Mycelial growth and exo-biopolymer production by submerged culture of various edible mushrooms under different media

AIMS: The effect of synthetic media on the submerged mycelial growth and exo-biopolymer production in various edible mushrooms was investigated in shake flask culture. METHODS AND RESULTS: Among 19 mushrooms examined, the relatively high yield in mycelial biomass and exo-biopolymer production was achieved in potato malt peptone (PMP) medium. In particular, Ganoderma lucidum NO. 1 and Phellinus linteus KCTC 6190 showed favourable growth in PMP medium with exo-biopolymer concentration of 1170 and 1520 mg l(-1), respectively. CONCLUSIONS: Enhanced exo-biopolymer production was achieved from Ganoderma lucidum NO. 1 and Phellinus linteus KCTC 6190 in a 5L batch fermentor, indicating approximately 5000 and 2410 mg l(-1), respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The exo-biopolymer production and mycelial growth from various mushrooms were found to be strongly controlled by different complex media.     

适合飞虱虫疠霉菌丝生产的液体培养基组分及发酵条件

蚜虫、飞虱、叶蝉等是威胁农业生产的刺吸式口器害虫,长期以来依赖化学农药防治,严重影响农产品的安全性和农业生态环境.探讨这类害虫的生物防治途径,减轻农产品化学农药的残留污染,一直受到国内外学者的关注.虫霉(En-tomophthorales)的许多种类具有优异的诱病杀虫能力,尤其通过主动弹射孢子而在害虫种群中高强度流行的特征,可在短期内迅速压低虫口密度,是极其重要的杀虫微生物资源[1～3].     

抗肿瘤银杏内生真菌Aspergillus oryzae YX-5发酵 培养基的优化

【目的】对一株具有抗肿瘤活性的银杏内生真菌Aspergillus oryzae YX-5的发酵培养基进行优化。【方法】以发酵后的菌体干重、粗提物质量和粗提物抗肿瘤活性为指标,通过单因素实验选出合适的碳源和氮源,再以选出的碳源、氮源以及K2HPO4、MgSO4·7H2O、KCl共5个因素进行正交实验,确定出最适宜的发酵培养基配方。【结果】YX-5的最佳发酵培养基配方为葡萄糖45 g/L,蛋白胨8 g/L,K2HPO4 0.5 g/L,MgSO4·7H2O 0.2 g/L,KCl 1 g/L,FeSO40.01 g/L。优化后菌体干重、代谢物的产量和抗肿瘤活性分别提高了41.88%、226.52%和19.31%。【结论】通过对米曲霉菌(Aspergillus oryzae)YX-5的发酵培养基进行优化,明显提高了粗提物产量和抗肿瘤活性,这对后期规模化发酵中减小发酵规模和降低工作量十分有利。     

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.     

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。     

Optimization of physical parameters for exo-biopolymer production in submerged mycelial cultures of two entomopathogenic fungi Paecilomyces japonica and Paecilomyces tenuipes

AIMS: In the present study, two different optimization techniques were used to determine the suitable operating parameters for exo-biopolymer production in submerged mycelial cultures of two entomopathogenic fungi Paecilomyces japonica and Paecilomyces tenuipes. METHODS AND RESULTS: First, the rotating simplex method, a nonstatistical optimization technique, was employed to obtain the best combination of physical parameters (viz. pH, agitation intensity, aeration rate) for maximum exo-biopolymer production by P. japonica in a batch bioreactor. The optimal combination was determined to be a pH of 8.06, an aeration of 3 vvm, without any impeller agitation, producing a 17-time increase in exopolymer production (34.5 g l(-1)) when compared with that achieved in unoptimized flask cultures. Second, the uniform design method, a statistical optimization technique, was employed to determine the best operating parameters for submerged culture of P. tenuipes. The optimal combination for mycelial growth was determined to be a pH of 4.88, an aeration of 2 vvm and an agitation of 350 rpm, while a pH of 4, an aeration of 2 vvm and an agitation of 150 rpm was best for exo-biopolymer production. CONCLUSIONS: The exo-biopolymer production in P. japonica optimized by the rotating simplex method was strikingly improved (max. 34.5 g l(-1)), and the exo-biopolymer production in P. tenuipes optimized by the uniform design method was also significantly increased (max. 3.4 g l(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: The successful application of these two different optimization techniques in this study implies that these methods are worthy of applying to other fermentation systems for the production of bioactive mycelial biomass and exo-biopolymers in liquid culture of higher fungi.     

