Biotransformation of Phlorizin by Eurotium cristatum to Increase the Antioxidant and Antibacterial Activity of Docynia indica Leaves.

Docynia indica is used as a plant resource for both medicine and food in minority areas of southwestern China and Southeast of Asia, especially Docynia indica leaves, which are often used as a kind of functional tea in daily life. In our previous research, it has found that D. indica is rich in polyphenols (mainly phlorizin (PHZ)). Although PHZ is the first polyphenolic competitive inhibitor of sodium-dependent glucose transporters (SGLTs) to be discovered, the promotion and application of PHZ are limited due to its extremely low bioavailability. As a kind of aglycons, phloretin (PHT) possesses a better bioavailability and bioactivity than PHZ. Therefore, the conversion of PHZ to PHT in D. indica leaves by the method of biotransformation can be applied to solve the above issue. In this study, Aspergillus niger and Eurotium cristatum were used to transform PHZ to PHT in D. indica. Compared with Aspergillus niger, Eurotium cristatum can cause the equimolar conversion of PHZ to PHT. However, Aspergillus niger resulted in the complete degradation of PHZ. In the process of deep fermentation, PHZ in D. indica leaves was gradually biotransformed into PHT, and its content was as high as ~ 12% after fermentation. With the increase of PHT content, the antioxidant and antibacterial activity of Docynia indica leaves increased. By the acute toxicity evaluation, it was confirmed that Docynia indica leaves and Eurotium cristatum fermented leaves were much safer. These results indicate that Eurotium cristatum fermentation has the ability to transform the functional compounds in Docynia indica leaves and increase the antioxidant and antibacterial activity of Docynia indica, thus making it a substitute for PHT and functional tea.

Structurally Diverse Polycyclic Salicylaldehyde Derivative Enantiomers from a Marine-Derived Fungus Eurotium sp. SCSIO F452.

To enlarge the chemical diversity of Eurotium sp. SCSIO F452, a talented marine-derived fungus, we further investigated its chemical constituents from a large-scale fermentation with modified culture. Four pairs of new salicylaldehyde derivative enantiomers, euroticins F-I (1-4), as well as a known one eurotirumin (5) were isolated and characterized. Compound 1 features an unprecedented constructed 6/6/6/5 tetracyclic structures, while 2 and 3 represent two new types of 6/6/5 scaffolds. Their structures were established by comprehensive spectroscopic analyses, X-ray diffraction, 13C NMR, and electronic circular dichroism calculations. Selected compounds showed significant inhibitory activity against α-glucosidase and moderate cytotoxic activities against SF-268, MCF-7, HepG2, and A549 cell lines.

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.

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.

Growth characterization and predictive behavior of Eurotium species in a feedstuff matrix.

Animal feeds are characterized by low water activity values. Nevertheless, fungal contamination with Eurotium species are quite common, causing nutritional depletion, spoilage and economic losses. The aim of this work was to assess Eurotium amstelodami, E. chevalieri, E. repens and E. rubrum growth in a feed matrix at different conditions of water activity (0.71-0.97) and temperature (5, 15, 25, 30 and 37°C). It was found that Eurotium species are able to grow in a wide range of water activity and temperature in a short period of time (7 days) and faster than in synthetic media. Rosso and probabilistic models were applied in order to determine the limiting and optimum growth conditions as well as growth probability at certain combinations of environmental factors. Both models provided an accurate fit to the cardinal parameters and good performance for growth/no growth cases. This is the first report assessing the growth parameters of Eurotium species directly in animal feed. Data obtained in the present study is useful to predict and avoid Eurotium species growth in animal feed.

Euroticins A and B, Two Pairs of Highly Constructed Salicylaldehyde Derivative Enantiomers from a Marine-Derived Fungus Eurotium sp. SCSIO F452.

