Julia Inés Fariña

Synergistic antifungal activity of statin–azole associations as witnessed by Saccharomyces cerevisiae - and Candida utilis -bioassays and ergosterol quantification

BACKGROUND Frequent opportunist fungal infections and the resistance to available antifungal drugs promoted the development of new alternatives for treatment, like antifungal drug combinations. AIMS This work aimed to detect the antifungal synergism between statins and azoles by means of an agar-well diffusion bioassay with Saccharomyces cerevisiae ATCC 32051 and Candida utilis Pr(1-2) as test strains. METHODS Synergistic antifungal effects were tested by simultaneously adding a sub inhibitory concentration (SIC) of statin (atorvastatin, lovastatin, pravastatin, rosuvastatin or simvastatin) plus a minimal inhibitory concentration (MIC) of azole (clotrimazole, fluconazole, itraconazole, ketoconazole or miconazole) to yeast-embedded YNB agar plates, and a positive result corresponded to a yeast growth inhibition halo higher than that produced by the MIC of the azole alone. Yeast cell ergosterol quantification by RP-HPLC was used to confirm statin-azole synergism, and ergosterol rescue bioassays were performed for evaluating statin-induced ergosterol synthesis blockage. RESULTS Growth inhibition was significantly increased when clotrimazole, fluconazole, itraconazole, ketoconazole and miconazole were combined with atorvastatin, lovastatin, rosuvastatin and simvastatin. Highest growth inhibition increments were observed on S. cerevisiae (77.5%) and C. utilis (43.2%) with a SIC of simvastatin plus a MIC of miconazole, i.e. 4 + 2.4 μg/ml or 20 + 4.8 μg/ml, respectively. Pravastatin showed almost no significant effects (0-7.6% inhibition increase). Highest interaction ratios between antifungal agents corresponded to simvastatin-miconazole combinations and were indicative of synergism. Synergism was also confirmed by the increased reduction in cellular ergosterol levels (S. cerevisiae, 40% and C. utilis, 22%). Statin-induced ergosterol synthesis blockage was corroborated by means of ergosterol rescue bioassays, pravastatin being the most easily abolished inhibition whilst rosuvastatin being the most ergosterol-refractory. CONCLUSIONS Selected statin-azole combinations might be viable alternatives for the therapeutic management of mycosis at lower administration doses or with a higher efficiency.

Structural stability of Sclerotium rolfsii ATCC 201126 β‐glucan with fermentation time: a chemical, infrared spectroscopic and enzymatic approach

Aims:  Sclerotium rolfsii ATCC 201126 exopolysaccharides (EPSs) recovered at 48 h (EPS I) and 72 h (EPS II) of fermentation, with differences in rheological parameters, hydrogel topography, salt tolerance, antisyneresis, emulsifying and suspending properties, were subjected to a polyphasic characterization in order to detect structural divergences.

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126

Aims:  Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126.

A critical assessment of a viscometric assay for measuring Saccharomycopsis fibuligera α-amylase activity on gelatinised cassava starch

A viscometric technique for measuring Saccharomycopsis fibuligera DSM-70554 α-amylase on gelatinised cassava starch aqueous solutions was assessed. The selected conditions for working over a reliable viscosity measurement range involved a starch concentration of 5% (w/v) and a shear rate of 0.168 1/s. Viscometric assay involved the determination of the slope of the decrease in viscosity with time of the starch solution consequent on enzyme addition. Thereafter, a calibration curve was constructed by plotting the slopes, expressed in arbitrary viscometric units (AVU), versus the corresponding absolute activity (in IU) of either the commercial α-amylase from Aspergillus oryzae (up to 0.1 IU) or the S. fibuligera DSM-70554 α-amylase (up to ca. 0.4 IU). The amount of enzyme expressed in absolute terms produced different liquefying activities according to the α-amylase tested, emphasising the necessity of this correlation to be carried out for the particular enzyme being measured. In this work, a linear relationship and a very good correlation factor were achieved for the calibration of both amylases. Likewise, α-amylase activities determined according to the conventional reducing sugar determination and the colorimetric assay with iodine were proportional to those viscometrically obtained, both for A. oryzae and S. fibuligera α-amylase, validating conversions between different units. The viscometric assay herein described showed to be specific and sensitive and, after its calibration, it allows to convert α-amylase measurements in absolute units thus facilitating future comparisons.

