INFLUENCE OF CULTURE SUBSTRATE pH ON CORDYCEPS MILITARIS MUSHROOM CORDYCEPIN CONTENT, GROWN ON DIFFERENT SOLID SUBSTRATES

Distribution of proteins and amino acids in milled and brown rice as affected by nitrogen fertilization and genotype

The relation between rice kernels and changes of free amino acid in them with elapsed storing-period was investigated by mean of paper partition chromatography. For paper chromatography, Toyo filter paper No. 51 and 80% phenol water solution chosen as solvent was used and also the developed spots were identified by Rf value determined previously with Azinomoto K. K.'s pure reagent. Ordinarily it was diclosed that free amino acid detected in extracts from flesh brown rice (Hervest;1960, Variety Norin No. 17.) were as follows; aspartic acid, glutamic acid, glycine, histidine, alanine and valine and that the decreases of above mentioned amino acids were generally found in older nice (Havest; 1957-1959), though both aspartrc acid and glutamic acid decreased more clearly than the others and the decrease of alanine was not shown clearly on map. In later stage of storage, however, now amino acids product by the degeneration of the proteins had been founded and presumably these amino acids were composed of asparagine, cystine and leucine. It was suggested that gradtial loss of the reserves in brown rice grain itself was caused for living such as maintenance of germinating ability, in the course of time after harvest and there was a general tendency that greater loss was observed in aspartic acid and glutamic acid included the brown rice and increase in leucine, asparagine and cystine was observed from unknown factors. It seems to be absolutely necessary to investigate glutamic acid, glycine and alanine as important amino acids included in white rice for the research and discussion on rice taste.

Cordycepin production in the submerged culture of Cordyceps militaris was demonstrated using hydrolyzed corn processing protein by-products, known as corn steep liquor hydrolysate (CSLH), as an alternative nitrogen source. The growth, metabolism, and cordycepin production of Cordyceps militaris were evaluated under various concentrations of CSLH induction. The results demonstrated that CSLH addition had positive effects on the growth and cordycepin production with various C. militaris strains. The optimum strain, C. militaris GDMCC5.270, was found to effectively utilize CSLH to promote mycelium growth and cordycepin production. Low concentrations of CSLH (1.5 g/L) in the fermentation broth resulted in 343.03 ± 15.94 mg/L cordycepin production, which was 4.83 times higher than that of the group without CSLH. This also enhanced the metabolism of sugar, amino acids, and nucleotides, leading to improved cordycepin biosynthesis. The increase in key amino acids, such as glutamic acid, alanine, and aspartic acid, in the corn steep liquor hydrolysate significantly enhanced cordycepin yield. The corn steep liquor hydrolysate was confirmed to be a cost-effective accelerator for mycelium growth and cordycepin accumulation in C. militaris, replacing partial peptone as a cheap nitrogen source. It serves as a suitable alternative for efficient cordycepin production at a low cost.

Extracts from powders of geminated brown rice (Oryza sativa L.), Moringa oleifera, and Cordyceps militaris were combined in various ratios and their health-beneficial properties were investigated. All combined extracts exerted the synergistic effects on antioxidant activity as well as inhibitions against α-amylase and α-glucosidase. Extract formulas T4 (70% germinated brown jasmine rice, 20% M. oleifera dry leaves, and 10% C. militaris) and T2 (80% germinated brown jasmine rice, 10% M. oleifera dry leaves, and 10% C. militaris) elicited the significantly highest antioxidant activity and the significantly highest inhibition against α-glucosidase, respectively. Both formulas exhibited the significantly highest inhibition against α-amylase. As compared to each other, extract formulas T4 and T2 possessed the higher glucose consumption-promoting and antiinflammatory activities, respectively. Both formulas were not toxic to normal RAW-264.7 and SW-480 human colon cancer cells. Bioactive compounds with health-supporting properties were identified using UHPLC-QTOF-MS/MS. These combined extracts can be promising therapeutic food.

