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Cordyceps chanhua , an important cordycipitoid medical mushroom with wide use in Asia, has gained attention for its bioactive component beauvericin (BEA), which is of medicinal value as a drug lead, but also of food safety risk. Recent observations by our group revealed a significant decrease of BEA content in C. chanhua when exposed to light, but the underlying regulatory mechanisms remain elusive. In this study, a comprehensive approach combining metabolomics and transcriptomics was employed to investigate the effects of white light on the secondary metabolism of C. chanhua for elucidation of the influence of light on BEA biosynthesis in this fungus. The result showed that the genes and metabolites involved in the synthesis of D-hydroxyisovaleric acid, a precursor of BEA synthesis, were down-regulated under light exposure, while those associated with the synthesis of phenylalanine, another precursor of BEA synthesis, were up-regulated leading to elevated phenylalanine levels. It suggested that the suppressive effect of light on BEA synthesis in C. chanhua occurred primarily through the inhibition of D-hydroxyisovaleric acid synthesis, while the enhanced phenylalanine biosynthesis likely directed towards other metabolic pathway such as pigment synthesis. These results contributed to a better understanding on how light modulates the secondary metabolism of C. chanhua and provided valuable guidance for optimizing BEA production in cultivation practices. [Display omitted] • Light inhibits the production of beauvericin (BEA) in Cordyceps chanhua. • Light reduces BEA by suppressing D-hydroxyisovaleric acid, a precursor. • Light boosts phenylalanine, another precursor, but changes its metabolic path. • Light exposure induces oxidative stress and stimulates the consumption of BEA. [ABSTRACT FROM AUTHOR]

Here, we report on a Cordyceps species entering into a multi-trophic, multi-kingdom association. Cordyceps cateniannulata , isolated from the stem of wild Coffea arabica in Ethiopia, is shown to function as an endophyte, a mycoparasite and an entomopathogen. A detailed polyphasic taxonomic study, including a multilocus phylogenetic analysis, confirmed its identity. An emended description of C. cateniannulata is provided herein. Previously, this species was known as a pathogen of various insect hosts in both the Old and New World. The endophytic status of C. cateniannulata was confirmed by re-isolating it from inoculated coffee plants. Inoculation studies have further shown that C. cateniannulata is a mycoparasite of Hemileia vastatrix , as well as an entomopathogen of major coffee pests; infecting and killing Hypothenemus hampei and Leucoptera coffeella. This is the first record of C. cateniannulata from Africa, as well as an endophyte and a mycoparasite. The implications for its use as a biocontrol agent are discussed. • An unprecedented case of multi-kingdom host-jumping is reported in Cordyceps. • This is the first record of C. cateniannulata from Africa and as an endophyte. • The fungus was isolated from the stem of wild Coffea arabica in Ethiopia. • Endophytic, mycoparasitic, and entomopathogenic abilities were proven experimentally. • The fungus offers novel biocontrol options for the management of coffee pests and diseases. [ABSTRACT FROM AUTHOR]

Recombinant Cordyceps militaris lipase (rCML) expressed in Komagataella phaffii is more highly N -mannosylated (Man 8–11 GlcNAc 2) than the native lipase. The lipase activity of rCML was enhanced compared to its deglycosylated form (drCML), but the increases in the V max values of rCML for substrates with chain lengths of C4:0–C14:0 were 1.8 to 2.9-fold greater than those of drCML, whereas the difference was narrower for C16:0–C18:0. Changes in acyl chain length selectivity correlated with the conformational changes rather than hydrophobicity changes induced by N -mannosylation, which may have altered active site accessibility by shifting the favorable binding locations depending on acyl chain length. Nevertheless, high N -mannosylation did not significantly affect the melting temperature of rCML compared to that of drCML (55.5 °C and 57.0 °C, respectively) during thermal unfolding, but alleviated harsh deactivation in the thermal-transition state (60–70 °C). Furthermore, high N -mannosylation enhanced the long-term storage stability at –20 °C, and loss of activity was 5.2 ± 1.1% and 32.8 ± 1.2% for rCML and drCML, respectively. Overall, the introduction of yeast-derived high mannose moieties endowed short- to medium-chain selectivity and improved the biochemical properties of recombinant lipase. [Display omitted] • Recombinant Cordyceps militaris lipase (rCML) from Komagataella phaffii is highly mannosylated. • Lipase activity of rCML was higher than its deglycosylated form, regardless of acyl chain length. • Notable variations in acyl chain length-dependent kinetic parameters were observed. • N -Mannosylation enhanced selectivity for short- to medium-chain fatty acids. • N -Mannosylation enhanced stability during thermal processing and long-term storage. [ABSTRACT FROM AUTHOR]

The study was the production of the probiotic cultured milk enriched with the edible mushroom crude extract and its evaluation to gain insight about the prebiotic potential of Cordyceps militaris. During the 28–day shelf life; microbial, physico–chemical, textural, and sensorial properties were investigated. The results indicated that the probiotic milk with extract was influential on values of gel firmness, color and sensorial attributes. The interaction between extract and probiotic culture, and hence increased acidity, has an impact on textural properties. Moreover, in terms of taste the extract left a dominant aroma in–mouth and bitterness in throat, and in terms of odor it suppressed the characteristic flavor of probiotic cultured milk. Consequently, the results suggested that extract showed a positive stimuli on growth and activity of probiotics, and it could be considered as potential prebiotic source as its impact had been evaluated on fermentation by a cultured model. [Display omitted] • Enrichment of cultured milk with mushroom extract affected gel fermentation. • C. militaris mushroom extract showed a positive effects on growth of probiotics. • C. militaris mushroom extract was the potential prebiotic source. [ABSTRACT FROM AUTHOR]

Dihydromyricetin (DMY) has poor solubility and stability in water, thus limiting its application. The aim of this study was to evaluate the effect of Cordyceps militaris polysaccharide (CMP) on the solubility, stability and antioxidant activity of DMY. The solubility assay showed that the solubility of DMY in water increased approximately 3 times. The stability of DMY was increased by 1.34 times as determined by thermostatic acceleration method. The phase solubility assay showed that DMY could form AL-type inclusion complex with CMP. Infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and thermogravimetric assays of the CMP-DMY composition revealed that the binding of CMP to DMY should be attributed to the macromolecular structure, surface porous structure of CMP, and the formation of non-covalent bonding interactions. The results of in vitro antioxidant assays showed that the CMP-DMY inclusion complex inhibited DPPH, ABTS, superoxide and hydroxyl radicals by 67.24 %, 99.89 %, 41.38 % and 35.25 %, respectively. Therefore, CMP can be used as a new material to help DMY dissolution in the future, and the inclusion complex should have good prospects for application in terms of ABTS radical scavenging efficacy. [Display omitted] • Cordyceps militaris (CMP) could improve the solubility of dihydromyricetin (DMY). • CMP could improve the stability of DMY. • CMP and DMY could form the AL type of inclusion complex in water. • The CMP-DMY inclusion complex has good performance in clearing ABTS free radical. [ABSTRACT FROM AUTHOR]

Cordyceps sinensis (Berk.) Sacc. (Ophiocordyceps sinensis) is an edible and medicinal fungus used as a natural superior tonic. It is considered as scarce fungus with a high market demand. Therefore, as an alternative, fermentation technology has been proposed to produce artificial cordyceps (fermented C. sinensis) to address the shortage of cordyceps resources for industrialization and commercial utilization. Numerous studies have proved that polysaccharides are the important bioactive substances in the fermented C. sinensis , but the research data lack systematic review. In this review, current relevant research data regarding the preparation (including extraction, fractionation, and purification), structural characterization (including molecular weight, monosaccharide composition, glycosidic bond type, structural and conformational features), bioactivities, structure-activity relationships (SAR) and applications of polysaccharides from different sources of fermented C. sinensis last decade were analyzed and discussed. The findings highlight that the most commonly employed methods for preparing fermented Cordyceps sinensis polysaccharides (FCSPs) involve water extraction and alcohol precipitation, combing with sophisticated chromatographic techniques such as ion exchange and gel permeation chromatography. From these processes, 34 different polysaccharides were identified including 5 glucans and 7 heteropolysaccharides that were thoroughly characterized. FCSPs exhibited a broad spectrum of biological activities, ranging from antioxidant and renal protective effects to immunomodulatory, antitumor, and hypolipidemic properties. The structure-activity relationships (SAR) demonstrated that key factors, such as molecular weight, monosaccharide composition and glucosidic bond types, play critical roles in determining the bioactivity of FCSPs. Nevertheless, there remain unknown elements that continue to influence SAR, leaving room for further exploration. Furthermore, the limitation of existing studies and some new perspectives for future investigations on FCSPs were proposed. [ABSTRACT FROM AUTHOR]

