200936149 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種自蟲草屬菌之真菌產生生物活性成分物質的 方法。 【先前技術】 滅 A 尊蛰、Cordyceps militaris)為冬备1茸餍、Cordyceps) 之模式種，其含蛹體及子實體之之藥材過去一向亦被視為冬蟲夏草入 藥’主要化學成分為腺甘（adenosine)、甘露醇（mannitol)、蟲草 素（cordycepin)及麥角固醇（ergosterol) (Liu etal., 1989) ° 蛹蟲草具抗腫瘤及狀陽作用，Liu等人(Liu，J.，Yang，S. J.，Yang，X.， Chen, Z. and Li, J. L. 1997. Anticarcinogenic effect and hormonal effect of Cordyceps militaris Link. Zhongguo Zhongyao Zazhi 22(2), 111-113, 129.)發現對於移殖肺癌及s-l8〇肉瘤細胞之小鼠，踊蟲草可以延長 其存活時間，對於正常之大鼠，蛹蟲草可以增加其血液中性激素可體 松（cortisone)與睪丸_ (testosterone)量，提升性器官重量， 具有類似雄性荷爾蒙作用。蜗蟲草已被臨床應用於治療癌症及老年性 痴呆症（揚企震及郭用莊.1995.蛹蟲草治療癌症療效初探中成藥 Π(5)’22-23;沈均及陶榮芬1999,蛹蟲草治療老年性痴呆療效觀察 臨床醫學8(12)4958-1959.)，對癌誘變劑-MMS所致BalbC小鼠脾細胞 DNA損傷具拮抗作用（鬱利平.1994.蛹蟲草對癌誘變劑所致 Balb C小鼠脾細胞DNA損傷的拮抗作用實用腫瘤學雜誌 8(2)，7-8.)。竭蟲草具鎮靜及性激素樣功能，劉等人（劉潔，揚旭及陳 正.1994.蠶蜗蟲草鎮靜及性激素樣作用的研究白求恩醫科大學學報 2|0(1),14 16.)以蜗蟲草水煎劑灌胃給藥’對小鼠具有鎮靜作用，能減 少=鼠自主活動次數’㈣戊巴比妥娜眠作用，抗驚厥，提高对疲 勞成力升高正常域血敎_及睪細含量增加大體重及包 4 200936149 皮腺、精囊、前列腺的重量，而且增加去勢囊—前列腺重量，具 有雄激素樣作用’其作用性質及強度與冬蟲夏草相似。 在化學組成上，蜗蟲草和冬蟲夏草中游離氨基酸的比較分析，張 等人（張甲生，丁長江及夏愛華.1994·蠶蜗蟲草和冬蟲夏草中游離氨 基酸的比較分析.白求恩醫科大學學報2〇⑴，Μ5.)採用氨基酸自動 分析儀測定喊草和冬蟲夏草巾17麟純級總量分別為16〇8. 5 及1875. 5mg/l〇〇g，其+11種人體必需、半必需氨基酸、1〇種藥效 氨基酸的含量總和，分別佔總含量的6〇· 4%、67.1%、57. 7%與68. 3%、 © 66· 0%、65. 7V為蛹草代替蟲草藥用提供了科學數據。在㈣大陸蛹 蟲草被開發為口服液（趙金星.1996·腎靈口服液的研製·中草藥通訊 27(6),335.)，輔蟲草菌絲體與冬蟲夏草之一般化學組成相近，但含有 較高量之蟲草素（彭國平，李紅陽及袁永泰·1996.冬蟲夏草與人工蛹蟲 草的成分比較.南京中醫學院學報12(5),26.)。蟲草素是一種嘌呤類 似物，被應用於治療白血症患者（F〇ss，F. M. 2000. Combination therapy with purine nucleoside analogs. Oncol. (Huntington). 14(6 Suppl 2)，31-35·; Kodama，E. N” McCaffrey，R· P.，Yusa，K· and Mitsuya， H. .2000. Antileukemic activity and mechanism of action of cordycepin ❹ against terminal deoxynucleotidyl transferase-positive (TdT+) leukemic cells.Biochemical Pharmacol. 59(3)，273-281.)，具抗細菌（Ahn, Y. J·, Park, S. J., Lee, S. G., Shin, S. C. and Choi, D. H. 2000. Cordycepin: Selective growth inhibitor derived from liquid culture of Cordyceps militaris against Clostridium spp.. J. Agri. Food Chem. 48(7), 2744-2748.)及抗真菌（Sugar, A. M. and McCaffrey, R. P. 1998. Antifungal activity of 3-deoxyadenosine (cordycepin). Antimicrobial Agents & Chemotherapy. 42⑹，1424-1427.)活性。蟲草素在包括一般 所認為常見之冬蟲夏草藥材之種源菌及多種蟲 200936149 草屬菌中並未被偵測到（81^〇，；\18.,^1^，2.：^，1^,1^.,1^11,11.丫· and Wang, J. J. 1994. Profiles of nucleosides and nitrogen bases in Chinese medicinal fungus Cordyceps sinensis and related species. Bot.200936149 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for producing a biologically active ingredient substance from a fungus of Cordyceps. [Prior Art] A species of Cordyceps militaris, Cordyceps, which contains steroids and fruiting bodies, has always been regarded as the main chemical component of Cordyceps sinensis. (adenosine), mannitol, cordycepin, and ergosterol (Liu et al., 1989) ° Cordyceps has anti-tumor and gonococcal effects, Liu et al. (Liu, J., Yang, SJ, Yang, X., Chen, Z. and Li, JL 1997. Anticarcinogenic effect and hormonal effect of Cordyceps militaris Link. Zhongguo Zhongyao Zazhi 22(2), 111-113, 129.) found for transplanting lung cancer and In mice with s-l8 sarcoma cells, Cordyceps sinensis can prolong its survival time. For normal rats, Cordyceps sinensis can increase the amount of sex hormone cortisone and testosterone, and increase the weight of sexual organs. Has a similar male hormone effect. Cordyceps sinensis has been clinically used in the treatment of cancer and senile dementia (Yang Qizhen and Guo Yongzhuang. 1995. The efficacy of Cordyceps militaris in the treatment of cancer. Chinese patent medicine Π (5) '22-23; Shen Jun and Tao Rongfen 1999, Cordyceps militaris Therapeutic effect of senile dementia on clinical medicine 8(12)4958-1959.), antagonistic effect on DNA damage of spleen cells of BalbC mice induced by cancer mutagen-MMS (Yu Liping. 1994. Cordyceps sinensis on cancer mutagen Antagonism of DNA damage in spleen cells of Balb C mice. Journal of Oncology 8(2), 7-8.). The sedative and sex hormone-like functions of Cordyceps sinensis, Liu et al. (Liu Jie, Yang Xu and Chen Zheng. 1994. Study on sedative and sex hormone-like effects of Cordyceps militaris. Journal of Bethune Medical University 2|0(1), 14 16.) Spirulina sinensis water-supplemental administration has a sedative effect on mice, which can reduce the number of spontaneous activities of rats. (4) Pentobarbital, anti-convulsion, increase the fatigue strength and increase the normal blood _ The fine content increases the body weight and the weight of the skin gland, seminal vesicle and prostate, and increases the weight of the castrated sac-prostate, which has an androgenic-like effect. Its action properties and intensity are similar to those of Cordyceps sinensis. Comparative analysis of free amino acids in Cordyceps militaris and Cordyceps sinensis in chemical composition, Zhang et al. (Zhang Jiasheng, Ding Changjiang and Xia Aihua. 