一株产紫杉醇内生真菌液体发酵工艺的优化

紫杉醇是一种高效、低毒、广谱的天然抗癌药物,可以有效地治疗乳腺癌、子宫癌等。近年来的研究发现,从植物内生真菌中发酵生产紫杉醇被证明是解决药源问题的有效途径。从分离到的420株内生真菌中筛选到一株产紫杉醇的内生真菌XC1-07为实验材料进行发酵条件的初步优化。结果表明：最适碳源、氮源分别是麦芽糖和NH4NO3;在含10g/L NH4NO3、90g/L麦芽糖、1.0g/L MgSO4、pH6的优化培养基中培养13d,紫杉醇的产量为1,124.34μg/L。为目前报道的植物内生真菌发酵生产紫杉醇最高的产量。     

一株苝产醌类光敏剂丝状真菌AL18的液体发酵工艺研究

为研究一株丝状真菌AL18产苝醌类光敏剂的液体发酵工艺.以马铃薯综合培养基为发酵培养基,采用单次单因素实验法,研究了液体摇瓶培养条件对苝醌类光敏剂产量的影响.实验结果表明液体摇瓶最适培养条件为250ml三角瓶装液量40ml,接种量7.5%,接种种龄40h,初始pH5.75,摇床转数180r/min,30℃振荡培养48h.在此培养条件下,采用单因素法筛选了发酵培养基中的碳源、氮源和无机离子,选用L9(34)对筛选到的绵白糖(A)、蛋白胨(B)、蚕蛹粉(C)、CuSO4·5H2O(D)进行了正交试验.经优化后的发酵培养基配方为马铃薯200g/L,绵白糖30g/L,蛋白胨3g/L,蚕蛹粉12.5g/L,磷酸二氢钾1g/L,硫酸镁0.5g/L,硫酸铜0.05g/L,VB1 100mg/L.对此发酵培养基配方进行了5次验证实验,苝醌类光敏剂的平均产量为1.21g/L.     

喇叭石蕊共生菌藻液体培养的研究

对喇叭石蕊共生菌、藻液体培养条件进行了研究。结果表明:共生菌生长在以40g/L肌醇为碳源、2g/LL-谷氨酰胺为氮源、起始pH值为7.0的LB液体培养基中,培养温度为20℃时表现最佳。其共生藻的生长在以160g/L葡萄糖为碳源、1.75g/LNaNO3为氮源、起始pH值为5.0的BBM液体培养基中,培养温度为20℃时表现最佳。     

拉曼被孢霉F5发酵生产γ-亚麻酸的研究

对拉曼被孢霉突变株F5发酵生产γ-亚麻酸的最适碳源、氮源、发酵时间及温度、无机盐离子添加、最适碳源浓度及补加碳源时间等发酵条件进行了研究探讨.最适发酵培养基组成为(g/L):葡萄糖100,酵母浸出粉4,蛋白胨1,K2HPO4 1,CaCl2 1×10-2,MgSO45×10-2,FeSO4 1×10-2,ZnSO4 7.5×10-3,CuSO4 0.5 × 10-5,MnSO4 2×10-3,pH 6.0.培养温度为25℃,140r/min振荡培养10天,培养8天后(即收获前2天)补加5%葡萄糖.发酵结果为:DC24.59g/L,TL 10.84g/L,TL/DC44.09%,GLA/TL10.67%,GLA产量为1156.63 mg/L. GLA产量较初始结果提高156.15%.该菌株已达到工业化生产菌株要求.     

