_B6_ 五、發明説明（） 發明之領域 本發明傺關於用於製備口服活傷寒疫苗之培餐方法， 更特定而言，為使用新穎生長培養基、保護培養基以及控 制培養溫度以使培養後細@細胞之收穫量增加以及冷凍乾 燥後免疫活性減毒活細菌之存活率高之培養方法。 先前技g之説明 傷寒熱，已知其病原菌為傷寒桿菌（Salmonella typhi) ，通常由污染的水或食物、擁擠地匾人之接觸而感染。 為了預防傷寒熱，通常使用非口服死@疫苗或口服活 薗疫苗。 目前，口服活菌疫苗之使用逐漸增加，因為死菌疫苗 雖具有高度安全性，但未顯示所欲之免疫活性。 活菌抑苗之製備方法與死菌疫苗者不同。活菌疫苗曾 以下列已知方法製造： (i)在液態或固態培養基中培養傷寒菌之減毒突變株 ,(ii)離心及冷凍乾燥，以及（iii)調配；而死菌疫苗藉 下列方法製造：（i)在液態或固態培養基中培養傷寒菌， (ii)用丙酮、酚或甲醛殺菌，以及（iii)調配。 經濟部+央標準局β工消费合作社印製 (請先閲讀背面之注意事項再塡寫本頁) 為了製造菌疫苗，通常使用傷寒桿菌減毒突變株Ty21a .其由傑曼尼爾（R. Germanier)及富勒（E. Furer)藉二段 突變處理自傷寒桿@Ty2中分離出。此等突變株之特擻為 無尿苷-二磷醛半乳糖-4-表異構酶之酶活性，其為生成細 菌細胞壁中脂多酷之必需中間物。所以，此等突變株實際 上無毒力，因為只生成不完全的細胞壁。 本紙張尺度逍用中曲B家樣準(CHS)甲4規格(210X297公龙) 203^^^ 八6 13 6 經濟部屮央標準局员工消贽合作社印製 五、發明説明（） 不過，此等突變株藉著存在半乳糖下合成脂多耱而顯 示卓越的免疫功效。過度供應半乳糖會造成其以半乳糖 -卜磷酸邇及UDP-半乳糖之形式蓄積，此將導致細菌溶解 Ο 1972年丨2月24日頒予傑曼尼爾等人之美國專利案第 3, 856, 935號掲示Ty21a菌株之突變，培養及生産。 下文為本案掲示之傷寒疫苗製法。 將被選擇之突變株Ty 21a於37¾下在振搖機器上之腦 心臓浸液中培養6·小時。在6, OOOg下離心，然後收取細 菌並將其懸浮在含有8%蔗糖，1.5%明膠及5%脱脂奶 粉之保護水性培餐基中。將懸浮液在小瓶中冷凍乾燥後， 細国藉下法製備：打開小瓶之一並接種在養份瓊脂斜面培 養基上，然後將其於371C下培育。將該細菌懸浮於生理食 鹽水溶液中並將所得懸浮液做為60 0毫升養分溶液之接種 物。該養分溶液藉下法製備：將28克酪蛋白水解物，10克 酵母菌萃取物，以及2克葡萄糖懸浮於1升蒸餾水中並將 PH值調至7.2。接種後將培養物移至25升批次之相同養分 溶液中並保持37°C歴1 2小時，同時以5升空氣/分鐘之速 率充氣。培養後，細菌細胞藉於6 000g下離心收取並將其 懸浮在上述水性保護培養基中並於小瓶内冷凍乾燥。 但根據上述方法培養之細菌於冷凍乾燥後只有少許存 活，因為Ty21a菌株對環境變化之抗性徹弱，所以冷凍乾 燥期間易被殺死。 發明之摘要 本紙張尺度逍用中國B家標準(CNS)甲4規格(210X297公货） -4 - (請先閲讀背而之注意事項再填寫本頁) Λ 6 13 6 經濟部屮央標準局貝工消t合作社印製 五、發明説明（） 本發明之目的為提供一種改良培餐方法，其使用新穎 生長培養基，保護培餐基以及控制培餐溫度，以使培養後 细菌细胞之收播量增加以及冷凍乾燥後免疫活性減毒活菌 之存活率高。本發明製備之生長培養基被設計成能使細菌 有效生長且藉著控制培養溫度使其易於適應琛境改變。本 發明製術之保護培養基被設計成能使細菌於冷凍乾燥期間 承受極快速冷凍，以及經冷凍乾燥之細菌能保存長時間而 不改變其性質。 使用新穎生長培餐基及保護培餐基之培餐方法得到之 口服傷寒疫苗顯示減毒細菌之存活率高以及對抗傷寒熱之 免疫活性高。 發明之詳細説明 根據本發明之培養方法之細節説明於下： 用於製備口服傷寒疫苗之傷寒菌株為傷寒桿菌之減毒 突變株Ty21a,其貯存於美國類型培養物收集所（“ATCC” ) ，貯存编號為ATCC- 3 345 9 ,其也貯存於韓國科技院 ("KIST”），貯存编號為KCTC- 24 24。上述被貯存之傷寒 菌株也可藉復活市售産物“Vivotif Berna”獲得， “Vivotif Berna”由瑞士百納血清S疫苗公司出售，其 為在復活培養基中之單一菌落，該復活培養基由腦-心浸 液瓊脂組成。 將單離自上述傷寒菌株單一群落之種_接種在10 0毫 升腦-心浸液（“ ΒΗΓ’）培養基上並將接種之菌株於301下 在振搖機器上培養18小時。將培養物在6, QGDG下離心10 (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度逍用中a B家標毕(CNS)甲4規格(210x297公址） Λ 6 13 6 經濟部中央標準局貝工消t合作社印製 五、發明説明（） 分鐘，然後收取細菌並將其懸浮在保護培養基中。保護培 養基含有8%乳糖，1%羧甲基纖維素，5%脱脂奶， 0.2%MgSO* · 7Ha〇以及1 %毅胺酸單鈉。 將上述懸浮液在安瓿中冷凍乾燥。經冷凍乾燥之安瓿 於製造活疫苗前貯存於4〜8C下。 細菌藉將安瓿打開並接種在BHI培養基上而製備。為 了培餐種豳，將接種細菌於30Ό下在振搖機器上培養18小 時。將如此所得之種菌接種在生産用生長培餐基上以行主 要培養，該生長培養基由37克BHI, 5克L-離胺酸，30克 山梨酵，1.5克KaHPO«, 0.5克MgS〇4.7Ha〇及1升蒸晡水 組成，且PH值調至7.0。在大量生産之情況，可將從生産 培養基得到之培餐物接種至250升發酵器内。在主要培餐 期間，培養溫度（為本發明主要待徽之一）應變化以使細薗 在冷凍乾燥期間能充分耐受快速溫度變化。在主要培養期 間培養溫度為（i)最初6小時為30Ϊ： , (H)接下來2小時 為25亡，以及（iii)最後14小時為20*C。 發酵器之充氣維持在0.5vvm(體積/體積/分鐘 培養完成時，細菌細胞藉在6000g下冷凍離心而收捎 上述保護培養基中2小時。 冷凍乾燥。最後將被冷凍乾 燥之細菌與乳糖混合.以使毎一克含有2.5X IV活菌， 然後將製得之疫苗充填入膠囊中，毎一膠囊之充填量為 〇 . 2克。將膠囊加腸衣以供口服使用。 本發明以下列實例更詳細說明。但是此等實例僅傺說 然後將其於〇〜4*0下懸 將該懸浮液在冷凍乾 (請先閲讀背面之注意事項再填寫本頁) 裝. 線· 本紙張尺度逍用中國Η家標準(CNS)甲4規格(210X297公*) - 6 - 2〇9^44 Λ 6 13 6 經濟部+央標準局貝Η消f合作杜印製 五、發明説明（） 明，非意欲限制本發明之範圍。 實例1 將得自 ATCC-33459, KCTC-2425 或 Vivotif Berna 之傷 寒桿薗減毒突變菌株Ty 2 la接種在100毫升BHI培養基中並 於30它下在振搖機器上培養18小時，然後將藉離心收獲之 細菌在安餌中冷凍乾燥。將做為種菌之冷凍乾燥細菌接種 在主要生長培養基上，該生長培養基由37克BHI, 5克L-離胺酸.30克山梨醇，1.5克KaHP0«, 0.5克MgS04.7Ha0 及1升蒸皤水組成且PH值調整至7.0。 為了進行主要培餐，將5升主要生産培養基移至10升 發酵器中。將發酵器以0.5VVm之速率充氣並在300rpm下攪 拌。主要培養期間之溫度為（i)最初6小時為301C, (ii) 接下來2小時為25它，以及（iii)最後14小時為20*C。 全部培養時間需要22小時。最後所得培養溶液之性質 顯示培養溶液之濁度在0D 660下為0.516Χ 20以及活菌數 目為 4. 