A high-molecular weight exopolysaccharide from the Cs-HK1 fungus: Ultrasonic degradation, characterization and in vitro fecal fermentation.

This work was to examine the impact of power ultrasound (US) on the molecular properties of a high-molecular weight (MW) exopolysaccharide (EPS) from the Cs-HK1 medicinal fungus and the utilization, and prebiotic function of the US-treated EPS fractions in human fecal microflora in vitro. The US treatment caused notable reduction of intrinsic viscosity, average MW and aggregate size of EPS in water but no significant changes in the molecular structure. The US-treated EPS fractions were consumed more rapidly by the fecal microflora, resulting in a higher total level of short chain fatty acids. They also affected the relative abundance in the microflora more beneficially than the original EPS. The results suggest that power US is effective for modifying and improving the prebiotic properties of high-MW polysaccharides.

Carotenoid Production by Caterpillar Medicinal Mushrooms, Cordyceps militaris (Ascomycetes), under Different Culture Conditions.

Cordyceps militaris, a well-known edible and medicinal species, is a potential source of natural carotenoids. The optimized conditions for carotenoid production are important for high-quality cultivation since carotenoid contents are the key factors for the appearance of fruiting bodies. In this study, carotenoid production by C. militaris under different culture conditions was compared. It was found that two-stage culture and fruiting body cultivation were suitable for the carotenoid production. Light is one of the important factors for carotenoid accumulation in this fungus and carotenoid can be produced by continuous shaking under light irradiation. Blue light with intensity of 17.3-64.3 lux can promote carotenoid production under the three tested culture methods. The carotenoid content was 4410.42 ± 30.83 and 3747.92 ± 178.13 µg g-1 under blue light intensity of 64.3 lux for the velum under two-stage culture and fruiting body cultivation, respectively. Oat medium may be suitable for the cultivation of fruiting bodies due to its high carotenoid production compared with other grains such as rice, wheat, and millet. Oxygen vector and oxidative stress induced by hydrogen peroxide can promote carotenoid production significantly under the two-stage culture. The carotenoid extracted from C. militaris showed strong antioxidant activity with IC50 value of 0.219 ± 0.01 mg ml-1, even higher than that of BHT, a positive control. This study will benefit the high-quality cultivation of this fungus and confirm that the carotenoid from C. militaris has the potential to be developed as a health food.

Improved Cordycepin Production by Cordyceps militaris KYL05 Using Casein Hydrolysate in Submerged Conditions.

Cordycepin, a beneficial bioactive product specifically found in Cordyceps, has received attention in various bioindustrial applications such as in pharmaceuticals, functional foods, and cosmetics, due to its significant functions. However, low productivity of cordycepin is a barrier to commercialization. In this study, Cordyceps militaris was mutated by UV irradiation to improve the cordycepin production. The highest producer KYL05 strain was finally selected and its cordycepin production was increased about 1.5-fold compared to wild type. In addition, the effects of culture conditions were fundamentally investigated. Optimal conditions were as follows: pH 6, temperature of 25 °C, shaking speed of 150 rpm, and culture time of 6 days. Effects of medium component on cordycepin production were also investigated by using various carbon and nitrogen sources. It was found that glucose and casein hydrolysate (CH) were most effective as carbon and nitrogen sources in cordycepin production (2.3-fold improvement) with maximum cordycepin production of about 445 mg/L. In particular, production was significantly affected by CH. These results should be of value in improving the efficiency of mass production of cordycepin.

Effect of radio-frequency heating on microbial load, flavor, color, and texture profiles of Cordyceps militaris.

BACKGROUND: Cordyceps militaris is a medicine and food dual-purpose mushroom extensively cultivated and consumed in East and Southeast Asia for centuries. However, it has an extremely short shelf life of 3-4 days at room temperature. C. militaris was pasteurized for 10, 20, and 30 min by radio-frequency (RF) at an electrode gap of 20 mm. The effect of RF heating on the microbial load, color, texture, and flavor attributes of C. militaris was evaluated and compared with those sterilized by conventional high-pressure steam. RESULTS: RF heating contributed to good heating uniformity, uniform temperature distribution, and significant decrease in total microbial load. C. militaris heated by RF exhibited unnoticeable total color difference compared with unpasteurized ones, while those sterilized by high-pressure steam presented undesired and unacceptable browning. Insignificant differences in hardness and chewiness were observed after RF heating. Improvements in mushroom-like flavor occurred after 10 and 20 min of RF heating. CONCLUSION: This study suggests that RF heating for pasteurization of edible mushrooms has promising prospects. Evaluation of RF heating on the taste and nutritional characteristics of edible mushrooms is needed in future work. © 2018 Society of Chemical Industry.

