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Black yeasts can survive extreme conditions in food production owing to their polyextremotolerant character. However, significant strain-to-strain variation in black yeast thermoresistance has been observed. In this study, we assessed the variability in tolerance to nonthermal interventions among a collection of food-related black yeast strains. Variation in tolerance to UV light treatment, high pressure processing, sanitizers, and osmotic pressure was observed within each species. The two strains previously shown to possess high thermotolerance, Exophiala phaeomuriformis FSL-E2-0572 and Exophiala dermatitidis YB-734, were also the most HPP tolerant, but were the least halotolerant. Meanwhile, Aureobasidium pullulans FSL-E2-0290 was the most UV and sanitizer tolerant, but had been shown to have relatively low thermoresistance. Fisher's exact tests showed that thermoresistance in black yeasts was associated with HPP tolerance and inversely with halotolerance, but no association was found with UV tolerance or sanitizer tolerance. Collectively, the relative stress tolerance among strains varied across interventions. Given this variation, a broad range of different food products are susceptible to black yeast spoilage. Additionally, different strains should be selected in challenge studies specific to the intervention. (1312/2000 characters).

Gray mold caused by Botrytis cinerea is a common postharvest disease in strawberries, reducing shelf life considerably. We investigated the potential of the yeast-like biocontrol fungus Aureobasidium pullulans (AP-SLU6) vectored by bumblebees (Bombus terrestris) in the Flying Doctors® system to inhibit the pathogen and increase the shelf life of harvested strawberries (cultivar Sonata). Using bumblebees as vectors of various biocontrol agents is becoming increasingly popular, but any potentially negative effects on bee performance have been understudied. Our results show that, over the 4-week period of the trial, the performance and activity of the bees were not negatively affected by A. pullulans. The bees successfully picked up the powder formulation; then, they carried and deposited it on the flowers. The vectoring of the biocontrol agent significantly reduced gray mold development on the harvested fruits by 45% and increased shelf life by 100% in comparison with control treatments. This suggests that the biocontrol fungus applied during flowering successfully reduced Botrytis infection and thus, effectively protected the fruits from gray mold. In addition, the bee-vectored application of the biocontrol agent was found to be significantly more effective than spray application because the latter may temporarily increase humidity around the flower, thereby creating a suitable environment for the pathogen to thrive. In summary, our study demonstrates that A. pullulans vectored by bumblebees can decrease gray mold infection and improve the shelf life of strawberries without adversely affecting the bees, thus providing a basis for the sustainable and efficient control of gray mold on strawberry. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

β-glucan, a plant polysaccharide, mainly exists in plant cell walls of oats, barley, and wheat. It is attracting attention due to its high potential for use as functional foods and pharmaceuticals. We have previously reported that low-molecular-weight Aureobasidium pullulans-fermented β-D-glucan (LMW-AP-FBG) could inhibit inflammatory responses by inhibiting mitogen-activated protein kinases and nuclear factor-κB signaling pathways. Bases on previous results, the objective of the present study was to investigate the therapeutic potential of LMW-AP-FBG in BALB/c mice intracutaneously transplanted with CT-26 colon cancer cells onto their backs. Daily intraperitoneal injections of LMW-AP-FBG (5 mg/kg) for two weeks significantly suppressed tumor growth in mice bearing CT-26 tumors by reducing tumor proliferation and inducing apoptosis as compared to phosphate buffer-treated control mice. In addition, LMW-AP-FBG treatment reduced the viability of CT-26 cells in a dose-dependent manner by inducing apoptosis with loss of mitochondrial transmembrane potential and increased activated caspases. Taken together, LMW-AP-FBG exhibits anticancer properties both in vivo and in vitro.

Antifungal agents inhibit the growth of or kill fungi by targeting various biochemical or biological processes.However, fungi continually develop mechanisms to evade antifungals. This doctoral thesis aimed to elucidate the antifungal resistance mechanisms of naturally occurring yeasts for the ultimate goal of discovering new plant protection strategies. The research focus was on the isolation, identification, and characterization of fungicide sensitivity of ubiquitous yeasts, molecular and functional characterization of the resistance mechanisms, and the application of these yeasts in combined disease control strategies. Chapter 1 provides an overview of the plant protection agents, the biology of yeasts, and the importance of yeasts in ecosystems. Fungicide use and its impacts, yeast interactions, and the practical importance of these interactions are given a particular focus. Chapter 2 documents the antifungal agents and reviews previously characterized antifungal mechanisms with a focus on four groups: demethylase inhibitors, the quinone outside inhibitors, anilinopyrimidines, and captan. Chapter 3 demonstrates the widespread distribution of naturally occurring yeasts with tolerance to commonly used antifungal agents. Using MALDI-TOF and Sanger sequencing of the ITS region, 376 isolates from 47 taxa were identified after isolation in the presence of fungicides. Among the taxa, isolates of the yeast-like fungus Aureobasidium pullulans were the most abundant. The sensitivity profiles of 30 strains of this species were investigated in microbroth sensitivity assays with captan (CPN), cyprodinil (CYP), and difenoconazole (DFN).These strains' minimum inhibitory concentrations (MIC50) recorded high tolerance of A. pullulansto CPN, CYP, and DFN. Chapter 4 describes the antifungal tolerance mechanisms in environmental and clinical A. pullulans. The study in this chapter built upon the CPN, CYP, and DFN sensitivity data for 30 A. pullulans strains, newly generated sensitivity data for clinical Aureobasidium isolates, and genome sequences of 46 strains in total to characterize tolerance mechanisms. GWAS and literature-based approaches predicted possible CPN, CYP, and DFN antifungal resistance mechanisms. SNP analysis within the coding regions reported 1767 genes involved in CYP tolerance of A. pullulans, and only four genes for CPN were predicted. The involvement in CYP resistance of five S. cerevisiae gene homologs of A. pullulans (STL1, LYS2, ENO1, HOL1, and CAC2) using the respective gene deletion strains was confirmed experimentally by agar spot assays. Through heterologous expression of predicted A. pullulans’ genes in S. cerevisiae and microbroth sensitivity assays, novel CYP tolerance mechanisms involving five newly predicted A. pullulans genes (FBD, GST, HP5, HP6, and HP8) were uncovered. Chapter 5 describes a protocol of competition assays to quantify the effect of biocontrol yeasts against plant pathogenic fungi on fruits. Chapter 6 demonstrates the synergistic interaction of CYP and a CYP-tolerant A. pullulans strain in controlling Botrytis spp. lesions on apples. The particular focus was on optimizing an artificial laboratory system for testing such synergism. The final chapter recapitulates the study and gives future perspectives for research on antifungal resistance mechanisms and the applications of yeast-fungicide combinations.

β-glucan derived from the black yeast Aureobasidium pullulans (A. pullulans) is one of the natural products attracting attention due to its high potential for application as a functional food and pharmaceutical. Our team of researchers obtained a highly soluble, low-molecular-weight β-glucan from the fermentation culture medium of A. pullulans via mechanochemical ball milling method, that is, the low-molecular-weight A. pullulans-fermented β-D-glucan (LMW-AP-FBG). We investigated the anti-inflammatory effect of LMW-AP-FBG using lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells) in the current study. LMW-AP-FBG altered LPS-stimulated inflammatory responses by reducing the release of inflammatory mediators such as nitric oxide (NO), interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. As well, the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways were downregulated by LMW-AP-FBG. Furthermore, LMW-AP-FBG markedly reduced LPS-induced expression of cell surface molecules, CD14, CD86, and MHC class II, which mediate macrophage activation. These findings suggest that LMW-AP-FBG can be used as an effective immune modulator to attenuate the progression of inflammatory disease.

