginate is used at a concentration of 0.1 to 5.0 (w / v)% based on the typhoid bacteria suspension, and is preferably used at 1.0 to 1.5 (w / v)%.

The typhoid bacteria suspension contains arabic gum, locust bean gum and xanthan gum as natural or synthetic polymers as additives to control the disintegration time of beads or granules so as to be immediate or sustained release. (Xanthan gum), gellan gum, guar gum, tragacanth gum, agar, pectin, carrageenan, gelatin, chitin or chitosan, carbopols, carboxymethylcellulose sodium , Hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, poloxamers, polyvinyl alcohol, etc. can be used alone or in combination of two or more, 0.1 to 20.0 (w) based on the typhoid bacteria suspension / v)% and preferably 0.5 to 2.0 (w / v)%.

In the above typhoid suspension, bentonite, calcium carbonate, calcium hydrogen phosphate, calcium sulfate, carboxymethyl cellulose calcium, microcrystalline cellulose, colloidal silicon oxide, croscarmellose sodium, and crospovidone are used to maintain the shape or structure of the beads or granules. , Povidones, cyclodextrins, low-substituted hydroxypropyl cellulose, magnesium oxide, magnesium stearate, polyethylene glycols, sodium starch glycolate, starch, talc, titanium oxide, etc. alone or as two or more structural substances It can be used by mixing, based on the typhoid bacteria suspension 0.1 to 20.0 (w / v)%, preferably to 1.0 to 5.0 (w / v)%.

Since the typhoid bacteria suspension may be killed by oxidative reaction during freeze-drying, tocopherol acetate, beta carotene, ascorbic acid and salts thereof, citric acid, etc., alone or two kinds, may be used as antioxidants. It can be used by mixing the above, it is used in 0.01 ~ 10.0 (w / v)% based on the typhoid bacteria suspension, preferably used in 0.1 ~ 1.0 (w / v)%.

Instead of the calcium chloride solution, polyvalent cation solutions such as calcium lactate, magnesium chloride (MgCl ₂ ), strontium chloride (SrCl ₂ ), barium chloride (BaCl ₂ ), and aluminum chloride (AlCl ₃ ) may be used as binding solutions. It can be used at a concentration of 0.1 to 2.0M based on the typhoid bacteria suspension, and preferably at a concentration of 0.2 to 0.4M.

The polyvalent cation solution may be added to the same concentration of the material used as the secondary stabilizer used in the typhoid suspension.

The pH of the polyvalent cation solution is adjusted to pH 7.0 ± 0.1 with citric acid, hydrochloric acid, tartaric acid, phosphoric acid, maleic acid, etc., and can be filtered with a general filter paper or 0.45㎛ filter. have.

EXAMPLE

Although the content of the present invention will be described in more detail with reference to examples, it is only described for the purpose of illustrating the present invention, and is not intended to limit the protection scope of the present invention.

<Example 1>

Attenuated ATCC-33459 strain, attenuated Salmonella typhi Ty21a, was inoculated in 100 ml BHI medium and shaken at 30 ° C for 18 hours, inoculated with 2% of the amount of the culture solution, and then cultured. The end point of was set to OD value of 2.5-3.0 at 610 nm. The number of viable cells was 5.2 × 10 ¹⁰ / ml. The cells were collected by centrifugation at 6000 g for 10 minutes, and the cells were collected and suspended in 8% solution of white sugar. Then, based on this suspension, white sugar and mannitol were added to 0.5M each. Sodium alginate was dissolved in an aqueous solution of 1.2 (w / v)% sodium alginate, and then dipped or sprayed in a 0.4 M calcium chloride solution (pH7.0) containing 0.5 M of sucrose and mannitol in the same volume as the typhoid suspension. Beads or granules were prepared, filtered, washed with sterile purified water, adapted for 1 hour at 0-4 ° C., evenly spread in a freeze drying pan, frozen at −20 ° C. for 4 hours, and lyophilized for 24 hours. Prepare coated microgranules or beads. The composition of Example 1 is as follows.

