培養基
（1）斜面和分離培養基(g／L)：可溶性淀粉10，(NH4)2SO4 2．5，NaC1 0．6，MgSO ·7H2O 1．2，K2HPO4·3H2O 3．0，CaCO3 3．0，瓊脂粉 20，pH 7．2～7．4，用蒸餾水配制。
種子培養基(g／L)：玉米淀粉25，花生餅粉1O，黃豆餅粉8．0，酵母粉5．0，酵母膏5．0，玉米漿4．0，CoCl2·6H2O 0．003，淀粉酶0．0025，pH7．O～7．2．用自來水配制。
發酵培養基(g／L)：玉米淀粉7O，花生餅粉1O，黃豆餅粉8．0，酵母粉5．0，酵母膏3．0，玉米漿2．2，CoCl2·6H2O 0．003，淀粉酶0．0025，pH7．O～7．2．用自來水配制。
（2）見筆記本
培養方法
從平板上挑取灰色豐滿的單菌落，轉接于斜面，28℃培養7～9 d．待長出豐富的灰色孢子后，轉接于種子培養液，220 r／min，28℃搖床培養28～30 h，接種5％于發酵培養液(250 ml三角瓶，裝50ml發酵液)，220 r／min，28℃搖床培養7～9 d，放瓶測定各指標。
3．誘導子制備
（1）藻多糖提取工藝
取海藻置于托盤放入恒溫培養箱低溫烘干，用電磨機打碎，使之成粉末狀。用天平稱取海藻粉50g放入500ml錐形瓶中加蒸餾水250ml放置于95℃水浴鍋中，并隔10min晃一次，4h后取出，冷卻到室溫。離心取上清液，在離心管中加無水乙醇，得到沉淀再次離心，取沉淀(多糖)，用蒸餾水沖洗數遍，于37度烘箱中烘干所得沉淀為粗多糖。
（2）1 多糖提取工藝紫菜一粉碎一紫菜干粉(40目)(加水(40-50倍)一微波加熱浸提-粗濾-離心(4000r/min,10min )-濃縮-45℃真空千燥一紫萊多糖（微波功率200w，加熱時間8min，水與紫菜液固重量比為50:1）
2 多糖含量的分析方法還原糖的測定:采用3.5一二硝基水楊酸比色法[51.總糖含量的測定:采用苯酚一硫酸比色法，以葡萄糖為標準品
3 紫菜多糖的計算多糖含量(%)=0.9x(總糖百分含量一還原糖百分含量)X100%，其中0.9是多糖的換算系數;紫菜多糖提取率(%)=紫菜多糖的含量/紫菜原料的質量x 100%.
（3）將用來制備誘導子的6種微生物分別接種到相應的液體培養基中進行搖床振蕩培養．其中，粗糙脈孢菌、紫紅曲霉在馬鈴薯葡萄糖液體培養基中25℃培養7 d；擲孢酵母、深紅酵母在麥芽汁液體培養基中25℃培養3 d；N89在營養肉湯培養基中28℃ 培養5 d；A05在蛋白胨查氏液體培養基中28℃ 培養10 d、分別收集培養好的菌體細胞，按Ayers 方法來制備誘導子．取2 g(濕重)的菌體細胞，用蒸餾水、100 mmol／L和500 mmol／L(pH 7．2)的PBS緩沖液分別洗滌2次，超聲破碎細胞，離心收集沉淀，沉淀再用500 mmol／L(pH 7．2)的PBS緩沖液和蒸餾水分別離心(4 000 r／min，5 min)洗滌8次，并重懸于蒸餾水中，0 1 MPa滅菌20 min后，置一20℃ 下儲存備。
4. 效價測定
（1）層析法分離純化，HPLC 法測定效價(任超，馬珦玻.阿維菌素B1a組分高產菌株誘變育種。生物技術通報，2005，4)
取發酵液5 ml，3 000 r／min，離心10 min，棄上清。加入丙酮2 ml，在旋渦式混合器上振蕩1 min，靜置10 min，重復3次。加入乙酸乙酯3 ml，振蕩1 min，3 000 r／min，離心10 min，上清液備用。
吸取4Oμl上清液點樣于GF254硅膠板上，然后層析。展層劑為：乙酸乙酯：三氯甲烷：二氯甲烷：無水:甲醇=9：9：2：1。層析后在紫外燈下觀察，將斑點處硅膠刮入離心管中，加入2 ml無水甲醇，在旋渦式混合器上振蕩1 min，靜置10 min后3 000 r／min，離心10 min。
HPLC分析采用C18反向柱，流動相為無水甲醇：水=85：15，流速1 ml／min，檢測波長244．6 nm。準確吸取5．0μl樣品濾液進樣，根據各組分的峰面積，對照標準曲線計算其含量，各組分之和即為總發酵單位。
（2）紫外分光光度法(對于斜面培養物)( 于秀蓮，何建勇，白秀峰. 阿維菌素產生菌的誘變育種. 沈陽藥科大學學報, 第21卷第3期)
將適量的斜面培養物鏟出置于離心管中，加入丙酮2 mL，浸泡后再加入乙酸乙酯2 mI ，在旋渦式混合器上震蕩2 min，3 000 r／min離心10 min，所得萃取液在754紫外分光光度計波長244 nm下，以甲醇為對照，測定樣品的光密度，根據預先制備的光密度一阿維菌素標準曲線，計算阿維菌素的效價。
（3）HPLC 法（同上）
色譜柱為kromasil Cl8(200 mm×4．6 ram)，流動相為甲醇-水(80：20)，流速1 mL/min。
取發酵液7 mL于離心管中。3 000 r/min離心10 min，棄去上清液。加入丙酮2 mL。在旋渦式混合器上震蕩2 min，靜置10 min，再加入乙酸乙酯5 mL，震蕩2 min，再以3 000 r/min離心10 min，得萃取液，用0．45 m的微孔濾膜過濾，準確吸取5．0 μl樣品濾液進樣，根據峰面積值進行計算
5．菌絲量（細胞干重），pH值
6．糖耗
總糖的測量：采用苯酚-硫酸比色法，以葡萄糖為標準品。
殘糖含量：采用3、5一二硝基水楊酸比色法。
7．電鏡、細胞膜通透性
8．活性氧(ROS)
9．質譜對比分析代謝物組變化
Enhanced production of avermectin B1a by medium optimization and glucose feeding with Streptomyces avermilitis
Xu Zhinan, Cen Peilin. Bioprocess Engineering 20 (1999) 67-71 @Springer-Verlag 1999
Culture condition and media
S. avermilitis IP 842 was maintained on YMS medium [12].Slant culture was incubated for 5±8 days at 28 _C. For avermectin fermentation in shake flasks, spores and mycelia were scalped from the agar surface (15 _ 150 mm test tube) in 5 ml sterile water. 