Eurotium Rubrum Research Articles

Development of a natural antifungal formulation for grated cheese and a microencapsulation approach using whey protein isolate and maltodextrin blend.

The antifungal activity of natural antimicrobials such as essential oils (EOs), citrus extracts, and other natural derivatives was evaluated against 10 fungal strains using minimum inhibitory concentration (MIC) analysis. Compounds having the highest inhibitory activity at the lowest concentrations were subsequently selected to evaluate the possible synergistic interactions by checkerboard method (FIC). The results showed that citrus extract A (CEA) and EOs rich in cinnamaldehyde had the highest inhibitory capacity against evaluated strains (Aspergillus niger, Aspergillus versicolor, Aureobasidium pullulans, Eurotium rubrum, Paecilomyces spp., Penicillium chrysogenum, Penicillium citrinum, Penicillium commune, Penicillium crustosum, and Penicillium roqueforti). The stability of the antifungal mixture was then optimized using lecithin and sucrose monopalmitate (SMP) as surfactants. Stability test showed that lecithin:SMP at HLB 10 maintains emulsion stability for 15 days of storage at 4°C. Encapsulation process for the loaded emulsion was optimized using whey protein isolate (WPI) and maltodextrin (MD) blend with ratios WPI:MD (1:2) and WPI:MD (1:3). The results showed that WPI:MD (1:3) led to a higher physicochemical stability (-40.5mV), encapsulation efficiency (91%), and antifungal activity (315ppm). Microencapsulation maintained the available active compounds content more prolonged with an average interval of 7 days compared to the nonencapsulated formulations during storage at 4°C.

Neoechinulins: Molecular, cellular, and functional attributes as promising therapeutics against cancer and other human diseases.

Neoechinulins are fungal and plant-derived chemicals extracted from Microsporum sp., Eurotium rubrum, Aspergillus sp., etc. Two analogues of neoechinulin, i.e., A and B, exerted extensive pharmacological properties described in this review. Neoechinulin is an indole alkaloid and has a double bond between C8/C9, which tends to contribute to its cytoprotective nature. Neoechinulin A exhibits protection to PC12 cells against nitrosative stress via increasing NAD(P)H reserve capacity and decreasing cellular GSH levels. It also confers protection via rescuing PC12 cells from rotenone-induced stress by lowering LDH leakage. This compound has great positive potential against neurodegenerative diseases by inhibiting SIN-1 induced cell death in neuronal cells. Together with these, neoechinulin A tends to inhibit Aβ42-induced microglial activation and confers protection against neuroinflammation. Alongside, it also inhibits cervical cancer cells by caspase-dependent apoptosis and via upregulation of apoptosis inducing genes like Bax, it suppresses LPS-induced inflammation in RAW264.7 macrophages and acts as an antidepressant. Whereas, another analogue, Neoechinulin B tends to interfere with the cellular mechanism thereby, inhibiting the entry of influenza A virus and it targets Liver X receptor (LXR) and decreases the infection rate of Hepatitis C. The present review describes the pharmaceutical properties of neoechinulins with notes on their molecular, cellular, and functional basis and their therapeutic properties.

Purification and Chemical Characterization of a Potent Acaricide and a Closely Related Inactive Metabolite Produced by Eurotium rubrum C47.

Mites are arthropods and some of them infest dry meat cured products and produce allergic reactions. Some mites, such as Tyrolichus casei, Tyrophagus putrescentiae, or Tyrophagus longior feed on filamentous fungi that grow during the meat curing process. Removal of mite infestation of meat products is extremely difficult and there are no adequate miticidal compounds. The filamentous fungus Eurotium rubrum growing on the surface of ham is able to exert a biocontrol of the population of mites due to the production of miticidal compound(s). We have purified two compounds by silica gel chromatography, gel filtration, semipreparative and analytical HPLC and determined their miticidal activity against T. casei using a mite feeding assay. Mass spectrometry and NMR analysis showed that these two compounds are prenylated salicilyl aldehydes with a C-7 alkyl chain differing in a double bond in the C-7 alkyl chain. Structures correspond to those of flavoglaucin and aspergin. Pure flavoglaucin has a miticidal activity resulting in more than 90% mite mortality whereas aspergin does not affect the mites. Both compounds were formed simultaneously by E. rubrum C47 cultures in different media suggesting that they are synthesized by the same pathway. Production of both compounds was higher in solid culture media and the products were associated with abundant formation of cleistothecia. In liquid cultures both compounds remained mainly cell-associated and only about 10% of the total compounds was released to the culture broth. This miticidal compound may be used to combat efficiently mite infestation in different habitats. These results, will promote further advances on the utilization of flavoglaucin in food preservation and in human health since this compound has antitumor activity.

