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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.

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.

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.

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.

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.

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.

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...

Pullulan has many useful characteristics but, its high cost limits its potential applications. In the present work, kitchen waste (KW), which otherwise has zero commercial value, was evaluated for the economical production of pullulan. Before fermentation, the KW was hydrolyzed into free sugars using an in-house produced cocktail of enzymes. During hydrolysis, 46 ± 3.5 g/l and 31 ± 2.2 g/l of total reducing sugars and glucose were released, respectively. Hydrolyzed kitchen waste was then used as substrate for fermentation by Aureobasidium pullulans MTCC 2013 yielding 20.46 ± 2.01 g/l pullulan. Further, effect of different nitrogen sources was evaluated and yeast extract (3%) was found to be the best, yielding (24.77 ± 1.06 g/l) exopolysaccharide (EPS). The pullulan produced from KW was characterized in terms of organoleptic properties, physical strength, Fourier-transform infrared spectroscopy (FTIR), and H nuclear magnetic resonance (H NMR) analysis. The results corroborated well with commercial pullulan. The biodegradable nature and water solubility of the film developed from pullulan was also confirmed. To the best of our knowledge, this is the first report on the validation of the biodegradability of in-house produced pullulan. Thus, kitchen waste appears to be a promising option for economical pullulan production. Additionally, the method may also prove to be helpful for managing the increasing load of municipal solid waste in an eco-friendly and scientific way.

A colour-variant strain of Aureobasidium pullulans FB-1 was used for the production of pullulan in a stirred tank reactor in a medium composed of sucrose, ammonium sulphate, yeast extract, K2HPO4, NaCl and MgSO4. Maximum pullulan production (4.8%, w/v) was obtained at an agitation rate of 300 rpm and aeration of 0.75 vvm in a stirred tank reactor (1 L) with a fermentation time of 6 days. Fourier transformed infrared and NMR spectrum of crude pullulan from A. pullulans FB-1 revealed solely α-(1→6) linked maltosyl units, in accord with the generally accepted structure of pullulan. Hydrolysis of crude pullulan, i.e. digestion with pullulanase also confirmed the identity as well as homogeneity of the produced pullulan. XRD analysis of crude pullulan verified its amorphous nature. Thermal analysis showed melting range of crude pullulan from 245.99–295.71 °C.

Bacterial cellulose (BC11 Uncommon abbreviation used: BC – Bacterial Cellulose) is a biomaterial produced by various strains of microorganisms. BC has improved strength and unique structural properties as compared to plant cellulose, thus has many usages in the food and pharmaceutical industries. In our previous study, a novel co-culture agitated fermentation of Komagataeibacter hansenii, a BC producer, with Aureobasidium pullulans, a producer of pullulan polysaccharide, had been demonstrated where the BC produced exhibited improved mechanical properties. Therefore, this study is undertaken to analyze BC production under different medium composition using response surface methodology (RSM) in shake-flasks and benchtop bioreactors. A verified local high point provided 22.4% higher BC production and 4.5- to 6- folds higher elastic moduli in shake-flasks and bioreactors compared to the baseline media. Overall, the study had revealed the potential of the co-culturing method to enhance BC production while maintaining the desired mechanical properties of BC produced in shake-flasks and larger scale bioreactors.

Objective In this pilot clinical study, we report the beneficial effects of beta glucans derived from two strains AFO-202 and N-163 of a black yeast Aureobasidium pullulans on the biomarkers for cytokine storm and coagulopathy in COVID-19 patients. Methods A total of 24 RT-PCR positive COVID-19 patients were recruited and randomly divided into three groups (Gr): Gr. 1 control (n=8) – Standard treatment; Gr. 2: Standard treatment + AFO-202 beta glucan (n=8); and Gr. 3, Standard treatment + combination of AFO-202 and N-163 beta glucans (n=8) for 30 days. Results There was no mortality or requirement of ventilation of the subjects in any of the groups. There was a decrease in D-Dimer values (751 ng/ml to 143.89 ng/ml) and IL-6 values (7.395 pg/ml to 3.16 pg/ml) in Gr. 1 in 15 days but the levels increased to abnormal levels on day 30 (D-Dimer: 202.5 ng/ml; IL-6 55.37 pg/ml); which steadily decreased up to day 30 in groups 2 (D-dimer: 560.99 ng/dl to 79.615; IL-6: 26.18 pg/ml to 3.41 pg/ml) and 3 (D-dimer: 1614 ng/dl to 164.25 ng/dl; IL-6: 6.25 pg/ml to 0.5 pg/ml). The same trend was observed with ESR. LCR and LeCR increased while NLR decreased significantly in Gr. 3. CD4+ and CD8+ T cell count showed relatively higher increase in Gr.3. There was no difference in CRP within the groups. Conclusion As these beta glucans are well known food supplements with a track record for safety, larger multi-centric clinical studies are recommended to validate their use as an adjunct in the management of COVID-19 and the ensuing long COVID-19 syndrome., Graphical Abstract

Salterns are hypersaline extreme environments with unique physicochemical properties such as a salinity gradient. Although the investigation of microbiota in salterns has focused on archaea and bacteria, diverse fungi also thrive in the brine and soil of salterns. Fungi isolated from salterns are represented by black yeasts (Hortaea werneckii, Phaeotheca triangularis, Aureobasidium pullulans, and Trimmatostroma salinum), Cladosporium, Aspergillus, and Penicillium species. Most studies on saltern-derived fungi gave attention to black yeasts and their physiological characteristics, including growth under various culture conditions. Since then, biochemical and molecular tools have been employed to explore adaptation of these fungi to salt stress. Genome databases of several fungi in salterns are now publicly available and being used to elucidate salt tolerance mechanisms and discover the target genes for agricultural and industrial applications. Notably, the number of enzymes and novel metabolites known to be produced by diverse saltern-derived fungi has increased significantly. Therefore, fungi in salterns are not only interesting and important subjects to study fungal biodiversity and adaptive mechanisms in extreme environments, but also valuable bioresources with potential for biotechnological applications.

