Effect of the salts of deep ocean water on the production of cordycepin and adenosine of Cordyceps militaris-fermented product

Cordyceps militaris is a type of entomogenous fungi and has been widely used as a medicinal fungus in Asia. Cordycepin produced by C. militaris has also been found to protect the liver. Moreover, deep ocean water (DOW) was proven to increase the functional compounds of functional fungi-fermented products. However, the regulation of the metals in DOW is still unclear. Therefore, this study investigated the effect of DOW and certain major ions on the production of cordycepin and adenosine of C. militaris. The results indicated that, compared with using ultra-pure water (UPW), using DOW to cultivate C. militaris in a submerged culture increases the production of biomass and adenosine (p < 0.05). In the results of solid culture, the concentration of DOW exhibits a dose effect on cordycepin production. DOW contains ions that can improve the effectiveness of cordycepin, such as Mg2+, Na+, Ca2+, Fe2+, and NO3−, whereas the ion Cl− features an inhibitory effect. Moreover, Mg2+, Na+, K+, Ca2+, Fe2+, and SO42−can increase the production of adenosine, whereas Cl− cannot. However, the synthetic water made from various types of sodium salts (MgCl2, NaCl, KCl, CaCl2, FeCl2) had nearly the same effect on cordycepin production as that of DOW.


Introduction
Deep ocean water (DOW) is characterized by low temperature, cleanness, and affluent production of inorganic nutrients and minerals. In recent years, because of its medicinal value, DOW has been extensively studied. Previous studies have noted that DOW has several health benefits, such as mitigating skin inflammation (Hataguchi et al. 2005), lowering blood lipids (Miyamura et al. 2004), reducing blood sugar (Hwang et al. 2009a), and reducing obesity (Hwang et al. 2009b). Moreover, DOW can be used to increase production of functional ingredients of microorganisms (Lee 2015). Studies have found that DOW can increase the functional ingredient production of monascin and ankaflavin of red mold dioscorea as well as triterpenoids, polysaccharide, and flavonoids of Antrodia camphorata (Lee et al. 2011;Wang et al. 2013a). Red mold dioscorea cultured by DOW lowers blood lipids and obesity more than that cultured by reverse osmosis water (ROW) and ultra-pure water (UPW) (Lee et al. 2011;Wang et al. 2013b). Moreover, compared with A. camphorata cultured by ROW, A. camphorata cultured by DOW protects the liver more effectively (Wang et al. 2013a). However, functional ions and the effects of ions in DOW on the growth and metabolites production of the functional fungi are still unclear incurrent. It should be an important topic for the application of DOW in future.
Recently, Cordyceps militaris has been regarded as an alternative to Ophiocordyceps sinensis. C. militaris has a similar functional composition to that of O. sinensis (Wang and Yao 2011). C. militaris also has antioxidant (Yu et al. 2006), antiinflammatory (Won and Park 2005), and antitumor effects (Yoo et al. 2004) as well as enhances sexual performance (Chang et al. 2008). According to relevant studies, the activity levels of the essential amino acids, cordycepin, Cordyceps polysaccharide, and superoxide dismutase of artificially cultured C. militaris are all higher than those of wild O. sinensis (Dong et al. 2012).
This study used C. militaris BCRC 32219 as the testing strain and focused on solid culture to examine how various culture mediums and concentrations of DOW affect the production of adenosine and cordycepin of C. militaris. This study also analyzed the effect of the major ions in DOW on the production of adenosine and cordycepin of C. militaris by adding only one single type of salt.

Chemicals
Yeast mold (YM) agar and broth were purchased from Difco Laboratories Co. (Detroit, MI, USA). Magnesium, sodium, potassium, calcium, zinc and iron standard solutions were purchased from Showa Chemical Co. (Tokyo, Japan). Ethanol (95 %) was purchased from Taiwan Tobacco and Liquor Co. (Taipei, Taiwan). The other chemicals purchased from Sigma Chemical Co. (St. Louis, MO, USA).