Sulfolobus acidocaldarius ATCC 33909麦芽寡糖基海藻糖合成酶在Bacillus subtilis中的重组表达和发酵优化

为实现Sulfolobus acidocaldarius ATCC 33909来源的麦芽寡糖基海藻糖合成酶(MTSase)基因tre Y在枯草芽孢杆菌(Bacillus subtilis)中的重组表达,以质粒p ET-24a(+)-tre Y为模板PCR扩增得到目的基因,并与表达载体pHY300PLK连接,转入表达宿主Bacillus subtilis CCTCC M 2016536中,重组菌在TB培养基中培养48 h后MTSase酶活达到17.5 U/m L;在此基础上对重组菌发酵条件进行优化,通过单因素实验(氮源种类、氮源复配、氮源浓度、碳源种类、葡萄糖浓度、初始pH、诱导温度)和正交实验(氮源浓度、葡萄糖浓度、初始pH、诱导温度)确定其摇瓶发酵产酶的最适培养基和培养条件为:氮源(工业蛋白胨∶棉籽粉=3∶1)48.0 g/L、葡萄糖为10.0 g/L、培养基初始pH为7.0,最适培养温度为30℃;在此条件下,MTSase的酶活可达41.5 U/m L,是优化前的2.4倍。     

一株产苝醌类光敏剂丝状真菌AL18的液体发酵工艺研究

为研究一株丝状真菌AL18产艹北醌类光敏剂的液体发酵工艺。以马铃薯综合培养基为发酵培养基,采用单次单因素实验法,研究了液体摇瓶培养条件对艹北醌类光敏剂产量的影响。实验结果表明:液体摇瓶最适培养条件为250ml三角瓶装液量40ml,接种量7.5%,接种种龄40h,初始pH5.75,摇床转数180r/min,30℃振荡培养48h。在此培养条件下,采用单因素法筛选了发酵培养基中的碳源、氮源和无机离子,选用L9(34)对筛选到的绵白糖(A)、蛋白胨(B)、蚕蛹粉(C)、CuSO4.5H2O(D)进行了正交试验。经优化后的发酵培养基配方为:马铃薯200g/L,绵白糖30g/L,蛋白胨3g/L,蚕蛹粉12.5g/L,磷酸二氢钾1g/L,硫酸镁0.5g/L,硫酸铜0.05g/L,VB1100mg/L。对此发酵培养基配方进行了5次验证实验,艹北醌类光敏剂的平均产量为1.21g/L。     

红豆杉内生真菌中巴卡亭Ⅲ产生菌的筛选及培养基的初步优化

目的:筛选出产巴卡亭Ⅲ的红豆杉内生真菌,为抗癌药物紫杉醇提供一个新的来源。方法:从云南红豆杉树皮中分离纯化出内生真菌,对其进行液体发酵培养,过滤,收集菌丝体并破碎,用二氯甲烷萃取,萃取液进行薄层层析,与巴卡亭Ⅲ标准品有相同比移值的样品再利用高效液型色谱与标样进行比照,进而筛选出产巴卡亭Ⅲ的内生真菌。同时以发酵培养基基础,对筛选出的真菌进行碳源和氮源的优化。并在此基础上探讨几种常见的紫杉烷类化合物代谢诱导剂乙酸钠、苯甲酸钠、苯丙氨酸和亮氨酸的浓度对巴卡亭Ⅲ积累的影响。结果:得到一株产巴卡亭Ⅲ的内生真菌NSZJ043,该菌株易培养,生长迅速,经鉴定属于曲霉属。NsZJ043产巴卡亭Ⅲ的培养条件的初步优化结果表明:最适碳源、氮源分别是蔗糖和蛋白胨;在含20g/L蔗糖、1．5g/L蛋白胨、0．1 mmol/L乙酸钠、0．01mmol/L苯甲酸钠的优化培养基中培养8d,巴卡亭Ⅲ含量为34．6μg/L。结论:筛选出一株产巴卡亭Ⅲ的内生真菌,其具有优良的发酵特性,巴卡亭Ⅲ前体物质苯甲酸钠和乙酸钠对巴卡亭Ⅲ的合成有促进作用;苯丙氨酸和亮氨酸对其含量影响不显著,优化后NSZJ043产巴卡亭Ⅲ含量有较大的提高。     