The production of cell-wall degrading enzymes (CWDE) such as cellulase and pectinase by Aureobasidium pullulans NAC8 through induction using orange peels was investigated for the potential ...

The fungi Aureobasidium pullulans, Mortierella humilis, Trichoderma harzianum and Phoma glomerata were used to investigate the formation of selenium- and tellurium-containing nanoparticles during growth on selenium- and tellurium-containing media. Most organisms were able to grow on both selenium- and tellurium-containing media at concentrations of 1 mM resulting in extensive precipitation of elemental selenium and tellurium on fungal surfaces as observed by the red and black colour changes. Red or black deposits were confirmed as elemental selenium and tellurium, respectively. Selenium oxide and tellurium oxide were also found after growth of Trichoderma harzianum with 1 mM selenite and tellurite as well as the formation of elemental selenium and tellurium. The hyphal matrix provided nucleation sites for metalloid deposition with extracellular protein and extracellular polymeric substances localizing the resultant Se or Te nanoparticles. These findings are relevant to remedial treatments for selenium and tellurium and to novel approaches for selenium and tellurium biorecovery.

Liamocins are biosurfactants produced by the fungus Aureobasidium pullulans. A. pullulans belongs to the black yeasts and is known for its ability to produce pullulan and melanin. However, the production of liamocins has not been investigated intensively. Initially, HPLC methods for the quantification of liamocin and the identification of liamocin congeners were established. Eleven congeners could be detected, differing in the polyol head groups arabitol or mannitol. In addition, headless molecules, so-called exophilins, were also identified. The HPLC method reported here allows quick and reliable quantification of all identified congeners, an often-overlooked prerequisite for the investigation of valuable product formation. Liamocin synthesis was optimized during cultivation in lab-scale fermenters. While the pH can be kept constant, the best strategy for liamocin synthesis consists of a growth phase at neutral pH and a subsequent production phase induced by a manual pH shift to pH 3.5. Finally, combining increased nitrogen availability with a pulsed fed-batch fermentation, cell growth, and liamocin titers could be enhanced. Here, the maximal titers of above 10 g/L that were reached are the highest reported to date for liamocin synthesis using A. pullulans in lab-scale fermenters.

Pullulan is a biopolymer used in food industry produced by Aureobasidium pullulans from starch. In the present work, sugarcane bagasse (SCB) hydrolysate was evaluated as an alternative substrate in fermentation process assisted by blue LED lights. The best fermentation conditions in Erlenmeyer flasks were 25.3 °C, stirring speed of 232 rpm and yeast extract concentration of 1.88 g/L, yielding 25.19 g/L of pullulan, that corresponded to yield of 0.48 g/g and 0.28 g/(L·h) of volumetric productivity. By using a column bubble photobioreactor, similar yield values were obtained. Thermal properties of the produced pullulan as glass transition (Tg) and melting (Tm) temperatures were 38 °C and 160 °C, respectively, which were similar to the corresponding values of commercial food grade pullulan. Therefore, SCB hydrolysate is a promising substrate to produce good quality pullulan (86% of purity) with application in food industry, besides to represent a new alternative for biorefineries.

Aureobasidium pullulans and A. melanogenum are black-yeast-like surface colonisers and are commonly encountered as contaminants in the hospital. The species are able to produce melanin which play a role in protection against environmental stress and irradiation. Aureobasidium melanogenum shows higher frequency in opportunistic infections compared to A. pullulans. Comparative pathogenicity of opportunistic black yeasts between Aureobasidium pullulans and A. melanogenum to explain the observed differences in frequency in infection. Degrees of melanisation and thermotolerance were measured, and virulence of strains from different sources was examined in Galleria mellonela and murine infection models. Aureobasidium melanogenum responds with increased melanisation to temperature stress and generally survives at 37°C, A. pullulans on average scored less on these parameters. In the murine model, differences between species were not significant, but the melanised A. melanogenum group showed the highest virulence. This result was not reproducible in Galleria mellonella larvae at 25°C. The A. melanogenum black group showed higher pathogenicity in murine model, indicating that the combination of melanisation and thermotolerance rather than species affiliation is instrumental. Galleria larvae did not survive very well at 37°C, and hence, this model is judged insufficient to detect the small virulence differences observed in Aureobasidium.

Cheese whey is an abundant and low-cost feedstock with lactose as its main component, but the inability to metabolize lactose prevents Aureobasidium pullulans from using cheese whey directly. In this study, Kluyveromyces marxianus was permeabilized to obtain nonviable but biocatalytic cells for lactose hydrolysis, and the mixed culture of A. pullulans and permeabilized K. marxianus was conducted for polymalic acid (PMA) production from cheese whey. In the mixed culture, PMA titer varied directly to β-galactosidase activity of K. marxianus, but inversely to cell viability of K. marxianus, and ethanol permeabilized K. marxianus was the most compatible with A. pullulans for PMA production. 37.8 g/L PMA was produced in batch fermentation, and PMA titer was increased to 97.3 g/L in fed-batch fermentation, with a productivity of 0.51 g/(L·h) and a yield of 0.56 g/g. This study paved an economical and environmentally friendly way for PMA production from cheese whey.

