1240751 五、 發明說明 ( 1 ) 技 術 領域 本 發明 爲 關 於 含有 活 細 胞或組 織 之膠囊 及 其應 用 0 更 詳 而 言 之， 本 發 明 關 於 含 有 來自微 生 物、植 物 或動 物 之 活 細 胞 或 組織 爲 塡 充 物 y 且 該 細胞或 組 織可繁 殖 之膠 囊 > 含有 該 膠 囊之 食 品 、 以 及 包含保存安 定 性高、 播 種於 土 壤 快 速 發 芽 之可再分化 的 植 物 細 胞組織 之 膠囊化人 工種 子 0 背 景 技術 白 古以 來 即 食 用 以 微 生 物使食 品 作用之 發 酵食 物 〇 代 表 之 發酵 食物 - 優 格 爲使 乳 酸菌 > 雙 岐桿 菌 (Bi f i d 〇 b a c t i r i um )等（ 以 下稱爲 乳 酸菌等 Z)於牛 奶 中 作 用 而 得 ，食 用 此 等 時 乳 酸 菌等進 入 腸內， 在 腸內 活 潑 地活 動 1 活潑 進 行 整 腸 作 用 〇 然而， 在 腸中存 活 地探 索 之 乳 酸 菌 等 僅爲 其 中 一 部 分而 已 ，其多 數 死於胃j 的1 強酸 〇 爲 解決 此 問 題 1 檢 討 將 乳酸菌 等 裝入腸 溶 性膠 囊 運 至 腸 中 (如特開平 8- 242763 號公報） 〇 但是運 用 此技 術 之 乳 酸 菌 等 爲冷 凍 乾 燥 株 冷 凍 乾燥乳 酸 菌等經 吸 收水 分 而 再 度 表 現活性 需 要 相 當 時 間 〇 以 此， 將 活 細 胞 送 至 腸 內而直 接 活化且 充 分發 揮 者 可 望 爲 使活 細 胞 到 達 腸 內 之技術。 另 一方 面 ， 生 物 科 技知 識驚人 的 進步影 響 醫藥 品 的 開 發 或 植 物的 改 良 等 各 種 領 域 。就植 物 領域而 言 ，嘗 試 將 可 再 分 化 之植 物 細 胞 組 織 (以下僅稱爲丨 '細胞組織」） 予 以 人 工 種 子 化。 般 而 言 1 將 細 胞組織 -3- 分 散、懸 浮 於多 糖 體 聚 1240751 五、發明說明（2) 合性低分子或連接性高分子之溶液中，藉由使其膠化，包 括膠內之細胞組織成形爲珠粒狀、平板狀、棒狀或纖維狀 ，進行人工種子化。 然而，如上述之膠內包含細胞組織之人工種子不耐於室 溫的乾燥，必須在製造後4日內播種而成爲問題。因此， 爲防止乾燥需冷藏保存或保存於液體中，僅可保存1個月 內，但在農舍儲藏庫或倉庫等特別無冷藏設備之乾燥場所 在保管上顯得困難。 爲了解決此乾燥之問題，嘗試以石蠟、蠘等塗於膠表面 。由此試驗中，僅確定改良保存安定性，但由於石蠟或蠟 無法穩定性地分解’即使播種於土中亦無法發芽之問題。 因此於播種時，必須一粒粒開洞而造成不便，無法獲得實 用之人工種子。 因此企求不僅用於作爲食品’亦可利用於醫藥用途、農 業用途等之廣泛範圍之封入活細胞且使其繁殖之技術。特 別就人工種子之領域中’企求至少在無特別冷藏設備之乾 燥場所中亦可保存3個月’且播種於土數日即可發芽之人 工種子。若獲得此人工種子’實際對廣泛之植物可予以適 用，可應用於植物細胞組織培養技術、選殖技術或游離病 毒技術相關、農業、林業、園藝業、花卉業等之廣泛範圍 〇 發明之揭示 本發明人等爲解決上述之課題潛心硏究之結果’發現可 1240751 五、 發明說明 ( 3) 製 造 包 含 懸 浮 於液體之活細胞或組織之膠囊，應用 於 食 品 及 農 業 領 域 而 完成本發明。 本 發 明 爲 包 含懸浮於液體之活細胞或組織之膠囊 y 提 供 該 液 體 內 之 細 胞或組織可增殖之膠囊。 較 佳 之 實 施 型態爲上述之膠囊爲無縫軟膠囊。 良 好 之 實 施 型態爲上述細胞或組織爲使用於食品 用 途 之 細 胞 或 植 物 組 織。 良 好 之 實 施 型態爲上述之細胞或組織爲選自乳酸菌（ 包 含 雙 岐 桿 菌 ) 、納豆菌、麵包酵母菌、釀造用酵母 菌 、 釀 造 用 絲 狀 菌 、 單細胞藻類、多細胞藻類、食用植物 及 食 用 植 物 組 織 之 群 中1或2以上之細胞。 本 發 明 又 關 於含有上述膠囊之食品，較佳含有上 述 膠 囊 之 食 品 爲 果 汁 飮料、果菜汁、健康飮料、加工乳、 豆 乳 果 凍 優 格 乳酸飮料、發酵乳、碳酸飮料、寡糖 飮 料 嘌 呤 〇 又 本 發 明 關 於包含懸浮於液體之可再分化的植物 細 胞 組 織 之 人 工 種 子 ，於該液體內保存細胞或組織之人工種子 〇 再 者 本 發 明 爲具有最內層、覆於該最內層之內皮 層 及 覆 於 該 內 皮 層 之外層三層或彼等以上之層構造的無 縫 軟 膠 -^r 曩 所 構 成 之 人 工種子，提供該最內層中含有可再分 化 之 植 物 細 胞 組 織 (細胞組織）、該內皮層以硬化油爲主成 分 之 內 皮 膜 所 構 成 該外層爲具有生物分解性之外皮膜的 人 工 種 子 0 -5- 1240751 五、發明說明（5) 較佳之情形爲包含於膠囊之液體（內容液）之比例爲3〇〇/〇 ’菌體密度高之情形，內容液之菌體密度亦可爲3 . 3 X 1〇1 1 個/毫升。以未膠囊化之游離菌培養至此高密度係爲困難 。又培養菌體後，濃縮爲此高密度之懸浮液的黏性甚高， 因此膠囊化以現今技術甚難達成，故本發明之有用性明顯 可見。 本發明中之「膠囊」宜爲具有外皮膜之構造，中間有可 封入細胞或組織之懸浮液或培養液之構造者，多半爲球形 ，亦可爲球形以外之形狀。 膠囊之外皮層可使用任何習知之膠囊，如自天然高分子 所形成之皮膜、或合成高分子皮膜。用於食品用途時較佳 爲天然高分子所形成之皮膜。 膠囊之形狀宜爲2層構造，亦可爲3層構造或其以上之 層構造，但較佳爲3層構造。 3層構造時較佳爲，具有活細胞或組織之液體爲最內層 ，包覆於此液體之中間層爲親油性膜，最外層視用途決定 具有可食性、易崩壞性（生物分解性）、腸溶性、不溶於水 性、生體適合性等之1或2以上之性質的外皮膜。 最內層（內容物）爲水、生理食鹽水、緩衝液、培養液、 或維持細胞生命之必要成分與細胞之液狀物質。 以下首先就食品用途之膠囊予以說明，其次說明人工種 子。 用於食品用途時，中間層之親油性膜使用供食用之各種 1240751 五、發明說明（7) 性方面較佳。 又內容物與內皮膜與外皮膜之組成比依膠囊粒徑而不同 ，但宜爲體積比10〜70: 10〜50: 5〜50，較佳爲30〜50: 25〜40 : 25〜40 ° 本發明之膠囊宜爲無縫軟膠囊。作爲無縫軟膠囊，疊層 構造之情形，可將親油性膜及外皮膜各自變薄。由此考量 ，細胞或組織增殖之必要物質較易於膠囊內部與外部間移 動，而使膠囊內之細胞或組織得以增殖。而且增殖之細胞 不會漏出於外部。 又視需要使用外皮層孔徑大而少抵抗物質移動之通透性 優良之膠囊外皮膜，或相反地使用孔徑小之物質移動抵抗 大之阻礙性高之膠囊皮膜，可視各用途做成外皮膜。又由 皮膜上持有電荷者，亦可爲具有帶電物質通過之選擇性， 可作爲具有多樣功能之膠囊。 可封入膠囊內之細胞或組織無特別之限制，如細菌、酵 母、黴菌、藻類、植物細胞、植物或動物組織，視各用途 使用爲佳。 用於食品用途之細胞或植物組織如乳酸菌（含雙岐桿菌） 、納豆菌、麵包酵母菌、釀造用酵母菌（含酒酵母菌、淸 酒酵母菌、味噌酵母菌、醬油酵母菌等）、釀造用絲狀菌（ 麴菌等）、單細胞藻類（如綠藻、螺旋藻等）、多細胞藻類（ 紫菜、昆布等）、食用植物及食用植物組織（如高麗人蔘等） 1240751 五、發明說明（8 ) 具有此等細胞或組織之膠囊以熟知此技藝者通常使用之 方法製造。三層構造之情形最佳爲無縫軟膠囊。無縫軟膠 囊之製法如特開平5 - 3 1 3 5 2號公報，食品加工技術’第1 5 卷，P.28-33，1995，或生物科技與工業’58冊，第7號 ，P . 3 1 - 3 4 ( 2000 )所記載。尤其使用三層噴嘴之滴下法使 中間層介有油狀物質（如硬化油）而做成者爲佳。 封入膠囊之細胞或組織的量由可存活或增殖之最小量經 過濾或離心收集之細胞或組織予以塡充至高密度量，但由 於本發明之特徵爲使膠囊內可進行細胞或組織培養增殖至 高密度，因此封入膠囊之細胞或組織的濃度以一般繼代培 養時之細胞或組織濃度即爲充足。 所得之無縫軟膠囊之平均粒徑依用途而異，爲0 . 1毫米 -1 0毫米。較佳爲0 · 2 - 8毫米。用於食用時，平均粒徑較 佳爲4毫米以下，更佳爲〇 . 1 - 2毫米。此無縫軟膠囊用於 食品時，通過咽喉時良好，可直接使用，亦可於膠囊表面 塗佈澱粉、澱粉分解物、果膠等糊劑或增黏劑。 所得之含有活細胞或組織之無縫軟膠囊可經由懸浮於適 當之培養基予以培養而增殖。尤其藻酸膠，在形成膠時需 要多價金屬離子之皮膜材料者，爲了維持膠強度宜添加必 要濃度之多價金屬離子於培養基中。因此，藻酸膠之情形 中，氯化鈣、氯化緦、氯化鋇、或氯化銨爲培養基量之 0 . 0 1 - 5重量％爲佳，較佳爲0 · 5 - 3重量％。 例如培養封入雙岐桿菌之膠囊時，由於膠囊內可培養增 -10 - 1240751 五、發明說明（9 ) 殖雙岐桿菌，因此膠囊內之活雙岐桿菌增加至數百億個/ 克膠囊以上之多，而由於膠囊內培養活細胞，以淸洗菌體 所失去之代謝產物（細菌素或多糖等）亦蓄積於膠囊內。因 此藉由攝取膠囊內增殖雙岐桿菌之每一膠囊，較冷凍乾燥 粉末可顯示立即且強力之整腸作用，且產生皮膚、關節滑 潤之效果。 又將活細胞封入之膠囊由於膠囊內可培養增殖乳酸菌， 藉由使用乳酸發酵生物反應而高效率生成乳酸，獲得未混 入菌體之培養液，亦容易回收乳酸。 本發明可使封入膠囊內之細胞或組織於所欲之培養基中 增殖。而後，含有此增殖之細胞或組織之膠囊使用於食品 等用途上。例如將乳酸菌（雙岐桿菌）、高麗人蔘組織等封 入膠囊，增殖後加入於果汁飮料、果菜汁、健康飮料、豆 乳、果凍、加工乳、優格、乳酸飮料、發酵乳、碳酸飮料 、寡糖飮料、嘌呤等，製造摻有本發明膠囊之食品。且本 發明中，爲果凍時包含膠型式之食品。又膠囊添加之食品 無此等之限制。 添加於食品之膠囊量無特定之限制，對1 〇〇克之食品宜 爲0.1-10克，較佳爲0.5-3克。 其次，就本發明之人工種子說明。本發明之人工種子爲 具有最內層、覆於該最內層之內皮層、及覆於該內皮層之 外層三層或彼等以上之層構造的無縫軟膠囊所構成之人工 種子。本發明之人工種子的橫切面模式圖如圖1所示。圖 -11- 1240751 五、發明說明（彳〇 ) 1爲三層構造之情形。最內層中含有細胞組織之不定胚。 此最內層中充滿維持不定胚生命之必要培養基成分與生長 調節劑之液體成分或膠。內皮層以硬化油爲主成分之內皮 膜（圖中以內皮膜（硬化油皮膜）表示）所構成。此硬皮油皮 膜可防止水分蒸發亦抑制氧通過。圖1中以外皮膜（明膠 皮膜）表示外層，其可保持物理強度及抑制氧通過。 尙且，種種學術文獻、專利文獻等記述中，將藻酸鈣膠 之單一球珠粒記載爲膠囊之情形，但此種膠囊即使明瞭其 後之製法亦與本發明之無縫軟膠囊完全不同。又本發明之 人工種子較佳爲表面乾燥，與濕潤之藻酸膠並不相同。 使用本發明之包含於最內層之細胞組織較佳使用不定胚 、不定芽、多芽體、莖頂、生長點、原軀樣本、不定根、 根毛等。亦可使用無病毒組織。 爲了維持細胞組織之生命，較佳使細胞組織懸浮於水、 生理時鹽水、緩衝液、培養液、或含有維持細胞組織再分 化（發芽）活性必要之成分的液狀物質或膠狀物質中（以下 稱爲總培養基）。培養基爲適於所用植物之培養基，如熟 知此技藝者通常使用之培養基如穆拉謝-思庫茲 (Murashige & Skoog)之基本培養基（1962年，以下稱爲「 MS」）或修飾其之培養基爲代表之培養基，但不限於此。 又亦可合倂添加一般培養用之植物激素、椰奶、酪蛋白水 解物或酵母萃取液等。且由播種後內皮膜崩壞至發芽所花 費之時間，受到微生物破壞時，亦可添加抑制微生物生長 -12- 1240751 五、發明說明（11) 用之抗菌物質。 內皮膜較佳使用以硬化油爲主成分之油脂。「以硬化油 爲主成分」爲含有單獨硬化油之情形或於硬化油中混合其 他油，調整爲所欲之性質者。較佳之內皮膜爲常溫下固體 之微生物分解性之硬化油。「常溫下固體之硬化油」爲熔 點約2(TC以上之硬化油。熔點30°C以上亦佳，40°C以上 亦佳，或5 0 °C以上亦佳。較佳使用熔點2 0 - 5 (TC之硬化油 。選擇任何硬化油時以考慮保存溫度、播種時間等予以決 定爲佳。 硬化油如中鏈脂肪酸之三酸甘油酯或二酸甘油酯等，例 如奶油、植物性奶油、短油或可可奶油等，但不限於此等 〇 / 此內皮膜爲耐水性，不僅爲形成水系物質之膠囊的必要 者，在防止含細胞組織之最內層水分揮發尙亦爲重要。 生物分解性或微生物分解性意指播種於土壤時藉由微生 物、其他生物分解或同化。硬化油藉由分解或同化，崩壞 內皮膜，將膠囊外之障礙予以去除，同時提高氧通透性， 成爲細胞組織可自休眠狀態甦醒、分化、增殖之環境。 外皮膜以水膨潤，爲生物分解性或微生物分解性者，但 較佳爲細胞組織分化、增殖，發芽至膠囊外時之障礙予以 去除。生物分解性或微生物分解性之外皮膜如蛋白質、多 糖體、生物分解性之塑體等。此等可各自單獨或2種以上 組合使用。1240751 V. Description of the Invention (1) Technical Field The present invention relates to capsules containing living cells or tissues and applications thereof. More specifically, the present invention relates to the inclusion of living cells or tissues from microorganisms, plants or animals as fillings. y Capsules in which the cells or tissues can reproduce > Foods containing the capsules, and encapsulated artificial seeds containing redifferentiable plant cell tissues that are highly stable and are sown in the soil and rapidly germinated 0 BACKGROUND ART Since ancient times Fermented food represented by microbes that act on foods 〇 Fermented foods represented by Yogurt are obtained by allowing lactic acid bacteria > bifid 〇bactiri um, etc. (hereinafter referred to as lactic acid bacteria, etc.) to act in milk, eat this Isochronous lactic acid bacteria enter the intestine and lively in the intestine. 1 Actively perform intestinal rectification. However, lactic acid that is actively exploring in the intestine Bacteria are only a part of them, and most of them die from the strong acid of the stomach j. To solve this problem 1 Review that lactic acid bacteria and the like are put into enteric capsules and transported to the intestine (such as Japanese Patent Application Laid-Open No. 8-242763) 〇 But use this The lactic acid bacteria and the like of the technology are freeze-dried strains and freeze-dried lactic acid bacteria and the like take a considerable time to regain their activity after absorbing water. In this way, live cells are sent to the intestine to be directly activated and fully exerted. technology. On the other hand, amazing advances in biotechnology know-how have affected various fields such as the development of pharmaceuticals or the improvement of plants. As far as the field of plants is concerned, try to artificially seed the re-dividable plant cell tissue (hereinafter simply referred to as the “cell tissue”). In general 1 Disperse and suspend the cell tissue-3- in polysaccharide poly 1240751 V. Description of the invention (2) Synthetic low-molecular or linking polymer solution, gel the gel, including the cells in the gel The tissue is formed into a bead shape, a flat plate shape, a rod shape, or a fibrous shape, and artificial seeding is performed. However, artificial seeds containing cell tissue as described above are not resistant to drying at room temperature and must be sown within 4 days after manufacture, which becomes a problem. Therefore, in order to prevent drying, it needs to be stored