[Display omitted] • The Kozak sequence 'CGGACC' could significantly enhance gene expression. • Methanol assimilation was enhanced by targeting CAT-G4S-AOX1-per2 to peroxisomes. • Overexpression of pgk and PpgapN could enhance the supply of ATP and NADPH. • Overexpression of purF , adss, and PpcpdB could enhance the supply of adenosine. • The CO 2-eq of Pichia pastoris was significantly lower than Cordyceps militaris. Cordycepin, a nucleoside analog, is widely used in medicine and health products. However, the production of cordycepin from Cordyceps militaris faces the challenges of low productivity and high rate of greenhouse gas emissions. In this study, by optimizing the cordycepin biosynthesis pathway through promoter combination, Kozak sequence, and enzyme fusion, enhancing the methanol assimilation capacity in peroxisomes, adjusting the synthesis of NADPH and ATP, and combining the enhanced supply of adenosine and 3′-AMP, the cordycepin high-yield strain Pp29 was constructed, which produced 1551.44 mg/L cordycepin by shake-flask fermentation. In fed-batch fermentation, Pp29 achieved the highest yield (8.11 g/L, 67.64 mg/g DCW, and 1.35 g/L/d) to date in 10 L fermenter, and the CO 2-eq emissions were 1.9–17.3 times lower than C. militaris and other yeast systems. This study provide basis for Pichia pastoris to be used as chassis cell for synthesizing cordycepin and other nucleoside analogs by methanol as carbon source. [ABSTRACT FROM AUTHOR]

Excessive exercise leads to body fatigue and destroys the balance of the oxidation/oxidation resistance system in the body, thus damaging the central nervous system and reducing learning and memory ability. Nrf2 is an important transcription factor that regulates the cell oxidative stress response. Therefore, the research and development of natural antioxidants with the effect of regulating Nrf2-related signalling pathways to improve central fatigue caused by body fatigue has application value. Methods and results: Cordyceps militaris polysaccharides were extracted, isolated and purified via DEAE-cellulose 52 and Sepharose CL-6B columns to obtain two saccharides, Cordyceps militaris acidic polysaccharides (CMPB) and CMPB-b. The results of behavioural tests showed that compared with the model group, the learning and memory abilities of the CMPB-H group (800 mg/kg) mice were remarkably improved in the dark avoidance and Morris water maze tasks (p < 0.01), and the levels of fatigue metabolites and oxidative stress in the body were obviously decreased (p < 0.01). The expression level of BDNF, PI3K, Nrf2 and HO-1 proteins in the hippocampus were significantly increased (p < 0.01). In vitro experiments, compared with the PC12 oxidative stress model group, CMPB-b high-dose group (100 μg/mL) had remarkably improved oxidative stress. CMPB-b also obviously promoted the phosphorylation of PI3K and AKT proteins (p < 0.01) and the nuclear translocation of Nrf2 (p < 0.01), and significantly increased the expression of HO-1 (p < 0.01). Conclusion: CMPB can alleviate the fatigue state of high-intensity swimming mice and improve the learning and memory impairment of exercise-fatigue mice by regulating the Nrf2-related signalling pathway. Its antioxidant active component CMPB-b exerts in vitro antioxidative neurological damage by the same mechanism. Our systematic studies provide strong supporting evidence for the future use of Cordyceps militaris acidic polysaccharides in health products to improve resistance to fatigue. [ABSTRACT FROM AUTHOR]

Nucleosides and polysaccharides are the main bioactive ingredients of Cordyceps genus. Nucleosides shows significant differences in different Cordyceps species. However, the differences of polysaccharides have not been decoded. Here, the structure characters of polysaccharides including molecular weight (Mw) distribution, compositional monosaccharides and glycosidic linkage types were compared in C. sinensis (CS) , C. militaris (CM) , silkworm-hosted C. militaris (SCM) and Cordyceps fermented products (CSF). Compositional monosaccharides including mannose, glucose and galactose, and 1,4-Glc p glycosidic linkage were found abundant in Cordyceps species. Chemometric analysis showed that Cordyceps exhibit significant differences in structural information especially glycosidic linkage types. Besides, polysaccharides in CS and CSF-4 had obviously strong capacity of stimulating phagocytic, NO production and cytokines secretion. Gray relational analysis and Pearson correlation analysis were performed to further investigate the relationship between key polysaccharide structure and immunomodulatory activities. The results indicated that polysaccharides with relatively large number of 1, 4-Glc p and Mw in range of 7.16 × 106 Da-7.99 × 107 Da and 1.43 × 104 D-6.94 × 105 Da probably contributed to its immunomodulatory activities. The chemical and biological evaluation of natural and various cultured cordyceps in this study is useful for understanding and regulating the quality of cultured Cordyceps. [Display omitted] [ABSTRACT FROM AUTHOR]

• SPE techniques reduce matrix effect and improve Nt/Ns pretreatment and purification. • MPs and silica-fiber coating are remarkable sorbents for miniaturised SPE forms. • Chromatographic methods employing various detectors are used to detect Nt/Ns. • HILIC, along with tandem mass spectrometry, acquires prominence in Nt/Ns detection. Cordyceps genus is entomopathogenic mushrooms that have traditionally been used in ethnomedicine in Asian countries. Nucleosides (Ns), nucleotide(Nt), Nucleobases (Nb) and their analogues play a critically physiological role and have a great potential in drug development, such as pentostatin and cordycepin (COR). Due to their significance bioactivity, several Nt/Ns were used as markers for quality evaluation for medicinal Cordyceps, including adenosine, inosine, guanosine, uridine and COR. Among them, COR is the most considerable adenosine analogue, exhibiting significant therapeutic potential and has many intracellular targets. Nt/Ns contains polar compounds and the phosphate groups of Nt deprotonate and carry negative charges with a broad range of pH values. Recent years, various advanced methods of extraction and separation, and nanomaterials have been developed to extract, isolate and determine these molecules, such as ultrasound-assisted extraction (UAE), Supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE) for the extraction, the solid phase extraction (SPE) methods (microextraction SPE (SPME), magnetic SPE (MSPE), and unique SPE materials based on the boronate affinity for the separation, and chromatography methods employing ultraviolet (UV), fluorescence, MS detection and electrospray ionization (ESI), along with matrix-assisted laser desorption/ ionization (MALDI) for the determination. COR derived from adenosine and its structure is very similar to that of 2′-deoxyadenosine (2′-dA) and adenosine, resulting in an incorrect identification, which will influence its therapeutic effects. Therefore, this review primarily focused on the characteristics of Nt/Ns, the advanced methods, strategies, nanomaterials for extracting and determining Nt/Ns (COR in particular) in Cordyceps spp, as well as the methods for distinguishing COR from its structure analogs. [ABSTRACT FROM AUTHOR]

Cordyceps sinensis , a traditionally prized medicinal fungus, contains polysaccharides as one of its main bioactive constituents, known for their significant immunomodulatory properties. In this study, we systematically investigated the composition and structure of Cordyceps sinensis polysaccharide, followed by an evaluation of its therapeutic effect on depression using a chronic restraint stress-induced depression model. The polysaccharide CSWP-2, extracted via hot water, precipitated with ethanol, and purified using DEAE-cellulose column chromatography from Cordyceps sinensis , is primarily composed of glucose, mannose, and galactose, with α -1,4-D-glucan as its major structural component. Behavioral tests, immunological profiling, metabolomics, and gut microbiota analyses indicated a notable ameliorative effect of CSWP-2 on depressive-like symptoms in mice. Furthermore, the action of CSWP-2 may be attributed to the modulation of the gut microbiome's abundance and its metabolic impacts, thereby transmitting signals to the host immune system and exerting immunomodulatory activity, ultimately contributing to its antidepressant effects. [Display omitted] • CSWP-2 with α -1,4 glucan as the main chain has antidepressant effects. • CSWP-2 may exert antidepressant effects through the gut-brain axis. • CSWP-2 modulates immune dysregulation and metabolic disorders in depression. [ABSTRACT FROM AUTHOR]

The fungal disease of Cordyceps militaris has become the primary disease during the cultivation of C. militaris. However, some of the causal organism remains unclear. During our investigation in 2020–2022, three fungal strains were isolated from both the mycelium and fruiting bodies of C. militaris in mushroom farms located in Changchun. The observed disease incidence was approximately 20%. White villous mycelium on the surface of hyphae or fruiting bodies of C. militaris , is one of the main features of the disease, which can cause slow growth of mycelium, delayed primordia differentiation, and growth failure. Finally, the white cotton flocculent mycelium covered the surface of the medium, and the C. militaris stopped differentiating and failed to produce the primordium, ultimately leading to the stunted growth and lodging of the fruiting body. The objective of this study was to identify the pathogen based on Koch's Postulates, thereby confirming its pathogenicity and characterizing it through morphological and molecular features. The phylogenetic analyses showed that they belong to the Trichoderma gamsii. This study presented the first evidence of T. gamsii caused fungal disease in C. militaris , providing a theoretical basis for the prevention and control measures. • The outbreak of fungal diseases in Cordyceps militaris cultivation poses a significant threat to production and quality. • The disease incidence was approximately 20%, which led to significant yield losses. • The pathogen responsible for white mildew disease was identified as Trichoderma gamsii. • This is the first report of Trichoderma gamsii causing white mildew disease in Cordyceps militaris in China. • The results facilitate the development of strategies for white mildew disease of Cordyceps militaris. [ABSTRACT FROM AUTHOR]

