Protective Effect of Eurotium cristatum Fermented Loose Dark Tea and Eurotium cristatum Particle on MAPK and PXR/AhR Signaling Pathways Induced by Electronic Cigarette Exposure in Mice.

Electronic-cigarette smoke (eCS) has been shown to cause a degree of oxidative stress and inflammatory damage in lung tissue. The aim of this study was to evaluate the repair mechanism of Eurotium cristatum fermented loose dark tea (ECT) and Eurotium cristatum particle metabolites (ECP) sifted from ECT after eCS-induced injury in mice. Sixty C57BL/6 mice were randomly divided into a blank control group, an eCS model group, an eCS + 600 mg/kg ECP treatment group, an eCS + 600 mg/kg ECT treatment group, an eCS + 600 mg/kg ECP prevention group, and an eCS + 600 mg/kg ECT prevention group. The results show that ECP and ECT significantly reduced the eCS-induced oxidative stress and inflammation and improved histopathological changes in the lungs in mice with eCS-induced liver injury. Western blot analysis further revealed that ECP and ECT significantly inhibited the eCS-induced upregulation of the phosphorylation levels of the extracellular Regulated protein Kinases (ERK), c-Jun N-terminal kinase (JNK) and p38mitogen-activated protein kinases (p38MAPK) proteins, and significantly increased the eCS-induced downregulation of the expression levels of the pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) proteins. Conclusively, these findings show that ECP and ECT have a significant repairing effect on the damage caused by eCS exposure through the MAPK and PXR/AhR signaling pathways; ECT has a better effect on preventing eCS-induced injury and is suitable as a daily healthcare drink; ECP has a better therapeutic effect after eCS-induced injury, and might be a potential therapeutic candidate for the treatment of eCS-induced injury.

Fintiamin: A diketopiperazine from the marine sponge-derived fungus Eurotium sp.

The fungus Eurotium sp., derived from the marine sponge Ircinia variabilis, was found to produce a diketopiperazine-indole alkaloid that we named fintiamin (1). Structural elucidation of 1 was achieved by extensive spectroscopic analysis including nuclear magnetic resonance spectroscopy and mass spectrometry. Compound 1 is a lipophilic terpenoid-dipeptide hybrid molecule that shows affinity for the cannabinoid CB1 receptor at low micromolar concentrations. Docking studies based on previous X-ray structures provide a plausible binding pose for compound 1 in the orthosteric binding site of the CB1 receptor.

Influence of Eurotium cristatum and Aspergillus niger individual and collaborative inoculation on volatile profile in liquid-state fermentation of instant dark teas.

The three instant dark teas were produced from instant green tea (IGT) by liquid-state fermentations using the microorganisms Eurotium cristatum (EFT), Aspergillus niger (AFT), and sequential inoculation of E. cristatum/A. niger (EAFT), respectively. The volatile compounds of four tea samples were extracted by headspace-solid phase microextraction (HS-SPME) and analyzed using gas chromatography-mass spectrometry (GC-MS) coupled with chemometrics. A total of 97 volatile compounds were tentatively identified to distinguish three fermented instant dark from IGT. Alcohols, acids, esters, ketones, aldehydes, and heterocyclics could be clearly distinguished by principal component analysis (PCA), venn diagram, heatmap analysis and hierarchical cluster analysis (HCA). Descriptive sensory analysis revealed that AFT had a moldy, woody and herbal aroma; EFT showed woody and herbal aroma; and EAFT smelled an herbal, sweet, minty and floral aroma. This study indicates that fermentation using different microorganisms is critical in forming unique aroma characteristics of instant dark teas.

Submerged fermentation of Ginkgo biloba seed powder using Eurotium cristatum for the development of ginkgo seeds fermented products.