樟芝产生三萜类化合物的液体培养条件的响应面法优化

樟芝(Antrodia cinnamomea)是一种非褶菌目、多孔菌科的多年生蕈菌类,樟芝子实体中含有多种生理活性物质包括三萜类化合物,但野生樟芝只生长在台湾特有的牛樟树树干腐朽的心材内壁和枯死倒伏的牛樟树表面,生长极其缓慢,很难获得,价格昂贵。本研究的目的是通过液体培养樟芝的菌丝体以产生三萜类化合物,用响应面法优化了樟芝产生三萜类化合物的液体发酵条件。首先对樟芝的液体发酵的培养基成分及培养条件进行了单因素优化,研究发现樟芝液体发酵产生三萜类化合物的最佳碳源和氮源分别为葡萄糖和酵母提取物,最佳浓度分别为100 g/L和9 g/L,优化的培养基配方为(g/L):葡萄糖100.0,酵母提取物9.0,Mg SO4·7H2O 0.5,KH2PO41.0。优化的培养条件为:初始pH值6.0,温度28℃,接种量10%(v/v),摇床转速180 r/min。在此基础上,用响应面法对樟芝液体发酵产生三萜类化合物影响最大的三个因素即碳源、氮源和培养温度进行了优化,结果优化的三种因素条件为:葡萄糖104.71 g/L,酵母提取物9.93 g/L,温度28.42℃。用优化的培养条件液体培养樟芝,其三萜类化合物的产量达到391.45 mg/L,比优化前的产量223.39 mg/L提高了75.23%,为进一步研究液体培养樟芝产生三萜类化合物奠定了基础。     

蛹虫草菌丝产虫草素液体培养条件的研究

通过对蛹虫草菌丝产虫草素液体培养条件的研究,明确蛹虫草菌丝产虫草素的适宜碳源及浓度,适宜氮源及浓度,最适pH值,最适培养温度,最适转速以及最适培养时间,以便应用于虫草素的工厂化生产。结果表明,蛹虫草菌丝产虫草素的条件：适宜碳源为D-果糖,最适浓度为10g/L;适宜氮源为蛋白胨,最适浓度为15g/L;最适初始pH为7,最适培养温度为24℃,最适转速为180r/min,最适培养时间为9d,其培养液虫草素含量可达到0.537g/L。     

拉曼被孢霉F_5发酵生产γ—亚麻酸的研究

对拉曼被孢霉突变株F5发酵生产γ—亚麻酸的最适碳源、氮源、发酵时间及温度、无机盐离子添加、最适碳源浓度及补加碳源时间等发酵条件进行了研究探讨。最适发酵培养基组成为 (g/L) :葡萄糖 1 0 0 ,酵母浸出粉 4 ,蛋白胨 1 ,K2 HPO4 1 ,CaCl2 1× 1 0 - 2 ,MgSO4 5× 1 0 - 2 ,FeSO4 1× 1 0 - 2 ,ZnSO4 7.5× 1 0 - 3,CuSO4 0 .5× 1 0 - 3,MnSO4 2× 1 0 - 3,pH 6.0。培养温度为 2 5℃ ,1 4 0r/min振荡培养 1 0天 ,培养 8天后 (即收获前 2天 )补加 5 %葡萄糖。发酵结果为 :DC 2 4 .5 9g/L ,TL 1 0 .84g/L ,TL/DC 4 4.0 9% ,GLA/TL 1 0 .67% ,GLA产量为 1 1 5 6.63mg/L。GLA产量较初始结果提高 1 5 6.1 5 %。该菌株已达到工业化生产菌株要求     

Laccase production by Monotospora sp., an endophytic fungus in Cynodon dactylon

The effects of the carbon and nitrogen sources, initial pH and incubation temperature on laccase production by the endophytic fungus Monotospora sp. were evaluated. The optimal temperature and initial pH for laccase production by Monotospora sp. in submerged culture were found to be 30 degrees C and 8.5, respectively. Maltose (2 g l(-1)) and ammonium tartrate (10 g l(-1)) were the most suitable carbon and nitrogen source for laccase production. Under optimal culture medium, the maximum laccase activity was determined to be 13.55 U ml(-1), which was approximately four times higher than that in basal medium. This is the first report on laccase production by an endophytic fungus.     

