Identification of Zinc Absorption Biomarkers in Muscle Tissue of Nile Tilapia Fed with Organic and Inorganic Sources of Zinc Using Metallomics Analysis.

The development of metallomics techniques has allowed for metallomics analysis of biological systems, enabling a better understanding of the response mechanisms for different stimuli, their relationship to metallic species, and the characterization of biomarkers. In this study, a metallomics analysis of the muscle tissue of Nile tilapia was used to aid the understanding of the molecular mechanisms involved in zinc absorption in this fish species when fed organic and/or inorganic sources of zinc and to identify possible biomarkers for the absorption of this micromineral. To accomplish this, the fish were separated into three groups of 24 g, 74 g, and 85 g initial weights, and each group, respectively, was fed a zinc-free diet (control group, G1), a diet containing zinc found in organic sources (treatment 1, G2), and a diet containing zinc from an inorganic source (treatment 2, G3). Two-dimensional polyacrylamide (2D PAGE) gel electrophoresis was used to separate the proteins of the muscle tissue. Subsequently, the expression profiles of protein spots in the samples where zinc was applied in different concentrations were compared, using the software ImageMaster 2D Platinum version 7.0, to identify proteins that were differentially expressed. The identified proteins were then exposed to atomic absorption spectrometry in a graphite furnace to determine zinc mapping and were subsequently characterized via electrospray ionization tandem mass spectrometry (ESI-MS/MS). The metallomic analysis identified 15 proteins differentially expressed and associated with zinc, leading to the conclusion that three metal-binding proteins presented as possible biomarkers of zinc absorption in fish.

Lipases obtained from orange wastes: Commercialization potential and biochemical properties of different varieties and fractions.

Brazil is the world's leading orange supplier for juice production purposes. However, the production process generates high amount of wastes, which leads to disposal problems. Orange wastes can be used for lipases production, incorporating the biorefinery concept into juice industries. Thus, the aim of the present study was to investigate the wastes of orange production chain as source of lipases based on different varieties (Pera, Hamlin, Valencia, and Natal), as well as on different fractions of wastes. The mass balance of the juice/wastes (2007-2016 crops) was evaluated, and lipases from different varieties and fraction were biochemically characterized. Overall, the wastes corresponded to approximately 43% of the fruit mass. All the fractions of all varieties showed lipase activity in emulsified olive oil and in p-nitrophenyl substrates. The highest lipase activities were obtained by Natal pulp in emulsified olive oil, Natal frit, and Hamlin peel in p-NPB and Hamlin frit in p-NPL and p-NPP. The bagasse, peel, and frit lipases from the different orange varieties showed optimum pH from 6.0 to 8.0 and optimal temperature from 30 °C to 60 °C. Thus, it is possible concluding that the orange processing for juice production purposes generates a large amount of wastes, which can be destined to profitable purposes as lipases production. Lipases produced by different fractions and varieties are biochemically diverse, enabling the application a wide range of processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2734, 2019.

Characterization of molecular biomarkers of mercury exposure to muscle tissue of Plagioscion squamosissimus and Colossoma macropomum from the Amazon region.

Mercury has the ability to bind to a variety of biomolecules, which can compromise its structure and functionality and thus promote its toxic effects. The aim of this study is to identify possible mercury biomarkers in muscle samples of Plagioscion squamosissimus (carnivorous fish) and Colossoma macropomum (omnivorous fish), from the Amazon region. The muscle proteome of fish species was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the total mercury concentrations in protein spots were determined by graphite furnace atomic absorption spectrometry (GFAAS). The protein spots containing mercury were characterized by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The mercury concentrations in the protein spots were in the range of 1.10 ±â€¯0.02-23.90 ±â€¯0.33 µg g-1. The proteins phosphoglycerate mutase 2 (P. squamosissimus), hemoglobin ß and cytochrome P450scc (C. macropomum), identified by ESI-MS/MS and showing the highest values of mercury concentration, may be considered possible mercury biomarkers.

Production of milk peptides with antimicrobial and antioxidant properties through fungal proteases.