• Temperature variations significantly influence the growth and metabolite production of C. militaris. • Responsive mechanisms in acclimatization to temperature conditions identified in C. militaris. • Low temperature induced transcriptional upregulation of lipid biosynthetic pathways indicating an adaptive metabolic response of C. militaris. Cordyceps militaris is a medicinal entomopathogenic fungus containing valuable biometabolites for pharmaceutical applications. Its genetic inheritance and environmental factors play a crucial role in the production of biomass enriched with cordycepin. While temperature is a crucial controlled parameter for fungal cultivation, its impacts on growth and metabolite biosynthesis remains poorly characterized. This study aimed to investigate the metabolic responses and cordycepin production of C. militaris strain TBRC6039 under various temperature conditions through transcriptome analysis. Among 9599 expressed genes , 576 genes were significantly differentially expressed at culture temperatures of 15 and 25 °C. The changes in the transcriptional responses induced by these temperatures were found in several metabolisms involved in nutrient assimilation and energy source, including amino acids metabolism (e.g., glycine, serine and threonine metabolism) and lipid metabolism (e.g., biosynthesis of unsaturated fatty acids and steroid biosynthesis). At the lower temperature (15 °C), the biosynthetic pathways of lipids, specifically ergosterol and squalene, were the target for maintaining membrane function by transcriptional upregulation. Our study revealed the responsive mechanisms of C. militaris in acclimatization to temperature conditions that provide an insight on physiological manipulation for the production of metabolites by C. militaris. [ABSTRACT FROM AUTHOR]

Lipase with unique regioselectivity is an attractive biocatalyst for elaborate lipid modification. However, the excavation of novel sn -2 regioselective lipases is difficult due to their scarcity in nature, with Candida antarctica lipase A (CALA) being the pronouncedly reported one. Here, we identified a novel CALA-like lipase from Cordyceps militaris (CACML7) via in silico mining. Through chiral-phase high-performance liquid chromatography, we determined that CACML7 displays sn -2 regioselectivity (>68 %) as does CALA, but exhibits distinctive chain length selectivity and bias against unsaturated fats. Notably, the curvature of the acyl-binding tunnel was expected to contribute to the 2.2-fold higher preference for cis -fatty acid (C18:1, cis- Δ9) over trans -fatty acid (C18:1, trans- Δ9) unlike trans -active CALA. Random pose docking of trioleoylglycerol (TOG) into the active site of a lid-truncated mutant of CACML7 revealed that TOG accepts a tuning fork conformation, of which the precise positioning of the reactive ester group towards the catalytic center was only favorable via sn -2 binding mode. The unique active site morphology, which we refer to as an "acyl-binding tunnel with a narrow entrance," may contribute to the sn -2 regioselectivity of CACML7. Our data provide an attractive model to better understand the mechanism underlying sn -2 regioselectivity. • Candida antarctica lipase A (CALA)-like lipase, CACML7, was discovered. • Like CALA, CACML7 exhibited sn-2 regioselectivity against trioleoylglycerol. • CACML7 was cis-active, whereas CALA was trans-active. • Trioleoylglycerol bound to CACML7 in tuning fork conformation. • Narrow tunnel entrance may contribute to the sn-2 regioselectivity of CACML7. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cordyceps protein alleviates lupus nephritis by inhibiting Th1 cell differentiation. • Cordyceps protein attenuates renal cell infiltration in mice with lupus nephritis. • Cordyceps protein prevents T cell migration into the kidney to alleviate kidney injury in MRL/ lpr mice. T cell infiltration and differentiation play a central part in the development of lupus nephritis (LN). Our prior research has indicated that protein, the primary active component of cordyceps (WCP), a traditional Chinese medicine, possesses properties that can enhance renal fibrosis and provide kidney protection. Nonetheless, the connection between WCP and T cell infiltration and differentiation in LN remains poorly understood. The objective of this research was to assess the immunomodulatory impacts of WCP in LN mice and elucidate the underlying mechanism through in vivo and in vitro investigations. To investigate the impact and mechanism of WCP in MRL/ lpr lupus-prone mice, WCP (1.5 g/kg/d), Bailing capsules (BC, 0.75 g/kg/d), and saline in equivalent quantities were administered to the mice over a period of 8 weeks. The therapeutic effects, T cell infiltration and differentiation of WCP on MRL/ lpr mice were verified through ELISA, Hematoxylin-eosin (H&E), Periodic Acid Schiff (PAS) staining, immunofluorescence, Luminex analysis and flow cytometry. The mechanism by which WCP alleviates LN was investigated using tissues of mice, T cells and Mouse Podocyte Clone-5 (MPC-5) cells by transcriptomics, Western blot (WB), and Real-time quantitative polymerase chain reaction (RT-qPCR). We found that WCP improved LN in MRL/ lpr mice by reducing urinary protein, creatinine, and serum auto antibodies, increasing complement 3 (C3) level, improving renal immunopathology and downregulating serum cytokines, including IFN-γ, IL-12, and RANTES. Notably, the infiltration of CD4+ and CD8+ T cells in the kidney was reduced by WCP. Similarly, the cell transwell co-culturation study showed that the WCP treated MPC-5 cells were weaker in inducing T cell migration. Consistent with this finding, our observations revealed that WCP could inhibit T cell-related chemokine expression in kidney and MPC-5 cells, as well as reduce the levels of TLR4, MYD88, phosphorylated-p38, phosphorylated-ERK, and phosphorylated-JNK. On the other hand, WCP was found to greatly inhibit the Th1 cells differentiation in vivo and in vitro. Cytokine-receptor induced Th1 cell differentiation pathway and PI3K-AKT pathway were the most enriched pathways based on differentially expressed genes (DEGs) enrichment analysis among different cell groups. Results from RT-qPCR and WB showed that WCP notably reduced the levels of IL-12, p-STAT4, IFN-γ, p-STAT1, p-PI3K, and p-AKT in T cells. WCP demonstrated positive immunomodulatory effects on LN disease, by decreasing the T cells infiltration through TLR4/MYD88/MAPK signaling pathway and inhibiting Th1 cells differentiation via IL-12-STAT4 and IFN-γ-STAT1 pathways, in addition to the PI3K-AKT pathway. [ABSTRACT FROM AUTHOR]

Cordyceps militaris , widely recognized as a medicinal and edible mushroom in East Asia, contains a variety of bioactive compounds, including cordycepin (COR), pentostatin (PTN) and other high-value compounds. This review explores the potential of developing C. militaris as a cell factory for the production of high-value chemicals and nutrients. This review comprehensively summarizes the fermentation advantages, metabolic networks, expression elements, and genome editing tools specific to C. militaris and discusses the challenges and barriers to further research on C. militaris across various fields, including computational biology, existing DNA elements, and genome editing approaches. This review aims to describe specific and promising opportunities for the in-depth study and development of C. militaris as a new chassis cell. Additionally, to increase the practicability of this review, examples of the construction of cell factories are provided, and promising strategies for synthetic biology development are illustrated. • C. militaris is a medicinal and edible mushroom. • C. militaris has the potential and possibility to be built as a cell factory. • Computational and advanced CRISPR technique were applied in C. militaris. • Metabolic engineering instructs in producing high-value compounds. • Synthetic biology facilitates the development of cell factory construction. [ABSTRACT FROM AUTHOR]

Fungi belonging to the Ascomycete genus Cordyceps are endoparasitoids and parasites, mainly of insects and other arthropods. Cordyceps militaris has been used as a therapeutic drug for cancer patients. However, the infection, parasitism, and fruiting body formation mechanisms of this fungus are still unknown. Based on our hypothesis that lectin(s) is involved in the interaction between the C. militaris fungi and insects, we partially purified and characterized a new lectin from C. militaris , designated CmLec4. In addition, we searched for substance(s) in the infected silkworm extracts that could bind to CmLec4, and succeeded in purifying the sex-specific storage protein 2 as a specific binding target. To examine function of the binding protein during the process of parasitism, we investigated the effect of recombinant CmLec4 on silkworms by inoculating the protein into silkworm pupae, and found that it significantly delayed emergence compared to the control. Furthermore, cmlec4 gene knockout strains constructed in this study produced markedly lower amounts of fruiting body than the wild-type strain. All the results revealed that the lectin CmLec4 produced by C. militaris would be involved in the infection into silkworm and fruiting body formation from the host. [ABSTRACT FROM AUTHOR]

This work investigated the protective effects of Cordyceps sinensis exopolysaccharide‑selenium nanoparticles (EPS-SeNPs) at different Se/EPS ratios (1/20, 1/3, 1/1, and 4/3) against 0.4 mM H 2 O 2 -induced oxidative stress in human hepatoma (HepG2) cells. Results revealed that EPS-SeNPs (0.1–4.0 μg/mL) promoted the proliferation of HepG2 cells with a cell viability of over 90% during H 2 O 2 stress. Different Se/EPS ratios of EPS-SeNPs provided protective effects against H 2 O 2 -induced oxidative stress in HepG2 cells by increasing cell viability, restoring cell and nucleus morphology, as well as reducing lactate dehydrogenase (LDH) levels. Particularly, EPS-SeNPs (Se/EPS = 1/1) with the smallest particle size showed the highest cell viability and the greatest inhibitory effect on LDH level. Besides, EPS-SeNPs also inhibited nitric oxide and reactive oxygen species (ROS) production, and increased mitochondrial membrane potential, superoxide dismutase (SOD), and catalase levels while reducing glutathione (GSH) content. Specially, EPS-SeNPs at Se/EPS ratio of 1/3 with smaller size showed the lowest ROS level and the highest antioxidant activities (SOD and GSH), implying that the ROS generation was inhibited by increasing enzymatic and non-enzymatic antioxidants. The enhanced protective effect of EPS-SeNPs (Se/EPS = 1/1 and 1/3) might be attributed to its smaller particle size. • EPS-SeNPs could protect human hepatoma cells against H 2 O 2 -induced oxidative stress. • EPS-SeNPs restored cell and nucleus morphology and cell membrane integrity. • EPS-SeNPs could suppress oxidative stress by improving SOD, CAT, and GSH levels. • Se/EPS = 1/3 had lowest ROS level and highest antioxidant activities (SOD and GSH). • The antioxidant effect of EPS-SeNPs was partly due to inhibition of ROS generation. [ABSTRACT FROM AUTHOR]