6X 101°¾ 升。 實例2 以與實例1相同之方式進行培養，不過主要培養之溫 度為（i)最初6〜8小時為301： , (ii)接下來之2〜4小時為 25Ό,以及（iii)最後12〜14小時為20〜25它。 最後所得培養溶液之性質顯示在0D 660處之濁度為 0.452X 20 — 0.516X 20,以及活菌之數目為 3.2X 101。/ 毫升。 實例3 (請先閲讀背面之注意事項再填寫本頁) 裝· 線- 本紙張尺/1逍用中B B家《準(CNS)肀4規格(210x297公龙) Λ 6 13 6 ‘二 五、發明説明（） 將實例1所得之培養溶液在6, OOOg下離心以及懸浮 在2.5升保護培養基中，該培養基由8%乳糖、1%羧甲 基纗維素，5%脱脂乳，〇·2% MgS04*7Ha0及1%毅胺 酸單鈉組成。讓在保護培養基中之懸浮液於Q〜41下靜置 2小時以增加對低溫之抗性。在冷凍乾燥器中，將抗性-增加懸浮液-701下冷凍2小時，然後在宾空狀態乾燥12 小時。 冷凍乾燥後活菌數目為6.8X 10*°/克。 實例4 以與實例3相同之方式進行冷凍乾燥，不過用1%明 膠取代1%羧甲基纖維素。冷凍乾燥後所得之活菌數目為 4, IX 101。/ 克。 (請先閲讀背面之注意事項再填寫本頁) 裝· -線· 經濟部中央標準局貝工消费合作社印製 本紙張尺度遑用中B B家搮準(CNS)甲4規格(210X297公:¢)_B6_ V. Description of the invention () Field of the invention The present invention relates to the method of preparing meals for preparing oral live typhoid vaccine, more specifically, the use of a novel growth medium, a protective medium, and a control of the culture temperature to make the cells fine after culture A method of cultivating an increase in the amount of harvest and a high survival rate of live bacteria with reduced immunological activity after freeze-drying. Description of previous techniques g Typhoid fever is known to be caused by Salmonella typhi, which is usually infected by contact with contaminated water or food or crowded plaques. In order to prevent typhoid fever, non-oral death @ vaccine or oral live barley vaccine is usually used. At present, the use of oral live bacteria vaccines is gradually increasing because the dead bacteria vaccines are highly safe, but they do not show the desired immune activity. The preparation method of live bacteria suppression vaccine is different from that of dead bacteria vaccine. Live bacterial vaccines have been manufactured by the following known methods: (i) cultivation of attenuated mutant strains of typhoid fever in liquid or solid medium, (ii) centrifugation and freeze-drying, and (iii) deployment; and dead bacteria vaccines are obtained by the following methods Manufacturing: (i) Cultivate typhoid bacteria in liquid or solid medium, (ii) Sterilize with acetone, phenol or formaldehyde, and (iii) Mix. Printed by the Ministry of Economic Affairs + Central Bureau of Standards, beta industrial consumer cooperative (please read the precautions on the back before writing this page). In order to make a bacterial vaccine, the attenuated mutant Ty21a of Typhoid fever is usually used. It is produced by German Neil (R. Germanier and E. Furer were isolated from typhoid fever @ Ty2 by two-stage mutation treatment. The speciality of these mutants is the absence of the enzyme activity of uridine-diphosphalgalactose-4-epi-isomerase, which is an essential intermediate for the production of liposomes in the cell walls of bacteria. Therefore, these mutants are practically non-virulent because only incomplete cell walls are produced. This paper is used in the Xiaoqu Zhongqu B Family Sample Standard (CHS) A 4 specifications (210X297 male dragon) 203 ^^^ August 6 13 6 Printed by the Employee Consumers Cooperative of the Central Standards Bureau of the Ministry of Economy V. Description of invention () However, These mutant strains show excellent immune effects due to the presence of synthetic lipopolysaccharide in the presence of galactose. Excessive supply of galactose will cause it to accumulate in the form of galactose-diphosphate and UDP-galactose, which will result in bacterial dissolution. 