201028477 六、發明說明： 【發明所屬之技術領域】 本發明涉及微生物發酵領域，具體地，涉及利用 循環式填充床反應1§發酵產生靈芝多糖的方法。 【先前技術】 • 按’靈《（Ganoderma lucidum( Leyss ex Fr.) Krast.) 屬於擔子菌綱、多孔菌科、靈芝屬高等真菌。中華傳統 醫學認為，靈芝味甘、性溫、無毒、可補心、肝、肺、 〇 脾、腎五脹之氣’具滋補強壯、固本扶正之功效。現代 醫學研究也表明，靈芝的藥理成分非常豐富，其中有效 成份包括多糖類（即：靈芝多糖）、三萜類（主要為靈 芝酸類）、靈芝多肽、16種氨基酸(其中含有七種人體必 需氨基酸）、蛋白質、甾類、甘露醇、香豆精苷、生物 鹼、有機酸（主含延胡索酸），以及微量元素Ge、P、 Fe、Ca、Mn、ZIl、等。靈芝對人體具有雙向調節作用， © 所治病種’涉及心腦企管、消化、神經、内分泌、呼吸、 運動等各個系統，尤其對腫瘤、肝臟病變、失眠以及衰 老的防治作用十分顯著。其主要有效成份為靈芝多糖、 靈芝酸具有抗癌和抗愛滋病等療效（Russell R，P aterson Μ.(2006) Ganoderma-A therapeutic fungal biofactory. PHytochemistry 67:1985-2001; Kun-Chieh Cheng, Hsuan-Cheng Huang, Jenn-Han Chen, et al. (2007) Ganoderma lucidum polysaccharides in human monocytic 5 201028477 leukemia cells: from gene expression to network construction. BMC Genomics. 8: 411. ； El-Mekkawy S,Meselhy MR, Nakamura N, Tezuka Y, Hattori M,Kakiuchi N,Shimotohno K,Kawahata T, Otake T.(1998) Anti -HIV 1 and anti-HIV-1-protease substances from «201028477 VI. Description of the Invention: [Technical Field] The present invention relates to the field of microbial fermentation, and in particular to a method for producing a ganoderma lucidum polysaccharide by a cyclic packed bed reaction. [Prior Art] • Ganoderma lucidum (Leyss ex Fr.) Krast. belongs to the genus Basidiomycetes, Polyporaceae, and Ganoderma lucidum. Chinese traditional medicine believes that Ganoderma lucidum is sweet, warm, non-toxic, can make up the heart, liver, lung, spleen, kidney and swell gas. It has the effect of nourishing strong and strengthening the body and strengthening the body. Modern medical research has also shown that Ganoderma lucidum is rich in pharmacological ingredients, including active polysaccharides (ie, Ganoderma lucidum polysaccharides), triterpenoids (mainly Ganoderma lucidum), Ganoderma lucidum peptides, and 16 amino acids (including seven essential amino acids). ), proteins, terpenoids, mannitol, coumarin, alkaloids, organic acids (mainly containing fumaric acid), and trace elements Ge, P, Fe, Ca, Mn, ZIl, and the like. Ganoderma lucidum has a two-way regulation effect on the human body. The disease treated by the disease involves various systems such as cardio-cerebral management, digestion, nerves, endocrine, respiration, and exercise, especially for the prevention and treatment of tumors, liver lesions, insomnia, and aging. Its main active ingredients are Ganoderma lucidum polysaccharides, and Ganoderma lucidum have anti-cancer and anti-AIDS effects (Russell R, P aterson Μ. (2006) Ganoderma-A therapeutic fungal biofactory. PHytochemistry 67: 1985-2001; Kun-Chieh Cheng, Hsuan- Cheng Huang, Jenn-Han Chen, et al. (2007) Ganoderma lucidum polysaccharides in human monocytic 5 201028477 leukemia cells: from gene expression to network construction. BMC Genomics. 8: 411. ; El-Mekkawy S, Meselhy MR, Nakamura N , Tezuka Y, Hattori M, Kakiuchi N, Shimotohno K, Kawahata T, Otake T. (1998) Anti -HIV 1 and anti-HIV-1-protease substances from «
Ganoderma lucidum. PHytochemistry 49:1651-1657 )。 在靈芝多糖的藥用價值被廣泛揭示的同時，靈芝產 〇 量和質量成為限制其應用的瓶頸。 目前主要利用發酵方法產生靈芝多糖和靈芝酸等 有效成份的方法，主要採用液體深層發酵。如中國大陸 專利公開號為CN1264743A(發明名稱：液體發酵法同時 生產靈芝多糖和靈芝酸的工藝）、中國大陸專利公開號 為CN1375557A (發明名稱：生物反應器中靈芝細胞兩 階段培養生產靈芝酸的方法）、中國大陸專利授權公告 ❹ 號為CN1141392C(發明名稱：中間補料發酵生產靈芝多 糖和靈芝酸的方法）、中國大陸專利公開號為 CN101235393A (發明名稱：促進靈芝酸和靈芝多糖生 物合成的發酵方法）等專利文獻公開了一系列通過液體 深層發酵產生靈芝多糖和靈芝酸的方法。但液體深層發 酵是採用游離細胞生產靈芝糖和靈芝酸，細胞密度低， 產物含量和產量低’這正是靈芝多糖和靈芝酸生產行業 的瓶頸。 6 201028477 為了解決上述問題，本發明人料了—種新龍環 式填充床反應器，細胞在發酵罐内處於固定狀熊，提高 細胞密度和耐受毒害能力，同時提高了細胞分:物的^ 量和含量，而且可多次重複發酵，直到發酵液中目的產 物達到理想含量，發賴纽循雜態，使發酵液能充 分而均勻的與新鮮钱接觸，提高整體溶氧效果，從而 提高發酵液中產生的靈芝多糖的含量。本發明將就利用 這種循環式填充床反應器進行發酵產生靈芝多糖的方 法進行描述。 【發明内容】 =發明的目的是提供—種利用新型循 反應器進㈣芝發酵產生蚊多㈣衫 靈芝多糖錄的產量和含量。 的“ ❹ 本發明是通過如下技術㈣f 環式填充床反應器包括路秘诚/ 树月所用循 道與所it、碰 發轉、魏W及通過通氣管 道接口和下管道接口，所所述發酵罐包括上管 述流通管上絲讀化=轉_安餘流通管，所 裝置和細胞固定化ΓΓ 述猶環系統包括管道 循環管道及咖刪罐外的 環管道上端與所述發 K W猶環液泵，所述循 述發酵罐之下管道接 ' 之上管道接σ連通，τ端與所 口連通’所述循環管道還包括成品 201028477 液管道及補充液管道，所述成品液管道一端與所述循環 管道連通’另一端為成品液出口，所述成品液管道與所 述循環管道的連接處設有換向閥門，所述補充液管道一 端與所述循環管道連通，另一端為補充液接口，所述補 充液管道與所述循環管道的連接處設有換向閥門，所述 細胞固定化裝置位於發酵罐内，包括一中轴，所述中轴 上少*裝至少一層以中軸為中心呈輻射狀朝向外的輻 條’本發明利用所述循環式填充床反應器發酵產生靈芝 多糖的方法包括如下步驟： (1) 菌液製備：將靈芝細胞在液體培養基中發酵 預培養至細胞幹質量達到0.6〜1.2mg/ml ; (2) 細胞固定化載體和培養液製備：取細胞固定 化載體’洗淨，按菌液體積與細胞固定化載體幹質量的 比例為6〜12ml : 1〜1.5mg，將細胞固定化載體安裝在 循環式填充床反應器細胞固定化裝置的輻條上，121^ 滅菌45〜60min ;同時滅菌配置好的培養液； (3) 細胞固定化：待循環式填充床反應器冷卻到 室溫’控制發酵罐内腔溫度為26-28°C，常壓，和通 氣狀態，將菌液從補充液接口導入循環管道，通過換向 閥門控制菌液流向，使菌液經循環管道進入流通管，& 通過霧化喷霧頭向細胞固定化載體喷射，從而使細胞固 定在細胞固定化載體上，菌液在發酵罐底部聚集，經下 8 201028477 管道接口再次進入循環管道，開始下—輪循環；通過循 環液泵控制菌液的流速，使每個霧化噴霧頭的喷射速率 為8〜12ml/min;本步驟⑶進行8〜12小時； ' (4)第一次發酵培養：控制發酵罐内腔溫度為26 . 28 C，常壓，和通氣狀態，將滅菌的培養液從補充液 接口導入循環管道’通過換向_控制培養液流向，使 培養液經循環管道進人流通管’並通過霧化喷霧頭向細 ® 胞固定化载體噴射’從而使固定在細胞固定化載體上的 細胞發酵，培養液在發酵罐底部聚集，經下管道接口再 次進入循環管道，進人下—輪循環，通過循環液泵控制 培養液的流速，使每個霧化噴霧頭的喷射速率為8〜 12ml/min ； (5) 收集成品：上述步驟（4)之發酵過程進行8 〜12小時後，通過換㈣門㈣發輕從成品液管道 流出收集，收集發酵液體積的6〇一8〇%作為成品儲存； (6) 循環發酵：補充與步驟（5)中所收集成品液 等體積賴鮮培魏’朗㈣發酵液混合，再重複上 述步驟(4)和步驟(5); (7) 重複上述步驟(4)、步驟(5)和步驟⑷，整個 發酵過程持續18—25天。 步驟⑴所用靈芝為紫芝（Ganoderma Sinense ) YTZ9kl CGMCC No.0742。 9 201028477 步騍（1)所述細胞幹質量達到0.7〜l.〇mg/ml。 利用循環式填充床反應器發酵產生靈芝多糖的方 法’所述多孔介質細胞固定化載體選自但不限於玉米 芯、人造海綿和絲瓜级。 步驟（2)所述菌液體積與細胞固定化載體幹質量 的比例為8〜1〇1»1 : 1〜l5mg。 步驟（3)所述喷霧頭的噴射速率為9〜llmi/min。 步驟（4)所述發酵液體積為發酵罐體積的4〇% — 80%。 本發明的有益效果為：（1)細胞處於固定狀態下， 進行液體深層發酵’增加了發酵的細胞密度和对受毒害 能力，同時提高了細胞分泌物的產量和含量，而且固定 化細胞可反復利用。（2)輪出的一部分發酵液不斷用於 產品開發；另一部分再與輸入反應器的細胞培養液混合 後再以恒定的速度輸入反應器，不但可以連續化生產產 品，而且提高了發酵液中產物的濃度〇 (3)同時，依賴 輸入反應器的培養液，使固定化細胞處於流化狀態，提 高了床内熱、質傳遞和氧溶量，有利於細胞的生長繁殖 和產物的生物合成。 【實施方式】 為了詳細說明循環式填充床反應器的技術内容，以 下結合實施方式並配合附圖作進一步說明。 201028477 首先結合圖示介紹本發明所利用的循環式填充床 反應器。 如圖1所示，圖1展示了本發明所涉及到的一種循 環式填充床反應器組成示意圖，包括發酵罐100、通氣 系統200以及循環系統300。 所述發酵罐100包括發酵罐頂蓋11〇、侧壁120、 底部130和内腔140。所述發酵罐頂蓋u〇外侧面上安 ❿ 裝有一調速電機111、呼吸閥112和視鏡燈113。呼吸 閥112上安裝一第一閥門U2i控制呼吸閥U2的開關。 所述發酵罐侧壁120外側安裝有一壓力檢測裝置121和 兩個觀察鏡122’側壁120内侧安裝有數根流通管123 ’ 在所述流通管123上安裝有數個霧化喷霧頭丨24，側璧 120没有通氣接口 125，連通所述通氣系統2⑼。所述 發酵罐上部還設有—上管道接σ 126，連通所述循環系 ❹ 統300 ’所述發酵罐底部130設有一下管遒接口 131， 也連通所述循環系統3〇〇。所述循環系統3〇〇包括細胞 固定化裝置Μ0和管道|置32〇。請配合參閱圖2，所 述細胞固定化裝置31〇包括中軸311和固定於中轴311 並以中轴為中心向外呈輻射狀排列的數排轉條犯，賴 條312上安裝有細胞固定化載體313，細胞固定化載禮 313為多孔介質，所述中軸311穿過所迷發酵罐谓策 削，與所述調速電機111連接，使整個細胞固定化裝 201028477 置310在發酵罐中懸空，調速電機111控制所述細胞固 定化裝置310在發酵罐内旋轉速率，所述霧化喷霧頭 124對安裝于頂蓋的細胞固定化裝置310喷射培養液或 發酵液。 所述管道裝置320包括循環管道321和循環管道 321上的循環液泵322，所述循環管道321上端與發酵 罐上管道接口 126連通，循環管道321下端與發酵罐下 ❺ 管道接口 131連通。 所述循環管道321還包括成品液管道325，所述成 品液管道325 —端與所述循環管道321連通，另一端為 成品液出口 326，所述成品液管道326與所述循環管道 的連接處設有第一換向閥門323。 所述循環管道321還包括補充液管道327，所述補 充液管道327 —端與所述循環管道321連通，另一端為 ❾ 補充液接口 328，所述補充液管道327與所述循環管道 321的連接處設有第二換向閥門324。 當然，也可把補充液接口 328直接設在發酵罐上 4，成品液出口 326直接設在發酵罐下部，或將補充液 接口 328設在發酵罐上部，成品液出口 326通過成品液 管道325與所述循環管道321連通，並在連接處設有換 向閥門323，或將成品液出口 326設在發酵罐下部，補 充液接口 328通過補充液管道327與所述循環管道321 12 201028477 連通，並在連接處設有換向閥門324。 所述循環管道321上還安裝有〆第二閥門329、一 細胞截留器330和一取樣口 331，所述第二閥門329、 細胞截留器330和取樣口 331依次位於發酵罐100底部 之下管道接口 131和第二換向閥門3^4之間。 所述通氣系統2〇〇包括螺茨風機201、空氣過濾系 統202、空氣冷卻系統203、通氣管遂204和通氣管道 ❹ 204上的第三閥門205，所述螺茨風機201、空氣過濾 系統202和空氣冷卻系統2〇3依次通過管道連通’所述 通氣管道204與所述發酵罐1〇〇侧壁之通氣接口 125連 通，並通過通氣管遒204向發酵罐内輪送空氣。 圖3展示了本發明涉及到的另一種循環式填充床 反應器的組成示意圖。所述反應器還包括與所述發酵罐 100、所述通氣系統200、所述循環系統300分別連通 © 的一滅菌罐400。所述滅菌罐4〇〇包括外壁410、夾層 420、内壁430和内腔440 ;外壁410和内壁430在滅 菌罐400頂部和底部連接一體，所述滅菌罐400頂部内 外壁連接處設有一通道411，並通過通道411與所述發 酵罐100之底部130下管道接口 131連通，使發酵罐 100内腔140和滅菌罐内腔440連通，所述通道411上 安裝一第四閥門412，第四閥門412的開關控制發酵罐 100内腔140和滅菌罐内腔440的連通和隔絕；所述通 13 201028477 道411在所述發酵罐1〇〇下管道接口 131和第四閥門 412之間設有一通道管道接口 413，所述通道管道接口 413通過循環管道321經第三換向闕門332連通補充液 管道327。