Abstract Fungi cause diverse, serious socio-economic problems, including biodeterioration of valuable products and materials that spawns a biocides industry worth ~$11 billion globally. To help combat environmental fungi that commonly colonise material products, this study tested the hypothesis that combination of an approved fungicide with diverse agents approved by the FDA (Food and Drug Administration) could reveal potent combinatorial activities with promise for fungicidal applications. The strategy to use approved compounds lowers potential development risks for any effective combinations. A high-throughput assay of 1280 FDA-approved compounds was conducted to find those that potentiate the effect of iodopropynyl-butyl-carbamate (IPBC) on the growth of Trichoderma virens; IPBC is one of the two most widely used Biocidal Products Regulations–approved fungicides. From this library, 34 compounds in combination with IPBC strongly inhibited fungal growth. Low-cost compounds that gave the most effective growth inhibition were tested against other environmental fungi that are standard biomarkers for resistance of synthetic materials to fungal colonisation. Trifluoperazine (TFZ) in combination with IPBC enhanced growth inhibition of three of the five test fungi. The antifungal hexetidine (HEX) potentiated IPBC action against two of the test organisms. Testable hypotheses on the mechanisms of these combinatorial actions are discussed. Neither IPBC + TFZ nor IPBC + HEX exhibited a combinatorial effect against mammalian cells. These combinations retained strong fungal growth inhibition properties after incorporation to a polymer matrix (alginate) with potential for fungicide delivery. The study reveals the potential of such approved compounds for novel combinatorial applications in the control of fungal environmental opportunists. Key points • Search with an approved fungicide to find new fungicidal synergies in drug libraries. • New combinations inhibit growth of key environmental fungi on different matrices. • The approach enables a more rapid response to demand for new biocides.

Made available in DSpace on 2021-06-25T11:46:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-29 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The objectives of the present study were to purify and assess the killer toxin effect produced by Aureobasidium pullulans under casual agents of green mold (Penicillum digitatum) and sour rot (Geotrichum citri-aurantii). Initially, different methods of protein precipitation were tested. The proteolytic activity and the presence of proteins acting on cell wall receptors, beta-1,3-glucanase and chitinase were determined, and toxin purification was conducted by Sephadex G-75 gel exclusion chromatography and cellulose chromatography (medium fibers). Subsequently, purification was confirmed by polyacrylamide gel electrophoresis, and the detection of killer activity was performed in solid YEPD-methylene blue buffered with citrate-phosphate (0.1 M, pH 4.6). Toxin identification was performed by liquid chromatography-mass spectrometry. The results showed that the best protein precipitation method was 2:1 ethanol (vol/vol ethanol/supernatant). It was possible to observe the presence of enzymes with proteolytic activity, including beta-1,3-glucanase and chitinase. During the purification process, it was verified that the killer toxin produced by the yeast has a low-molecular-weight protein belonging to the ubiquitin family, which presents killer activity against P. digitatum and G. citri-aurantii. Julio de Mesquita Filho Paulista State Univ, Grad Agr Microbiol Program, Agr & Veterinarian Fac, Sao Paulo, Brazil Univ Ctr Herminio Ometto Fdn FHO, Grad Program Biomed Sci, Sao Paulo, Brazil Sylvio Moreira Citriculture Ctr IAC, Rodovia Anhanguera,Km 158,CP 04, BR-13470970 Cordeiropolis, SP, Brazil Julio de Mesquita Filho Paulista State Univ, Grad Agr Microbiol Program, Agr & Veterinarian Fac, Sao Paulo, Brazil FAPESP: 2014/25067-3

The coproduction of polymalic acid (PMA) and liamocins, two important metabolites secreted by Aureobasidium pullulans, from two waste by-products from the xylitol and gluconate industries was investigated in shake flasks and fermentors, confirming that waste xylose mother liquor (WXML) could be utilized as an economical feedstock without any pretreatment. Gluconate could strengthen carbon flux and NADPH supply for the synergetic biosynthesis of PMA and liamocins. High PMA and liamocin titers of 82.9 ± 2.1 and 28.3 ± 2.7 g/L, respectively, were obtained from the coupled WXML and waste gluconate mother liquor (WGML) in batch fermentation, with yields of 0.84 and 0.25 g/g, respectively. These results are comparable to those obtained from renewable feedstocks. Economic assessment of the process revealed that PMA and liamocins could be coproduced from two by-products at costs of $1.48/kg or $0.67/kg (with liamocins credit), offering an economic and sustainable process for the application of waste by-products.

Organotin compounds are applied in several industrial reactions and can present antifungal and antibacterial activities. Incorrect handling and storage practices of biodiesel and diesel–biodiesel blends can lead to microbial development, impacting its final quality. Concerning this problem, this work investigated the antimicrobial action of two organotin catalysts used in biodiesel production with four isolated microroorganisms (Bacillus pumilus, Pseudomonas aeruginosa, Pseudallescheria boydii, and Aureobasidium pullulans) and a pool of microorganisms (ASTM E1259 standard practice). Samples of soybean biodiesel with different concentrations of dibutyl tin dilaurate (catalyst 1) and di-n-butyl-oxo-stannane (catalyst 2) were prepared and added of mineral medium. The pool of microorganisms was inoculated and incubated at 30 °C and final biomass was weighted after 14 days. Thereafter, soybean biodiesel with catalyst 2 was used. Fungal biomass was weighted, and plate count was used to assess bacterial growth. Results show that catalysts 1 and 2 presented no inhibitory activity on the pool of microorganisms evaluated. A slight inhibitory activity was observed for B. pumilus and A. pullulans growth, but not for P. boydii, P. aeruginosa, or the pool of microorganisms. All experiment exhibited acidification higher than sterile control. Infrared analysis show less microbiological degradation products in the tin-protected fuel with ASTM inoculum. These results suggest that these tin-based catalysts show no toxic effect on native microbial population and a slight effect on some isolated microbial population in laboratory scale and for the first time shows that these organotin compounds can be employed safely as biodiesel catalyst.

