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.     

Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris

The influence of initial pH value, various nitrogen sources, plant oils, and modes of propagation (shake-flask and static culture) on the production of biomass, exopolysaccharide (EPS), adenosine and, in particular, cordycepin, by Cordyceps militaris CCRC 32219 were investigated. Optimal conditions for mycelial growth, EPS and cordycepin production were observed at relatively low pH. Amongst organic sources, yeast extract (YE) was favorable for EPS and cordycepin production, while corn steep powder (CSP) was favorable for adenosine production. A lower C/N ratio was favorable for adenosine and cordycepin production; however, too low a C/N ratio led to diminished production. All plant oils tested stimulate mycelial growth and EPS production of C. militaris, but they did not show much effect on the adenosine and cordycepin production. A two-stage fermentation process by combining shake-flask fermentation with static culture significantly enhanced cordycepin production. A Box–Behnken experimental design was employed to optimize the production of cordycepin, which showed that the optimum conditions to produce cordycepin by C. militaris CCRC 32219 were at pH 6, YE concentration of 45 g/l and 8.0 day of the shake culture followed by 16 days of the static culture. Under the optimized conditions, the maximum production (2214.5 mg/l) of cordycepin was obtained, which is much higher than those reported up to date.     

Optimization of fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05

The optimal fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05 were investigated. The medium components and fermentation conditions were optimized according to the one at a time method, while the concentration of medium components was determined by the orthogonal matrix method. The results showed that the optimal fermentation medium was as follows: sucrose 3.5% (w/v), peptone 0.5%, yeast extract 0.2%, K(2)HPO(4) 0.1%, and MgSO(4) 0.05%. The suitable fermentation conditions were as follows: initial pH 7.0, temperature 25 degrees C, medium volume 75 mL/250 mL, inoculum volume 5% (v/v), time 5d. In such optimal nutrition and environmental conditions, the maximal mycelial yield was 2.124 g/100 mL after 4 day's fermentation, while maximal water-soluble exo-polysaccharides production reached 2.144 g/L after 5 day's fermentation.     

Optimization of culture medium for cordycepin production using Cordyceps militaris mutant obtained by ion beam irradiation

The effect of medium components on cordycepin production by Cordyceps militaris mutant obtained by ion beam irradiation was investigated. According to the response surface analysis using a central composite design for the prospective mutant G81-3, the predicted optimal concentrations of glucose as the carbon source and the yeast extract as the nitrogen source were 86.2 g/l and 93.8 g/l, respectively, and 6.84 g/l cordycepin was obtained. To date, this is the highest value for cordycepin production. The optimal concentrations of glucose and yeast extract for cordycepin production of the mutant was much higher than that of control (wild strain) and the cordycepin production was 2.79 times higher. Therefore, this new mutant will be a promising strain for future higher cordycepin production at industrial levels.     

Optimization of fermentation medium for Cordyceps militaris high producing strain

在单因素试验初步确定高产蛹虫草菌株发酵培养基的基础上,以蛹虫草菌丝体中腺苷含量为指标,进行11因素2水平Plackett - Burman试验设计试验,结合多元一次回归模型和F检验方法,筛选出发酵培养基中影响显著的组分酵母浸粉、蔗糖和维生素B1,采用旋转中心组合设计方法对这三个组分进行进一步优化,结合多元二次回归模型和响应面分析,获得高产蛹虫草菌株的最佳培养基(g/L):蔗糖18.85、蛋白胨10、酵母浸粉18.97、KH2 PO4 3、MgSO4 3、维生素B10.235、ZnCl2 0.011、(NH4)2SO4 10.验证试验结果表明蛹虫草腺苷得率较单因素优化获得的发酵培养基提高了26.91％.     

Optimisation of submerged culture conditions for the production of mycelial biomass and exopolysaccharide byPleurotus nebrodensis

The optimisation of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) fromPleurotus nebrodensis. The optimal temperature and initial pH for both mycelial growth and EPS production in shake flask cultures were 25 °C and 8.0, respectively. Maltose was found the most suitable carbon source for both mycelial biomass and EPS production. Yeast extract was favourable nitrogen source for both mycelial biomass and EPS production. Optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth and EPS production was as follows: 200 g l^?1 bran, 25 g l^?1 maltose, 3 g l^?1 yeast extract, 1 g l^?1 KH₂PO₄, 1 g l^?1 MgSO₄ 7H₂O. Under the optimal conditions, the mycelial biomass (4.13 g l^?1) and EPS content (2.40 g l^?1) ofPleurotus nebrodensis was 2.3 and 3.6 times compared to the control with basal medium respectively.     

