Functional characterization and structural insights of three cutinases from the ascomycete Fusarium verticillioides.

Cutinases are serine esterases that belong to the α/ß hydrolases superfamily. The natural substrates for these enzymes are cutin and suberin, components of the plant cuticle, the first barrier in the defense system against pathogen invasion. It is well-reported that plant pathogens produce cutinases to facilitate infection. Fusarium verticillioides, one important corn pathogens, is an ascomycete upon which its cutinases are poorly explored. Consequently, the objective of this study was to perform the biochemical characterization of three precursor cutinases (FvCut1, FvCut2, and FvCut3) from F. verticillioides and to obtain structural insights about them. The cutinases were produced in Escherichia coli and purified. FvCut1, FvCut2, and FvCut3 presented optimal temperatures of 20, 40, and 35 °C, and optimal pH of 9, 7, and 8, respectively. Some chemicals stimulated the enzymatic activity. The kinetic parameters revealed that FvCut1 has higher catalytic efficiency (Kcat/Km) in the p-nitrophenyl-butyrate (p-NPB) substrate. Nevertheless, the enzymes were not able to hydrolyze polyethylene terephthalate (PET). Furthermore, the three-dimensional models of these enzymes showed structural differences among them, mainly FvCut1, which presented a narrower opening cleft to access the catalytic site. Therefore, our study contributes to exploring the diversity of fungal cutinases and their potential biotechnological applications.

Enhancement of the Functional Properties of Mead Aged with Oak (Quercus) Chips at Different Toasting Levels.

Consumers increasingly prefer and seek functional beverages, which, given their characteristics, provide important bioactive compounds that help prevent and treat chronic diseases. Mead is a traditional fermented alcoholic beverage made from honey solution. The aging process of mead with oak chips is innovative and bestows functional characteristics to this beverage. Thus, in this study, we sought to develop and characterize a novel functional beverage by combining the health benefits of honey with the traditional aging process of alcoholic beverages in wood. Phenolic compounds, flavonoids, and antioxidant capacity were analyzed in mead using oak chips at different toasting levels and aged for 360 days. LC-ESI-QTOF-MS/MS was used to analyze the chemical profile of different meads. Over time, the aging process with oak chips showed a higher total phenolic and flavonoid content and antioxidant capacity. Eighteen compounds belonging to the classes of organic acids, phenolic acids, flavonoids, and tannins were identified in meads after 360 days. Our findings revealed that the addition of oak chips during aging contributed to p-coumaric, ellagic, abscisic, and chlorogenic acids, and naringenin, vanillin, and tiliroside significantly impacted the functional quality of mead.

Separation of microbial oil produced by Mortierella isabellina using polymeric membranes.

The objective of this work was to concentrate, through a membrane separation process, the fatty acids from oil/solvent mixture. The oil was obtained by ultrasound-assisted extraction from freeze-dried cells of Mortierella isabellina. The concentration of the fatty acids was investigated using flat-sheet polymer membranes of ultrafiltration and nanofiltration. The effects of temperature and pressure were evaluated by the retention of the fatty acids. Oil retentions between 45.23 and 58.20% to ultrafiltration membrane and 43.50 and 56.00% to nanofiltration membrane were observed. The best condition for the ultrafiltration membrane was 4 bar and 40 °C and for nanofiltration membrane was 12 bar and 50 °C. The oil contains a high concentration of oleic acid and palmitic acid that is a desirable property for the biodiesel production. The results showed the applicability of this technology in the solvent recovery step whereas the oil recovered contains a high concentration of fatty acids.

Production of metabolites with antioxidant activity by Botryosphaeria dothidea in submerged fermentation.

Antioxidants are able of inhibiting free radicals and play an important role in human diet, protection of foods, anti-aging cosmetics, among others. Fungi have been highlighted in production of metabolites with biological activity, such as antioxidant. The main objective of this study was to select a fungus from resources of Brazilians Pampa and Atlantic Forest biomes with hability for production of antioxidants by submerged fermentation. The scavenging activity of the compounds was determined against radical 2.2-diphenyl-1-picrylhydrazyl (DPPH), superoxide and hydroxyl. Botryosphaeria dothidea exhibited higher potential for the production of antioxidants, and its supernatant showed percentage inhibition values of 94.47, 94.87 and 89.78% against DPPH, superoxide and hydroxyl radicals and IC50 of 0.206 mg/mL. The identification of the volatile compounds present in the broth verified the presence of the hexahydropyrrolizin-3-one; 1.2-benzenedicarboxylic acid bis(2-methylpropyl) ester and 3.6-bis(2-methylpropyl)-2.5-piperazinedione. According to this study, Botryosphaeria dothidea presents great potential to produce compounds with antioxidant activity.