Two pairs of salicylaldehyde derivative enantiomers, euroticins A and B (1 and 2), were isolated from a marine-derived fungus Eurotium sp. SCSIO F452. Compound 1 possesses a highly constructed 6/6/6/5/7 pentacyclic structure featuring an unprecedented 2,11-dioxatricyclo[5.3.1.04,8]undecane core. Compound 2 represents the first example of 6/6/6/6 tetracyclic salicylaldehyde derivative. Their structures were established by spectroscopic analyses, X-ray diffraction, and electronic circular dichroism (ECD) and 13C NMR calculations. Compounds (+)-2 and (-)-2 exhibited remarkable antioxidative activities.

Amount of Eurotium sp. in Chinese Liupao tea and its relationship with tea quality.

OBJECTIVE: Eurotium sp. are the sexual states of the genus Aspergillus, and their ascospore is a spherical closed capsule with a golden colour. The growth of Eurotium sp. during tea production is a key step in achieving the unique quality of dark tea. This study aimed to evaluate the relationship between Eurotium sp. amount and Liupao tea quality. METHODS AND RESULTS: The amounts of Eurotium sp. in 26 differently aged Liupao tea samples from several factories were studied. Indicators related to the quality of Liupao tea were investigated. The amounts of Eurotium sp. were divided into 0, 105 and 106 levels, and quantitative real-time polymerase chain reaction (qPCR) was performed. Using ultrahigh-performance liquid chromatography and high-performance liquid chromatography, the amounts of emodin and physcion were determined to be closely related to the amount of Eurotium sp. Emodin was not found or occurred in minimal amounts in all raw Liupao tea samples. By contrast, physcion was found in Liupao tea at the 106 level of Eurotium sp. Liupao tea samples with varying levels of Eurotium sp. also exhibited evident differences in aroma and chromaticity. Result of the Pearson correlation test showed that the amount of Eurotium sp. plays a key role in creating the unique quality of Liupao tea. CONCLUSION: The amount of Eurotium sp. in dark tea detected via qPCR can be used as a quantitative quality indicator for evaluating dark tea. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides an efficient method for identifying the different qualities of dark tea and addressing quality control issues in fermenting dark tea.

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.

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.

Eurotiumins A⁻E, Five New Alkaloids from the Marine-Derived Fungus Eurotium sp. SCSIO F452.

Three new prenylated indole 2,5-diketopiperazine alkaloids (1⁻3) with nine known ones (5⁻13), one new indole alkaloid (4), and one new bis-benzyl pyrimidine derivative (14) were isolated and characterized from the marine-derived fungus Eurotium sp. SCSIO F452. 1 and 2, occurring as a pair of diastereomers, both presented a hexahydropyrrolo[2,3-b]indole skeleton. Their chemical structures, including absolute configurations, were elucidated by 1D and 2D NMR, HRESIMS, quantum chemical calculations of electronic circular dichroism, and single crystal X-ray diffraction experiments. Most isolated compounds were screened for antioxidative potency. Compounds 3, 5, 6, 7, 9, 10, and 12 showed significant radical scavenging activities against DPPH with IC50 values of 13, 19, 4, 3, 24, 13, and 18 µM, respectively. Five new compounds were evaluated for cytotoxic activities.

Ectopic expression of fungal EcGDH improves nitrogen assimilation and grain yield in rice.

NADP(H)-dependent glutamate dehydrogenases (GDH) in lower organisms have stronger ammonium affinity than those in higher plants. Here we report that transgenic rice overexpressing the EcGDH from Eurotium cheralieri exhibited significantly enhanced aminating activities. Hydroponic and field tests showed that nitrogen assimilation efficiency and grain yields were markedly increased in these transgenic plants, especially at the low nitrogen conditions. These results suggest that EcGDH may have potential to be used to improve nitrogen assimilation and grain yield in rice.

High molecular weight genomic DNA mini-prep for filamentous fungi.

