Optimization of lipase production using fungal isolates from oily residues.

Lipases are triacylglycerol hydrolases that catalyze hydrolysis, esterification, interesterification, and transesterification reactions. These enzymes are targets of several industrial and biotech applications, such as catalysts, detergent production, food, biofuels, wastewater treatment, and others. Microbial enzymes are preferable for large scale production due to ease of production and extraction. Several studies have reported that lipases from filamentous fungi are predominantly extracellular and highly active. However, there are many factors that interfere with enzyme production (pH, temperature, medium composition, agitation, aeration, inducer type, and concentration, etc.), making control difficult and burdening the process. This work aimed to optimize the lipase production of four fungal isolates from oily residues (Penicillium sp., Aspergillus niger, Aspergillus sp., and Aspergillus sp.). The lipase-producing fungi isolates were morphologically characterized by optical and scanning electron microscopy. The optimal lipase production time curve was previously determined, and the response variable used was the amount of total protein in the medium after cultivation by submerged fermentation. A complete factorial design 32 was performed, evaluating the temperatures (28 °C, 32 °C, and 36 °C) and soybean oil inducer concentration (2%, 6%, and 10%). Each lipase-producing isolate reacted differently to the conditions tested, the Aspergillus sp. F18 reached maximum lipase production, compared to others, under conditions of 32 °C and 2% of oil with a yield of 11,007 (µg mL-1). Penicillium sp. F04 achieved better results at 36 °C and 6% oil, although for Aspergillus niger F16 was at 36 °C and 10% oil and Aspergillus sp. F21 at 32 °C and 2% oil. These results show that microorganisms isolated from oily residues derived from environmental sanitation can be a promising alternative for the large-scale production of lipases.

Maltodextrin and Gum Arabic-Based Microencapsulation Methods for Anthocyanin Preservation in Juçara Palm (Euterpe edulis Martius) Fruit Pulp.

The juçara fruits (Euterpe edulis Martius), native to the Atlantic Forest, are rich in anthocyanins. To preserve the anthocyanins in juçara fruit pulp, this study aimed to evaluate the effectiveness of microencapsulation by spray drying and freeze drying with maltodextrin (dextrose equivalent 16.5 to 19.5) and gum arabic in different proportions. The obtained microparticles were characterized by quantifying the total polyphenol and anthocyanin contents, by performing differential scanning calorimetry, thermogravimetry, and infrared spectroscopy and by using scanning electron microscopy to analyze the morphology of the particles. The total amount of polyphenols in the fruit pulp was 750 ± 16.7 mg GAE/100 g of the freeze-dried sample. The total anthocyanins in the fruit pulp was 181.25 ± 5.36 (mg/100 g). The microparticles were formed by employing maltodextrin and gum arabic in a 1:1 proportion as the polymeric matrix; the mixtures of pulp and polymeric matrix were prepared in proportions of 2:3 and 2:1, preserving up to 83.69% of the anthocyanin content. Lyophilization of the 2:1 mixture resulted in an anthocyanin content of 116.89 ± 4.43 (mg/100 g), whereas lyophilization of the 2:3 mixture resulted in 151.68 ± 1.39 (mg/100 g) anthocyanin content, which did not differ from the value obtained by spray drying the 2:3 mixture (150.76 ± 5.79 (mg/100 g)). Thermal analyses showed that the microparticles obtained by freeze drying at a ratio of 2:3 presented greater resistance to degradation with increasing temperature. The incorporation of the pulp in the polymeric matrix was demonstrated by IR analyses. Microparticles obtained by freeze drying showed the formation of various-sized flakes, whereas those obtained by spray drying were spherical in shape. Microencapsulation is a possible alternative for improving the stability of the anthocyanins in this fruit.

Barcode-style lateral flow immunochromatographic strip for the semi-quantitative detection of ochratoxin A in coffee samples.

Ochratoxin A (OTA) is a mycotoxin contaminating agricultural products produced by fungi, associated with important toxic effects. Thus, the development of fast, sensitive, and economical approaches for OTA detection is crucial. In this study, a barcode-style lateral flow assay for the semi-quantitative detection of OTA in coffee samples was developed. To achieve this goal, a BSA-OTA complex was immobilized in three test zones to compete with OTA molecules in the sample for binding with anti-OTA antibodies labeled with gold nanoparticles. Different concentrations of OTA in the sample produced distinct colour patterns, allowing semi-quantification of the analyte. The assay exhibited high sensitivity, with a limit of detection of 2.5 µg.L-1, and high reproducibility, with variation coefficient values between 2% and 13%. Moreover, the colour patterns obtained in the analysis with coffee samples were similar to the results obtained with standard OTA solutions, demonstrating a reliable applicability in real samples.