滅菌罐4〇〇底部設有一出口 414，出口 414 連通所述循環管道321下端；所述滅菌罐400内壁430 上安裝一文丘裏管431，所述文丘裏管431開口朝向所 述滅菌罐400底部，並穿過所述滅菌罐内、外壁與通氣 ❹ 系統200之另一通氣管道206連通’所述滅菌罐400内 壁430上還安裝一壓力檢測裝置432，檢測滅菌罐400 内腔440的壓力。 通氣管道206上設有第五閥門207 ’控制通氣系統 200是否向滅菌罐内腔440通氣’所述通氣管道206在 空氣冷卻系統203和第三閥門205之間連通通氣管道 204。 G 失廣420通過一通氣管道421與所述發酵罐内腔 140連通’通氣管道421上安裝有第六和第七闕門423 和424，一壓力檢測裝置425分別與滅菌罐夾層420和 通氣管道421之第六和第七閥門423和424之間連通， 檢測滅菌罐夾層420的氣壓。所述滅菌罐400還安裝有Ganoderma lucidum. PHytochemistry 49:1651-1657 ). While the medicinal value of Ganoderma lucidum polysaccharides has been widely revealed, the amount and quality of Ganoderma lucidum production has become a bottleneck limiting its application. At present, a method for producing active ingredients such as Ganoderma lucidum polysaccharide and Ganoderma lucidum by a fermentation method is mainly used, and liquid deep fermentation is mainly used. For example, the Chinese Patent Publication No. CN1264743A (the name of the invention: the process of simultaneous production of Ganoderma lucidum polysaccharide and Ganoderma lucidum by liquid fermentation), and the Chinese Patent Publication No. CN1375557A (Inventive name: Two-stage culture of Ganoderma lucidum cells in a bioreactor produces ganoderic acid Method), China Mainland Patent Licensing Announcement No. CN1141392C (Invention Name: Method for Production of Ganoderma Lucidum Polysaccharide and Ganoderma Acid by Intermediate Feed Fermentation), Mainland China Patent Publication No. CN101235393A (Invention Name: Promoting Biosynthesis of Ganoderma Acid and Ganoderma Lucidum Polysaccharide The patent document discloses a series of methods for producing ganoderma lucidum polysaccharide and ganoderic acid by liquid submerged fermentation. However, deep liquid fermentation is the production of ganoderma lucidum and ganoderic acid using free cells, with low cell density and low product content and yield. This is the bottleneck in the ganoderma lucidum polysaccharide and ganoderic acid production industries. 6 201028477 In order to solve the above problems, the inventors have envisioned a new-ring-type packed bed reactor in which cells are fixed in a fermenter, which increases cell density and tolerance to toxicity, and at the same time increases cell fractions. ^ Quantity and content, and the fermentation can be repeated many times until the desired product in the fermentation broth reaches the desired content, which makes the fermentation liquid fully and evenly contact with fresh money, improving the overall dissolved oxygen effect, thereby improving The content of the ganoderma lucidum polysaccharide produced in the fermentation broth. The present invention will be described in terms of a method of producing a ganoderma lucidum polysaccharide by fermentation using such a circulating packed bed reactor. SUMMARY OF THE INVENTION The purpose of the invention is to provide a production and content of a variety of Ganoderma lucidum polysaccharides produced by a new type of reactor. The invention is based on the following technology: (4) The f-ring packed bed reactor includes the method used by the road secrets/trees and the it, the hair transfer, the Wei W, and the interface through the vent pipe and the lower pipe. The tank includes the upper tube on the flow tube, the wire reading = the _ _ _ flow tube, the device and the cell immobilization ΓΓ 犹 犹 包括 system includes the pipe circulation pipe and the upper end of the ring pipe outside the coffee can and the KW ring a liquid pump, the pipeline below the fermenting tank is connected to the upper side of the pipeline, and the τ end is connected with the mouth. The circulating pipeline further comprises a finished product 201028477 liquid pipeline and a supplementary liquid pipeline, and the finished liquid pipeline has one end and The other end of the circulation pipe is connected to the product liquid outlet, and the connection between the product liquid pipe and the circulation pipe is provided with a reversing valve, one end of the replenishing liquid pipe is connected with the circulation pipe, and the other end is a replenishing liquid. An interface, a reversing valve is disposed at a junction of the replenishing liquid pipe and the circulation pipe, and the cell fixing device is located in the fermenting tank, and includes a central axis, and the central axis is less than * at least one layer is installed The method of fermenting the ganoderma lucidum polysaccharide by the circulating packed bed reactor comprises the following steps: (1) preparation of the bacterium solution: pre-culturing the ganoderma lucidum cells in the liquid medium to the cells The dry mass reaches 0.6~1.2mg/ml; (2) Preparation of cell immobilization carrier and culture solution: Take the cell immobilization carrier 'washing, according to the ratio of the volume of the bacterial solution to the dry mass of the cell immobilized carrier is 6~12ml : 1 ~1.5mg, the cell immobilization carrier is mounted on the spokes of the circulating packed bed reactor cell immobilization device, sterilized for 45~60min; and the culture solution is sterilized at the same time; (3) Cell immobilization: to be recycled The packed bed reactor is cooled to room temperature. The temperature of the inner chamber of the fermenter is controlled to be 26-28 ° C, atmospheric pressure, and aeration state. The bacterial liquid is introduced into the circulation pipeline from the replenishing liquid interface, and the flow direction of the bacteria is controlled by the reversing valve. The bacterial liquid enters the flow tube through the circulation pipe, and is sprayed to the cell immobilization carrier through the atomizing spray head, so that the cells are fixed on the cell immobilization carrier, and the bacterial liquid gathers at the bottom of the fermenter. After the next 8 201028477 pipe interface enters the circulation pipe again, starting the lower-wheel cycle; controlling the flow rate of the bacteria liquid through the circulating liquid pump, so that the spray rate of each atomizing spray head is 8~12ml/min; this step (3) is carried out 8~ 12 hours; ' (4) First fermentation culture: control the temperature of the inner chamber of the fermenter to 26.28 C, atmospheric pressure, and aeration state, and introduce the sterilized culture solution from the replenishing liquid interface into the circulation pipe. The culture medium flows, and the culture solution is introduced into the flow tube through the circulation pipe and sprayed to the fine cell immobilization carrier through the atomizing spray head to ferment the cells immobilized on the cell immobilization carrier, and the culture solution is in the fermenter. The bottom gathers, enters the circulation pipeline again through the lower pipeline interface, enters the lower-wheel cycle, and controls the flow rate of the culture fluid through the circulating liquid pump, so that the spray rate of each atomizing spray head is 8~12ml/min; (5) Collection Finished product: After the fermentation process of the above step (4) is carried out for 8 to 12 hours, it is collected from the finished liquid pipeline by changing the (four) door (four), and the volume of the fermentation liquid is collected as the finished product storage; (6) circulation Fermentation Supplementing with the volume of the product liquid collected in the step (5), mixing the volume of the lyophilized Wei'lang (four) fermentation broth, and repeating the above steps (4) and (5); (7) repeating the above steps (4), (5) and In step (4), the entire fermentation process lasts for 18-25 days. The ganoderma used in the step (1) is Ganoderma Sinense YTZ9kl CGMCC No. 0742. 