ABSTRACTCordyceps militaris has been a keystone in combating myriad health problems with innumerable far-reaching therapeutic effects. The present study focuses on effect of fermentation conditions such as (pH, temperature and incubation time) and solid-state fermentation (SSF) using solid substrates (wheat, oat and rice) on production of cordycepin. Temperature, pH and incubation time was found to have a direct effect on cordycepin production. The best possible combination of temperature, pH and incubation time was found to be 25°C, 5.5 and 21 days, respectively, for maximum cordycepin production. SSF of solid substrate medium culture leads to the production of cordycepin. Among the solid substrates, rice medium had highest cordycepin production (814.60 mg/g) followed by oat and wheat medium (638.85 and 565.20 mg/g, respectively). This method provides an effective way for increasing the cordycepin production at a large scale. This study could have a wide application in other fermentation processes at industrial level.

Chapter 14 - Comparison of the Maillard-Derived Aroma Volatiles of Cooked Milled and Brown Rice

Changes in the concentrations of gamma-aminobutyric acid (GABA), soluble calcium ions, glutamic acid, and the activity of glutamate decarboxylase (GAD) were investigated in non-germinated vs. germinated brown rice. Brown rice was germinated for 72 h by applying each of the following solutions: (1) distilled water, (2) 5 mM lactic acid, (3) 50 ppm chitosan in 5 mM lactic acid, (4) 5 mM glutamic acid, and (5) 50 ppm chitosan in 5 mM glutamic acid. GABA concentrations were enhanced in all of the germinated brown rice when compared to the non-germinated brown rice. The GABA concentration was highest in the chitosan/glutamic acid that germinated brown rice at 2,011 nmol/g fresh weight, which was 13 times higher than the GABA concentration in the non-germinated brown rice at 154 nmol/g fresh weight. The concentrations of glutamic acid were significantly decreased in all of the germinated rice, regardless of the germination solution. Soluble calcium and GAD were higher in the germinated brown rice with the chitosan/glutamic acid solution when compared to the rice that was germinated in the other solutions. GAD that was partially purified from germinated brown rice was stimulated about 3.6-fold by the addition of calmodulin in the presence of calcium. These data show that the germination of brown rice in a chitosan/glutamic acid solution can significantly increase GABA synthesis activity and the concentration of GABA.

The amino acid contents of wheat and rice as well as their final products produced by commercial milling were determined quantitatively by the amino acid analyzer. Less quantities of lysine, glycine, arginine, alanine, and aspartic acid were found in the flour, whereas more levels of these amino acids were found in the wheat grain. Conversely, more proline, phenylalanine and glutamic acid were found in the flour, and less amounts of these amino acids were found in the wheat grain. The proportion of the other amino acids were slightly affected by milling. On the other hand, less amounts of aspartic acid, serine, glutamic acid, and proline were found in rice bran and germ, whereas more levels of these amino acids were found in brown and milled rice. Conversely, rice bran and germ contained more threonine and cystine than brown and milled rice. The proportions of the other amino acids were slightly affected by milling, however, high content of lysine in germ was notable.