1994. Comparative analysis of free amino acids in Cordyceps militaris and Cordyceps sinensis. Journal of Bethune Medical University 2〇(1), Μ5.) Using the automatic amino acid analyzer to determine the total purity of the cypress and the Cordyceps sinensis 17 linings are 16 〇 8.5 and 1875. 5 mg / l 〇〇 g, which +11 kinds of essential, semi-essential amino acids, 1 〇 The total content of medicinal amino acids is 6〇·4%, 67.1%, 57.7% and 68.3%, © 66·0%, 65. 7V, respectively. Scientific data. In (4) the mainland Cordyceps militaris was developed as an oral liquid (Zhao Jinxing. 1996·Jinling Oral Liquid Development·Chinese Herbal Medicine 27(6), 335.), the general chemical composition of Cordyceps sinensis and Cordyceps sinensis is similar, but contains High amount of Cordyceps (Peng Guoping, Li Hongyang and Yuan Yongtai·1996. Comparison of the composition of Cordyceps sinensis and artificial Cordyceps militaris. Journal of Nanjing University of Traditional Chinese Medicine 12 (5), 26.). Cordycepin is a guanidine analogue used in the treatment of patients with leukocytosis (F〇ss, FM 2000. Combination therapy with purine nucleoside analogs. Oncol. (Huntington). 14 (6 Suppl 2), 31-35·; Kodama, E. N" McCaffrey, R. P., Yusa, K. and Mitsuya, H. .2000. Antileukemic activity and mechanism of action of cordycepin ❹ against terminal deoxynucleotidyl transferase-positive (TdT+) leukemic cells. Biochemical Pharmacol. 59(3 ), 273-281.), with antibacterial (Ahn, Y. J., Park, SJ, Lee, SG, Shin, SC and Choi, DH 2000. Cordycepin: Selective growth inhibitor derived from liquid culture of Cordyceps militaris against Clostridium Spp.. J. Agri. Food Chem. 48(7), 2744-2748.) and antifungal (Sugar, AM and McCaffrey, RP 1998. Antifungal activity of 3-deoxyadenosine (cordycepin). Antimicrobial Agents & Chemotherapy. 42(6) , 1424-1427.) Activity. Cordycepin was not detected in the provenances of the Cordyceps sinensis, which is generally considered to be common, and various insects 200936149. ^〇,;\18.,^1^,2.:^,1^,1^.,1^11,11.丫· and Wang, JJ 1994. Profiles of nucleosides and nitrogen bases in Chinese medicinal fungus Cordyceps sinensis And related species. Bot.
Bull. Acad· Sin. 35(4)，261-267.)。 與游離細胞系統比較，一般固定化細胞具有（i)提供高細胞密 度培養（2)易於連續操作（3)簡化下游分離回收程序（4)可提供 較適當之微環境條件，使細胞代謝率提高（5)結合高細胞濃度與高 流速操作可有高的單位容量生產力（6)可避免剪力造成之細胞傷害 (7)降低培養系統被汙染之可能性（8)可將細胞做最有效率之利用 (9)疋一種多酵素之生物系統（1〇)細胞具有較高之遺傳安定性 (Dervakos, G. A. and Webb, C. 1991. On the merits of viable-cell immobilization. Biotechnol. Adv. 9(4)： 559-612.; Walsh, P. K. and Malone, D. M. 1995. Cell growth patterns in immobilization matrices. Biotechnol· Adv. 13(1): 13-43.)。擔體適性之選擇與條件與菌體細胞關 係密切，尤其是材料之安全性與可塑性。幾丁聚醣由於其製備來源來 自天然物、存量豐富、低毒性及具有生物相容性之優點，許多微生物 在菌體細胞生長與特定代謝物生產時所需之營養成分不盡相同。 【發明内容】 本發明係關於一種自蛹蟲草菌產生蟲草素的方法，包含： (a) 將菌株接種於馬鈴薯洋菜培養基上，並至於恆溫培養箱中培養； (b) 以幾丁聚醣珠粒固定菌體細胞； ° ’ (c) 將幾丁聚醣珠粒移至含碳源及氮源之一階培養基中培養； (d) 將幾丁聚醣珠粒移至含碳源及氮源之二階培養基中培養； (e) 將該真菌所產生之生物活性成分物質以高效液相層析儀分析定 量。 200936149 利用本發断狀方法職生之蟲草餘量可切。 本發明所述之方法所用以固定菌體細胞之幾丁聚膽珠粒為交聯 化或未交聯化之幾丁聚醣珠粒。幾了聚醣珠粒的製備方法以及交聯化 方法詳述於以下實施例二。 在較佳的實施例中’該幾丁聚醣珠粒為交聯化之幾丁聚酷珠粒。 ° 本發賴狀綠顧關定_之方式_闕丁聚醣珠粒 包埋菌體細胞（包埋式）或以幾丁聚醋珠粒吸附菌體細胞（固定式）， 其中該幾丁聚醣珠粒固定菌體細胞之時間為6〜18小時。包埋式係將 蛹蟲草菌與不同濃度之滅賴丁聚醣溶液於無_作台中等量混 合，再滴入於絲化納溶液中，而形成含菌體之幾丁聚醣珠粒；吸附 式固定化方式則是直接將幾丁聚醣珠粒製成後，直接置於菌液中。 本發明所述之方法所用之一階培養基及二階培養基之碳源可來 自葡萄糖、乳糖、蔗糖或果糖；氮源可來自酵母菌萃出物、玉米漿、 ® 硫酸錢或氣化銨。培養基之組成成分詳述於以下實施例一中。 在較佳的實施例中，一階培養基碳源來自葡萄糖；氮源來自酵母 菌萃出物；二階培養基碳源來自蔗糖；氮源來自玉米漿。 利用本發明所述之方法培養蛹蟲草菌時，可進一步利用1N氫氧 化鈉控制培養液之pH值，使其維持恆定狀態，其中該—階培養基或 二階培養基之恆定酸鹼值可調控在pH 6. 〇〜7_ 〇之範圍内。 7 200936149 在較佳的實施例中，一階培養基酸驗值為pH 6.5，二階培養基酸 鹼值為pH 6. 0。 【實施方式】 實施例一 菌株 而7如响（BCRC 32219)購自食品工業研究所生物資源 ❹ 保存中心。 菌株保存邀錐_ 菌株保存於-18°C冷凍櫃及一般冰箱中，每個月以馬鈴薯洋菜培 養基(PDA)培養基繼代維持。 種菌製備 將菌株接種於PDA上於24。(：恆溫培養箱中培養1〇天，將長有 菌落之PDA切下約5x5 mm大小之方塊，接種至含1〇〇 mi下述種菌 Ο 基礎培養基中，於250 ml燒瓶中於28°C恆溫震盪培養箱中，以250 rpm轉速震盪培養4天，計算其生質量。 基礎培養基組成(舞*公升） 葡萄糖（glucose) 30克；硫酸銨（(NH4)2S〇4) 2.36克；磷酸二 氫钟（KH2P〇4) 0.022 克；硫酸鎮（MgS〇4.7H2〇) 〇415 克；氯化妈 (CaCl2’2H20 ) 0.026 克；氣化納（NaC1) 〇.〇15 克；硫酸銅 (CuS〇4.5H2〇) 〇·〇4 毫克；硫酸鐵（FeS04.7H20) 1.1 毫克；氣化錳 (MnC!2’4H2〇) 0.H 毫克；硫化鋅（ZnS〇4.7H2〇) 〇25 毫克；培養 200936149 液 pH 4.2 〇 一階培卷生產叛虚焙卷基組成(gU1) 葡萄糖（glucose ) 30克；酵母萃出物（yeast extracts ) 2.36克；鱗 酸二氫鉀（KH2P〇4) 0.022 克；硫酸鎂（MgS04.7H20) 0.415 克；氣 化鈣（CaCl2.2H20) 0.026克；氣化納（NaCl) 0.015克；硫酸銅 (CuS04.5H20 ) 0.04 毫克；硫酸鐵（FeS04.7H20) 1.1 毫克；氣化錳 (MnCl2,4H20) 0.14 毫克；硫化鋅（ZnS04-7H20) 0.25 毫克；培養 液 pH 4.2。 