BACKGROUND: Ginkgo biloba seeds are well known for the significant curative effects on relieving cough and asthma. However, the development of products from ginkgo seeds still falls behind at present, resulting in a great waste of ginkgo seeds' resource. In this work, submerged fermentation of ginkgo seed powder using Eurotium cristatum was studied to investigate its feasibility as a new processing method. RESULTS: To promote the growth of E. cristatum, the optimum fermentation medium was 80.0 g L-1 of ginkgo seed powder with addition of 5.0 g L-1 calcium chloride (CaCl2 ), 4.0 g L-1 magnesium sulfate (MgSO4 ), 1.25 g L-1 zinc sulfate (ZnSO4 ) and 0.65 g L-1 iron(II) sulfate (FeSO4 ). The optimum fermentation conditions were pH 5.8 ± 0.1, inoculum size 5.1 × 106 CFU mL-1 , liquid medium volume 100 mL in 250-mL Erlenmeyer flask and fermentation 4 days. Through fermentation, the production of lovastatin in fermentation broth could reach up to 32.97 ± 0.17 µg mL-1 and the total antioxidant capacity was improved by more than two-fold. In addition, 40.15% of the ginkgotoxin in ginkgo seed powder was degraded while the entire degradation of ginkgolic acids was obtained. Moreover, fermented ginkgo seed powder suspension presented pleasant fragrances, and the activities of amylase and protease were enhanced to 11.30 ± 0.10 U mL-1 and 23.01 ± 0.20 U mL-1 , respectively. CONCLUSIONS: Submerged fermentation using E. cristatum could significantly enhance the functional value and safety of ginkgo seed powder, and had great potential to become a novel processing method for the development of ginkgo seeds fermented products. © 2020 Society of Chemical Industry.

A novel predict-verify strategy for targeted metabolomics: Comparison of the curcuminoids between crude and fermented turmeric.

Curcuminoids are the major bioactive constituents of turmeric, the application of which are limited by the poor bioavailability. In this study, turmeric was fermented by the Monascus purpureus and Eurotium cristatum fungi to enhance its bioavailability. To explore the variations in the curcuminoids contents in fermented turmeric, a targeted predict-verify strategy was established. For targeted analysis of curcuminoids, a compound library containing all possible curcuminoids based on their structural skeleton was predicted and built for targeted scanning. Then, the MS data were automatically matched with the predicted library to verify the corresponding curcuminoids. As a result, 115 curcuminoids (48 novel compounds and 14 compounds reported in turmeric for the first time) were fully characterized in crude and fermented turmeric. Among these curcuminoids, 31 were newly generated in fermented turmeric. The established predict-verify strategy allows for an efficient and automatic metabolomic analysis to screen for curcuminoids with potentially better bioavailability.

Insight into effects of isolated Eurotium cristatum from Pingwu Fuzhuan brick tea on the fermentation process and quality characteristics of Fuzhuan brick tea.

BACKGROUND: Pingwu Fuzhuan brick tea is a type of post-fermented tea manufactured from leaves of the tea plant, Camellia sinensis var. sinensis, the quality of which is influenced by numerous factors, especially microorganisms. Currently, there is little research on the effect of microorganisms on the fermentation and quality characteristics of Pingwu Fuzhuan brick tea. Investigation of the main fungus in this tea and its effect on the fermentation process and tea quality can provide insights into the manufacturing of 'western road' border-selling tea and could lay the foundation for the popularization of Pingwu Fuzhuan brick tea. RESULTS: The main 'golden flower fungus' in Pingwu Fuzhuan brick tea was isolated and identified as Eurotium cristatum (GenBank accession number: MF800948.1; strain PW-1). Compared with natural fermentation, PW-1 inoculated fermentation accelerated biotransformation of phenolic compounds, which provided tea samples with better taste and tea infusion color. The proportions of velvety and sweet-tasting amino acids increased after 16-day fermentation with PW-1. Alcohols were the most abundant volatiles, with 40.13% and 39.43% content in NF16d and IF16d tea samples, respectively. Orthogonal partial least-squares discriminant analysis (OPLS-DA) and hierarchical clustering analysis (HCA) further revealed that naturally fermented and PW-1 fermented teas were significantly different. CONCLUSION: Strain PW-1 plays an important role in the fermentation process of Fuzhuan brick tea. Considering fermentation efficiency and tea quality, fermentation inoculated with E. cristatum PW-1 can be applied in the manufacturing of 'western road' border-selling tea. © 2020 Society of Chemical Industry.

A study of surface moulds and mycotoxins in Croatian traditional dry-cured meat products.