30 ml of typhoid bacteria suspension (8% solution per bag)

5.1g (0.5M) per bag

Mannitol 2.7 g (0.5 M)

Sodium alginate 0.36 g (1.2%)

Calcium chloride (0.4M) 1.33g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, containing 5.1g per bag, 2.7g of mannitol)

<Example 2>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution per bag)

5.1g (0.5M) per bag

Mannitol 2.7 g (0.5 M)

Sodium alginate 0.36 g (1.2%)

Arabian gum 0.6g (2.0%)

Calcium chloride (0.4M) 1.33g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, containing 5.1g per bag, 2.7g of mannitol)

<Example 3>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution of lactose)

5.1g (0.5M) per bag

Mannitol 2.7 g (0.5 M)

Sodium alginate 0.42 g (1.4%)

Arabian gum 0.3g (1.0%)

Dry Vitamin E 50% Powder 0.3g (1.0%)

Calcium chloride (0.4M) 1.33g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, containing 5.1g per bag, 2.7g of mannitol)

<Example 4>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution of lactose)

5.1g (0.5M) per bag

Sorbitol 2.7g (0.5M)

Sodium alginate 0.42 g (1.4%)

Arabian gum 0.6g (2.0%)

Calcium chloride (0.4M) 1.33g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, containing 5.1g per bag, 2.7g sorbitol)

Example 5

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution per bag)

Trehalose 5.7g (0.5M)

Glycerine 1.4 g (0.5 M)

Sodium alginate 0.36 g (1.2%)

Talc 0.3 g (1.0%)

Microcrystalline cellulose 0.3g (1.0%)

Calcium lactate (0.2M) 1.30g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, 5.1g trehalose, 2.7g glycerin)

<Example 6>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution of lactose)

Adonitol 2.3g (0.5M)

Lactose 5.1g (0.5M)

Sodium alginate 0.36 g (1.2%)

0.6g (2.0%) corn starch

Ascorbic Acid 30mg (0.1%)

Calcium lactate (0.2M) 1.30g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, 2.3g of adonitol, 5.1g of lactose)

<Example 7>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution per bag)

Glycerin 0.83 g (0.3 M)

Xylitol 1.37g (0.3M)

Sodium alginate 0.36 g (1.2%)

Xanthan gum 0.15g (0.5%)

Microcrystalline cellulose 0.6g (2.0%)

Calcium chloride (0.4M) 1.33 g / 30 ml of sterile water (adjusted to pH 7.0 with phosphoric acid, 0.83 g of glycerin, 1.37 g of xylitol)

<Example 8>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution of lactose)

5.1g (0.5M) per bag

Mannitol 2.7 g (0.5 M)

Sodium alginate 0.36 g (1.2%)

Agar 0.15g (0.5%)

Colloidal Silicon Dioxide (Aerosil) 0.3g (1.0%)

Beta Carotene 30mg (0.1%)

Calcium Lactate (0.2M) 1.30g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, containing 5.1g per bag and 2.7g of mannitol)

Example 9

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution per bag)

Trehalose 5.7g (0.5M)

Sodium alginate 0.36 g (1.2%)

Agar 0.15g (0.5%)

Titanium oxide 0.3 g (1.0%)

Citric acid 30mg (0.1%)

Calcium Lactate (0.2M) 1.30g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, including 5.7g of trehalose)

<Example 10>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution of lactose)

Sorbitol 1.6g (0.3M)

Xylitol 1.4g (0.3M)

Sodium alginate 0.30 g (1.0%)

Pectin 0.15 g (0.5%)

Magnesium Stearate 0.6g (2.0%)

Calcium chloride (0.4M) 1.33g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, 1.6g of sorbitol, 1.4g of xylitol)

<Example 11>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution per bag)

5.1g (0.5M) per bag

Glycerin 0.83g (0.3M)

Sodium alginate 0.36 g (1.2%)

Pectin 0.15 g (0.5%)

Calcium Dihydrogen Phosphate 0.3g (1.0%)