The preparation of DOW, SW, and various salts water
The concentrated DOW purchased from the Taiwan Yes Deep Ocean Water Co. (Hualien, Taiwan) was pumped from a depth of 670 m in the Pacific Ocean near the Eastern Taiwan and processed though the electrodeionization. According to our previous study, DOW including 20.65 mg/L Mg 2+ was defined as onefold DSW (Wang et al. 2013a). In this study, 30-fold DOW (including 619.5 mg/L Mg 2+ ) was prepared by the dilution of concentrated DOW (including 43,400 mg/L Mg 2+ ) with UPW. The concentrations of the trace elements and minerals in 30X DOW included 619.5 mg/L Mg 2+ , 327 mg/L Na + , 132 mg/L K + , 5.13 mg/L Ca 2+ , 8.55 µg/L Fe 2+ , 1.156 mg/L nitrate, 649 mg/L sulfate, and 1.898 g/L chloride.
In the preparation of the various metals salts solution, Mg(NO 3 ) 2 , NaCl, KCl, CaCO 3 , and (NH 4 ) 2 Fe(SO 4 ) 2 solution were prepared according to the equal Mg 2+ , Na + , K + , Ca 2+ , Fe 2+ concentrations in 10X-DOW, respectively. The five salts solutions were further mixed for the preparation of the synthetic water of mixed salts (Salt-SW). In the preparation of the sodium salts solution, NaNO 3 , Na 2 SO 4 , Na 3 PO 4 and NaCl solutions were prepared according to equal SO 4 2− , PO 4 3− , NO 3 − , and Cl − concentration in 30X or 60X DOW, respectively. These four sodium salts solutions were mixed for the preparation of a synthetic water of mixed sodium salts (Na-SW). In the preparation of the nitrate salts solution Mg(NO 3 ) 2 , NaNO 3 , KNO 3 , Ca(NO 3 ) 2 , Fe(NO 3 ) 2 ·6H 2 O solutions were prepared according to equal Mg 2+ , Na + , K + , Ca 2+ , and Fe 2+ concentrations in 30X DOW, respectively. In the preparation of the chloride salts solutions, MgCl 2 , NaCl, KCl, CaCl 2 , FeCl 2 solutions were prepared according to equal Mg 2+ , Na + , K + , Ca 2+ , and Fe 2+ concentrations in 30X DOW, respectively. These five chloride salts solutions were further mixed for the preparation of a synthetic water of mixed chloride salts (Cl-SW).

Microorganism and seed cultures
C. militaris BCRC 32219 was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan). C. militaris was maintained on YM agar at 24 °C and transferred to fresh medium for 10 days intervals. Seed cultures were prepared by transferring a loopful of colony from YM agar slant into a 500-mL Hinton flask containing 100 mL medium (3 g/L yeast extract, 5 g/L malt extract, 10 g/L peptone, 3 g/L dextrose). The cultures were incubated at 28 °C and 100 rpm for 5 days. After that, inoculum sizes 5 % was transferred to submerged or solid cultured substrate.

Submerged fermentation of C. militaris in DOW or UPW
Submerged fermentation was carried out using a 500-mL Hinton flask containing 100-mL medium (3 g/L yeast extract, 5 g/L malt extract, 10 g/L peptone, 3 g/L dextrose in UPW or DOW). The cultures were incubated at 28 °C for 5 days at 100 rpm. After submerged culture, mycelium and filtrate were separated using filter paper. The mycelium was dried by freeze dryer and then weighted. The dried mycelium powder and fresh filtrate was analyzed for intracellular and extracellular β-1,3 glucan, respectively.

Solid fermentation of C. militaris in DOW or ROW
Thirty grams oat substrates was soaked in 30 mL UPW, DOW, SW, or various salts water, and then was autoclaved for 20 min at 121 °C in a 500-mL glass bottle. After being cooled, the substrate was inoculated with a 10 % (v/w) seed culture medium. The inoculated substrate was cultured at 24 °C for 20 days in a dark incubator. After dark culture, C. militaris was then cultured at 14 °C under a 12 h light:12 h dark cycle (light on at 6:00) for 60 days. After fermentation, the crushed and dried product was used for the experiments.

Determination of β-1,3 glucan
The selective aniline blue reaction was employed to detect the existence of β-1,3 glucan. The method described by the previous studies (Wood and Fulcher 1984;Young and Jacobs 1998) were followed with some modifications. The sample was dissolve with 0.3 N NaOH and stirred at ambient temperature for completely dissolve. The pH of the sample solution was then adjusted to 11.5 ± 0.1 by adding 1 N HCl and the volume was made to 10 mL using Na 2 HPO 4 -NaOH buffer (pH 11.5 containing 0.5 M NaCl). A 0.2 mL of sample was reacted with 0.1 mL aniline blue (1 mg/mL) for 2 h at ambient temperature. The excitation and emission wavelength were set at 395 and 495 nm, respectively.

Determination of cordycepin and adenosine
The powder of C. militaris-fermented product (0.1 g) was extracted respectively with 1 mL of methanol at 50 °C for 1 h. The extracts (10 %, w/v) were further filtered with 0.45 μm pore size filter and analyzed by HPLC (Model L-2130, Hitachi Co., Tokyo, Japan) on a C 18 column (25 cm × 4.6 mm i.d., 5 μm, Luna ® , Phenomenex, Torrance, CA, USA) using the gradient elution. HPLC was performed according to the method described previously (Yu et al. 2007) in triplicate. Cordycepin and adenosine were separated by gradient elution using the mobile phase with the composition of water-methanol (95.0/5.0 to 58.4/41.6 in 20 min, v/v). The flow rate was set at 0.8 mL/min. Cordycepin and adenosine were detected using a photodiode array detector (Model L-2455 DAD, Hitachi Co.) set at 260 nm and full wavelength.