refrigerated or stored in liquid. It can only be stored for one month, but it is difficult to store in dry places such as farmhouse storages or warehouses without refrigeration facilities. In order to solve this drying problem, try to apply wax to the surface of the glue with paraffin. In this test, only improved storage stability was determined, but paraffin or wax could not be decomposed stably. Even if it was sown in soil, it could not germinate. Therefore, when seeding, it is necessary to open holes one by one to cause inconvenience, and it is impossible to obtain practical artificial seeds. Therefore, a technique for enclosing and propagating a wide range of cells not only as a food product but also for a wide range of medical and agricultural uses has been sought. Especially in the field of artificial seeds, 'the artificial seeds which are required to be stored for at least 3 months in a dry place without special refrigerating equipment' and are sown in soil for a few days to germinate. If this artificial seed is obtained, it can actually be applied to a wide range of plants, and can be applied to a wide range of plant cell tissue culture technology, selection technology or free virus technology, agriculture, forestry, horticulture, flower industry, etc. As a result of intensive research to solve the above-mentioned problems, the present inventors found that 1240751 can be used. V. Description of the invention (3) Production of capsules containing living cells or tissues suspended in a liquid, which is applied to the fields of food and agriculture to complete the present invention. The present invention is a capsule containing living cells or tissues suspended in a liquid. A capsule that provides cells or tissues in the liquid to proliferate. A better implementation type is that the aforementioned capsules are seamless soft capsules. A good implementation type is that the above cells or tissues are cells or plant tissues used for food. A good embodiment is that the above-mentioned cells or tissues are selected from the group consisting of lactic acid bacteria (including bifidobacteria), natto bacteria, baker's yeast, brewing yeasts, brewing filamentous fungi, unicellular algae, multicellular algae, and edible plants. And 1 or more cells in a group of edible plant tissues. The present invention also relates to foods containing the capsules, preferably the foods containing the capsules are fruit juice extract, fruit and vegetable juice, healthy seasoning, processed milk, soy milk jelly yoghurt lactic acid seasoning, fermented milk, carbonated seasoning, oligosaccharide seasoning purine. The invention relates to an artificial seed comprising redifferentiable plant cell tissue suspended in a liquid, and the artificial seed containing cells or tissues is stored in the liquid. Furthermore, the present invention has an innermost layer, an inner endothelial layer covering the innermost layer, and The artificial seed consisting of seamless soft gum- ^ r 构造 structure that covers the outer layer of the endothelial layer three layers or above, provides the innermost layer containing re-differentiable plant cell tissue (cell tissue), The endothelial layer is composed of an endothelial membrane containing hardened oil as its main component. The outer layer is an artificial seed with a biodegradable outer membrane. 0 -5- 1240751 V. Description of the invention (5) A liquid contained in a capsule is preferred (content Liquid) ratio of 300 / 〇 'high cell density Cell density case, the content liquid may also be 3. 3 X 1〇1 1 / ml. It is difficult to cultivate unencapsulated free bacteria to this high density. After culturing the bacterial cells, the concentration of this high-density suspension is very high, so the encapsulation is difficult to achieve with current technology, so the usefulness of the present invention is clearly visible. The "capsule" in the present invention is preferably a structure having an outer membrane, and a structure in which a suspension or culture liquid can be enclosed in cells or tissues. Most of the structures are spherical, and can also have shapes other than spherical. The outer layer of the capsule can be any conventional capsule, such as a film formed from a natural polymer, or a synthetic polymer film. When used for food applications, it is preferably a film made of a natural polymer. The shape of the capsule is preferably a two-layer structure or a three-layer structure or more, but a three-layer structure is preferred. For a three-layer structure, it is preferable that the liquid with living cells or tissues is the innermost layer, and the middle layer covered with this liquid is a lipophilic film. The outermost layer is edible and easily collapsed (biodegradability) depending on the application. ), Enteric, insoluble in water, biocompatible, etc. 1 or more properties of the outer membrane. The innermost layer (content) is water, physiological saline, buffer, culture medium, or liquid components necessary for maintaining the life of cells and cells. In the following, capsules for food use are explained first, and then artificial seeds are explained. For food use, the lipophilic film of the middle layer is used for various foods. 1240751 V. Description of the invention (7) It is better in terms of properties. The composition ratio of the contents to the endothelial membrane and the outer membrane varies depending on the size of the capsule, but it is preferably a volume ratio of 10 to 70: 10 to 50: 5 to 50, preferably 30 to 50: 25 to 40: 25 to 40. ° The capsule of the present invention is preferably a seamless soft capsule. In the case of a seamless soft capsule having a laminated structure, the lipophilic film and the outer film can be thinned. From this consideration, the necessary substances for cell or tissue proliferation are easier to move between the inside and outside of the capsule, so that the cells or tissue in the capsule can proliferate. And the proliferating cells will not leak out. If necessary, use a capsule film with a large outer pore size and less permeability to resist material movement, or a capsule film with a small pore size and high resistance to movement, which can be made into an outer film according to various uses. The person who holds the charge on the membrane can also have the selectivity of passing the charged substance, and can be used as a capsule with various functions. There are no special restrictions on the cells or tissues that can be enclosed in the capsule, such as bacteria, yeasts, molds, algae, plant cells, plant or animal tissues, depending on the application. Cells or plant tissues for food use, such as lactic acid bacteria (including bifidobacteria), natto bacteria, baker's yeast, brewing yeast (including wine yeast, wine yeast, miso yeast, soy sauce yeast, etc.), Filamentous fungi (yeast fungi, etc.), single-cell algae (such as green algae, spirulina, etc.), multi-cell algae (seaweed, kombu, etc.), edible plants and edible plant tissues (such as Korean mandarin ducks, etc.) 1240751 V. Description of the invention (8) Capsules having such cells or tissues are manufactured by a method generally used by those skilled in the art. A three-layer structure is best for seamless soft capsules. The method of making seamless soft capsules is disclosed in JP-A-H5-31-3125, Food Processing Technology 'Vol.15, P.28-33, 1995, or Biotechnology and Industry' Vol.58, No.7, P As described in 3 1-3 4 (2000). In particular, it is preferable to use a three-layer nozzle dropping method in which the intermediate layer is interposed with an oily substance (such as hardened oil). The amount of cells or tissue enclosed in the capsule is filled with a minimum amount of viable or proliferative cells or tissue collected by filtration or centrifugation to a high density, but because the feature of the present invention is that the cell or tissue can be proliferated in the capsule Density, so the concentration of cells or tissues enclosed in the capsule is sufficient as the concentration of cells or tissues in general subculture. The average particle size of the obtained seamless soft capsules varies depending on the application and ranges from 0.1 mm to 10 mm. It is preferably 0. 