The beneficial immunomodulation effects of a biological response modifier glucan (BRMG) produced by two strains of Aureobasidium pullulans, AFO-202 and N-163, have already been reported. Herein, we compared their efficacy on immune-inflammatory parameters in Sprague Dawley (SD) rats. This study was performed on four groups of healthy SD rats, n=6 in each group: Group 1, euthanised on Day 0 for baseline values; Group 2, control (drinking water); Group 3, AFO-202 beta glucan, 200 mg/kg/day; and Group 4, N-163 beta glucan, 300 mg/kg/day. The neutrophil to lymphocyte ratio (NLR) decreased and leukocyte-to C-reactive protein ratio (LeCR) increased in Group 3 (AFO-202) at 15 and 29 days whereas the lymphocyte to C-reactive protein ratio (LCR) increased in group 4 (N-163), within the normal physiological range. These promising results warrant further investigations in larger numbers of healthy and diseased models to develop appropriate strategies for balancing immune system dysregulation.

Xylooligosaccharides (XOS) are non-digestible and fermentable oligomers that stand out for their efficient production by enzymatic hydrolysis and beneficial effects on human health. This study aimed to investigate the influence of the main reaction parameters of the beechwood xylan hydrolysis using crude xylanase from Aureobasidium pullulans CCT 1261, thus achieving the maximum XOS production. The effects of temperature (40 to 50 °C), reaction time (12 to 48 h), type of agitation, substrate concentration (1 to 6%, w/v), xylanase loading (100 to 300 U/g xylan), and pH (4.0 to 6.0) on the XOS production were fully evaluated. The most suitable conditions for XOS production included orbital shaking of 180 rpm, 40 °C, and 24 h of reaction. High contents of total XOS (10.1 mg/mL) and XOS with degree of polymerization (DP) of 2-3 (9.7 mg/mL), besides to a high percentage of XOS (99.1%), were obtained at 6% (w/v) of beechwood xylan, xylanase loading of 260 U/g xylan, and pH 6.0. The establishment of the best hydrolysis conditions allowed increasing both the content of total XOS 1.5-fold and the percentage of XOS by 9.4%, when compared to the initial production (6.7 mg/mL and 89.7%, respectively). Thus, this study established an efficient enzymatic hydrolysis process that results in a hydrolysate containing XOS with potential prebiotic character (i.e., rich in XOS with DP 2-3) and low xylose amounts.

In this study, cost-effective substrates such as cassava starch, corn steep liquor (CSL) and soybean meal hydrolysate (SMH) were used for pullulan production by Aureobasidium pullulans CCTCC M 2012259. The medium was optimized using response surface methodology (RSM) and artificial neural network (ANN) approaches, and analysis of variance indicated that the ANN model achieved higher prediction accuracy. The optimal medium predicted by ANN was used to produce high molecular weight pullulan in high yield. SMH substrates increased both biomass and pullulan titer, while CSL substrates maintained higher pullulan molecular weight. Results of kinetic parameters, key enzyme activities and intracellular uridine diphosphate glucose contents revealed the physiological mechanism of changes in pullulan titer and molecular weight using different substrates. Economic analysis of batch pullulan production using different substrates was performed, and the cost of nutrimental materials for CSL and SMH substrates was decreased by 46.1% and 49.9%, respectively, compared to the control using glucose and yeast extract as substrates, which could improve the competitiveness of pullulan against other polysaccharides in industrial applications.

To improve β-1,3-1,6-D-glucan (β-glucan) production by Aureobasidium pullulans, an Agrobacterium tumefaciens-mediated transformation method was developed to screen a mutant A. pullulans CGMCC 19650. Based on thermal asymmetric-interlaced PCR detection, DNA sequencing, BLAST analysis, and quantitative real-time PCR assay, the T-DNA was identified to be inserted in the coding region of mal31 gene, which encodes a sugar transporter involved in pullulan biosynthesis in the mutant. The maximal biomass and β-glucan production under batch fermentation were significantly increased by 47.6% and 78.6%, respectively, while pullulan production was decreased by 41.7% in the mutant, as compared to the parental strain A. pullulans CCTCC M 2012259. Analysis of the physiological mechanism of these changes revealed that mal31 gene disruption increased the transcriptional levels of pgm2, ugp, fks1, and kre6 genes; increased the amounts of key enzymes associated with UDPG and β-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored β-glucan production. However, the T-DNA insertion decreased the transcriptional levels of ags2 genes, and reduced the biosynthetic capability to form pullulan, resulting in the decrease in pullulan production. This study not only provides an effective approach for improved β-glucan production by A. pullulans, but also presents an accurate and useful gene for metabolic engineering of the producer for efficient polysaccharide production. KEY POINTS: • A mutant A. pullulans CGMCC 19650 was screened by using the ATMT method. • The mal31 gene encoding a sugar transporter was disrupted in the mutant. • β-Glucan produced by the mutant was significantly improved.

The results of a study of the light chestnut soil of an ampelocenosis located in a zone with a semi-desert-steppe regime and a low hydrothermal coefficient are presented. Analysis of the chemical composition revealed that the soil is nonsaline, saturated with bases, and deficient in individual plant nutrients. The study of the taxonomic composition of yeasts, which can act as a model group of microorganisms in the study of soil-microbiological processes, has shown the ability of this soil to play the role of a biological reservoir for storage and distribution in the natural cycle of various physiological groups of yeasts. The yeast complex included 11 species: 2 basidiomycetes and 9 ascomycetes. The yeasts Aureobasidium pullulans and Lachancea thermotolerans were significantly dominant. The yeasts were isolated by a direct plating and enrichment cultivation; the species identification of pure cultures was based on the analysis of the nucleotide sequences of the rDNA region. The accumulation of allochthonous yeasts, as well as other groups of microorganisms, influencing the biodiversity of the ampelocenosis soil, expands the potential for the participation of mycobiota in soil processes.

Although intestinal fungi play important roles in host health and disease, the composition and diversity of fungal communities remain poorly reported in fish. In this study, fungi in the fore-, mid-, and hind-intestine of tilapia (Oreochromis mossambicus) and bighead carp (Aristichthys nobilis) from Hongchaojiang Reservoir in Guangxi, China were investigated by ITS sequencing. Based on this, we obtained 1 763 478 high-quality tags, which clustered into 1 089 operational taxonomic units (OTUs). In total, 404 OTUs were annotated, of which 310, 68, and 26 belonged to Ascomycota, Basidiomycota, and other, respectively. Results show significant differences in the community composition of intestinal fungi between tilapia and bighead carp but not within their different intestinal segments. Furthermore, 154 of the 404 annotated OTUs were considered reliable and were classified into three trophic modes and nine guilds. The three trophic modes consisted of 108 OTUs of saprotrophic fungi, 41 OTUs of pathotrophic fungi, and five OTUs of symbiotrophic fungi. The top three most abundant OTUs overall (i.e., Otu000002, Scopulariopsis acremonium; Otu000018, Alternaria palandui; Otu000034, Aureobasidium pullulans) showed lower abundance in the hind-intestinal segments of bighead carp, suggesting uneven distribution of these fungi in this species. In addition, saprotrophic and pathotrophic fungi were markedly decreased in the hind-intestine. It is indicated that the fungal community was not only related to host species specificity but also to the respective physiological functions of different intestinal segments. These findings provide valuable information on the composition, structure, and potential function of the intestinal fungi community associated with different intestinal segments in tilapia and bighead carp under natural conditions, thus highlighting the importance of fungi as an integral part of the intestinal microbiota in maintaining host health.

