Proteomics analysis of enzyme systems and pathway changes during the moromi fermentation of soy sauce mash.

BACKGROUND: During the brewing of soy sauce, the conversion of multiple substances is driven by various microorganisms and their secreted enzyme systems. Soy sauce mash is an important source of enzyme systems during moromi fermentation, but the changes of enzyme systems in soy sauce mash during moromi fermentation are poorly understood. In order to explore the predominant enzyme systems existing during moromi fermentation and to explain the characteristics of the enzyme system changes, an enzymatic activities assay and 4D-label-free proteomics analysis were conducted on soy sauce mash at different stages of fermentation. RESULTS: The activities of hydrolytic enzymes in soy sauce mash decreased continuously throughout the fermentation process, while most of the characteristic physicochemical substances in soy sauce mash supernatant had already accumulated at the early stage of fermentation. Four hydrolytic enzymes were found to be positively correlated with important physicochemical indexes by principal component analysis and Pearson correlation analysis. The proteomics analysis revealed three highly upregulated enzymes and two enzymes that were present in important metabolic pathways throughout the fermentation process. Furthermore, it was found that Aspergillus oryzae was able to accumulate various nutrients in the soy sauce mash by downregulating most of its metabolic pathways. CONCLUSION: Enzymes present with excellent properties during the moromi fermentation period could be obtained from these results. Meanwhile, the characterization of the metabolic pathways of microorganisms during the moromi fermentation period was revealed. The results provide a basis for more scientific and purposeful improvement of moromi fermentation in the future. © 2024 Society of Chemical Industry.

Cordyceps militaris polysaccharides modulate gut microbiota and improve metabolic disorders in mice with diet-induced obesity.

BACKGROUND: Cordyceps militaris is an edible and medicinal fungus, and its polysaccharides are among its main pharmacological components. They can display immunomodulation, anti-oxidation, anti-inflammation, anti-hypolipidemic, and other functions. The anti-obesity effect of C. militaris polysaccharides (CMP) is not yet fully understood, however. RESULTS: In this study, a CMP diet intervention was applied over a 4 week period to mice with obesity induced by a high-fat diet (HFD), followed by profiling of obesity-induced dyslipidemia, low-grade inflammation, and gut dysbiosis. The results suggested that CMP could significantly reduce HFD-induced obesity, alleviate obesity-induced hyperlipidemia and insulin resistance, and ameliorate systemic inflammation, showing a promising ability to protect mice from obesity. Further analyses revealed that CMP could regulate obesity-induced gut dysbiosis by restoring the phylogenetic diversity of gut microbiota. It could also increase the relative abundance of short-chain fatty acid (SCFA)-producing bacteria, while down-regulating the level of bacteria that were positively related to the development of obesity. A correlation analysis showed that Helicobacter, Allobaculum, Clostridium XVIII, Parabacteroides, Ligilactobacillus, Faecalibaculum, Adlercreutzia, and Mediterraneibacter were positively related to obese phenotypes. CONCLUSION: This study highlights the potential of CMP as a prebiotic agent to protect obese individuals from metabolic disorders and gut dysbiosis. © 2022 Society of Chemical Industry.

Purification and structural characterization of a novel natural pigment: cordycepene from edible and medicinal mushroom Cordyceps militaris.

In the present work, a novel cordycepic pigment was successfully isolated and identified from Cordyceps militaris, as well as named as cordycepene (C14H17N1O4), according to the long unsaturated conjugated polyene structural characteristic. Cordycepene is sensitive to light, high temperature (≥ 60 °C), and acidic condition (pH ≤ 3), but possesses high stability against metal ions, and under alkaline and neutral conditions. Cordycepene shows a comparable DPPH (1,1-diphenyl-2-picrylhydrazyl) radical-scavenging activity at higher concentration (≥ 2 mg/mL) to vitamin C. Cordycepene promotes the growth of HSF (human skin fibroblast cell) after incubation for 72 h, and has an ability to repair the UV light-treated HSF cells. In addition, cordycepene increases the antioxidant activity (SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase) and decreases MDA (malondialdehyde) level, indicating that cordycepene inhibits the photochemical senescence of HSF by enhancing the antioxidant defense system. The discovery of cordycepene can provide a basis for research on light incubation and the accumulation of yellow pigment (carotenoids) from C. militaris.

