Regular articleAnalysis of exopolysaccharide production patterns of Cordyceps militaris under various light-emitting diodes
Introduction
Cordyceps militaris, a Chinese medicinal mushroom, is an entomopathogenic fungus belonging to the class Ascomycetes. This particular Cordyceps species has been used for medicinal purposes in China, Japan, Korea and other Asian countries. C. militaris has been reported to possess many biological and pharmacological activities, including anti-inflammatory, anti-oxidative, anti-tumor, anti-proliferative and anti-diabetic. These were generally attributed to the presence of bioactive components like adenosine, cordycepin and exopolysaccharides (EPS) [1], [2], [3].
EPS are a class of biopolymers that are secreted extracellularly by mushrooms in order to withstand adverse conditions [4]. EPS produced by some mushrooms were reported to possess anti-tumor activities [5], [6], [7], [8]. EPS from Antrodia cinnamomea mycelia with a molecular weight higher than 100 kDa were reported to give an immune modulatory effect against angiogenesis [9]. Lavi et al. proposed that low molecular weight polysaccharides (less than 145 kDa) from Pleurotus ostreatus display various antioxidant activities that are capable of inducing apoptosis in colon cancer cells, thereby inhibiting their proliferation [10]. Sun et al. indicated that EPS from Porphyridium cruentum with a low molecular weight of 6.5 kDa exhibited greater antioxidant activity than EPS with a molecular weight higher than 61 kDa [11]. Due to their health and medical benefits, EPS are widely used in the food, biotechnology and pharmaceutical industries [12].
Different approaches to EPS production from C. militaris have been reported in previous studies. Shih et al. reported that the best nitrogen source for EPS production was obtained from a submerged culture of C. militaris CCRC32219 with yeast extract [1]. Cui et al. proposed a two-stage culture method for EPS production [13] and the highest levels were obtained in medium containing Mg2+ [14]. Lin et al. reported on the use of ultraviolet mutagenesis to derive a mutant strain, C. militaris SU5-08, which had the ability to give a higher EPS production [15]. Although the above studies all aimed for high EPS production, none focused on the effects of light on C. militaris growth and EPS production in a submerged culture.
Light, a kind of energy, affects the growth and metabolism of many plants and microalgae [16]. Danesi et al. reported that the production of biological compounds in Spirulina platensis, such as carotenoid, phycocyanin and chlorophyll A, were dramatically affected by the use of various light sources [17]. In some cases, light wavelength may also influence fungal growth and metabolites. Some fungi, such as Aspergillus nidulans, A. fumigatus, Gibberrella moniliformis, Ustilago maydis and Cryptococcuus neoformans, were reported to carry photochrome, a photoreceptor that senses red and far-red light [18]. Furthermore, some pigment-producing filamentous fungi are affected by light wavelength. Velmurugan et al. found that red light enhanced the pigment yield of Monascus purpureus and Penicillium purpurogenum while in a fermentation broth [19]. Dong et al. discovered that some bioactive components in the fruiting bodies of C. militaris, such as adenosine, carotenoid and cordycepin, were significantly enhanced by the regulation of the light wavelength [20]. For instance, light with a short wavelength stimulated the production of carotenoids, suggesting that C. militaris is a light sensitive fungus; its growth and metabolite production may be influenced by illumination conditions[20].
Although the effect of light on the growth of C. militaris fruiting bodies during solid-state cultivation has been observed [20], few studies examined the kinetics data on the growth and metabolite production of C. militaris in an illuminated submerged culture. For the kinetic study, the logistic equation was used to estimate the mycelial growth of fungi like Armillaria luteo-virens Sacc. and Pleurotus mutilis, while the Luedeking-Piret equation was used to predict the production of metabolites like EPS and pleuromutilin [21], [22].
To date, studies of C. militaris during submerged cultivation focused mainly on growth factors, biomass production and metabolite production. None have focused on the effects of light on biomass or metabolite production. In this study, light-emitting diodes (LEDs) with various wavelengths were applied to a submerged culture of C. militaris. The effects of light wavelength on mycelial growth and EPS production were then investigated. A modified logistic equation and the Luedeking-Piret equation were used to simulate mycelial biomass and EPS production. The quantitative analyses of light wavelength on biomass and EPS production rates during cultivation were also examined.
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Strain and cultivation conditions
C. militaris (BCRC 33735) was purchased from the Culture Collection Research Center at the Food Industry Research and Development Institute (Hsinchu, Taiwan). The strain was cultured on a Potato Dextrose Agar (PDA) plate at 25 °C for 7 days. The seed medium (100 ml) containing 4% (w/v) glucose, 1% yeast extract, 0.05% KH2PO4, 0.05% K2HPO4, 0.05% MgSO4·7H2O and 0.01% FeSO4·7H2O (pH adjusted to 6.0) was inoculated with two units of cutter square (2cm × 2 cm) mycelium. Cultivation was carried out on a
Light effect on hyphal growth in a solid-state culture
LEDs with different wavelengths were used on C. militaris in a solid-state culture in order to observe hyphal growth (Table 1). According to our data, green and red lights gave the best growth rates at 4.33 and 4.37 mm/day, respectively, very close to the results under dark conditions. Blue and white lights, however, yielded lower growth rates of 3.59 and 3.39 mm/day, respectively. As seen in Fig. 1, the hyphae of C. militaris grown slowly under white, yellow and blue lights became thicker than
Conclusion
In this study, various wavelengths of LEDs were used to examine biomass and EPS production during C. militaris cultivation. The experimental results demonstrated that while both could be enhanced under illuminated conditions, the largest biomass growth rate could be obtained using red LED light. In contrast, the comparatively poor growth under yellow light was likely due to the lack of yellow photoreceptors in C. militaris cells. The logistic rate equations predicted the larger biomass growth
Acknowledgement
This work was supported by the research grant provided by the Ministry of Science and Technology, R.O.C (Grant No. MOST103-2221-E-005-071-MY3).
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2021, International Journal of Biological MacromoleculesCitation Excerpt :The exopolysaccharides are crucial bioactivity ingredients of C. militaris and have attracted attentions of researchers. Numerous literatures paid attentions on the enhancement of production through fermentation conditions, submerged culture process, metal ions and surfactants, various lights and so on [48–51]. Some literatures focused on the exploration of the bioactivities of exopolysaccharides produced by C. militaris.