Degradation of Amaranth azo dye in water by heterogeneous photo-Fenton process using FeWO4 catalyst prepared by microwave irradiation.

FeWO4 particles were synthesized by a simple, rapid and facile microwave technique and their catalytic properties in heterogeneous photo-Fenton reaction were evaluated. This material was employed in the degradation of Amaranth azo dye. Individual and interactive effects of operational parameters such as pH, dye concentration and H2O2 dosage on the decolorization efficiency of Amaranth dye were evaluated by 2(3) central composite design. According to characterization techniques, a porous material and a well-crystallized phase of FeWO4 oxide were obtained. Regarding the photo-Fenton reaction assays, up to 97% color and 58% organic carbon removal were achieved in the best experimental conditions. In addition, the photo-Fenton process maintained treatment efficiency over five catalyst reuse cycles to indicate the durability of the FeWO4 catalyst. In summary, the results reveal that the synthesized FeWO4 material is a promising catalyst for wastewater treatment by heterogeneous photo-Fenton process.

Separation and purification of fructooligosaccharides on a zeolite fixed-bed column.

Fructooligosaccharides (FOS), a well-known prebiotic product, are obtained by enzymatic synthesis and consist of a mixture of mono- and disaccharides. In this work, a methodology for their separation and purification was developed using a zeolite fixed-bed column. The effects of column temperature (40-60°C), eluent flow rate (0.10-0.14 mL/min), injected to bed volume percent ratio (2.6-5.1%), and ethanol concentration in the eluent (40-60%, v/v) were investigated using a fractionary factorial design (2(4-1)), having the separation efficiency and purity as target responses. Additional experiments were performed as well, where the temperature and ethanol concentration were studied in a central composite design (2(2)). In this work, the zeolite fixed-bed column was shown to be a good alternative for FOS purification, allowing a FOS purity of 90% and separation efficiency of 6.86 between FOS and glucose, using an eluent at 45°C with 60% ethanol concentration.

Removal of hazardous pharmaceutical dyes by adsorption onto papaya seeds.

Papaya (Carica papaya L.) seeds were used as adsorbent to remove toxic pharmaceutical dyes (tartrazine and amaranth) from aqueous solutions, in order to extend application range. The effects of pH, initial dye concentration, contact time and temperature were investigated. The kinetic data were evaluated by the pseudo first-order, pseudo second-order and Elovich models. The equilibrium was evaluated by the Langmuir, Freundlich and Temkin isotherm models. It was found that adsorption favored a pH of 2.5, temperature of 298 K and equilibrium was attained at 180-200 min. The adsorption kinetics followed the pseudo second-order model, and the equilibrium was well represented by the Langmuir model. The maximum adsorption capacities were 51.0 and 37.4 mg g(-1) for tartrazine and amaranth, respectively. These results revealed that papaya seeds can be used as an alternative adsorbent to remove pharmaceutical dyes from aqueous solutions.

Adsorption of leather dye onto activated carbon prepared from bottle gourd: equilibrium, kinetic and mechanism studies.

Activated carbon prepared from bottle gourd has been used as adsorbent for removal of leather dye (Direct Black 38) from aqueous solution. The activated carbon obtained showed a mesoporous texture, with surface area of 556.16 m(2) g(-1), and a surface free of organic functional groups. The initial dye concentration, contact time and pH significantly influenced the adsorption capacity. In the acid region (pH 2.5) the adsorption of dye was more favorable. The adsorption equilibrium was attained after 60 min. Equilibrium data were analyzed by the Langmuir, Freundlich, Dubinin-Radushkevich and Temkin isotherm models. The equilibrium data were best described by the Langmuir isotherm, with maximum adsorption capacity of 94.9 mg g(-1). Adsorption kinetic data were fitted using the pseudo-first-order, pseudo-second-order, Elovich and intraparticle diffusion models. The adsorption kinetic was best described by the second-order kinetic equation. The adsorption process was controlled by both external mass transfer and intraparticle diffusion. Activated carbon prepared from bottle gourd was shown to be a promising material for adsorption of Direct Black 38 from aqueous solution.

Biomolecules in modern and sustainable agriculture.