Cv. Arbequina fruits were inoculated during one day with suspensions of Aureobasidium pullulans, Epicoccum nigrum or both species (104 spores/g olives), isolated from olive leaves, aiming to increase the phenolic content of the olive oils. Oils were extracted from non-inoculated (control) and inoculated olives, being evaluated for their oxidative stability, and phenolic composition. Compared to the control oils, those extracted from inoculated olives showed higher oxidative stability (+1629%), phenolic acids contents (up to+37%), phenolic alcohols (up to+101%), flavonoids (up to+154%) and secoiridoid derivatives (up to + 12%). These positive effects could be attributed to the hydrolytic activity of enzymes secreted by the studied fungi on the fruit cellular membranes and extracted phenolics, increasing the extractability and, consequently, altering the phenolic concentration. Considering the two fungi species, E. nigrum inoculation had a more significant positive impact on the contents of tyrosol, oleuropein, and ligstroside derivatives, showing an inhibition effect regarding the oleacein formation. Contrary, A. pullulans inoculation mainly resulted into an accentuated increase of luteolin and oleacein contents (3 and 12-times higher compared with control oils, respectively). Overall, no enhanced positive synergetic effect could be attributed to the inoculation with a suspension containing both fungi. The significant changes observed on the phenolic contents, due to the fungal inoculation, allowed the successful discrimination of the four types of extracted oils using an electronic tongue. This device acts as an artificial taste device allowing anticipating an impact at the oils basic taste sensations due to the fungi inoculation., The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for fnancial support by national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020), CEB (UIDB/04469/2020), REQUIMTE-LAQV (UIDB/50006/2020) units and SusTEC (LA/P/0007/2020); to Project “GreenHealth—Digital strategies in biological assets to improve well being and promote green health” (Norte-01–0145-FEDER-000042) and to Project AgriFood XXI (NORTE-01–0145-FEDER-000041), funded by the European Regional Development Fund under the scope of Norte2020 ‐ Programa Operacional Regional do Norte. Ítala M.G. Marx acknowledges the Ph.D. grant (SFRH/BD/137283/2018) pro vided by FCT. Nuno Rodrigues thanks to National funding by FCT-Foundation for Science and Technology, P.I., through the institutional scientifc employment program-contract, info:eu-repo/semantics/publishedVersion

Bacterial cellulose (BC) is a biopolymer produced by different microorganisms, but in biotechnological practice, Komagataeibacter xylinus is used. The micro- and nanofibrillar structure of BC, which forms many different-sized pores, creates prerequisites for the introduction of other polymers into it, including those synthesized by other microorganisms. The study aims to develop a cocultivation system of BC and prebiotic producers to obtain BC-based composite material with prebiotic activity. In this study, pullulan (PUL) was found to stimulate the growth of the probiotic strain Lactobacillus rhamnosus GG better than the other microbial polysaccharides gellan and xanthan. BC/PUL biocomposite with prebiotic properties was obtained by cocultivation of Komagataeibacter xylinus and Aureobasidium pullulans, BC and PUL producers respectively, on molasses medium. The inclusion of PUL in BC is proved gravimetrically by scanning electron microscopy and by Fourier transformed infrared spectroscopy. Cocultivation demonstrated a composite effect on the aggregation and binding of BC fibers, which led to a significant improvement in mechanical properties. The developed approach for “grafting” of prebiotic activity on BC allows preparation of environmentally friendly composites of better quality.

In this study, crude and partially purified xylanase from Aureobasidium pullulans CCT 1261 were characterized and applied in beechwood xylan hydrolysis to obtain xylooligosaccharides (XOS). Fractional precipitation (0–30 %/30–60 %) with ammonium sulfate allowed partial purification of xylanases in 6.8-fold and recovery of 69.4 %. Both extracts exhibited optimum activity at 50 °C and pH 4.5, and greater stability at 40 °C within the studied range. The thermostability parameters (kd, t1/2, ΔH*, ΔG*, ΔS*) of the extracts indicated the enzyme denaturation by the increment in temperature from 40 °C to 60 °C; its process was endothermic and led to protein unfolding in a more random structure. Crude xylanase showed a lower value of the Michaelis-Menten constant (25 mg/mL) than the partially purified enzyme (50 mg/mL). In hydrolysis reactions, total XOS contents (7.7 mg/mL and 7.9 mg/mL) and XOS yields (25.7 % and 26.5 %) were equal statistically using the crude or partially purified extracts, differing only in the amount of xylose released. The hydrolysates were mainly composed of potential prebiotics, i.e., xylobiose (50.9 % and 53.8 %) and xylotriose (30.9 % and 32.8 %). Based on these results, the use of crude and partially purified xylanase from A. pullulans showed a promising perspective for xylan hydrolysis to produce XOS.

Xylanases are hydrolase enzymes mainly produced by microorganisms and applied in several industrial segments. In this paper, the effects of different pretreatments of oat hulls were evaluated on the production of xylanase by Aureobasidium pullulans CCT 1261 under submerged cultivation. Oat hulls were subjected to several pretreatments: alkaline (0.5, 1, 2, 3, and 4% of NaOH, w/v), alkaline hydrogen peroxide (7.5% of H2O2, v/v), ultrasound-assisted alkaline (1% of NaOH, w/v, 90 W, 20 kHz), and sequential alkaline and ultrasound procedures. Both untreated and pretreated oat hulls were used as substrates for xylanase production by the strain CCT 1261. Changes caused by the pretreatments were observed through the characterization of oat hulls (i.e. lignocellulosic composition and scanning electron microscopy) and then related to the enzymatic production. Sequential alkaline and ultrasound pretreatments on oat hulls promoted the highest xylanase production (30.2 U/mL) and productivity (0.42 U/mL.h) in comparison to the other strategies evaluated. The sequential pretreatment of oat hulls also increased xylanase production more than 11-fold when compared to the untreated substrate. The effectiveness of the sequential procedure, and consequent increase in xylanase production, were attributed to the physical modification of the biomass surface by the ultrasound waves, as well as the lignin removal and hemicellulose maintenance by the alkaline pretreatment with 1% NaOH. Sequential pretreatment of oat hulls allowed the production of microbial xylanases in a more sustainable way using an agricultural by-product as substrate.

Cucumber mosaic virus (CMV), the most prevalent virus in chili pepper (Capsicum annuum L.), negatively affects chili pepper production in South Korea. In this study, foliar spraying with β-glucans obtained from the mycelial walls of the yeast-like fungus Aureobasidium pullulans inhibited CMV infection of chili pepper if applied before virus inoculation. At three concentrations, β-glucans from A. pullulans significantly ameliorated CMV symptoms in treated chili pepper; the effect was greater in plants treated with 0.01% β-glucans than 0.005% or 0.001% β-glucans. Double antibody sandwich enzyme-linked immunosorbent assay showed that these β-glucans treatments resulted in 1.7- to 10-fold reductions in CMV accumulation in the treated chili pepper. The glucans did not act directly on the virus and did not interfere with virus disassembly or replication. Foliar spraying with 0.01% β-glucans from A. pullulans at 24 hr intervals for 3 days significantly increased plant height, the total number of fruit, and the fresh weight of chili pepper fruit. However, the stem diameter of chili pepper treated with β-glucans did not increase significantly. These results indicate that foliar spraying with β-glucans from A. pullulans acts an antiviral agent against CMV infection and stimulates chili pepper growth.

Seed sprouts are nutrient-rich components, and their use in human and animal diets have increased in recent times. Microbial fermentation of seed sprouts not only improves the nutrient digestibility but also elevates the nutritional characteristics by lowering the level of antinutritional factors (ANFs). This study evaluates the effect of sprouting followed by fungal fermentation on the nutritional characteristics of canola seed sprouts. Canola seeds were soaked overnight and sprouted over a 6-day period. Dried and ground seed sprouts were subjected to submerged fermentation for 5 days using three fungal strains (Aureobasidium pullulans, Trichoderma reesei, Neurospora crassa). Results showed an improvement in proteins and reduction in ANFs of sprouts when compared to seeds. Fermentation with T. reesei resulted in higher protein titers, while N. crassa fermented sprouts exhibited slight reduction in crude fiber. Sprouting prior to fermentation helped in enhancing the protein solubility of canola seed and reducing the overall fermentation duration. Based on these findings it could be concluded that sprouting prior to fermentation can potentially lead to the production of nutritionally enriched canola based high protein ingredient. However, further research needs to be conducted to develop a mechanism for minimizing the oil loss during seed sprouting process.