2-8 mm. When used for food, the average particle size is preferably 4 mm or less, and more preferably 0.1-2 mm. This seamless soft capsule is good for food passing through the throat and can be used directly. The surface of the capsule can also be coated with a paste, such as starch, starch degradation product, pectin, or thickener. The obtained seamless soft capsules containing living cells or tissues can be proliferated by culturing and suspending in an appropriate medium. In particular, alginic acid gums, if a film material of polyvalent metal ions is needed to form the gum, it is desirable to add the polyvalent metal ions to the medium in a necessary concentration in order to maintain the strength of the gum. Therefore, in the case of alginate, calcium chloride, gadolinium chloride, barium chloride, or ammonium chloride is preferably 0.01 to 5% by weight, and more preferably 0.5 to 3% by weight. . For example, when cultivating a bifidobacteria-encapsulated capsule, -10-1240751 can be cultured in the capsule. V. Description of the invention (9) Bifidobacterium spp., The number of viable bifidobacteria in the capsule has increased to tens of billions per gram capsule. There are many metabolites (bacteriocins or polysaccharides, etc.) lost by washing the cells due to the cultivation of living cells in the capsules. Therefore, by ingesting each capsule of the bifidobacterium in the capsule, it can show an immediate and powerful intestinal remodeling effect than freeze-dried powder, and produce smooth skin and joint effects. In addition, the capsules enclosing live cells can grow lactic acid bacteria in the capsules, and use lactic acid fermentation to generate lactic acid with high efficiency, and obtain culture liquid without mixing with bacteria, and it is easy to recover lactic acid. The present invention allows cells or tissues enclosed in a capsule to proliferate in a desired medium. Thereafter, the capsule containing the proliferating cells or tissues is used for food and the like. For example, lactobacillus (Bifidobacterium), Korean Tissue, etc. are enclosed in capsules, and after proliferation, they are added to fruit juice, fruit and vegetable juice, healthy seasoning, soy milk, jelly, processed milk, yogurt, lactic acid seasoning, fermented milk, carbonated seasoning, Glucosides, purines, etc., to produce foods incorporating the capsules of the present invention. In addition, in the present invention, a jelly type food is included in the jelly. There are no such restrictions on foods added in capsules. The amount of capsules to be added to food is not particularly limited, and it is preferably 0.1-10 g, preferably 0.5-3 g, for 100 g of food. Next, the artificial seeds of the present invention will be described. The artificial seed of the present invention is an artificial seed composed of a seamless soft capsule having an innermost layer, an innermost layer covering the innermost layer, and three or more outer layers covering the innermost layer. A schematic cross-sectional view of the artificial seed of the present invention is shown in FIG. 1. Figure -11- 1240751 V. Description of the Invention (彳 〇) 1 is a three-layer structure. The innermost layer contains adventitious embryos of cellular tissue. This innermost layer is filled with liquid components or gums necessary for the maintenance of adventitious embryo life and growth regulators. The endothelial layer is composed of an endothelium membrane (shown as an endothelium membrane (hardened oil film)) as the main component of hardened oil. This crusty oil film prevents moisture from evaporating and inhibits the passage of oxygen. The outer film (gelatin film) in Fig. 1 represents the outer layer, which can maintain physical strength and inhibit oxygen passage. Moreover, in various descriptions of academic literature, patent literature, etc., a single spherical bead of calcium alginate is described as a capsule, but even if the subsequent method is clear, the capsule is completely different from the seamless soft capsule of the present invention. . Also, the artificial seeds of the present invention are preferably dry on the surface, which is different from the wet alginate. For the cell tissue contained in the innermost layer of the present invention, adventitious embryos, adventitious buds, polybuds, stem tops, growth points, protozoan samples, adventitious roots, root hairs, etc. are preferably used. Virus-free tissue can also be used. In order to maintain the life of the cell tissue, it is preferable to suspend the cell tissue in water, physiological saline, buffer, culture medium, or a liquid or gelatinous substance containing components necessary to maintain the redifferentiation (germination) activity of the cell tissue ( Hereinafter referred to as total medium). The medium is a medium suitable for the plants used, such as the medium commonly used by those skilled in the art such as the basic medium of Murashige & Skoog (1962, hereinafter referred to as "MS") or modified The medium is a representative medium, but is not limited thereto. Alternatively, plant hormones, coconut milk, casein hydrolysate, or yeast extract can be added in combination. In addition, from the time it takes for the endothelial membrane to collapse to germinate after sowing, it can also be added to inhibit the growth of microorganisms when it is damaged by microorganisms. -12-12751751 V. Description of the invention (11) Antibacterial substances used. The endothelial membrane preferably uses a fat containing a hardened oil as a main component. The "mainly hardened oil" refers to a case where a hardened oil is contained alone or another oil is mixed with the hardened oil and adjusted to a desired property. A preferred endothelial membrane is a solid microbial degradable hardened oil at room temperature. "Solid hardened oil at room temperature" is a hardened oil with a melting point of about 2 ° C or higher. A melting point of 30 ° C or higher is preferred, a temperature of 40 ° C or higher is preferred, or a temperature of 50 ° C or higher is preferred. A melting point of 20- 5 (TC hardening oil. When selecting any hardening oil, it is better to consider storage temperature, sowing time, etc .. Hardening oils such as triglycerides or diglycerides of medium chain fatty acids, such as cream, vegetable cream, Short oil or cocoa butter, etc., but not limited to this. This endothelial membrane is water-resistant and is not only necessary for forming capsules of water-based substances, it is also important to prevent evaporation of moisture in the innermost layer of cell-containing tissues. Biodegradation Sexual or microbial degradability means that when seeded in the soil, it is decomposed or assimilated by microorganisms and other organisms. By decomposing or assimilating, the hardened oil breaks down the endothelial membrane, removes obstacles outside the capsule, and improves oxygen permeability, and becomes Cell tissue can be awake, differentiate, and proliferate in a dormant state. The outer membrane is swollen with water and is biodegradable or microbial decomposable, but preferably the cell tissue is differentiated, proliferated, and germinated into capsules. Remove external obstacles. Biodegradable or microbially degradable coatings such as protein, polysaccharides, biodegradable plastics, etc. These can be used individually or in combination of two or more.
