响应面法优化蛹虫草与厚朴双向液体发酵工艺

为提高蛹虫草液体发酵胞外多糖含量和菌丝体生物量,以厚朴为药性基质,对蛹虫草进行双向液体发酵。在单因素实验的基础上,应用Box-Behnken实验设计,对发酵过程关键影响因素进行优化。结果表明,在厚朴添加量5g、接种量15.5mL、发酵温度25℃的条件下发酵9d,蛹虫草双向液体发酵产物中胞外多糖含量为3.11mg/mL,菌丝体生物量为18.81mg/mL。各发酵因素中,发酵液胞外多糖含量受接种量影响最大,菌丝体生物量则主要受发酵温度影响。优化所得发酵工艺可行性高、周期短、生产过程可控,为进一步提高人工培育蛹虫草质量、增加其关键活性产物的产量提供了参考。     

4种虫草相关真菌菌丝体粗多糖的生物活性评价

本研究以细脚棒束孢、蛹虫草、蝉棒束孢和球孢白僵菌的菌丝体粗多糖为对象,分析4种虫草相关真菌菌丝体粗多糖含量与生物量的相关性,并进一步对其抗氧化能力和抗肿瘤活力进行评价。液体发酵结果表明,蝉棒束孢MF12、MF13和蛹虫草MF27、MF1的菌丝体粗多糖含量（>40mg/g）显著高于其他菌株,蝉棒束孢MF11、MF13和蛹虫草MF27菌丝体生物量（>12g/L）显著高于其他菌株,但相关性分析表明,4种虫草相关真菌10个菌株菌丝体的多糖含量与生物量之间没有显著相关性;抗氧化活性表明,蛹虫草MF27、MF1和球孢白僵菌MF10菌丝体粗多糖具有良好的体外抗氧化活性,其EC₅₀均小于0.9mg/mL;抗肿瘤活性表明,蛹虫草MF1、MF28、MF27和球孢白僵菌MF10菌丝体粗多糖在体外能有效抑制HepG-2细胞增殖,其IC₅₀均小于1.5mg/mL。综上,蛹虫草MF27、MF1和球孢白僵菌MF10虫草菌株具有良好的开发和应用潜力。     

优良性状蚕蛹虫草的筛选及高产虫草素液态发酵条件优化

蛹虫草是重要的食药用真菌,虫草素为其主要活性成分,在抗肿瘤、抗菌、降血糖等方面具有较为突出的功效。蛹虫草菌株间的形态及环境条件差异,对菌株次级代谢产物虫草素产生影响显著。本研究对不同来源的6株蛹虫草菌株（YCC-B、YCC-C、YCC-H、YCC-W、YCC-Y、CGMCC 3.4655）,从蚕蛹体培养子实体性状,液体发酵条件（培养天数、培养方式、外源金属离子等）和传代稳定性等方面筛选优良性状菌株,提高其发酵合成虫草素的能力及稳定性。结果表明,蛹虫草菌株YCC-W在蚕蛹子实体出草及菌体液体发酵产虫草素上综合表现优良,传代稳定;液体发酵培养基中添加外源金属离子Mn²⁺作为酶的辅基,可以促进虫草素合成;采用振荡-静置相结合的混合发酵培养方式,可以避免单纯振荡培养溶氧量大、菌丝体生长旺盛,而虫草素产生不佳的问题。先振荡培养3d后静置培养至25d时,菌株YCC-W合成虫草素含量最高,可达(874.13±24.25)μg/mL,且稳定性良好。为进一步开发菌种及扩大规模生产提供参考。     

锌富集对蛹虫草菌丝体内虫草素、腺苷含量的影响

为了解蛹虫草菌丝体对锌的富集特性,研究锌富集对蛹虫草菌丝体内虫草素、腺苷含量的影响,通过在液体培养基中添加不同质量浓度的锌离子(0~35 g/L),探讨其对蛹虫草菌丝生物量、菌丝体内锌积累量,以及锌的富集对菌丝体内虫草素、腺苷含量产生的影响。结果表明:在0~35 g/L锌离子的梯度范围内,蛹虫草菌丝生物量与锌质量浓度呈显著负相关,锌质量浓度35 g/L为蛹虫草菌丝生长极限浓度。锌质量浓度40.0 g/L及以上菌丝生长受到完全抑制。菌丝体内锌的积累量随锌质量浓度的增加而显著升高,锌质量浓度为35.0 g/L时锌积累量可达到193.87 mg/g(干重)。蛹虫草菌丝体内腺苷的含量随锌质量浓度的增加而降低,在锌质量浓度为5 g/L时降幅显著,腺苷含量仅为对照组的17.24%,之后腺苷含量变化趋势趋于水平。腺苷含量的降低可能是因为锌的富集干扰了蛹虫草菌丝体内初生代谢的正常进行。虫草素的含量随锌质量浓度的增加而显著降低,可能是由于其直接前体腺苷含量的降低,或是Zn离子的加入,使得某些被刺激的酶和基因通过转录因子影响了虫草素的合成。     