We have successfully identified the transcription factor Cmr1 from the fungus Aureobasidium pullulans Hit-lcy3T, which regulates melanin biosynthesis genes. Bioinformatics analysis revealed that the Cmr1 gene encodes a protein of 945 amino acids, containing two Cys2His2 zinc finger domains and a Zn(II)2Cys6 binuclear cluster domain located at the N-terminus of Cmr1. To investigate the function of the Cmr1 gene, we performed gene knockout and overexpression experiments. Our results showed that Cmr1 is a key regulator of melanin synthesis in Hit-lcy3T, and its absence caused developmental defects. Conversely, overexpression of Cmr1 significantly increased the number of chlamydospores in Hit-lcy3T and improved melanin production. RT-qPCR analysis further revealed that overexpression of Cmr1 enhanced the expression of several genes involved in melanin biosynthesis, including Cmr1, PKS, SCD1, and THR1. Melanin extracted from the Hit-lcy3T was characterized using UV and IR spectroscopy. Furthermore, we assessed the antioxidant properties of Hit-lcy3T melanin and found that it possesses strong scavenging activity against DPPH·, ABTS·, and OH·, but weaker activity against O2−·. These findings suggest that Hit-lcy3T melanin holds promise for future development as a functional food additive.

The microbiota of winter wheat seeds from the North-East of Ukraine was studied by a biological method. Its considerable variability is established over three years (2017–2019). The effect of the treatment agents on most microorganisms of wheat seed microbiota in Ukraine, rather than on its genera and species, is shown. It has been proven that fungicides deleted some species and did not affect the development of others. Chemicals replaced some species or genera of fungi with others or even other microorganisms. Biological seed treatment (Phytosporin-M) has caused less microbiota change than chemical treatment (Maxim 0.25 FS, Rostock, Kinto Duo). Fungicides have replaced the dominance of Alternaria spp. (2017 – 57.8%, 2018 – 63.5%) for the dominance of yeast (Rostock – 54%) and Aureobasidium pullulans (Maxim 0.25 FS – 84.2%) in 2017, bacteria (Maxim 0.25 FS – 72.3%, Rostock – 53.8%) – in 2018. A. pullulans dominated in the microbiota of winter wheat seeds in 2019. The highest amount of A. pullulans was noted for the treatment of seeds by Phytosporin-M (85.9%). The biological seed treatment reduced the amount of Nigrospora spp. and Alternaria spp. Several times (3 and 5, respectively), chemical agents did not give Nigrospora spp. germination reduced the amount of A. pullulans, Alternaria spp. in 2019. Maxim 0.25 FS, Rostock 50%, and Kinto Duo delayed seed germination and seedling development on agar medium and soil, whereas Phytosporin-M – on the contrary, promoted the growth of seedlings and significantly exceeded control. Keywords: seed microbiota, winter wheat, seed treatment, seed-borne fungi, fungicides References Adhikari, P., Khatri-Chhetri, G., Shrestha, S., & Marahatta, S. (2016). Study on prevalence of mycoflora in wheat seeds. Turkish Journal of Agriculture - Food Science and Technology, 4 (1), 31–35. https://doi.org/10.24925/turjaf.v4i1.31-35.509 Adhikari, P., Khatri-Chhetri, G., Shrestha, S., & Marahatta, S. (2018). In-vitro study on prevalence of mycoflora in wheat seeds. Journal of the Institute of Agriculture and Animal Science, 33, 27–34. https://doi.org/10.3126/jiaas.v33i0.20679 Andersen, B. Nielsen, K.F., Fernandez, P.V, & Patriarca, A. (2015).Characterization of Alternaria strains from Argentinean blueberry, tomato, walnut and wheat. International Journal of Food Microbiology, 196, 1–10. https://doi.org/10.1016/j.ijfoodmicro.2014.11.029 Bankina, B., Bimsteine, G., Neusa-Luca, I., Roga, A., & Fridmanis, D. (2017). What influences the composition of fungi in wheat grains? ActaAgrobotanica, 70(4), 1726. https://doi.org/10.5586/aa.1726 Baybakova, E.V., Nefed'eva, E.E., & Belopukhov, S.L. (2016). Assessment of the influence of modern protectants on the germination of seeds and growth of seedlings of grain crops. Proceedings of Universities. Applied Chemistry and Biotechnology, 6(3), 57–64 (in Russian).

The combined biochemical impact of carbon, nitrogen and temperature on the biosynthesis of the antifungal volatile organic compounds (VOCs): ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol produced by Aureobasidium pullulans A1 and A3 was investigated using a Box-Behnken experimental design and response surface methodology (RSM). Normalized peak areas derived from solid phase micro extraction-gas chromatography-mass spectrometry (SPME-GC-MS) analysis, indicated that initial carbon content had a significant influence on the biosynthesis of ethanol and alcohols with greater than three carbon atoms. This result suggests a dominant activity of the A. pullulans anabolic pathway to biosynthesize three higher alcohols via de novo biosynthesis of amino acids from sugar metabolism. Low concentrations of carbon (3-13 g l-1 ) with nitrogen as both ammonium and amino acids in the growth medium resulted in a higher number of significant linear and quadratic relationships. Nitrogen availability and growth temperature had significant negative linear and quadratic correlations with VOCs biosynthesis in most instances. Isolate-dependant metabolic response was evident for all abiotic parameters tested on alcohol production. The findings of this study offer new perspectives to improve the production of key antifungal compounds by antagonists in biological control systems.

Sequancing of pullulanase from the fungus Aureobasidium pullulans isolated from Egypt soil; Genomic DNA of pullulanase was determined for the first time using PCR, according to Baser program, Pullulanase nucleotide collection from Aureobasidium pullulans was blasted which showed similarity using NCBI significant alignment with Aureobasidium namibiae CBS 147.97 hypothetical protein partial mRNA and 46 % with Aureobasidium pullulans JQ624241 and AF470619; Identified sequenced fragment was 2051 bp. and G+C content is 50.5% with molecular mass 63 KDa.

Pullulan is an imperative microbial exo-polymer commercially produced by yeast like fungus Aureobasidium pullulans. Its structure contains maltosyl repeating units which comprises two α-(1 → 4) linked glucopyranose rings attached to one glucopyranose ring through α-(1 → 6) glycosidic bond. The co-existence of α-(1 → 6) and α-(1 → 4) glycosidic linkages endows distinctive physico-chemical properties to pullulan. It is highly biocompatible, non-toxic and non-carcinogenic in nature. It is extremely resistant to any mutagenicity or immunogenicity. The unique properties of pullulan make it a potent candidate for biomedical applications viz. drug delivery, gene delivery, tissue engineering, molecular chaperon, plasma expander, vaccination, etc. This review highlights the potential of pullulan in biomedical research and development.

Pepaya adalah buah klimakterik yang memiliki umur simpan pendek dan potensi gangguan penyakit antraknosa pada saat tahap pascapanen yang disebabkan oleh patogen Colletotrichum gloeosporioides. Tujuan dari penelitian ini adalah mengkaji keefektifan beberapa spesies khamir antagonis untuk memperpanjang umur simpan dan mengendalikan penyakit antraknosa pada pascapanen buah pepaya Callina. Penelitian ini dilaksanakan di kebun pepaya Desa Kanaga, Kabupaten Lebak, Banten pada November 2018 sampai Juli 2019 dan Laboratorium Pascapanen AGH, IPB pada bulan Maret sampai dengan Juli 2019. Penelitian ini menggunakan rancangan kelompok lengkap teracak dengan perlakuan lima jenis khamir yaitu Cryptococcus albidus Yp, Aureobasidium pullulans Dmg 30 DEP, Rhodotorula minuta Dmg 16 BEP, Candida tropicalis Lm 13 BE, dan Pseudozyma hubeiensis Dmg 18 BEP, dan sebagai pembanding perlakuan fungisida berbahan aktif azoksistrobin serta tanpa perlakuan sebagai kontrol. Hasil penelitian menunjukkan penggunaan khamir Candida tropicalis, Aureobasidium pullulans, dan Cryptococcus albidus dapat memperpanjang umur simpan buah pepaya Callina masing-masing 12.6, 12.4, dan 12.2 hari, lebih lama dibandingkan kontrol yang hanya 7 hari, dan efektif mengendalikan penyakit antraknosa dengan tingkat penekanan secara berturut-turut 58.33%, 54.17%, dan 50.00%, selama penyimpanan. Penggunaan khamir antagonis tidak mempengaruhi mutu fisik dan kimia buah pepaya Callina. Kata kunci: buah klimakterik, Colletotrichum gloeosporioides, pascapanen