Phenotype responses to abiotic stresses, asexual reproduction and virulence among isolates of the entomopathogenic fungus Cordyceps javanica (Hypocreales: Cordycipitaceae).

Selecting entomopathogenic fungal isolates with resilience to environmental stresses, optimal mass production characteristics, and with high virulence to target pests favors the development of mycopesticides. A case in point, Cordyceps (= Isaria) javanica has been extensively investigated for non-chemical control of whiteflies worldwide. We phylogenetically characterized 11 native C. javanica isolates from Northeastern and Central Brazil. These isolates were screened for tolerance to heat-shock, UV-B radiation, osmotic and oxidative stresses, as well as conidial production on cereal grain and insecticidal activity against the whitefly Bemisia tabaci (MEAM 1) in the laboratory. All isolates were pathogenic to whiteflies and significant (3-fold) differences in median lethal concentration were observed among isolates. Furthermore, pronounced differences among isolates were found for stress factors and conidial production. Using principal component analysis, our results highlighted three major clusters formed by isolates (i) resistant to osmotic and oxidative stress, (ii) resilient to UV-B, and (iii) with high virulence, conidial production and heat tolerance. Overall, isolate CG1228 performed best based on multi-stress resistance, mass production and virulence attributes in the laboratory. This study highlights the importance of exploring natural variation in entomopathogenic fungi for selection of appropriate isolates for effective biocontrol of insect pests coupled with mass production characteristics and abiotic stress tolerances.

Effects of microwave-assisted pulse-spouted bed freeze-drying (MPSFD) on volatile compounds and structural aspects of Cordyceps militaris.

BACKGROUND: Microwave-assisted pulse-spouted bed freeze-drying (MPSFD) uses microwave heating to substitute the customary conduction heating during freeze drying (FD), resulting in high-quality dried samples with a short drying time. The aim of this study was to determine the effect of MPSFD on volatile compounds and structural aspects of Cordyceps militaris. In order to highlight the effect of MPSFD, FD (recognized for its high-quality products) and hot-air dying (HD; the most commonly used) were also evaluated using headspace solid-phase micro-extraction combined with gas chromatography-mass spectrometry and mid-Fourier transform infrared spectroscopy. RESULTS: Sixty-two components were obtained by FD, 62 compounds by MPSFD and 39 by HD. FD and MPSFD exhibited much better effects on the retention of volatile components of C. militaris than HD. Principal component analysis, hierarchical clustering analysis and E-nose analysis indicated that the volatile compounds of C. militaris dried by FD and MPSFD were similar. The spectral features of FD and MPSFD samples were also similar, whereas the HD sample presented a clearly different spectral feature around a wavenumber of 1640 cm-1 . CONCLUSION: MPSFD exhibited a beneficial effect on aroma and structure retention, with shorter drying time and lower energy consumption. © 2018 Society of Chemical Industry.

Heat and light stresses affect metabolite production in the fruit body of the medicinal mushroom Cordyceps militaris.