Effect of agitation intensity on the exo-biopolymer production and mycelial morphology in Cordyceps militaris

AIMS: The influence of agitation intensity on Cordyceps militaris morphology and exo-biopolymer production was investigated in a 5 litre stirred vessel using a six-blade Rushton turbine impeller. METHODS AND RESULTS: The mycelial morphology of C. militaris was characterized by means of image analysis, which included mean diameter, circularity, roughness and compactness of the pellets. The morphological parameters of the pellets grown under different stirring conditions were significantly different, which correspondingly altered exo-biopolymer production yields. CONCLUSIONS: The compactness of the pellets was found to be the most critical parameter affecting exo-biopolymer biosynthesis; more compact pellets were formed at 150 rev min(-1) with maximum exo-biopolymer production (15 g l(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study suggest that morphological change of pellets is a good indicator for identifying the cell activity for exo-biopolymer production.     

高产蛹虫草菌株发酵培养基的研究

在单因素试验初步确定高产蛹虫草菌株发酵培养基的基础上,以蛹虫草茵丝体中腺苷含量为指标,进行11因素2水平Plackett—Burman试验设计试验,结合多元一次回归模型和F检验方法,筛选出发酵培养基中影响显著的组分酵母浸粉、蔗糖和维生素B1,采用旋转中心组合设计方法对这三个组分进行进一步优化,结合多元二次回归模型和响应面分析,获得高产蛹虫草菌株的最佳培养基（g／L）：蔗糖18．85、蛋白胨10、酵母浸粉18．97、KH2PO,3、MgSO4 3、维生素Bl0．235、ZnCl20．011、（NH4）2S0410。验证试验结果表明蛹虫草腺苷得率较单因素优化获得的发酵培养基提高了26．91％。     

Optimization of a solid culture medium based on Monochamus alternatus for Cordyceps militaris fruiting body formation

Monochamus alternatus (Coleoptera: Cerambycidae; M. alternatus), popularly known as the Japanese pine sawyer, is a vector of pinewood nematode (Bursaphelenchus xylophilus) that causes pine wilt disease. A solid medium culture with M. alternatus produced Cordyceps militaris fruiting bodies with the longest strips and the highest biological efficiency. Supplementing the original form of M. alternatus with oats resulted in slightly enhanced fruiting body production. The original form of M. alternatus showed higher production than its powder form. The solid culture medium was optimized using a response surface methodology, and the optimal medium contained the following: 8·5 g per bottle of M. alternatus and 11·5 g per bottle of oats mixed with 22·4 ml of water in a 300-ml cylindrical plastic bottle. The optimal culturing period for the fruiting body formation was 37·1 days. Under these conditions, a fruiting body dry weight of 38·0 g per bottle (actual value) was attained. The fruiting body produced using a solid culture medium based on M. alternatus had a cordycepin content of about 25 µg g⁻¹. The solid culture medium containing M. alternatus is highly efficient and eco-friendly, and its effectiveness in large-scale fruiting body production from C. militaris has been demonstrated.     

Optimization of submerged culture condition for the production of mycelial biomass and exopolysaccharides by Agrocybe cylindracea

The optimization of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) from Agrocybe cylindracea ASI-9002 using the statistically based experimental design in a shake flask culture. Both maximum mycelial biomass and EPS were observed at 25 degrees C. The optimal initial pH for the production of mycelial biomass and EPS were found to be pH 4.0 and pH 6.0, respectively. Subsequently, optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth was as follows: maltose 80 g/l, Martone A-1 6 g/l, MgSO4 x 7H2O 1.4 g/l, and CaCl2 1.1 g/l; for EPS production: maltose 60 g/l, Martone A-1 6 g/l, MgSO4 x 7H2O 0.9 g/l, and CaCl2 1.1 g/l. Under the optimal culture condition, the maximum EPS concentration achieved in a 5-l stirred-tank bioreactor indicated 3.0 g/l, which is about three times higher than that at the basal medium.     

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。     

Optimization of submerged culture requirements for the production of mycelial growth and exopolysaccharide by Cordyceps jiangxiensis JXPJ 0109

AIMS: The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. METHODS AND RESULTS: The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min(-1), seed age 4-8 days, inoculum size 2.5-7.5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26 degrees C and pH 5 and at 28 degrees C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl2 > glycerol > MgSO4 > KH2PO4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH2PO4, 0.2 g MgSO4, and 0.5 g CaCl2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH2PO4, and 0.5 g CaCl2 in 1 l of distilled water. CONCLUSIONS: Under the optimal culture requirements, the maximum exopolysaccharide production reached 3.5 g l(-1) after 10 days of fermentation, while the maximum production of mycelial growth achieved 14.5 g l(-1) after 8 days of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.     