二階培卷生產暮礎培卷基組成(gL、 蔗糖（sucose) 30 克；玉米漿（com steep liquor) 2.36 克；碟酸 二氫鉀（KH2P〇4) 0.022 克；硫酸鎂（MgS04.7H20) 0.415 克；氣化 鈣（CaCl2.2H20) 0.026 克；氣化納（NaCl) 0.015 克；硫酸銅 (CuS04.5H20) 0.04 毫克；硫酸鐵（FeS04.7H20) 1.1 毫克；氣化錳 (MnCl2.4H20) 0.14 毫克；硫化鋅（ZnS04.7H20) 0.25 毫克；培養 液 pH 4.2。 實施例二 細胞固定化 農T聚醣玫粒（ChitosanBead)芻借 將80 g之80%去乙醯度幾丁聚醣溶於1 l含1〇% (w/v)聚乙稀 20000 (p〇lyethylene 20000 )之 7〇/〇 ( w/v )醋酸（聊加 add )溶液中， 以針筒將上述溶液滴入含5% (w/v)氫氧化納（s〇dium hydr〇xide) 之20% (v/v)乙醇溶液中形成珠粒，幾丁聚醣珠粒以無菌水清洗兕 9 200936149 分鐘。 A聯化幾 聚醣珠敕 f Cross-linked Chitosan Bead ; CCR)寧j 備 將幾丁聚醣珠粒置於l〇 m〇l%之乙二醇二縮水甘油醚（ethylene glycol diglyddyl ether ; EDGE)溶液中，7〇。(：熱水浴 3 小時。 玉幾丁聚醣玦教匍，盥公析Bull. Acad. Sin. 35(4), 261-267.). Compared with the free cell system, generally immobilized cells have (i) provide high cell density culture (2) easy to operate continuously (3) simplify downstream separation and recovery procedures (4) can provide more appropriate microenvironment conditions, improve cell metabolic rate (5) High unit cell concentration and high flow rate operation can have high unit capacity productivity (6) can avoid cell damage caused by shear force (7) reduce the possibility of contamination of culture system (8) can make cells most efficient The use of (9) a multi-enzyme biological system (1〇) cells has a high genetic stability (Dervakos, GA and Webb, C. 1991. On the merits of viable-cell immobilization. Biotechnol. Adv. 9 ( 4): 559-612.; Walsh, PK and Malone, DM 1995. Cell growth patterns in immobilization matrices. Biotechnol· Adv. 13(1): 13-43.). The choice and conditions of the suitability of the support are closely related to the cell-cell relationship, especially the safety and plasticity of the material. Chitosan is derived from natural sources, abundant in stock, low in toxicity and biocompatible. Many microorganisms have different nutrients required for cell growth and specific metabolite production. SUMMARY OF THE INVENTION The present invention relates to a method for producing cordycepin from Cordyceps militaris, comprising: (a) inoculating a strain on potato acacia medium and culturing in a constant temperature incubator; (b) using chitosan The beads are fixed to the cells; ° ' (c) the chitosan beads are transferred to a medium containing a carbon source and a nitrogen source; (d) the chitosan beads are moved to a carbonaceous source and Cultured in a second-order medium of nitrogen source; (e) Quantifying the bioactive component material produced by the fungus by high performance liquid chromatography. 200936149 The amount of Cordyceps can be cut by using this hairline method. The chitin beads used to immobilize the bacterial cells of the method of the present invention are cross-linked or uncrosslinked chitosan beads. The preparation method of the several polysaccharide beads and the crosslinking method are detailed in the following Example 2. In a preferred embodiment, the chitosan beads are crosslinked chitosan beads. ° The method of _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The time during which the beads are fixed to the cells is 6 to 18 hours. The embedding type is to mix the Cordyceps militaris with different concentrations of the lysine solution in a medium amount, and then drip into the silk crystallization solution to form a chitosan beads containing the cells; The immobilization method is to directly form the chitosan beads and directly place them in the bacterial liquid. The carbon source of the first-order medium and the second-order medium used in the method of the present invention may be derived from glucose, lactose, sucrose or fructose; the nitrogen source may be derived from yeast extract, corn syrup, sulphuric acid or ammonium sulphate. The composition of the medium is detailed in Example 1 below. In a preferred embodiment, the first-order medium carbon source is derived from glucose; the nitrogen source is derived from the yeast extract; the second-order medium carbon source is derived from sucrose; and the nitrogen source is derived from corn syrup. When the Cordyceps militaris is cultured by the method of the present invention, the pH of the culture solution can be further controlled by using 1N sodium hydroxide to maintain a constant state, wherein the constant pH value of the medium-order medium or the second-order medium can be regulated at pH. 6. Within the range of 〇~7_ 〇. 7 200936149 In a preferred embodiment, the acidity of the first-order medium is pH 6.5 and the pH of the second-order medium is pH 6.0. [Embodiment] The first example strain and the 7th ring (BCRC 32219) were purchased