Statistical analysis
Data are expressed as mean ± standard deviation. Analysis of variance by Duncan's test and Pearson's productmoment correlation coefficient test were determined using SPSS version 10.0 software (SPSS Institute, Inc., Chicago, IL, USA). Differences with p < 0.05 were considered statistically significant.

Effect of deep ocean water on the production of mycelium and certain functional ingredients under submerged culture
This study investigated the effect of DOW on the production of C. militaris mycelium and certain functional ingredients of C. militaris under submerged culture. As presented in Table 1, compared with ultrapure water (UPW), DOW enhanced the production of C. militaris mycelium by 23 % (p < 0.05), which significantly increased the production of adenosine by 18 % (p < 0.05). However, DOW did not significantly affect the production of intracellular and extracellular β-1,3-glucan (p > 0.05). Moreover, under the submerged culture, the experimental results of this study revealed that no cordycepin was produced in the fermentative liquid and mycelium of UPW and DOW.

Effect of various deep ocean water concentrations on production of cordycepin and adenosine in Cordyceps militaris-fermented products
Cordycepin cannot be produced by cultivating C. militaris in submerged culture. Therefore, this study focused on solid culture and explored the effect of various factors on production of the functional ingredients of C. militaris. For this phase, the DOW of various concentrations was used as the water source for C. militaris-fermented production. The effect of DOW on the production of adenosine and cordycepin was also analyzed. Figure 1a shows that after 60 days' fermentation, the C. militaris-fermented products with UPW, 10X-DOW, or 20X-DOW sources had increased cordycepin production; however, the increase was lower than that of the C. militaris-fermented products with the 30X-DOW source. When the fermentation was extended to 80 days, the C. militarisfermented product with the 30X-DOW source had the highest cordycepin production. This cordycepin production was higher than that of the 20X-DOW, 10X-DOW, and UPW sources by 22 % (p < 0.01), 26 % (p < 0.01), and 64 % (p < 0.01), respectively, displaying a dose effect. Figure 1b presents the production state of adenosine. When DOW of various concentrations was used as the water source for C. militaris-fermented production, the production of adenosine at various time points did not differ significantly (p > 0.05).

Effect of the major metal ions of deep ocean water on production of cordycepin and adenosine of Cordyceps militaris-fermented products
DOW is rich in the concentration of Mg 2+ , Na + , K + , Ca 2+ , Zn 2+ , and Fe 2+ , which is possible to enhance the production of the functional metabolites. Therefore, the effect of the major metal ions in DOW on the production of cordycepin and adenosine were further analyzed Table 1 Effect of DOW on the biomass, cordycepin, adenosine, intracellular β-1,3-glucan, and extracellular β-1,3-glucan production of C. militaris in submerged culture Data are presented as mean ± SD (n = 3).
* Indicated the significant difference (p < 0.05) as compared with UPW. a Under detection limit.

Effect of various sodium salts on production of cordycepin and adenosine of Cordyceps militaris-fermented products
According to above results (Fig. 2a), NaCl solution significantly suppressed the production of cordycepin. This section further discusses the effect of various sodium salts on production of cordycepin and adenosine. According to the comparison in Fig. 3, at the 30-fold sodium salt concentration, DOW, Na-SW, NaNO 3 , Na 2 SO 4 , Na 3 PO 4 , and NaCl, the C. militaris-fermented products with the DOW source and NaNO 3 respectively had 52 and 46 % higher cordycepin production than the C. militarisfermented products with UPW (p < 0.01). However, after fermentation, NaCl increased cordycepin production by 58 % (p < 0.001). Moreover, DOW, Na-SW, and NaNO 3 enhanced adenosine production by 60, 74, and 104 % (p < 0.05), respectively. Nevertheless, the selection of DOW, Na 2 SO 4 , Na 3 PO 4 , and NaCl did not affect the production of adenosine. At a concentration of 60-fold, the DOW, Na-SW, NaNO 3 , Na 2 SO 4 , and Na 3 PO 4 groups exhibited a decline in cordycepin production. However, in contrast to the UPW group, the NaCl group showed a significant 70 % decrease in the production of cordycepin (p < 0.001). Furthermore, DOW and Na 2 SO 4 increased adenosine production (p < 0.05), whereas adenosine production decreased in the other groups.

Fig. 1 Effect of various concentration of DOW on the cordycepin (a)
and adenosine (b) production of C. militaris-fermented oats in solid culture. Data are presented as mean ± SD (n = 3). ** indicated the significant difference (p < 0.01) as compared with UPW.