虫草素高产菌株的筛选及不同添加物对虫草素产量的影响研究

通过对14株蛹拟青霉菌株进行摇瓶液体发酵培养试验,筛选虫草素产量最高的蛹虫草菌株,并确定了虫草素的最佳收获期;比较8种营养添加物对虫草发酵过程中虫草素积累的影响,筛选出最佳的营养添加物,以提高液体发酵生产虫草素的产量。结果表明：蛹虫草CM001号菌株的虫草素产量最高,蛹虫草菌在第10天细胞量达到最大值20.44mg/mL,虫草素含量在第13天达到最大值73.81mg/L,70%以上的虫草素分布在发酵液中。其中分别添加腺苷、腺嘌呤、丙氨酸、L-天冬氨酸、甘氨酸5种物质,对虫草素合成的促进作用均较明显,均能有效提高蛹虫草液体发酵虫草素的产量,特别是添加1g/L的腺嘌呤能使10号菌株的虫草素产量提高7.09倍。     

假蜜环菌发酵工艺的优化研究

目前,国内普遍采用亮菌固体发酵工艺生产亮菌制剂。采用小型发酵罐液体深层发酵和液体静置发酵对亮菌发酵工艺进行优化研究。液体深层发酵采用28℃,200 r/min,通气量1∶1 v/v·m,培养7 d,亮菌干重为16.55g/L,其中菌丝体中多糖含量为5.42%,蛋白含量为1.75%;液体静置发酵采用500 mL三角瓶,28℃,150 r/min,摇瓶3 d后静置发酵14 d,亮菌干重可达10.69 g/L,发酵液中多糖含量为1.016 g/L,蛋白含量为0.320 g/L。对液体静置发酵进一步研究发现其发酵液中亮菌甲素含量可达3.118 mg/L。由此可见,两种发酵方式在保证生物量和活性成分的前提下,缩短了发酵周期,均优于传统的固体发酵工艺,值得工业生产借鉴。     

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。     

不同培养条件和前体对蛹虫草液体发酵产虫草素的影响

蛹虫草能产生虫草素等多种活性物质。为考察不同液体发酵方式及添加前体物质对虫草素积累的影响,选用蛹虫草08Y1菌株,通过光照振荡、光照静置、黑暗振荡、黑暗静置四种培养条件和添加前体物质（腺嘌呤1g/L+甘氨酸16g/L）,发酵16d后检测虫草素和腺嘌呤含量。结果表明：08Y1菌株在黑暗振荡培养条件下,虫草素积累达1,015.0mg/L,腺嘌呤利用率达98.5%,说明黑暗振荡培养并添加前体物质是提高虫草素产量的有效方法。     

蛹虫草对锌的耐性与富集特征

在培养基内添加不同量的锌,研究其对蛹虫草子实体的形成、子实体和菌丝体生物量、子实体多糖含量和葡萄糖含量的影响,以及蛹虫草子实体和菌丝体对锌的富集能力。结果表明锌对上述各项都有影响。液体培养条件下,锌浓度在453906mg/L范围内可以促进菌丝体生长,锌浓度超过4077mg/L时,菌丝生长受到抑制。培养基锌的浓度在4077mg/L以下时,蛹虫草菌丝体锌的富集量随着液体培养基锌浓度的提高而提高。固体培养条件下,锌含量在226453mg/kg范围内可以促进蛹虫草子实体生长,并且在此含量范围内,蛹虫草子实体中葡萄糖含量较高。培养基锌含量在680906mg/kg时,子实体多糖含量较高。培养基锌含量在2038mg/kg以下时,蛹虫草子实体中锌的富集量随着培养基锌含量的提高而提高,在培养基锌含量为2038mg/kg时,子实体中锌的含量达到28570mg/kg(干重)。     