Biological control of mites by xerophile Eurotium species isolated from the surface of dry cured ham and dry beef cecina

The aim of this article is to study the possible biological control of mites by fungi that form part of the normal microbiota of these meat products. Some yellow/orange pigmented fungi growing on the ham surface decreased the proliferation of mites; therefore, we isolated from ham and cecina xerophilic yellow/orange coloured fungal strains that were identified as members of the genus Eurotium (recently reclassified as Aspergillus section Aspergillus). Using molecular genetic tools, we have identified 158 strains as Eurotium rubrum (Aspergillus ruber), Eurotium repens (Aspergillus pseudoglaucus) and Eurotium chevalieri (Aspergillus chevalieri). Two strains, E. rubrum C47 and E. rubrum C49, showed strong miticidal activity. The toxic compound(s) are associated with the formation of cleistothecia. In synchronized mite development experiments, we observed that all stages of the mite lifecycle were inhibited by the E. rubrum C47 strain. In addition, we searched for miticidal activity in 13 culture collection Eurotium strains isolated from different habitats, and found that only one, Eurotium cristatum NRRL 4222 (Aspergillus cristatus) has a strong miticidal activity. These fungal strains have proliferated on the surface of ham and cecina for decades, and possibly have acquired miticidal activity as a resistance mechanism against fungivores. Biological control of infecting mites by favouring growth of E. rubrun C47, in place of the normal mixed population of Aspergillus and Penicillium, is an attractive approach to control mite infestations.

The bioactivity of bacterium and fungi living associate with the sponge Reniera sp. against multidrug-resistant Staphylococcus aureus and Escherichia coli

Abstract. Trianto A, Nirwani, Susanti O, Maesaroh D, Radjasa OK. 2019. The bioactivity of bacterium and fungi living associate with the sponge Reniera sp. against multidrug-resistant Staphylococcus aureus and Escherichia coli. Biodiversitas 20: 2302-2307. The study aimed to isolate and identify the sponge-associated microorganisms producing the antibacterial substances. The sponge Reniera sp. was collected by hand during skin diving in Karimunjawa Islands, Indonesia. The microbial symbionts were isolated with the dilution method and screened with the overlay method against the MDR S. aureus and E. coli. The bacterium was cultured in Zobell medium, while the fungi were cultured in malt extract broth (MEB) medium. The isolates were identified based on the molecular method. A total of 46 bacteria and 43 fungi were isolated, which 7 bacteria and 20 fungi exhibited antibacterial activity against the MDR E. coli and S. aureus strains. The molecular identification revealed that the active isolates close to Pseudoalteromonas maricaloris (99%), Aspergillus nomius (96%), Eurotium rubrum (99%), and Penicillium sp. (100%). Fractionation of K.J.16.U extract gave a fraction that active to the S. aureus and E. coli strains at concentration 150 and 15 µg disk-1. The fraction K.J.16.U.1.4.4 exhibited stronger activity than that exhibited by chloramphenicol at 150 µg disk-1. The sponge Reniera sp. collected from Karimunjawa Islands comprise bacterial and fungal isolates produced antibacterial compounds that inhibited the growth the MDR E. coli and S. aureus strains.

Fungal stains on paper: is what you see what you get?

Mitigation of fungal biodeterioration on paper documents and artworks represents a challenge to conservators worldwide. Numerous lists of fungal species have been identified from paper collections, but are those species responsible for the respective biodeterioration phenomena or just environmental contaminants? The present work was aimed at obtaining an association between specific fungal stains and causative fungal species. 23 stains from three paper documents were sampled. Fungal structures observed in situ with optical and scanning electron microscopy were compared with the identification of isolates by molecular biology tools. Correlation between the observed fungal structures and the identified fungal isolates was achieved, varying from 13% to 64% of the samples within the three studied documents. Grey/black and dark brown stains were associated with Chaetomium globosum, C. murorum, Penicillium chrysogenum, P. commune, Myxotrichum deflexum and Stachybotrys chartarum. Eurotium rubrum was isolated from a foxing stain and Penicillium citrinum was identified on light orange stains.