———. 1240751 五、發明說明（13) 膠囊。自所得之膠囊脫去凝固油後，經由轉筒乾燥獲得乾 燥膠囊化不定胚之人工種子。以細胞組織不死之低溫下進 行乾燥。 所得之膠囊化人工種子的粒徑依組織細胞大小而有不同 ，但爲1毫米-1 2毫米。較佳爲3毫米-1 0毫米。封入膠 囊之細胞組織數亦根據組織大小決定，可由1至數個，亦 考慮再分化率（發芽率），以可顯示人工種子適當性質之數 予以封入爲佳。植物亦可，較佳爲1 - 4個。 由此所得之本發明之人工種子可於乾燥之儲藏庫等室溫 下維持發芽能力之狀態，保存3個月以上，尤其無保存於 冷藏庫或低溫水中之必要。若保存於1 〇°C以下之冷藏保存 庫中，可長期保存6個月以上。 本發明之膠囊化人工種子包含可再分化之植物細胞組織 (細胞組織）之中空無縫軟膠囊，硬化油之內皮層’生物分 解性之膠（蛋白質、多糖體）之外皮層、生物分解性塑體類 等所構成，使表面乾燥而得。經由此構成’限制膠囊內氧 的供給，可使膠囊內之細胞組織以一種休眠狀態生存，而 且抑制水分蒸發可於常溫下保存2個月。又經由將膠囊播 種於土’水與土中微生物作用以分解外皮膜’更分解內皮 膜之硬化油’提高氧通透性，自休眠狀態中E醒’移至發 芽狀態。 !實施例 以下將已實施例具體說明本發明，但此等並不限定本發 -15- 1240751 五、發明說明（14) 明之範圍。 實施例1 :添加活雙岐桿菌膠囊之果汁飮料 將長雙岐桿囷 Bifidobacterium longum JCM7050 之菌 落-2以接種環植菌於脫脂牛奶1 5%、酵母萃取液〇 . 4%、蔔 萄糖3%、pH 6.5之液體的脫脂牛奶培養基l〇〇mi，37它下 以無液體包裝（三菱瓦斯化學公司製）於厭氧狀態之容器中 以60 1： pm緩慢振盪培養15小時。由於進行過程中pH値 下降，故以pH自動調節機以5M-NaOH水溶液維持於pH 5 · 5。1 5小時培養後之菌數於BL瓊脂培養基（日水製藥公 司製）上24小時厭氧培養，計算菌落數，計得2X 1〇9個/ 毫升。將此菌液於15%脫脂牛奶水溶液以0.01 %胰晦F(天 野製藥公司製）於4 5 °C處理1 5小時外，同上述組成之脫脂 牛奶培養基（以下稱爲「經胰晦處理之脫脂牛奶」）稀釋 100倍爲膠囊之內容液。 表1 部位 成分 構成比 (重量％) 內容物 雙岐桿菌液 30 內皮膜 維生素E油 30 外皮膜 2%瓊脂溶液 40 膠囊全體之構成比 其次以袠1之處方，使用三重管噴嘴之無縫軟膠囊製造 機以液中滴下法，製造裝入此內容液之三層構造膠囊（平 -1 6 - 1240751 五、發明說明（15) 均粒徑1 · 8毫米）。外皮膜以瓊脂作成，形成膠囊滴與固 化於硬化液中進行。 將此膠囊1 00克裝入經胰晦處理之脫脂牛奶培養基1公 升，將氮氣85%、氫氣10%、二氧化碳氣體5%之混合氣體（ 住有精化公司製）以100毫升/分之流量吹入，以攪拌翼於 5 0 r pm緩慢攪拌，調整至pH 5 . 5並於3 7 °C培養3 0分鐘。 培養後之膠囊內活菌數爲3 X 1 0 1 ()個/克膠囊（濕重）。 將此膠囊表面以滅菌蒸餾水洗淨後，以2克添加於透明 之葡萄果汁100毫升，每100毫升之葡萄（mascut)果汁中 有活雙岐桿菌600億個之多的膠囊，製作葡萄果汁飮料。 添加此有活雙岐桿菌膠囊之葡萄果汁飮料不會因雙岐桿菌 特有之惡味而使味道變差，爲透明而美味之飮料。含有此 膠囊之果汁飮料具有大量活雙岐桿菌，被期待具有整腸作 用。 實施例2 :添加活乳酸菌膠囊之果菜汁 將乳酸乳球菌L a c t 〇 c 〇 c c u s 1 a c t i s〗C Μ 7 6 3 8以C M G瓊 脂培養基（酵母萃取液0.5%、多肽0.5%、NaCl 0.5%、葡 萄糖1%、瓊脂2%)培養之菌落1以接種環植菌於脫脂牛奶 12%、酵母萃取液〇.4%、葡萄糖3%、pH6.5之液體脫脂牛 奶培養基2 0毫升，於3 7 °C靜置培養1 5小時。培養1 5小 時後之菌數爲2 X 1 09個/毫升。將此液體以相同之脫脂牛 奶培養基稀釋500倍作爲膠囊之內容液。 其次，同實施例1製成封入此內容液之三層構造之膠囊 -17- 1240751 五、發明說明（彳6) (平均粒徑1 . 8毫米）。 將所得之膠囊100克以含有0.01 %蛋白晦P (天|?製—疏 公司製）之12%脫脂牛奶水溶液於40t處理15小時以外， 裝入上述相同組成之脫脂牛奶培養基（以下稱爲「經^ & 晦P處理之乳酸菌用脫脂牛奶」）5 0 0毫升，以5 Μ - N a Of·} zK 溶液調至pH 5.2，同時於371：以50 jrpni振盪培養2天。 培養後之膠囊內活菌數爲6 X 1 0 1 ^個/克膠囊（濕重），顯示 膠囊內之乳酸菌顯著的增加。 由此培養之膠囊以滅_蒸飽水洗淨後，以1克/ 1 Q Q毫升^ 之比例加入果菜汁中。此果菜汁不僅活乳酸菌含量多，# 彼等之惡味，膠囊通過咽喉時味道亦佳，爲美味之飮料。 因此，飮用以往未考量之果菜汁可攝取大量之乳酸菌。 實施例3 :添加高麗人蔘培養細胞膠囊之健康飮料 於含有蔗糖 3%與吲哚-3 -乙酸（IAA) 1毫升/升之 Murashige-Skoog液體培養基（pH 5.6)中繼代培養高麗人 蔘（Panax ginseng C. A. Meyer)之細胞 5 克（濕重），裝 入5 00毫升錐形瓶中，添加相同培養基100毫升，於25T： 、1 2 0 r pm振盪培養2週，獲得游離之細胞集塊。再將此游 離細胞集塊以8 0 // m孔徑之耐綸網過濾，通過此等之細的 細胞集塊再以20 # m孔徑之耐綸網過濾，收集在網上殘餘 之細胞集塊。將此細胞集塊5克（濕重）懸浮於同上述組成 之Murashige-Skoog液體培養基100毫升中，作爲膠囊之 內容液。 -18- 1240751 五、發明說明（17) 無縫軟膠囊化之進行除使細胞懸浮液之內容液以振盪機 振動同時進行以外，其他同實施例1進行之。取出具有所 得高麗人蔘細胞之膠囊1 0克，裝入5 0 0毫升錐形瓶中， 添加相同組成之Murashige-Skoog液體培養基1〇〇毫升， 於25 °C、50 rpm培養3週。膠囊內高麗人蔘增殖而充滿於 膠囊內。將此培養所得之膠囊2克以滅菌蒸餾水洗淨後， 添加使膠囊浮游用之肽0 . 05克於健康飮料（森下仁丹公司 製之商品名「超力源」相同之組成，未加入高麗人蔘萃取 液者）50毫升，調製成加入活高麗人蔘細胞之健康飮料。 此健康飮料不僅含有活高麗人篸之紐胞，亦無苦味或惡味 ，通過咽喉時亦佳，爲美味之飮料。 實施例4 :添加納豆菌膠囊之豆乳飮料 於粉碎之大豆粉100克中加入蔗糖5克、NaCl 1克、及 水500毫升，於121°C滅菌30分鐘，離心獲得上淸液（以 下稱爲大豆粉萃取培養基）。將此大豆粉萃取培養基1 〇毫 升裝入5 0毫升之錐形瓶，將納豆菌（B a c i 1 1 u s s u b t i 1 i s IFO 13169)之菌落-1以接種環接種，於30°C、120rpm振 盪培養1 5小時。將此培養液全量加入大豆粉萃取培養基 9 0毫升中，均勻懸浮後作爲膨囊化之內容液。 與實施例1相同予以膠囊化，將所得之膠囊1 00克加入 於大豆粉萃取培養基400毫升，於30°C、100 r pm培養24 小時。培養後之膠囊內增殖納豆菌形成黏質物。菌數爲3 X 1 09個/克膠囊（濕重）。將此膠囊3克以蒸餾水洗淨後添 -19- 1240751 五、發明說明（18) 加於豆乳1 00毫升中。此爲少有納豆菌臭味而美味之飮料 ，可一同攝取豆乳及納豆菌之黏質物。 實施例5 :膠囊化雙岐桿菌之培養方法 將以貫施例1相Μ方法製成之B i f i d 〇 b a c t e r i u m 1 ο n g a m JCM7050膠囊塡充於具保溫套之100毫升圓筒狀柱。於外 部培養槽中加入1公升胰晦處理之脫脂牛奶培養基，自膠 囊塡充柱之下方往上方以1 0毫升/分流入培養基，再於培 養槽中來回循環。培養槽以pH自動調節機、1 0M氨水維持 於pH 5.5，爲保持於厭氧狀態，吹入氮氣85%、氫氣10% 、二氧化碳氣體5%之混合氣體（住有精化公司製）。混合氣 體於最初12小時以100毫升/分，12-30小時以20毫升/ 分吹入。於37°C培養30小時，膠囊內之膠囊活菌數爲5 X 1 0 1 °個/克膠囊之多。 以此柱培養之方法並未使膠囊內受到物理性損害而使膠 囊內可高密度培養雙岐桿菌時，減少雜菌感染之機會。又 收集膠囊時，膠囊不易損壞，可於混合爲食品之柱一同搬 運。因此，培養時及培養後之處理極爲容易，適於以工業 等級培養膠囊內之雙岐桿菌。 實施例6 :冷凍乾燥菌體之膠囊化與培養 將 Bifidobacterium Ion gam JCM7050 以同實施例 1 之 脫脂牛奶培養基厭氧培養，所得之菌體以4°C、8000 rpm 離心20分鐘予以收集，加入菌體1 〇倍量之蒸餾水攪拌分 解，予以冷凍乾燥。將此冷凍乾燥菌體裝入無液體包裝， -20- 1240751 五、發明說明（19) 於厭氧狀態之密閉容器中冷藏保存之處極爲穩定。取0. i 克此冷凍乾燥菌體懸浮於1公升之脫脂牛奶培養基中，作 爲膠囊內容物。此懸浮液之活菌數爲5 X 1 〇7個/毫升。 其次，以下表2所示之配方，以空中滴下法製成封有內 容液之無縫軟膠囊（平均粒徑爲3 . 5毫米）。 表2 咅位 成分 構成比 (重量％) 內容物 雙岐桿菌液 30 內皮膜 維生素E油 30 外皮膜 4%藻酸鈉溶液 40 凝固液 3%氯化鈉水溶液 *膠囊全體之構成比 將所得之膠囊1 00克置入經胰晦處理之脫脂牛奶培養基 1公升中’於無液體包裝、厭氧狀態容器中，以5M Na〇H 水溶液調整爲pH 5.5同時於371振盪培養2日。培養後 之膠囊內活菌數爲2 X 1 0 1 ()個/克膠囊（濕重），被認爲膠囊 內有增殖現象。 實施例7 :添加活雙岐桿菌膠囊之乳酸菌飮料 將乳酸菌 Lactococcus lactis ICM7 6 3 8 以 CMG 瓊脂培 養基（酵母萃取液0.5%、多肽0.5%、NaCl 〇.5%、葡萄糖 1 %、瓊脂2% )培養，獲得菌落。將此接種環植菌於脫脂牛 奶12%、酵母萃取液〇 · 4%、葡萄糖3%、調整至PH6 . 5之液 -21 - 1240751 五、發明說明（2〇) 體脫脂牛奶培養基20毫升中，於3 7 °C靜置培養1 5小時後 ’菌數爲6X109個/ ¾升。 將此培養液全量以4 °C、8 0 0 0 X g離心2 0分鐘，去除上 淸液，於菌體中加入40毫升食鹽水，以攪拌器攬拌，再 以8 0 0 0 X g離心2 0分鐘，收集菌體。再重複1次相同之操 作獲得洗淨之菌體。於此洗淨菌體全量加入透明之蘋果汁 40毫升’以攪拌器均勻分散。將懸浮所得菌體之蘋果汁 〇 . 5毫升添加於透明之蘋果汁1 〇〇毫升。此乳酸菌懸浮蘋 果汁中之活菌數爲1X107個/毫升，大致上透明。 另一方面，同實施例1之膠囊化，製成含有培養之雙岐 桿菌之膠囊2克，添加於上述所得之乳酸菌懸浮蘋果汁 1 〇〇毫升中。裝有此活雙岐桿菌之膠囊的雙岐桿菌之活菌 數爲30 0億個/克膠囊。裝有裝入此活雙岐桿菌膠囊之活 乳酸菌的蘋果汁爲「乳等省令」規格基準所形成之乳酸飮 料，但與習知之乳酸菌飲料完全不同，大致上透明且每 1 00毫升可攝取600億個活雙岐桿菌，爲口味良好乳酸菌 飮料。 實施例8 :膠囊化人蔘不定胚之人工種子 人蔘培養細胞及不定胚之製作依據使用人蔘根之「植物 組織細胞培養」（1 979年，原田、駒嶺編，理工學社 )p . 9 1 - 1 04記載之方法爲準。將液體培養基製作之不定胚 以網孔5 0 0 // m與8 5 0 # m耐綸網予以篩選，得到5 0 0 A m -8 5 0 v m大小之不定胚。所得之不定胚爲心型至魚雷型之形 -22- 1240751 五、發明說明（21) 狀。將此等之不定胚懸浮於未含有激素而含有脫乙醯殼多 糖0 . 2%之MS培養基中，作爲膠囊內容物。 膠囊化係以熔點32°C之三酸甘油酯硬化油（Farmazol B-1 1 5 ’日本油脂公司製）作爲內皮膜，2 2%明膠溶液作爲外 皮膜’以三重管噴嘴之無縫軟膠囊製造機依液中滴下法進 行之。調節放入一個個內容液之不定胚的泵。於凝固油中 進行形成膠囊滴及固化，作成三層構造之膠囊。自所得之 活膠囊脫去凝固油後微乾，獲得膠囊化不定胚之人工種子 。乾燥後之粒徑爲7毫米。 另一方面，製作藻酸膠珠粒包埋人蔘不定胚作爲比較例 (比較例1 )。即以實施例8製作所篩選的不定胚懸浮於含 3%(w/v)藻酸鈉之MS培養基中，滴入於50mM氯化鈣溶液 ，製成包埋不定胚之藻酸鈣膠珠粒（粒徑約5毫米）。此與 報告之習知人工種子相同。 保存及發芽試驗 將上述不定胚膠囊化之人工種子（實施例8 )與以藻酸鈣 膠珠粒包埋不定胚之膠珠粒於20°C、濕度65%之恆溫器中 保存3個月。於保存第3日，藻酸鈣膠珠粒即相當乾燥而 珠粒變小，1週後不定胚亦乾燥，爲乾皺狀態。另一方面 ’膠囊化人工種子即使於1週後亦不被認爲外觀有所變化 〇 製成人工種子之後，直接於1個月後、2個月後、3個 月後，於未經土壤殺菌之苗圃土盆中各100粒播種於表面 -23- 1240751 五、發明說明（22) 2公分深處，予以澆水。播種後，置入2 51培養器中，以 3 OOOLlu每日照明16小時，以及兩天澆水1次，使其生長 。計算3週後各發芽數之結果如表3。 表3 人蔘人工種子之發芽試驗結果 保存期間 製造後 1個月 2個月 3個月 實施例8 95 75 72 68 比較例1 96 0 〇 〇 數字顯示各播種1 〇〇粒之發芽數。 表3所示，製造後之發芽率中實施例8及比較例1大致 相同。然而保存1個月以上之習知型式的人工種子之比較 例1完全未有發芽。對於此點，本發明之膠囊化人工種子 以保存3個月後仍有68%之高發芽率。 由此結果，本發明之膠囊化不定胚關於人工種子之保存 安定性，顯示優於習知藻酸膠珠粒包埋不定胚型式之人工 種子。 實施例9 :膠囊化草莓葉芽之人工種子 _ 由栽培之草莓取出之莖頂組織置於含有蔗糖丨〇克/升及 茱萸烷膠2克/升作爲膠化劑之MS培養基，於暗處25 °C培 養丨4日後，轉移至含有苄腺嘌呤0 . 2毫克/升及蔗糖1 0 克/升之MS培養基中，以25 °C每日以2000Lux照明16小 -24- 1240751 五、發明說明（23) 時，並以150 rprn迴轉振盪培養。培養30曰後’將所得之 葉芽打開，以網孔丨毫米及1 . 7毫米之耐綸網篩選’取得 1 - 1 . 7毫米大小之葉芽。將取得之葉芽懸浮於未含激素之 MS液體培養基’作成膠囊化內容物。 除調節送入葉芽懸浮液之泵流量外’其他進行同實施例 8之膠囊化。將所得膠囊予以乾燥獲得膠囊化草莓葉芽之 人工種子。乾燥後之粒徑爲8毫米。 另一方面，以藻酸膠珠粒包埋草莓葉芽作爲比較（比較 例2 )。以實施例9製作所篩選之草莓葉芽’同比較例1之 方法製成包埋草莓葉芽之藻酸鈣膠珠粒（粒徑約5毫米）。 此相同於習知之人工種子。 所得之草莓葉芽人工種子（實施例9 )及藻酸膠珠粒包埋 草莓葉芽（比較例2 )之保存及發芽試驗同實施例8進行之 。結果如表4所示。 表4 草莓人工種子之發芽試驗結果 保存期間 製造後 1個月 2個月 3個月 實施例9 91 71 68 63 比較例2 94 0 0 〇 數字顯示各播種丨〇〇粒之發芽數。 大致相 表4所示，製造後之發芽率實施例9及比較例2 -25- 1240751 五、發明說明（25 ) 製作表5所示之植物不定胚、頂芽、或不定根’進行同 實施例8之膠囊化，乾燥後，3個月間於20°C、濕度65% 之恆溫器中保存後，各1 0 0粒播種於同實施例8之土盆中 ，1 - 3個月中，同實施例8之生長，觀察其發芽。結果如 表5所示。 表5 各植物人工種子之發芽試驗結果 實施例 植物 細胞組織 粒徑（毫米） 發芽數 11 天冬門 不定胚 7 71 12 報春花 不定胚 8 68 13 鐵百合 頂芽 9 53 14 康乃馨 不定胚 8 62 15 薔薇 不定根 9 51 16 杉樹 不定根 10 37 數字顯示各播種1 00粒之發芽數。 表5之結果亦顯示本發明之膠囊化人工種子的優良保存 安定性。 產業上可利用性 本發明之膠囊爲活細胞或組織可於膠囊內部增殖，而不 溶於胃酸之膠囊。尤其使用乳酸菌（含雙岐桿菌）時，由於 可將大量活菌送至腸中，而迅速顯現強力之整腸作用。再 者，包含本發明之可再分化之植物細胞組織的膠囊化人工 -27- 1240751 五、發明說明（26) 種子於常溫之乾燥狀態中保存安定性高，且播種於土壤經 由膠囊皮膜膨潤及微生物作用而崩壞，可提供快速發芽之 人工種子。可應用於植物細胞組織培養技術、選殖技術或 無病毒技術相關者，農業、林業、園藝業、花卉葉等廣泛 之應用。 -28- 1240751 申請曰期 2001. 6. 28 案 號 90115736 類 別 Α〇ίΗ%^ (以上各欄由本局填註） （94年2月18日修正) II專利說明書 =名稱 中 文 含有活細胞或組織之膠囊 英 文 CAPSULE COMPRISING LIVING CELLS OR TISSUES 姓 名 1. 淺田雅宣（浅田雅宣） 2. 波多野由美 3. 釜口良誠 4. 春原秀基 一 發明义 一、創作人 國 籍 1 .〜4.曰本 住 、居所 1.〜4.日本國大阪府大阪市中央區玉造1 丁目1番30號 森下仁丹株式会社內 姓 名 (名稱） 森下仁丹股份有限公司 (森下仁丹株式会社） 國 籍 曰本 三、申請人 住、居所 (事務所） 曰本國大阪府大阪市中央區玉造1 丁目1番30號 代 姓 表人 名 岡崎康雄 1240751 际Τ ϊθ 五、發明說明（4) 良好之實施型態爲乾燥上述之外皮層。 良好之實施型態爲上述之可再分化之植物細胞組織選自 不定胚、不定芽、多芽體、莖頂、生長點、原球莖體 (protocorm)、不定根、根毛之群。 又良好之實施型態爲上述之內皮膜於常溫下爲固體之微 生物分解性之硬化油。 另一良好之實施型態爲上述之外皮膜爲選自蛋白質、多 糖體、及生物分解性塑體之微生物分解性的外皮膜。 圖示簡單說明 圖1爲本發明人工種子之橫切面模式圖。 爲實施本發明之最佳型態 自習知以來，由於膠囊化水系物質之困難，僅將乾燥細 胞膠囊化，但使封閉於膠囊中之細胞或組織增殖係爲不可 能之事，故亦全無試驗之事。然而，本發明人等最先成功 地將水系物質膠囊化，儘管細胞或組織亦封入膠囊內卻不 死亡且可增殖，遂完成本發明。 根據本發明，如習知僅有冷凍乾燥之乳酸菌、雙岐桿菌 等可送達而只能得到緩慢地整腸作用，但藉由送達活乳酸 菌、雙岐桿菌等，可得到立即且強力之整腸作用等之效果 。再者由於膠囊內之細胞或組織可增殖，可得到具有較高 菌體密度（如約101()個-1011個/克膠囊）之膠囊，因此除上 述食品用途外，亦可廣用於生物反應用途、醫藥用途、醫 療用途、人工種子用途等之用途。 1240751 五、發明說明（6) 油脂類、脂肪酸類、糖之脂肪酸酯類等，但較佳使用動物 性油脂、植物性油脂、彼等之生物學或化學處理之油脂等 。具體如各種天婦羅油、沙拉油、熔點3 5 °C以下之硬化油 、維生素E、小麥胚芽油、胡麻油、可可油、牛油、植物 奶油、短油（shortening oi 1 )、蔗糖脂肪酸酯等，但不限 於此。使用此等親油性材料可得包括水系物質之膠囊。 本發明之膠囊用於食品用途時，可食性外皮膜爲天然性 高分子皮膜，如明膠、瓊脂、果膠、藻酸、鹿角菜膠 (caHageen)、熱凝膠（curdl an )、澱粉、茱萸烷膠、葡甘 露聚糖或此等之混合物，或視需要，較佳於此等中添加蛋 白質、糖蛋白、黏多糖、糖、糖醇、多價醇等之物質所得 之高分子皮膜。添加之天然高分子之具體例如，阿拉伯膠 、支鏈澱粉、葡聚糖、黃色素膠、昆蟲蜂膠、膠原蛋白、 酪蛋白等。 再者，視需要可選自口中溶解性之外皮膜、胃溶解性之 外皮膜、小腸溶解性之外皮膜、大腸溶解性之外皮膜、不 溶性之被排出之外皮膜等。腸溶性之外皮膜如爲明膠或瓊 脂與果膠組合所得之外皮膜。外皮膜考慮成形性等亦可添 加甘油等。 良好之組合爲三層構造之情形，表爲中間層（內皮膜）-外皮膜，與維生素E油-瓊脂、小麥胚芽油-藻酸鈉、蔗糖 酯-明膠、甘油中鏈脂肪酸酯-鹿角菜膠等。