蛹虫草菌产生的胞外多糖及其理化性能和抗氧化活性的初步研究

蛹虫草菌Cordycepsmilitaris在液体发酵条件下可产生胞外多糖,其粗多糖的蛋白含量在14．31％左右,氨基酸含量为13．8％,共有17种氨基酸,水分5％左右,多糖含量大约占80％。蛹虫草菌胞外多糖具有良好的增稠性、触变性、抗盐耐热和对广泛pH的稳定性能。蠕虫草菌丝体及其胞外多糖具有一定的抗氧化活性,在60℃的促进氧化条件下放置7天,0．05％含量的菌丝体及其胞外多糖降低油脂氧化率分别达到18．8％和19．8％。     

A comparative study on the production of exopolysaccharides between two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis in submerged mycelial cultures

AIMS: The present study comparatively investigates the optimal culture conditions for the production of exopolysaccharides (EPS) and cordycepin during submerged mycelial culture of two entomopathogenic fungi Cordyceps militaris and Cordyceps sinensis. METHODS AND RESULTS: Fermentations were performed in flasks and in 5-l stirred-tank fermenters. In the case of C. militaris, the highest mycelial biomass (22.9 g l(-1)) and EPS production (5 g l(-1)) were achieved in a medium of 40 g l(-1) sucrose, 5 g l(-1) corn steep powder at 30 degrees C, and an initial pH 8.0. The optimum culture conditions for C. sinensis was shown to be (in g l(-1)) 20 sucrose, 25 corn steep powder, 0.78 CaCl2, 1.73 MgSO4.7H2O at 20 degrees C, and an initial pH 4.0, where the maximum mycelial biomass and EPS were 20.9 and 4.1 g l(-1) respectively. Cordycepin, another bioactive metabolite, was excreted at low levels during the early fermentation period (maximum 38.8 mg l(-1) in C. militaris; 18.2 mg l(-1) in C. sinensis). CONCLUSIONS: The two fungi showed different nutritional and environmental requirements in their submerged cultures. Overall, the concentrations of mycelial biomass, EPS and cordycepin achieved in submerged culture of C. militaris were higher than those of C. sinensis. SIGNIFICANCE AND IMPACT OF THE STUDY: C. militaris and C. sinensis are representative insect-born fungi which have been longstanding and widely used as traditional medicines in eastern Asia. Comparative studies between two fungi are currently not available and this is the first report on the optimum medium composition for submerged culture of C. sinensis.     

利用扁桃斑鸠菊叶发酵生产蛹虫草胞外多糖的条件及其抗氧化活性

蛹虫草胞外多糖具有增强免疫力、抗疲劳等药理活性,有极高的保健价值。为高效地获取蛹虫草胞外多糖,本研究通过向发酵培养基中添加适量的扁桃斑鸠菊叶粉末,来提高蛹虫草发酵液中胞外多糖的产量,并对优化得到的胞外多糖红外吸收光谱和化学抗氧化活性进行了研究。实验结果表明,液体发酵最优条件为:扁桃斑鸠菊叶粉末添加量8 g/L、发酵时间9 d、pH 6.5、接种量5.0 mL,在此条件下,蛹虫草胞外多糖的产量可达(5.24±0.28) mg/mL,与未添加扁桃斑鸠菊叶的空白组相比,胞外多糖产量提高了约205.20%;红外分析与抗氧化活性实验结果显示,扁桃斑鸠菊叶对蛹虫草生产的胞外多糖结构和活性影响较小。该研究结果表明扁桃斑鸠菊叶能够有效地提高蛹虫草胞外多糖的产量,为蛹虫草胞外多糖的高效生产提供了新思路。     

Optimization of submerged culture process for the production of mycelial biomass and exo-polysaccharides by Cordyceps militaris C738

AIMS: The objective of the present study was to determine the optimal culture conditions for mycelial biomass and exo-polysaccharide (EPS) by Cordyceps militaris C738 in submerged culture. METHODS AND RESULTS: The optimal temperatures for mycelial biomass and EPS production were 20 degrees C and 25 degrees C, respectively, and corresponding optimal initial pHs were found to be 9 and 6, respectively. The suggested medium composition for EPS production was as follows: 6% (w/v) sucrose, 1% (w/v) polypeptone, and 0.05% (w/v) K2HPO4. The influence of pH on the fermentation broth rheology, morphology and EPS production of C. militaris C738 was carried out in a 5-l stirred-tank fermenter. The morphological properties were comparatively characterized by pellet roughness and compactness by use of image analyser between the culture conditions with and without pH control. The roughness and compactness of the pellets indicated higher values at pH-stat culture (pH 6.0), suggesting that larger and more compact pellets were desirable for polysaccharide production (0.91 g g(-1) cell d(-1). CONCLUSIONS: Under the optimized culture conditions (with pH control at 6), the maximum concentration of biomass and EPS were 12.7 g l(-1) and 7.3 g l(-1), respectively, in a 5-l stirred-tank fermenter. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effect of pH on fungal morphology and rheology presented in this study can be widely applied to other mushroom fermentation processes.     