Hypolipidemic effect of an Exo-biopolymer produced from a submerged mycelial culture of Hericium erinaceus

The hypolipidemic effect of an exo-biopolymer produced from a submerged mycelial culture of Hericium erinaceus was investigated in dietary-induced hyperlipidemic rats. Hypolipidemic effects were proportionally increased with the increasing concentration of the exo-biopolymer for oral administration. The exo-biopolymer, at the dose of 200 mg/kg body weight, substantially reduced the plasma total cholesterol (32.9%), LDL cholesterol (45.4%), triglyceride (34.3%), phospholipid (18.9%), atherogenic index (58.7%), and hepatic HMG-CoA reductase activity (20.2%). It increased the plasma HDL cholesterol level (31.1%) as compared to the control group. The molecular mass of this exo-biopolymer measured by HPLC was under 40 kDa. Total sugar and protein contents were 91.2 and 8.8%, respectively. The sugar and amino acid compositions of the exo-biopolymer were analyzed in detail.     

九州虫草菌丝体对Mn的耐性及富集

重金属耐性真菌的研究是生物修复的重要研究内容。本文研究了九州虫草（Cordyceps kyusyuensis）对于Mn的耐性及富集。在液体培养基中添加不同浓度（0—60 g/L）的Mn离子,测定其菌丝生物量、菌丝Mn含量、菌丝抗氧化酶活性和过氧化水平以及菌体细胞离子交换量、Mn在细胞中的分布的变化情况。实验结果表明九州虫草菌丝生物量与Mn浓度呈显著负相关,Mn浓度60 g/L为九州虫草菌丝生长极限浓度。菌丝中Mn含量随培养基中Mn浓度的增大而显著升高,10 g/L Mn时,菌丝细胞中Mn积累量达到细胞干重的1.0013%。九州虫草菌丝中过氧化产物丙二醛（MDA）、可溶性蛋白（SP）含量、可溶性糖浓度与培养基中Mn浓度呈负相关,实验组与对照组差异显著。抗氧化酶（过氧化氢酶（CAT）、过氧化物酶（POD）、超氧化物歧化酶（SOD））活性随着培养基中Mn浓度增大而显著升高,但变化趋势不同。九州虫草菌丝细胞不可溶性组分中Mn的量（91.51%—98.6%）显著高于可溶部分（1.40%—8.49%）。九州虫草菌丝细胞壁离子交换量（CEC）随着培养基中Mn浓度的升高变化不明显。说明在九州虫草菌丝对Mn的富集过程中,其细胞壁、细胞膜和细胞器对于Mn结合发挥了主要作用,细胞质中可溶性成分对Mn的结合发挥次要作用。在Mn的胁迫下,增强抗氧化酶系统的协同作用以清除大量自由基是细胞对锰耐性的重要机制。     

不同培养条件对中国被毛孢胞内核苷类组分的影响

采用紫外分光光度法,对冬虫夏草Cordyceps sinensis无性型——中国被毛孢Hirsutella sinensis液体摇瓶培养时胞内核苷类组分的形成积累条件进行了研究。结果表明:菌丝体中核苷类成分形成积累的最适摇瓶培养条件是碳源为葡萄糖:氮源为酵母浸出粉:微量元素为MgSO_4·7H_2O和KH_2PO_4;起始pH值6．5:摇瓶装液量100mL/500mL:摇床转数180r/min:接种量10％。正交试验结果表明最佳培养基配方为葡萄糖2％,酵母浸出粉3％,奶粉1％,MgSO_4·7H_2O 0．1％,KH_2PO_40．2％。培养条件优化后,菌丝体中总核苷的含量可达6．5748mg/g,比优化前提高了33．6％。     

桑木层孔菌液体培养条件的研究

对桑木层孔菌(Phellinus mori)液体发酵条件进行了研究,以生物量和胞外多糖为指标,通过L16(45)和L9(34)正交表进行了两次正交试验,筛选出桑木层孔菌最适液体培养条件为:麦芽糖30 g/L,酵母浸粉和蛋白胨15 g/L(质量比2 1),KH2PO4和CaCl25.5 g/L(质量比1 1),初始pH6.0;通过单因素试验筛选出最适装液量为120 mL/250 mL,最适接种量为10%。在此条件下液体发酵培养7 d后,桑木层孔菌生物量达到23.375 g/L,胞外多糖产量达到3.993 g/L。     