其中以維生素 E油-2〜4%瓊脂、小麥胚芽油-2〜4%藻酸鈉所得之膠囊物 1240751 «———. 1240751 V. Description of the invention (13) Capsule. After the coagulated oil was removed from the obtained capsules, the dried seeds were obtained from dried capsules with adventitious embryos through drum drying. The cells were dried at a low temperature where the tissues did not die. The particle size of the obtained encapsulated artificial seeds varies depending on the size of the tissue cells, but it is 1 mm to 12 mm. It is preferably 3 mm to 10 mm. The number of cells and tissues encapsulated in the capsule is also determined according to the size of the tissue, and can be from one to several. The re-differentiation rate (germination rate) is also considered. It is better to enclose the number that shows the proper properties of the artificial seeds. Plants are also possible, preferably 1-4. The artificial seeds of the present invention thus obtained can maintain the state of germination at room temperature, such as in a dry storage room, and can be stored for more than 3 months, especially without the need to be stored in a refrigerator or low-temperature water. If stored in a cold storage below 10 ° C, it can be stored for more than 6 months. The encapsulated artificial seed of the present invention comprises hollow seamless soft capsules of redifferentiable plant cell tissues (cell tissues), the outer layer of hardened oil's biodegradable gel (protein, polysaccharide), outer cortex, and biodegradability It is made of plastic body and the like, and the surface is dried. By constituting the 'limitation of oxygen supply in the capsule, the cellular tissues in the capsule can survive in a dormant state, and the evaporation of water can be suppressed, and it can be stored at room temperature for 2 months. The capsule is seeded in the soil, and the microorganisms in the soil act to decompose the outer membrane. The hardened oil that decomposes the endothelial membrane enhances oxygen permeability and moves from the dormant state to the budding state. EXAMPLES The present invention will be described in detail in the following examples, but these do not limit the scope of the present invention (14). Example 1: Fruit juice with live bifidobacteria capsules. Colonies of Bifidobacterium longum JCM7050-2 were inoculated to inoculate cyclophyte in skimmed milk 15%, yeast extract 0.4%, and glucose 3 %, Liquid skimmed milk culture medium at pH 6.5 100 mi, 37 under a liquid-free package (manufactured by Mitsubishi Gas Chemical Co., Ltd.) in an anaerobic container and cultured slowly at 60 1: pm for 15 hours. As the pH was decreased during the process, the pH was maintained at a pH of 5 · 5.1 with a 5M-NaOH aqueous solution using an automatic pH adjuster. The bacterial count after 15-hour incubation was anaerobic on a BL agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) for 24 hours. Cultivate, calculate the number of colonies, and calculate 2X109 cells / ml. This bacterial solution was treated in a 15% skimmed milk aqueous solution with 0.01% pancreatic F (manufactured by Amano Pharmaceutical Co., Ltd.) at 45 ° C for 15 hours. Skim milk ") diluted 100 times into the contents of the capsule. Table 1 Composition ratio of parts (% by weight) Contents Bifidobacterium solution 30 Endothelium membrane vitamin E oil 30 Outer membrane membrane 2% agar solution 40 The composition of the entire capsule is next to 袠 1, seamless using a triple tube nozzle The capsule making machine uses the drip-in-liquid method to manufacture a three-layer structured capsule filled with the content liquid (flat-1 6-1240751 V. Description of the invention (15) average particle diameter 1 · 8 mm). The outer membrane is made of agar, forming capsule drops and curing in a hardening solution. One hundred grams of this capsule was filled with 1 liter of skim milk culture medium treated with pancreatic obesity, and a mixed gas (manufactured by Sumitomo Chemical Co., Ltd.) of 85% nitrogen, 10% hydrogen and 5% carbon dioxide gas was used at a flow rate of 100 ml / min Blow in, stir slowly with a stirring wing at 50 r pm, adjust to pH 5.5, and incubate at 37 ° C for 30 minutes. The number of viable bacteria in the capsules after the culture was 3 X 1 0 1 () capsules per gram (wet weight). After the surface of this capsule was washed with sterilized distilled water, 2 g of it was added to 100 ml of transparent grape juice. Each 100 ml of mascut juice contained as many as 60 billion viable bifidobacteria capsules to make grape juice sauce. . The grape juice condiment with this live bifidobacteria capsule will not deteriorate the taste due to the peculiar odor of bifidobacteria. It is a transparent and delicious condiment. Juice extract containing this capsule has a large amount of live Bifidobacterium and is expected to have an intestinal rectifying effect. Example 2: Fruit and vegetable juice added with live lactic acid bacteria capsules. Lactococcus lactis L act 〇c occus 1 actis CM 7 6 3 8 CMG agar medium (yeast extract 0.5%, polypeptide 0.5%, NaCl 0.5%, glucose 1%, agar 2%) cultured colonies 1 to inoculate ring-planted bacteria in skimmed milk 12%, yeast extract 0.4%, glucose 3%, pH 6.5 liquid skim milk medium 20 ml, at 37 ° C was allowed to stand for 15 hours. After 15 hours of incubation, the number of bacteria was 2 × 109 cells / ml. This liquid was diluted 500 times with the same skimmed milk culture medium as the contents of the capsule. Next, a three-layered capsule containing the content liquid was prepared in the same manner as in Example 1. -17-1240751 V. Description of the invention (彳 6) (average particle diameter 1.8 mm). 100 g of the obtained capsules were treated with a 12% skimmed milk aqueous solution containing 0.01% protein P (manufactured by Shuji Co., Ltd.) at 40t for 15 hours, and then charged into a skim milk medium of the same composition (hereinafter referred to as " ^ &Amp; Skim milk treated with skimmed milk "500 ml of skim milk") was adjusted to a pH of 5.2 with a 5 M-Na · · zK solution, and cultured at 371: 50 jrpni with shaking for 2 days. The number of viable bacteria in the capsules after cultivation was 6 X 1 0 1 ^ capsules / g capsules (wet weight), showing a significant increase in lactic acid bacteria in the capsules. The capsules thus cultured were washed with distilled water, and then added to the fruit and vegetable juice at a ratio of 1 g / 1 Q Q ml ^. This fruit and vegetable juice not only has a lot of live lactic acid bacteria, # their nasty taste, the capsule also tastes good when passing through the throat, which is a delicious ingredient. Therefore, a large amount of lactic acid bacteria can be ingested with fruit and vegetable juice that has not been considered in the past. Example 3: Healthy Korean Cellulose with Cell Culture Capsules Addition of Murashige-Skoog Liquid Medium (pH 5.6) containing 3% sucrose and indole-3 -acetic acid (IAA) 1 ml / L. 5 g (wet weight) of cells (Panax ginseng CA Meyer), put into a 500 ml Erlenmeyer flask, add 100 ml of the same medium, and shake and culture at 25T: 1200 r pm for 2 weeks to obtain a free cell set Piece. This free cell mass was filtered through a nylon mesh with a hole diameter of 80 / m. After passing through these fine cell masses, it was filtered through a nylon mesh with a hole diameter of 20 m to collect the remaining cell mass on the mesh. . 5 g (wet weight) of this cell aggregate was suspended in 100 ml of Murashige-Skoog liquid medium having the same composition as the inner liquid of the capsule. -18- 1240751 V. Description of the invention (17) Seamless soft encapsulation is carried out in the same manner as in Example 1 except that the contents of the cell suspension are simultaneously shaken by a shaker. 10 g of the capsules with the obtained Korean pupae cells were taken out, put into a 500 ml conical flask, 100 ml of Murashige-Skoog liquid medium of the same composition was added, and cultured at 25 ° C and 50 rpm for 3 weeks. The Korean pupae in the capsule multiply and fill the capsule. After washing 2 g of the cultured capsules with sterilized distilled water, 0.05 g of the peptide used to float the capsules was added to the healthy ingredients (the same product name "Super Power Source" manufactured by Morishita Rendan Co., Ltd., without adding Koreans).