3rd, U.S. Patent Case No. 3 issued to German Neil et al. , No. 856, 935 shows the mutation, culture and production of Ty21a strain. The following is the typhoid vaccine preparation method shown in this case. The selected mutant strain Ty 21a was cultured in the brain and heart infusion on shaking machine for 6 hours at 37¾. Centrifuge at 6,000 g, then collect the bacteria and suspend them in a protective aqueous base containing 8% sucrose, 1.5% gelatin and 5% skimmed milk powder. After freeze-drying the suspension in a vial, Xiguo is prepared by the following method: one of the vials is opened and inoculated on the nutrient agar slant medium, and then incubated at 371C. The bacteria were suspended in physiological saline solution and the resulting suspension was used as an inoculum of 600 ml of nutrient solution. The nutrient solution was prepared by the following method: 28 g of casein hydrolysate, 10 g of yeast extract, and 2 g of glucose were suspended in 1 liter of distilled water and the pH was adjusted to 7.2. After inoculation, the culture was transferred to a 25-liter batch of the same nutrient solution and kept at 37 ° C for 12 hours, while being inflated at a rate of 5 liters of air / minute. After cultivation, the bacterial cells were collected by centrifugation at 6,000 g and suspended in the above-mentioned aqueous protective medium and freeze-dried in vials. However, the bacteria cultivated according to the above method survived only a little after freeze-drying. Because the Ty21a strain is completely resistant to environmental changes, it is easy to be killed during freeze-drying. Summary of the invention The paper size is free to use China B Standard (CNS) A4 specifications (210X297 public goods) -4-(please read the precautions before filling out this page) Λ 6 13 6 Ministry of Economic Affairs Bureau of Standards Printed by Beigongxiaot Cooperative Society 5. Description of the invention () The purpose of the present invention is to provide an improved meal cultivation method, which uses a novel growth medium, protects the meal cultivation base and controls the meal cultivation temperature, so that the bacterial cells can be broadcasted after cultivation The increase of the amount and the survival rate of the attenuated live-immunized bacteria after freeze-drying are high. The growth medium prepared by the present invention is designed to allow bacteria to grow efficiently and to easily adapt to changes in the environment by controlling the cultivation temperature. The protective medium of the present invention is designed to allow bacteria to withstand extremely rapid freezing during freeze-drying, and freeze-dried bacteria can be stored for a long time without changing their properties. The oral typhoid vaccine obtained using the novel growth culture base and the protection culture base culture method shows a high survival rate of attenuated bacteria and a high immune activity against typhoid fever. Detailed description of the