Coumarins with an unprecedented tetracyclic skeleton and coumarin dimers from chemically engineered extracts of a marine-derived fungus

The unprecedented tetracyclic coumarin derivatives 1 and 2 and the coumarin dimers 3–5 were isolated from chemically engineered extracts (coumarin dimerization of natural extract) of the marine-derived fungus Eurotium rubrum. The structures of these compounds were established using NMR, MS and IR methods. The absolute configuration of 1 was determined by ECD calculations. The unprecedented tetracyclic coumarin skeleton was generated by domino-Knoevenagel-Diels-Alder reactions. Compounds 1–5 showed tyrosinase inhibitory activity (IC50 = 1.7, 1.2, 4.9, 1.8 and 2.9 μM, respectively). The isolated coumarin derivatives 1–5 were not observed by HPLC analysis in crude extracts of E. rubrum, suggesting that chemically engineered extract generated these new coumarin derivatives with tyrosinase inhibitory activity.

Effect of water activity and temperature on the growth of Eurotium species isolated from animal feeds

BackgroundXerophilic fungi represent a serious problem due to their ability to grow at low water activities causing the spoiling of low and intermediate moisture foods, stored goods and animal feeds, with the consequent economic losses. AimsThe combined effect of water activity and temperature of four Eurotium species isolated from animal feeds was investigated. MethodsEurotium amstelodami, Eurotium chevalieri, Eurotium repens and Eurotium rubrum were grown at 5, 15, 25, 37 and 45°C on malt extract agar adjusted with glycerol in the range 0.710–0.993 of water activities. ResultsThe cardinal model proposed by Rosso and Robinson (2001) was applied to fit growth data, with the variable water activity at fixed temperatures, obtaining three cardinal water activities (awmin, awmax, awopt) and the specific growth rate at the optimum aw (μopt). A probabilistic model was also applied to define the interface between growth and no-growth. The cardinal model provided an adequate estimation of the optimal aw to grow and the maximum growth rate. The probabilistic model showed a good performance to fit growth/no-growth cases in the predicted range. ConclusionsThe results presented here could be applied to predict Eurotium species growth in animal feeds.

Anthraquinones from the saline-alkali plant endophytic fungus Eurotium rubrum.

Saline-alkali plant endophytes similar as marine mangrove plant-derived ones are a big member of microbes inhabiting in tissues of plants without causing obvious disease to their hosts, and they must adapt to the extreme environment of high osmolarity and nutrient deprivation in plant, which are different from terrestrial plant endophytes. Owing to the unique bio-environments, endophytes originated from mangrove plants are being considered as a new resource of natural product research and diverse secondary metabolites with a wide range of bioactivities have been isolated.1, 2, 3 Compared with those of marine mangrove plant-derived endophytes, chemical investigation of saline-alkali plant-derived endophytic fungi have just begun, and only several natural products were isolated from these unique environmental fungi.4,5,6,7Suaeda salsa L., an important model halophytic plant, is distributed at different saline-alkali soil especially at seaside. This plant possesses the ability to absorb salt from saline soils and accumulate them in the plant tissues. Some results revealed that the total salt contents in shoot and root were 23–27% and 10–12%, respectively.8 During our ongoing chemical investigation from unique fungal species as sources of new bioactive natural products, a halo-tolerant endophytic fungus Eurotium rubrum isolated from salt-tolerance wild plant S. salsa L. collected from ‘BoHai’ seaside was chosen to chemical investigation. Previous reports revealed that E. rubrumwas frequently isolated from mangrove plants, and different secondary metabolites with a wide range of bioactivities have been isolated from this endophytic fungus.9, 10, 11, 12, 13 Purification of the solid culture extract of this fungus led to isolate a new anthraquinone named rubrumol (1) with poly-hydroxyl groups together with four known analogs catenarin (2),14 rubrocristin (3),15 emodin (4)16 and 2-methyleurotinone (5).15 In this paper, the structural elucidation for the new compound rubrumol (1) (Figure 1) and bioactivities for compounds 1–5 were present. Several errors on the coupling constants of the known coniothyrinone A (6) were pointed out in this note.17

Steroids from an Endophytic Eurotium rubrum Strain

A new steroid, 3β,5α-dihydroxy-10α-methyl-6β-acetoxy-ergosta-7,22-diene (1), and ten known steroids (2–11), were isolated from the cultures of Eurotium rubrum, an endophytic fungus isolated from the marine green alga Codium fragile. Structures of these compounds were unambiguously established by spectroscopic techniques and by comparison with literature values. These steroids exhibited low activity to inhibit acetylcholinesterase and no activity against plant pathogenic fungi Colletotrichum lagenarium and Fusarium oxysporium.