Effects of thermal, alkaline and ultrasonic treatments on scleroglucan stability and flow behavior

Aqueous solutions (0.2%, w/v) of scleroglucans from Sclerotium rolfsii ATCC 201126 from different cultivation time or purification protocol (EPS I, EPS II, EPSi) as well as a commercial scleroglucan (LSCL) exhibited different sensitivity against thermal (65, 95 and 150°C), ultrasonic (1, 5 and 10 min; 20% amplitude) or alkaline (0.01-0.2 N NaOH) treatments. Scleroglucan triple helix usually showed signs of denaturation at 150°C or with 0.2 NaOH with a pronounced decrease in apparent viscosity and loss of pseudoplastic behavior. Differences in sensitivity could be noted depending on the scleroglucan sample, which may be likely related to polysaccharide conformational features, and these latter to production and/or downstream processing conditions. Transmission electron microscopy showed scleroglucan topologies in accordance with thermal and alkaline denaturation. Size exclusion chromatography of control scleroglucans revealed elution profiles compatible with macromolecular aggregates which tended to diminish or disappear as thermal, alkali or sonication treatments progressed. Scleroglucan granule dissolution process took ∼8-14 s, according to DIC-light microscopy, and showed to be facilitated by addition of NaOH.

Microbial production of scleroglucan and downstream processing.

Synthetic petroleum-based polymers and natural plant polymers have the disadvantage of restricted sources, in addition to the non-biodegradability of the former ones. In contrast, eco-sustainable microbial polysaccharides, of low-cost and standardized production, represent an alternative to address this situation. With a strong global market, they attracted worldwide attention because of their novel and unique physico-chemical properties as well as varied industrial applications, and many of them are promptly becoming economically competitive. Scleroglucan, a β-1,3-β-1,6-glucan secreted by Sclerotium fungi, exhibits high potential for commercialization and may show different branching frequency, side-chain length, and/or molecular weight depending on the producing strain or culture conditions. Water-solubility, viscosifying ability and wide stability over temperature, pH and salinity make scleroglucan useful for different biotechnological (enhanced oil recovery, food additives, drug delivery, cosmetic and pharmaceutical products, biocompatible materials, etc.), and biomedical (immunoceutical, antitumor, etc.) applications. It can be copiously produced at bioreactor scale under standardized conditions, where a high exopolysaccharide concentration normally governs the process optimization. Operative and nutritional conditions, as well as the incidence of scleroglucan downstream processing will be discussed in this chapter. The relevance of using standardized inocula from selected strains and experiences concerning the intricate scleroglucan scaling-up will be also herein outlined.

Scleroglucan compatibility with thickeners, alcohols and polyalcohols and downstream processing implications.

Thickening capacity and compatibility of scleroglucan with commercial thickeners (corn starch, gum arabic, carboxymethylcellulose, gelatin, xanthan and pectin), glycols (ethylene glycol and polyethylene glycol), alcohols (methanol, ethanol, 1-propanol and isopropanol) and polyalcohols (sorbitol, xylitol and mannitol) was explored. Exopolysaccharides (EPSs) from Sclerotium rolfsii ATCC 201126 and a commercial scleroglucan were compared. Compatibility and synergism were evaluated taking into account rheology, pH and sensory properties of different thickener/scleroglucan mixtures in comparison with pure solutions. S. rolfsii ATCC 201126 EPSs induced or increased pseudoplastic behaviour with a better performance than commercial scleroglucan, showing compatibility and synergy particularly with corn starch, xanthan, pectin and carboxymethylcellulose. Compatibility and a slight synergistic behaviour were also observed with 30% (w/v) ethylene glycol whereas mixtures with polyethylene glycol (PEG) precipitated. Scleroglucan was compatible with polyalcohols, whilst lower alcohols led to scleroglucan precipitation at 20% (v/v) and above. PEG-based scleroglucan downstream processing was compared to the usual alcohol precipitation. Downstream processed EPSi (with isopropanol) and EPS-p (with PEG) were evaluated on their yield, purity, rheological properties and visual aspect pointing to alcohol downstream processing as the best methodology, whilst PEG recovery would be unsuitable. The highest purified EPSi attained a recovery yield of ~23%, similar to ethanol purification, with a high degree of purity (88%, w/w vs. EPS-p, 8%, w/w) and exhibited optimal rheological properties, water solubility and appearance. With a narrower molecular weight distribution (M(w), 2.66×10(6) g/mol) and a radius of gyration (R(w), 245 nm) slightly lower than ethanol-purified EPSs, isopropanol downstream processing showed to be a proper methodology for obtaining a refined-grade scleroglucan.