Purification of high quality genomic DNA (gDNA) from filamentous fungi suitable for whole genome sequencing has previously involved many steps. Here, we report a simple and easy-to-follow mini-preparation protocol for high molecular weight (∼20kb) gDNA from filamentous fungi including Aspergillus and Eurotium. This comprehensive protocol includes graphic step-by-step instructions for inoculation, homogenization, and purification of gDNA. The most critical step is a thorough 3-5min homogenization of the freeze-dried mycelium using a motorized hand-held homogenizer with a mini spatula inserted. Approximately 20mg of the fine mycelial powder is then subjected to a modified procedure for the DNeasy Plant Mini Kit (Qiagen). This Qiagen spin column protocol avoids precipitation, dryness, and resuspension of gDNA, which can cause shearing and loss of gDNA. Final gDNA yields from ∼20mg of fine mycelial powder are 8 to 20µg with a consistent 260/280nm absorbance ratio of ∼1.9. All 30 gDNA samples we purified using our method were of high molecular weight (∼20kb). Whole genome sequencing of these DNA samples resulted in 160-260 X coverage with 2×150 reads using NextSeq 500. These gDNAs are also of a suitable quality for Southern blotting and PCR-based amplification of various genes in filamentous fungi.

Indolediketopiperazine Alkaloids from Eurotium cristatum EN-220, an Endophytic Fungus Isolated from the Marine Alga Sargassum thunbergii.

Four new indolediketopiperazine derivatives (1-4), along with nine known congeners (5-13), were isolated and identified from the culture extract of Eurotium cristatum EN-220, an endophytic fungus obtained from the marine alga Sargassum thunbergii. The structures of thesecompounds were elucidated on the basis of extensive spectroscopic analysis and the absolute configurations of compounds 1-4 were established by NOESY experiments and by chiral HPLC analyses of their acid hydrolysates. The absolute configuration of C-8 (a quaternary carbon substituted with a hydroxyl group) in 5 of preechinulin class was firstly determined by electronic circular dichroism (ECD) calculations. All these compounds were evaluatedfor brine shrimp (Artemia salina) lethality and nematicidal activity as well as antioxidativeand antimicrobial potency.

Asperflavin, an Anti-Inflammatory Compound Produced by a Marine-Derived Fungus, Eurotium amstelodami.

In the present study, 16 marine-derived fungi were isolated from four types of marine materials including float, algae, animals and drift woods along with the coast of Jeju Island, Korea and evaluated for anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW 24.7 cells. The broth and mycelium extracts from the 16 fungi were prepared and the broth extract (BE) of Eurotium amstelodami (015-2) inhibited nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells without cytotoxicity. By further bioassay-guided isolation, three compounds including asperflavin, neoechinulin A and preechinulin were successfully isolated from the BE of E. amstelodami. It was revealed that asperflavin showed no cytotoxicity up to 200 µM and significantly inhibited LPS-induced NO and PGE2 production in a dose-dependent manner. In the western blot results, asperflavin suppressed only inducible NOS (iNOS), but COX-2 were slightly down-regulated. Asperflavin was also observed to inhibit the production of pro-inflammatory cytokines including TNF-α, IL-1ß, and IL-6. In conclusion, this study reports a potential use of asperflavin isolated from a marine fungus, E. amstelodami as an anti-inflammatory agent via suppression of iNOS and pro-inflammatory cytokines as well as no cytotoxicity.

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.

Conditional changes enhanced production of bioactive metabolites of marine derived fungus Eurotium rubrum.

Metabolites of marine derived fungus Eurotium rubrum MPUC136 differed between cultivation on wheat medium and Czapek-Dox agar medium. Melanin synthesis inhibitory activity of crude extract of culture on wheat medium showed stronger activity than that of crude extract of culture on Czapek-Dox agar medium. A new diketopiperazine compound isoechinulin D (1) and eight reported diketopiperazines (2-9) were isolated from the crude extract of wheat medium. The structure of 1 was established using NMR, MS and IR methods. 2-5 inhibited melanogenesis using B16 melanoma cells (IC50=68, 2.4, 83, 9.1µM each). Structure-Activity-Relationships of diketopiperazines (1-10) indicated the importance of the prenyl groups at C-2, C-5 and C-7, the vinyl group at C-12 to C-25 and the sp2 carbons at C-8 and C-9. Isolated compounds (1-9) were not or slightly observed from the extracts of Czapek-Dox agar medium by HPLC analysis, suggesting that different cultivation processes could affect metabolism and enhance bioactivities.

