As confirmed in previous studies, DOW can enhance production of the functional ingredients of red mold dioscorea such as monascin and ankaflavin as well as reduce the production of hepatotoxin, nephrotoxin, and citrinin (Lee et al. 2011). DOW can also promote production of A. camphorata mycelium as well as increase production of the functional ingredients of triterpenoids, polysaccharide, and flavonoids (Wang et al. 2013a). This study focuses on submerged culture to investigate the effect of DOW on production of C. militaris mycelium and the functional ingredients of C. militaris. Table 1 demonstrates that DOW promoted the production of C. militaris mycelium more than UPW did and significantly increased the production of adenosine (p < 0.05). This result agrees with those of previous studies except for DOW significantly affecting the production of intracellular and extracellular β-1,3-glucan (p < 0.05), a phenomenon that was not observed in this study.
Moreover, this study addressed the effect of DOW of various concentrations on the production of cordycepin and adenosine in C. militaris-fermented products. In this study, cordycepin production increased as the concentration of DOW increased. The content of cordycepin in the 30X-DOW solution reached the highest point on the 80th day of fermentation (p < 0.01). The DOW of various concentrations did not significantly enhance the production of adenosine (p > 0.05). The results were compared with the SW results. Mg(NO3)2 promoted cordycepin production (p < 0.05), NaCl significantly suppressed cordycepin production (p < 0.001), and KCl reduced cordycepin production. However, NaCl and KCl had Cl−, whereas CaCO3 and (NH4)2Fe(SO4)2 had no significant effect on cordycepin production (p > 0.05). Furthermore, SW can increase cordycepin production to a degree similar to that of DOW. (NH4)2Fe(SO4)2 increases adenosine production (p < 0.05). However, the other four salt solution did not have this effect (p > 0.05). SW can also increase adenosine production to a degree similar to that of DOW.
The results illustrated that all the metal ions in DOW can either increase or not affect cordycepin production. However, the production of cordycepin in NaCl solution significantly decreases after fermentation. Na+ constitutes a large proportion of DOW. This study compared the effects of DOW and various sodium salt solutions with concentrations equal to that of DOW on the production of cordycepin and adenosine. At the 30-fold sodium salt concentration, NO3
− increased the production of cordycepin (p < 0.01) and adenosine (p < 0.05) the most; however, it had no effect on adenosine production. SO4
2− and PO4
− had no effect on the production of cordycepin and adenosine. SW did not significantly increase cordycepin production but significantly increased the production of adenosine (p < 0.05). At the 60-fold sodium salt concentration, the production of cordycepin and adenosine with SW, NO3
−, and PO4
− decreased. At this concentration, the cordycepin production with SO4
2− declined as well, but the adenosine production increased significantly (p < 0.05). Concurrently, Cl- of the 60-fold concentration significantly reduced cordycepin production (p < 0.001). The NO3
−, SO4
2−, PO4
−, and Cl− used in this study refer to sodium salts. The NaCl solution reduced cordycepin production, whereas the NaNO3 solution increased cordycepin production. Therefore, we conclude that NO3
− can increase cordycepin production, that Cl− may reduce cordycepin production, and that NaCl solution has no effect on adenosine production. Nevertheless, the NaNO3 solution promoted adenosine production. At the 60-fold sodium salt concentration, only the Na2SO4 solution enhanced adenosine production. Therefore, NO3
− and SO4
2− may enhance adenosine production, whereas Cl− does not affect adenosine production.
Regarding to the effect of nitrates of various compositions on the production of cordycepin and adenosine (Fig. 4), Mg(NO3)2, NaNO3, Ca(NO3)2, and Fe(NO3)2 significantly increased cordycepin production (p < 0.01). However, NaNO3 did not have this effect (p > 0.05). Mg(NO3)2, NaNO3, KCl, Ca(NO3)2, and Fe(NO3)2 significantly increases adenosine production (p < 0.001). Because equal amounts of NO3
− produces equal effects, Mg2+, Na+, Ca2+, and Fe2+ are estimated to be primarily responsible for increasing production of cordycepin and adenosine. In addition, K+ is likely to significantly increase adenosine production.
According to Fig. 5, Cl− can suppress cordycepin production. This study used various chloride salts to investigate the effect of metal ions on the production of cordycepin and adenosine. All the chloride salts, MgCl2, NaCl, and KCl significantly reduced cordycepin production (p < 0.05). Furthermore, CaCl2 and FeCl2 reduced cordycepin production. Therefore, the SW composed of the five chloride salts significantly reduced cordycepin production (p < 0.05). Furthermore, MgCl2, NaCl, KCl, and FeCl2 significantly increased adenosine production (p < 0.05). However, CaCl2 did not increase adenosine production (p > 0.05). SW increased adenosine production (p < 0.05). The addition of Mg(NO3)2, NaNO3, KNO3, Ca(NO3)2, or Fe(NO3)2 can significantly enhance cordycepin production. However, in all the chloride salts, MgCl2, NaCl, KCl, CaCl2, and FeCl2 either significantly reduced cordycepin production or caused a declining trend in cordycepin production. Therefore, Cl− might contribute to reducing cordycepin production in C. militaris-fermented products.