<p class="Default"><strong>Summary: </strong> qualitative composition and quantitative content of 17 amino acids, including 7 essential were defined in <em>Flammulina</em><em> velutipes</em> medicinal mushroom’s biomass dry powders. Biomass powders were received in static conditions and by submerged culture.</p><p class="Default"><strong>Key words:</strong> biomass, <em>Flammulina velutipes</em>, cultivation in static conditions, submerged culture, aminoacids, liquid chromatography.</p><p class="Default"><strong> </strong></p><p><strong>Introduction</strong><strong>. </strong>The medicinal mushroom <em>Flammulina (F.) velutipes</em> is widely used in non-traditional and officinal Eastern medicine as immunomodulatory, antitumor, antioxidant, antihypertensive, antibacterial, antifungal, antiviral agent [5; 6].</p><p>Polysaccharide content of <em>F. velutipes</em> biomass was identified in previous studies [7]. Continuing work on the study of bioactive substances of medicinal mushroom we conducted determination of biomass dry powders amino acid composition.</p><p>Amino acids are widely used in medicine for the treatment of neuropsychiatric disorders, some diseases of the gastrointestinal tract and liver, for the prevention of atherosclerosis, to increase immunity and to inhibit tumor development, etc. [1 - 4].</p><p>Thus, the purpose of this work was to determine and compare amino acids qualitative composition and quantitative content in <em>F. velutipes</em> biomass dry powders.</p><p><strong>Materials and methods</strong><strong></strong></p><p>The objects of our study were two types of medicinal mushroom <em>F. velutipes</em> biomass dry powders. They were obtained by two methods of microbiological synthesis – cultivation in static conditions and by submerged culture.</p><p>Water suspension with waste of CO<sub>2</sub>-extraction – Amaranthus flour (60 g/l) as a submerged culture medium for <em>F. velutipes</em> biomass production was explored. Mycelia were grown in 500 ml of microbial mattress at 26 ±1°C and pH value 6,0. Biomass was dried in a vacuum freeze dryer Cryodos-500 (Terrasa,Spain).</p><p>Amino acid composition was studied on an automatic amino acid analyzer T-339 («Mikrotechna», Prague, Czech Republic) by ion-exchange liquid chromatography at the laboratory of the Palladin Institute of biochemistry of the National Academy of Sciences of Ukraine.</p><p><strong>Results and discussion</strong></p><p>17 amino acids, including 7 essential (valine, isoleucine, leucine, lysine, methionine, threonine, phenylalanine), 2 conditionally essential (arginine and histidine) and 8 replaceable amino acids (alanine, aspartic acid, glycine, glutamic acid, proline, serine, tyrosine, cystine) were identified in <em>F. velutipes</em> biomass dry powders. Biomass dry powders obtained by different methods of microbiological synthesis (cultivation in static conditions and by submerged culture) do not differ in amino acids qualitative composition.</p><p>However, the biomass dry powder of medicinal mushroom that was obtained in static conditions contains 1.41 times more amino acids (essential to 1.27 times more; replaceable – 1.46 times more).</p><p><strong>Conclusions</strong></p><p>1. Using ion-exchange liquid chromatography in <em>F. velutipes</em> biomass dry powders were identified 17 amino acids, including 7 essential, 2 conditionally essential and 8 replaceable ones. Biomass dry powders were obtained by cultivation in static conditions and by submerged culture. The dominant of the essential amino acids in both investigated powders are leucine, of replaceable ones – glutamic and aspartic acid.</p>2. Cultivation of medicinal mushroom <em>F. velutipes</em> in static conditions allows to obtain in 1.41 times more amino acids compared to the submerged culture.

This study investigated the potential mechanism of Cordyceps militaris(CM) against non-small cell lung cancer(NSCLC) based on serum untargeted metabolomics. Specifically, Balb/c nude mice were used to generate the human lung cancer A549 xenograft mouse model. The tumor volume, tumor weight, and tumor inhibition rate in mice in the model, cisplatin, Cordyceps(low-, medium-, and high-dose), and CM(low-, medium-, and high-dose) groups were compared to evaluate the influence of CM on lung cancer. Gas chromatography-mass spectrometry(GC-MS) was used for the analysis of mouse serum, SIMCA 13.0 for the compa-rison of metabolic profiles, and MetaboAnalyst 5.0 for the analysis of metabolic pathways. According to the pharmacodynamic data, the tumor volume and tumor weight of mice in high-dose CM group and cisplatin group decreased as compared with those in the model group(P<0.05 or P<0.01). The results of serum metabolomics showed that the metabolic profiles of the model group were significantly different from those of the high-dose CM group, and the content of endogenous metabolites was adjusted to different degrees. A total of 42 differential metabolites and 7 differential metabolic pathways were identified. In conclusion, CM could significantly inhibit the tumor growth of lung cancer xenograft mice. The mechanism is the likelihood that it influences the aminoacyl-tRNA biosynthesis, the metabolism of D-glutamine and D-glutamate, metabolism of alanine, aspartate, and glutamate, metabolism of glyoxylate and dicarboxylic acid, biosynthesis of phenylalanine, tyrosine, and tryptophan, arginine biosynthesis as well as nitrogen metabolism. This study elucidated the underlying mechanism of CM against NSCLC from the point of metabolites. The results would lay a foundation for the anticancer research and clinical application of CM.