Sesame seed oil cake (SSOC) was applied as a novel substrate for production of high-added value pullulan by Aureobasidium pullulans KY767024 (a melanin-deficient strain). The optimization of production conditions by central composite design showed that the maximum production yield (54.50 g pullulan/kg substrate) was obtained under optimum conditions (Liquid-solid ratio (LSR) of 1.7 v/w, inoculum size of 7 mL, and temperature of 23 °C), which was confirmed by the experimental data (52.50 ± 0.73 g/kg). The obtained pullulan had an average-molecular weight of 358.736 kDa. The present peaks in the FT-IR spectrum validated that the obtained purified precipitate was composed of pullulan. Also, the XRD pattern and TGA analysis showed that the produced pullulan had a fully amorphous structure with high thermal stability. In addition, the pullulan solutions had a different flow behavior in different concentrations and with an increase in concentration, their behavior changed from Newtonian to pseudoplastic.

Polymalic acid (PMA), a homopolymer of L-malic acid (MA) generated from a yeast-like fungus Aureobasidium pullulans, has unique properties and many applications in food, biomedical, and environmental fields. Acid hydrolysis of PMA, releasing the monomer MA, has become a novel process for the production of bio-based MA, which currently is produced by chemical synthesis using petroleum-derived feedstocks. Recently, current researches attempted to develop economically competitive process for PMA and MA production from renewable biomass feedstocks. Compared to lignocellulosic biomass, PMA and MA production from low-value food processing wastes or by-products, generated from corn, sugarcane, or soybean refinery industries, showed more economical and sustainable for developing a MA derivatives platform from biomass biorefinery to chemical conversion. In the review, we compared the process feasibility for PMA fermentation with lignocellulosic biomass and food process wastes. Some useful strategies for metabolic engineering are summarized. Its changeable applicability and future prospects in food and biomedical fields are also discussed.

Aureobasidium pullulans is a yeast-like fungus that produces volatile organic compounds (VOCs) with antifungal properties. VOCs have the potential to trigger the production of intracellular reactive oxygen species (ROS), lipid peroxidation and electrolyte loss in microorganisms. The relationship among A. pullulans VOCs, induced ROS accumulation and electrolyte leakage was investigated in Botrytis cinerea and Alternaria alternata in vitro. Exposure to a mixture of A. pullulans VOCs: ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol, resulted in electrolyte leakage in both B. cinerea and A. alternata. Fluorescence microscopy using 2′,7′-dichlorofluorescein diacetate indicated triggered ROS accumulation in exposed fungal mycelia and the presence of the superoxide radical was evident by intense red fluorescence with dihydroethidium. Partial inhibition of enzymes of the mitochondrial respiratory chain complex I of B. cinerea and A. alternata by pre-treatment with rotenone reduced ROS accumulation in hypha exposed to A. pullulans VOCs and reversed the VOCs inhibition of fungal growth. Scanning electron micrographs revealed that B. cinerea and A. alternata hypha exposed to A. pullulans VOCs had altered cell wall structures. Our findings give insights into the potential mechanisms involved in the antifungal properties of A. pullulans in the suppression of B. cinerea and A. alternata growth in vitro.

The content of pullulan and melanin in 500 mutants of Aureobasidum pullulans obtained by ultraviolet mutagenesis were examined and statistically analyzed, and a strong positive correlation was found between them. The result was further confirmed by culturing wild type strain As3.3984 in different media. Then we constructed melanin-deletion mutant As-Δalb1 and pullulan-deletion mutant As-Δpul. As-Δalb1 was a melanin-free strain with the yield of pullulan decreased by 41.01%. The supplementation of melanin in the culture of As-Δalb1 increased the production of pullulan. As-Δpul synthesized neither pullulan nor melanin and recovered melanin synthesis by adding pullulan to the medium. The results suggested that high concentration- of pullulan induced morphological transformation and synthesis of melanin, and melanin promoted the synthesis of pullulan. The pullulan biosynthetic genes, upt, pgm, ugp, and pul, were down-regulated, while the negative regulatory gene of pullulan synthesis, creA, was up-regulated by melanin deficiency.

The effects of several surfactants on the biosynthesis of β-1,3-D-glucan (β-glucan) and pullulan by Aureobasidium pullulans CCTCC M 2012259 were investigated, and Triton X-100 was found to decrease biomass formation but increase β-glucan and pullulan production. The addition of 5 g/L Triton X-100 to the fermentation medium and bioconversion broth significantly increased β-glucan production by 76.6% and 69.9%, respectively, when compared to the control without surfactant addition. To reveal the physiological mechanism underlying the effect of Triton X-100 on polysaccharides production, the cell morphology and viability, membrane permeability, key enzyme activities, and intracellular levels of UDPG, NADH, and ATP were determined. The results indicated that Triton X-100 increased the activities of key enzymes involved in β-glucan and pullulan biosynthesis, improved intracellular UDPG and energy supply, and accelerated the transportation rate of precursors across the cell membrane, all of which contributed to the enhanced production of β-glucan and pullulan. Moreover, a two-stage culture strategy with combined processes of batch fermentation and bioconversion was applied, and co-production of β-glucan and pullulan in the presence of 5 g/L Triton X-100 additions was further improved. The present study not only provides insights into the effect of surfactant on β-glucan and pullulan production but also presents a feasible approach for efficient production of analogue exopolysaccharides. KEY POINTS: • Triton X-100 increased β-glucan and pullulan production under either batch fermentation or bioconversion. • Triton X-100 increased the permeability of cell membrane and accelerated the transportation rate of precursors across cell membrane. • Activities of key enzymes involved in β-glucan and pullulan biosynthesis were increased in the presence of Triton X-100. • Intracellular UDPG levels and energy supply were improved by Triton X-100 addition.