The effect of SW on the production of cordycepin and adenosine varied depending on the combination of sodium salts. As shown in Fig. 3, Na-SW composed of various sodium salts had no significant effect on cordycepin production but enhanced adenosine production. The increase of cordycepin production might have been attributed to NaNO3. Moreover, NaCl suppressed the production of cordycepin, and the effect of Na2SO4 and NaPO4 on cordycepin production was not significant. Therefore, the combined effect of the four sodium salts rendered the resultant Na-SW least effective for promoting cordycepin production. Moreover, NaNO3 enhanced adenosine production, whereas Na2SO4, NaPO4, and NaCl had no effect on adenosine production. Hence, the combination of these four sodium salts resulted in the Na-SW that significantly promoted adenosine production. Figure 5a, b show that the Cl-SW of various combinations of chloride salts suppressed cordycepin production and promoted adenosine production. This Cl-SW effect might have occurred because MgCl2, NaCl, and KCl were all active in suppressing cordycepin production, and CaCl2 and FeCl2 were not significantly associated with cordycepin production. Therefore, the Cl-SW comprising the five chloride salts reduced cordycepin production. Moreover, except for CaCl2, the other chloride salts increased adenosine production. Therefore, the Cl-SW increased adenosine production. As confirmed in Figs. 2 and 5 demonstrate that Salt-SW increased the production of cordycepin and adenosine, and the Mg(NO3)2 sodium salt should be the functional salt for increasing the production of cordycepin and adenosine. The results of fermentation in various chloride salt mediums revealed that NaCl and KCl suppressed cordycepin production but enhanced adenosine production. This result is verified in Fig. 5a, b. However, CaCO3 and (NH4)2Fe(SO4)2 least enhanced cordycepin production, whereas (NH4)2Fe(SO4)2 increased adenosine production (Fig. 2). Figure 4a, b imply that Ca2+ and Fe2+ are likely to enhance the production of cordycepin and adenosine. Hence, the Salt-SW comprising Mg(NO3)2, NaCl, KCl, CaCO3, and (NH4)2Fe(SO4)2 enhanced production of cordycepin and adenosine. Furthermore, Salt-SW produced by combining sodium and chloride salts increased adenosine production to the same extent that DOW did. However, the Na-SW comprising only sodium salts increased cordycepin production to nearly the same extent that DOW did.
The study by Cui and Zhang (2012) noted that the addition of Mg2+ and Mn2+ in the cultivation of C. militaris in submerged culture significantly promoted C. militaris mycelium and extracellular polysaccharide production. Moreover, adding sodium selenite into the solid culture mediums for C. militaris cultivation significantly increased the production of cordycepin, cordyceps acid, cordyceps polysaccharide, and organic selenium of the C. militaris fruit body compared with the control group that had no sodium selenite. This production increase was proportional to the concentration of sodium selenite (Dong et al. 2012). The addition of 10 mM Ca2+ in Ganoderma liquid culture mediums can significantly increase the production of Ganoderma acid (Xu and Zhong 2012). In summary, compared with UPW, DOW can more effectively promote the growth of C. militaris mycelium and enhance the production of cordycepin, its functional ingredient. Moreover, the concentration of DOW has a dose effect on cordycepin production. The enhancing effect of DOW on cordycepin production is mainly attributed to its ions such as Mg2+, Na+, Ca2+, Fe2+, and NO3
−. However, the Cl− in DOW suppresses cordycepin production. For adenosine, Mg2+, Na+, K+, Ca2+, Fe2+ and SO4
2− have the ability of increasing its production, but such an ability is not observed for Cl−. Furthermore, the DOW ions can increase cordycepin production. This ability might be induced by the pressure of the C. militaris growth environment, which forces C. militaris to enter the secondary metabolic phase and begin producing secondary metabolites. This ability could stem from ions in DOW that serve as enzyme or protein cofactors in cultivating C. militaris and produce cordycepin to promote the growth of mycelium and production of the functional ingredients of C. militaris. The effect of Na-SW on the production of cordycepin and adenosine varies depending on the combination of sodium salts. Therefore, the production of cordycepin can be increased by adding nitrates and reducing the production of Cl− in DOW. Moreover, chloride salts, nitrates, and SO4
2− can be added to DOW to increase adenosine production.
This study employed DOW to cultivate C. militaris in submerged and solid culture and investigated the effect of DOW on the production of C. militaris’ fermentative products, namely cordycepin and adenosine. The results demonstrated that using 30X-DOW as a water source to produce C. militaris-fermented products can significantly increase the production of cordycepin. This cordycepin production is 65 % higher than the C. militaris-fermented products of the UPW water solution (p < 0.001). The concentration of DOW also has a dose effect on cordycepin production. Moreover, the Mg2+, Na+, Ca2+, Fe2+, and NO3
− in DOW increase cordycepin production; however, the Cl− in DOW exhibits an opposite effect. Mg2+, Na+, K+, Ca2+, Fe2+, and SO4
2− can increase adenosine production, but Cl− cannot. The Cl-SW containing MgCl2, NaCl, KCl, CaCl2, FeCl2 can not only increase adenosine production but also promote cordycepin production to nearly the same extent that DOW does.