Optimization of submerged culture conditions for mycelial polysaccharide production in Cordyceps pruinosa

Cordyceps pruinosa is an entomogenous fungus noteworthy for its various bioactivities. The influence of synthetic medium and cultural conditions on polysaccharides production was investigated in shake flask culture. In the present study, optimal medium and submerged culture conditions were investigated using an orthogonal layout. Media and cultural conditions including potato starch 2% (w/v), sucrose 2.5%, soybean 0.5%, beef extract 0.5%, yeast extract 0.1%, KCl 0.02%, K₂HPO₄ 0.1%, MgSO₄·7H₂O 0.05%, pH 7.0, inoculum size 5%, medium capacity 50 ml/250 ml flask, dispersant 15 beads, culture time 7 days were employed. In fermentation medium, sucrose, beef extract and yeast extract were replaced with molasses of sucrose, groundnut and Vitamin B complex, respectively. Under optimal culture conditions, the yield of polysaccharides production was 9.51 g l⁻¹ after 54 h of fermentation in a 25 l fermenter, which was approximately twice as high as that in shake flask cultures. In addition the entire period of fermentation was shorted to around 1/4 of flask culture time (9 days). Thus, it will meet closely the requirements of industrial fermentation scale of polysaccharides production in C. pruinosa.     

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.     

培养基和栽培方式对蛹虫草子实体活性成分的影响

采用麦粒（W）、麦粒加蚕蛹粉（WW）、大米（R）、大米加蚕蛹粉（RW）的配料栽培方式,以及蚕蛹（CY）活体接种栽培方式培养蛹虫草子实体,比较蛹虫草子实体中多糖及核苷、游离糖醇和小分子糖类含量。结果表明：培养基和栽培方式影响蛹虫草多糖含量及其单糖组成和分子量分布,多糖含量最高的为蚕蛹活体接种培养的处理（CY）,多糖含量为6.19%,其他处理的多糖含量仅为CY的44.4%-62.8%;蚕蛹（CY）上培养的蛹虫草子实体中核苷类成分含量最高,在麦粒上培养获得的子实体中核苷含量显著高于在大米上的处理,并且麦粒添加蚕蛹粉后培养的子实体中尿苷、鸟苷、N⁶-(2-羟乙基)腺苷含量显著上升,大米添加蚕蛹粉后培养的子实体中尿苷、虫草素、N⁶-(2-羟乙基)腺苷含量显著上升;配料栽培的4个处理,其子实体中海藻糖含量为20.07%-23.40%,蚕蛹活体接种的处理（CY）海藻糖含量显著降低,为8.41%;添加蚕蛹粉后子实体中甘露醇含量显著降低。对各成分含量的数据进行归一化处理后进行蛹虫草品质的综合评价,在蚕蛹上培养的蛹虫草综合评分最高,麦粒为主要培养基培养的蛹虫草品质好于大米为主要培养基的,且添加蚕蛹粉的处理优于未添加的处理。     

Enhancement of cordycepin production from Cordyceps militaris culture by epigenetic modification

Biotechnology Letters - Cordycepin (3′-deoxyadenosine) is a nucleoside analogue and biosynthesised by Cordyceps militaris, an entomopathogenic fungus. In this study, an epigenetic modifier...     

Comparison of culture methods on exopolysaccharide production in the submerged culture of Cordyceps militaris and process optimization

Aims: To improve exopolysaccharides (EPS) production of Cordyceps militaris (C. militaris), effects of different culture method on mycelial biomass and EPS production in the submerged culture of C. militaris were investigated. Methods and Results: A new two‐stage fermentation process for EPS production of C. militaris was designed in this work. Central composite design (CCD) was utilized to optimize the two‐stage fermentation process. The results showed that the two‐stage fermentation process for EPS production was superior to other culture method (conventional static culture and shake culture). CCD revealed that the optimum values of the test variables for EPS production were shaken for 140 h followed by 130‐h static culture. The maximum EPS production reached 3·2 g l^?1 under optimized two‐stage culture and was about 2·3‐fold and 1·6‐fold in comparison with those of original static culture and shake culture. Conclusions: It was indicated that a new two‐stage culture method obtained in this work possessed a high potential for the industrial production for EPS of C. militaris. Significance and Impact of the Study: The fundamental information obtained in this work is complementary to those of previous investigations on the submerged culture of C. militaris for the production of bioactive metabolites.     

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。