Aureobasidium pullulans is the most abundant and ubiquitous species within the genus and is also considered a core component of the grape juice microflora. So far, a small number of other Aureobasidium species have been reported, that in contrast to A. pullulans, appear far more constrained to specific habitats. It is unknown whether grape juice is a reservoir of novel Aureobasidium species, overlooked in the course of conventional morphological and meta-barcoding analyses. In this study, eight isolates from grape juice taxonomically classified as Aureobasidium through ITS sequencing were subjected to whole-genome phylogenetic, synteny and nucleotide identity analyses, which revealed three isolates to likely represent newly discovered Aureobasidium species. Analyses of ITS and metagenomic sequencing datasets show that these species can be present in grape juice samples from different locations and vintages. Functional annotation revealed the Aureobasidium isolates possess the genetic potential to support growth on the surface of plants and grapes. However, the loss of several genes associated with tolerance to diverse environmental stresses suggest a more constrained ecological range than A. pullulans.

Abstract Aureobasidium pullulans is a ubiquitous and widely distributed fungus in the environment, and exhibits substantial tolerance against toxic metals. However, the interactions between metals and metalloids with the copious extracellular polymeric substances (EPS) produced by A. pullulans and possible relationships to tolerance are not well understood. In this study, it was found that mercury (Hg) and selenium (Se), as selenite, not only significantly inhibited growth of A. pullulans but also affected the composition of produced EPS. Lead (Pb) showed little influence on EPS yield or composition. The interactions of EPS from A. pullulans with the tested metals and metalloids depended on the specific element and their concentration. Fluorescence intensity measurements of the EPS showed that the presence of metal(loid)s stimulated the production of extracellular tryptophan-like and aromatic protein-like substances. Examination of fluorescence quenching and calculation of binding constants revealed that the fluorescence quenching process for Hg; arsenic (As), as arsenite; and Pb to EPS were mainly governed by static quenching which resulted in the formation of a stable non-fluorescent complexes between the EPS and metal(loid)s. Se showed no significant interaction with the EPS according to fluorescence quenching. These results provide further understanding of the interactions between metals and metalloids and EPS produced by fungi and their contribution to metal(loid) tolerance. Key points • Metal(loid)s enhanced production of tryptophan- and aromatic protein-like substances. • Non-fluorescent complexes formed between the EPS and tested metal(loid)s. • EPS complexation and binding of metal(loid)s was dependent on the tested element. • Metal(loid)-induced changes in EPS composition contributed to metal(loid) tolerance.

In this study, a new strain suspected to be Aureobasidium pullulans was isolated from trees leaves. The molecular characterization and the resulting phylogenetic tree showed that the isolated strain was A. pullulans. Also, the results of methylation analysis, monosaccharide composition, FTIR, NMR and XRD confirmed that the obtained exo-polysaccharide from the mentioned strain was pullulan. The pullulan production optimization by central composite design (CCD) indicated that the maximum yield obtained under optimum conditions (pH of 6.5, sucrose concentration of 5.5% (w/v) and yeast extract concentration of 0.1% (w/v)) was 51.4 ± 0.50 g/L. The produced pullulan had an average molecular weight (Mw) of 2.07 × 105 g.mol−1 based on gel permeation chromatography results. The decomposition temperature (Td) of the produced pullulan was ~300 °C and also, the resulting pullulan had a Newtonian flow behavior in a wide range of concentrations.

Made available in DSpace on 2022-04-29T08:45:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Introduction. Some studies have reported the occurrence of microorganisms isolated from water. Considering these microorganisms, fungi are known to occur ubiquitously in the environment, including water, and some are pathogenic and may cause health problems, especially in immunocompromised individuals. The aim of this study was to identify fungi in hospital water samples and to correlate their presence with the concentration of free residual chlorine. Methods. Water samples (100 mL) were collected from taps (n = 74) and water purifiers (n = 14) in different locations in a university hospital. Samples were filtered through a nitrocellulose membrane and placed on Sabouraud dextrose agar and incubated for 24 hours at 30°C. Fungi were identified according to established methods based on macroscopic and microscopic characteristics (filamentous) and physiological tests (yeasts). Free chlorine residual content was measured at the time of sample collection. Results. Seventy species of fungi were identified in the water samples and about 56% of the water samples contained culturable fungi. Cladosporium oxysporum, Penicillium spinulosum, and Aspergillus fumigatus were the most common filamentous fungi. Aureobasidium pullulans and Candida parapsilosis were the most common yeasts. Chemical analyses revealed that free residual chlorine was present in 81.8% of the samples within recommended concentrations. Among samples from water purifiers, 92.9% showed low levels of free residual chlorine (

Liamocins are a class of biosurfactants with growing interest. However, methods for identification and quantification of liamocins on the molecular level are lagging behind. Therefore, we developed a chromatographic separation based on supercritical fluid chromatography (SFC) for liamocins and structurally related exophilins. The different congeners could be separated on a charge modulated hydroxyethyl amide functionalized silica-based column. Coupling to high-resolution mass spectrometry (MS) revealed four exophilin species and four liamocin species with mannitol and arabitol as polyol head group in a sample of the yeast-like fungus Aureobasidium pullulans (A. pullulans). In contrast to a recently published reversed phase high-performance liquid chromatography (HPLC) method, the different subclasses (exophilins, mannitol liamocins and arabitol liamocins) were additionally separated by means of SFC. The structures were confirmed by their accurate masses and tandem mass spectrometry (MS/MS). A complementary quantification method was developed using SFC coupled to charged-aerosol detection (CAD) to overcome the disadvantages of quantification by means of MS without authentic standards. A flow compensation by varying the make-up flow was used to obtain a constant composition of the mobile phase during detection and to ensure a stable detector response. The concentrations of the individual liamocin species were determined using an external calibration with n-octyl-β-d-glycopyranoside. The total amount of these concentrations agrees with the dry weight of an aliquot of the heavy oil. The developed SFC-based method has the advantage of shorter analysis time and superior selectivity compared to the previously published LC separation. In brief, the here presented SFC hyphenations enable comprehensive analysis of liamocin biosurfactants providing identification and absolute quantification of individual congeners.