Sodium Ascorbate 30mg (0.1%)

Calcium lactate (0.2M) 1.30g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, containing 5.1g per bag, 0.83g glycerin)

<Example 12>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% lactose)

Mannitol 2.7 g (0.5 M)

Adonitol 2.3g (0.5M)

Sodium alginate 0.36 g (1.2%)

Pectin 0.30g (1.0%)

Microcrystalline cellulose 0.6g (2.0%)

Calcium chloride (0.4M) 1.33g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, 2.7g mannitol, 2.3g adonitol)

Example 13

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution of lactose)

Trehalose 3.4g (0.3M)

Lactose 3.1g (0.3M)

Sodium alginate 0.36 g (1.2%)

Gelatin 0.15 g (0.5%)

Talc 0.3 g (1.0%)

Tocopherol Acetate 50% Powder 0.3g (1.0%)

Calcium chloride (0.4M) 1.33 g / sterile water 30 ml (adjusted to pH 7.0 with phosphoric acid, 3.4 g trehalose, 3.1 g lactose)

<Example 14>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution per bag)

5.1g (0.5M) per bag

Mannitol 2.7 g (0.5 M)

Sodium alginate 0.36 g (1.2%)

Gelatin 0.15 g (0.5%)

Low Substituted Hydroxypropyl Cellulose 0.6g (2.0%)

Calcium chloride (0.4M) 1.33g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, containing 5.1g per bag, 2.7g of mannitol)

<Example 15>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution of lactose)

Glycerin 0.83g (0.3M)

Sorbitol 1.6g (0.3M)

Sodium alginate 0.36 g (1.2%)

Hydroxypropylcellulose 0.6g (2.0%)

Titanium oxide 0.3 g (1.0%)

Calcium lactate (0.2M) 1.30g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, 0.83g glycerin, 1.6g sorbitol)

<Example 16>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution per bag)

Trehalose 3.4g (0.3M)

Adonitol 2.3g (0.5M)

Sodium alginate 0.36 g (1.2%)

Hydroxypropylcellulose (HPC) 0.3g (1.0%)

Polyethylene glycol 6000 0.6 g (2.0%)

Calcium chloride (0.4M) 1.33 g / sterile water 30 ml (adjusted to pH 7.0 with phosphoric acid, 3.4 g of trehalose, 2.3 g of adonitol)

<Example 17>

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution per bag)

Xylitol 2.3g (0.5M)

Sodium alginate 0.36 g (1.2%)

Hydroxypropylmethylcellulose (HPMC) 0.3g (1.0%)

Bentonite 0.3g (1.0%)

Calcium Carbonate 0.3g (1.0%)

Beta Carotene 30mg (0.1%)

Calcium chloride (0.4M) 1.33g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, 2.3g of xylitol)

Example 18

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution of lactose)

5.1g (0.5M) per bag

Glycerin 1.4g (0.5M)

Sodium alginate 0.42 g (1.4%)

Hydroxypropyl Methyl Cellulose (HPMC) 0.15g (0.5%)

Microcrystalline cellulose 0.6g (2.0%)

Calcium lactate (0.2M) 1.30g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, containing 5.1g per bag, glycerin 1.4g)

Example 19

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid suspension (8% solution per bag)

Glycerin 1.4g (0.5M)

Trehalose 3.4g (0.3M)

Sodium alginate 0.36 g (1.2%)

Poloxamer 188 0.15g (0.5%)

0.6g (2.0%) corn starch

Ascorbic Acid 30mg (0.1%)

Calcium chloride (0.4M) 1.33g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, 1.4g glycerin, 3.4g trehalose)

Example 20

Typhoid vaccine oral beads or granules were prepared in the same manner as described in Example 1 with the following composition.