2 ml of such mixed suspension was used to inoculate 30 ml seed medium in 250 ml Erlenmeyer flask. The seed medium contained (g/l in water) soluble corn starch 30; yeast meal 15.0; corn steep liquor 5.0; KH2PO4, 0.4; PEG 2000, 2.5; pH 7.2 before autoclaving. After 24 h of incubation at 28.C on a shaker operating at 220 rpm, 4 ml of the seed culture was used to inoculate 40 ml of CSYC medium (Corn starch, Soybean meal, Yeast meal, Corn steep liquor) in 500 ml Erlenmeyer flask. Medium CSYC consisted of (g/l in water) corn starch, 60; soybean meal, 10.0; yeast meal, 15.0; corn steep liquor, 5.0; KH2PO4, 0.5; PEG 2000, 2.5; pH 7.2 before autoclaving. Corn starch was liquidated with 0.05% a-amylase before addition. After 5 days of incubation at 28 _C on a shaker operating at 220 rpm, the broth was taken for analysis of avermectin B1a and total avermectins (AVMs). It should be noted here that the seed medium and fermentation medium CSYC have been preliminarily optimized for high avermectin production [13].
In order to investigate the effects of metal elements on B1a production, CoCl2; FeSO4; ZnSO4 and MgSO4 were added to the control medium respectively. Different PEG (1000, 2000 and 4000) were added in the control medium to study the effect of PEG on B1a.
Fermentation studies in 10 liter fermentors
An optimized complex production medium was used for batch and fed-batch cultivation of S. avermilitis IP 842. Two hundred ml of 24 h seed culture was used to inoculate 6 liters of production medium in a 10-liter Braun Biostat B fermentor. During all the experiments, the agitation speed and air flow rate were adjusted as required, in order to keep the dissolved oxygen (DO) level above 20% of saturation. DO was monitored by a sterilizable electrode. The pH of fermentation medium was 7.2 before autoclaving. Samples were taken to measure packed mycelium volume, total sugar and reducing sugar, total avermectins (AVMs) and avermectin B1a. Two different modes of glucose feeding were tested in the fed-batch fermentations according to a predetermined schedule.
Analytical methods
Mycelia growth
Mycelia growth was estimated by measuring packed mycelium volume. After the centrifugation of 10 ml of fermentation broth at 4500 rpm for 15 min, the packed volume was measured. ?(DCW)
(2)The total sugar
The total sugar concentration in the culture broth was measured by the phenol-sulfuric acid method [14].
(3)Reducing sugar
Reducing sugar concentration in the culture medium was determined with 3,5-dinitrosalicyclic acid reagent method with glucose as a standard [15].
(4)The quantities of total avermectins
Equal volume of methanol was added to the sample and the mixture was shaken for 30 min to extract avermectins from the mycelia. A 10ul sample of supernatant of methanol extract was examined by HPLC. A nova-pak C18 column (Waters Ltd, USA: 3.9 mm in inner diameter, 150 mm in length) was developed with methanol water (85:15 [vol/vol]) at a flow rate of 0.8 ml/min in ambient conditions. The quantities of total avermectins (AVMs) were calculated from the integration value at 246 nm using an authentic sample of avermectin B1a as a standard [12]. The B1a proportion among avermectins was calculated as a ratio of B1a peak area and the total area at 246 nm.
誘導子的制備
待查：南昌霉素檢測 涂國全，高勇生．江西農業大學學報．1990，15：90～96
注：阿維菌素產生菌的菌落特征很不穩定，存在嚴重的自然分化現象，在不同菌落形態的分化菌株中，其中產灰色孢子的菌株能產生阿維菌素，但即使經單孢子選出的灰色菌落經傳代后，仍分化出不產阿維菌素的白色和光禿菌落，因此誘變處理后應從分離培養基上挑取產抗能力強的灰色孢子進行發酵培養，以平衡經常出現的發酵單位不穩定現象。