This review presents scientific findings which indicate biomolecules are excellent candidates for the development of biopesticides. Efforts are being done to find routes to increase their concentrations in the cultivation media because this concentration facilitates applications, storage, and transportation. Some of these routes are co-fermentation and ultrasound-assisted fermentation. Ultrasonication increases metabolite production and growth rates by improvement of cell permeability and nutrient uptake rates through cell membranes. For example, 24% increase in the enzymatic activity of cellulases produced by Trichoderma reesei in solid-state fermentation was achieved with ultrasonication. Also, chitinase and ß-1,3-glucanase productions were stimulated by ultrasound in Beauveria bassiana cultivation, presenting positive results. The common parameters evaluated in the production of biomolecules by ultrasound-assisted fermentation are the duty cycle, time of application, power, energetic density, and how long the sonication is maintained in the fermentation media. Many successful cases are reported and discussed, which include the final formulation of bioproducts for agricultural applications. In this trend, nanotechnology is a promising tool for the development of nanoformulations. Nanoemulsification, green synthesis, biosynthesis, or biogenic synthesis are technologies used to produce such nanoformulations, allowing the controlled release of control agents, as well as the delivery of biomolecules to specific targets.

Strategy to increase the lipid stability of the microbial oil produced by Umbelopsis isabellina for food purposes: Use of microencapsulation by external ionic gelation.

Oleaginous microorganisms, including the fungus Umbelopsis isabellina, have emerged as a biotechnological alternative to obtain polyunsaturated fatty acid-rich oils, which are strongly linked to energy purposes (biofuel) than the food industry. Considering the composition of microbial oil and its use by the food industry, it is necessary to investigate strategies that increase its lipid stability. Ergo, this pioneering study aimed to microencapsulate the oil produced by Umbelopsis isabellina and evaluate its oxidative stability throughout the storage period against factors such as temperature and luminosity. The microbial oil was microencapsulated through the external ionic gelation technique, producing an encapsulation efficiency of 80% and proving to be a suitable method because it maintained oil composition. Combining microencapsulation and refrigerated storage led to the best effects on storage time, increasing the evaluated lipid stability through the peroxide values and conjugated diene formation. Moreover, saturated and monounsaturated fatty acid content increased, and polyunsaturated fatty acid content decreased during storage for both the free and microencapsulated oil, regardless of storage temperature, although microencapsulation reduced the changes. The results primarily demonstrate how microencapsulation prolongs the oxidative stability and unsaturated fatty acid content of the microbial oil by reducing its reactions to external environmental factors, thus facilitating its use in the food industry.

Chemical study, antioxidant activity, and genotoxicity and cytotoxicity evaluation of Ruellia angustiflora.

Ruellia angustiflora (Acanthaceae) is known as flower-of-fire, and its leaves are traditionally employed to promote wound healing. This study was aimed at extracting and characterizing the chemical constituents of the extracts of R. angustiflora obtained by ultrasound-assisted and supercritical fluid extractions, and subsequently investigate their antioxidant potential and cyto-genotoxicity. The extract obtained by ultrasound (UAE-EtOH) was characterized by ultraperformance liquid chromatography-mass spectrometer (UPLC-MS), and the extract obtained via supercritical fluid (SFE-CO2) by gas chromatograph-mass spectrometer (GC-MS). The antioxidant potential was verified by the antiradical activity against the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), and the cyto-genotoxicity evaluation to test cell viability and DNA damage was performed in peripheral blood mononuclear cells (PBMC) cultures. The SFE-CO2 extract presented some fatty acids, triterpenes, tetraterpenes, tocopherols and phytosterols. The UAE-EtOH extract contained phenolic acids and flavonoids, and showed the highest antioxidant capacity. Neither extract was genotoxic or cytotoxic at the tested concentrations.

Chitinase production by Trichoderma koningiopsis UFSMQ40 using solid state fermentation.

The chitinases have extensive biotechnological potential but have been little exploited commercially due to the low number of good chitinolytic microorganisms. The purpose of this study was to identify a chitinolytic fungal and optimize its production using solid state fermentation (SSF) and agroindustry substrate, to evaluate different chitin sources for chitinase production, to evaluate different solvents for the extraction of enzymes produced during fermentation process, and to determine the nematicide effect of enzymatic extract and biological control of Meloidogyne javanica and Meloidogyne incognita nematodes. The fungus was previously isolated from bedbugs of Tibraca limbativentris Stal (Hemiptera: Pentatomidae) and selected among 51 isolated fungal as the largest producer of chitinolytic enzymes in SSF. The isolate UFSMQ40 has been identified as Trichoderma koningiopsis by the amplification of tef1 gene fragments. The greatest chitinase production (10.76 U gds-1) occurred with wheat bran substrate at 55% moisture, 15% colloidal chitin, 100% of corn steep liquor, and two discs of inoculum at 30 °C for 72 h. Considering the enzymatic inducers, the best chitinase production by the isolated fungus was achieved using chitin in colloidal, powder, and flakes. The usage of 1:15 g/mL of sodium citrate-phosphate buffer was the best ratio for chitinase extraction of SSF. The Trichoderma koningiopsis UFSMQ40 showed high mortality of M. javanica and M. incognita when applied to treatments with enzymatic filtrated and the suspension of conidia.