The effect of Zn on the Zn accumulation and biosynthesis of amino acids in mycelia of Cordyceps sinensis

Zinc is an essential trace element in the human body and it participates in various pathways of metabolism. Cordyceps sinensis is a wellknown traditional Chinese medicine that contains cordycepin, cordycepic polysaccharides, proteins, vitamins, trace elements, and many other biological active materials. In this study, we cultured C. sinensis in liquid medium containing Zn ions and then analyzed the biomass, the ratios of total Zn accumulation, and the organic Zn content in the mycelia. The results showed that when the media contain 150 mg/L Zn, the biomass of mycelia of C. sinensis reached 10.7 g/L and the Zn content present in the mycelia reached 4875.1 microg/g. The percentage of Zn accumulated in the mycelia was 34.05% of the total Zn in the media, of which the organic Zn accounted for 76.33%. The results also revealed that the content of total amino acid (TAA) and essential amino acid (EAA) in the mycelia were increased substantially TAA and EAA in the Zn-treated mycelia were 11.1% and 15.2% higher, respectively, than that in the mycelia without Zn treatment. These results will be useful for elucidation of the physiological mechanism of Zn effects on the biological metabolism in the mycelia of C. sinensis.     

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.     

Establishment of cultivating strategy for highly aggregated mycelia of Morchella esculenta in a stirred-tank bioreactor

Mycelia of Morchella esculenta were found to aggregate rapidly in a submerged culture, which caused the decrease in dispersed mycelia and the problem of diffusion limitation. The effect of different agitation schemes on the growth of mycelia was investigated in a stirred-tank bioreactor. At the constant speed of 100 or 300?rpm, rapid aggregation caused the biomass concentration to drop to zero in 30?h, which was even worse than achieved under static culture. Intermittent agitation maintained a higher mycelium fragment concentration for 48?h and enhanced the biomass concentration to 4.73?g/L at 120?h. The operation with a polytron connection disrupted effectively mycelium aggregation, thus increasing the specific growth rate, biomass concentration and maximum productivity to 0.0613 1/h, 7.73?g/L and 0.0878?g/L?h at 88?h, respectively. Moreover, logistic equations and genetic algorithm (GA) were used for the simulation of biomass growth and estimation of all kinetic coefficients. The operating strategy developed in this study could be used for the production of highly aggregated mycelia, which could also achieve a high cell-density culture in a stirred tank reactor.     

β-蜕皮激素对蛹虫草液体培养菌丝体生长及代谢产物的促进作用

The effects of β-ecdysone on mycelial dry weight (MDW) and the yield of extracellular polysaccharide (EPS), intracellular polysaccharide (IPS) and cordycepin of Codyceps militaris in submerged cultivation was investigated. Adding 12mg/L of β-ecdysone to the culture medium, the MDW, the secretion volume of EPS per unit of MDW and total yield of EPS were increased by 18.5%, 181.7% and 63.2%, respectively. The yield of IPS was enhanced by 29.2%, when 1.2mg/L of β-ecdysone was added. When 4mg/L of β-ecdysone was provided, the amount of cordycepin per unit of MDW and the total yield of cordycepin were increased by 65.4% and 84.7%, respectively. These results demonstrated that β-ecdysone promoted the yield of MDW, EPS, IPS and cordycepin of Codyceps militaris mycelia, though this effect varied among different components. Significantly, β-ecdysone showed a more positive role in secondary metabolite (cordycepin) accumulation than in primary metabolite (IPS) production and growth of mycelia. Furthermore, β-ecdysone stimulated the secretion of EPS.     

β-蜕皮激素对蛹虫草液体培养菌丝体生长及代谢产物的促进作用(英文)

The effects of β-ecdysone on mycelial dry weight (MDW) and the yield of extracellular polysaccharide (EPS), intracellular polysaccharide (IPS) and cordycepin of Codyceps militaris in submerged cultivation was investigated. Adding 12mg/L of β-ecdysone to the culture medium, the MDW, the secretion volume of EPS per unit of MDW and total yield of EPS were increased by 18.5%, 181.7% and 63.2%, respectively. The yield of IPS was enhanced by 29.2%, when 1.2mg/L of β-ecdysone was added. When 4mg/L of β-ecdysone was provided, the amount of cordycepin per unit of MDW and the total yield of cordycepin were increased by 65.4% and 84.7%, respectively. These results demonstrated that β-ecdysone promoted the yield of MDW, EPS, IPS and cordycepin of Codyceps militaris mycelia, though this effect varied among different components. Significantly, β-ecdysone showed a more positive role in secondary metabolite (cordycepin) accumulation than in primary metabolite (IPS) production and growth of mycelia. Furthermore, β-ecdysone stimulated the secretion of EPS.     