from the Food Industry Research Institute Biological Resources ❹ Preservation Center. The strains were preserved in a -18 °C freezer and a general refrigerator, and were maintained subcultured each month with potato amaranth culture medium (PDA) medium. Inoculation preparation The strain was inoculated on a PDA at 24. (: Incubate for 1 day in a constant temperature incubator, cut the colony of PDA with a size of about 5x5 mm, and inoculate it into the following medium containing 1 〇〇mi, in a 250 ml flask at 28 °C. The culture medium was shaken at 250 rpm for 4 days in a constant temperature shaking incubator to calculate the biomass. Basic medium composition (dance * liter) glucose 30 g; ammonium sulfate ((NH4) 2S 〇 4) 2.36 g; Hydrogen clock (KH2P〇4) 0.022 g; sulfuric acid town (MgS〇4.7H2〇) 〇415 g; chlorination mother (CaCl2'2H20) 0.026 g; gasification nano (NaC1) 〇.〇15 g; copper sulfate (CuS 〇4.5H2〇) 〇·〇4 mg; iron sulfate (FeS04.7H20) 1.1 mg; manganeseated manganese (MnC! 2'4H2〇) 0.H mg; zinc sulfide (ZnS〇4.7H2〇) 〇25 mg; Culture 200936149 liquid pH 4.2 〇 first-stage culture to produce remnant baking base composition (gU1) glucose 30g; yeast extracts 2.36g; potassium dihydrogen phosphate (KH2P〇4) 0.022 g ; magnesium sulfate (MgS04.7H20) 0.415 g; calcium carbonate (CaCl2.2H20) 0.026 g; gasified sodium (NaCl) 0.015 g; copper sulfate (CuS04.5H20) 0.04 mg; iron sulfate (FeS04.7H20) 1.1 mg; manganeseated manganese (MnCl2, 4H20) 0.14 mg; zinc sulfide (ZnS04-7H20) 0.25 mg; culture solution pH 4.2. Second-order culture of crucible production gL, sucose 30 g; corn steeple (com steep liquor) 2.36 g; potassium dihydrogen potassium (KH2P〇4) 0.022 g; magnesium sulfate (MgS04.7H20) 0.415 g; calcium carbonate (CaCl2.2H20) 0.026 g; gasified sodium (NaCl) 0.015 g; copper sulfate (CuS04.5H20) 0.04 mg; iron sulfate (FeS04.7H20) 1.1 mg; manganeseated manganese (MnCl2.4H20) 0.14 mg; zinc sulfide (ZnS04.7H20) 0.25 mg; culture solution pH 4.2. Example 2 Cell-immobilized Agro-T-grain rosette (ChitosanBead) by dissolving 80 g of 80% deacetylated chitosan in 1 l containing 1% (w/ v) Polyethylene 20,000 (p〇lyethylene 20000) in 7〇 / 〇 (w / v) acetic acid (Talk plus add) solution, the above solution was dropped into the 5% (w / v) sodium hydroxide by syringe Beads were formed in 20% (v/v) ethanol solution of (s〇dium hydr〇xide), and chitosan beads were washed with sterile water for 369 200936149 minutes. A-linked chitosan beads cross-linked Chitosan Bead; CCR) Ning j prepared to place chitosan beads in l〇m〇l% ethylene glycol diglycidyl ether (ethylene glycol diglyddyl ether; EDGE In the solution, 7〇. (: Hot water bath for 3 hours. Jade chitosan 玦 teaching, 盥 盥
❹ 以不同濃度幾丁聚醣溶液d-6%)滴入於3%氫氧化納溶液中’ 形成之幾丁聚醣珠粒（平均直徑25公釐）以無菌水清洗30分鐘。 將製備完成之幾丁聚醣珠粒於掃描式電子顯微鏡下觀察，每種處理量 測10個樣品’計算其平均孔隙大小。 玉聚醣珠粒倉載菌絲艚令詈對蟲箪素生產之截響 將濃度6.71 mg/mL蛹蟲草菌與不同濃度（1_6。/〇)滅菌幾丁聚醣 溶液於無菌操作台中等量混合，滴入於3%氫氧化納溶液中，以形成 含菌體之幾T魏絲（包埋法），每處理三重複，以無菌水製成每 ml含20個珠粒之懸浮液。將含菌體之幾丁聚醣珠粒懸浮液置於一階 ^養基中（IV: IV)，於耽恆溫震蘆培養箱中，以％ _轉速震 盈培養6天，分析其幾丁聚醣珠粒平均孔隙、菌絲體負載量、培養液 中蟲草素產量及pH值。 丁聚醣珠粒輿不同蘭液接種遒 將不同孔隙大小幾丁聚醣珠粒於交聯化前後接種不同濃度菌液 每種處理_重複），丨2小時後同前述之濃度與比例’置於—階培養 基中’每處理三重複’於耽十亙溫震藍培養箱中，以5〇rpm轉速震 盈培養6天’以分析其培養液中蟲草素產量。 200936149 間蟲草素生成之影释 將幾丁聚料粒分別以包埋及吸附方式製備，包埋式固定化方式 如前所述，吸附式固定化方式則是直接將幾丁聚醣珠粒製成後，直接 置於菌液中不同時間（6、12及18小時），每處理三重複。將幾丁聚 醣珠粒移出，同前述之濃度與比例，置入於一階培養基中，於2yc 恆溫震盪培養箱中，以5〇 rpm轉速震盪培養1〇天，每兩天取樣一 次’以HPLC分析其培養液中蟲草素產量。 養基中碳源與皇源種類對交聯化幾丁聚醣珠救备截茴絲、艚及蛊笪 素之影響 將一階培養基組成配方中之碳源（葡萄糖）與氮源（硫酸敍）分 別以其他碳源（蔗糖、果糖及乳糖）及其他氮源（酵母抽出物、玉米 漿及氣化銨）替代，以18小時菌絲體吸附式固定化處理之交聯化幾 丁聚醣珠粒100 ml ( 10珠粒/ml )，於250 ml燒瓶中以28°C恆溫震盪 培養箱中，以250 rpm轉速震盪培養8天，將經18小時菌絲體吸附 式固定化處理之交聯化幾丁聚醣珠粒中菌絲體負載量及培養液蟲草 素含量，上述每種處理三重複。 彡階培養對交聯化^負載菌絲體及蟲箪音之影顰 將6%濃度所製備之幾丁聚醣珠粒交聯化後，以50〇 mL之 14.35mg/mL 濃度菌液（1〇〇/〇接種量）與 500 mL (含 5 個 CCB/mL) 基礎培養基混合，於28。(：恨溫震盪培養箱中，以50 Φ111轉速震盪培 養72小時，於無菌操作台中取出吸附菌體之細胞固定化CCB，再將 其置於含三公升一階培養液之5 L酸酵槽中培養96小時，培養條件 (溫度：28°C、攪拌速率：50 rpm、通氣量：1 VVM)。將一階培養 11 200936149 液流出並同時獻二階培絲，繼觀料相同轉條件,η 時。-階培·二階培養期間，分別以i N 4氧化納饋入控。制使择養 液pH於培養期間維持穩定狀態，討三種培養液pH (阳7 〇、5 及ΡΗ6.0)對於幾丁聚醣珠粒負載菌絲體及蟲草素之影響上 處理三重複。 a ^ _幾丁聚醋珠来立中菌絲體負載量 〇 ❹ 將不含菌絲體之幾丁聚醣珠粒於培養前先行秤重（A)，將接菌 之細胞固疋化幾丁聚酷珠粒於培養6天後取出秤重（B)，「八七」即 為幾丁聚醣珠粒中菌絲體負載量。 