Use of a sequential strategy of experimental design to optimize the inulinase production in a batch bioreactor.

A potential application of inulinase in the food industry is the production of fructoligosaccharides (FOS) by the transfructosilation of sucrose. The FOSs present many interesting functional properties besides their ability to increase the shelf-life and flavor of many products. The use of an industrial medium represents a good alternative to producing inulinase at low cost, since the activity may improve, or at least remain the same, as that obtained using a synthetic medium. This work was an optimization study of the inulinase production by Kluyveromyces marxianus NRRL Y-7571 using industrial pre-treated culture medium in a bioreactor employing a sequential strategy of experimental design. Initially, a Plackett-Burman (Screening Design) design was used, where the studied variables were molasses, corn steep liquor, yeast extract concentration, and agitation and aeration rates. After the analysis of the effects, a central composite rotational design (CCRD) was carried out. The optimized condition for the inulinase production was: 250 g/l of molasses, 80 g/l of corn steep liquor, 6 g/l of yeast extract, 300 rpm of agitation and 1.5 vvm aeration rate, which resulted in an enzymatic activity of 1,317 +/- 65 U/ml.

Potentiality of the Phoma sp. inactive fungal biomass, a waste from the bioherbicide production, for the treatment of colored effluents.

The potentiality of Phoma sp. inactive fungal biomass, waste from the bioherbicide production, was evaluated for the treatment of colored effluents containing Acid Red 18 (AR 18) dye. The batch experiments were performed to evaluate the following parameters: pH of the solution (2-10), dye concentration (50-200 mg L-1), adsorbent dose (0.5-2.5 g L-1), contact time (0-180 min) and temperature (298-328 K). The batch experiments using a synthetic dye solution revealed that Phoma sp. was efficient at pH of 2.0, 298 K and using a dosage of 1.25 g L-1. The process was fast, being the equilibrium reached within 180 min. The maximum value of biosorption capacity was 63.58 mg g-1, being the process favorable and exothermic. From the fixed bed assays, breakthrough curves were obtained, presenting a mass transfer zone of 7.08 cm and breakthrough time of 443 min. Phoma sp. was efficient to decolorize a simulated effluent, removing more than 90% of the color. From the obtained results, it can be concluded that Phoma sp. inactive biomass is a low-cost option to treat colored effluents in continuous and discontinuous biosorption modes. These indicate that Phoma sp. of inactive biomass is an option for the treatment of colored effluents.

New perspectives for weeds control using autochthonous fungi with selective bioherbicide potential.

The prospection of bioherbicides has been an alternative to weed control, aiming at mitigating chemical risks to human, animal and environmental health due to extreme use of synthetic herbicides. In the present study, various fungi were isolated from plants with symptoms of fungal diseases for bioherbicide purposes against weeds (Urochloa plantaginea, Euphorbia heterophylla and Bidens pilosa). Fungi isolated were identified by molecular methods and enzymatic products obtained by fungi fermentation (cellulase, lipase, peroxidase, and amylase) were quantified. Bioherbicide selectivity study was performed on crops (soybean and corn), as well as on resistant weeds. Among the isolated fungi, Fusarium oxysporum, Fusarium ploriferatum, and Trichoderma koningiopsis presented bioherbicide potential. T. koningiopsis, in particular, presented the highest effect on Euphorbia heterophylla (popular name - Mexican fire plant), causing up to 60% of foliar damage, without presenting phytotoxicity against corn crop. New perspectives for weeds control and their use in corn crops were prospected, considering the bioherbicide selectivity described in this study.

Development of nanoemulsions containing Physalis peruviana calyx extract: A study on stability and antioxidant capacity.

The aim of this study was to develop and evaluate the physicochemical and antioxidant stability of nanoemulsions containing a Physalis peruviana calyx extract (CPp-NE) and free extracts under different storage conditions (7 and 25 °C) and with absence or incidence of light for 120 days. The calyx extracts were prepared with ethanol 60% and characterized for later preparation of the nanoemulsions by spontaneous emulsification. The formulations presented nanometric sizes, low polydispersity index, negative zeta potential, acid pH, rutin content (11 µg·mL-1), and encapsulation efficiency of 85%. Regarding the stability, the droplet size and PdI of the CPp-NE stored at refrigeration temperature in the dark, room temperature in the dark, and refrigeration temperature with light incidence were stable for 120 days and with no visible changes in the formulations. The antioxidant capacity was related to the reducing capacity, and the best results were found for nanoemulsions stored at room temperature and in absence of light. In addition, CPp-NE presented higher antioxidant and reducing capacity in relation to the free extracts.