Plasmonic immunosensors based on spoon-shaped waveguides for fast and on-site ultra-low detection of ochratoxin A in coffee samples.

The high toxicity and occurrence of ochratoxin A (OTA) in grains and foods has been a growing concern due to the impacts on health and the economy in many countries. In this sense, simplified devices with high sensitivity and specificity for local monitoring are enthusiastically pursued. In this work, we report for the first time the detection of ochratoxin A in coffee samples using a spoon-shaped waveguide immunosensor. The biosensor was built with the surface of the spoon-shaped waveguide covered by a 60 nm layer of gold to enable the SPR phenomenon. The measurements indicated a linear relationship between the change in the SPR phenomenon values and the OTA concentration in the range from 0.2 ppt to 5 ppt. When analyzed in coffee samples, the biosensor was highly selective and did not suffer matrix interference. The developed biosensor represents a promising analytical device for coffee quality analyses, as it is portable, simple, and suitable for onsite detection of target analytes.

Label-free electrochemical immunosensor for Ochratoxin a detection in coffee samples.

Ochratoxin A (OTA) is a nephrotoxic and carcinogenic mycotoxin frequently found in coffee, which directly impacts human health and the economy of many countries. For this reason, there has been a growing need for simple and sensitive tools for the on-site detection of this mycotoxin. In this study, we developed a label-free impedimetric immunosensor to detect OTA. The biosensor was built on a thin-film gold electrode evaporated on glass substrtes, modified with a self-assembled cysteamine monolayer and anti-OTA antibodies. Atomic force microscopy and Microspectroscopy RAMAN confirmed the successful functionalization of the electrodes. The biosensor performance was evaluated by electrochemical impedance spectroscopy and the measurements indicated a linear relationship between the change in the impedance values and the OTA concentration in the range from 0.5 to 100 ng mL-1 with a limit of detection of 0.15 ng mL-1. The biosensor was highly selective and did not suffer matrix interference when analyzed in coffee samples. Furthermore, considering the small sample volumes, the short time required for analysis, and the possibility of miniaturization, the developed biosensor represents a promising analytical device for on-site coffee quality analyses.

Localized Surface Plasmon Resonance-Based Nanosensor for Rapid Detection of Glyphosate in Food Samples.

In this study, we developed a biosensor based on the localized surface plasmon resonance (LSPR) phenomenon of gold nanoparticles (AuNPs) to detect the widely used herbicide glyphosate in food samples. To do so, either cysteamine or a specific antibody for glyphosate were conjugated to the surface of the nanoparticles. AuNPs were synthesized using the sodium citrate reduction method and had their concentration determined via inductively plasma coupled mass spectrometry. Their optical properties were analyzed using UV-vis spectroscopy, X-ray diffraction, and transmission electron microscopy. Functionalized AuNPs were further characterized via Fourier-transform infrared spectroscopy, Raman scattering, Zeta potential, and dynamic light scattering. Both conjugates succeeded in detecting the presence of glyphosate in the colloid, although nanoparticles functionalized with cysteamine tended to aggregate at high concentrations of the herbicide. On the other hand, AuNPs functionalized with anti-glyphosate functioned at a broad concentration range and successfully identified the presence of the herbicide in non-organic coffee samples and when it was added to an organic coffee sample. This study demonstrates the potential of AuNP-based biosensors to detect glyphosate in food samples. The low-cost and specificity of these biosensors make them a viable alternative to current methods for detecting glyphosate in foodstuffs.

Lateral flow assay applied to pesticides detection: recent trends and progress.

Devices based on lateral flow assay (LFA) have been gaining more and more space in the detection market mainly due to their simplicity, speed, and low cost. These devices have excellent sensing format versatility and make these strips an ideal choice for field applications. The COVID-19 pandemic boosted the democratization of this method as a "point of care testing" (POCT), and the trend is that these devices become protagonists for the monitoring of pesticides in the environment. However, designing LFA devices for detecting and monitoring pesticides in the environment is still a challenge. This is because analytes are small molecules and have only one antigenic determinant, which makes it difficult to apply direct immunoassays. Furthermore, most LFA devices provide only qualitative or semi-quantitative results and have a limited number of applications in multi-residue analysis. Here, we present the state of the art on the use of LFA in the environmental monitoring of pesticides. Based on well-documented results, we review all available LFA formats and strategies for pesticide detection, which may have important implications for the future of monitoring pesticides in the environment. The main advances, challenges, and perspectives of these devices for a direction in this field of study are also presented.

Impact of immobilization strategies on the activity and recyclability of lipases in nanomagnetic supports.