蔘 extract solution) 50 ml, prepared into healthy ingredients with live Korean 蔘 cells. This healthy condiment not only contains the cells of living Korean concubines, but also has no bitterness or bad taste. It is also good when passing through the throat, making it a delicious condiment. Example 4: Soymilk extract with Natto bacteria capsules added to 100 grams of crushed soybean flour, 5 grams of sucrose, 1 grams of NaCl, and 500 ml of water, sterilized at 121 ° C for 30 minutes, and centrifuged to obtain the upper liquid (hereinafter referred to as Soy flour extraction medium). 10 ml of this soybean powder extraction medium was filled into a 50 ml Erlenmeyer flask, and colonies of Natto (B aci 1 1 ussubti 1 is IFO 13169) were inoculated with an inoculating loop, and cultured at 30 ° C with shaking at 120 rpm 1 5 hours. The entire amount of this culture solution was added to 90 ml of the soybean powder extraction medium, and it was evenly suspended to serve as a content solution for expansion. Encapsulation was performed in the same manner as in Example 1. 100 g of the obtained capsules were added to 400 ml of soybean powder extraction medium, and cultured at 30 ° C and 100 r pm for 24 hours. Natto bacteria are propagated in the capsules after culture to form slime. The number of bacteria was 3 X 10 09 capsules per gram (wet weight). After washing 3 g of this capsule with distilled water, add -19-1240751. V. Description of the invention (18) Add to 100 ml of soybean milk. This is a delicious sauce with little natto odor and can be taken together with soy milk and the slime of natto. Example 5: Culture method of encapsulated bifidobacteria B i f i d o b a c t e r u m 1 ο n g a m JCM7050 capsules prepared by the method of Phase 1 of Example 1 were filled into a 100 ml cylindrical column with a thermal insulation cover. Add 1 liter of pancreas-treated skim milk culture medium to the external culture tank, and flow into the culture medium from the bottom of the capsule filling column to the top at 10 ml / min, and then circulate back and forth in the culture tank. The culture tank was maintained at pH 5.5 with an automatic pH adjuster and 10M ammonia water. In order to maintain an anaerobic state, a mixed gas of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide gas (manufactured by Sumitomo Chemical Co., Ltd.) was blown. The mixed gas is blown in at a rate of 100 ml / min for the first 12 hours and 20 ml / min for 12-30 hours. Incubate at 37 ° C for 30 hours. The number of viable bacteria in the capsules is as many as 5 X 10 1 ° capsules per gram. When the column culture method does not cause physical damage to the capsules, so that bifidobacteria can be cultured in the capsules at a high density, reducing the chance of bacterial infection. When collecting capsules, the capsules are not easy to be damaged and can be transported together in the column mixed with food. Therefore, handling during and after culturing is extremely easy, and it is suitable for culturing bifidobacteria in capsules on an industrial scale. Example 6: Encapsulation and culture of freeze-dried bacteria. Bifidobacterium Ion gam JCM7050 was anaerobic cultured with the skimmed milk medium of Example 1. The resulting bacteria were centrifuged at 4 ° C and 8000 rpm for 20 minutes to collect. Add bacteria. 10 times the volume of distilled water was stirred to decompose and freeze-dried. Put this freeze-dried bacteria into liquid-free packaging. -20-1240751 V. Description of the invention (19) It is extremely stable in cold storage in a closed container in an anaerobic state. 0.1 g of this freeze-dried bacterial cell was suspended in 1 liter of skim milk medium as a capsule content. The viable count of this suspension was 5 X 107 cells / ml. Secondly, the formula shown in Table 2 below was used to form a seamless soft capsule (average particle size of 3.5 mm) with a content liquid sealed by an aerial dropping method. Table 2 Composition ratio of niches (wt%) Contents Bifidobacterium liquid 30 Endothelium membrane vitamin E oil 30 Outer membrane 4% sodium alginate solution 40 Coagulation liquid 3% sodium chloride aqueous solution * Composition ratio of the whole capsule One hundred grams of the capsules were placed in 1 liter of pancreas-treated skim milk medium in a liquid-free, anaerobic container, adjusted to a pH of 5.5 with a 5M NaOH solution, and shake-cultured at 371 for 2 days. The number of viable bacteria in the capsule after culture was 2 X 1 0 1 () capsules per gram (wet weight), and it was considered that there was proliferation in the capsule. Example 7: Lactic acid bacteria supplemented with live Bifidobacterium capsules Lactobacillus lactis ICM7 6 3 8 was cultured in CMG agar medium (yeast extract 0.5%, polypeptide 0.5%, NaCl 0.5%, glucose 1%, agar 2%) Cultivate to obtain colonies. This inoculated loop plant was placed in 12% skim milk, 0.4% yeast extract, 3% glucose, and a liquid adjusted to pH 6.5-21-1240751. V. Description of the invention (20) Body skim milk culture medium 20 ml After 15 hours of incubation at 37 ° C, the number of bacteria was 6X109 cells / ¾L. Centrifuge the entire amount of the culture solution at 4 ° C, 8 0 0 0 X g for 20 minutes, remove the supernatant, add 40 ml of saline to the bacteria, stir with a stirrer, and then add 8 0 0 0 X g Centrifuge for 20 minutes to collect bacterial cells. Repeat the same operation once more to obtain washed bacteria. Here, the whole amount of the bacterial cells was washed, and 40 ml of transparent apple juice was added to it, and dispersed evenly with a stirrer. 0.5 ml of apple juice in suspension of the obtained bacterial cells was added to 100 ml of transparent apple juice. The number of live bacteria in this lactic acid bacteria suspension apple juice is 1 × 107 / ml, which is almost transparent. On the other hand, it was encapsulated in the same manner as in Example 1 to obtain 2 g of a capsule containing the cultured bifidobacteria, which was added to 100 ml of the lactic acid bacteria suspension apple juice obtained above. The number of viable bacteria of the bifidobacteria contained in the capsules of the viable bifidobacteria was 3 billion capsules per gram. The apple juice containing the live lactic acid bacteria contained in this live bifidobacterium capsule is a lactic acid sauce formed according to the "milk and other provincial regulations" standard, but it is completely different from the conventional lactic acid bacteria drink, which is roughly transparent and can ingest 600 per 100 ml. 100 million live bifidobacteria, a good taste for lactic acid bacteria. Example 8: Capsules of artificial seeds of adventitious embryos and culture of adventitious embryos are based on "plant tissue cell culture" using human centipede roots (1979, edited by Harada, Kuling, Science and Technology Institute) p. The method described in 9 1-1 04 shall prevail. The adventitious embryos made from the liquid culture medium were screened with nylon meshes of 5 0 // // m and 8 5 0 # m, and adventitious embryos with a size of 500 A m -8 5 0 v m were obtained. The obtained indefinite embryo is in the shape of heart to torpedo -22-1240751 V. Description of invention (21). These adventitious embryos were suspended in an MS medium containing no hormone and deacetylated polysaccharide 0.2% as capsule contents. The encapsulation is a seamless soft capsule with a triple tube nozzle using a triglyceride hardened oil (Farmazol B-1 1 5 'made by Nippon Oil & Fats Co., Ltd.) with a melting point of 32 ° C and a 22% gelatin solution as an outer film. The manufacturing machine performs this by dripping in liquid. Adjust the pump that puts the adventitious embryos into the contents. Capsules are formed and solidified in solidified oil to form a three-layered capsule. The