」 盖草素〔cordvcepin)HPTX：分析定詈 HPLC操作條件：几 Chitosan beads (average diameter 25 mm) formed by dropping d-6% of different concentrations of chitosan solution into 3% sodium hydroxide solution were washed with sterile water for 30 minutes. The prepared chitosan beads were observed under a scanning electron microscope, and 10 samples were measured for each treatment to calculate the average pore size. The cutoff of the production of worms in the corn gluten-loaded mycelium 将 将 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 The mixture was added dropwise to a 3% sodium hydroxide solution to form a few T-weses containing the cells (embedding method), and three suspensions per treatment were used to make a suspension containing 20 beads per ml in sterile water. The chitosan-bearing bead suspension containing the cells was placed in the first-order nutrient medium (IV: IV), and cultured in a constant temperature shake-reed incubator for 6 days at % _ rpm for analysis for several days. The average pore size of the polysaccharide beads, the mycelium loading, the production of cordycepin in the culture solution, and the pH value. Inoculation of chitosan beads with different blue liquids, chitosan beads with different pore sizes were inoculated with different concentrations of bacterial solution before and after cross-linking, and each concentration was compared with the above-mentioned concentration and ratio. In the medium-order medium, 'three replicates per treatment' was cultured in a 震十亘 Wen Zhenlan incubator at 5 rpm for 6 days to analyze the production of cordycepin in the culture solution. 200936149 Interpretation of Cordycepin Formation The chitosan particles were prepared by embedding and adsorption, and the immobilization method was as described above. The adsorption immobilization method was directly made of chitosan beads. After the formation, it was directly placed in the bacterial liquid at different times (6, 12 and 18 hours), and each treatment was repeated three times. The chitosan beads were removed and placed in the first-order medium in the same concentration and ratio as above, and shaken in a 2 yc constant temperature incubator at a speed of 5 rpm for 1 day, sampling every two days. The production of cordycepin in the culture solution was analyzed by HPLC. The effect of carbon source and huangyuan in the nutrient base on the cross-linked chitosan beads to protect the sputum, sputum and sputum. The carbon source (glucose) and nitrogen source (sulphate) in the first-order medium were respectively Cross-linked chitosan beads immobilized by 18 hours of mycelium adsorption by other carbon sources (sucrose, fructose and lactose) and other nitrogen sources (yeast extract, corn syrup and ammonium hydride) 100 ml (10 beads/ml), shaken in a 250 ml flask at a constant temperature of 28 °C, shake culture at 250 rpm for 8 days, and cross-link the 18-hour mycelium adsorption-type immobilization treatment. The mycelium loading in the chitosan beads and the content of the culture liquid cordycepin were repeated three times for each of the above treatments. The effect of the sputum culture on the cross-linked mycelium and the worm sounds. The chitosan beads prepared at the 6% concentration were cross-linked, and the bacterial solution was concentrated at a concentration of 14.35 mg/mL at 50 〇mL. 1〇〇/〇 inoculum) mixed with 500 mL (containing 5 CCB/mL) basal medium at 28. (: In a hatched incubator, incubate at 50 Φ111 for 72 hours, remove the cell-adhered CCB from the adsorbed cells in the aseptic table, and place it in a 5 L acid fermentation tank containing three liters of first-order medium. The culture was carried out for 96 hours, and the culture conditions (temperature: 28 ° C, stirring rate: 50 rpm, aeration: 1 VVM). The first-order culture 11 200936149 liquid was discharged and the second-order rice was simultaneously dispensed, followed by the same transfer condition, η During the second-stage culture, the feed was controlled by i N 4 and the pH of the culture solution was maintained during the culture period. The pH of the three culture solutions (positive 7 〇, 5 and ΡΗ 6.0) was obtained. For the effect of chitosan beads loaded mycelium and cordycepin, the treatment was repeated three times. a ^ _ diced vinegar beads to establish the mycelium load 〇❹ will not contain mycelium chitosan The beads were weighed before the culture (A), and the cells of the inoculated cells were solidified and the beads were taken out for 6 days, and the weight was taken out. (B), "Eight seven" was the chitosan beads. Middle mycelial load. ”cordvcepin HPTX: Analytical 詈 HPLC operating conditions:
HPLC 機型：ShimadzuLC-lOA 管柱:Waters Cl8 reverse Nova-pak column 3.9*300 mm, particle size 4 um 沖提液流速：0.7ml/min 偵測波長:254 nm，UV range 0.08 積分儀:Shimadzu LC-6A 沖提液：甲醇15% (溶劑A) 磷酸二氫鉀0.02M (溶劑B) 經0.45μιη硝酸纖維薄膜（MFSCorp.,CA)過攄 12HPLC model: Shimadzu LC-lOA column: Waters Cl8 reverse Nova-pak column 3.9*300 mm, particle size 4 um Eluent flow rate: 0.7 ml/min Detection wavelength: 254 nm, UV range 0.08 Integrator: Shimadzu LC -6A Eluent: Methanol 15% (Solvent A) Potassium Dihydrogen Phosphate 0.02M (Solvent B) After 0.45μιη nitrocellulose film (MFSCorp., CA)