Production of red pigment by submerged culture of Paecilomyces sinclairii

AIMS: From a survey of submerged culture of edible mushrooms, a high pigment-producing fungus Paecilomyces sinclairii was selected and its optimal culture conditions investigated. METHODS AND RESULTS: The optimal culture conditions for pigment production were as follows: inoculum age, 3 d; temperature, 25 degrees C; initial pH, 6.0; carbon source, 1.5% (w/v) soluble starch; nitrogen source, 1.5% (w/v) meat peptone. Although addition of 10 mmol l(-1) CaCl2 to the culture medium slightly increased pigment production, most of the bio-elements examined had no notable or detrimental effect on pigment production. CONCLUSIONS: Under the optimal conditions obtained in the flask culture tested, a ninefold increase in pigment production (4.4 g l(-1)) was achieved using a 5(-l) batch fermenter. Paecilomyces sinclairii secreted water-soluble red pigment into the culture medium. The pigment colour was strongly dependent on the pH of the solution: red at pH 3-4, violet at pH 5-9 and pink at pH 10-12. SIGNIFICANCE AND IMPACT OF THE STUDY: The high concentration of pigment (4.4 g l(-1)) produced by P. sinclairii demonstrates the possibility of commercial production of pigment by this strain, considering its relatively high production yield and light stability.     

绿僵菌无纺布菌条的制作(I)液体种子培养条件

研究了金龟子绿僵菌菌株M a83液体培养最佳营养及环境条件,为无纺布培养提供良好的液体种子。以生物量为指标,通过液体摇瓶试验确定了绿僵菌M a83菌株液体种子培养参数,对不同的氮源、碳源及其浓度和初始pH进行单因素分析。结果表明,绿僵菌M a83菌株液体种子培养最佳营养条件为4%黄豆粉,2%白砂糖,0.5%MgSO4.7H2O,0.05g/LKC l,0.1g/L FeSO4.7H2O,1.0g/L KH2PO4,pH6.3~6.5。70L发酵罐培养,干生物量第三天可达35g/L,显著优于筛选前培养基(SDA,28g/L,a=0.05)。     

茴香酸产生菌发酵条件的优化

为提高微生物降解反式茴脑获得茴香酸的产量,对假单胞菌Pseudomonas sp.NT2的发酵参数进行优化,以提高降解过程的转化率。利用单因素试验考察碳氮源种类及浓度、反式茴脑添加量、发酵温度、接种量、初始pH以及装液量对茴香酸生成量、反式茴脑降解率的影响,通过Plackett-Burman试验和最陡爬坡试验确定影响茴香酸生成量的显著因素并获取中心点,最后采用Box-Behnken模型进行响应面优化得到最佳发酵条件并验证。结果表明氯化铵浓度、初始pH和装液量是显著影响因素,最佳发酵条件为:柠檬酸钠10 g/L,氯化铵1.26 g/L,反式茴脑添加量1%,发酵温度30℃,接种量4%,初始pH 7.9,装液量42 mL/250 mL。优化后茴香酸生成量为7.24 g/L,为优化前的3.5倍,茴香酸摩尔生成率为80.72%,反式茴脑降解率为89.81%,分别比优化前提高了270.28%和97.78%。综上,假单胞菌NT2是生物转化生产茴香酸的潜力菌株。响应面优化可以显著提高反式茴脑的降解率和茴香酸产量,这为大规模生产茴香酸奠定了基础。     

氮源对灵芝菌丝体液态深层发酵合成灵芝三萜的影响

以沪农灵芝1号为供试菌株,葡萄糖作为碳源,用硫酸铵、氯化铵、鱼粉蛋白胨、胰蛋白胨和酵母粉作为氮源,研究不同种类氮源对灵芝菌丝体液态深层发酵过程的影响。首先,确定了N-10酵母自溶粉作为发酵的氮源,降低了发酵的复杂性和不确定性;其次,考察N-10酵母自溶粉不同浓度对灵芝菌丝体发酵合成灵芝三萜过程中菌丝体的生物量、葡萄糖消耗、灵芝三萜产量等方面的影响,确定了N-10酵母自溶粉的适宜添加浓度。在此基础上,采用响应面中心组合设计,对4因素最佳水平范围进行研究,结果表明,葡萄糖、N-10酵母自溶粉、磷酸二氢钾和七水硫酸镁的含量分别为31.06g/L、2.76g/L、1.77g/L和1.99g/L时,灵芝三萜的理论产量为21.166g/kg干菌丝体,实际发酵产量提高到21.153g/kg干菌丝体。与原工艺相比,新工艺的灵芝三萜产量提高了6.22%。     