Mycoflora and mycotoxins in finished fish feed and feed ingredients from smallholder farms in East Africa

A total of 52 samples of finished fish feeds and ingredients were collected from smallholder farmers in Kenya, Tanzania, Rwanda and Uganda, and analyzed. Culture and molecular techniques were used to identify fungal isolates from the feedstock, and mycotoxin profiles were determined using liquid chromatography–tandem mass spectrometry. The most prevalent fungal species recovered in the samples was Asperigillus flavus (54.5%). Other fungal species recovered from the samples were Aspergillus tamarii (9.1%), Mucor velutinosus (9%), Phoma sp. (6.1%), Aspergillus niger (6%), Eurotium rubrum (3%) and Penicillium chrysogenum (3%). Fourteen mycotoxins were identified: aflatoxins B1, B2, G1 and G2, fumonisin B1 and B3, deoxynivalenol (DON) and acetyldeoxynivalenol (sum of 3-ADONand 15-ADON), ochratoxin A, roquefortine C, alternariol, T-2 toxin, and nivalenol. DON (92.9%), aflatoxins (64.3%) and fumonisins (57.1%) were the most prevalent within locally manufactured feeds, while no contamination was found in imported feed. Samples from Kenya were the most contaminated with aflatoxin (maximum 806.9μg·kg−1). The high levels of aflatoxin and trichothecene type A and B contamination found in this study point to potential risks to fish performance and to the health of consumers of the fish and derived products.

Biocontrol of ochratoxigenic moulds (Aspergillus ochraceus and Penicillium nordicum) by Debaryomyces hansenii and Saccharomycopsis fibuligera during speck production

Speck is a meat product obtained from the deboned leg of pork that is salted, smoked and seasoned for four to six months. During speck seasoning, Eurotium rubrum and Penicillium solitum grow on the surface and collaborate with other moulds and tissue enzymes to produce the typical aroma. Both of these strains usually predominate over other moulds. However, moulds producing ochratoxins, such as Aspergillus ochraceus and Penicillium nordicum, can also co-grow on speck and produce ochratoxin A (OTA). Consequently, speck could represent a potential health risk for consumers. Because A. ochraceus and P. nordicum could represent a problem for artisanal speck production, the aim of this study was to inhibit these mould strains using Debaryomyces hansenii and Saccharomycopsis fibuligera. Six D. hansenii and six S. fibuligera strains were tested in vitro to inhibit A. ochraceus and P. nordicum. The D. hansenii DIAL 1 and S. fibuligera DIAL 3 strains demonstrated the highest inhibitory activity and were selected for in vivo tests. The strains were co-inoculated on fresh meat cuts for speck production with both of the OTA-producing moulds prior to drying and seasoning. At the end of seasoning (six months), OTA was not detected in the speck treated with both yeast strains. Because the yeasts did not adversely affect the speck odour or flavour, the strains are proposed as starters for the inhibition of ochratoxigenic moulds.

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.

Occurrence of fungi on deteriorated old dluwang manuscripts from Indonesia.

Dluwang was made from the bark of the paper mulberry (Broussonetia papyrifera Vent.). Old Javanese manuscripts were handwritten on dluwang. Fungi samples from deteriorated dluwang manuscripts from the libraries of royal palaces in Indonesia and from the faculty library of Universitas Indonesia were collected by using cotton swabs and adhesive tape. Fungal isolates were obtained by the culture-dependent method. Identification of 38 fungal isolates was carried out by the molecular method using the internal transcribed spacer (ITS) regions of ribosomal DNA. Phenotypic description of the isolates was obtained by classic culturing and direct microscopic observation. The results showed that fungi from the genera of Aspergillus and Penicillium were commonly found on old dluwang manuscripts. According to the molecular identification, species obtained were Aspergillus awamori, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus jensenii, Aspergillus niger, Aspergillus pulvericola, Aspergillus ruber (Eurotium rubrum), Aspergillus versicolor, Penicillium citrinum, Penicillium oxalicum, Penicillium rubens, Pseudocercospora chiangmaiensis, and Trichoderma longibrachiatum. The isolated fungi were xerophilic and cellulolytic in nature. All isolates were able to grow on dluwang paper, 29 isolates (76%) were able to utilize carboxymethyl cellulose (CMC), and 18 isolates (47%) were able to utilize CMC and microcrystalline cellulose (MCC). The present study showed that the deterioration of the written heritage was potentially caused by diverse fungi.