Cordyceps militaris is a highly valued edible and medicinal fungus due to its production of various metabolites, including adenosine, cordycepin, N6-(2-hydroxyethyl)-adenosine, and carotenoids. The contents of these metabolites are indicative of the quality of commercially available fruit body of this fungus. In this work, the effects of environmental abiotic factors, including heat and light stresses, on the fruit body growth and metabolite production in C. militaris were evaluated during the late growth stage. The optimal growth temperature of C. militaris was 20 °C. It was found that a heat stress of 25 °C for 5-20 days during the late growth stage significantly promoted cordycepin and carotenoid production without affecting the biological efficiency. Light stress at 6000 lx for 5-20 days during the late growth stage significantly promoted cordycepin production but decreased the carotenoid content. Both heat and light stresses promoted N6-(2-hydroxyethyl)-adenosine production. In addition, gene expression analysis showed that there were simultaneous increases in the expression of genes encoding a metal-dependent phosphohydrolase (CCM_04437) and ATP phosphoribosyltransferase (CCM_04438) that are involved in the cordycepin biosynthesis pathway, which was consistent with the accumulation of cordycepin during heat stress for 5-20 days. A positive weak correlation between the cordycepin and adenosine contents was observed with a Pearson correlation coefficient of 0.338 (P < 0.05). The results presented herein provide a new strategy for the production of a superior quality fruit body of C. militaris and contribute to further elucidation of the effects of abiotic stress on metabolite accumulation in fungi.

Effect of Pulsed Light Irradiation on Bioactive, Nonvolatile Components and Antioxidant Properties of Caterpillar Medicinal Mushroom Cordyceps militaris (Ascomycetes).

The caterpillar medicinal mushroom Cordyceps militaris contains many bioactive components, such as adenosine, cordycepin, and polysaccharides. In this study, C. militaris was exposed to 0, 3, 6, or 9 pulses of light irradiation to estimate changes in vitamin D2, bioactive compounds, nonvolatile taste components, and antioxidant properties. In addition, we compared the components and properties of C. militaris mycelia and solid waste medium that had been treated with pulsed light (PL) irradiation. Overall, PL irradiation of C. militaris increased the vitamin D2 content and increased the total amino acid levels 9-48%; the antioxidant properties of the mycelia treated with 0 pulses and of the solid waste medium treated with 3 pulses all exhibited lower half-maximal effective concentrations. Therefore, PL irradiation affected the amounts of bioactive compounds, but the irradiated samples still contained intense umami taste and a sufficient amount of antioxidant components.

Photoperiodic Responses and Characterization of the Cmvvd Gene Encoding a Blue Light Photoreceptor from the Medicinal Caterpillar Fungus Cordyceps militaris (Ascomycetes).

Light is a necessary environmental factor for production of conidia and pigment, formation of stroma, and development of Cordyceps militaris, a well-known edible and medicinal mushroom. In this study, an obvious rhythm loop was observed in certain strains of C. militaris under conditions of alternating 12-hour intervals of dark and light. A possibly related gene, Cmvvd, the homologue of the blue-light photoreceptor of Neurospora crassa, was cloned from the genome of C. militaris. The protein CmVVD is predicted to be 203 amino acids in length and is characterized by the presence of a light, oxygen, or voltage domain. Analysis of the CmVVD sensor domain (light, oxygen, or voltage) suggested that it is a blue-light receptor. Cysteine 108 is essential for the in vivo function of VIVID (VVD) in N. crassa photoadaptation. However, proline is in this position instead in all of the tested CmVVD proteins, suggesting that CmVVD may have a different function or may function in ways different from VVD in N. crassa. Genetic variation analysis of CmVVD in 6 representative strains indicated that 3 informative sites exist. Cmvvd messenger RNA was able to be induced by light, and the expression level increased over 10 times after irradiation and was maintained at high levels in the nascent fruiting body. The light-induced expression of Cmvvd was abolished in Cmwc-1 mutants, suggesting that the expression of Cmvvd is dependent on the photoreceptor CmWC-1 or on a functional CmWC-1/WC-2 complex. This article will help to open the still-unexplored field of circadian rhythms for this fungus.

Effects of Illumination Pattern during Cultivation of Fruiting Body and Bioactive Compound Production by the Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes).

We investigated the effects of light intensity in the 3 cultivation stages separately-the mycelium colonization stage, the primordial initiation stage, and the fruiting stage (in order)-on fruiting body and bioactive compound production by Cordyceps militaris. In the mycelium colonization stage, rice substrates were incubated in a spawn running room at 23°C. During the primordial initiation stage, C. militaris was grown at 18°C and illuminated 12 hours/day. In the fruiting stage the temperature was 23°C, with illumination provided 12 hours/day. The highest fruiting body yield and biological efficiency were 4.06 g dry weight/bottle and 86.83%, respectively, under 1750 ± 250 lux during the second and third stages. The cordycepin content was highest during the second and third stages under 1250 ± 250 lux. The mannitol and polysaccharide contents were highest under 1250 ± 250 and 1750 ± 250 lux during the primordial initiation stage and the fruiting stage, respectively. Thus, with controlled lighting, C. militaris can be cultivated in rice-water medium to increase fruiting body yield and bioactive compound production.