Xerophilic species of Aspergillus, Penicillium and Eurotium genera from surfaces of dry-cured traditional meat products (TMPs) can cause mycotoxin contamination during uncontrolled household processing. The aim of this study was to investigate into surface moulds growing on Croatian prosciuttos and fermented sausages produced in different climate regions using different technologies (n = 160), and to relate the occurrence of aflatoxin B1 (AFB1) and ochratoxin A (OTA) to their presence. The results revealed the Penicillium (79%) to be the dominating contaminating mould, while Aspergillus (11%), Eurotium (7%) and Mucor (4%) species were present in a significantly lower number of isolates, with higher prevalence and greater diversity in prosciuttos than in sausages, relative of the production technology and regional climate. OTA contamination (14% of samples) was significantly more frequent than that with AFB1 (8% of samples), with OTA concentration rising to the maximal 6.86 µg/kg, whereas AFB1 concentrations were slightly higher than, or around, the limit of quantification of the method in use, with the maximal value of 1.92 µg/kg. The presence of AFB1 in absence of toxicogenic moulds, observed in some samples, can be attributed to contaminated spices used in TMP production or an indirect contamination via a carry-over effect.

Eurotium Cristatum Fermented Okara as a Potential Food Ingredient to Combat Diabetes.

Type 2 diabetes mellitus (T2DM) is a chronic disease, and dietary modification is a crucial part of disease management. Okara is a sustainable source of fibre-rich food. Most of the valorization research on okara focused more on the physical attributes instead of the possible health attributes. The fermentation of okara using microbes originated from food source, such as tea, sake, sufu and yoghurt, were explored here. The aim of this study is to investigate fermented okara as a functional food ingredient to reduce blood glucose levels. Fermented and non-fermented okara extracts were analyzed using the metabolomic approach with UHPLC-QTof-MSE. Statistical analysis demonstrated that the anthraquinones, emodin and physcion, served as potential markers and differentiated Eurotium cristatum fermented okara (ECO) over other choices of microbes. The in-vitro α-glucosidase activity assays and in-vivo mice studies showed that ECO can reduce postprandial blood glucose levels. A 20% ECO loading crispy snack prototype revealed a good nutrition composition and could serve as a fundamental formulation for future antidiabetes recipe development, strengthening the hypothesis that ECO can be used as a novel food ingredient for diabetic management.

Marine Fungi from the Sponge Grantia compressa: Biodiversity, Chemodiversity, and Biotechnological Potential.

The emergence of antibiotic resistance and viruses with high epidemic potential made unexplored marine environments an appealing target source for new metabolites. Marine fungi represent one of the most suitable sources for the discovery of new compounds. Thus, the aim of this work was (i) to isolate and identify fungi associated with the Atlantic sponge Grantia compressa; (ii) to study the fungal metabolites by applying the OSMAC approach (one strain; many compounds); (iii) to test fungal compounds for their antimicrobial activities. Twenty-one fungal strains (17 taxa) were isolated from G. compressa. The OSMAC approach revealed an astonishing metabolic diversity in the marine fungus Eurotium chevalieri MUT 2316, from which 10 compounds were extracted, isolated, and characterized. All metabolites were tested against viruses and bacteria (reference and multidrug-resistant strains). Dihydroauroglaucin completely inhibited the replication of influenza A virus; as for herpes simplex virus 1, total inhibition of replication was observed for both physcion and neoechinulin D. Six out of 10 compounds were active against Gram-positive bacteria with isodihydroauroglaucin being the most promising compound (minimal inhibitory concentration (MIC) 4-64 µg/mL) with bactericidal activity. Overall, G. compressa proved to be an outstanding source of fungal diversity. Marine fungi were capable of producing different metabolites; in particular, the compounds isolated from E. chevalieri showed promising bioactivity against well-known and emerging pathogens.

On-line screening and identification of free radical scavenging compounds in Angelica dahurica fermented with Eurotium cristatum using an HPLC-PDA-Triple-TOF-MS/MS-ABTS system.

Eurotium cristatum, a beneficial fungus isolated from Fuzhuan tea, was used to ferment Angelica dahurica for the first time. The antioxidant capacities of the extracts before and after fermentation were compared using ABTS, DPPH and FRAP assays. The results showed that the antioxidant capacities of the extracts acquired using organic solvents were greater after fermentation. Moreover, based on a comparison of the HPLC chromatograms, the chemical composition of Angelica dahurica changed substantially during fermentation. To further understand the changes in its antioxidant constituents, an on-line HPLC-PDA-Triple-TOF-MS/MS-ABTS system was employed. Twelve antioxidants belonging to three different classes were detected and identified, and their antioxidant capacities were preliminarily evaluated. The results indicated that the substances produced during the fermentation of Eurotium cristatum played important roles in enhancing the antioxidant capacity.

Assessment of Ni accumulation capability by fungi for a possible approach to remove metals from soils and waters.