9 201028477 The cell dry mass of step (1) reaches 0.7~l.〇mg/ml. Method for producing Ganoderma lucidum polysaccharide by fermentation in a circulating packed bed reactor The porous medium cell-immobilized carrier is selected from, but not limited to, corn cob, artificial sponge and loofah grade. The ratio of the volume of the bacterial solution to the dry mass of the cell-immobilized carrier in the step (2) is 8 to 1 〇 1»1 : 1 to 15 mg. The spraying rate of the spray head in the step (3) is 9 to llmi/min. The volume of the fermentation liquid in the step (4) is from 4% to 80% of the volume of the fermenter. The beneficial effects of the invention are as follows: (1) the cell is in a fixed state, and the liquid submerged fermentation increases the density of the fermented cells and the ability to be poisoned, and at the same time increases the yield and content of the cell secretion, and the immobilized cells can be repeated. use. (2) A part of the fermentation broth that is rotated is continuously used for product development; the other part is mixed with the cell culture solution input to the reactor and then input into the reactor at a constant rate, which not only continuously produces the product, but also improves the fermentation broth. The concentration of the product 〇(3), at the same time, depends on the culture solution input into the reactor, so that the immobilized cells are in a fluidized state, which improves the heat, mass transfer and oxygen solubility in the bed, and is beneficial to cell growth and reproduction and biosynthesis of the product. . [Embodiment] In order to explain in detail the technical contents of the circulating packed bed reactor, the following description will be further described with reference to the accompanying drawings. 201028477 First, a circulating packed bed reactor utilized in the present invention will be described with reference to the drawings. As shown in Fig. 1, Fig. 1 is a schematic view showing the composition of a circulating packed bed reactor according to the present invention, including a fermentor 100, a venting system 200, and a circulation system 300. The fermentor 100 includes a fermentor can lid 11 , a side wall 120 , a bottom portion 130 , and a lumen 140 . An outer surface of the fermenter top cover u is equipped with a speed regulating motor 111, a breathing valve 112 and a mirror lamp 113. A first valve U2i is mounted on the breathing valve 112 to control the opening of the breathing valve U2. A pressure detecting device 121 and two observation mirrors 122 are disposed on the outer side of the fermentor side wall 120. A plurality of flow tubes 123 are mounted on the inner side of the side wall 120. A plurality of atomizing spray heads 24 are mounted on the flow tube 123. The 璧120 has no venting interface 125 that communicates with the venting system 2 (9). The upper portion of the fermenter is further provided with an upper pipe connection σ 126, which communicates with the circulation system 300 ′. The fermentor bottom 130 is provided with a lower pipe joint interface 131, which also communicates with the circulation system 3〇〇. The circulatory system 3 includes a cell immobilization device Μ0 and a tube|tube 32〇. Referring to FIG. 2, the cell immobilization device 31 includes a middle shaft 311 and a plurality of rows of rotating strips fixed to the central axis 311 and arranged radially outwardly around the central axis. The cell 312 is mounted with a cell fixing. The carrier 313, the cell immobilization carrier 313 is a porous medium, and the central axis 311 passes through the fermenter, and is connected to the speed control motor 111, so that the entire cell is fixed and placed in the fermenter. The speed regulating motor 111 controls the rotation rate of the cell immobilization device 310 in the fermenter, and the atomizing spray head 124 sprays the culture liquid or the fermentation liquid to the cell immobilization device 310 attached to the top cover. The piping device 320 includes a circulating conduit 321 and a circulating fluid pump 322 on the circulating conduit 321 . The upper end of the circulating conduit 321 is in communication with the upper tank interface 126 of the fermenter, and the lower end of the circulating conduit 321 is in communication with the fermentor lower conduit interface 131. The circulation pipe 321 further includes a product liquid pipe 325, the end of the product liquid pipe 325 is in communication with the circulation pipe 321, and the other end is a product liquid outlet 326, and the joint of the product liquid pipe 326 and the circulation pipe A first reversing valve 323 is provided. The circulation pipe 321 further includes a replenishing liquid pipe 327, the replenishing liquid pipe 327 is connected to the circulation pipe 321 at one end, and the other end is a replenishing liquid port 328, and the replenishing liquid pipe 327 and the recirculation pipe 321 A second reversing valve 324 is provided at the joint. Of course, the replenishing liquid interface 328 can also be directly disposed on the fermenter 4, the product liquid outlet 326 is directly disposed in the lower portion of the fermenting tank, or the replenishing liquid interface 328 is disposed in the upper portion of the fermenting tank, and the product liquid outlet 326 is passed through the product liquid pipe 325 and The circulation pipe 321 is connected, and a reversing valve 323 is provided at the connection, or the product liquid outlet 326 is disposed at a lower portion of the fermenter, and the replenishing liquid port 328 is connected to the circulation pipe 321 12 201028477 through the replenishing liquid pipe 327, and A reversing valve 324 is provided at the joint. The circulation conduit 321 is further provided with a second valve 329, a cell trap 330 and a sampling port 331, and the second valve 329, the cell trap 330 and the sampling port 331 are sequentially located under the bottom of the fermentor 100. Between the interface 131 and the second reversing valve 3^4. The venting system 2A includes a slotting fan 201, an air filtering system 202, an air cooling system 203, a venting port 204, and a third valve 205 on the venting port 205, the slotting fan 201, the air filtering system 202 The air cooling system 2〇3 is in turn communicated through the conduits. The venting duct 204 communicates with the venting port 125 of the sidewall of the fermentor 1 and delivers air to the fermentor through the vent 204. Fig. 3 is a schematic view showing the composition of another circulating packed bed reactor according to the present invention. The reactor also includes a sterilization tank 400 that communicates with the fermentor 100, the venting system 200, and the circulatory system 300, respectively. The sterilization tank 4 includes an outer wall 410, an interlayer 420, an inner wall 430 and an