Gluconic acid (GA) has many applications such as in the food and pharmaceutical industry. Aureobasidium pullulans P25 strain is able to produce high levels of Ca2+-GA. The genome length, GC content and the gene number of this yeast were found to be 30.97 Mb, 50.28% and 10,922, respectively. The pathways for gluconic acid biosynthesis were annotated. Glucose oxidase (Gox) sequences from different strains of A. pullulans were highly similar but were distinct from those of other fungi. The glucose oxidase had two FAD binding sites and a signal sequence. Deletion of the GOX gene resulted in a strain that showed no Gox activity and that was unable to produce Ca2+-GA. Overexpression of the GOX gene in strain P25 generated strain GA-6 that produced 200.2 ± 2.3 Ca2+-GA g/l and 2480 U/mg of Gox activity. The productivity of Ca2+-GA was 2.78 g/l/h and the yield was 1.1 g/g.

Background Extracellular polymeric substances isolated from Aureobasidium pullulans (EAP), containing specifically 13% β-1,3/1,6-glucan, have shown various favorable bone-preserving effects. Textoria morbifera Nakai (TM) tree has been used as an ingredient in traditional medicine and tea for various pharmacological purposes. Thus, the present study was aimed to examine the synergistic anti-osteoporotic potential of mixtures containing different proportions of EAP and TM compared with that of the single formulations of each herbal extract using bilateral ovariectomized (OVX) mice, a renowned rodent model for studying human osteoporosis. Methods Thirty five days after bilateral-OVX surgery, 9 combinations of EAP:TM (ratios = 1:1, 1:3, 1:5, 1:7, 1:9, 3:1, 5:1, 7:1, 9:1) and single separate formulations of EAP or TM were supplied orally, once a day for 35 days at a final concentration of 200 mg/kg. Variations in body weight gains during the experimental periods, as well as femur weights, bone mineral density (BMD), bone strength (failure load), and mineral content (calcium [Ca] and inorganic phosphorus [IP]) following sacrifice were measured. Furthermore, histomorphometric and histological profile analyses of serum biochemical parameters (osteocalcin content and bone specific alkaline phosphatase [bALP] activity) were conducted following sacrifice. Femurs histomorphometric analyses were also conducted for bone resorption, structure and mass. The results for the mixed formulations of EAP:TM and separate formulations were compared with those of risedronate sodium (RES). Results The EAP:TM (3:1) formulation synergistically enhanced the anti-osteoporotic potential of individual EAP or TM formulations, possibly due to enhanced variety of the active ingredients. Furthermore, the effects of EAP:TM were comparable to those of RES (2.5 mg/kg) treatment. Conclusion The results of this study suggest that, the EAP:TM (3:1) combination might act as a new pharmaceutical agent and/or health functional food substance for curing osteoporosis in menopausal women. Electronic supplementary material The online version of this article (10.1186/s12906-018-2362-y) contains supplementary material, which is available to authorized users.

Microbial oils can be used for biodiesel production and fumaric acid (FA) is widely used in the food and chemical industries. In this study, the production of lipids and FA by Aureobasidium pullulans var. aubasidani DH177 was investigated. A high initial carbon/nitrogen ratio in the medium promoted the accumulation of lipids and FA. When the medium contained 12.0% glucose and 0.2% NH4NO3, the yeast strain DH177 accumulated 64.7% (w/w) oil in its cells, 22.4 g/l cell biomass and 32.3 g/l FA in a 5-L batch fermentation. The maximum yields of oil and FA were 0.12 g/g and 0.27 g/g of consumed sugar, respectively. The compositions of the produced fatty acids were C14:0 (0.6%), C16:0 (24.9%), C16:1 (4.4%), C18:0 (2.1%), C18:1 (57.6%), and C18:2 (10.2%). Biodiesel obtained from the extracted oil burned well. This study provides the pioneering utilization of the yeast strain DH177 for the integrated production of oil and FA.

Polymalic acid (PMA) is a linear anionic polyester composed of l-malic acid monomers, which have potential applications as drug carriers, surgical suture, and biodegradable plastics. In this study, a novel strain of Aureobasidium pullulans var. melanogenum GXZ-6 was isolated and identified according to the morphological observation and deoxyribonucleic acid internal-transcribed spacer sequence analysis, and the product of PMA was characterized by FT-IR, 13C-NMR, and 1H-NMR spectra. The PMA titer of GXZ-6 reached 62.56 ± 1.18 g L−1 with productivity of 0.35 g L−1 h−1 using optimized medium with addition of metabolic intermediates (citrate and malate) and inhibitor (malonate) by batch fermentation in a 10-L fermentor. Besides that the malate for PMA synthesis in GXZ-6 might mainly come from the glyoxylate cycle, based on results, citrate, malate, malonate, and maleate increased while succinate and fumarate inhibited the production of PMA, which was different from that of other A. pullulans. This study provided a potential strain and a simple metabolic control strategy for high-titer production of PMA and shared novel information on the biosynthesis pathway of PMA in A. pullulans.