Effect of UV-B Irradiation on Physiologically Active Substance Content and Antioxidant Properties of the Medicinal Caterpillar Fungus Cordyceps militaris (Ascomycetes).

Ultraviolet-B (UV-B) light irradiation is a well-known technique for converting vitamin D2 from ergosterol in mushroom fruit bodies. Mushrooms are a natural and nonanimal food source of vitamin D2. We studied the effect of UV-B light irradiation on the amount of vitamin D2 and physiologically active substances in Cordyceps militaris and their antioxidant properties. After UV-B irradiation for 2 hours, the vitamin D2 content of freshly harvested C. militaris fruiting bodies, mycelia, whole submerged culture (WSC), and homogenized submerged culture (HSC) increased from 0 to 0.03 to 0.22 to 1.11 mg/g, but the ergosterol content was reduced from 1.36 to 2.50 to 1.24 to 2.06 mg/g, respectively. After UV-B irradiation, the amount of adenosine, cordycepin, and ergothioneine of fruiting bodies dramatically increased 32-128%, but the polysaccharide content slightly decreased 36%. The reverse trends were observed in mycelia, WSC, and HSC. UV-B irradiation could reduce the effective concentrations at 50% of fruiting bodies for ethanolic and hot water extracts in reducing power, scavenging, and chelating abilities, whereas mycelia, WSC, and HSC of ethanolic extracts increased effective concentrations at 50% in reducing power, scavenging, and chelating abilities. UV-B irradiation slightly increased flavonoid content (10-56%) and slightly affected total phenol content.

Effect of ultrasonic treatment on structure and antitumor activity of mycelial polysaccharides from Cordyceps gunnii.

Taking mycelial polysaccharides from Cordyceps gunnii (C. gunnii) as the study subject, the effect of ultrasonic power, time and concentration of polysaccharides on antitumor activity of the polysaccharides was investigated. The ultrasonic processing condition of the polysaccharides was optimized by using orthogonal test design, and determined to be 400 W, 15 min and 1g/L. The change of structures of polysaccharides before and after ultrasonic treatment was also studied. Results show that ultrasonic treatment did not change the characteristic attribute of polysaccharides from C. gunnii. The composition of monosaccharide residues and the category of glycosidic bond have not been changed. But the molecular weight and intrinsic viscosity was reduced, and the alpha-helicity was enhanced after ultrasonic treatment. It was possible that ultrasonic treatment is an effective way for enhancing antitumor activity of polysaccharides.

NMR and GC-MS based metabolic profiling and free-radical scavenging activities of Cordyceps pruinosa mycelia cultivated under different media and light conditions.

Variation of metabolic profiles in Cordyceps pruinosa mycelia cultivated under various media and light conditions was investigated using 1H nuclear magnetic resonance (NMR) analysis and gas chromatography mass spectrometry (GC-MS) with multivariate statistical analysis. A total of 71 metabolites were identified (5 alcohols, 21 amino acids, 15 organic acids, 4 purines, 3 pyrimidines, 7 sugars, 11 fatty acids, and 5 other metabolites) by NMR and GC-MS analysis. The mycelia grown in nitrogen media and under dark conditions showed the lowest growth and ergosterol levels, essential to a functional fungal cell membrane; these mycelia, however, had the highest levels of putrescine, which is involved in abiotic stress tolerance. In contrast, mycelia cultivated in sabouraud dextrose agar with yeast extract (SDAY) media and under light conditions contained relatively higher levels of fatty acids, including valeric acid, stearic acid, lignoceric acid, myristic acid, oleic acid, palmitoleic acid, hepadecenoic acid, and linoleic acid. These mycelia also had the highest phenolic content and antioxidant activity, and did not exhibit growth retardation due to enhanced asexual development caused by higher levels of linoleic acid. Therefore, we suggested that a light-enriched environment with SDAY media was more optimal than dark condition for cultivation of C. pruinosa mycelia as biopharmaceutical or nutraceutical resources.