Background: Apples often experience postharvest damage due to being attacked by mold organisms. Several groups of molds such as Aspergillus sp., Penicilium expansum, Botrytis cinerea, and Venturia sp. can cause a serious postharvest disease exhibited as watery regions where areas of blue-green tufts of spores develop. Current methods using fungicides to control pathogenic fungi can cause resistance if applied in the long term. An alternative procedure using yeast as a biological agent has been found. Objective: The aim of this study is to screen potential yeast, which has the ability to inhibit the growth of Aspergillus brasielensis (isolate A1) and Aspergillus flavus section flavi (isolate A17) isolated from apple fruits. Methods: Antagonism test using YMA dual culture medium using in vitro assays and ITS rDNA identification were performed. Results: The result showed that 3 out of 19 yeast isolated from Cerbera manghas L, T1, T3 and T4, demonstrated the potential ability as a biocontrol agent. ITS rDNA identification demonstrated that T1 has a similarity to Rhodotorula mucilaginosa while T3 and T4 were identified as Aureobasidium sp. nov. The 3 isolates exhibited the ability to reduce the growth of A. brasiliensis sensu lato better than dithane 0.3% with a Disease Incidence (DI) of 100% and a Disease Severity (DS) value of 45%. Only isolate T1 and T3 were able to reduce decay symptoms in apples inoculated with A. flavus sensu lato (with DO and DS were 100% and 25%, respectively) compared to dithane pesticides 0.3%. Conclusion: This study indicated that competition between nutrients occurs between pathogenic molds and under-yeast in vitro and in vivo conditions. However, further studies in the future might be able to elucidate the ‘killer’ activity and interaction with the pathogen cells and the bio-product production using Rhodotorula mucilaginosa and Aureoubasidium namibiae strains to control postharvest diseases.

O fungo Colletotrichum truncatum (Schwein.) Andrus & W.D. Moore é um importante fitopatógeno em várias culturas de interesse econômico, inclusive na soja (Glycine max (L.) Merril), causando a doença conhecida como antracnose. Apesar do controle químico ser o método de controle mais usado pelos produtores rurais, o controle biológico se apresenta como uma alternativa sustentável para o manejo desta doença. Esta pesquisa avaliou o potencial do fungo Aureobasidium pullulans (de Bary & Lowenthal) G. Arnaud, originário do bioma Caatinga, no controle de C. truncatum. Foi avaliado o efeito inibitório sobre o crescimento micelial do patógeno a partir de compostos voláteis (in vitro), além de testes in vivo. Foram analisadas as variáveis germinação, índice de velocidade de germinação (IVG), massa seca e sanidade das sementes. Nos ensaios in vitro, os compostos voláteis produzidos pelo antagonista não apresentaram ação inibitória expressiva a C. truncatum. Nos testes in vivo, foi observada a ação inibitória de A. pullulans a C. truncatum em sementes de soja, por meio dos tratamentos preventivo e curativo. O tratamento preventivo proporcionou maior efetividade em relação ao tratamento curativo, evidenciando média superior de IVG (41,84) e menor incidência do patógeno (16,00%). Desse modo, verificam-se as perspectivas do uso de A. pullulans no biocontrole de C. truncatum, especialmente, quando se realiza o tratamento preventivo. Este é o primeiro relato de A. pullulans sobre C. truncatum. Palavras-chave: Antagonismo Microbiano. Controle Biológico. Fungos. Fitopatógeno. Abstract The fungus Colletotrichum truncatum (Schwein.) Andrus & W.D. Moore is an important phytopathogen in several crops of economic interest, including soybean (Glycine max (L.) Merril), causing a disease known as anthracnose. Although chemical control is the most used control method by farmers, biological control presents itself as a sustainable alternative for the management of this disease. This study evaluated the potential of Aureobasidium pullulans (de Bary & Lowenthal) G. Arnaud, originating from Caatinga biome vegetation, in the control of C. truncatum. The inhibitory effect on the mycelial growth of the pathogen was evaluated from volatile compounds produced by the antagonist (in vitro) in addition to the in vivo tests. The following variables were analyzed, germination, germination speed index (GSI), dry mass and seed health. In the in vitro assays the volatile compounds produced by the antagonist presented no significant inhibitory action against C. truncatum. In in vivo tests, an inhibitory action of A. pullulans and C. truncatum was observed in soybean seeds, through preventive and curative treatments. The preventive treatment provided greater effectiveness in relation to the curative treatment, showing higher average of GSI (41.84) and lower incidence of the pathogen (16.00%). Thus, the perspectives of the use of A. pullulans in the biocontrol of C. truncatum are verified, especially when the preventive treatment is carried out. This is the first report of A. pullulans on C. truncatum. Keywords: Microbial Antagonism. Biological Control. Fungi. Phytopathogen.

Extracellular rhodanese obtained from Aureobasidium pullulans was employed in both free and immobilized forms for the biodegradation of cyanide present in cassava processing mill effluent (CPME). Crosslinking with glutaraldehyde (at an optimum concentration of 5% v/v) before entrapment in alginate beads resulted in the highest immobilization yield of 94.5% and reduced enzyme leakage of 1.8%. Rhodanese immobilized by cross-linking before entrapment (cbe) retained about 46% of its initial activity after eight cycles of catalysis compared to the entrapment in alginate alone (eaa) which lost more than 79% after the fifth catalytic cycle. A cross-examination of thermodynamic ( Δ G d * , Δ S d * , Δ H d * ) kinetic ( k d , t 1 / 2 , D and z - values ) parameters at 30-70 °C showed that cbe displayed a higher resistance to thermal inactivation when compared to the free enzyme (fe) and (eaa). The efficiency of cyanide biodegradation from the CPME by the fe, eaa and cbe were 55, 62, and 74% respectively after 6 h. Biodegradation of cyanide using the cbe was monitored using FTIR spectroscopy. Rhodanese immobilized via cbe had a higher resistance to thermal denaturation over other enzyme forms. Hence, this makes cbe adaptable for large-scale detoxification of cyanide from CPME.

With the COVID-19 pandemic causing huge threat to public health and definite treatment modalities and preventive vaccines yet to be arrived at, some of the key indicators of relevance to its prognosis have started emerging. One such independent predictor of outcome has been fasting plasma glucose (FPG) at the time of admission. Earlier, co-morbidities such as diabetes also have been reported to have a risk of relatively increased mortality due to COVID-19. In this background, we herein report on the beneficial effects of Biological response modifier glucan (BRMG) secreted by the black yeast Aureobasidium pullulans AFO-202 which has been proven to bring under control blood sugar levels in human subjects and also has potential in enhancing & regulating the immune parameters in relevance to COVID-19. We further recommend that this BRMG be tried in clinical studies of COVID-19 to provide a prophylactic effect for validation.

Background: The alleviating effects of diabetic nephropathy and hepatopathy of β-glucan were evaluated in this study. Objective: The anti-diabetic effects of β -glucan from Aureobasidium pullulans were assessed in a streptozotocin (STZ)-induced rat diabetes model at 62.5 and 125 mg/kg doses. In addition, the possibility of changes in the effects of β-glucan according to the severity of diabetes was also assessed at one dosage (62.5 mg/kg): severe, >360 mg/dL; slight, 130-200 mg/dL. Methods: Test articles were administered orally to STZ-induced diabetic rats from 21 days after STZ dosing for 4 weeks. Each of five or six female rats per group was selected using blood glucose levels at 21 days after STZ dosing. Changes in body weight were recorded during the study, along with blood glucose, blood urea nitrogen, creatinine, and serum aspartate aminotransferase and alanine aminotransferase levels. On the day of sacrifice, livers and kidneys were weighed and observed microscopically for changes in the percentage of degenerative regions and numbers of degenerative tubules in the kidney. Results: β-glucan showed no hypoglycemic effects in the STZ-induced diabetic rat model. However, it had favorable effects on decreasing diabetic complications related to diabetic nephropathy and hepatopathy. Conclusion: Based on the results of this study, it was concluded that β-glucan showed favorable effects in decreasing diabetic complications in STZ-induced rat diabetes model.