Abandoned industrial sites and mines may constitute possible hazards for surrounding environment due to the presence of toxic compounds that may contaminate soils and waters. The possibility to remove metal contaminants, specifically nickel (Ni), by means of fungi was presented exploiting a set of fungal strains isolated from a Ligurian dismissed mine. The achieved results demonstrate the high Ni(II) tolerance, up to 500 mg Ni l-1, and removal capability of a Trichoderma harzianum strain. This latter hyperaccumulates up to 11,000 mg Ni kg-1, suggesting its possible use in a bioremediation protocol able to provide a sustainable reclamation of broad contaminated areas.

Anti-inflammatory activity of questinol isolated from marine-derived fungus Eurotium amstelodami in lipopolysaccharide-stimulated RAW 264.7 macrophages.

In the present study, an anthraquinone derivative, questinol was successfully isolated from the broth extract of the marine-derived fungus Eurotium amstelodami for the first time. The structure of questinol was determined based on the analysis of the MS and NMR spectral data as well as comparison of those data with the published data. Moreover, the anti-inflammatory effect of questinol in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was investigated. The results showed that questinol did not exhibit cytotoxicity in LPS-stimulated RAW 264.7 cells up to 200 µM. Questinol could significantly inhibit NO and PGE2 production at indicated concentrations. Questinol was also found to inhibit the production of pro-inflammatory cytokines, including TNF-α, IL-1ß, and IL-6. Furthermore, the western blot analysis showed that questinol suppressed the expression level of iNOS in a dose-dependent manner. However, questinol could slightly inhibit the expression of COX-2 at the concentration of 200 µM. Therefore, our study suggests that questinol might be selected as a promising agent for the prevention and therapy of inflammatory disease.

Eurothiocin A and B, sulfur-containing benzofurans from a soft coral-derived fungus Eurotium rubrum SH-823.

Two new sulfur-containing benzofuran derivatives, eurothiocin A and B (1 and 2), along with five known compounds, zinniol (3), butyrolactone I (4), aspernolide D (5), vermistatin (6), and methoxyvermistatin (7), were isolated from the cultures of Eurotium rubrum SH-823, a fungus obtained from a Sarcophyton sp. soft coral collected from the South China Sea. The new compounds (1 and 2) share a methyl thiolester moiety, which is quite rare among natural secondary metabolites. The structures of these metabolites were assigned on the basis of detailed spectroscopic analysis. The absolute configurations of 1 and 2 were determined by comparison of the experimental and calculated electronic circular dichroism (ECD) data. Compounds 1 and 2 exhibited more potent inhibitory effects against α-glucosidase activity than the clinical α-glucosidase inhibitor acarbose. Further mechanistic analysis showed that both of them exhibited competitive inhibition characteristics.

PTP1B inhibitory secondary metabolites from marine-derived fungal strains Penicillium spp. and Eurotium sp.

The selective inhibition of PTP1B has been widely recognized as a potential drug target for the treatment of type 2 diabetes and obesity. In the course of screening for PTP1B inhibitory fungal metabolites, the organic extracts of several fungal species isolated from marine environments were found to exhibit significant inhibitory effects, and the bioassay-guided investigation of these extracts resulted in the isolation of fructigenine A (1), cyclopenol (2), echinulin (3), flavoglaucin (4), and viridicatol (5). The structures of these compounds were determined mainly by analysis of NMR and MS data. These compounds inhibited PTP1B activity with 50% inhibitory concentration values of 10.7, 30.0, 29.4, 13.4, and 64.0 micrometer, respectively. Furthermore, the kinetic analysis of PTP1B inhibition by compounds 1 and 5 suggested that compound 1 inhibited PTP1B activity in a noncompetitive manner, whereas compound 5 inhibited PTP1B activity in a competitive manner.

Molecular identification of Aspergillus and Eurotium species isolated from rice and their toxin-producing ability.

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp. infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. niger) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it doesn't work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.

Variable beta-glucans production by different states of Eurotium amstelodami explains differences in inflammatory responses in airway cells.