30 ml of typhoid bacteria suspension (8% solution of lactose)

Adonitol 2.3g (0.5M)

Lactose 3.1g (0.3M)

Sodium alginate 0.36 g (1.2%)

Poloxamer 188 0.15g (0.5%)

Talc 0.6 g (2.0%)

Calcium chloride (0.4M) 1.33g / 30ml sterile water (adjusted to pH 7.0 with phosphoric acid, 2.3g of adonitol, 3.1g of lactose)

Experimental Example 1 (survival rate test)

Survival was measured for the typhoid vaccine oral beads or granules of Examples 1 to 20 above. In other words, the bead or granule was stirred at 300 rpm for 30 minutes in artificial gastric juice of pH 3.0 using dissolution test apparatus 1 of the dissolution test method of the Korean Pharmacopoeia General Test Method, and then transferred to artificial intestine (pH 6.8 phosphate buffer solution) for dissolution test. After stirring for 2 hours at 500 rpm with the apparatus 2, this eluate is taken out and the number of viable cells is measured (A). Separately, after stirring at 500 rpm for 2 hours in dissolution test apparatus 2 in the artificial intestine solution, the eluate is taken an appropriate amount and the number of viable bacteria is measured (B).

Survival Rate (%) = A / B × 100

The results of the survival test are as described in Table 1 below.

In the results of Table 1, typhoid vaccine oral beads or granules overcome instability in gastric juice, all showing a high viable cell count and viability of 10 ⁹ or more per g. Therefore, the typhoid vaccine beads or granules of the present invention are orally administrable, overcome the instability caused by gastric acid, and are thought to be agents that enable high incidence of typhoid vaccine in the intestine.

Experimental Example 2 (antibody production test)

Ty21a antibody production confirmation test (ELISA TEST) was performed on Example 2, Example 3, Example 4, Example 9, and Example 10, in which the survival rate and the number of viable cells were excellent in Experimental Example 1, and the results were obtained. Are listed in Table 2 below.

In other words, it was tested by the antibody detection method of typhoid fever strain, Blank was to add 10% FBS to RPMI 1640 medium, the control group was orally administered to mice three times at 48 hours intervals with 0.2ml physiological saline. Specimens were orally administered (1 × 10 ⁷ Cells / 0.2ml) to mice three times at 48 h intervals with microenterally coated beads, and blood samples were analyzed by taking 0.7ml after 10 days from the last oral administration. . The reaction time after the addition of the OPD substrate was 13 minutes. As a result of antibody production, microenterally coated beads showed 5 to 10 times antibody production rate compared to the control group.

Experimental Example 3 (Attack Test)

Oral typhoid vaccine oral granules prepared according to Example 3 were orally administered to 20 rats three times at 48-hour intervals, followed by intraperitoneal injection of wild typhoid bacteria to 15 rats 4 weeks after the last oral administration. Separately, phosphate buffer solution (pH 7.4) was orally administered to 10 rats three times at 48-hour intervals, and 4 weeks after the last oral administration, wild typhoid bacteria were intraperitoneally injected into five rats. The final number of survivors is the number of survivors 10 days after the intraperitoneal injection of wild typhoid bacteria. The test results showed a high survival rate of 93% in the test group while the survival rate of wild typhoid bacteria after the vaccination was 0% in the control group as shown in Table 3 below.

In the present invention, the conventional oral typhoid vaccine enteric capsules cannot be taken in children, and the conventional capsules contain the capsules themselves due to the death of live bacteria and hot air due to prolonged exposure to high temperatures when the capsules are coated with an enteric polymer during manufacture. As about 12% of the moisture is dried, the capsule tends to break, and stability during storage is shortened, and when taken, the capsule is broken by the above exercise in the stomach, or the micropores are coated when the coating is carried out on the binding site of the capsule or the lower part. This method relates to a new oral typhoid vaccine enteric bead or granule preparation method that overcomes the disadvantages of capsules that have low intestinal delivery rate of live bacterial vaccine by penetrating gastric acid into the micropores during gastrointestinal passage. Lymphocytes in the mucous membrane of the small intestine by increasing the survival rate In one of the Peyer's patch area antibody production rate is higher vaccine effect was significantly increased in accordance with the dose was possible even for children is a high incidence of typhoid ages.