inner cavity 440. The outer wall 410 and the inner wall 430 are integrally connected at the top and the bottom of the sterilization tank 400, and a passage 411 is provided at the top inner and outer wall joints of the sterilization tank 400. And communicating with the bottom pipe 130 of the bottom of the fermentor 100 through the passage 411, the inner chamber 140 of the fermentor 100 and the inner chamber 440 of the sterilization tank are connected, and a fourth valve 412 is installed on the passage 411, and the fourth valve is installed. The switch of 412 controls the communication and isolation of the inner chamber 140 of the fermentor 100 and the inner chamber 440 of the sterilization tank; the passage 13 201028477 lane 411 is provided with a passage between the lower tank interface 131 and the fourth valve 412 of the fermentor 1 The pipe interface 413 communicates with the replenishing liquid pipe 327 through the circulation pipe 321 via the third reversing door 332. An outlet 414 is disposed at the bottom of the sterilization tank 4, and an outlet 414 is connected to the lower end of the circulation duct 321; a venturi 431 is mounted on the inner wall 430 of the sterilization tank 400, and the venturi 431 is opened toward the bottom of the sterilization tank 400. And passing through the inner and outer walls of the sterilization tank and the other ventilation duct 206 of the vent system 200. A pressure detecting device 432 is further mounted on the inner wall 430 of the sterilization tank 400 to detect the pressure of the inner chamber 440 of the sterilization tank 400. A fifth valve 207' is provided on the venting conduit 206 to control whether the venting system 200 vents to the sterilizing tank lumen 440. The venting conduit 206 communicates between the air cooling system 203 and the third valve 205 to the venting conduit 204. G lost 420 is in communication with the fermenter lumen 140 through a venting duct 421. The venting duct 421 is provided with sixth and seventh steps 423 and 424, a pressure detecting device 425 and a sterilizing tank interlayer 420 and a venting duct, respectively. The sixth and seventh valves 423 and 424 of 421 are in communication to detect the gas pressure of the sterilizing tank interlayer 420. The sterilization can 400 is also installed with
洛，所述夹層420内裝有水，所述電力裝置 電力裝I 433加熱所述夾廣420内水’對滅菌腔440進行滅菌’ 當通氣管道421上的第六和第七閥門423、424打開時， 14 201028477 所述發酵罐100的發酵腔140也被滅菌，當第六閥門 423關關社 ’第七閥門424打開時，僅滅菌腔440被滅菌。 所述滅菌罐400還安裝有壓力檢測裝置432、溫度檢測 裝置 4、PH檢測裝置435和溶解氧檢測裝置436，檢 測所述減菌腔楊内發酵液的參數。 以下對本發明涉及到的循環式填充床反應器的工 作方式做進—步說明。 第種循環式填充床反應器的工作過程： 〜、滅菌： 打開發酵管100之頂蓋110,向細胞固定化裝置31〇 之輻條312上安裝上預處理好的細胞固定化載體313， 關上頂蓋11G，對反鮮進行滅菌。 ―、接種：參照圖4，箭頭所示為細胞原液的流向 > 反應器冷卻到室溫’將製備好的細胞原液通過補充 液接口 328 >主入補充液管道327，旋轉第二換向闊門 324 ’使細胞原液經過第二換向閥門流向循環液栗 322 ’並利用循環液泵322提供的動力，經循環管道流 向第-換向間門323,旋轉第一換向閱門切，使細胞原 液通過管道流向發酵罐1()()内的流通管123，通過流通 g 123經霧化噴霧頭124，向所述細胞固定化載體313 喷射細胞原液，細胞在細胞固定化載體313上被固定。 15 201028477 細胞原液在發酵罐底部130聚集，並經發酵罐底部之下 管道接口 131再次進入循環管道321，依次經第二閥門 329、細胞截留器330、取樣口 331和第二換向閥門324 ’ 進入下一個循環。細胞原液往復循環流動’使大量細胞 固定到細胞固定化載體313上，當接種量達到預定值， 停止循環。 此進程中細胞原液的流向：補充液接口 328—補充 ❹ 液管道327—第二換向閥門324—循環液泵322—第一 換向閥門323—流通管123—霧化喷霧頭124—細胞固 定化载體313—發酵罐底部130下管道接口 131—第二 閥門329—細胞截留器330—取樣口 331 —第二換向閥 門324—循環液泵322。 三、第一次發酵培養：參照圖5 ’箭頭所示為培養 液的流向圖。 G 培養液滅菌後，通過補充液接口 328注入補充液管 道327 ’旋轉第二換向閥門324，使培養液經過第二換 向闕門324流向循環液泵322，並利用循環液泵322提 供的動力，經循環管道流向第一換向閥門323，旋轉第 一換向閥門323,使培養液通過管道流向發酵罐内的 流通管123，通過流通管123經霧化噴霧頭124，向所 述細胞固定化載體313喷射培養液，細胞在細胞固定化 載體313上被固定。培養液在發酵罐底部13〇聚集，並 16 201028477 經發酵罐底部之下管道接口 131再次進入循環管道 321，依次經第二閥門329、細胞截留器330、取樣口 331和第二換向閥門324，進入下一個循環。 培養液流向：補充液接口 328—補充液管道327 — 第二換向閥門324 —循環液泵322—第一換向閥門323 一流通管123—霧化喷霧頭124—細胞固定化載體 313—發酵罐底部130管道接口 131—第二閥門329—細 ❹ 胞截留器330—取樣口 331—第二換向閥門324。 四、循環發酵培養並同時收集部分成品：參照圖6， 箭頭所示為成品液的流向圖。 收集成品：當細胞發酵到一定時間，從取樣口 331 取樣，檢測發酵液所含成份達到目的值，停止第一輪發 酵。旋轉第一換向閥門323,使發酵液流向成品液出口 326，收集成品。 G 成品流向：成品一下管道接口 131—第二閥門329 —細胞截留器330—取樣口 331—第二換向閥門324 — 循環液泵322-第一換向閥門323—成品液管道325 — 成品液出口 326。 循環發酵：也請參照圖5，箭頭所示也代表混合液 的流向圖。在收集成品中仍保留部分發酵液在發酵罐 100中。向發酵罐中補充新鮮培養液。培養液滅菌後， 通過補充液接口 328注入補充液管道327，旋轉第二換 17 201028477 向閥門324 ’使培養液經過第二換向閥門324流向循環 液泵322,並利用循環液泵322提供的動力，經循環管 道流向第一換向閥門323，旋轉第一換向閥門323,使培 養液通過管道流向發酵罐100内的流通管123，通過流 通管123經霧化噴霧頭124’向所述細胞固定化載體313 喷射堉養液。培養液在發酵罐底部13〇聚集，並與聚集 在那裏的剩餘發酵液混合，混合液並經發酵罐底部之下 ❹ 管道接口 131再次進入循環管道32卜依次經第二閥門 329、細胞截留器33g、取樣σ 331和第二換向閥門似， 進入下一個循環。 /扣口液抓向·培養液—補充液接口 328—補充液管 道327—第一換向閥門324~德環液泵322-第-換向 間門323—流通管123—霧化嘴霧頭124—細胞固定化 載體313〜發酵罐底部13〇下管道接口 131處，培養液 〇 與發酵罐内剩餘發酵液混合一出口 414—第二閥門329 —細胞截留器31取樣口 33卜第二換向閥門似。 重複上述第三和第四步驟’直到檢測發酵液中目的 成份到達目的值。 五、收集成品：收集成品，停止發酵。 々值得提出的是，在整個細胞生長和發酵的過程中， 通氣系統一直通過管道向發酵罐提供細胞生長繁殖所 需要的氣體，並通過呼吸閥排出廢氣。 201028477 从下結合第一實施例對本發明利用循環式填充床 反應器發酵產生靈芝多糖的方法，做進一步詳細描逃。 (1) 菌液製備：將靈芝細胞在液體培養基中發酵 預培養至細胞幹質量達到〇 8mg/ml; (2) 細胞固定化載體和培養液製備：取細胞固定 化載體，洗淨，按菌液體積與細胞固定化載體幹質量的 比例為10ml: l.2mg，將細胞固定化載艎安裝在循環式 填充床反應器細胞固定化裝置的輻條上，12rc滅菌β 〜60min ;同時滅菌配置好的培養液； (3) 細胞固定化：待循環式填充床反應器冷卻到 室溫，控制發酵罐内腔溫度為26_28°c，常壓，和通 氣狀態，將菌液從補充液接口導入循環管道，通過換向 閥門控制菌液流向，使菌液經循環管道進入流通管，並 通過霧化喷霧頭向細胞固定化載體喷射，從而使細胞固 定在細胞固定化載體上，菌液在發酵罐底部聚集，經下 管道接口再次進入循環管道，開始下一輪循環；通過循 環液泵控制菌液的流速’使每個霧化噴霧頭的噴射速率 為10ml/min ;本步驟（3)進行1〇小時； (4) 第一次發酵培養·控制發酵罐内腔溫度為26 〜28°C ,常壓，和通氣狀態，將滅菌的培養液從補充液 接口導入循環管道’通過換向閥門控制培養液流向，使 培養液經循環管道進入流通管’並通過霧化喷霧頭向細 19 201028477 胞固定化載體喷射，從而使W在細胞Μ化載體上的 細胞發酵，培養液在發酵罐底部聚集，經下管道接口再 次進入循環管道’進人下-輪循環，通過循環液果控制 培養液的流速，使每個霧化噴霧頭的喷射速率為 l〇ml/min ； ⑸收集H上述㈣（4)之發酵過程進行 10個小時後’通過換向閥門控制發酵液從成品液管道 流出收集，㈣發酵液體積的8G%作為成品儲存； ⑷循環發酵：補充與步驟（5)中所收集成品液 等體積的新鮮培養液，與剩餘的發酵液混合，再重複上 述步驟(4)和步驟(5); (7)重複上述步驟(4)、步驟(5)和步驟（6)，整個 發酵過程持續20天。 第二種循環式填充床反應器的工作過程： ―、滅菌：參照圖7’箭頭所示為培養液的流向圖。 打開發酵管100之頂蓋110,向細胞固定化裝置31〇 之輻條312上安裝上預處理好的細胞固定化載體313， 關上頂蓋110。打開通氣管道421上的閥門423，424， 使減菌罐夾層420和發酵罐内腔140，保持氣流暢通。 將配置好的培養液通過補充液接口 328注入補充液管 道327，旋轉第三換向閥門332和第二換向閥門324， 並打開第二閥門329,使培養液經過第三換向閥門 201028477 332，流入循環管道，再經第二換向闕門324流向取樣 口 331，並通過取樣口 331和細胞截留器330到達滅菌 罐底部之出口 414 ’經出口 進入滅菌罐内腔440， 打開電力裝置450 ’對發酵罐1〇〇内腔140和滅菌罐内 腔340及其内腔内細胞載體313和培養液進行滅菌。 培養液的流向為：補充液接口 328 —補充液管道 327—第二換向閥門332—第二換向閥門324一取樣口 ❹ 331〜細胞截留器33〇_第二閥門奶—滅菌罐底部之 出口 414 一滅菌罐内腔440。 -接種.參照圖8 ’箭頭所示為細胞原液的流向 圖。 V卻到至,皿’關閉通氣管道切上的闕η似和通 上的閥門412，使發酵罐内腔14〇和滅菌罐夾層 41 _ 44Q _ ’㈣備好的細胞原液通過補充液 28注入補充液管道327，旋轉第三換向閥門332 第換向閥門324,使細胞原液經過第三換向闕門 332和第二換向閥門324流向循環液泵如，並利用循 環液粟322提供的動力，經循環管道流向第一換向闕門 323,旋轉第—換向_ 323，使細麵液通過管道流向 2酵罐刚内的流通管123，通過流通管123經霧化喷 霧頭124，向所述細胞固定化栽趙313賴細胞原液， 細胞在細胞固定化載體313上被固定。細胞原液在發酵 21 201028477 罐底部130聚集，並通過通道411流向第三換向閥門 332，進入下一個循環。細胞原液往復循環流動，使大 量細胞固定到細胞固定化載體上。當接種量達到預定 值，停止循環。 細胞原液的流向：補充液接口 328 —補充液管道 327 —第三換向閥門332—第二換向閥門324 —循環液 泵322 —第一換向閥門32—流通管123—霧化喷霧頭 φ 124—細胞固定化載體313—發酵罐底部130—通道411 —第三換向閥門332。 三、第一次發酵培養：參照圖9，箭頭所示為發酵 液的流向圖。 培養液冷卻到室溫，開始發酵。打開第二閥門329 和通道411上的閥門412，培養液經滅菌罐底部之出口 之414，依次經過第二閥門329、細胞截留器330、取 ❿ 樣口 331到第二換向閥門324,旋轉第二換向閥門324, 使培養液通過管道流向循環液泵322，並利用循環液泵 322提供的動力，經循環管道流向第一換向閥門323,旋 轉第一換向閥門323,使培養液通過管道流向發酵罐100 内的流通管123，通過流通管123經霧化喷霧頭124， 向所述細胞固定化載體313喷射培養液，培養液在發酵 罐底部130聚集，並通過通道411流入滅菌罐100内腔 140，然後再經過滅菌罐底部出口 414，進入下一個循 22 201028477 環。為了避免循環中可能存在的細胞堵塞出口 414，打 開閥門207，通過通氣管道206向文丘裏管332通氣， 使滅菌罐300内的液體處以翻滾狀態，從而避免出口 414堵塞。 發酵液流向：培養液一出口之414—第二閥門329 —細胞截留器330—取樣口 331 —第二換向閥門324 — 循環液泵322—第一換向閥門323—流通管123—霧化 ❹ 喷霧頭124—細胞固定化載體313 —發酵罐底部130聚 集一通道411一滅菌罐100内腔140 —出口 414。 四、第二次循環發酵培養：參照圖10,箭頭所示 為成品的流向圖。 收集成品：當細胞發酵到一定時間，從取樣口 331 取樣，檢測發酵液所含成份達到目的值，停止第一輪發 酵。關閉通道411閥門412，使發酵罐内腔140和滅菌 ❹ 罐内腔440隔絕《旋轉第一換向閥門323,使發酵液流向 成品出口 326，收集成品。 成品流向：成品一出口 414—第二閥門329—細胞 截留器330—取樣口 331—第二換向閥門324 —循環液 泵322—第一換向閥門323—成品液管道325—成品液 出口 326。 循環發酵：也請參照圖9，箭頭所示也代表混合液 的流向圖。