coagulated oil was removed from the obtained live capsules and dried slightly to obtain artificial seeds of encapsulated adventitious embryos. The particle size after drying was 7 mm. On the other hand, alginate beads were used as a comparative example (Comparative Example 1). That is, the adventitious embryo screened in Example 8 was suspended in an MS medium containing 3% (w / v) sodium alginate, and was dropped into a 50 mM calcium chloride solution to prepare calcium alginate gel beads embedded in adventitious embryo (Particle size is about 5 mm). This is the same as the reported artificial seed. Preservation and germination test The above-mentioned adventitious embryo-encapsulated artificial seed (Example 8) and the adventitious embryo-embedded bead with calcium alginate gel beads were stored in a thermostat at 20 ° C and 65% humidity for 3 months . On the third day of storage, the calcium alginate gel beads were quite dry and the beads became small, and the adventitious embryos were also dried after 1 week, and they were in a dry wrinkled state. On the other hand, 'encapsulated artificial seeds are not considered to change in appearance even after 1 week. 0 After being made into artificial seeds, directly after 1 month, 2 months, 3 months, without soil. 100 seeds each in sterilized nursery soil pots are sown on the surface -23-1240751 V. Description of the invention (22) 2 cm deep and watered. After sowing, place them in a 2 51 incubator, illuminate 16 000 Llu for 16 hours a day, and water them once every two days to allow them to grow. The results of calculating the number of germinations after 3 weeks are shown in Table 3. Table 3 Results of germination test of human artificial seeds. Storage period 1 month 2 months 3 months after manufacture Example 8 95 75 72 68 Comparative Example 1 96 0 0 0 The number of 100 germinated seeds per sown was numerically displayed. As shown in Table 3, the germination rate after production was substantially the same in Example 8 and Comparative Example 1. However, Comparative Example 1 of a conventional type of artificial seed stored for more than one month did not germinate at all. In this regard, the encapsulated artificial seeds of the present invention still have a high germination rate of 68% after 3 months of storage. From this result, the preservation stability of the encapsulated adventitious embryos of the present invention with respect to artificial seeds was shown to be superior to the artificial seeds of the conventional alginate beads-embedded adventitious embryo type. Example 9: Artificial seeds of encapsulated strawberry leaf buds_ The stem-top tissue taken from cultivated strawberries was placed in MS medium containing sucrose and 0 g / L of dogwood gum as gelling agents, in a dark place 25 After 4 days of incubation at ° C, transfer to MS medium containing 0.2 mg / L of benzyl adenine and 10 g / L of sucrose, and illuminate at 25 ° C with 2000 Lux for 16 hours -24-1240751. 5. Description of the invention (23), and cultured at 150 rprn with rotary shaking. After 30 days of cultivation, 'the obtained leaf buds were opened and screened with a nylon mesh of 1.7 mm and 1. 7 mm' to obtain leaf buds having a size of 1 -1.7 mm. The obtained leaf buds were suspended in MS liquid medium containing no hormones to prepare encapsulated contents. The encapsulation was carried out in the same manner as in Example 8 except that the pump flow rate into the leaf bud suspension was adjusted. The obtained capsules were dried to obtain artificial seeds of encapsulated strawberry leaf buds. The particle size after drying was 8 mm. On the other hand, strawberry leaf buds were embedded with alginate beads for comparison (Comparative Example 2). Calcium alginate beads (particle size: about 5 mm) in which strawberry leaf buds were encapsulated were prepared in the same manner as in Comparative Example 1 using strawberry leaf buds screened in Example 9. This is the same as the conventional artificial seeds. The preservation and germination tests of the obtained strawberry leaf bud artificial seeds (Example 9) and alginate beads embedded strawberry leaf buds (Comparative Example 2) were performed in the same manner as in Example 8. The results are shown in Table 4. Table 4 Germination test results of strawberry artificial seeds Storage period 1 month 2 months 3 months after manufacture Example 9 91 71 68 63 Comparative Example 2 94 0 0 0 0 The number of seeds germinated by each seed is shown numerically. Approximately as shown in Table 4, Example 9 and Comparative Example 2 after production. 25-251240751 V. Description of the invention (25) The adventitious embryos, apical buds, or adventitious roots of the plants shown in Table 5 were produced, and the same examples were performed. Capsules of 8 were dried and stored in a thermostat at 20 ° C and 65% humidity for 3 months, and 100 grains each were sown in the earthen pot of the same Example 8 for 1 to 3 months. The growth of Example 8 was observed for germination. The results are shown in Table 5. Table 5 Results of germination test of artificial seeds of each plant Example of plant cell tissue particle size (mm) Number of germination 11 Asparagus adventitious embryo 7 71 12 Primula adventitious embryo 8 68 13 Iron lily top bud 9 53 14 Carnation adventitious embryo 8 62 15 Adventitious root of rose 9 51 16 Adventitious root of fir tree 10 37 The number shows the germination number of 100 seeds per seed. The results in Table 5 also show the excellent preservation stability of the encapsulated artificial seeds of the present invention. Industrial Applicability The capsule of the present invention is a capsule in which living cells or tissues can proliferate inside the capsule and are insoluble in gastric acid. In particular, when lactic acid bacteria (including Bifidobacterium) are used, a large amount of live bacteria can be sent to the intestine, and a powerful intestinal rejuvenating effect is quickly exhibited. Furthermore, the encapsulated artificial -27-1240751 containing the redifferentiable plant cell tissue of the present invention V. Description of the invention (26) The seeds are stable in a dry state at normal temperature, and are sown in the soil through the capsule film to swell and It breaks down due to the action of microorganisms, which can provide artificial seeds that germinate quickly. Can be applied to plant cell tissue culture technology, selection technology or virus-free technology, agriculture, forestry, horticulture, flower leaf and other widely used applications. -28- 1240751 Application date 2001. 6. 28 Case No. 90115736 Class A〇ίΗ% ^ (The above columns are filled by the Office) (Amended on February 18, 1994) II Patent Specification = Name Chinese contains living cells or tissues Capsule COMPOSING LIVING CELLS OR TISSUES in English Capsule Name 1. Asada Masahiro (Asada Masahiro) 2. Hatano Yumi 3. Keiguchi Higuchi 4. Haruharu Haraichi Inventor Yiyi 1. Nationality of the creator 1. ~ 4. Residence and residence . ~ 4. Morishita Rendan Co., Ltd., No. 30, Tamade 1, Chuo-ku, Osaka City, Osaka Prefecture, Japan. Morishita Rendan Co., Ltd. (Morishita Rendan Co., Ltd.) Nationality Japanese Version 3. Applicant's residence, domicile ( Office) It is said that the name of the surname, No. 30, Yuzao, Chuo-ku, Osaka City, Osaka Prefecture, Japan, is 30, and the surname is Okazaki Yasuo, 1240751. Τ ϊ 五 Ⅴ. Description of the invention (4) A good implementation is to dry the outer skin. A good embodiment is that the above-mentioned redifferentiable plant cell tissue is selected from the group consisting of adventitious embryos, adventitious buds, polybuds, stem tops, growth points, protocorm, adventitious roots, and root hairs. A good embodiment is the microbiodegradable hardened oil whose endothelial membrane is solid at normal temperature. Another good embodiment is that the outer film is a microorganism-decomposable outer film selected from the group consisting of proteins, polysaccharides, and biodegradable plastids. Brief description