Claims (11)

0.1 to 10.0 (w / v)% sodium alginate was introduced into the typhoid suspension, which was incubated with ATCC-33459, a strain of attenuated typhoid typhoid (Salmonella typhi Ty21a), and the stabilizer during lyophilization was 0.1 to 5.0 M. Or added in combination of two or more thereof, the additive of natural or synthetic polymers alone or in combination of two or more at a concentration of 0.1 to 50.0 (w / v)%, and the antioxidant of 0.01 to After adding alone or in mixture of two or more at a concentration of 20.0 (w / v)%, finely sprayed or instilled in 0.05 to 5.0 M of a polyvalent cation solution, beads were prepared and then 1 to 5 at 0 to 4 ° C. Oral typhoid vaccine enteric-coated granules or beads prepared by low temperature adaptation for a period of time, freezing for 1 to 24 hours at -20 to -70 ° C and lyophilizing for 1 to 48 hours. The method of claim 1, wherein the granules or beads are prepared using sodium alginate at a concentration of 0.1 to 5.0 (w / v)% based on the typhoid bacteria suspension. The method of claim 1, wherein as a stabilizer for lyophilization, sucrose, mannitol, dextrose, fructose, glycerin, lactose, galactose, maltose, sorbitol, xylitol, trehalose, adonitol Method for preparing granules or beads using adonitol or the like alone or in a mixture of two or more at a concentration of 0.1 to 2.0 M based on the typhoid bacteria suspension The method of claim 1, wherein the aqueous solution of sodium alginate is added as an additive to natural or synthetic polymers such as Arabic gum, Locust bean gum, Xanthan gum, Gellan gum, Guar gum. Guar gum, Tragacanth gum, agar, pectin, carrageenan, gelatin, chitin or chitosan, carbopol, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxy Method for producing granules or beads using propyl methyl cellulose, poloxamers, polyvinyl alcohol, etc. alone or in combination of two or more at a concentration of 0.1 to 20.0 (w / v)% based on the typhoid bacteria suspension The aqueous solution of sodium alginate according to claim 1, wherein the aqueous solution of sodium alginate is bentonite, calcium carbonate, calcium dihydrogen phosphate, calcium sulfate, carboxymethyl cellulose calcium, microcrystalline cellulose, colloidal silicon oxide, croscarmellose sodium, crospovidones, povidone , Cyclotextine, low-substituted hydroxypropyl cellulose, magnesium oxide, magnesium stearate, polyethylene glycols, sodium starch glycolate, starch, talc, titanium oxide, etc. / v)% to produce granules or beads, alone or in combination of two or more at a concentration of The method of claim 1, wherein the aqueous sodium alginate solution is used alone or in a concentration of 0.01 to 10.0 (w / v)% of tocopherol acetate, beta carotene, ascorbic acid and salts thereof, citric acid and the like as an antioxidant, or How to prepare granules or beads by mixing two or more kinds The method according to claim 1, wherein the polyvalent cation solution is used to prepare granules or beads using calcium chloride, calcium lactate, magnesium chloride, strontium chloride, barium chloride, aluminum chloride and the like at a concentration of 0.1 to 2.0 M based on the typhoid suspension. The method of claim 1, wherein the granules or beads are prepared by adding the stabilizer of claim 3 to the polyvalent cation solution. The method of claim 1, wherein the pH of the polyvalent cation solution is adjusted to pH 7.0 ± 0.1 with citric acid, hydrochloric acid, tartaric acid, phosphoric acid, maleic acid, etc., and is used for preparing granules or beads after general filtration or 0.45 μm filtration. The granules or beads prepared according to claim 1 are filtered, washed with sterile purified water, adapted for 1 to 2 hours at 0-4 ° C. for low temperature, frozen at −20 to −70 ° C. for 1 to 4 hours, and 12 to 24 hours. Freeze-drying for time to make the final product A method according to claim 10, wherein during the lyophilization of granules or beads, the method is immersed in an aqueous solution of the stabilizer used in claim 3 and lyophilized to form a final product.