Removal efficiency of Cr6+ by indigenous Pichia sp. isolated from textile factory effluent.

Resistance of the indigenous strains P. jadinii M9 and P. anomala M10, to high Cr6+ concentrations and their ability to reduce chromium in culture medium was studied. The isolates were able to tolerate chromium concentrations up to 104 μg mL−1. Growth and reduction of Cr6+ were dependent on incubation temperature, agitation, Cr6+ concentration, and pH. Thus, in both studied strains the chromium removal was increased at 30°C with agitation. The optimum pH was different, with values of pH 3.0 and pH 7.0 in the case of P. anomala M10 and pH 7.0 using P. jadinii M9. Chromate reduction occurred both in intact cells (grown in culture medium) as well as in cell-free extracts. Chromate reductase activity could be related to cytosolic or membrane-associated proteins. The presence of a chromate reductase activity points out a possible role of an enzyme in Cr6+ reduction.

Editorial: Microbial Exopolysaccharides: From Genes to Applications.

The Research topic “Microbial exopolysaccharides from genes to applications” covers 12 articles dealing with the highly diverse class of microbial exopolysaccharides (EPSs). Many bacteria, archaea, yeast, and filamentous fungi are able to produce EPSs under different conditions. These biopolymers significantly differ in monomer composition, substituent decoration, degree and type of branching as well as molecular weight. Therefore, both chemical diversity and functionality of biopolymers is enormous. Their natural roles range from adhesives, to storage compounds, protective hulls, as well as pathogenicity factors. The complete field of putative natural applications is not fully understood up to now. A similar situation is observed for the different biosynthetic pathways. Only minimal information is available for EPS biosynthesis in fungi and similarly, little is known for cyanobacterial and archaeal polysaccharide synthesis routes. In addition to these challenges from the biological point of view, the current methods for polysaccharide analysis are still limited due to many different constraints, which in conjunction make exopolysaccharides a challenging topic of study. One of these limitations is the low achievable EPS concentration often caused by their high viscosity, which, for example, lowers the efficiency of NMR analysis. Despite, all the remaining challenges and obstacles concerning the study of molecular processes underlying formation of EPS and their chemical characterization, many aspects of this highly diverse class of biopolymers are already known sustaining them as biomolecules of industrial interest. In the series of articles presented in this book, the authors provide an overview of the different fields involved in microbial EPS production, characterization, and applications. Particular emphasis is directed toward the molecular mechanisms of EPS biosynthesis and modification as well as their regulation. Additionally, the production of fungal EPSs is also explored to show the potential use of these currently less understood microbial biopolymers. Furthermore, the problems and future perspectives related to polysaccharide characterization are summarized and described. Finally, the various aspects of microbial polysaccharide application covering a wide range of uses are also discussed. In summary this research topic deals with the following aspects. Effective microbial EPS production is based on the identification of novel and efficient production strains. Therefore, Ruhmann et al. describe and compare the different available methods to identify EPS producers, including the characterization of monomer composition of these polysaccharides. They finally disclose the most promising and currently available novel techniques, as the main input for boosting the exploration and discovery of new EPS-producers in the short term. The review of Schmid et al. gives a comprehensive overview of the different biosynthetic pathways known for bacterial EPS-producers and compares in detail the different kinds of EPS. It summarizes the regulatory mechanisms of bacterial EPS production and describes present and future engineering strategies toward tailor-made EPS variants. The minireview of Becker goes much deeper into this topic, with a specific focus on xanthan and succinoglycan biosynthesis. It includes challenges and perspectives in combinatorial assembly of the biosynthetic pathways in order to obtain tailored variants. The enhanced bacterial persistence due to enzymatic EPS modifications is described in the review of Whitfield et al. They describe the effect of different enzymatic activities, which are involved in the modification of different EPSs such as alginate, Pel polysaccharide as well as a nitrogen-containing EPS, and link them to their biological function with respect to enhanced survival of the producing microorganisms, either in pure