Bioactive peptides can provide health benefits due to different mechanisms. The aims of the present study are to produce bioactive peptides from bovine and goat milk subjected to the proteolytic activity of Aspergillus oryzae and Aspergillus flavipes enzymes, as well as to assess their putative antimicrobial and antioxidant activity. Bioactive peptides were successfully generated from proteases of fungi cultivated in solid-state fermentation. The generated peptides were effective against all tested bacteria and fungi. There was antioxidant activity, up to 92.5% DPPH reduction and ORAC stabilization at 52.5 µmol µL-1 of Trolox Equivalent. The generation of milk-specific sequences peptides in the samples was obtained through 2D-PAGE fractioning followed by mass spectrometry (ESI-MS/MS). Based on results in the present study, milk bioactive peptides presenting broad antimicrobial action and antioxidant activity spectra can be cost-effectively produced through solid-state fermentation. The herein addressed approach can be valuable for the pharmaceutical and food industries.

Improvement of lipase obtaining system by orange waste-based solid-state fermentation: production, characterization and application.

Lipases are an economic important group of biocatalysts that can be produced by some fungal under solid-state fermentation. Orange wastes are source of lipases and potential substrates for lipases production. This work assessed 19 fugal strains cultivated in Citrus sinensis cv. Hamlin orange wastes (peel, frit and core) for production of lipases in order to generate compounds with antioxidant, antimicrobial and cytotoxic properties. Fifteen of those fungi grew and produced lipases, mainly the Aspergillus brasiliensis [National Institute of Quality Control (INCQS) 40036]/frit system, which showed 99.58 U/g total lipase. The substrate with the highest production of lipase was frit with 26.67 and 78.91 U/g of total lipases produced on average by the 15 microorganisms. Aspergillus niger 01/frit (33.53 U/g) and Aspergillus niger (INCQS 40015)/frit (34.76 U/g) systems showed the highest specificity values in all the herein tested synthetic substrates with 4, 12 and 16 carbons. Analysis of the fatty acid profile of hydrolysis products obtained in the most prominent systems applied to corn and sunflower oils showed: palmitic acid, linoleic acid, oleic acid, and stearic acid. These acids showed antioxidant capacity of up to 58% DPPH (2,2-diphenyl-1-pierylhydrazyl) radical reduction and antibacterial activity against Escherichia coli, Listeria monocytogenes, Pseudomonas aureginosa, Salmonella Enteritidis and Staphylococcus aureus, as well as cytotoxicity to SCC9 cells (squamous cancer cells).

Bioremediation of cooking oil waste using lipases from wastes.

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases.

Novel inexpensive fungi proteases: Production by solid state fermentation and characterization.

A comparative study was carried out for proteases production using agroindustrial residues as substrate for solid state fermentation (SSF) of several fungal strains. High protease production was observed for most of the microorganisms studied, as well as very different biochemical characteristics, including activities at specific temperatures and a wide range of pH values. The enzymes produced were very different regarding optimum pH and they showed stability at 50 °C. Aspergillus oryzae showed stability at all pH values studied. Penicillium roquefortii and Aspergillus flavipes presented optimum activity at temperatures of 50 °C and 90 °C, respectively. Lyophilized protease from A. oryzae reached 1251.60 U/g and yield of 155010.66 U/kg of substrate. Therefore, the substrate as well as the microorganism strain can modify the biochemical character of the enzyme produced. The high protease activity and stability established plus the low cost of substrates, make these fungal proteases potential alternatives for the biotechnological industry.

Obtaining lipases from byproducts of orange juice processing.

The presence of lipases was observed in three byproducts of orange juice processing: peel, core and frit. The enzymes were characterised biochemically over a wide pH range from neutral (6-7) to alkaline (8-9). The optimal temperature for the activity of these byproducts showed wide range at 20°C to 70°C, indicating fairly high thermostability. The activities were monitored on p-NP-butyrate, p-NP-laurate and p-NP-palmitate. For the first time, lipase activity was detected in these residues, reaching 68.5 lipase U/g for the crude extract from fractions called frit.