Cmfhp Gene Mediates Fruiting Body Development and Carotenoid Production in Cordyceps militaris.

Cordyceps militaris fruiting bodies contain a variety of bioactive components that are beneficial to the human body. However, the low yield of fruiting bodies and the low carotenoid content in C. militaris have seriously hindered the development of the C. militaris industry. To elucidate the developmental mechanism of the fruiting bodies of C. militaris and the biosynthesis mechanism of carotenoids, the function of the flavohemoprotein-like Cmfhp gene of C. militaris was identified for the first time. The Cmfhp gene was knocked out by the split-marker method, and the targeted gene deletion mutant ΔCmfhp was obtained. An increased nitric oxide (NO) content, no fruiting body production, decreased carotenoid content, and reduced conidial production were found in the mutant ΔCmfhp. These characteristics were restored when the Cmfhp gene expression cassette was complemented into the ΔCmfhp strain by the Agrobacterium tumefaciens-mediated transformation method. Nonetheless, the Cmfhp gene had no significant effect on the mycelial growth rate of C. militaris. These results indicated that the Cmfhp gene regulated the biosynthesis of NO and carotenoids, the development of fruiting bodies, and the formation of conidia. These findings potentially pave the way to reveal the developmental mechanism of fruiting bodies and the biosynthesis mechanism of carotenoids in C. militaris.

Transcriptome Analysis of Cordyceps militaris Reveals Genes Associated With Carotenoid Synthesis and Identification of the Function of the Cmtns Gene.

Cordyceps militaris, a valuable edible and medicinal fungus, has attracted increasing attention because of its various bioactive ingredients. However, the biosynthetic pathway of C. militaris carotenoids is still unknown due to lack of transcriptome information. To uncover genes related to the biosynthesis of C. militaris carotenoids, the transcriptomes of mycelia CM10_D cultured under dark conditions and mycelia CM10_L cultured under light exposure conditions were sequenced. Compared with mycelia CM10_D, 866 up-regulated genes and 856 down-regulated genes were found in mycelia CM10_L. Gene ontology (GO) analysis of differentially expressed genes (DEGs) indicated that DEGs were mainly classified into the "metabolic process," "membrane," and "catalytic activity" terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs suggested that DEGs were mainly enriched in "metabolic pathways," "MAPK signaling pathway-yeast," and "biosynthesis of secondary metabolites." In addition, the carotenoid content of the Cmtns gene deletion mutant (ΔCmtns) was significantly lower than that of the wild-type C. militaris CM10, while the carotenoid content of the complementary strain (ΔCmtns-c) of the Cmtns gene was not significantly different from that of C. militaris CM10, suggesting that the Cmtns gene significantly affected the biosynthesis of carotenoids in C. militaris. These results potentially pave the way for revealing the biosynthetic pathway of carotenoids and improving carotenoids production in C. militaris.

Increasing of the Contain of Carotenoids in Caterpillar Mushroom, Cordyceps militaris (Ascomycetes) by Using the Fungal Elicitors Cultivation.