β-poly(L-malic acid) (PMLA) has attracted industrial interest for its potential applications in medicine and other industries. For a sustainable PMLA production, it requires replacing/reducing the CaCO3 usage, since the residual CaCO3 impeded the cells’ utilization, and a large amount of commercially useless gypsum was accumulated. In this study, it was found that more glucose was converted into CO2 using soluble alkalis compared with CaCO3 usage. Moreover, since the high ion strength and respiration effect of soluble alkalis also inhibited PMLA production, they could not effectively replace CaCO3. Furthermore, comparing the fermentations with different neutralizers (soluble alkali vs. CaCO3), it was found that the differential genes are mainly involved in the pathway of starch and sucrose metabolism, pentose and glucuronate interconversions, histidine metabolism, ascorbate and aldarate metabolism, and phagosome. In detail, in the case with CaCO3, 562 genes were downregulated and 262 genes were upregulated, and especially, those genes involved in energy production and conversion were downregulated by 26.7%. Therefore, the irreplaceability of CaCO3 was caused by its effect on the PMLA metabolic pathway rather than its usage as neutralizer. Finally, a combined pH shift control strategy with CaCO3 addition was developed. After the fermentation, 64.8 g/L PMLA and 38.9 g/L biomass were obtained with undetectable CaCO3 and less CO2 emission. • The effect of CaCO3 on PMLA metabolic pathway resulted in its irreplaceability. • A pH shift control strategy with CaCO 3 addition was developed. • Undetectable CaCO 3 and less CO 2 emission were detected with the new strategy.

Background: Poly-L-malic acid (PLMA) comprises aliphatic polyester polymers with broad applications in pharmaceutical industries. The fungal microorganisms are among the best natural sources recruited to supply L-malic acid (MA) as a precursor of PLMA. In this study, we investigated MA production ability of 7 clinical isolated of the fungus Aureobasidium pullulans. Materials and Methods: Seven clinical isolates of A. pullulans acquired from Westerdijk Fungal Biodiversity Institute were studied, and the isolate with the highest total MA production was selected for the optimization process. We tried to optimize the output by applying different concentrations of CaCO3 in fungus medium (1.5%, 3%, and 6%) and various incubation temperatures (27°C, 32°C, and 37°C) during 3, 7, and 14 days. Results: Intra-strains variation was significantly strong (P0.05). Conclusion: Overall, we obtained the highest MA production in Sabouraud dextrose agar (SDA)medium with 3% CaCO3 at 27°C after 7 days of incubation. Our study indicated that the fermentation period and CaCO3 concentration significantly alter MA production in A. pullulans var. melanigenum.

For the first time, the composition of micromycetes communities was investigated in both photic and aphotic entrance zones of seven caves in Montenegro: Obodska Pecina, Golubinja Pecina, Velustica Pecina, Vrbacka jama, Jama ER-1, Njegos Pecina, and an unnamed cave. In total, 72 species of micromycetes were identified: 46 species from illuminated photic zones of the caves, including 43 species on phototrophs, 10 species from the air, 15 species on substrates, and 50 species from unlit aphotic zones, including 35 species from the air and 37 species on substrates. The most frequently isolated species were Acrodontium salmoneum, Aureobasidium pullulans, Cladosporium oxysporum, Mucor racemosus f. racemosus, Penicillium chrysogenum, and Rhizopus stolonifer. The similarity between micromycetes composition of different biotopes was evaluated using the Jaccard index and cluster analysis. The greatest similarity was observed in the communities isolated from the air of the photic zones of the caves.

Aureobasidium pullulans LB83 was evaluated for cellulase production under submerged fermentation conditions. Different process variables such as carbon sources (corn cob, sugarcane bagasse, and sug...

Cellulose is the most abundant renewable carbon source found in nature and is of great interest to various industries, including the food industry. Generally, microorganisms can be used to convert ...

Low-temperature red winemaking is mainly used for enhancing the wine aroma; however, colour and phenolic extraction is reduced under this condition. This work evaluated the effects of the pectinoly...

The influence of the structure and method of grassmixture sowing on the biological activity and mycological structure of reclaimed soil on different research plots is considered. It is noted that there is constant mutual beneficial biotic correlation between microflora and plants, which is reflected in the growth of physiological groups of microorganisms number in rhizosphere. Root excretions of plants specifically effect on microorganisms development of root zone and biochemical activity of soil. The research has revealed the advantage of increasing the number of bacteria using mineral nitrogen and streptomycetes in variant of grassmixture: Phalaris arundinacea, Dactylis glomerata, Trifolium pratense, both for row sowing and for scattering. On experimental samples of grassmixtures mineralization processes are noted to be more intensive, respectively, the level of microflora activity is much higher compared to design composition. The average indicators of mineralization coefficients on the samples of experimental grassmixtures indicate that mineralization processes are more intense, what means that the level of microflora activity is higher compared to control variant. Judging by the average indicators of oligotrophic coefficient on reclaimed lands after ilmenite ores extraction, both oligotrophic and eurytrophic microorganisms can develop quite actively, that proves instability of the soil coenosis. The meteorological conditions of research years had a decisive influence on microbial biomass indicators. However, during 2014-2017, the agrocenoses of experimental variants in terms of microbial biomass prevail the agrocenosis of the design grassmixture composition (data for 2017 year). Carbon dioxide emission on the experimental plots in 2017 are higher than control variant, that indicates a higher level of microbiological activity in rhizosphere of experimental grassmixtures on reclaimed plots. In accordance to dynamics of mineralization indicators, we observe changes of oligotrophic coefficient indicators. During the development of agrocenosis of experimental variants, the agrocenosis of design composition of grassmixture is proved to prevail in terms of microbial biomass. The research shows that the growth of plant species diversity also contributes to mycological structure of soils enrichment on disturbed soils of Yavoriv and Novyj Rozdil sulfur quarries. It is noted that the highest biological diversity of 50 species of micromycetes with a high frequency of Aureobasidium pullulans (23.1 %), Trichoderma lignorum (12.5 %) are characterized by disturbed lands of Novyj Rozdil sulfur quarry, on which mixed stands are created. Slightly less diverse (43 species) is appropriate for mycobiota of soils with a high frequency of Penicillium digitatum (31.5 %), Aureobasidium pullulans (18.5 %) in Yavoriv sulfur quarry, where the species composition of stands is significantly simplified.

Epiphytic yeasts were isolated from different cultivars of apples and lemons and identified by a combination of PCR-RFLP of 5.8S rRNA region and sequencing of D1/D2 domain of the 26S rRNA gene. Among 69 isolates, Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 strains showed the greatest antagonistic activity against two significant apple and lemon postharvest pathogens, Penicillium expansum DSM62841 (blue mold) and Penicillium digitatum DSM2750 (green mold), after preliminary screening. Yeasts were applied as single and mixed cultures with two different cell concentrations of 106 and 108 cells/ml in the present study. It was determined that antagonistic activity of two yeast strains studied emerged with a combination of several mechanisms of action including competition for space and nutrients, production of volatile organic compounds (VOCs), secretion of extracellular lytic enzymes and inhibition of fungal spore germination. The highest inhibition of mycelial growth on P. expansum DSM62841 and P. digitatum DSM2750 (83.4% and 74.7%, respectively) was achieved by utilization of single culture of A. pullulans GE17. Otherwise, the application of mixed culture at the ratio of 108 cells/ml inhibited spore germination of both pathogens from 86% to 95%. Results of this study suggest that an increase in yeast cell concentrations positively affected their biocontrol activity against blue and green molds. According to the results, employing single culture of M. guilliermondii KL3 did not exhibit effective antagonistic activity against blue and green molds. However, utilization of A. pullulans GE17 alone and mixed culture showed succesfull controlling against both P. expansum DSM62841 and P. digitatum DSM2750.