200936149 實施例三 —击交聯化處理幾丁聚醣珠粒平均孔隙對包埋式固定 H蛹蟲单_羞缝缓直載量、蟲箪素產量及培養液油值之聚等 幾丁聚糖濃度與未交聯化處理幾丁聚醣珠粒平均孔隙對包埋式 f定化蛹蟲草_體貞載量、蟲草素產量及培養液pH值之影響如表 一所不。結果顯示隨著幾丁聚糖濃度之增加，其所形成幾丁聚糖珠粒 中平均孔隙直徑則較小。菌絲體負載量及蟲草素產量以5%幾丁聚糖 濃度所形叙幾了雜雜（平均孔隙餘6· 21//m)最高，分別為 4. 66 mg/g及〇. 18 g/L，其次為4%幾丁聚糖濃度所形成之幾丁聚糖 珠粒（平均孔隙直# 8 21//ffl)，其麟體承負量及蟲草素產量分別 為4. 56 mg/g及〇. μ g/L〇 5%幾丁聚糖濃度所形成之幾丁聚糖珠粒 蟲草素產量0.18 g/L為未經固定化處理蛹蟲草醱酵培養對照組蟲草 素產量0. 09 g/L之兩倍《在實驗中，幾丁聚糖珠粒所形成之平均孔 隙直徑介於6.21-1G.14_，其g絲體負載量（4 47--4.66 mg/g) 及蟲草素產量（G. 15 g/L-G.18 g/L)較高，顯示以幾丁聚糖 濃度所製備之幾丁聚糖珠粒較適於菌體之固定化。不同平均孔隙直徑 幾丁聚糖珠粒菌絲趙醋酵培養液之pH值並無顯著差異。 ί二严二聚县醣^?與未交、聯化處理幾丁聚酶珠粒平均孔隙對包埋式固定化蜗 蟲草（接種量6.71—_L) _體貞锻、蟲草素產量及培養液pH值以 幾丁聚醣珠粒 幾丁聚醣濃度平均孔隙 (%) ("m) 1 14.35 2 12.85 3 10.14 菌絲體負載 量（mg/g) 蟲草素（g/L) 培養液pH值 3.25 0.11 .. 3.36 3.98 0.13 3.26 4.47 0.15 3.14 13200936149 Example 3—Break-crosslinking treatment of chitosan beads average pores on the embedded fixed H worms _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The effects of sugar concentration and average pore size of un-crosslinked chitosan beads on the embedded 定 蛹 蛹 _ _ 、 、 、 、 、 、 、 、 、 、 、 、 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 The results show that as the concentration of chitosan increases, the average pore diameter in the chitosan beads formed is smaller. The mycelial load and the cordycepin yield were the highest in the 5% chitosan concentration (the average pore residue was 6.21/m), which was 4.66 mg/g and 〇. 18 g, respectively. / /, followed by a 4% chitosan concentration of chitosan beads (average pore straight # 8 21 / / ffl), the core load and the cordycepin yield were 4. 56 mg / g and 〇. μ g / L 〇 5% chitosan concentration of chitosan beads avermectin yield 0.18 g / L for the unfixed treatment of Cordyceps militaris fermentation control control cordycepin yield 0. Double of 09 g/L "In the experiment, the average pore diameter formed by chitosan beads is between 6.211-1G.14_, its g filament loading (4 47--4.66 mg/g) and Cordyceps The yield of the prime (G. 15 g/LG.18 g/L) was higher, indicating that the chitosan beads prepared at a chitosan concentration were more suitable for immobilization of the cells. The pH values of chitosan beads mycelium vinegar fermentation broth were not significantly different. ί二严二聚县糖^? and uncrossed, combined treatment of chitosan beads average pores on the embedded immobilized Cordyceps sinensis (inoculation amount 6.71-_L) _ body forging, cordycepin production and culture pH value of chitosan beads chitosan concentration average pores (%) ("m) 1 14.35 2 12.85 3 10.14 Mycelial load (mg/g) Cordycepin (g/L) Culture medium pH Value 3.25 0.11 .. 3.36 3.98 0.13 3.26 4.47 0.15 3.14 13
❹ 200936149 ，高，其次為接種量14· 35mg/mL。不論幾丁聚糖珠粒交聯化前或後， 同接種量下絲能使蟲草素產量提高，可能為過高接種量影響培 養基中營養物f之傳送。幾丁聚糖珠粒置於1Q _之乙二醇二縮水 甘油醚（ethylene glycol diglycidyl ether ; EDGE)溶液中，以 70°C熱水浴3小時交聯化後，其平均孔_小。與幾丁聚糖珠粒交聯 化前比較，交聯化後之幾丁聚糖珠粒其蟲草素產量較高。以1〇 42 mg/mL接種量為例，平均孔隙直徑6.21am交聯化前幾丁聚糖珠粒蟲 草素產量0· 21 g/L，交聯化後幾丁聚糖珠粒蟲草素產量則提高為〇. 27 g/L，顯示交聯化後幾丁聚糖珠粒明顯有益於菌絲體蟲草素生成。 表二接種不同蛹蟲草菌液濃度對交聯化前後包埋式固定化處理之 孔隙幾丁聚醣珠粒生產蟲草素之影響 τ ~ 蟲箪素（g/L 1 幾丁聚醣珠粒接藉晉（mg/mL) 交聯化處理 平均孔隙（/zm) 6/71 10.42 14.35 200936149 後 10.14 8.21 6.21 9.87 7.91 5.83 0.15 0.16 0.18 0.21 0.21 0.24 0.17 0.18 0.21 0.24 0.25 0.27 0.14 0.13 0.16 0.23 0.23 0.25 ❹ ❹ 酷擔丄ΐ:二定於孔隙5· 83"m交聯化幾丁聚 草菌培養期間蟲草素生成疋之=式(與如時/嫩聯化幾丁聚酷輕^ 固料hir α 影響（如表三所示）。結果顯示於包埋式 «生減草最佳蚊化時間為12小時，其次為6小時；菌 菌體最養時間為第6天。於吸附式固定化處理下， 則為18小時’其次為12小時；_體生成蟲草 化處理菌^體生^ ^ 8天。與包埋式固定化處理比較，吸附式固定 卻顯著較i。最紅騎__較長，但越草素產量 式固定==草=_間12小時情況下，吸附 化處理蟲草素產Ϊ 〇 =素二。_^ (〇.79g/L) 產量（0.26 g/L)高達三倍。 疋化處理取同蟲卓素 15 200936149 表三不同細胞固定化方式與時間對交聯化幾丁聚醣珠粒蛹蟲草菌培養期間蟲 草素生成之影響 Ο❹ 200936149, high, followed by an inoculum of 14.35mg/mL. Regardless of whether the chitosan beads are cross-linked before or after, the same inoculum can increase the production of cordycepin, which may affect the transmission of nutrient f in the medium. The chitosan beads were placed in a solution of 1Q _ ethylene glycol diglycidyl ether (EDGE) and the average pore size was small after cross-linking in a hot water bath at 70 ° C for 3 hours. Compared with the chitosan beads before cross-linking, the cross-linked chitosan beads had higher cordycepin yield. Taking the inoculum size of 1〇42 mg/mL as an example, the average pore diameter of 6.21am was pre-crosslinked, and the yield of chitosan beads was 0. 