Identification of antifungal peptides produced by Lactobacillus plantarum IS10 grown in the MRS broth

The use of secondary metabolites of lactic acid bacteria for preservation of foods is increasingly gaining interest to the food industry to replace synthetic preservatives. In this study, the cell free supernatant containing peptides obtained from Lactobacillus plantarum IS10 was fractionated by size exclusion chromatography using sephadex G-25, and tested against Aspergillus flavus MD3, Penicillium roqueforti MD4 and Eurotium rubrum MD5. Among the fractions, fraction number 10 showed 60% antifungal activity at a concentration of 0.02 mg peptide/mL. Four novel peptides out of twenty peptides obtained from fraction 10 were identified and determined by de novo sequencing. Peptide FPSHTGMSVPPP with a net charge +1, hydrophobicity ratio 58% and molecular weight of 1253 was further studied. The selected peptide showed a good activity at a concentration of 5 mg/mL against selected fungi and poor activity at low concentrations. This work indicates that L. plantarum IS10 has the capability of producing peptides which are affective against spoilage fungi.

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.

Neoechinulin B and its analogues as potential entry inhibitors of influenza viruses, targeting viral hemagglutinin

A class of prenylated indole diketopiperazine alkaloids including 15 new compounds namely rubrumlines A-O obtained from marine-derived fungus Eurotium rubrum, were tested against influenza A/WSN/33 virus. Neoechinulin B (18) exerted potent inhibition against H1N1 virus infected in MDCK cells, and is able to inhibit a panel of influenza virus strains including amantadine- and oseltamivir-resistant clinical isolates. Mechanism of action studies indicated that neoechinulin B binds to influenza envelope hemagglutinin, disrupting its interaction with the sialic acid receptor and the attachment of viruses to host cells. In addition, neoechinulin B was still efficient in inhibiting influenza A/WSN/33 virus propagation even after a fifth passage. The high potency and broad-spectrum activities against influenza viruses with less drug resistance make neoechinulin B as a new lead for the development of potential inhibitor of influenza viruses.

Mycobiota associated with the rhodophyte alien species Asparagopsis taxiformis (Delile) Trevisan de Saint‐Léon in the Mediterranean Sea

AbstractThis study is the first to investigate and characterize the mycobiota associated with the alien species Asparagopsis taxiformis, a rhodophyte classified as one of the ‘100 worst invasive species’ in the Mediterranean Sea by the Hellenic Center for Marine Research, as it threatens biodiversity. Fungal endophyte and epiphyte communities were investigated on algal specimens from two sampling sites on the island of Linosa (Marine Protected Area Isole Pelagie, Italy). 87% of the 24 specimens that were analysed for epiphytes displayed a microfungal colonization. No endophytes were found. Only a small amount of microfungi was found to be associated with this alga. Only five fungal taxa were isolated; two of which are sporadically associated with the alga, while three, Eurotium rubrum, Cladosporium cladosporioides and Cladosporium pseudocladosporioides, seem to be related to A. taxiformis. This scarcity could be related to algal chemical composition.

Genomic adaptations of the halophilic Dead Sea filamentous fungus Eurotium rubrum.

The Dead Sea is one of the most hypersaline habitats on Earth. The fungus Eurotium rubrum (Eurotiomycetes) is among the few species able to survive there. Here we highlight its adaptive strategies, based on genome analysis and transcriptome profiling. The 26.2 Mb genome of E. rubrum shows, for example, gains in gene families related to stress response and losses with regard to transport processes. Transcriptome analyses under different salt growth conditions revealed, among other things differentially expressed genes encoding ion and metabolite transporters. Our findings suggest that long-term adaptation to salinity requires cellular and metabolic responses that differ from short-term osmotic stress signalling. The transcriptional response indicates that halophilic E. rubrum actively counteracts the salinity stress. Many of its genes encode for proteins with a significantly higher proportion of acidic amino acid residues. This trait is characteristic of the halophilic prokaryotes as well, supporting the theory of convergent evolution under extreme hypersaline stress.