Effect of various nitrate salts on production of cordycepin and adenosine in Cordyceps militaris-fermented products
As shown in Fig. 3, at the 30-fold sodium salt concentration, only NaNO 3 could enhance cordycepin and adenosine production. Therefore, this section focuses on the concentration of NaNO 3 and discusses the effect of various combinations of nitrate salts on the production of cordycepin and adenosine. The 30X concentration of Mg 2+ , Na + , K + , Ca 2+ , Fe 2+ in Mg(NO 3 ) 2 , NaNO 3 , KNO 3 , Ca(NO 3 ) 2 , Fe(NO 3 ) 2 ·6H 2 O were equal to that in 30X-DOW. According to Fig. 4a, DOW, Mg(NO 3 ) 2 , NaNO 3 , Ca(NO 3 ) 2 , and Fe(NO 3 ) 2 had significantly increased cordycepin production (p < 0.01). However, KNO 3 did not exhibit such an increase (p < 0.05). Moreover, Fig. 4b illustrates that Mg(NO 3 ) 2 , NaNO 3 , KNO 3 , Ca(NO 3 ) 2 , and Fe(NO 3 ) 2 significantly increased adenosine production (p < 0.001), whereas 30X-DOW moderately increased adenosine production (p < 0.05). , and Cl − concentration to that in 30X or 60X DOW, respectively. Na-SW was a mixture of the four sodium salt solutions. Data are presented as mean ± SD (n = 3). *, **, *** indicated the significant difference (p < 0.05, p < 0.01, p < 0.001, respectively) as compared with UPW. Figure 4 illustrates that at the 30-fold sodium salt concentration, NaNO 3 enhanced cordycepin production, but Na 2 SO 4 and NaPO 4 did not enhance cordycepin production. However, NaCl significantly reduced cordycepin production (p < 0.001). Therefore, this section uses solutions with equal metal ion concentrations to that in 30X-DOW to discuss the effect of various chloride salts on production of cordycepin and adenosine. As shown in Fig. 5a, MgCl 2 , NaCl, and KCl significantly reduced cordycepin production (p < 0.05), and cordycepin production in the CaCl 2 and FeCl 2 solutions declined. The Cl-SW solution produced by combining all five chloride salts significantly reduced cordycepin production (p < 0.05).

Effect of various chloride salts on production of cordycepin and adenosine in Cordyceps militaris-fermented products
The adenosine production results are shown in Fig. 5b. DOW and Cl-SW enhanced the production of adenosine (p < 0.05). Furthermore, MgCl 2 , NaCl, KCl, and FeCl 2 significantly enhanced the production of adenosine (p < 0.05), whereas CaCl 2 had no significant effect on adenosine production (p < 0.05). However, Cl-SW enhanced adenosine production (p < 0.05).

Discussion
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 Fig. 4 Effect of various nitrate salts on the cordycepin (a) and adenosine (b) production of C. militaris-fermented oats in solid culture. The 30X concentration of Mg 2+ , Na + , K + , Ca 2+ , Fe 2+ in Mg(NO 3 ) 2 , NaNO 3 , KNO 3 , Ca(NO 3 ) 2 , Fe(NO 3 ) 2 ·6H 2 O were equal to that in 30X-DOW. Data are presented as mean ± SD (n = 3). *, **, *** indicated the significant difference (p < 0.05, p < 0.01, p < 0.001, respectively) as compared with UPW.
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(NO 3 ) 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 CaCO 3 and (NH 4 ) 2 Fe(SO 4 ) 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. (NH 4 ) 2 Fe(SO 4 ) 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, NO 3 − increased the production of cordycepin (p < 0.01) and adenosine (p < 0.05) the most; however, it had no effect on adenosine production. SO 4 2− and PO 4 − 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, NO 3 − , and PO 4 − decreased. At this concentration, the cordycepin production with SO 4 2− declined as well, but the adenosine production increased significantly (p < 0.05). Concurrently, Clof the 60-fold concentration significantly reduced cordycepin production (p < 0.001). The NO 3 − , SO 4 2− , PO 4 − , and Cl − used in this study refer to sodium salts. The NaCl solution reduced cordycepin production, whereas the NaNO 3 solution increased cordycepin production. Therefore, we conclude that NO 3 − can increase cordycepin production, that Cl − may reduce cordycepin production, and that NaCl solution has no effect on adenosine production. Nevertheless, the NaNO 3 solution promoted adenosine production. At the 60-fold sodium salt concentration, only the Na 2 SO 4 solution enhanced adenosine production. Therefore, NO 3 − and SO 4 2− may enhance adenosine production, whereas Cl − does not affect adenosine production.
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 NaNO 3 . Moreover, NaCl suppressed the production of cordycepin, and the effect of Na 2 SO 4 and NaPO 4 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, NaNO 3 enhanced adenosine production, whereas Na 2 SO 4 , NaPO 4 , 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