invention The details of the cultivation method according to the present invention are described below: The typhoid strain used for preparing the oral typhoid vaccine is the attenuated mutant strain Ty21a of typhoid bacillus, which is stored in the American Type Culture Collection ("ATCC"), The storage number is ATCC- 3 345 9, which is also stored in the Korea Institute of Science and Technology (" KIST ”), and the storage number is KCTC-24 24. The above-mentioned stored typhoid strains can also be borrowed to revive the commercially available product“ Vivotif Berna ” Obtained, "Vivotif Berna" is sold by Swiss Bainer Serum S vaccine company, which is a single colony in the resurrection medium, which is composed of brain-heart infusion agar. Isolate from the above single species of typhoid fever strain _ It was inoculated on 100 ml of brain-heart infusion ("BHΓ ') medium and the inoculated strain was cultured on a shaking machine at 301 for 18 hours. Centrifuge the culture at 6, QGDG for 10 (please read the precautions on the back before filling in this page). This paper is used in the standard a B home standard (CNS) A 4 specifications (210x297 public address) Λ 6 13 6 Economy The Ministry of Central Standards Bureau Beigongxiaot Cooperative printed five minutes of invention description () minutes, then collected bacteria and suspended it in a protective medium. The protective medium contains 8% lactose, 1% carboxymethyl cellulose, 5% nonfat milk, 0.2% MgSO * · 7Ha〇, and 1% monosodium probamate. The above suspension was freeze-dried in ampoules. Freeze-dried ampoules are stored at 4 ~ 8C before making live vaccines. Bacteria are prepared by opening and inoculating ampoules on BHI medium. In order to cultivate rice seeds, the inoculated bacteria were cultured on a shaking machine for 18 hours at 30 °. The inoculum thus obtained was inoculated on the production growth culture base for main culture. The growth medium consisted of 37 grams of BHI, 5 grams of L-lysine, 30 grams of sorbic acid, 1.5 grams of KaHPO «, 0.5 grams of MgS〇4.7 Ha〇 and 1 liter of distilled water, and the pH value was adjusted to 7.0. In the case of mass production, the cultured food obtained from the production medium can be inoculated into a 250-liter fermentor. During the main meal cultivation period, the cultivation temperature (which is one of the main features of the present invention) should be changed so that the scallion can fully tolerate rapid temperature changes during freeze-drying. The cultivation temperature during the main cultivation period is (i) 30Ϊ for the first 6 hours: (H) 25 deaths for the next 2 hours, and (iii) 20 * C for the last 14 hours. The aeration of the fermenter is maintained at 0.5vvm (volume / volume / minute when the culture is completed, the bacterial cells are collected in the above-mentioned protective medium by freeze centrifugation at 6000g for 2 hours. Freeze drying. Finally, the freeze-dried bacteria and lactose are mixed Each gram contains 2.5X IV live bacteria, and then the prepared vaccine is filled into capsules, and the filling amount of each capsule is 0.2 grams. The capsule is added with enteric coating for oral use. Detailed