21 g/L. The yield of chitosan beads after cross-linking The increase was 〇. 27 g/L, indicating that the chitosan beads after cross-linking were significantly beneficial to the mycelium production. Table 2: Effect of inoculating different concentration of Cordyceps militaris liquid on the production of cordycepin by chitosan beads in the embedded immobilization treatment before and after cross-linking τ ~ worm (g/L 1 chitosan beads) By jin (mg/mL) cross-linking treatment average pore (/zm) 6/71 10.42 14.35 200936149 after 10.14 8.21 6.21 9.87 7.91 5.83 0.15 0.16 0.18 0.21 0.21 0.24 0.17 0.18 0.21 0.24 0.25 0.27 0.14 0.13 0.16 0.23 0.23 0.25 ❹ ❹ Cool 丄ΐ: two fixed in the pores of the 5·83"m cross-linked sylvestre sphaeroides during the culture of Cordyceps sinensis = (as with the time / tender joints, chitin, condensed light ^ solid hir α effect (As shown in Table 3). The results show that the optimal mosquitoing time for the embedded grass is 12 hours, followed by 6 hours; the most common time for the bacteria is the sixth day. Under the adsorption immobilization treatment , then 18 hours' followed by 12 hours; _ body-generated cordyceps treatment bacteria ^ body birth ^ ^ 8 days. Compared with the embedded immobilization treatment, adsorption-type fixation is significantly better than i. The most red riding __ longer , but the yield of oxalicin is fixed == grass = _ between 12 hours, the adsorption treatment of cordycepin Ϊ 〇 = 素二_^ (〇.79g/L) Yield (0.26 g/L) up to three times. Sputum treatment with worms 15 200936149 Table 3 Different cell immobilization methods and time pairs of cross-linked chitosan beads The effect of cordycepin formation during the culture of Cordyceps militaris
固定化處理 培養時間（day) 蟲草素（ε/ιηη 細胞固定化時Pd (h、 6 12 18 包埋式 2 0.15 0.18 0.14 4 0.18 0.23 0.16 6 0.20 0.26 0.13 8 0.16 0.23 0.09 10 0.11 0.21 0.04 吸附式 2 - - 4 0.01 0.03 0.05 6 0.52 0.63 0.67 8 0.71 0.73 0.79 10 0.68 0.72 0.76 邊查基碳源輿fej康種類董蟲箪菌絲體自盤詈、蟲苴音漆暑盥边 通值之影響 依據前述實驗結果’選擇以吸附式（18小時）培養8小時固定 化交聯化幾丁聚醣珠粒’做為後續相關研究之基材。培養基中碳源與 氮源種類對於交聯化幾丁聚醣珠粒菌絲體承負量及蟲草素生成之影 響如表四所示。結果顯示四種培養基碳源中，以葡萄糖為碳源之交聯 化幾丁聚醣珠粒中有最高之菌絲體負載量（7. 23 mg/g)，其次分別為 乳糖、蔗糖與果糖，其菌絲體負載量分別為6.14呢/g、5.19 mg/g f 4· 21mg/g ;蟲草素產量則以蔗糖為碳源之交聯化幾丁聚醣珠粒最 间（1.17g/L) ’其次為乳糖（〇. 96 g/L)，葡萄糖與果糖則相對較低， 200936149 分別為0.82 g/L與0.81 g/L。四種培養基氮源中，以酵母萃出物為 氮源之交聯化幾丁聚醣珠粒中有最高之菌絲體負載量（7.68 mg/g)， 其次為玉米漿（7. 41 mg/g)、硫酸錢與氣化銨其菌絲體負載量則相對 較低，分別為3. 26 mg/g、及3. 47 mg/g ;蟲草素產量則以玉米漿為 氮源之交聯化幾丁聚醣珠粒最高（1.19 g/L)，其次為酵母萃出物 (0. 94 g/L)，硫酸錄與氣化銨亦相對較低，分別為0. 64 g/L與0. 58 g/L。比較培養基中礙源與氮源，交聯化幾丁聚聽珠粒菌絲體承負量 較高，培養液中pH相對較低。 表四培養基破源與氮源種類對蛹蟲草菌絲體負載量、蟲草素產量與培養液pH值 之影響 培養基碳源與氮源菌絲體負 載量 種類 (mg/g) 蟲草素（g/L) 培養液pH值 碳源 葡萄糖 7.23 0.82 3.14 秦擧培養基碳源與夢禮種類對蜗蟲草菌产丨_負載量、蟲草 素與培養液pH值 果糖 6.14 0.81 3.35 乳糖 4.21 0.96 4.94 氮源 酵母萃出物 7.68 0.94 3.07 玉米漿 7.41 1.19 3.22 硫酸錢 3.26 0.64 5.53 氣化銨 3.47 0.58 5.42 一階輿二階饋料策略結合培養液pH對於蛹蟲箪菌絲體I載量、蟲蕈 素產量之影響 17 Ο 〇 200936149 :據前述之實驗結果’培養基中碳源與氮源分別為軸糖 負載量提高;培養基中碳源與氮源分別為游: I、、未漿較有利於蟲草素產量提高。以葡萄糖與酵母萃出物做 基中之碳源與氮源有最高菌絲體負載量，以此為—階培養基 j =培養，以餘與騎雜躲養基t之概與氮源有最冑蟲草素產 :二此為二階培養基進行蟲草素生產。並於培養過程以0·1 __ :1培養基pH於不同之悝定狀態。一階與二階饋料策略結合培 =對於蛹蟲草菌絲體負載量、蟲草素產量之影響如表五所示。於L 2養及^階培树，與未㈣随料基pH之韻触較，控制 ^基二種蚊pH (6 G、6 5及7 ())之菌_負載量及蟲草素產 =局於未控恤定pH培養基之_、體承負量及鮮素產量，顯示 ^ J培養基恆疋pH有助於交聯化幾丁聚醣珠粒吸附式固定化菌絲體 其蟲草素生成。在-階麟策略時，㈣養基pH控制於& 5時蟲 素產量1. 53 g/L最高，其次分別為pH 6. 〇之L 47 g/L及pH 7. 〇 ^ L 23 g/L。在二階饋料策略時，以培養基pH控制於& 〇時蟲草素 ^ 2. 76 g/L最高，其次分別為pH 6 5之2 48 g/L及pH 7 〇之 捭8g/Le運用二階培養所得之蟲草素產量皆較一階培養時為高。於 二:基恆定ρΗ 6· 〇時，二階培養所得之蟲草素產量2. 76 g几為_階 。養所得之蟲草素產量L 47 g/L之188倍，其菌絲體負載量1〇· 9? §。與未經固定化處理蛹蟲草醱酵培養對照組蟲草素產量〇. 09g/L 較本發明最後所得最高產量2. 76 g/L為對照組產量之30. 67倍。 表五~ 之影％'階與二階鑛策略結合培她pH對_蟲草騎載量、蟲草素產量 地策略Immobilization treatment culture time (day) Cordycepin (ε/ιηη cell immobilization Pd (h, 6 12 18 embedded type 2 0.15 0.18 0.14 4 0.18 0.23 0.16 6 0.20 0.26 0.13 8 0.16 0.23 0.09 10 0.11 0.21 0.04 adsorption type) 2 - - 4 0.01 0.03 0.05 6 0.52 0.63 0.67 8 0.71 0.73 0.79 10 0.68 0.72 0.76 The basis of the cross-value of the sputum-based carbon source 舆fej Kang type The above experimental results 'selected adsorption-type (18 hours) culture for 8 hours to immobilize cross-linked chitosan beads' as a substrate for subsequent research. Carbon and nitrogen source types in the medium for cross-linking The effects of glycan bead mycelium and cordycepin production are shown in Table 4. The results showed that among the four medium carbon sources, the highest in the cross-linked chitosan beads with glucose as the carbon source. The mycelial load (7.22 mg/g) was followed by lactose, sucrose and fructose, and the mycelium loading was 6.14%/g, 5.19 mg/gf 4·21 mg/g, respectively. Cross-linked chitosan beads with sucrose as the carbon source 1.17g/L) 'Secondly lactose (〇. 96 g/L), glucose and fructose are relatively low, 200936149 are 0.82 g/L and 0.81 g/L, respectively. Among the four medium nitrogen sources, yeast extract The cross-linked chitosan beads with nitrogen source have the highest mycelium loading (7.68 mg/g), followed by corn syrup (7. 