of the drawings Fig. 1 is a schematic cross-sectional view of an artificial seed according to the present invention. In order to implement the best form of the present invention, since it is difficult to encapsulate water-based substances, only dry cells are encapsulated, but it is impossible to make the cell or tissue proliferation system enclosed in the capsule impossible, so there is no such thing. Test things. However, the present inventors succeeded in encapsulating water-based substances successfully, and although the cells or tissues were also sealed in the capsules, they did not die and proliferate, and completed the present invention. According to the present invention, it is known that only freeze-dried lactic acid bacteria, bifidobacteria, etc. can be served, and only a slow intestinal rectification effect can be obtained. Effect and so on. In addition, because the cells or tissues in the capsule can proliferate, capsules with higher cell density (such as about 101 ()-1011 capsules / g capsules) can be obtained, so in addition to the above food applications, it can also be widely used in biological Uses for reaction, medicine, medical, artificial seed, etc. 1240751 V. Description of the invention (6) Fats and oils, fatty acids, fatty acid esters of sugar, etc., but animal fats, vegetable fats and fats, biologically or chemically treated fats, etc. are preferably used. Specific examples include various tempura oils, salad oils, hardened oils with a melting point below 35 ° C, vitamin E, wheat germ oil, flax oil, cocoa butter, butter, vegetable cream, shorting oi 1 and sucrose fatty acids Esters and the like, but are not limited thereto. Capsules containing water-based substances can be obtained using these lipophilic materials. When the capsule of the present invention is used for food, the edible outer film is a natural polymer film such as gelatin, agar, pectin, alginic acid, caHageen, curdl an, starch, dogwood Alkyl gum, glucomannan, or a mixture thereof, or a polymer film obtained by adding proteins, glycoproteins, mucopolysaccharides, sugars, sugar alcohols, polyvalent alcohols, and the like, as needed, is preferable. Specific examples of the added natural polymers include acacia, amylopectin, dextran, xanthan gum, insect propolis, collagen, casein, and the like. Furthermore, if necessary, it may be selected from the group consisting of a mouth-soluble outer film, a stomach-soluble outer film, a small-intestine-soluble outer film, a large-intestine-soluble outer film, and an insoluble excreted outer film. The enteric outer film is, for example, a gelatin film or a combination of agar and pectin. Glycerin may be added to the outer film in consideration of moldability. A good combination is a three-layer structure, the table shows the middle layer (endothelium)-outer membrane, and vitamin E oil-agar, wheat germ oil-sodium alginate, sucrose ester-gelatin, glycerol medium chain fatty acid ester-antlers Vegetable gum and so on. Among them, capsules obtained from vitamin E oil-2 ~ 4% agar, wheat germ oil-2 ~ 4% sodium alginate 1240751 «
五、發明說明（12) 蛋白質如明膠、膠原蛋白等，但不限於此。此等可各自 單獨或2種以上組合使用。 多糖體較佳使用形成膠之多糖。此多糖類如瓊脂、鹿角 菜膠、阿拉伯膠、茱萸烷膠、黃色素膠、果膠、藻酸等，但 不限於此。此等可各自單獨或2種以上組合使用。 生物分解性之塑體類可爲聚乳酸、聚羥基丁酸、彼等之 混合物等但不限於此。此等可各自單獨或2種以上組合使 用。 又必要時，於上述生物分解性物質中添加糖、糖醇、多 價糖醇、支鏈澱粉、脫乙醯殼多糖等，可改變皮膜性質者 〇 特佳之皮膜可爲，如乾燥後，氧阻礙性高之明膠。 本發明之膠囊化人工種子，視需要，可形成四層以上之 構造，可選擇材質具有各種皮膜性質者。又亦可在膠囊表 面塗佈藥劑等。 無縫軟膠囊之一般製法於上述食品用途中，完整說明， 具體而言，如以液體培養基或固體培養基形成之細胞組織 (如不定胚）中取得適當大小之不定胚，懸浮於培養基作爲 膠囊之內容物（最內層），內膜層使用常溫下固體之硬化油 ，外皮膜以適當濃度之上述生物分解性物質之溶液（如22% 明膠溶液）作成，以三重管噴嘴之無縫軟膠囊製造機以液 中滴下法造粒。此時，調整裝入一個個內容液之不定胚之 泵。於凝固油中進行形成膠囊滴與固化，作成三層構造之 -14- 1240751 五、發明說明（24 ) 同。保存1個月以上之習知型式的人工種子之比較例2完 全未有發芽，但本發明之膠囊化人工種子被認爲有高發芽 率，保存3個月後仍維持60%以上之發芽率。 由此結果，關於本發明之膠囊化人工種子之保存安定性 明顯優於習知藻酸膠珠粒包埋型式之人工種子。 實施例10:膠囊化蝴蝶蘭之原球莖體（protocorm)的人工 種子 將白花黃枝之蝴蝶蘭花莖腋芽形成之原球莖體打開，以 1.5毫米及3毫米之耐綸網予以篩選，獲得1.5-3毫米大 小之原球莖體。將所得之原球莖體懸浮於含有偏下性肥料 (N/ P / K = 6.5:6: 19) 6 克 / 升、蔗糖 15 克 / 升、脫 乙醯殼多糖0.2%之pH 5.6培養基，作成三層膠囊之內容 物。 調節輸送此原球莖體懸浮液之泵，除一原軀體包於一膠 囊中外，其餘同實施例8進行膠囊化。乾燥所得膠囊，獲 得蝴蝶蘭之原球莖體的乾燥膠囊化人工種子。乾燥後之粒 徑爲9毫米。 將此人工種子於1 5°C暗處保存2個月後，於未經土壤殺 菌之苗圃土盆中各播種100粒於表面2公分深處，予以澆 水。播種後，置入25°C培養器中，以3000Lux每日照明 1 6小時，以及兩天澆水1次，使其生長。計算2個月後之 發芽數爲100粒中發芽63粒。 實施例11 - 1 6 -26-V. Description of the invention (12) Proteins such as gelatin, collagen, etc., but not limited thereto. These can be used alone or in combination of two or more kinds. As the polysaccharide, a gum-forming polysaccharide is preferably used. The polysaccharides are, but are not limited to, agar, carrageenan, acacia gum, rutacanthin, xanthan gum, pectin, alginic acid, and the like. These can be used individually or in combination of 2 or more types. The biodegradable plastics may be, but are not limited to, polylactic acid, polyhydroxybutyric acid, and mixtures thereof. These can be used alone or in combination of two or more kinds. When necessary, sugar, sugar alcohol, polyvalent sugar alcohol, amylopectin, chitosan, etc. can be added to the above biodegradable substances, which can change the properties of the film. A particularly good film can be, for example, after drying, oxygen High barrier gelatin. The encapsulated artificial seed of the present invention can be formed into a structure with more than four layers as required, and the material can be selected to have various coating properties. Alternatively, the surface of the capsule may be coated with a medicine or the like. The general manufacturing method of seamless soft capsules is fully explained in the above food use. Specifically, if an appropriate embryo of an appropriate size is obtained from a cell tissue (such as an advent embryo) formed by a liquid medium or a solid medium, it is suspended in the medium as a capsule. The content (innermost layer), the inner film layer uses solid hardened oil at room temperature, the outer film is made of a solution of the above-mentioned biodegradable substance (such as a 22% gelatin solution) with an appropriate concentration, and a seamless soft capsule with a triple tube nozzle The manufacturing machine granulates by dripping. At this time, adjust the pumps that are filled with adventitious embryos. The formation of capsule drops and solidification in coagulated oil to form a three-layer structure -14-12740751 V. Description of the invention (24) Same. Comparative Example 2 of conventional types of artificial seeds stored for more than one month did not germinate at all, but the encapsulated artificial seeds of the present invention are considered to have a high germination rate, and a germination rate of 60% or more is maintained after 3 months of storage . From this result, the preservation stability of the encapsulated artificial seeds of the present invention is significantly better than that of the conventional alginate beads-embedded artificial seeds. Example 10: Artificial seeds of encapsulated protocorm of Phalaenopsis open the protocorm body formed by axillary buds of Phalaenopsis orchid stems with white flowers and yellow branches, and screen with 1.5 mm and 3 mm nylon meshes to obtain 1.5- 3 mm protocorm body. The obtained protocorm body was suspended in a pH 5.6 medium containing 6 g / L of sub-fertilizer fertilizer (N / P / K = 6.5: 6: 19), sucrose 15 g / L, and chitosan 0.2% to prepare The contents of a three-layer capsule. The pump for transporting the suspension of the protocorm body was adjusted, except that a protosome was enclosed in a capsule, and the rest was encapsulated in the same manner as in Example 8. The obtained capsules were dried to obtain dried encapsulated artificial seeds of the protocorm body of Phalaenopsis. The grain diameter after drying was 9 mm. After the artificial seeds were stored in a dark place at 15 ° C for 2 months, 100 seeds were sown in a soil pot of a nursery without soil sterilization to a depth of 2 cm on the surface and watered. After sowing, it was placed in a 25 ° C incubator, illuminated with 3000 Lux daily for 16 hours, and watered once for two days to grow. The number of germinations after 2 months was calculated to be 63 out of 100. Example 11-1 6 -26-