Cordyceps sinensis (C. sinensis) is a widely used and highly valuable traditional Chinese medicine. Several dipeptides have been detected in C. sinensis , but current scientific knowledge of its chemical makeup remains limited. In this study, an improved approach that integrates offline two-dimensional liquid chromatography (2D LC) separation, precursor ion list, library screening, and diagnostic ion filtering was established to systematically screen and characterize dipeptides in C. sinensis. Offline 2D LC integrating hydrophilic interaction LC and reverse phase separations was established to eliminate interference and identify the target dipeptides. A library containing the potential 400 dipeptides was created, and a precursor ion list with all theoretical precursor ions was adopted to trigger the MS/MS scan with high sensitivity. To identify dipeptides, the type and connection sequence of amino acids were determined according to the product ions. Ile and Leu residues were differentiated for the first time according to the characteristic ion at m/z 69.07. Ultimately, 170 dipeptides were identified or tentatively characterized from C. sinensis , and most are reported for the first time in this species herein. In addition, the identified dipeptides were also applied for discrimination among the three Cordyceps species, and 11 markers were identified. The obtained results provide a deeper understanding of the chemical basis of C. sinensis. [Display omitted] • An enhanced targeted identification strategy is presented. • It involves offline 2D-LC/MS, in-house library searching and diagnostic product ion filtering. • 170 dipeptides were reported from Cordyceps sinensis. • Characteristic ion was proposed for differentiation between Ile and Leu. • Three Cordyceps species were differentiated. [ABSTRACT FROM AUTHOR]

A new type of acidic exopolysaccharide (AESP-II) was extracted and separated from the fermentation broth of Cordyceps militaris (C. militaris), which was further purified to elucidate its structural characteristics and immunological activity. AESP-II was confirmed to be an acidic pyranose with a molecular weight of 61.52 kDa, which consisted of mannose, glucuronic acid, rhamnose, galactose acid, N -acetyl-galactosamine, glucose, galactose and arabinose with a molar ratio of 1.07: 5.38: 1: 3.14: 2.23: 15: 6.09: and 4.04. Animal experiment results verified that AESP-II can significantly promote the proliferation of spleen T and B lymphocytes in mice with immune injury caused by cyclophosphamide (CTX). In particular, the promotion of B lymphocytes presented a dose-effect relationship. In addition, the levels of the cytokines IL-2, IL-4, and IFN-γ, which are mainly secreted by T lymphocytes, and immunoglobulin IgG, IgM and IgA, which are mainly secreted by B lymphocytes, were increased after AESP-II treatment. The above results suggest that fluid immunity is involved in the immunomodulatory function of AESP-II. Simultaneously, AESP-II was detected significantly to promote the phosphorylation expression of p38 kinase (p38), extracellular regulated protein kinases (ERK) and c-Jun N-terminal kinase (JNK) by Western blot, further suggesting that the activation of the MAPK signaling pathway mediates the immunoregulatory function of AESP-II. • AEPS-II was a kind of low molecular weight acidic polysaccharide. • AEPS-II belongs to a kind of amorphous pyranose form. • AEPS-II can reverse the immune damage caused by cyclophosphamide and promote the proliferation of T and B lymphocytes in spleen. • AEPS-II activates mouse B cells through MAPK signaling pathways. • AEPS-II could potentially serve as an effective immunomodulator. [ABSTRACT FROM AUTHOR]

Cordyceps militaris is a well-known traditional Chinese medicine. Studies have demonstrated that the polysaccharides of C. militaris have various bioactivities. However, their mechanisms of action remain unclear. We previously purified a water-soluble polysaccharide CM1 from C. militaris and found that it has a cholesterol efflux improving capacity. This study further investigates the effect of CM1 in anti-atherosclerosis and its underlying mechanism in apolipoprotein E-deficient mice. Our data indicated that CM1 significantly decreased the total cholesterol and triglyceride in the plasma of mice, and decreased lipid deposition and formation of atherosclerotic plaque in a dose-dependent manner. Integrated bioinformatics analysis revealed that CM1 interacted with multiple signaling pathways, including those involved in lipid metabolism, inflammatory response, oxidoreductase activity and fluid shear stress, to exert its anti-atherosclerotic effect. Molecular technology analysis showed that CM1 enhanced the expression of proteins involved in lipid metabolism, reduced the expression of intercellular adhesion molecule-1 and tumor necrosis factor-α in the aorta, and decreased the content of oxidative products by enhancing the activities of antioxidant enzymes. Microarray analysis and biochemical data indicated that CM1 can improve lipid metabolism, reduce inflammation and oxidative stress. Taken together, CM1 could be used for the treatment of hyperlipidemia and atherosclerotic cardiovascular diseases. • The bioactive CM1 alleviated formation of atherosclerotic lesions in apoE −/− mice. • The underlying mechanisms was elucidated by integrated bioinformatics analysis. • The mechanisms of action of CM1 was further verified by other molecular methods. • These data explained the bioactivities of the polysaccharides from C. militaris. [ABSTRACT FROM AUTHOR]

An eco-friendly and fast HPLC method was developed for the determination of adenosine, inosine, guanosine and uridine in Cordyceps and related products (fermented mycelia of Hirsutella sinensis and Paecilomyces hepiali). The sample was ultrasonically extracted using 0.5% phosphoric acid solutions for 2.5 min. Sample separation was performed on a Poroshell SB-Aq column (50 mm × 4.6 mm, 2.7 μm) using eco-friendly mobile phase consisting of formic acid and ammonium formate aqueous solution at a flow rate of 1.0 mL·min−1. The detection wavelength was 260 nm. The developed HPLC method showed good linearity with correlation coefficients of 1.0000 in the test range. Good precision, repeatability and stability of this method were also observed (RSD ≤ 2.81%). The recovery ranged from 91.84%−105.19% (RSD ≤ 2.59%). Compared with reported methods, the current method did not use harmful organic solvent and took only 10.5 min. It obtained a high eco-score of 91 by the "Analytical Eco-Scale" tool. The developed method is eco-friendly and fast, which is suitable for the quality evaluation of Cordyceps and related products. [ABSTRACT FROM AUTHOR]

Hirsutella sinensis , the anamorph of Ophiocordyceps sinensis (syn. Cordyceps sinensis), is a great substitute for precious and rare wild Cordyceps sinensis to effectively treat a variety of lung and kidney diseases. In this study, an α-glucan (named as HSWP-2a) was obtained by hot water extraction, DEAE-cellulose separation, and Sepharose CL-6B purification from H. sinensis mycelia. Different from known α-glucans, HSWP-2a is an α-(1 → 4)-D-glucan that branched at O -6, O -3, or O -2 with a terminal 1-linked α-D-Glc p as side chain, with an average molecular weight of 870.70 kDa. Immunological tests showed that HSWP-2a could remarkably enhance the phagocytosis of macrophages and increase the production of NO, IL-1β, IL-6, and TNF-α, via activating the p38, JNK, and NF-κB signaling pathways. Moreover, HSWP-2a could significantly promote splenic lymphocyte proliferation. Taken together, HSWP-2a may be potentially utilized as a natural immunomodulatory agent. • A water-soluble polysaccharide (HSWP-2a) was isolated from Hirsutella sinensis mycelia. • HSWP-2a was an α-(1 → 4)-D-glucan that branched at O -6, O -3, or O -2 with a terminal 1-linked α-D-Glc p as side chain. • HSWP-2a exhibited remarkable immune-enhancing activities on RAW264.7 cells through the p38, JNK, and NF-κB signaling pathways. • HSWP-2a could significantly promote the proliferation of T and B lymphocytes. [ABSTRACT FROM AUTHOR]

Crude Cordyceps militaris polysaccharides (CMPs) have been proven to be effective in alleviating harmful effects of a long-term high-fat diet (HFD), but there are fewer related studies on identifying the health-regulating abilities of certain subcomponents of CMPs. This study aimed to figure out the main component of CMPs in anti-obesity and to further explore its regulatory effects in vivo. Results showed among three main subcomponents (CMP40, CMP60 and CMP80) of CMPs, the large molecular weight CMP40 (with average molecular weight of more than 100 kDa) has shown superior in vitro hypoglycemic and hypolipidemic capacities. The simultaneous intervention of CMP40 indicated that it could reduce the body weight and white adipose tissue mass/area in the HFD mice. Serum parameters also suggested a decrease in the cholesterol and triglyceride level in the CMP40 group. These results might be due to the down-regulation of lipid synthesis and the up-regulation of lipolysis pathways. Besides, CMP40 could alleviate the inflammatory condition, such as the lower LPS and inflammatory cytokines levels, in the HFD mice in a dose-dependent manner. It could also restore gut health by increasing the SCFAs level, regulating gut microbiota structure, and repairing intestinal tight junction. Results also confirmed safe in CMP40 consumption since there were no side-effects on a high concentration addition of CMP40 (8%) to a regular diet. These findings suggested the promising anti-obesity effect of CMP40 and provided a theoretical basis of its further application in food industry. • Cordyceps militaris polysaccharides-CMP40 with more than 100 kDa was obtained. • CMP40 showed superior in vitro hypoglycemic and hypolipidemic capacities. • CMP40 alleviated lipid metabolism, dyslipidemia, inflammation and gut disorder caused by high-fat diet. • A high concentration addition of CMP40 (8%) to a regular diet was confirmed safe. [ABSTRACT FROM AUTHOR]