保留部分發酵液，旋轉第一換向閥門323, 23 201028477 使發酵液流向發酵罐100並保存在發酵罐100中。待滅 菌罐内腔340發酵液全部流出後，旋轉第三換向閥門 332和第二換向閥門324,向補充液接口 328注入配置好 的培養液，使培養液經過三換向閥門332和第二換向閥 門324，流向細胞截留器501，進入滅菌罐内腔340進 行滅菌。待培養液冷卻到室溫，打開通道411閥門412, 剩餘發酵液和培養液混合。旋轉第二換向閥門324和 @ 第一換向閥門323，辱合液依次經過滅菌罐出口 414、 細胞截留器330、取樣口 331、第二換向閥門324、循 環液泵322和第一換向閥門323，進入發酵罐侧壁之流 通管123，通過流通管123經霧化喷霧頭124，向所述 細胞固定化載體313喷射培養液，進入下一個發酵循 環。 混合液流向：在滅菌罐中，發酵罐内剩餘發酵液與 〇 滅菌罐内培養液混合一出口之414—第二閥門329—細 胞截留器330—取樣口 331—第二換向閥門324—循環 液泵322-第一換向閥門323 —流通管123—霧化喷霧 頭124—細胞固定化載體313—發酵罐底部130聚集一 通道411-滅菌罐100内腔140—出口 414。 重複上述三，四步驟，直到檢測發酵液中目的成份 到達目的值。 收集成品：收集成品，停止發酵。 24 201028477 值得提出的是’在整個細胞生長和發酵的過程中， 通氣系統’一直通過管道向發酵罐提供細胞生長繁殖所 需要的氣體，並通過呼吸閥，排出廢氣。 以下結合第二實施例對本發明利用循環式填充床 反應器發酵產生靈芝多糖的方法，做進一步詳細描述： (1)菌液製備：將靈芝細胞在液體培養基中發酵 預培養至細胞幹質量達到1 0mg/ml; 瘳 細胞固定化載體和培養液製備：取細胞固定 化載體，洗淨’按菌液體積與細胞固定化載體幹質量的 比例為10ml: 1.5mg’將細胞固定化載體安裝在循環式 填充床反應器細胞固定化裝置的輻條上，12ir滅菌耗 〜60min ;同時滅菌配置好的培養液； (3) 細胞固定化：待循環式填充床反應器冷卻到 室溫’控制發酵罐内腔溫度為26—28。(：，常壓，和通 ❹ 氣狀態’將菌液從補充液接口導入循環管道，通過換白 閥門控制菌液流向，使菌液經循環管道進入流通管，並 通過霧化喷霧頭向細胞固定化載體喷射，從而使細胞固 定在細胞固定化載體上，菌液在發酵罐底部聚集，經下 管道接口再次進入循環管道’開始下—輪循環；通過猶 環液泵控制菌液的流迷’使每個霧化噴霧頭的喷射連率 為10ml/min ;本步雜（3 )進行10小時； (4) 第一次發酵培養·控制發酵罐内腔溫度為2 25 201028477 — 28t：，常壓，和通氣狀態，將滅菌的培養液從補充液 接口導入循環管道，通過換向閥門控制培養液流向，使 培養液經循環管道進入流通管，並通過霧化噴霧頭向細 胞固定化载體喷射，從而使固定在細胞固定化載體上的 細胞發酵’培養液在發酵罐底部聚集，經下管道接口再 次進入循環管道，進入下一輪循環，通過循環液果控制 培養液的流速’使每個霧化喷霧頭的喷射速率為 φ 10ml/min ; (5) 收集成品：上述步驟（4)之發酵過程進行 10個小時後’通過換向閥門控制發酵液從成品液管道 流出收集，收集發酵液體積的70%作為成品儲存； (6) 循環發酵：補充與步驟（5)中所收集成品液 等體積的新鮮培養液，與剩餘的發酵液混合，再重複上 述步驟(4)和步驟(5); 〇 (7)重複上述步驟(4)、步驟(5)和步驟（6)，整個 發酵過程持續20天。 上述實施例，只是本發明的較佳實施例，並非用來 限制本發明實施範圍，故凡以本發明權利要求所述的構 造、特徵及原理所做的等效變化或修飾，均應包括在本 發明權利要求範圍之内。 26 201028477 【圖式簡單說明】 圖1為本發明利用循環式填充床反應器發酵產生 靈芝多糖的方法中所用循環式填充床反應器的組成示 意圖。 圖2為圖1所示循環式填充床反應器之細胞固定 化裝置除去發酵罐頂蓋的俯視圖。 圖3為本發明利用循環式填充床反應器發酵產生 ❹ 靈芝多糖的方法中所用另一種循環式填充床反應器的 組成示意圖。 圖4為本發明利用循環式填充床反應器發酵產生 靈芝多糖的方法之第一實施例，圖中展示了細胞原液 的循環流向。 圖5展示了第一實施例中的培養液或發酵液與培 養液的混合液的循環流向。 ❹ 圖6為展示了第一實施例中的成品的流向。 圖7為本發明利用循環式填充床反應器發酵產生 靈芝多糖的方法之第二實施例，圖中展示了培養液的 流向。 圖8展示了第二實施例中細胞原液的循環流向。 圖9展示了第二實施例中發酵液或發酵液與培養 液的混合液的循環流向。 圖1〇展示了第二實施例中成品的流向。 27 201028477 【主要元件符號說明】Luo, the interlayer 420 is filled with water, and the power device power device I 433 heats the water 420 to sterilize the sterilization chamber 440. The sixth and seventh valves 423 on the ventilation duct 421, When 424 is opened, 14 201028477 the fermentation chamber 140 of the fermentor 100 is also sterilized, and when the sixth valve 423 is closed, the seventh chamber 424 is opened, and only the sterilization chamber 440 is sterilized. The sterilization tank 400 is further provided with a pressure detecting device 432, a temperature detecting device 4, a pH detecting device 435, and a dissolved oxygen detecting device 436, and detects parameters of the fermentation liquid in the sterilizing chamber. The following is a description of the operation of the circulating packed bed reactor of the present invention. Working process of the first circulating packed bed reactor: ~, sterilization: opening the top cover 110 of the fermentation tube 100, mounting the pretreated cell immobilization carrier 313 on the spokes 312 of the cell immobilization device 31, and closing the top Cover 11G to sterilize the fresh-keeping. ―, inoculation: Referring to Fig. 4, the arrow indicates the flow direction of the cell stock solution> The reactor is cooled to room temperature. The prepared cell stock solution is passed through the replenishing liquid interface 328 > the main replenishing liquid pipe 327, and the second reversing direction is rotated. The wide door 324' flows the cell stock solution through the second reversing valve to the circulating liquid pump 322' and uses the power provided by the circulating liquid pump 322 to flow through the circulation duct to the first commutating door 323 to rotate the first reversing door. The cell stock solution is flowed through a pipe to the flow tube 123 in the fermentor 1 () (), and the cell stock solution is sprayed onto the cell-immobilized carrier 313 by passing the g 123 through the atomizing spray head 124, and the cells are on the cell-immobilized carrier 313. be fixed. 15 201028477 The cell stock solution is collected at the bottom 130 of the fermenter and re-enters the circulation conduit 321 through the conduit interface 131 below the bottom of the fermentor, sequentially passing through the second valve 329, the cell trap 330, the sampling port 331 and the second reversing valve 324' Go to the next cycle. The cell stock solution is reciprocatingly circulated to fix a large number of cells to the cell-immobilized carrier 313, and when the inoculum amount reaches a predetermined value, the circulation is stopped. Flow direction of the cell stock solution in this process: replenishing fluid interface 328 - replenishing sputum pipe 327 - second reversing valve 324 - circulating fluid pump 322 - first reversing valve 323 - flow tube 123 - atomizing spray head 124 - cells The immobilization carrier 313 - the fermentor bottom 130 lower pipe interface 131 - the second valve 329 - the cell trap 330 - the sampling port 331 - the second reversing valve 324 - the circulating fluid pump 322. 3. First fermentation culture: Refer to the arrow diagram of Figure 5 for the flow diagram of the culture solution. After the G culture solution is sterilized, the replenishing liquid pipe 327 is injected through the replenishing liquid port 328 to rotate the second reversing valve 324, and the culture liquid is passed through the second reversing switch 324 to the circulating liquid pump 322, and is supplied by the circulating liquid pump 322. The power flows through the circulation pipe to the first reversing valve 323, rotates the first reversing valve 323, and the culture liquid flows through the pipe to the flow pipe 123 in the fermenter, and passes through the atomizing spray head 124 through the flow pipe 123 to the cell. The immobilization carrier 313 ejects the culture solution, and the cells are fixed on the cell immobilization carrier 313. The culture solution is collected at the bottom of the fermenter 13 and is again introduced into the circulation conduit 321 through the conduit interface 131 below the bottom of the fermentor, sequentially passing through the second valve 329, the cell trap 330, the sampling port 331 and the second reversing valve 324. , enter the next cycle. Culture fluid flow direction: replenishing liquid interface 328 - replenishing liquid pipe 327 - second reversing valve 324 - circulating liquid pump 322 - first reversing valve 323 a flow pipe 123 - atomizing spray head 124 - cell immobilization carrier 313 - Fermentor bottom 130 conduit interface 131 - second valve 329 - fine sputum trap 330 - sampling port 331 - second reversing valve 324. 