Beta-1,3 Glucanases e quitinases: aplicação na lise de leveduras e inibição de fungos / Beta-1,3 glucanases and chitinases: application in the yeast cell lysis and fungi inhibition

Objetivou-se, no presente trabalho, a aplicação de β-1,3 glucanases e quitinases da linhagem Cellulosimicrobium cellulans 191 na lise de leveduras e inibição de fungos, respectivamente. O delineamento experimental mostrou que as melhores condições para a lise de Saccharomyces cerevisiae KL-88 pela β-1,3 glucanase foi pH 6,5 e 35ºC. As células de leveduras incubadas por 10 h em frascos sem agitação mostraram-se mais susceptíveis à lise pela ação da enzima. Foi obtido maior lise da levedura quando a suspensão de células foi submetida ao tratamento com β-1,3 glucanase e cisteína 1mM. A enzima invertase intracelular ou ligada à célula de S. cerevisiae KL-88 e K. marxianus NCYC 587 foi extraída após tratamento da suspensão celular com β-1,3 glucanase, sendo que o tratamento prévio das leveduras com a enzima aumentou a susceptibilidade das células à lise com ultra-som. A preparação de quitinase foi capaz de formar halos de inibição de alguns fungos.

The aim of this work was the application of β-1,3 glucanases and chitinases by Cellulosimicrobium cellulans 191 strain on yeast cell lysis and fungi inhibition, respectively. The experimental design study showed that the best conditions to Saccharomyces cerevisiae KL-88 lysis by β-1,3 glucanase extract were pH 6,5 and 35ºC. This study also demonstrated that the yeast cells were more susceptible to lysis after 10 h of cultivation in flasks without agitation. Lysis activity was increased when S. cerevisiae KL-88 cell suspension was treated with β-1,3 glucanase and cystein 1mM. The enzyme invertase of S. cerevisiae KL-88 and Kluyveromyces marxianus NCYC 587 was extracted after treatment of cell suspension with β-1,3 glucanase and the previous treatment of yeasts with the enzyme, increased the susceptibility to lysis when ultrasonic treatment was used. The chitinase presented growth inhibition halos for some of the fungi.

Toluene gas phase biofiltration by Paecilomyces lilacinus and isolation and identification of a hydrophobin protein produced thereof.

Paecilomyces lilacinus consumed toluene as the sole carbon source in a gas-phase biofilter packed with perlite obtaining an average elimination capacity of 50 g m(-3) h(-1), a removal efficiency of 53%, and a final biomass of 31.6 mg biomass g dry support(-1). Hydrophobin proteins from the mycelium produced in the biofilter were purified by formic acid extraction and precipitated by electrobubbling, and the molecular weight was found to be 10.6 +/- 0.3 kDa. The peptide mass fingerprinting analysis of the purified hydrophobin by matrix-assisted laser desorption/ionization time-of-flight resulted in the identification of two peptides that presented high homology with sequences of class I hydrophobin proteins from other ascomycetous fungi when compared against the National Center for Biotechnology Information database. The yield of hydrophobin (PLHYD) from P. lilacinus was 1.1 mg PLHYD g biomass(-1). These proteins modified the hydrophobicity of Teflon by lowering the contact angle from 130.1 (+/-2) degrees to 57.0 (+/-5) degrees supporting hot sodium dodecyl sulfate washing. This work is the first report about biodegradation of toluene by the nematophagous fungus P. lilacinus in a gas-phase biofilter and the identification of its hydrophobin protein.

Production of glucosyltransferase by Erwinia sp. using experimental design and response surface methodology

A glicosiltransferase obtida pela linhagem Erwinia sp. é uma enzima intracelular que catalisa a conversão de sacarose em isomaltulose. A isomaltulose é um dissacarídeo redutor, não cariogênico e comercialmente utilizado em alimentos como substituto da sacarose. A metodologia de superfície de resposta e planejamento fatorial composto central-23 foram utilizados para otimizar o meio de cultivo para a producão de glicosiltransferase de Erwinia sp. em frascos sob agitacão a 200 rpm e 30ºC. As três variáveis independentes envolvidas no estudo foram o melaco de cana de acúcar, a água de maceracão de milho e o extrato de levedura Prodex Lac SD. As análises estatísticas dos resultados mostraram que, dentro da faixa estudada das concentracões dos componentes de meio de cultivo, todas as variáveis apresentaram efeito significativo na producão de glicosiltransferase. O meio de cultivo otimizado foi composto de 100 gL-1 de melaco de cana de acúcar, 60 gL-1 de água de maceracão de milho e 8 gL-1 de extrato de levedura Prodex Lac SD, apresentando atividade de glicosiltransferase de 6.65 U mL-1.