The widespread attention garnered by the utilization of prebiotics for the regulation of intestinal flora has not yet extended to the exploration of edible fungi protein hydrolysates or peptides that possess potential prebiotic functions. In this study, Cordyceps militaris (C. militaris) ZA17-C4 mycelium protein hydrolysates were evaluated for their potential as prebiotics and thermo-protectors for Lactobacillus fermentum (L. fermentum) J26. The results showed that the addition of 0.5% C. militaris ZA17-C4 mycelium albumin hydrolysates and 1% glutenin hydrolysates promoted the proliferation of L. fermentum J26. Furthermore, the addition of 1% 5–10 kDa C. militaris ZA17-C4 mycelial albumin hydrolysate had a significant protective effect on L. fermentum J26 under the condition of treatment at 65 °C for 20 min. L. fermentum J26 viable counts were increased by three orders of magnitude compared to the control. Meanwhile, the addition of 1% of 5–10 kDa C. militaris ZA17-C4 mycelial albumin hydrolysate had a protective effect on the cell membrane of L. fermentum J26. Subsequently, several new peptides were identified using LC-MS/MS. These peptides may have potential probiotic thermo-protectants effects. [ABSTRACT FROM AUTHOR]

Cordyceps militaris Link. (C. militaris) with strong anti-hyperuricemia effects could be detected in mice urine and serum after oral administration as we previously proved. In this study, the mechanism and molecular effects of anti-hyperuricemia by C. militaris in gouty nephropathy (GN) mice were firstly investigated, and its key active structure was further explored. The results showed C. militaris could significantly reduce the uric acid (UA) level and related inflammatory factors. Meanwhile, C. militaris maintain the normal dynamic balance of UA by inhibiting hepatic xanthine oxidase (XO) and regulating the expression of renal transport proteins ABCG2, GLUT9 and URAT1. Then we employed network pharmacology to theoretically determine two active components in C. militaris , cordycepin and ergosterol, that act on GN. Moreover, the molecular docking and molecular dynamic simulation results showed cordycepin and ergosterol could stably bind to XO and cyclooxygenase-2 (COX-2) due to hydrogen bonding and hydrophobic interactions. This study could provide theoretical evidence for utilization of C. militaris in hyperuricemia-management functional foods. • Cordycepin and ergosterol were two active components in C. militaris. • Cordycepin and ergosterol alleviated gouty nephropathy by acting on XO and COX-2. • C. militaris could reduce serum uric acid level and promoted uric acid excretion. • C. militaris better relieved inflammatory response compared to allopurinol. • C. militaris might be a potentially functional food to mitigate gouty nephropathy. [ABSTRACT FROM AUTHOR]

Ustiloxins I–M (1 – 5), five undescribed cyclopeptides bearing a 15-membered macrocyclic skeleton, were isolated from Cordyceps militaris. The structures of 1 and 5 were identified by spectroscopic and crystallographic methods, whereas the structures of 2 – 4 were assigned by spectroscopic and computational approaches. Biological evaluation of all the compounds toward human triple-negative breast cancer cells revealed that compounds 4 and 5 are toxic with IC 50 values of 64.29 μM and 28.89 μM, respectively. Five undescribed cyclopeptides were isolated from the fungus Cordyceps militaris. Compounds 45 and exhibit moderate cell toxic activity toward MDA-MB-231 cells. [Display omitted] • Five undescribed cyclopeptides were isolated from Cordyceps militaris. • 1 – 5 both consist of four amino acids and possess a 15-membered ring system. • Compounds 4 and 5 show potential cytotoxic activity. [ABSTRACT FROM AUTHOR]

• Forty critical compounds were identified through MS2 based GNPS matching. • SSF-LGPT significantly lowered lipid content of C.elegans. • SSF-LGPT prolonged the lifespan and healthspan of C. elegans , • SSF-LGPT fermented on the 10th day was the optimum product. Microbial tea products are widely welcomed for their unique quality and characteristics. In this study, Cordyceps militaris (C. militaris), a medicinal and edible fungus, was used to ferment Lu'an GuaPian tea (LGPT) by solid-state fermentation (SSF) and thus developed a new deeply processed tea product (SSF-LGPT) with less bitterness and a distinct herbal aroma. SSF-LGPT (100 μg/mL) showed α -glucosidase inhibitory activity and significantly lowered fat content (34 % inhibition rate) in Caenorhabditis elegans (C. elegans). SSF-LGPT also exhibited antioxidant activity in vitro , prolonged the lifespan, delayed the age-related decline of phenotype, and enhanced stress resistance to heat, oxidative stress, and UV irradiation in C. elegans. Together, C. militaris fermentation of LGPT improves the flavor, hypoglycemic and fat-lowering activities. This study provides a new alternative approach to comprehensively utilize resourceful green tea via C. militaris fermentation. [ABSTRACT FROM AUTHOR]

[Display omitted] • Cordyceps militaris resisted Pb stress mainly relying on protein glutathione (PB-SH). • The genes of ugp was the key enzyme under the stress of heavy metal. • The domesticated Cordyceps militaris could alleviate oxidative stress caused by Pb2+. In this study, we compared the response mechanism of ordinary Cordyceps militaris and domesticated one to Pb2+ stress by exploring the reduced glutathione content (GSH), biomass, expression level of enzymes related to polysaccharide and enzyme activity of GSH-PX, SOD and CAT. Results showed that the GSH was mainly concentrated in intracellular, and the content of protein GSH (PB SH) was higher than non-protein GSH (NP SH), which revealed that the Cordyceps militaris resisted heavy metal mainly relying on PB SH. Besides, the biomass level of domesticated group was always higher than that in the ordinary group. In addition, the domesticated group had a higher enzyme activity of PGM, PGI and UGP. And the genes of ugp was the most critical enzyme under the stress of heavy metal. Further more, the domesticated Cordyceps militaris could alleviate the oxidative stress caused by Pb2+, and exercise the function of relieving Pb2+ poisoning. Owing to its preferable response mechanism, domesticated Cordyceps militaris revealed good prospect in removing heavy metals from organisms. [ABSTRACT FROM AUTHOR]

Cordycepin, which has great immunomodulatory activities such as anticancer, antifungal, antivirus, antileukemia and lipid-lowering ones, is the secondary metabolite of Cordyceps militaris (C. militaris). Liquid submerged fermentation is the common cultivation process to produce cordycepin. To optimize the fermentation process and improve production, monitoring the cordycepin secretion in the fermentation is essential. The measurement based on chromatography-mass spectrometry methods is generally involved in the complex sample pretreatments and time-consuming separation, so more rapid and convenient methods are required. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is more attractive for faster and direct detection. Therefore, MALDI-MS detection combined with isotope-labeled internal standard was applied to the measurement of cordycepin content in the fermentation broth and mycelium. This method made accurate quantification of cordycepin in the range of 5–400 μg/mL with a relative standard deviation of 5.6%. The recovery rates of fermentation samples after the 1, 13, and 25 days were 90.15%, 94.27%, and 95.06%, respectively. The contents of cordycepin in the mycelium and fermentation broth were 136 mg/g and 148.39 mg/mL on the 20th culture day, respectively. The cordycepin secretion curve of the liquid fermentation of C. militaris was real-time traced over 25 days. [Display omitted] • A rapid quantification method of cordycepin based on MALDI-MS is proposed. • The quantification relies on the stable isotope standard method. • Rapid determination of the cordycepin content in the liquid fermentation broth of Cordyceps militaris without pre-treatment. • Monitoring the fermentation state of C. militaris fermentation broth is benefit to improve the yield of cordycepin. [ABSTRACT FROM AUTHOR]

Hyperlipidemia is one major cause of atherosclerosis, which is a basic pathological change of cardiovascular diseases. Polysaccharide is a water-soluble component with lipid-lowering effects. In this study, alkaline-extracted polysaccharides were obtained from the fruiting body of C. militaris. Polysaccharides were purified via anion exchange and size exclusion chromatography. Their structural characteristics were investigated via chemical and spectroscopic methods. CM3I was mainly composed of →4)α-D-Glc p (1 → glycosyls and differed from starch due to the presence of →4,6)β-D-Glc p (1 → glycosyls. CM3II was characterized by its backbone, which was composed of →4)-β-D-Man p (1 → 6)-α-D-Man p (1 → 6)-β-D-Man p (1 → linked glycosyls, and especially the presence of O -methyl. Moreover, CM3II exhibited powerful anti-atherosclerotic effects via lowering plasma lipid levels in apolipoprotein E -deficient mice. The underlying mechanisms were attributed to its promoting effect on LXRα and inhibitory effect on SREBP-2. Collectively, CM3I and CM3II are different from the previously reported polysaccharides from C. militaris , and CM3II has a potential application in hypolipidemia and anti-atherosclerosis. • Two polysaccharides were obtained from the alkaline extraction of C. militaris. • CM3I is a starch like polysaccharide mainly composed of →4)α-D-Glc p (1 → glycosyls. • CM3II is a novel heteropolysaccharide characterized by the presence of OCH 3. • CM3II exhibited anti-atherosclerotic and lipid-lowering effects in apoE −/− mice. • CM3II promoted LXRα/ABCG8 pathway and inhibited SREBP-2 expression in mice liver. [ABSTRACT FROM AUTHOR]