Photo morphogenesis and photo response of the blue-light receptor gene Cmwc-1 in different strains of Cordyceps militaris.

Light is a necessary environmental factor for stroma formation and development of Cordyceps militaris, a well-known edible and medicinal fungus. In this study, photo morphogenesis and the blue-light receptor gene were studied using five representative strains of C. militaris. The results suggest that light was essential for colony pigmentation and could promote conidia production. Cmwc-1, the homologe of the blue-light photoreceptor of Neurospora crassa, was cloned from the genome of C. militaris by Hi-tail PCR. The protein CmWC-1 was characterized by the presence of the LOV and PAS domains and a GATA-type Znf domain. Genetic variation analysis of Cmwc-1 in different strains showed that 15-bp deletions occurred in three strains that resulted in 5-Gln deletions in the transcription activation domain. Phylogenetic analysis based on the Sordariomycetes WC-1-like proteins suggested that the sequence of WC-1 could be used as a candidate marker for phylogenetic analysis in fungi. Cmwc-1 mRNA was light inducible and the expression level increased significantly after irradiation in all tested strains. The sequence of CmWC-1 and the relative expressions responding to irradiation in degenerate and albino strains were similar as the cultivated one. This report will help to open the still-unexplored field of stroma development for this fungus.

Alteration of media composition and light conditions change morphology, metabolic profile, and beauvericin biosynthesis in Cordyceps bassiana mycelium.

Metabolic alterations of Cordyceps bassiana mycelium were investigated under the following culture medium and light conditions: dextrose agar supplemented with 0.5% yeast extract (SDAY) medium with light (SL), SDAY medium without light (SD), nut medium without light (ND), and iron-supplemented SDAY medium without light (FD). The levels of asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, ornithine, and proline were significantly higher under SD and SL conditions. The levels of most of the alcohols, saturated fatty acids, unsaturated fatty acids, fatty acid esters, sterols, and terpenes were higher under the ND condition than in the other conditions, but beauvericin was not detectable under the ND condition. The FD condition was favorable for the enhanced production of aminomalonic acid, malic acid, mannonic acid, and erythritol. Thus, the metabolic characteristics of C. bassiana can be manipulated by varying the cultivation conditions, rendering this fungus potentially favorable as a nutraceutical and medicinal resource.

Effects of selenium and light wavelengths on liquid culture of Cordyceps militaris Link.

To investigate the effects of selenium and light wavelengths on the growth of liquid-cultured Cordyceps militaris and the main active components' accumulation, culture conditions as selenium selenite concentrations and light of different wavelengths were studied. The results are: adenosine accumulation proved to be significantly selenium dependent (R(2) = 0.9403) and cordycepin contents were determined to be not significantly selenium dependent (R(2) = 0.3845) but significantly enhanced by selenium except for 20 ppm; there were significant differences in cordycepin contents, adenosine contents, and mycelium growth caused by light wavelengths: cordycepin, blue light > pink light > daylight, darkness, red light; adenosine, red light > pink light, darkness, daylight, blue light; and mycelium growth, red light > pink light, darkness, daylight > blue light. In conclusion, light wavelength had a significant influence on production of mycelia, adenosine, and cordycepin, so lightening wavelength should be changed according to target products in the liquid culture of C. militaris.

Production of cordycepin by a repeated batch culture of a Cordyceps militaris mutant obtained by proton beam irradiation.