description. However, these examples are only said, and then suspend it under 〇 ~ 4 * 0. The suspension is freeze-dried (please read the precautions on the back before filling this page). Thread China Η Family Standard (CNS) A4 specifications (210X297 g *)-6-2〇9 ^ 44 Λ 6 13 6 The Ministry of Economic Affairs + Central Standards Bureau Bei Η Xiaof cooperation du printing 5. Invention description () Ming, non It is intended to limit the scope of the present invention. Example 1 Attenuated typhoid typhimurium strain Ty 2 la from ATCC-33459, KCTC-2425 or Vivotif Berna was inoculated in 100 ml of BHI medium and shaken on the machine at 30 ° Incubate for 18 hours, and then collect the bacteria harvested by centrifugation Freeze-dried in the bait. The freeze-dried bacteria used as inoculum were inoculated on the main growth medium. The growth medium consisted of 37 g BHI, 5 g L-lysine, 30 g sorbitol, 1.5 g KaHP0 «, 0.5 g MgS04 .7Ha0 and 1 liter of steamed water and the pH value is adjusted to 7.0. For the main meal, 5 liters of the main production medium is transferred to a 10-liter fermentor. The fermentor is aerated at a rate of 0.5VVm and stirred at 300 rpm The temperature during the main cultivation period is (i) 301C for the first 6 hours, (ii) 25 ° C for the next 2 hours, and (iii) 20 * C for the last 14 hours. The total cultivation time is 22 hours. The final culture solution The properties showed that the turbidity of the culture solution was 0.516X 20 at 0D 660 and the number of viable bacteria was 4.6X 101 ° ¾ liter. Example 2 The cultivation was carried out in the same manner as in Example 1, but the main cultivation temperature was (i) The first 6 to 8 hours are 301: (ii) the next 2 to 4 hours are 25Ό, and (iii) the last 12 to 14 hours are 20 to 25. The nature of the resulting culture solution finally shows turbidity at 0D 660 The degree is 0.452X 20-0.516X 20, and the number of viable bacteria is 3.2X 101 / Ml. Example 3 (Please read the precautions on the back before filling in this page) Pack · Thread-This paper ruler / BB's "quasi (CNS) 肀 4 specifications (210x297 male dragon) Λ 6 13 6 ' 25. Description of the invention () The culture solution obtained in Example 1 is centrifuged at 6,000 g and suspended in 2.5 liters of protective medium, which consists of 8% lactose, 1% carboxymethyl quinoline, 5% skim milk, 〇.2% MgS04 * 7Ha0 and 1% Yisine monosodium. Let the suspension in the protection medium stand at Q ~ 41 for 2 hours to increase the resistance to low temperature. In a freeze dryer, the resistance-increasing suspension-701 was frozen for 2 hours, and then dried in a vacant state for 12 hours. The number of viable bacteria after freeze-drying was 6.8X 10 * ° / g. Example 4 Lyophilization was carried out in the same manner as Example 3, except that 1% gelatin was substituted for 1% carboxymethyl cellulose. The number of viable bacteria after freeze-drying was 4, IX 101. / Gram. (Please read the precautions on the back and then fill out this page) Installation ·-Line · Printed by the Beigong Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. The paper size is used in the BB Family Standard (CNS) A 4 specifications (210X297 public: ¢ )