Isolation, Stereochemical Study, and Antioxidant Activity of Benzofuranone Derivatives from a Mangrove-derived Fungus Eurotium rubrum MA-150.

Enantiomers of a 2-benzofuran-1(3H)-one derivative [(-)- and (+)-] and four known analogs () were isolated and identified from the culture extract of Eurotium rubrum MA-150, a fungus obtained from the mangrove-derived rizospheric soil. Their structures were established by detailed interpretation of nuclear magnetic resonance (NMR) data and the structure of (±)- was confirmed by single-crystal X-ray diffraction analysis. The absolute configuration of the enantiomers (-)- and (+)- was determined by means of online high-performance liquid chromatography - electronic circular dichroism (HPLC-ECD) measurements and time-dependent Density Functional Theory - electronic circular dichroism (TDDFT-ECD) calculations. Compounds (±)- as well as and exhibited potent DPPH radical scavenging activities with IC50 values of 1.23, 2.26, and 3.99 µg/mL, respectively. Chirality 28:581-584, 2016. © 2016 Wiley Periodicals, Inc.

[Effect of CGMCC No. 8730 Eurotium cristatum fermented dark tea extract on body weight and blood lipid in two dyslipidemia rat models].

OBJECTIVE: To investigate the effect of the CGMCC No. 8730 Eurotium cristatum fermented dark tea on body weight and blood lipid. METHODS: 1. Egg yolk emulsion rat model 40 male Sprague-Dawley( SD) rats were randomly divided into 5groups, given distilled water or the Eurotium cristatum fermented dark tea extract by intragastric administration for 11 days, then fasted and intraperitoneal injected with distilled water or egg yolk emulsion. The next day rats were taken blood and tested for blood lipid. Body weight were recorded regularly. 2. High-fat diet induced rat model 50 male SD rats were randomly divided into 5 groups fed with normal or 60 kcal% high-fat diet, given distilled water or the Eurotium cristatum fermented dark tea extract byintragastric administration for 9 weeks. Blood lipid was tested at baseline( 1st week) and endpoint( 9th week), and body fat ratio was calculated from perirenal and epididymal fat weight at endpoint. Body weight, food intake and energy intake was weekly recorded. RESULTS: 1. Body weight and serum triglycerides of 2500 mg / kg Eurotium cristatum fermented dark tea extract group were significantly decreased foregg yolk emulsion rat model. 2. Body weight, serum triglycerides, food intake and energy intake of 250 mg / kg and 750 mg / kg Eurotium cristatum fermented dark tea extract group were significantly decreased forhigh-fat diet induced rat model. CONCLUSION: The CGMCC No. 8730 Eurotium cristatum fermented dark tea extract decreases body weight andserum triglycerides for SD rats.

Rubrumazines A-C, Indolediketopiperazines of the Isoechinulin Class from Eurotium rubrum MA-150, a Fungus Obtained from Marine Mangrove-Derived Rhizospheric Soil.

Three new indolediketopiperazine alkaloids of the isoechinulin type, rubrumazines A-C (1-3), each possessing an oxygenated prenyl group either at C-7 (1 and 2) or at C-5 (3), along with 13 related analogues (4-16), were isolated and identified from a culture extract of Eurotium rubrum MA-150, a fungus obtained from mangrove-derived rhizospheric soil collected from the Andaman Sea coastline, Thailand. Their structures were established by detailed interpretation of NMR spectroscopic and mass spectrometry data analysis. The structure and absolute configuration of compound 1 were confirmed by X-ray crystallographic analysis, thus providing the first characterized crystal structure of an isoechinulin-type alkaloid. All isolated compounds were evaluated for brine shrimp lethality and antibacterial activity.

Total synthesis and structural revision of (+)-cristatumin C.

Naturally occurring (+)-cristatumin C, a bis-pyrrolidinoindoline diketopiperazine alkaloid isolated from Eurotium cristatum EN-220, is the 2R,3R,11S,15R,2'R,3'R,11'S,15'S enantiomer, as confirmed by total synthesis.