cultures or in biofilms. The contribution of Ertesvag especially focuses on alginate modifying enzymes, and discusses how the different modifications influence on material properties of the respective alginate variants. This review represents a comprehensive overview of enzymatic tools suitable for tailoring alginate polysaccharides. In the original research article of Jachlewski et al. different techniques are presented for the targeted isolation of various EPS and further polymeric compounds, such as DNA and proteins, from biofilms of extremophilic archaea. The authors present a combined approach of proteome and EPS analyses, which provides further insight into the composition and functionality of extremophile biofilms and alludes to the potential of biofilms in future applications. The special class of microbial EPSs produced by lactic acid bacteria is described in detail by Torino et al. The authors give a comprehensive outlook of either capsular or exopolysaccharides produced by lactobacilli and summarize their traditional and novel applications in food and beverage manufacturing. To complete the landscape, the authors provide a wide description of relevant EPS characteristics and the enzymes involved in their biosynthesis. The section of rare-sugar-containing EPSs, such as fucose or rhamnose, is described by Roca et al. These polymers are uncommon or at least, rarely identified up to now, and open new frontiers for special applications. In this brief review, EPSs containing rare sugars as well as the respective producing strains are presented, along with the cultivation conditions influencing their monomer pattern. Additionally, the authors focus on their downstream processing and discuss the applications of these special polymers in various fields such as e.g., cosmetics, foodstuff, pharmaceuticals, and biomedical applications. An overview of current and future biomedical applications of microbial EPSs is given by Moscovici. This article explores the various EPS applications starting from the first tested medical applications, such as the use of dextran as plasma expander, up to the latest innovations in the field, like micro- and nanoparticle-based EPS formulations. The complete scleroglucan production process, one of the few fungal representative EPSs commercially available, is revisited by Castillo et al. In this comprehensive review, they describe the complete fermentative production process as well as some downstream processing clues which have influence on the final EPS properties. The utilization of non-conventional complex media as efficient carbon-sources for sustainable production of EPS is also outlined and discussed. Putative future solutions for resolving one of the main obstacles of EPS characterization, i.e., the efficient determination of the EPS structure by NMR analysis, are outlined in the article by Larsen and Engelsen. They present a simple but very efficient combination of NMR spectroscopy and molecular modeling as an efficient EPS analytical tool. This approach might be efficiently used to determine the chemical and three-dimensional EPS structures in fast and reliable way in the near future. Further, insights into the microbial EPS structure characterization by using novel imaging technologies at various length scales are summarized by Lilledahl et al. The authors present the use of different high-resolution microscopic techniques along with the combination of different approaches in order to support the elucidation of structural features of isolated and secreted EPSs in the cell environment. In conclusion, these contributions summarize important aspects related to microbial biopolymer research. The technical limitations for analyzing/characterizing microbial EPSs, the urgent need to advance our understanding on EPS biosynthetic pathways, as well as the relevance of producing tailor-made EPSs, are widely explored and discussed employing various representative examples. On the other hand, a range of current and future applications of microbial EPSs is presented, which either already, or in the near future, will contribute to a biobased industry. In summary, a wide readership with interest in bio-polysaccharides and their promising future is expected to find in this research topic a clear overview assessing the currents gaps in our understanding of EPS while already taking advantage of the current knowledge in the field of microbial polysaccharide research, thus identifying still unmet needs informing future R&D programmes.

Isolation of a laccase-coding gene from the lignin-degrading fungus Phlebia brevispora BAFC 633 and heterologous expression in Pichia pastoris

Isolate and characterize a laccase‐encoding gene (lac I) of Phlebia brevispora BAFC 633, as well as cloning and expressing cDNA of lac I in Pichia pastoris. And to obtain a purified and characterized recombinant laccase to analyse the biotechnological application potential.