Extraction of inulinase obtained by solid state fermentation of sugarcane bagasse by Kluyveromyces marxianus NRRL Y-7571.

Production of inulinase by solid state fermentation always involves an extraction step, which dictates enzyme recovery yield and is related to cultivation conditions and control of process parameters. This work is focused on the study of extraction conditions aiming to maximize yield of an inulinase obtained by solid state fermentation of sugar cane bagasse and Kluyveromyces marxianus NRRL Y-7571. Kinetics of extraction was followed varying the kind of solvent used. After determining the best solvent, an experimental design was carried out to study the effect of the solid/liquid ratio (1:10-1:20), extraction temperature (20-53 degrees C), and stirring rate (50-177 rpm). Results showed that maximum yield was obtained when sodium acetate buffer 0.1 M pH 4.8 was used, using a solid/liquid ratio of 1:10, at 53 degrees C and 150 rpm for 40 min.

Importance of Lupinus albescens in agricultural and food-related areas: A review.

The purpose of this review is to assist readers in understanding the importance of Lupinus albescens to nature, farmers, and scientists. L. albescens is mostly found in Argentina, Uruguay, Paraguay, and in "Campanha, Litoral and Missões" regions of State of Rio Grande do Sul (Brazil). Therefore, this review presents information and discussion on this plant that can encourage novel studies in a near future for exploring evermore the biological and physicochemical properties of L. albescens. The plant presents adaptive characteristics of soils with low content of nutrients, being an important plant for the recovering of degraded areas. In the last few years, there was an increase in scientific interest for exploring its chemical composition and biological activities. All plant matrices (i.e., roots, leaves, seeds, and stalks) are rich in antioxidant and antifungal compounds, especially stigmasterol. For example, the extracts obtained from the roots are reported with more than 50 wt% stigmasterol and 25 wt% ergosterol. Furthermore, the extracts present remarkable fungicide effects, especially against Fusarium oxysporum and Fusarium verticillioides.

First report of the production of a potent biosurfactant with α,ß-trehalose by Fusarium fujikuroi under optimized conditions of submerged fermentation.

Biosurfactants have many advantages over synthetic surfactants but have higher production costs. Identifying microorganisms with high production capacities for these molecules and optimizing their growth conditions can reduce cost. The present work aimed to isolate and identify a fungus with high biosurfactant production capacity, optimize its growth conditions in a low cost culture medium, and characterize the chemical structure of the biosurfactant molecule. The fungal strain UFSM-BAS-01 was isolated from soil contaminated with hydrocarbons and identified as Fusarium fujikuroi. To optimize biosurfactant production, a Plackett-Burman design and a central composite rotational design were used. The variables evaluated were pH, incubation period, temperature, agitation and amount of inoculum in a liquid medium containing glucose. The partial structure of the biosurfactant molecule was identified by nuclear magnetic resonance spectrometry. F. fujikuroi reduced surface tension from 72 to 20mNm-1 under the optimized conditions of pH 5.0, 37°C and 7 days of incubation with 190rpm agitation. The partial identification of the structure of the biosurfactant demonstrated the presence of an α,ß-trehalose. The present study is the first report of the biosynthesis of this compound by F. fujikuroi, suggesting that the biosurfactant produced belongs to the class of trehalolipids.

Inulinase production by Kluyveromyces marxianus NRRL Y-7571 using solid state fermentation.

Inulinase is an enzyme relevant to fructose production by enzymatic hydrolysis of inulin. This enzyme is also applied in the production of fructo-oligosaccharides that may be used as a new food functional ingredient. Commercial inulinase is currently obtained using inulin as substrate, which is a relatively expensive raw material. In Brazil, the production of this enzyme using residues of sugarcane and corn industry (sugarcane bagasse, molasses, and corn steep liquor) is economically attractive, owing to the high amount and low cost of such residues. In this context, the aim of this work was the assessment of inulinase production by solid state fermentation using by Kluyveromyces marxianus NRRL Y-7571. The solid medium consisted of sugar cane bagasse supplemented with molasses and corn steep liquor. The production of inulinase was carried out using experimental design technique. The effect of temperature, moisture, and supplements content were investigated. The enzymatic activity reached a maximum of 445 units of inulinase per gram of dry substrate.