Cordycepin (3'-deoxyadenosine) is one of the most versatile metabolites of Cordyceps militaris due to its broad spectrum of biological activity. In our previous study, the C. militaris mutant G81-3, which produces higher levels of cordycepin, was obtained by high-energy proton beam irradiation. In this study, the effects of adenosine on cordycepin production in a surface liquid culture of the mutant and the wild type strains were investigated. For the mutant strain, the optimum dose of adenosine yielded a 30% increase in cordycepin production; the maximum levels of production with adenosine and without adenosine were 8.6g/l and 6.7 g/l, respectively. In contrast, the increase due to adenosine supplementation for the wild type strain was only 15% (3.1g/l with adenosine and 2.7 g/l without adenosine). Furthermore, a repeated batch culture, an efficient production method, was carried out to eliminate the relatively long lag phase of the mutant culture. Over four cycles, both the mutant and the wild type strain maintained a production level of more than 85% of that of the initial cycle. As a result, the disadvantage of the mutant was successfully overcome, resulting in a productivity (0.48 g/(ld)) higher than that of the batch culture (0.29 g/(ld)). The productivity for cordycepin obtained in this study is the highest reported value to date, and this method could be applied to large-scale production of cordycepin at industrial levels.

[High-yielding cordycepin in Cordyceps militaris modified by low-energy ion beam].

Cordycepin has the effect of anti-tumor, immunomodulation, anti-inflammation and so on. In order to make use of Cordyceps militaris better, we implanted different doses of low-energy ion beam into C. militaris, chose best cordycepin extracting technology, and determined cordycepin content in strains before and after ion beam implantation by UV spectrophotometry. Results showed that the best dose of low-energy ion beam was 2.60 x 10(15) ions/cm2, the best conditions of microwave assisted ultrasonic extraction technology were as follows: 70% ethanol as the solvent, microwave power as 200 W, extraction time as 110 s, Material - liquid ratio as 1: 240. We chose 15 strains with high-yielding cordycepin, of which cordycepin content was up to (11.924 +/- 0.063) mg/g, which was a nearly 30% increase compared with the original strain.

A new approach for improving cordycepin productivity in surface liquid culture of Cordyceps militaris using high-energy ion beam irradiation.

AIMS: To obtain a higher cordycepin production using Cordyceps militaris mutant obtained by a new mutagenesis technique called 'ion beam'. METHODS AND RESULTS: Successful irradiation of C. militaris NBRC 9787 by a proton beam with high energy was performed, and 30 classes of 8-azaadenine- and 28 classes of 8-azaaguanine-resistant mutants were obtained on mutant screening, of which seven classes were selected as promising preliminary mutants having an antibacterial ability as an index of cordycepin production. In a surface liquid culture technique, some of the 8-azaadenine-resistant mutants gave a better performance for the cordycepin productivity; in contrast, among the 8-azaaguanine-resistant mutants, it was shown that mutant no. G81-3 was much better than the control in the metabolic rate of glucose and the cordycepin productivity. In primary optimization using the enriched medium, the cordycepin production was 3.1 and 1.8 g l(-1) on 21-day culture for mutant no. G81-3 and the control, respectively. The cordycepin production obtained by the mutant was 72% more than the control. CONCLUSIONS: The mutant obtained by proton beam irradiation had higher productivity of cordycepin than that of the control. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant obtained by irradiation had a superior production performance of cordycepin, and therefore, it could be used in the realm of applied industrial biotechnology for the large-scale production of cordycepin.

Quantification of ultraviolet-C irradiation induced cyclobutane pyrimidine dimers and their removal in Beauveria bassiana conidiospore DNA.

Ultraviolet (UV) radiation-induced DNA damage leading to entomopathogenic fungal inactivation is commonly measured by viability counts. Here we report the first quantification of UV-induced cyclobutane pyrimidine dimers (CPD) in DNA of the entomopathogenic fungus, Beauveria bassiana. Changes in the mobility of UV-C irradiated DNA were resolved with CPD specific bacteriophage T4 endonuclease V and alkaline agarose gel electrophoresis. The maximum number of CPD formed in B. bassiana DNA in vitro by UV-C irradiation was 28 CPD/ 10 kb after 720 J/m2 dose. The maximum number of CPDs formed in B. bassiana conidiospore DNA irradiated in vivo was 15 CPD/10 kb after 480 J/m2 dose and was quantified from conidiospores that were incubated to allow photoreactivation and nucleotide excision repair. The conidiospores incubated for photoreactivation and nucleotide excision repair showed decreased number of CPD/10 kb DNA and a higher percent survival of conidiospore populations than conidiospores not allowed to repair.