As a natural pigment, cordycepic carotenoids have many bioactive functions, such as antiinflammation, anticancer, and antioxidation. In addition, the good coloring of this hydrophilic pigment enables it to have wide application in the food industry. This study investigated five species of fungal elicitors, namely, Rhodotorula glutinis, Saccharomyces cerevisiae, Monascus ruber, Blakeslea trispora, and Flammulina velutipes, to evaluate their effects on carotenoid accumulation in Cordyceps militaris. Results showed that all fungal elicitors, except Rh. glutinis, have no positive effect on the biosynthesis of cordycepic carotenoids. The Rh. glutinis elicitor remarkably stimulated the accumulation of carotenoids with a 13.72% increase compared with the control. Subsequently, the entire Rh. glutinis elicitor (part NHK) was divided into three parts, namely, exopolysaccharide (EPS) (part E), mixture of EPS and protein (part PE), and other components (part O), to analyze their effects on carotenoid accumulations. Results showed that part O may be the effective component that remarkably stimulates the biosynthesis of carotenoids with a 26% increase compared with the control. This research demonstrated that Rh. glutinis elicitor can effectively increase the content of natural carotenoids in C. militaris, and provided an important reference for the development and utilization of carotenoid industrialization.

Efficient CRISPR-Cas9 Gene Disruption System in Edible-Medicinal Mushroom Cordyceps militaris.

Cordyceps militaris is a well-known edible medicinal mushroom in East Asia that contains abundant and diverse bioactive compounds. Since traditional genome editing systems in C. militaris were inefficient and complicated, here, we show that the codon-optimized cas9, which was used with the newly reported promoter Pcmlsm3 and terminator Tcmura3, was expressed. Furthermore, with the help of the negative selection marker ura3, a CRISPR-Cas9 system that included the Cas9 DNA endonuclease, RNA presynthesized in vitro and a single-strand DNA template efficiently generated site-specific deletion and insertion. This is the first report of a CRISPR-Cas9 system in C. militaris, and it could accelerate the genome reconstruction of C. militaris to meet the need for rapid development in the fungi industry.

Effect of pH on the rheological properties of borate crosslinked hydroxypropyl guar gum hydrogel and hydroxypropyl guar gum.

pH is an important factor affecting the performance of polymer fluid. The rheological properties of hydroxypropyl guar gum (HPG) base fluid and the structural strength, rheological properties, viscoelastic properties and thixotropy properties of HPG gel depend largely on the pH values. For the base fluid, an apparent viscosity-increasing effect was observed over the pH range from 7 to 11, and the apparent viscosity gradually decreased at pH 11.5-14, exhibiting electrostatic repulsion behavior and steric effects. For the HPG gel, at pH 7-12.5, the gel possessed higher apparent viscosity, higher elastic modulus (G'), lower tanδ (the ratio of the viscous modulus to the elastic modulus) and an "8"-shaped hysteresis loop, indicating stronger gel structure strength and the elastic dominant property. At pH 13-13.5, the gel samples exhibited the transition from a pseudoplastic fluid to a Newtonian fluid, and their viscosity, elastic modulus decreased but tanδ increased with the increase in pH values, exhibiting gradually weakened elastic properties. When the pH was 14, the gel mainly exhibited viscous characteristics.

Intrinsic viscosity and rheological properties of natural and substituted guar gums in seawater.

The intrinsic viscosity and rheological properties of guar gum (GG), hydroxypropyl guar (HPG) and carboxymethyl guar (CMG) in seawater and the effects of shear rate, concentration, temperature and pH on these properties were investigated. An intrinsic viscosity-increasing effect was observed with GG and HPG in seawater (SW) compared to deionized water (DW), whereas the intrinsic viscosity of CMG in seawater was much lower than that in DW due to a screening effect that reduced the repulsion between the polymer chains. Regardless of the functional groups, all sample solutions was well characterized by a modified Cross model that exhibited the transition from Newtonian to pseudoplastic in the low shear rate range at the concentrations of interest to industries, and their viscosity increased with the increase in their concentration but decreased with the increase in temperature. In contrast to nonionic GG or HPG, anionic CMG had a slightly decreased viscosity property in SW, exhibiting polyelectrolyte viscosity behavior. The α value in the zero-shear rate viscosity vs. concentration power-law equation for the samples gave the order of CMG>HPG>GG while the SW solution of CMG had the lowest viscous flow activation energy and exhibited a strong pH-dependent viscosity by a different shear rate.