41 mg/g), sulfuric acid and ammonium sulfate. The mycelium load was relatively low, 3.26 mg/g and 3. 47 mg/g, respectively. The cordycepin yield was the highest in the cross-linked chitosan beads with corn syrup as the nitrogen source (1.19). g/L), followed by yeast extract (0. 94 g / L), sulfuric acid recorded and ammonium sulfate are also relatively low, respectively 0.64 g / L and 0. 58 g / L. Comparison medium The source of the interference and the nitrogen source, the cross-linked chitosan aureus mycelium has a higher negative load, and the pH in the culture solution is relatively lower. Table 4: The source of the medium and the source of the nitrogen source, the mycelium load of Cordyceps militaris, Cordyceps Effect of prime yield and pH value of culture medium Medium and nitrogen source Mycelium load type (mg/g) Cordycepin (g/L) Culture medium pH carbon source glucose 7.23 0.82 3.14 Qinju medium carbon source and dream Ceremony Grass 丨 丨 _ load, cordycepin and culture solution pH fructose 6.14 0.81 3.35 lactose 4.21 0.96 4.94 nitrogen source yeast extract 7.68 0.94 3.07 corn syrup 7.41 1.19 3.22 sulfuric acid money 3.26 0.64 5.53 vaporized ammonium 3.47 0.58 5.42 first-order The effect of the second-order feeding strategy combined with the pH of the culture solution on the mycelium I load and the production of the insecticidal substance of the larvae of the larvae 17 Ο 〇 200936149 : According to the above experimental results, the carbon source and the nitrogen source in the medium are respectively the loading of the shaft sugar Increase; the carbon source and nitrogen source in the medium are respectively: I, and the unslurry is more conducive to the increase of the production of cordycepin. The highest mycelium load is used as the carbon source and nitrogen source in the base of glucose and yeast extract, so as the medium-order medium j = culture, and the nitrogen source has the most Cordycepin production: This is a second-order medium for cordycepin production. And in the culture process with 0·1 __: 1 medium pH in different determination states. Combined with the first-order and second-order feeding strategies, the effects on the mycelial load and cordycepin production of Cordyceps militaris are shown in Table 5. In the case of L 2 and the cultivar of the cultivar, the pH of the two species of mosquitoes (6 G, 6 5 and 7 ()) is controlled by the rhyme of the pH (6), and the cordyceps production = In the uncontrolled shirtding pH medium, body weight and fresh product yield, it is shown that the constant pH of the J medium helps the cross-linked chitosan bead adsorption-fixed mycelium to produce cordycepin. In the -step lining strategy, (iv) the pH of the nucleus was controlled at & 5 when the nymphalin production was 1.53 g/L, followed by pH 6. 47L 47 g/L and pH 7. 〇^ L 23 g /L. In the second-order feeding strategy, the medium pH was controlled at & 虫 虫 虫 虫 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. 2. The yield of the obtained cordycepin was higher than that of the first-order culture. When the base is constant ρΗ 6· 〇, the yield of cordycepin obtained by the second-order culture is 2.76 g, which is _ order. The yield of cordycepin produced is 188 times that of L 47 g/L, and the mycelium load is 1〇·9? §. The yield of cordycepin was 〇. 09g/L compared with the non-immobilized treatment of Cordyceps militaris fermentation control. The highest yield of 2.76 g/L was 30.67 times of the control group. Table 5~ The shadow of the '' and the second-order mining strategy combined with her pH to _ Cordyceps riding capacity, cordycepin production strategy
一階 200936149 6.5 10.31 1.53 6.0 9.23 1.47 對照組 7.23 1.16 二階b 7.0 10.38 2.38 6.5 10.68 2.48 6.0 10.97 2.76 對照組 - 10.35 1.69 a :以一階培養液培養至第8天。 ❹ b :以一階培養液（pH維持於pH 6.0)培養至第4天，再以二階培養液培養至第 8天。 【圖式簡單說明】 無 【主要元件符號說明】 無 19First order 200936149 6.5 10.31 1.53 6.0 9.23 1.47 Control group 7.23 1.16 Second order b 7.0 10.38 2.38 6.5 10.68 2.48 6.0 10.97 2.76 Control group - 10.35 1.69 a : Incubate in the first order culture medium until day 8. ❹ b : The culture was carried out until the fourth day with the first-order culture solution (pH maintained at pH 6.0), and then cultured to the eighth day with the second-order culture solution. [Simple diagram description] None [Main component symbol description] None 19