The crude β-xylosidase from Aureobasidium pullulans CBS 135684 was immobilized on the surface of the modified spent expanded perlite via (3-aminopropyl) triethoxy-silane and glutaraldehyde cross-linking reaction. Over 80% of the initial free enzyme activity was detected on the bound enzyme. The immobilized β-xylosidase (204.7 ± 6.5 U g-1 of perlite) exhibited transxylosylation activity when sugars from black liquor xylan hydrolysate and a variety of alcohols (C1-6) were used as donor and acceptor molecules, respectively, yielding a number of alkyl xylosides. The synthesized hexyl xyloside exhibited some interesting properties comparable with those of a commercial hexyl glucoside, including an antioxidant activity (IC50 = 8.9 ± 2.0 mg mL-1), wetting time (43 s), and critical micelle concentration value (252 mmol L-1). Optimization for hexyl xyloside synthesis was performed via statistical analysis using a Box and Behnken design. The maximum predicted yield of hexyl xyloside (499.0 ± 1.1 mg g-1 xylan hydrolysate) was obtained with 10% (w/v) xylan hydrolysate, 10 U g-1 immobilized β-xylosidase, and 13% (v/v) hexanol at 70 °C, pH 6.0 for 4 h, and it was validated by the following experimental yield (498.9 ± 4.8 mg g-1 xylan hydrolysate). The maximum production yield remained relatively stable after the immobilized enzyme was reused for 12 cycles with a 471.5 ± 15.1 mg g-1 hexyl xyloside yield (94.4% of the first cycle). Thus, the immobilized β-xylosidase has a high potential as a robust catalyst for application in alkyl xyloside production.

Bacteria, yeasts and microscopic fungi, which are live and grow normally on the grape surfaces, are important for winemaking. The aim of this study was to investigate the microbiota of the Tokaj wines grapes of Slovak origin. The total bacterial count ranged from 2.48 log cfu/g to 4.52 log cfu/g. Malt Extract Agar (MEA) with acid base indicator bromocresol green was used for cultivation of yeast and microscopic fungi and the number of this microorganisms ranged from 1.11 log cfu/g to 4.16 log cfu/g. We found following bacterial species: Staphylococcus epidermidis, Micrococcus luteus, Paenibacillus pasadenensis and Sphingomonas sp. Identified yeasts species included Candida sorbose, Kloeckera apiculata, Metschnikowia pulcherrima, Rhodotorula glutinis, Rhodotorula mucilaginosa and Saccharomyces cerevisiae. From microscopic fungi were isolated: Alternaria sp., Arthrinium sp., Aspergillus niger, Aureobasidium pullulans, Botrytis cinerea, Cladosporium sp., Eurotium sp., Geotrichum candidum, Penicillium expansum, Penicillium sp. and Rhizopus sp. The most dominant species of Tokaj grape were Kloeckera apiculata and Metchnikowia pulcherrima.

Chitin-glucan complex (CGC) copolymer is a cell wall component in fungi and yeasts, used in medicine and cosmetics applications. This study investigated the production of CGC from Aureobasidium pullulans KSY-0516, which is used in the industrial production of β-1,3/1,6-glucan. The amino acid composition of the A. pullulans fermentation by-products was determined using an amino acid analyzer. The total amount of amino acid was 6.67 g/100 g of by-product. Additionally, the recovery of the β-glucan complex from the A. pullulans fermentation by-products was 8.44 g/100 g of by-product using ethanol precipitation. The CGC yield was determined from the by-products after yeast fermentation. The recovery of CGC was 14.83% for the yeast cell wall obtained by alkaline extraction. The CGC and β-glucan were characterized using Fourier transform infrared analysis, and compared with crab shell chitin and laminarin. This study aimed to examine the potential, as a useful substrate for biotechnology, by producing CGC from yeast cultivation by-products.

This study involved the design and fabrication of airlift fermenters for the mass propagation of a consortium of three microorganisms (Pseudomonas aeruginosa, Pseudomonas fluorescence and Aureobasidium pullulans). The efficacy of the blend in the bioremediation of simulated crude oil polluted water systems environments was also tested with a view to generating more data on crude oil degrading microorganisms for bioremediation purposes. The organisms were separately grown on a gyratory shaker at 120 rpm and at 30 °C until notable growths were observed. Equal volume of the grown organisms (500 ml) of mixed culture was used as inoculum for the 4.5 L of medium in the 7 L airlift fermenter. Thereafter, this was transferred into the 45 litres medium in the 70 L fermenter until significant growth was observed at room temperature. Culture broths were withdrawn at intervals for the determination of biomass and residual hydrocarbon concentrations. The product from the 70 L fermenter was introduced into simulated polluted water systems from which samples were withdrawn weekly. Analyses for residual hydrocarbon contents were carried out using appropriate analytical techniques. The results showed that 78.5% hydrocarbon removal was observed in the 7 L fermenter after 72 h, while that of the 70 L fermenter was 97.8% after 180 h of fermentation. Polluted water sample treated with the microbes experienced 96.4% removal after 10 weeks of treatment. It was observed further that the application of NPK fertilizer biostimulant aided microbial activities in the removal of petroleum hydrocarbons than urea, cow dung and poultry droppings. The study has demonstrated that effective bioremediation of crude oil polluted water systems could be achieved through the application of biostimulants with mass propagated crude oil degrading organisms using airlift fermenter systems. Keywords: Pseudomonas aeruginosa; Pseudomonas fluorescence; Aureobasidium pullulans; Airlift fermenter; Bioremediation; Biostimulation; Bioaugmentation.

This study aimed to investigate the efficacy and safety of the exopolymers from Aureobasidium pullulans (EAP) in subjects with mild-to-moderate atopic dermatitis. A total of 68 subjects who visited Semyung Oriental Medical Center from November 16, 2015, to September 8, 2016, were included in the study. The subjects were treated orally with EAP (250 mg/day; experimental group) or placebo (control group) for 12 weeks. A series of clinical examinations, hematological assessments, blood biochemistry, urinalysis, and skin assessments were conducted at baseline and after the study period to evaluate efficacy and safety. The primary endpoint was the Severity Scoring of Atopic Dermatitis (SCORAD) index. A significantly greater reduction of SCORAD index as a primary endpoint showed in the treatment group than in the placebo group (p = 0.0053) during the treatment period. The secondary endpoints of serum interferon-γ levels, skin hydration, and transepidermal water loss showed significant changes over time within the EAP group. Other secondary endpoints showed no statistically significant changes. Safety evaluations identified no abnormal changes within the experimental or placebo groups. These experiments indicated that it was safe to ingest this dose of EAP orally for 12 weeks and that this treatment could help to alleviate atopic dermatitis.

The interest in pullulan produced from industrial wastes has substantially increased due to its industrial applications. The major constraint in pullulan production is expensive and low yield. In this study, attempts were made on the evaluation of media components for maximum pullulan production from Aureobasidium pullulans MTCC 1991 using cassava waste in solid-state fermentation. A statistical approach was scrutinized to evaluate the effects of yeast extract, K2HPO4, (NH4)2SO4, MgSO4⋅7H2O, and NaCl on pullulan production. The optimal conditions eliciting the maximum pullulan yield (6.45 g pullulan/L of culture broth) were found to be 0.6 g/L yeast extract, 3 g/L K2HPO4, 0.2 g/L (NH4)2SO4, 0.3 g/L MgSO4⋅7H2O, and 1 g/L NaCl. The produced pullulan was extracted using the solvent precipitation method and characterized by Fourier transform infrared spectroscopy and proton nuclear magnetic resonance spectroscopy spectroscopic analysis that confirmed pullulan with linear α (1–6) linkage. The results revealed that cassava waste could be used as an effective carbon source for the production of pullulan.