Lactobacillus pentosus K1-23 was selected from among 25 lactic acid bacterial strains owing to its high inhibitory activity against several pathogenic bacteria, including Escherichia coli, Salmonella typhimurium, S. gallinarum, Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium perfringens, and Listeria monocytogenes. Additionally, among 13 strains of Aureobasidium spp., A. pullulans NRRL 58012 was shown to produce the highest amount of β-glucan (15.45 ± 0.07%) and was selected. Next, the optimal conditions for a solid-phase mixed culture with these two different microorganisms (one bacterium and one yeast) were determined. The optimal inoculum sizes for L. pentosus and A. pullulans were 1% and 5%, respectively. The appropriate inoculation time for L. pentosus K1-23 was 3 days after the inoculation of A. pullulans to initiate fermentation. The addition of 0.5% corn steep powder and 0.1% FeSO₄ to the basal medium resulted in the increased production of lactic acid bacterial cells and β-glucan. The following optimal conditions for solid-phase mixed culture were also statistically determined by using the response surface method: 37.84°C, pH 5.25, moisture content of 60.82%, and culture time of 6.08 days for L. pentosus; and 24.11°C, pH 5.65, moisture content of 60.08%, and culture time of 5.71 days for A. pullulans. Using the predicted optimal conditions, the experimental production values of L. pentosus cells and β-glucan were 3.15 ± 0.10 × 10⁸ CFU/g and 13.41 ± 0.04%, respectively. This mixed culture may function as a highly efficient antibiotic substitute based on the combined action of its anti-pathogenic bacterial and immune-enhancing activities.

Killer toxin resistant 6 (Kre6) and its paralog, suppressor of Kre null 1 (Skn1), are thought to be involved in the biosynthesis of cell wall β-(1 → 6)-D-glucan in baker's yeast, Saccharomyces cerevisiae. The Δkre6Δskn1 mutant of S. cerevisiae and other fungi shows severe growth defects due to the failure to synthesize normal cell walls. In this study, two homologs of Kre6, namely, K6LP1 (Kre6-like protein 1) and K6LP2 (Kre6-like protein 2), were identified in Aureobasidium pullulans M-2 by draft genome analysis. The Δk6lp1, Δk6lp2, and Δk6lp1Δk6lp2 mutants were generated in order to confirm the functions of the Kre6-like proteins in A. pullulans M-2. The cell morphologies of Δk6lp1 and Δk6lp1Δk6lp2 appeared to be different from those of wild type and Δk6lp2 in both their yeast and hyphal forms. The productivity of the extracellular polysaccharides, mainly composed of β-(1 → 3),(1 → 6)-D-glucan (β-glucan), of the mutants was 5.1-17.3% less than that of wild type, and the degree of branching in the extracellular β-glucan of mutants was 14.5-16.8% lower than that of wild type. This study showed that the gene disruption of Kre6-like proteins affected the cell morphology, the productivity of extracellular polysaccharides, and the structure of extracellular β-glucan, but it did not have a definite effect on the cell viability even in Δk6lp1Δk6lp2, unlike in the Δkre6Δskn1 of S. cerevisiae.

Here, we report an efficient endoglucanase from Aureobasidium pullulans (termed ApCel5A) was expressed in Pichia pastoris. ApCel5A shows two different enzyme activities of endoglucanase (1270 U/mg) and mannanase (31.2 U/mg). Through engineering the signal peptide and fed-batch fermentation, the enzyme activity of endoglucanase was improved to 6.63-folds, totally. Its efficient synergism with Celluclast 1.5 L, excellent tolerance to low pH (2.5), cholate and protease suggests potential application in bioresources, food and feed industries. Site-directed mutagenesis experiments present that ApCel5A residues Glu245 and Glu358 are key catalytic sites, while Asp118, Asp122, Asp198 and Asp314 play an auxiliary role. More importantly, ApCel5A display high degradation efficiency of glucan and glucomannan substrates by using tetrasaccharide contained reducing end of glucose residue as an intermediate. This study elucidated the effective methods to improve an endoglucanase expression and detailed catalytic mechanism for degradation of various substrates, which provides a new insight for endoglucanase application.

Poly(β-l-malic acid) (PMA) production by Aureobasidium pullulans HA-4D was carried out through solid-state fermentation (SSF) using agro-industrial residues. Maximum PMA production (75.4mg/g substrate) was obtained from a mixed substrate of sweet potato residue and wheat bran (1:1, w/w) supplemented with NaNO3 (0.8%, w/w) and CaCO3 (2%, w/w), with an initial moisture content of 70% and inoculum size of 13% (v/w) for 8days. Repeated-batch SSF was successfully conducted for 5 cycles with a high productivity. The scanning electron microscopy showed that the yeast-like cells of A. pullulans HA-4D could grow well on the solid substrate surface. Moreover, the cost analysis showed that the unit price of PMA in SSF was much lower than that of SmF. This is the first report on PMA production via SSF, and this study provided a new method to produce PMA from inexpensive agro-industrial residues.

Surfactants are amphiphilic molecules with large industrial applications produced currently by chemical routes mainly derived from oil industry. However, biotechnological process, aimed to develop new sustainable process configurations by using favorable microorganisms, already requires investigations in more details. Thus, we present a novel approach for biosurfactant production using the promising yeast Aureobasidium pullulans LB 83, in stirred tank reactor. A central composite face-centered design was carried out to evaluate the effect of the aeration rate (0.1-1.1min-1) and sucrose concentration (20-80g.L-1) in the biosurfactant maximum tensoactivity and productivity. Statistical analysis showed that the use of variables at high levels enhanced tensoactivity, showing 8.05cm in the oil spread test and productivity of 0.0838cm.h-1. Also, unprecedented investigation of aeration rate and sucrose concentration relevance in biosurfactant production by A. pullulans in stirred tank reactor was detailed, demonstrating the importance to establish adequate conditions in bioreactors, aimed to scale-up process.