Optimization of submerged culture conditions for the mycelial growth and exo-biopolymer production by Cordyceps militaris

AIMS: The objective of the present study was to determine the optimal culture conditions for exo-biopolymer production by Cordyceps militaris in shake flask culture. METHODS AND RESULTS: The optimal temperature and initial pH for both mycelial growth and exo-biopolymer production by Cordyceps militaris in shake flask culture were found to be 20 degrees C and 6.0, respectively. Sucrose (40 g x l(-1)) and corn steep powder (10 g x l(-1)) were the most suitable carbon and nitrogen source for both mycelial growth and exo-biopolymer production. CONCLUSION: Under optimal culture conditions, the maximum exo-biopolymer concentration in a 5-l jar fermenter indicated 10.3 g x l(-1), which was approximately three times higher than that in shake flask culture. SIGNIFICANCE AND IMPACT OF THE STUDY: This process can have a significant impact on the industrial scale when sucrose and corn steep powder were used as carbon and nitrogen source.     

蛹虫草蓝光诱导两步法发酵产类胡萝卜素

采用黑暗摇瓶发酵和蓝光照射静置培养的两步培养法,进行蛹虫草(Cordyceps militaris L.)液体发酵产类胡萝卜素的蓝光诱导。结果表明蛹虫草在2d的黑暗培养和5d的蓝光照射静置培养后,其类胡萝卜素的含量可达到最高值558.4μg/gFW。而以黑暗摇瓶培养2d后,进行不同时间的蓝光照射静置培养。结果表明,蓝光照射最初2d,蛹虫草类胡萝卜素含量变化不明显,随后快速增加,并在第5天达到最大值558.4μg/gFW,随后类胡萝卜素的含量并无明显变化。通过研究解决了蛹虫草液体发酵产类胡萝卜素的培养过程中蓝光的给光问题。     

Production and in vitro antioxidant activity of exopolysaccharide by a mutant, Cordyceps militaris SU5-08

Cordyceps militaris SU5-08 was derived from an initial strain (C. militaris SU5) by ultraviolet mutagenesis of protoplasts, and the extraction parameters for C. militaris SU5-08 exopolysaccharide (EPS) produced during submerged culture were optimized. The extraction rate of EPS was 1919.16±165.27 mg/l, which was 120.38±11.36% higher than that of C. militaris SU5. The in vitro scavenging effects of EPS of C. militaris SU5-08 on hydroxyl, superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals at a dosage of 5 g/l were 63.64±3.52%, 75.27±5.16%, and 6.46±5.03%, respectively. The reducing power of EPS of C. militaris SU5-08 was 0.21±0.01. The results suggest that the EPS of C. militaris SU5-08 can be used as a potential antioxidant which enhances adaptive immune responses.     

蛹虫草子实体中海藻糖含量的测定

为分析食用菌中海藻糖的含量,以蛹虫草子实体为材料,比较了提取溶剂、提取方式及提取时间等条件对海藻糖提取效果的影响,确定海藻糖分析的前处理方法为：1g子实体粉中加入100mL 90%乙醇热回流提取1h。采用高效液相色谱法测定海藻糖,优化后的色谱条件为：SUGAR SP0810柱（300mm×8mm）,超纯水洗脱,流速0.5mL/min,柱温70℃,示差折光检测器检测,进样量10μL。方法学考察结果表明,该方法准确度高,稳定性、精密度、重现性好,对海藻糖标准品的检测特异性良好,适用于蛹虫草子实体中海藻糖含量的     

泰山虫草C-2最优液体培养条件筛选

在泰山虫草资源调查过程中,采集、分离获得一株泰山虫草菌(Cordycepssp.)。对其进行人工驯化栽培,发现其生长周期短,发菌快,适应性强。利用正交试验设计法筛选其最佳液体发酵培养基及条件,用SAS数据处理软件系统对菌丝生物量、多糖进行方差分析,并测定发酵液中超氧化物歧化酶(SOD)、过氧化物酶(POD)等酶的活性。结果表明,泰山虫草最优的菌丝体发酵培养基组合是:马铃薯20%、蔗糖2%、豆粉2%、胶体几丁质0.5%、无机盐0.1%KH2PO41、2 h光照。