Aspergillus flavus is widely known as an aflatoxin-producing fungus that frequently contaminates feed and affects livestock, which leads to severe health problem for animal and human. Biological agents have been proven to prevent this contamination since they can produce metabolites which have antagonistic activity. In this study, phylloplane yeasts isolated from Moringa oleifera leaf have shown an ability to inhibit the growth of Aspergillus flavus UNJCC F-30 collected from chicken feed. This research was conducted in three stages: (1) yeast isolation (leaf washing and direct method), followed by (2) antagonistic test using dual culture method, and (3) molecular identification using D1/D2 region of 26S rDNA. In the first stage, 38 yeast isolates have been succesfully obtained. These isolates were of different colors: peach pigment (60.5%), the non-pigmented yeast (26.5%), cream (10%), and orange (3%). Antagonistic activity against A. flavus UNJCC F-30 was tested based on growth, sporulation, and the presence of clear zones. Screening result showed that 12 yeast isolates are capable of inhibiting A. flavus UNJCC F-30. Among them, 4 isolates with the code K4, K10, K15, and K26 showed the highest antagonist ability. Molecular identification resulted that the 4 isolates show a similar identity with Aureobasidium pullulans UWFP 993 (100%), Aureobasidium melanogenum QCC:M017/17 (99%), Aureobasidium melanogenum QCC:M017/17 (100%) and Rhodotorula taiwanensis CBS:11729 (99%), respectively. Isolate K10 exhibited the highest percentage of inhibition activity among all isolates which is potential for application as biocontrol agent against A. flavus. As A. pullulans is a common yeast found on leaf surfaces of many Indonesian flora, therefore it can be considered as safe and alternative to reduce fungal contamination from A. flavus in feed chicken.

Determination of creatinine concentration in biological fluids is becoming increasingly relevant as a clinical test. Various chemical and physico-chemical methods have been proposed to determine creatinine concentration in biological samples, including various modifications of the well-known Jaffe color reaction, although it has several drawbacks, namely, a low specificity and sensitivity to positive and negative interference from related substances. In addition, the available methods are time consuming, require a complicated sample preparation procedure, expensive reagents, highly qualified personnel and, most importantly, do not allow real-time analysis. Therefore, increasing attention has been paid to enzymatic methods for the determination of creatinine, particularly, using microbial creatinine deiminase. In this regard, the search for new microorganisms – effective producers of specific creatinine deiminases – is an actual task for microbial enzymology and analytical biotechnology. We have analyzed creatinine deiminase activity for 10 strains of different molds: Botrytis aclada , Botrytis allii 100(5), Monilinia fructicola , Sporobolomyces salmonicolor , Totula sp., Stachybotris chartarum , Pichiapini , Aspergillus oryzea , Trichoderma lignorum , Aspergillus glaucus , Trichothecium roseum , Aureobasidium pullulans . Among the strains tested, the enzyme activity was detected in the following species: M. fructicola, A. pullulans, B. allii, T. roseum, and C. g lobosum , although only two fungi, B. allii and C. globosum , were able to use creatinine as the sole nitrogen source. Creatinine deiminase activity in cell free extracts of B. allii and C. globosum , grown in glucose medium supplemented with creatinine (as the sole carbon and nitrogen source, respectively) was 2.5 and 1.5 times higher, related to that for the strains grown in glucose-sodium nitrate medium. Therefore, the fungi B. allii and C. globosum can be used for isolation of creatinine deiminase to be used in construction of biosensors selective to creatinine.

Bioorganic asymmetric reduction of carbonyl compounds is one of the most important fundamental and practical reactions for producing chiral alcohols. The stereoselective bioreduction of prochiral ketones of benzofuran derivatives in the presence of yeast-like fungus Aureobasidium pullulans contained in the antifungal Boni Protect agent was studied. Biotransformations were carried out under moderate conditions in an aqueous and two-phase system and without multiplication of the bioreagent. Despite similar chemical structure, each of the used ketone has been reduced with varying efficiency and selectivity. One of the reasons for these results is the presence of a whole set of oxidoreductases in A. pullulans cells that are sensitive to the smallest changes in the structure of prochiral substrate. The unsymmetrical methyl ketones were biotransformed with the highest selectivity. Aureobasidium pullulans microorganism is less effective in the reduction of unsymmetrical halomethyl ketones. The presence of a heteroatom in the alkyl group significantly decreases the selectivity of the process. Finally, as a result of the preferred hydride ion transfer from the dihydropyridine ring of the cofactor to the carbonyl double bond on the re side, secondary alcohols of the S and R configuration were obtained with moderate to high enantioselectivity (55-99%).

This study evaluates the responses of different tomato cultivars to disease initiation by cell-free culture filtrates (CFCFs) of selected fungal pathogens. Twenty-one days old culture filtrates of four fungal pathogens; Alternaria triglochinicola, Aureobasidium pullulans, Pythium ultimum and Sclerotium rolfsii grown on Potato Sucrose Broth were tested against four tomatoes; Roma Savanna, Tropimech, Riogrande and Roma VF. Seed germination was inhibited by filtrates of Alternaria triglochinicola and Pythium ultimum. Plant heights were lower in Roma Savanna seedlings treated with filtrates of Sclerotium rolfsii (3.27 cm), and Alternaria triglochinicola (3.30 cm); Riogrande seedlings treated with filtrates of Pythium ultimum (2.22 cm) and Alternaria triglochinicola (2.46 cm), compared to controls (3.7 cm and 2.56 cm). Roma Savanna and Riogrande cultivars showed 25% and 50% necrotic responses respectively to Alternaria triglochinicola, while Roma VF showed 50.00% response to Aureobasidium pullulans. Differences in foliar symptoms induced by CFCFs of different fungal pathogens on the studied tomato cultivars were significant (P ≤ 0.05).

Fungal strain PA-2 was isolated from infected poplar leaves from the Ping'an District, Haidong City of Qinghai Province, China. Based on the culture characteristics and the internal transcribed spacer (ITS) sequence of its 16S rDNA, the strain was identified as Aureobasidium pullulans. The culture and metabolites of strain PA-2 showed high herbicidal potential to five tested weeds Galium aparine var. tenerum, Chenopodium album, Malva crispa, Polygonum lapathifolium and Avena fatua. For the in vitro test, 5 days after the detached leaves were inoculated with PA-2 culture, all leaves infected by the hyphae and became black and rotten. For the in vivo test, a metabolite filtrate of PA-2 culture was sprayed over the living weed plants, and five days after inoculation, the weed plants became withered and necrotic. Seven days after inoculation, the fresh weight reductions of G. aparine var. tenerum, C. album, M. crispa, P. lapathifolium and A. fatua were 87.25, 78.46, 82.25, 62.11, and 80.27%, respectively. Galium aparine var. tenerum and M. crispa exhibited significant reductions in fresh weight. The bio-safety test on the five crops showed no significant plant height reductions, which was also observed for wheat (Triticum aestivum), faba bean (Vicia faba), and barley (Hordeum vulgare). By contrast, oilseed rape (Brassica napus) and pea (Pisum sativum) exhibited light spots but no significant reductions in plant height. These results indicated that A. pullulans could be a potential microbial herbicide for the control of the target weeds in crops. Optimization of the carbon and nitrogen sources for cultural media and substances for solid-state fermentation indicated that PA-2 had better colony growth and spore production with the optimal carbon source of glucose (C4), nitrogen source of soybean flour (N2), and the optimal substance was wheat bran. The results in this study provide useful information for the development of A. pulluans PA-2 as an herbicide for bio-control of the weed.

Aureobasidium pullulans YTP6-14 was demonstrated to be an excellent multiple biosurfactant producer utilizing cheap carbon sources available in Thailand, including glycerol and cassava flour hydrolysate. A. pullulans YTP6-14 maximally produced 1.81 g/l biosurfactant in an aqueous layer (BS-AQ) in a medium containing glycerol, and 7.37 or 6.37 g/l biosurfactant in a heavy oil layer (BS-HO) in cassava flour hydrolysate or a glucose containing medium, respectively. Each BS-AQ and BS-HO had critical micelle concentration values of 41.32 mg/l and 13.51 mg/l, and both biosurfactants formed a stable food oil emulsion and reduced the amount of biofilms formed by Streptococcus sobrinus and Streptococcus mutans. BS-AQ and BS-HO were mainly composed of liamocins or exophilins and massoia lactone, respectively.

En el presente trabajo se estudia la interacción entre Saccharomyces cerevisiae y Aureobasidium pullulans en la fermentación de mosto de uva, evaluando en primer lugar la cinética fermentativa en un mosto concentrado y posteriormente, la fermentación en mosto de la variedad Verdejo, analizando al finalizar el vino obtenido y la producción de aromas, Grado en Enología