Eurotium amstelodami, a mold frequently identified in housing and farm air samples, is a suspected cause of respiratory diseases such as allergic alveolitis, atopic asthma, and organic dust toxic syndrome. This fungus is present in the air in three different states (ascospores, conidia, and hyphae). The aim of this study was to test in vitro the differential inflammatory response of airway cells exposed to 1,3 betaglucanase-treated protein extract (BGPE), from E. amstelodami ascospores, conidia, and hyphae. Confluent cells from the A549 cell line were inoculated with calibrated BGPE issued from the three fungal forms. The levels of eight cytokines and chemokines involved in inflammatory responses were measured after 8 h of exposure. Beta-d-glucan (BDG) was quantified in total fungal extract as well as in the BGPE from the three fungal states. Hyphal BGPE were the only ones to induce a marked inflammatory response and they contain higher quantities of BDG. The present study adds to the growing body of evidence that beta-glucan from fungal hyphae play a crucial role in respiratory diseases.

Optimization of physico-chemical and nutritional parameters for a novel pullulan-producing fungus, Eurotium chevalieri.

AIMS: To isolate the novel nonmelanin pullulan-producing fungi from soil and to optimize the physico-chemical and nutritional parameters for pullulan production. METHODS AND RESULTS: A selective enrichment method was followed for the isolation, along with development of a suitable medium for pullulan production, using shake flask experiments. Pullulan content was confirmed using pure pullulan and pullulanase hydrolysate. Eurotium chevalieri was able to produce maximum pullulan (38 ± 1·0 g l(-1) ) at 35°C, pH 5·5, 2·5% sucrose, 0·3% ammonium sulfate and 0·2% yeast extract in a shake flash culture medium with an agitation rate of 30 rev min(-1) for 65 h. CONCLUSIONS: The novel pullulan-producing fungus was identified as E. chevalieri (MTCC no. 9614), which was able to produce nonmelanin pullulan at from poorer carbon and nitrogen sources than Aureobasidium pullulans and may therefore be useful for the commercial production of pullulan. SIGNIFICANCE AND IMPACT OF THE STUDY: Eurotium chevalieri could produce pullulan in similar amounts to A. pullulans. Therefore, in future, this fungus could also be used for commercial pullulan production, because it is neither polymorphic nor melanin producing, hence its handling during pullulan fermentation will be easier and more economical.

Natural anthraquinone derivatives from a marine mangrove plant-derived endophytic fungus Eurotium rubrum: structural elucidation and DPPH radical scavenging activity.

There is considerable interest in the isolation of potent radical scavenging compounds from natural resources to treat diseases involving oxidative stress. In this report, four new fungal metabolites including one new bisdihydroanthracenone derivative (1, eurorubrin), two new seco-anthraquinone derivatives [3, 2-O-methyl-9-dehydroxyeurotinone and 4, 2-Omethyl- 4-O-(alpha-D-ribofuranosyl)-9-dehydroxyeurotinone], and one new anthraquinone glycoside [6, 3-O-(alpha-D-ribofuranosyl)- questin], were isolated and identified from Eurotium rubrum, an endophytic fungal strain that was isolated from the inner tissue of the stem of the marine mangrove plant Hibiscus tiliaceus. In addition, three known compounds including asperflavin (2), 2-O-methyleurotinone (5), and questin (7) were also isolated and identified. Their structures were elucidated on the basis of spectroscopic analysis. All of the isolated compounds were evaluated for 1,1-diphenyl-2-picrylhydrazyl(DPPH) radical scavenging activity.

Antioxidants produced by Eurotium herbariorum of filamentous fungi used for the manufacture of karebushi, dried bonito (Katsuobushi).

Extracts prepared by culturing ten filamentous fungi from Aspergillus and Eurotium species isolated from dried bonito (katsuobushi) were examined for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity. The extracts prepared by culturing E. herbariorum NE-1 and NE-4, which are used in the molding process for the manufacture of karebushi (a kind of katsuobushi), were shown to have higher activity than the others. Five antioxidants were isolated from the extracts and identified as isodihydroauroglaucin (IDAG), auroglaucin (AG), dihydroauroglaucin (DAG), tetrahydroauroglaucin (TAG), and flavoglaucin (FG) by (1)H-NMR, (13)C-NMR, and EI-MS analyses. Compared with alpha-tocopherol, the isolated antioxidants exhibited high antioxidative activity for the radical scavenging capacity of DPPH and superoxide, but low activity for inhibiting the autoxidation of docosahexaenoic acid (DHA). The isolated antioxidants were produced by the Eurotium species, but not by the Aspergillus species. DAG and TAG exhibited higher radical scavenging capacity than the other antioxidants and were abundantly contained in the extracts of E. herbariorum NE-1 and NE-4.