One novel alkali-extracted polysaccharide, CM3-SII, was obtained from the fruiting body of C. militaris via column chromatography. Its structural characteristics were investigated via chemical and spectroscopic methods. The backbone of CM3-SII was composed of →4)-β-D-Man p (1→, →6)-β-D-Man p (1→, and →6)-α-D-Man p (1→ glycosyls, and branching at the O -4 positions of →6)-β-D-Man p (1→ glycosyls with β-D-Gal p , (1→2) linked-β-D-Gal f , and →2,6)-α-D-Man p (1→ residues. Furthermore, O -6 and O -2 positions of the →2,6)-α-D-Man p (1→ residues were substituted with methyl and β-D-Gal p , respectively. This polysaccharide significantly enhanced the intracellular protein expression of low-density lipoprotein receptor and proprotein convertase subtilisin/kexin type 9 (PCSK9) via regulating sterol regulatory element-binding protein 2 in hepatoma Huh7 cells. Of note, CM3-SII significantly decreased PCSK9 secretion at the concentration of 200 μg/mL. Collectively, CM3-SII is different from the previously reported alkali-extracted polysaccharides isolated from the fruiting body of C. militaris , and it may have potential application in hypolipidemia or as a pharmaceutical additive. • A novel alkali-extracted polysaccharides CM3-SII was purified from C. militaris. • CM3-SII is different from the previously reported polysaccharides from C. militaris. • CM3-SII enhanced the expression of LDLR in Huh7 cells via regulating SREBP-2. • Of note, CM3-SII significantly decreased PCSK9 secretion in hepatic Huh7 cells. [ABSTRACT FROM AUTHOR]

Iron-enriched Cordyceps militaris was obtained by adding FeSO 4 solution to the mycelia for biotransformation. The polysaccharide-iron (III) was extracted by water extraction and alcohol precipitation. High performance liquid chromatography showed that the crude polysaccharide-iron (III) had three components. The second component was purified by Sephadex G-150 and named as CPS-iron-II. The average molecular weight of CPS-iron-II was 44.136 kDa. The content of iron was 2.73%. The monosaccharide composition analysis indicated that the CPS-iron-II was composed of rhamnose, arabinose, galactose, glucose, mannose, galacturonic acid with percentage ratio of 0.94:3.12:27.01:36.62:30.20:2.12. The results of methylation analysis revealed that the CPS-iron-II was made of →2)-β-D-Glc p -(1→, with →2, 4)-α-D-Glc p -(1→ highly branched. Congo-red test showed that CPS-iron-II can cause flocculation of Congo red solution. The anti-oxidative analysis showed that antioxidant activity of CPS-iron-II was almost equal to that of Vc. The manuscript provided a new way for the preparation of polysaccharide-iron(III) from Cordyceps militaris. • Iron-enriched Cordyceps militaris mycelia were prepared through biotransformation. • CPS-iron-II was isolated from iron-enriched mycelia. • The structures of CPS-iron-II were accurately characterized. • The CPS-iron-II had good antioxidant activity. [ABSTRACT FROM AUTHOR]

A novel neutral exopolysaccharide (EPS-III) was isolated from culture broth of Cordyceps militaris (C. militaris). The EPS-III was a homogeneous polysaccharide with M w of 1.56 × 103 kDa. The yield of EPS-III from culture broth was 123.2 ± 3.1 mg/L and the sugar content was 93.32 ± 0.87%. The backbone of EPS-III was mainly consisted of →4)-α-D-Gal p -(1→, while →3, 6)-α-D-Man p -(1→, →4)-α-D-Man p -(1→, →3)-β-D-Gal p -(1→ and →3)-α-D-Glc p -(1→ were distributed in the backbone or in the branch chains. The EPS-III had helix structure when dissolved in weak alkaline solution. It also had branched and intertwined form on the surface. The inhibition of α-glucosidase significantly increased as the increase of purity of exopolysaccharides. The EPS-III had effective inhibition on the α-glucosidase with dose-effect relationship. Besides, the results of hypoglycemic activity analysis in vivo indicated that EPS-III can alleviate weight loss, reduce plasma glucose concentration, improve glucose tolerance, protect immune organs and repair dyslipidemia to relieve diabetes in STZ-induced diabetic mice. The manuscript first studied the hypoglycemic activity of exopolysaccharide of by C. militaris , proving and promoting the application value of culture broth. The structure characterization of EPS-III laid experimental foundations on the exploration of structure-activity relationship. • A novel exopolysaccharide (EPS-III) of Cordyceps militaris was isolated. • The yield of EPS-III from culture broth was 123.2 ± 3.1 mg/L. • The chemical structure of EPS-III was accurately characterized. • The EPS-III has effective inhibition on α-glucosidase. • The EPS-III can relieve diabetes in STZ-induced diabetic mice. [ABSTRACT FROM AUTHOR]

In this study, the purified polysaccharide (DCP-I) was extracted from Cordyceps militaris domesticated with Pb2+. After that, the structural feature and mechanism of lead resistance of DCP-I were investigated using novel approaches. The results showed that the average molecular weight of DCP-I was 1.206 × 103 kDa and mainly consist of Rhamnose, Galactose, Glucose, Galacturonic acid and Glucuronic acid in a molar ratio of 0.130:47.687:40.784:1.795:0.48. Besides, the main chain of DCP-I was composed by →6)-Gal p -(1→, →4)-Glc p -(1→ and →1,4)-Glc p -(6→, while the side chain was →1)-Rha f- (2→ and D-Glc p -(1→, and the DCP-I contained Alacturonic acid and Glucuronic acid. In addition, the result of Congo red test showed that DCP-I did not exist triple-helical structures. SEM, EDX and XPS analyses results showed that the functional groups of DCP-I related to C, H and O (-OH, -COOH and -C=O) could combined with Pb2+effectively. The adsorption processes were described by the Pseudo-second-order kinetic model (R2 = 0.9978) and Langmuir isotherm (R2 = 0.9979) for Pb2+ indicating that adsorption process of DCP-I to Pb2+ was a kind of single molecular layer chemical adsorption. • A novel polysaccharide DCP-I was isolated from Cordyceps militaris domesticated with Pb2+. • The structure of the DCP-I was accurately characterized. • The adsorption process of DCP-I to Pb2+ was single molecular layer chemical adsorption. • The functional groups of -OH, -COOH and -C=O could combined with Pb2+ effectively. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungi (EPF) produce multiple mycotoxins, which play an essential role in improving fungal pathogenesis and virulence. To characterize various mycotoxins from the crude methanol extract of Cordyceps fumosorosea , a major EPF against various insect pests, we performed ultra-performance liquid chromatography coupled to quadrupole time of flight mass spectrometer (UPLC-QTOF MS) technique, and all compounds were identified through molecular mass and formulae. Bassianolide was assessed against the nymphs and adults of Diaphorina citri reared on healthy and Huánglóngbìng (HLB)-diseased Citrus spp. Plants under laboratory conditions. Overall, 17 compounds were identified from the fungal extract and categorized into three groups, i.e. (1) alkaloids (Isariotins A-C), (2) peptides (Bassianolide, Beauverolides, Beauvericin A, Isaridins and Destruxin E) and (3) polyketide (Tenuipyrone). The detected beauverolides (B, C, F, I, Ja) from C. fumosorosea were novel mycotoxins, and their detection intensity was the highest in the fungal extract. Furthermore, bassianolide caused more than 70% and 80% mortality of D. citri nymphs and adults after two days of application, respectively. After three days of chemical application, all nymphal and adult populations of D. citri were killed by bassianolide. However, the mortality rates of both populations, nymphs and adults, were higher on HLB-diseased plants as compared to healthy plants. Image 1 • Seventeen mycotoxins were identified by mass and formulae via liquid chromatography-mass spectrometry. • Mycotoxins were classified into three groups; 1) alkaloids, 2) peptides, 3) polyketide. • Methanol extract of Cordyceps fumosorosea was enriched with novel beuverolides. • Bassianolide caused more than 70% mortality of citrus psyllid within two days. • Mortality of adult and nymph was higher on huanglongbing-diseased plants. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN), a complication of diabetes mellitus, has been the leading cause of death in people with chronic kidney disease. This study was conducted to examine the potential health benefits of Cordyceps cicadae polysaccharides (CCP) on kidney injury and renal interstitial fibrosis that occur in DN rats. First, a DN model was established using SD rats fed with a high-fat diet for 8 weeks, then injected with STZ (35 mg/kg) intraperitoneally. The rats were then supplemented with CCP (75, 150 and 300 mg/kg) for 4 weeks. The results indicated that CCP improve insulin resistance and glucose tolerance in DN rats. Furthermore, CCP intervention significantly suppressed the inflammation, renal pathological changes and renal dysfunction, slowing down the progression of renal interstitial fibrosis. Moreover, high-throughput pyrosequencing of 16S rRNA suggested that CCP modulated the dysbiosis of gut microbiota by enhancing the relative abundance and proliferation capacity of probiotics. In vitro, CCP can markedly decrease LPS-induced inflammatory cytokine levels and TGF-β1-induced fibroblast activation. In summary, the results provided evidence that CCP exerted a beneficial effect on tubulointerstitial fibrosis in DN rats by possibly suppressing the inflammatory response and modulating gut microbiota dysbiosis, via blocking the TLR4/NF-κB and TGF-β1/Smad signaling pathway. Unlabelled Image [ABSTRACT FROM AUTHOR]