Soil inorganic amendments produce safe rice by reducing the transfer of Cd and increasing key amino acids in brown rice

Cordycepin, an important active substance in Cordyceps militaris, possesses antiviral and other beneficial activities. In addition, it has been reported to effectively promote the comprehensive treatment of COVID-19 and thus has become a research hotspot. The addition of naphthalene acetic acid (NAA) is known to significantly improve the yield of cordycepin; however, its related molecular mechanism remains unclear. We conducted a preliminary study on C. militaris with different concentrations of NAA. We found that treatment with different concentrations of NAA inhibited the growth of C. militaris, and an increase in its concentration significantly improved the cordycepin content. In addition, we conducted a transcriptome and metabolomics association analysis on C. militaris treated with NAA to understand the relevant metabolic pathway of cordycepin synthesis under NAA treatment and elucidate the relevant regulatory network of cordycepin synthesis. Weighted gene co-expression network analysis (WGCNA), transcriptome, and metabolome association analysis revealed that genes and metabolites encoding cordycepin synthesis in the purine metabolic pathway varied significantly with the concentration of NAA. Finally, we proposed a metabolic pathway by analyzing the relationship between gene-gene and gene-metabolite regulatory networks, including the interaction of cordycepin synthesis key genes; key metabolites; purine metabolism; TCA cycle; pentose phosphate pathway; alanine, aspartate, and glutamate metabolism; and histidine metabolism. In addition, we found the ABC transporter pathway to be significantly enriched. The ABC transporters are known to transport numerous amino acids, such as L-glutamate, and participate in the amino acid metabolism that affects the synthesis of cordycepin. Altogether, multiple channels work together to double the cordycepin yield, thereby providing an important reference for the molecular network relationship between the transcription and metabolism of cordycepin synthesis.

Cordyceps militaris, a medicinal fungus rich in cordycepin, shows promise in treating diseases such as cancer, respiratory issues, and COVID-19. This study examines the impact of different Taiwanese rice varieties on its solid-state fermentation, focusing on optimizing cordycepin production. The results indicated that the cordycepin yield was indeed affected by the type of rice used. In terms of the fruiting bodies, germ rice resulted in the highest yield (13.1 ± 0.36 mg/g), followed by brown rice (11.9 ± 0.26 mg/g). In the rice culture medium (RCM), brown rice led to the highest yield (4.77 ± 0.06 mg/g). Using gas chromatography-mass spectrometry and untargeted metabolomics, the study identifies four key volatile components linked to cordycepin, providing insights into developing functional rice porridge products. These findings are significant for advancing cordycepin mass production and offering dietary options for older individuals.

To explore the differences in protein quality among classic medicinal entomopathogenic fungi and to evaluate their metabolic adaptability, we analyzed the amino acid composition and proteomic characteristics of Cordyceps sinensis (CS), Cordyceps militaris (CM), and Cordyceps cicadae (CC). Quantitative analysis showed CM contained the highest crude protein and lysine, methionine, threonine, and valine. CS adapted to high-altitude hypoxia and exhibited lower protein but elevated leucine, isoleucine, and histidine contents, which may contribute to membrane stabilization and oxidative stress resistance. CC displayed higher non-essential amino acids such as arginine, proline, and tyrosine, reflecting active nitrogen metabolism. Four-dimensional data-independent acquisition (4D-DIA) proteomics identified 495 differentially expressed proteins (DEPs). Compared with CS, CM and CC displayed upregulated glutamate oxaloacetate transaminases 2 (GOT2), glutamate dehydrogenase (GDH), and argininosuccinate synthase 1 (ASS1) coordinately regulate nitrogen flux through the alanine-aspartate-glutamate metabolic network and urea cycle, supporting metabolic intermediate replenishment for energy metabolism. The upregulation of branched-chain keto acid dehydrogenase E1 subunit alpha (BCKDHA) and acyl-CoA dehydrogenase short/branched chain (ACADSB) in CM and CC facilitated the integration of branched-chain amino acid catabolism with the TCA cycle, explaining species-specific differences in protein content. This study presents the first application of 4D-DIA proteomics to compare CS, CM, and CC, providing insights into quality divergence mechanisms in medicinal fungi.

Cordyceps militaris alleviates COPD by regulating amino acid metabolism, gut microbiota and short chain fatty acids.