4. Cycling fermentation culture and collecting part of the finished product at the same time: Referring to Figure 6, the arrow shows the flow diagram of the product liquid. Collecting the finished product: When the cells are fermented for a certain period of time, samples are taken from the sampling port 331 to detect that the components contained in the fermentation broth reach the target value, and the first round of fermentation is stopped. The first reversing valve 323 is rotated to flow the fermentation liquid to the product liquid outlet 326 to collect the finished product. G Finished product flow: finished product pipe interface 131 - second valve 329 - cell trap 330 - sampling port 331 - second reversing valve 324 - circulating fluid pump 322 - first reversing valve 323 - product liquid pipe 325 - product liquid Exit 326. Cyclic fermentation: See also Figure 5, which also shows the flow pattern of the mixture. A portion of the fermentation broth remains in the fermentor 100 in the collected product. Add fresh culture to the fermenter. After the culture solution is sterilized, the replenishing liquid pipe 327 is injected through the replenishing liquid interface 328, and the second exchange 17 201028477 is rotated to the valve 324' to pass the culture liquid to the circulating liquid pump 322 through the second reversing valve 324, and is supplied by the circulating liquid pump 322. The power flows through the circulation pipe to the first reversing valve 323, rotates the first reversing valve 323, and the culture liquid flows through the pipe to the flow pipe 123 in the fermentor 100, and passes through the flow pipe 123 through the atomizing spray head 124'. The cell-immobilized carrier 313 sprays the nutrient solution. The culture solution is collected at the bottom of the fermenter 13 and mixed with the remaining fermentation liquid accumulated there, and the mixed liquid is again introduced into the circulation pipe 32 through the bottom of the fermenter ❹ pipe interface 131, and then passed through the second valve 329 and the cell trap. 33g, sampling σ 331 and the second reversing valve, enter the next cycle. / buckle liquid grasping · culture liquid - replenishing liquid interface 328 - replenishing liquid pipe 327 - first reversing valve 324 ~ de ring liquid pump 322 - first - reversing door 323 - flow pipe 123 - atomizing nozzle fog head 124—cell immobilization carrier 313~fermenter bottom 13 under the tube interface 131, the culture solution is mixed with the remaining fermentation liquid in the fermenter, an outlet 414—second valve 329—cell interceptor 31 sampling port 33 Like a valve. The above third and fourth steps are repeated until the target component in the fermentation broth is detected to reach the target value. 5. Collect the finished product: collect the finished product and stop the fermentation. It is worth mentioning that during the whole process of cell growth and fermentation, the ventilation system always supplies the fermenter with the gas required for cell growth and reproduction through the pipeline, and exhausts the gas through the breathing valve. 201028477 The method for producing Ganoderma lucidum polysaccharide by fermentation in a circulating packed bed reactor according to the present invention will be further described in detail below in connection with the first embodiment. (1) Preparation of bacterial solution: Ganoderma lucidum cells are pre-cultured in liquid medium until the cell dry mass reaches mg8 mg/ml; (2) Preparation of cell-immobilized carrier and culture solution: Take the cell-immobilized carrier, wash, according to the bacteria The ratio of the liquid volume to the dry mass of the cell-immobilized carrier is 10 ml: 1.2 mg, and the cells are fixed and mounted on the spokes of the circulating packed bed reactor cell immobilization device, and 12 rc is sterilized for β to 60 min; (3) Cell immobilization: The circulating bed reactor is cooled to room temperature, and the temperature of the inner chamber of the fermenter is controlled to be 26_28 ° C, atmospheric pressure, and aeration state, and the bacterial liquid is introduced into the circulation from the replenishing liquid interface. The pipeline controls the flow of the bacterial liquid through the reversing valve, so that the bacterial liquid enters the circulation tube through the circulation pipeline, and is sprayed to the cell immobilization carrier through the atomizing spray head, so that the cells are fixed on the cell immobilization carrier, and the bacterial liquid is fermented. The bottom of the tank gathers, enters the circulation pipeline again through the lower pipeline interface, and starts the next cycle; the flow rate of the bacterial liquid is controlled by the circulating liquid pump to make the injection rate of each atomizing spray head 10 ml/min. This step (3) is carried out for 1 hour; (4) The first fermentation culture, the temperature of the fermentation chamber is controlled to be 26 to 28 ° C, atmospheric pressure, and aeration, and the sterilized culture solution is introduced from the replenishing liquid interface. The circulation pipe 'controls the flow direction of the culture liquid through the reversing valve, and the culture liquid enters the circulation pipe through the circulation pipe' and is sprayed to the cell immobilization carrier by the atomizing spray head, thereby causing the cells on the cell deuteration carrier. Fermentation, the culture solution accumulates at the bottom of the fermenter, enters the circulation pipeline again through the lower pipeline interface, enters the lower-wheel cycle, and controls the flow rate of the culture fluid through the circulating liquid fruit, so that the spray rate of each atomization spray head is l〇ml (5) Collecting the fermentation process of H (4) (4) above for 10 hours, 'control the fermentation liquid from the product liquid pipeline through the reversing valve to collect, (4) 8G% of the volume of the fermentation liquid as the finished product storage; (4) Cycle fermentation: supplement Mixing the fresh liquid of the same volume as the product liquid collected in the step (5) with the remaining fermentation liquid, and repeating the above steps (4) and (5); (7) repeating the above steps (4) and (5) ) and step (6), The entire fermentation process lasts for 20 days. The working process of the second circulating packed bed reactor: ―, Sterilization: The flow diagram of the culture solution is shown by the arrow in Fig. 7'. The top cover 110 of the fermentation tube 100 is opened, and the pretreated cell immobilization carrier 313 is attached to the spokes 312 of the cell immobilization device 31, and the top cover 110 is closed. The valves 423, 424 on the venting duct 421 are opened to maintain the sterilizing tank interlayer 420 and the fermenter chamber 140 to maintain a smooth air flow. The configured culture solution is injected into the replenishing liquid pipe 327 through the replenishing liquid interface 328, the third reversing valve 332 and the second reversing valve 324 are rotated, and the second valve 329 is opened to pass the culture liquid through the third reversing valve 201028477 332. , flowing into the circulation pipe, and then flowing to the sampling port 331 through the second reversing switch 324, and passing through the sampling port 331 and the cell trap 330 to the outlet 414 of the bottom of the sterilization tank, and entering the sterilization chamber 440 through the outlet, opening the electric device 450 'Sterilization tank 1 inner chamber 140 and sterilization tank inner chamber 340 and the inner cell carrier 313 and the culture solution are sterilized. The flow direction of the culture liquid is: replenishing liquid interface 328 - replenishing liquid pipe 327 - second reversing valve 332 - second reversing valve 324 - sampling port 331 - cell retentate 33 〇 _ second valve milk - bottom of sterilization tank The outlet 414 is a sterilization canister lumen 440. - Inoculation. Referring to Figure 8, the arrow shows the flow pattern of the cell stock solution. V is approaching, the dish 'closes the venting of the venting pipe and the valve 412 on the valve, so that the fermenter inner cavity 14 灭菌 and the sterilizing tank interlayer 41 _ 44Q _ ' (4) prepared cell stock solution is injected through the replenishing solution 28 The replenishing liquid pipe 327 rotates the third reversing valve 332 to the reversing valve 324 to flow the cell stock solution through the third reversing switch 332 and the second reversing valve 324 to the circulating liquid pump, for example, and provides the circulating liquid slurry 322. The power flows through the circulation pipe to the first reversing door 323, and rotates the first-reverse _323, so that the fine surface liquid flows through the pipe to the flow pipe 123 in the 2 fermenter, and passes through the flow pipe 123 through the atomizing spray head 124. The cells were fixed to the cells, and the cells were fixed on the cell-immobilized carrier 313. The cell stock is in the fermentation 21 201028477 The tank bottom 130 collects and flows through the passage 411 to the third reversing valve 332 to enter the next cycle. The cell stock reciprocates and circulates, allowing a large number of cells to be immobilized on the cell-immobilized carrier. When the inoculum reaches a predetermined value, the cycle is stopped. Flow direction of cell stock solution: replenishing liquid interface 328 - replenishing liquid pipe 327 - third reversing valve 332 - second reversing valve 324 - circulating liquid pump 322 - first reversing valve 32 - flow pipe 123 - atomizing spray head φ 124 - cell immobilization carrier 313 - fermentor bottom 130 - channel 411 - third reversing valve 332. Third, the first fermentation culture: Referring to Figure 9, the arrow shows the flow diagram of the fermentation broth. The culture solution was cooled to room temperature and fermentation was started. The second valve 329 and the valve 412 on the passage 411 are opened, and the culture liquid passes through the outlet 414 of the bottom of the sterilization tank, and sequentially passes through the second valve 329, the cell trap 330, the sample port 331, and the second reversing valve 324, and rotates. The second reversing valve 324 allows the culture liquid to flow through the pipeline to the circulating liquid pump 322, and flows to the first reversing valve 323 through the circulation pipe by the power provided by the circulating liquid pump 322, and rotates the first reversing valve 323 to make the culture liquid. The flow tube 123 flows through the pipe to the flow pipe 123 in the fermenter 100, and the culture liquid is sprayed onto the cell immobilization carrier 313 through the atomization spray head 124 through the flow pipe 123. The culture liquid is collected at the bottom 130 of the fermenter and flows through the passage 411. The inner cavity 140 of the canister 100 is sterilized, and then passed through the bottom outlet 414 of the sterilization canister to enter the next cycle of 22 201028477. In order to prevent cells that may be present in the circulation from clogging the outlet 414, the valve 207 is opened and the venturi tube 332 is vented through the venting conduit 206 to cause the liquid in the sterilizing tank 300 to be tumbling to avoid clogging of the outlet 414. Flow of fermentation broth: 414 of culture solution - second valve 329 - cell trap 330 - sampling port 331 - second reversing valve 324 - circulating fluid pump 322 - first reversing valve 323 - flow tube 123 - atomization喷雾 Spray head 124 - cell immobilization carrier 313 - Fermentor bottom 130 collects a channel 411 - a sterilizer 100 lumen 140 - an outlet 414. Fourth, the second cycle of fermentation culture: Referring to Figure 10, the arrow shows the flow diagram of the finished product. Collecting the finished product: When the cells are fermented for a certain period of time, samples are taken from the sampling port 331 to detect that the components contained in the fermentation broth reach the target value, and the first round of fermentation is stopped. The channel 411 valve 412 is closed to isolate the fermenter lumen 140 from the sterilizing canister lumen 440. "Rotating the first reversing valve 323 to allow the fermentation broth to flow to the finished product outlet 326 to collect the finished product. Finished product flow direction: finished product outlet 414 - second valve 329 - cell trap 330 - sampling port 331 - second reversing valve 324 - circulating liquid pump 322 - first reversing valve 323 - product liquid pipe 325 - product liquid outlet 326 . Cyclic fermentation: Please also refer to Figure 9, which also shows the flow pattern of the mixture. A portion of the fermentation broth is retained, and the first reversing valve 323, 23 201028477 is rotated to flow the fermentation broth to the fermentor 100 and stored in the fermentor 100. After the fermentation liquid in the inner chamber 340 of the sterilization tank is completely discharged, the third reversing valve 332 and the second reversing valve 324 are rotated, and the configured culture liquid is injected into the replenishing liquid interface 328, so that the culture liquid passes through the three reversing valve 332 and the first The two reversing valve 324 flows to the cell trap 501 and enters the sterilization chamber lumen 340 for sterilization. After the culture solution was cooled to room temperature, the passage 411 valve 412 was opened, and the remaining fermentation liquid and the culture liquid were mixed. Rotating the second reversing valve 324 and the @ first reversing valve 323, the sterilizing liquid sequentially passes through the sterilization tank outlet 414, the cell trap 330, the sampling port 331, the second reversing valve 324, the circulating liquid pump 322, and the first exchange To the valve 323, the flow tube 123 entering the side wall of the fermenter is passed through the atomizing spray head 124 through the flow tube 123, and the culture liquid is sprayed onto the cell-immobilized carrier 313 to proceed to the next fermentation cycle. Mixing liquid flow direction: In the sterilization tank, the remaining fermentation liquid in the fermenter is mixed with the culture liquid in the sterilization tank, and an outlet 414 - second valve 329 - cell trap 330 - sampling port 331 - second reversing valve 324 - circulation The liquid pump 322 - the first reversing valve 323 - the flow tube 123 - the atomizing spray head 124 - the cell immobilization carrier 313 - the fermentor bottom 130 gathers a channel 411 - the sterilization chamber 100 lumen 140 - the outlet 414. Repeat the above three or four steps until the target component in the fermentation broth reaches the target value. Collect the finished product: collect the finished product and stop the fermentation. 