培养条件对双歧杆菌EPS各组分产量及比例的影响

【目的】动物双歧杆菌RH产生的胞外多糖(exopolysaccharides, EPS)经阴离子交换柱层析可获得EPSa和EPSb两个组分。得到可提高EPS的总产量, 尤其是EPSb产量的最佳培养基和培养条件。【方法】对培养基类型、氮源、碳源、碳源浓度、培养基初始pH值、培养温度和时间对双歧杆菌EPSa和EPSb产量的影响进行分析。【结果】在初始pH值调整为7.0的含5%蔗糖的PTYG培养基上, 在35 °C温度下厌氧培养60 h时动物双歧杆菌RH的EPSa和EPSb产量分别为0.982±0.003 g/L和0.312±0.001 g/L。【结论】在上述条件下EPS总产量高且可获得较多的EPSb。     

Fusarium solani G6, a novel vitexin-producing endophytic fungus: characterization,yield improvement and osteoblastic proliferation activity

The study aimed to characterize a novel vitexin-producing endophytic fungus Fusarium solani G6 from Cajanus cajan, improve its capability for producing vitexin and evaluate its osteoblastic proliferation activity. A total of 153 endophytic fungi, classified into 6 genera, were isolated from C. cajan. Among them, only one strain, endophyte G6 identified as Fusarium solani, was found to produce vitexin. After the optimization of fermentation conditions, the highest vitexin yield (18.72 mg/L) for the strain was observed in PDB liquid medium containing 20.54 g/L of glucose and 8.90 g/L of ammonium sulfate, at an initial medium pH of 5.1 and at 28 °C for 6 days of cultivation. Moreover, the fungal vitexin exhibited notable osteoblastic proliferation stimulating activity. A novel vitexin-producing endophytic fungus F. solani G6 was characterized from C. cajan for the first time. The findings highlighted its potential use for large-scale production of vitexin and might have a promising use as therapeutic agent for osteoporosis.

     

Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.     

Improvement in raw sago starch degrading enzyme production from Acremonium sp. endophytic fungus using carbon and nitrogen sources

Attempts were made to improve the growth of endophytic fungus Acremonium sp. and its raw sago starch degrading enzyme (RSSDE) production using different nitrogen and carbon sources at varying pH values and temperatures. It was observed that growth and enzyme activity levels were highest with peptone and sodium nitrate as the nitrogen sources and raw sago starch as the carbon source of which the optimum concentrations were 0.5 g/l, 3 g/l, and 20 g/l, respectively. Cell growth and RSSDE production reached their optimum at pH 5.0 and incubation temperature of 30 degrees C. Under these conditions, the enzyme production was significantly increased by 19- to 22-folds compared to the activity obtained in the original basal medium.     

前体及诱导子和发酵条件对烟曲霉TMS-26产紫杉醇发酵体系优化

紫杉醇是一种广谱的抗癌药物,因其具有独特的抗癌机制、良好的抗癌效果和供不应求的市场等特征而备受关注。紫杉醇具有重大经济效益,但产量受到制约,价格极为昂贵,通过内生真菌发酵法生产紫杉醇能在一定程度上缓解其来源困难的问题。在产紫杉醇内生真菌TMS-26发酵液中添加前体物质和诱导子,并通过对接种量、装液量、初始pH和发酵时间等条件进行优化研究。单因素及正交试验表明在PDB培养基中加入苯丙氨酸20mg/L、苯甲酸钠30mg/L、乙酸钠8g/L、甘氨酸15mg/L、CuSO₄ 0.05mg/L、H₂O₂ 6mmol/L、3,5-二硝基水杨酸15mg/L时能有效提高紫杉醇产量,比优化前增产46.64%,达到446.28µg/L,并且发现最适菌株TMS-26的发酵条件为pH7.5、接种量5%、装液量120mL/250mL、发酵时间为10d。