Different strains of the saprophytic yeast-like fungus Aureobasidium pullulans (Ascomycota: Dothideales) exhibit different biochemical characteristics, while their ubiquitous occurrence across diverse habitats and environmental conditions makes them an easily accessible source for biotechnological exploitation. They are useful in agricultural and industrial applications. Their antagonistic activities against postharvest pathogens make them suitable bioagents for the postharvest preservation of fruits and vegetables, while they possess antimicrobial activities against bacteria and fungi. Additionally, A. pullulans appears to be a potent source of single-cell protein. Many strains of A. pullulans harbor a wide range of industrially important enzymes, while the trademark exopolysaccharide pullulan that they produce has been extensively studied and is currently used in many applications. They also produce poly (β-L-malic acid), heavy oil liamocins, siderophore, and aubasidan-like β-glucan which are of interest for future applications. Ongoing studies suggest that A. pullulans holds many more interesting properties capable of further potential biotechnological applications.

To test the inactivation of the antibiotic, virginiamycin, by laccase-induced culture supernatants of Aureobasidium pullulans. Fourteen strains of A. pullulans from phylogenetic clade 7 were tested for laccase production. Three laccase-producing strains from this group and three previously identified strains from clade 5 were compared for inactivation of virginiamycin. Laccase-induced culture supernatants from clade 7 strains were more effective at inactivation of virginiamycin, particularly at 50 °C. Clade 7 strain NRRL Y-2567 inactivated 6 µg virginiamycin/ml within 24 h. HPLC analyses indicated that virginiamycin was degraded by A. pullulans. A. pullulans has the potential for the bioremediation of virginiamycin-contaminated materials, such as distiller’s dry grains with solubles (DDGS) animal feed produced from corn-based fuel ethanol production.

The amylase from Aureobasidium pullulans Cau 19 was purified by ammonium sulfate precipitation and Sephadex G-100 chromatography with a 9.25-fold increase in specific activity as compared to crude enzyme. Km and turn over values of the enzyme were 6.25 mg/mL and 5.0 × 102/min, respectively. Effect of different metal ions on the purified enzyme was investigated; 1 mM calcium (Ca) and cobalt (Co) enhanced enzyme activity by twofold; copper (Cu) had no effect on the activity of the enzyme. Mercury (Hg) 1 mM caused 90% inactivation whereas iron (Fe) and manganese (Mn) caused 10 to 16% inhibition. Amylase from A. pullulans Cau 19 was bioconjugated to gold nanoparticles synthesized using the biomass of A. pullulans Cau 19. Fourier transform infrared spectroscopy confirmed the conjugation of the enzyme to the gold nanoparticles. Though, only 20% of the added enzyme was adsorbed/conjugated on gold nanoparticles, 80% of the adsorbed activity could be estimated in the assay. The conjugated enzyme exhibited better tolerance to a broad pH range of 3.0–9.0 and higher temperatures compared with native enzyme.

Aims The objective of this study was to determine the optimal pretreatment and fungal strain to reduce glucosinolates (GLS), fiber, and residual sugars while increasing the nutritional value of canola meal. Methods and Results Submerged incubation conditions were used to evaluate four pretreatment methods (extrusion, hot water cook, dilute acid, and dilute alkali) and three fungal cultures (Aureobasidium pullulans Y-2311-1, Fusarium venenatum NRRL-26139, and Trichoderma reesei NRRL-3653) in hexane extracted (HE) and cold pressed (CP) canola meal. Conclusions The combination of extrusion pretreatment followed by incubation with T. reesei resulted in the greatest overall improvement to HE canola meal, increasing protein to 51.5%, while reducing NDF, GLS, and residual sugars to 18.6%, 17.2 μM.g−1, and 5% w.w−1, respectively. Extrusion pretreatment and incubation with F. venenatum performed the best with CP canola meal, resulting in 54.4% protein while reducing NDF, GLS, and residual sugars to 11.6%, 6.7 μM.g−1, and 3.8% w.w−1, respectively. Significance and Impact of Study The work is significant in that it provides a method of reducing glucosinolates (up to 98%) and neutral detergent fiber (up to 65%) while increasing the protein content (up to 45%) of canola meal. This novel pretreatment and submerged incubation process could be used to produce a canola product with higher nutritional value for livestock consumption. This article is protected by copyright. All rights reserved.

Aureobasidium pullulans is an increasingly attractive host for bio-production of pullulan, heavy oil, polymalic acid, and a large spectrum of extracellular enzymes. To date, genetic manipulation of A. pullulans mainly relies on time-consuming conventional restriction enzyme digestion and ligation methods. In this study, we present a one-step homologous recombination-based method for rapid genetic manipulation in A. pullulans. Overlaps measuring >40bp length and 10μg DNA segments for homologous recombination provided maximum benefits to transformation of A. pullulans. This optimized method was successfully applied to PKSIII gene (encodes polyketide synthase) knock-out and gltP gene (encodes glycolipid transfer protein) knock-in. After disruption of PKSIII gene, secretion of melanin decreased slightly. The melanin purified from disruptant showed lower reducing capacity compared with that of the parent strain, leading to a decrease in exopolysaccharide production. Knock-in of gltP gene resulted in at least 4.68-fold increase in heavy oil production depending on the carbon source used, indicating that gltP can regulate heavy oil synthesis in A. pullulans.