24 201028477 It is worth mentioning that during the whole process of cell growth and fermentation, the aeration system always supplies the fermenter with the gas required for cell growth and reproduction through the pipeline, and exhausts the gas through the breathing valve. The method for producing the ganoderma lucidum polysaccharide by the circulating packed bed reactor according to the present invention will be further described in detail below with reference to the second embodiment: (1) Preparation of the bacterial solution: the ganoderma lucidum cells are pre-cultured in a liquid medium until the cell dry mass reaches 1 0mg/ml; preparation of sputum cell immobilization carrier and culture solution: take the cell immobilization carrier, wash the 'by volume of the bacterial solution and the dry mass of the cell-immobilized carrier is 10ml: 1.5mg' to install the cell-immobilized carrier in the cycle On the spokes of the packed bed reactor cell immobilization device, 12 ir sterilization consumption is ~60 min; at the same time, the sterilized culture solution is sterilized; (3) Cell immobilization: the circulating bed reactor is cooled to room temperature to control the fermenter The chamber temperature is 26-28. (:, normal pressure, and through-gas state) The bacteria liquid is introduced into the circulation pipeline from the replenishing liquid interface, and the flow of the bacterial liquid is controlled by the whitening valve, so that the bacterial liquid enters the circulation pipe through the circulation pipe, and passes through the atomizing spray head. The cell-immobilized carrier is sprayed, so that the cells are fixed on the cell-immobilized carrier, and the bacterial liquid is collected at the bottom of the fermenter, and enters the circulation pipe again through the lower pipe interface to start the lower-wheel cycle; the flow of the bacterial liquid is controlled by the liquid circulation pump. The fan's injection rate of each atomizing spray head is 10ml/min; this step (3) is carried out for 10 hours; (4) The first fermentation culture and control the temperature of the fermenter chamber is 2 25 201028477 — 28t: , normal pressure, and aeration state, the sterilized culture solution is introduced into the circulation pipeline from the replenishing liquid interface, and the flow direction of the culture liquid is controlled by the reversing valve, so that the culture liquid enters the circulation tube through the circulation pipeline, and is fixed to the cells through the atomizing spray head. The carrier is sprayed, so that the cells immobilized on the cell-immobilized carrier are fermented. The culture solution is collected at the bottom of the fermenter, and enters the circulation pipe again through the lower pipe interface, and enters the next cycle. The circulating liquid fruit controls the flow rate of the culture solution to make the injection rate of each atomizing spray head φ 10 ml/min; (5) Collecting the finished product: the fermentation process of the above step (4) is carried out for 10 hours, The valve controls the fermentation liquid to be collected from the product liquid pipeline, and collects 70% of the volume of the fermentation liquid as a finished product storage; (6) Cyclic fermentation: supplements the same volume of fresh culture liquid with the product liquid collected in the step (5), and the remaining fermentation Mixing the liquid, repeating the above steps (4) and (5); 〇 (7) repeating the above steps (4), (5) and (6), the entire fermentation process lasts for 20 days. The above embodiment is only The preferred embodiments of the invention are not intended to limit the scope of the invention, and the equivalents and modifications of the embodiments of the invention are intended to be included in the scope of the appended claims. 26 201028477 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view showing the composition of a circulating packed bed reactor used in a method for producing a polysaccharide of Ganoderma lucidum by a circulating packed bed reactor according to the present invention. A top view of the top of the fermenter is removed from the cell immobilization apparatus of the bed reactor. Fig. 3 is a schematic view showing the composition of another circulating packed bed reactor used in the method for producing a polysaccharide of Ganoderma lucidum by a circulating packed bed reactor. 4 is a first embodiment of the method for producing a ganoderma lucidum polysaccharide by fermentation in a circulating packed bed reactor, wherein the circulation of the cell stock solution is shown. Figure 5 shows the culture solution or fermentation broth and culture in the first embodiment. Circulating flow of the liquid mixture. ❹ Figure 6 is a flow chart showing the flow of the finished product in the first embodiment. Figure 7 is a second embodiment of the method for producing a polysaccharide of Ganoderma lucidum by a circulating packed bed reactor according to the present invention. The flow direction of the culture solution is shown. Fig. 8 shows the circulation flow of the cell stock solution in the second embodiment. Fig. 9 is a view showing the circulation flow of the fermentation liquid or the mixed liquid of the fermentation liquid and the culture liquid in the second embodiment. Figure 1A shows the flow direction of the finished product in the second embodiment. 27 201028477 [Main component symbol description]
發酵罐 100 發酵罐頂蓋 110 調速電機 111 呼吸閥 112 第一閥門 1121 視鏡燈 113 發酵罐侧壁 120 壓力檢測裝置 121 觀察鏡 122 流通管 123 霧化喷霧頭 124 通氣接口 125 上管道接口 126 發酵罐底部 130 下管道接口 131 發酵罐内腔 140 通氣系統 200 螺茨風機 201 空氣過濾系統 202 空氣冷卻系統 203 通氣管道 204 第三閥門 205 通氣管道 206 第五閥門 207 循環系統 300 細胞固定化裝置310 中轴 311 輻條 312 細胞固定化載體313 管道裝置 320 循環管道 321 循環液泵 322 第一換向閥門 323 第二換向閥門 324 成品液管道 325 成品液出口 326 補充液管道 327 補充液接口 328 第二閥門 329 細胞截留器 330 取樣口 331 第三換向閥門 332 28 201028477 滅菌罐 400 外壁 410 通道 411 第四閥門 412 通道管道接口 413 出π 414 夾層 420 通氣管道 421 第六閥門 423 第七閥門 424 壓力檢測裝置 425 内壁 430 文丘裏管 431 壓力檢測裝置 432 電力裝置 433 溫度檢測裝置 434 pH檢測裝置 435 溶解氧檢測裝置436 滅菌罐内腔 440 29Fermentor 100 Fermentation tank top cover 110 Speed control motor 111 Breathing valve 112 First valve 1121 Mirror light 113 Fermentor side wall 120 Pressure detecting device 121 Viewing mirror 122 Flow tube 123 Atomizing spray head 124 Ventilation interface 125 Upper pipe connection 126 Fermentor bottom 130 Lower tubing interface 131 Fermentor lumen 140 Ventilation system 200 Stables blower 201 Air filtration system 202 Air cooling system 203 Ventilation duct 204 Third valve 205 Ventilation duct 206 Fifth valve 207 Circulatory system 300 Cell immobilization unit 310 Center shaft 311 Spokes 312 Cell immobilization carrier 313 Piping device 320 Circulating pipe 321 Circulating fluid pump 322 First reversing valve 323 Second reversing valve 324 Product liquid pipe 325 Product liquid outlet 326 Replenishing liquid pipe 327 Replenishing fluid interface 328 2 valve 329 cell trap 330 sampling port 331 third reversing valve 332 28 201028477 sterilization tank 400 outer wall 410 channel 411 fourth valve 412 channel pipe interface 413 out π 414 interlayer 420 ventilation pipe 421 sixth valve 423 seventh valve 424 pressure Detection device 425 inner wall 430 Wenqiu Tube 431 Pressure detecting device 432 Power unit 433 Temperature detecting unit 434 pH detecting unit 435 Dissolved oxygen detecting unit 436 Sterilization tank chamber 440 29