基于人工神经网络-遗传算法的樟芝发酵培养基优化

采用优化模型对药用丝状真菌樟芝的复杂发酵过程进行建模,并获得最优发酵培养基组成.对樟芝发酵过程中的形态变化过程进行了观察,并分别采用人工神经网络(ANN)和响应面法(RSM)对樟芝发酵过程进行建模,同时采用遗传算法(GA)优化了发酵培养基组成.结果表明,ANN模型比RSM模型具有更好的实验数据拟合能力和预测能力,GA计算得到樟芝生物量理论最大值为6.2 g/L,并获得发酵最佳接种量及培养基组成:孢子浓度1.76× 105个/mL,葡萄糖29.1 g/L,蛋白胨9.4 g/L,黄豆粉2.8 g/L.在最佳培养条件下,樟芝生物量为(6.1±0.2)g/L.基于ANN-GA的优化方法可用于优化其他丝状真菌的复杂发酵过程,从而获得生物量或活性代谢产物.     

The use of additives as the stimulator on mycelial biomass and exopolysaccharide productions in submerged culture of Grifola umbellata

In this study, various additives including organic acids, alcohols, vegetable oils, surfactants and polymers were added in the cultural medium to investigate their stimulatory effects on Grifola umbellate mycelia growth and exopolysaccharide (EPS) production. It was found that the commonly used stimulatory additives, effective in other mushrooms’ cultures, exhibited negative results in Grifola umbellata submerged culture. In contrast, the polymer additive, polyethylene glycol (PEG), displayed an effective stimulatory effect on both biomass and EPS productions. With the addition of PEG8 (molecular weight: 8,000 Da), the mycelial biomass production at day 12 was increased from 4.69 to 6.30 g/L, accounting for a 34% increase. Meanwhile, the EPS production was enhanced from 0.478 to 0.767 g/L, accounting for 60% increase.     

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)。     

复合有机氮源对灵芝三萜液态深层发酵的影响

以灵芝为材料,在前期研究基础上,以不同酵母粉作为氮源,研究复合有机氮源对灵芝三萜液态深层发酵的影响。首先,由单因素实验考察3种不同的酵母粉对灵芝菌丝体合成灵芝三萜的影响,确定酵母粉的最适宜浓度范围。在此基础上,根据中心组合实验设计,对3种酵母粉分别采用2种复合和3种复合的方式,优化复合有机氮源的最佳组合配比。结果表明：当基础培养基中添加6.6g/L的酵母粉N-1与6.6g/L的酵母粉N-2时,灵芝三萜产量可达0.478g/L（理论产量为0.485g/L）,比添加单一酵母粉N-1、N-2、N-3分别提高了21%、139%、103%,其氮源用量为两种组合时最低。当基础培养基中酵母粉N-1、N-2与N-3添加量分别为5.07g/L、3.78g/L、7.63g/L时,灵芝三萜产量达0.514g/L（理论产量为0.510g/L）,比单因素对照组分别提高了30%、157%和74%。本研究优化的复合氮源添加方式可明显提高灵芝菌丝体液态深层发酵生产灵芝三萜的产量,为其规模化液态深层发酵的生产提供科学数据。     

Optimization of nutritional requirements and feeding strategies for clavulanic acid production by Streptomyces clavuligerus

The present work reports the nutritional requirements and environmental conditions for submerged culture of Streptomyces clavuligerus for clavulanic acid production using orthogonal matrix method (Taguchi L(16) design) and also fed-batch fermentation for clavulanic acid production by feeding glycerol, arginine and threonoine to the fermentation medium intermittently. Clavulanic acid production was increased by 18% with the span of feeding glycerol and reached a maximum at 1.30mg/ml with 120h glycerol feeding as compared to 1.10mg/ml in the control. The production also increased with the span of feeding amino acids and reached a maximum of 1.31 and 1.86mg/ml with feeding arginine and threonine, respectively in 120h. There was an overall increase of 18% and 9% in clavulanic acid production with arginine and threonine feeding as compared to the respective controls (1.10 and 1.70mg/ml, respectively).