In the study, the β -glucan content, the primary structure and the antioxidant capacity of polysaccharides in Cordyceps militaris cultivated with different initial growth pH were evaluated. Meanwhile, the mechanism of β -glucan biosynthesis was investigated by RNA-Seq. Based on the results, C. militaris growing at an initial growth pH of 5–7 (CMsA) was distinguished from C. militaris growing at an initial growth pH of 8–9 (CMsB) and their unigenes showed the comparable expression. The mean of β -glucan content of CMsB group was 32.7% (w/w), 10% higher than that of CMsA. The results of RNA-seq showed 1088 differentially expressed genes between CMsA and CMsB groups. Furthermore, oxidative phosphorylation-related Gene ontology terms were up-regulated in CMsB groups. In addition, the results of structural analysis (FTIR spectrum, monosaccharide composition, periodate oxidation) and bioactivity evaluation speculated that C. militaris polysaccharides possessed higher β -(1 → 6)-glucan content and antioxidant activities in CMsB groups. Unlabelled Image • Initial growth pH can apparently affect the content of β -glucan. • Oxidative phosphorylation-related genes were up-regulated in alkaline conditions. • CMPs from C. militaris (in alkaline conditions) possessed more β -(1 → 6)-glucan. • CMPs from C. militaris (in alkaline conditions) had greater antioxidant activities. [ABSTRACT FROM AUTHOR]

• Growth of C. militaris mycelium was influenced by addition of Na 2 SeO 3. • Metabolites of C. militaris including thiol and polysaccharide were also affected. • Activities of enzymes including CAT, SOD, GSH-PX, PGM, PGI and UGP enhanced. The main objective of our study was to illuminate effects of sodium selenite (Na 2 SeO 3) on growth, metabolism and enzyme activities of Cordyceps militaris. By adding Na 2 SeO 3 in fermentation medium (7 mg/L), it was found that mycelium of C. militaris was stronger with more plump spores and its biomass was higher accompanied by the maximum (13.19 g/L) at 6 d of culture. Besides, Na 2 SeO 3 also caused the enhancement of total thiol (T-SH), non-protein thiol (NP-SH) contents and the biosynthesis of Se-polysaccharide (Se-CMP) with discriminatory molecular weight, optical rotation, UV–vis and FT-IR spectra. Activities of antioxidase including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and enzymes involved in polysaccharide synthesis including phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), UDPG-pyrophosphorylase (UGP) enhanced to different extent. Their corresponding genes were also up-regulated but cat gene (encoding CAT). Se-enrichment of C. militaris provided a good way for desirable biomass and polysaccharide biosynthesis in further research. [ABSTRACT FROM AUTHOR]

Cordyceps cicadae is a traditional Chinese medicine with high nutritional value and biological activities. Previously, we reported on the antioxidant activity associated with the polysaccharides from Cordyceps cicadae (CP). To further explore which of the fraction of CP had the greatest potency, in here, the in vitro antioxidant and in vivo anti-aging activities of the fractions CP30–CP80 of CP were evaluated. The in vitro antioxidant activity results revealed that all the fractions (i.e. CP30–CP80) were potent with CP70 as the most potent. Notably, CP70 prolonged the lifespan of Drosophila (P < 0.05), increased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) (P < 0.01), and inhibited the formation of malondialdehyde (MDA) (P < 0.01). Additionally, CP70 upregulated the expression level of antioxidant-related genes CAT , SOD1 and MTH in Drosophila (P < 0.05). These results indicated that CP70 may prolong the lifespan of Drosophila through the up-regulation of the expression level of antioxidant-related genes CAT , SOD1 and MTH in Drosophila. Thus, polysaccharides from Cordyceps cicadae possess significant antioxidant and anti-aging activities, and could be explored as a new dietary supplement to slow down the aging process. [ABSTRACT FROM AUTHOR]

Tumour-associated macrophages (TAMs) inhibit the killing effect of T lymphocytes on tumour cells through the immunocheckpoint programmed death ligand-1 (PD-L1)/programmed death-1 (PD-1) axis. TAMs-targeted therapy i s a promising approach that could be used to reverse the immunosuppressive tumour microenvironment. Here, we further report CMPB90-1, a novel natural polysaccharide from Cordyceps militaris , could function as an anti-tumour modulator that resets TAMs from a tumour-promoting M2 phenotype to a tumour-killing M1 phenotype. This process involves reversing the functional inhibition of T lymphocytes by inhibiting the PD-L1/PD-1 axis between TAMs and T lymphocytes. Mechanistically, the membrane receptor of CMPB90-1 binding to M2 macrophages was identified by tandem mass spectrometry. CMPB90-1 converts immunosuppressive TAMs via binding to toll-like receptor 2 (TLR2), which causes the release of Ca2+ and the activation of p38, Akt and NF-κB, or ERK. This process then leads to the polarization of TAMs from M2 phenotype to the M1 phenotype. In vivo experiment shows that CMPB90-1 is able to polarize TAMs into the M1 phenotype and has anti-tumour effects with improved safety. Additionally, the anti-tumour effects of CMPB90-1 in vivo depend on the phenotypic conversion of TAMs. The results demonstrated that CMPB90-1 could be developed as a potential immune-oncology treatment reagent. Unlabelled Image • Membrane receptors of CMPB90-1 binding to TAMs were identified by protein mass spectrometry and bioinformatics analysis. This process involves inhibiting the PD-L1/PD-1 axis between TAMs and T lymphocytes. [ABSTRACT FROM AUTHOR]

This study was to assess the anti-inflammatory potential of exopolysaccharide (EPS) produced by a medicinal fungus Cordyceps sinensis Cs-HK1. The EPS was isolated from the Cs-HK1 mycelial fermentation broth by ethanol precipitation and purified by deproteinization and dialysis. The EPS had a total sugar content of 74.8% and a maximum average molecular weight (MW) over 107 Da, and consisted mainly of glucose and mannose, and a small amount of galactose and ribose. In THP-1 and RAW264.7 cell cultures, EPS significantly inhibited lipopolysaccharide (LPS)-induced inflammatory responses of the cells including the release of NF-κB and several pro-inflammatory factors such as NO, TNF-α and IL-1β. In the murine model of LPS-induced acute intestinal injury, the oral administration of EPS to the animals effectively suppressed the expression of major inflammatory cytokines TNF-α, IL-1β, IL-10 and iNOS and alleviated the intestinal injury. The results suggest that the Cs-HK1 EPS has notable anti-inflammatory activity and can be a potential candidate for further development of new anti-septic therapeutics. To the best of our knowledge, this is the first report on the anti-inflammation of an EPS from C. sinensis fungal fermentation. • High MW exopolysaccharide (EPS) was isolated from Cs-HK1 mycelial culture broth. • EPS inhibited LPS-induced NF-κB, NO and inflammatory cytokines in both cell and animal models. • The inhibiting activities of EPS were dose-dependent with relatively low IC50 values. • EPS alleviated LPS-induced acute intestinal injury in animal model. [ABSTRACT FROM AUTHOR]

The natural form of wild edible fungus is the fruiting body. The cultivation of fruiting bodies from sexual reproduction requires strict conditions and long periods. Some literatures have paid attentions on the mycelia prepared with liquid fermentation to alter fruiting bodies. Cordyceps militaris (C. militaris) is a kind of precious edible fungus. The polysaccharide is an important active ingredient in C. militaris. The manuscript aimed to evaluate the feasibility of alternative of mycelia to fruit bodies with studies of polysaccharides from C. militaris of different developmental stages. The two polysaccharides were separated. The chemical structure and inhibitory activity on α-glucosidase of polysaccharides were explored. The results indicated that the structure and inhibitory activity on α-glucosidase of polysaccharides with different developmental stages had significant differences. The polysaccharides from fruiting bodies had better inhibitory activity on α-glucosidase. It demonstrated that the mycelia of C. militaris from asexual reproduction with liquid fermentation can't be an effective substitute for fruiting bodies from sexual reproduction, from the perspective of polysaccharides. • Polysaccharides were separated from C. militaris with different developmental stages. • Two polysaccharides had significant differences in chemical structure. • Polysaccharide from fruiting bodies had better inhibitory activity on α-glucosidase. • Mycelia of C. militaris can't be an effective substitute for fruiting bodies. [ABSTRACT FROM AUTHOR]

• First study reporting cloning and sequencing of fungal fibrinolytic protease gene. • Recombinant fibrinolytic enzyme from Cordyceps militaris was expressed extracellularly in Pichia pastoris. • Recombinant enzyme degraded fibrin clot. • Homology-based 3D structure of CmFE was built using urokinase-type plasminogen activator as template. • 3D model provided in-depth insight into the catalytic pocket, substrate binding sites and disulfide bridges. Fibrinolytic enzymes are important thrombolytic agents for blood-clotting disorders like cardiovascular diseases. Availability of novel recombinant fibrinolytic enzymes can overcome the shortcomings of current thrombolytic drugs. With the objective of facilitating their cost-effective production for therapeutic applications and for gaining deeper insight into their structure-function, we have cloned and expressed the first fibrinolytic protease gene from Cordyceps militaris. Cordyceps militaris fibrinolytic enzyme (CmFE) has one open reading frame of 759 bp encoding "pre-pro-protein" of 252 amino acids. Recombinant CmFE was expressed as 28 kDa extracellular enzyme in Pichia pastoris which was capable of degrading fibrin clot. A structure homology model of CmFE was developed using urokinase-type plasminogen activator. The active site contains catalytic triad His41, Asp83, Ser177 and consensus sequence of GDSGG. The substrate binding residues are Asp (171), Gly (194) and Ser (192). Its trypsin-like specificity is determined by the critical Asp171 in S1 subsite. The "oxyanion hole" is formed by backbone amide hydrogen atoms of Gly-175 and Ser-177. CmFE contains six conserved cysteines forming three disulfide linkages. This is the first study describing cloning, expression and prediction of structure-function relationship of a mushroom fibrinolytic protease. Hence it has great relevance in application of fibrinolytic enzymes as thrombolytic agents. [ABSTRACT FROM AUTHOR]

In this study, we extracted the polysaccharides from C. militaris fruiting bodies (CFIPs), mycelial intracellular polysaccharides (CMIPs), and fermentation broth extracellular polysaccharides (CFEPs) to investigate their physicochemical properties, antioxidant capacities, and effects on oxazolone-induced zebrafish ulcerative colitis (UC). Our results revealed differences in monosaccharide composition and surface structure among CFIPs, CMIPs, and CFEPs. The molar ratios of glucose to mannose in CFIPs, glucose to xylose in CMIPs, and xylose to glucose in CFEPs were 7.57: 1.6, 7.26: 1.81, and 5.44: 2.98 respectively. Moreover, CFEPs exhibited significantly (p < 0.05) higher chemical antioxidant capacity compared to CMIPs and CFIPs. Surprisingly, CFEP treatment didn't show a significant effect in protecting against H 2 O 2 -induced oxidative damage in RAW 264.7 cells. After 3 d of treatment, the levels of ROS, MDA, and MPO in the CFIPs group exhibited a significant (p < 0.05) reduction by 37.82 %, 68.15 %, and 22.77 % respectively. Additionally, the ACP and AKP increased by 60.33 % and 96.99 %. Additionally, C. militaris polysaccharides (CMPs) were found to effectively improve UC by activating the MyD88/NF-κB signaling pathway in vivo. These findings confirm the distinct physicochemical properties of these three types of CMP and their potential for development into antioxidant-rich anti-inflammatory health foods. [Display omitted] • The monosaccharide composition and structure of the three polysaccharides exhibited variations. • CFIP inhibits ROS and MPO activities while enhancing SOD and LZM activities. • CMPs effectively ameliorated UC by activating the MyD88/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Asymmetric synthesis of chiral alcohols by biocatalysts holds promising prospect. In this study, a medicinal Cordyceps fungus, namely Tolypocladium inflatum , was adopted as a novel biocatalyst for the synthesis of chiral alcohols. Among the assessed prochiral ketone substrates, T. inflatum exhibited the best catalytic activity towards 1-(4-bromophenyl)−2,2,2-trifluoroethanone (BPFO), yielding a valuable pharmaceutical intermediate (S)−1-(4-bromophenyl)−2,2,2-trifluoroethanol ((S)-BPFL) with >99.9 % enantiomeric excesses (ee). For more efficient biosynthesis of (S)-BPFL, cyclodextrins or surfactants were introduced to the reaction system as additives to strengthen this bioprocess. Among which methyl β-cyclodextrin (MCD) improved cell membrane permeability and alleviated product inhibition, while Triton X-100 enhanced BPFO solubility. Furthermore, synergistic promotion for BPFO bioreduction by MCD and Triton X-100 was observed. Under the optimal reaction conditions, 50 mM BPFO was converted into (S)-BPFL with 89.1 % yield and >99.9 % ee in 12 h, showing 16-fold increase of space-time yield in the developed MCD/Triton X-100-buffer system compared to neat buffer solution. This study illustrated the potential of the enantioselective preparation of chiral alcohols mediated by Cordyceps fungus. Additionally, the biocatalytic efficiency was significantly improved in the constructed synergistic reaction system, and the underlying mechanism was also explored. [Display omitted] • Medicinal Cordyceps T. inflatum was firstly applied for chiral alcohols synthesis. • T. inflatum -catalyzed reduction was intensified in MCD/Triton X-100-buffer system. • MCD and Triton X-100 exhibited synergistic promotion for BPFO bioreduction. • Space-time yield of (S)-BPFL was increased by 16 times in the synergistic system. [ABSTRACT FROM AUTHOR]

[Display omitted] • Infrared data benefits herbal medicine detection. • Federated learning: privacy-preserving model training. • Deep learning: advanced neural networks for C.sinensis analysis. • Preprocessing: improves data quality for accurate analysis. The modeling and analysis of infrared spectrum data have gained significant advantages in the field of rapid detection of herbal medicine. However, extracting valuable insights and patterns from diverse chemical information relies on large-scale datasets and advanced algorithms. Unfortunately, due to privacy concerns, data sharing between different companies is often limited in practice. In such scenarios, federated learning emerges as a novel solution. Federated learning allows different regions or companies to jointly train models without sharing sensitive data, thereby protecting privacy. To address the quality control challenges related to Cordyceps sinensis(C.sinensis), this paper proposes a federated learning and deep learning method based on infrared spectroscopy data of C.sinensis, and then evaluates their performance on the C.sinensis authenticity detection and multi-classification tasks. In the classification tasks of 'part', 'phase', 'region', and 'True or False' (TorF), the mean MCC of the best model is 0.8805, 0.5865, 0.7004, and 0.9555 respectively. Experimental results show that the method proposed in this paper is an effective C.sinensis quality control method and it also provides solutions and ideas for privacy protection issues involving drug monitoring. [ABSTRACT FROM AUTHOR]

In this study, we extracted and isolated two polysaccharides with molecular weights of 2.28 × 104 Da and 2.71 × 103 Da from Cordyceps militaris , named CMP-90 and CMP-E, respectively. Structural characterization revealed that CMP-90 consists of six glycosidic bonds, Glc p -(1→, Gal p -(1→, →3)-Glc p -(1→, →6)-Glc p -(1→, Man p -(1→, and →4,6)-Man p -(1→. CMP-E consists of →4)-Gal p -(1→, →4)-Glc p -(1→, →6)-Glc p -(1→, Glc p -(1→, →3)-Glc p -(1→, →2,3,6)-Glc p -(1→, and →2)-Glc p -(1→ glycosidic bonds. Animal experiments demonstrated that both polysaccharides were able to increase immune cell viability, regulate the ratio of lymphocyte subpopulations, and increase the levels of serum cytokines (TNF-α, IL-2, and IFN-γ) by improving the status of immune organs in loaded tumor-bearing mice. CMP-90 and CMP-E were also able to activate caspase-mediated apoptosis to cause apoptosis of S180 cells through the Wnt/β-catenin pathway, which was blocked at the G1 phase. The above experimental results suggest that Cordyceps militaris polysaccharides have the potential to become a functional agent for sarcoma. [Display omitted] • CMP-90 and CMP-E were purified from Cordyceps militaris. • The structures of the two Cordyceps militaris polysaccharides were compared. • CMP-90 and CMP-E presented superior anti-tumor activity in vivo. • C. militaris polysaccharides induces cell apoptosis through Wnt/β-catenin pathway. [ABSTRACT FROM AUTHOR]

Cordyceps militaris , a medicinal and edible fungus, has garnered significant interest for its polysaccharides, known for their diverse physiological activities and health benefits. The purpose of this study was to extract polysaccharides (CM-1) from Cordyceps militaris fruiting bodies using hot water extraction and column chromatography, and evaluate its structural characteristics, antioxidant capacity, and in vivo absorption effects. Additionally, the study aimed to assess its protective effects against carbon tetrachloride (CCl 4) induced liver injury in mice. The results showed that CM-1 significantly decreased the expression levels of serum ALT, AST, TNF-α, IL-6, and IL-1β. Pre-treatment with CM-1 also increased the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Furthermore, histopathological examination of liver tissues confirmed the protective effects of CM-1 against acute liver injury induced by CCl 4. These results indicate CM-1 promising potential as a dietary supplement for liver injury prevention and treatment. [ABSTRACT FROM AUTHOR]

In this study, Cordyceps militaris matrix was employed for the first time to fabricate a biodegradable food packaging. Carmine and Ag@CuBTC were introduced to cross-link with mycelium and were uniformly dispersed within the matrix to enhance the water resistance, antimicrobial, and antioxidant properties of the bio-films. The bio-film displayed high biodegradability, with nearly 100 % degradation achieved after three weeks. The bio-film exhibited exceptional resistance to oxidation (49.30 % DPPH and 93.94 % ABTS•+), as well as effective inhibitory capabilities against E. coli and S. aureus , respectively. The composite film maintained a high CO 2 /O 2 selective permeability, which was advantageous for mitigating fruit metabolism and extending shelf life. Simultaneously, food preservation experiments confirmed that these bio-films can decelerate the spoilage of fruits and effectively prolong the shelf-life of food. The experimental findings indicated that the prepared Bio-R-Ag@Cu film held promise as an environmentally friendly biodegradable material for food packaging. [ABSTRACT FROM AUTHOR]