The use of enzymes immobilized on nanomagnetic supports has produced surprising results in catalysis, mainly due to the increase in surface area and the potential for recovery and reuse. However, the meticulous control of the process and difficulties in reproducibility have made industrial-scale applications unfeasible. Furthermore, the role of conjugation strategies in the catalytic activity and recycling of catalysts is unclear. Therefore, the objective of this study was to compare the conjugation of enzymes on nanomagnetic supports through physical adsorption (naked) or covalent bonding with mercaptopropyltrimethoxysilane (MPTS) and aminopropyltriethoxysilane (APTS) ligands. The free lipase obtained from Rhizomucor miehei was used as a model enzyme. Total protein and enzyme activity were determined using spectrophotometry (UV-Vis) and the p-nitrophenyl palmitate (p-NPP) hydrolysis method. The results indicated that a more significant enzyme surface loading does not always mean better immobilization success. The physical adsorption binding strategy had higher surface loading and low catalytic activity. On the other hand, covalent coupling with free NH2 had an excellent catalytic activity with very low surface loading. Finally, we show that recyclability can be improved with conjugation mediated by disulfide bonds. The findings presented here are essential for developing nanoconjugates with high enzymatic activity, which can guarantee the success of several industrial applications.

Physicochemical characterization of monazite sand and its associated bacterial species from the beaches of southeastern Brazil.

Beaches with monazitic sands show high natural radiation, and the knowledge of this radiation is fundamental to simulate the effects of natural terrestrial radiation on biological systems. Monazite-rich sand from a beach in the southeastern Brazil were collected and analyzed by X-ray fluorescence, X-ray diffraction, and magnetic susceptibility. The natural terrestrial radiation of the beach sand showed a positive correlation with the Th and Y elements, which are closely associated with Ce, Nd, Ca, and P, suggesting that this grouping is mainly associated with local natural radiation. Based on the sand characterization, a physical simulator of natural gamma radiation was built with parameters similar to those of the monazite beach sand, considering areas with high natural radiation levels. The simulation revealed that the natural radiation of the monazite sands has a significant effect on reducing the growth of the bacteria strains of E. coli and S. aureus present in the beach sand, with a reduction of 23.8% and 18.4%, respectively.

Nanomagnetic approach applied to microalgae biomass harvesting: advances, gaps, and perspectives.

Microalgae biomass is a versatile option for a myriad of purposes, as it does not require farmable land for cultivation and due of its high CO2 fixation efficiency during growth. However, biomass harvesting is considered a bottleneck in the process because of its high cost. Magnetic harvesting is a promising method on account of its low cost, high harvesting speed, and efficiency, which can be used to improve the results of other harvesting methods. Here, we present the state of the art of the magnetic harvesting method. Detailed approaches involving different nanomaterials are described, including types, route of synthesis, and functionalization, variables that interfere with harvesting, and recycling methods of nanoparticles and medium. In addition to discussing the overall perspectives of the method, we provide a guideline for future research.

Rapid and sensitive detection of ochratoxin A using antibody-conjugated gold nanoparticles based on Localized Surface Plasmon Resonance.

The regulation of tolerable levels of ochratoxin A in food for human and animal consumption has been defined in some countries. To meet these levels, simpler, more efficient, and faster analytical methods are being developed to facilitate the identification of this dangerous contaminant in food. Here, we combined gold nanoparticles (AuNPs) with anti-ochratoxin A (OTA) IgG to detect elementary levels of OTA based on Localized Surface Plasmon Resonance. AuNPs were prepared with trisodium citrate and characterized by UV-visible spectroscopy, X-ray, dynamic light scattering, and transmission electron microscopy. The conjugation of AuNPs to IgG anti-OTA was confirmed by bathochromic shift (UV-vis) and RAMAN spectroscopy. The sensitivity of the nanosensor was investigated by measuring LSPR band λmax shifts. Our results suggest this assay is highly sensitive, with a lower detection limit of about 0.001 pg mL-1. The LSPR nanosensor reduced detection limits by roughly 10 times compared to other methods. We demonstrated that the approach investigated here is a rapid and sensitive method for OTA detection.

Impact of conjugation strategies for targeting of antibodies in gold nanoparticles for ultrasensitive detection of 17ß-estradiol.

Antibody-coated nanoparticles have recently attracted considerable attention, with the focus falling on diagnostics. Nevertheless, controlled antibody bioconjugation remains a challenge. Here, we present two strategies of bioconjugation with the aim of evaluating the best approach for the coupling of antibodies on the surface of nanomaterials in an oriented way. We employed electrostatic interaction (physical adsorption) and covalent conjugation in the orientation of antibodies on the metallic surface as coupling methods, and their influence on the detection of 17ß-estradiol was addressed with localized surface plasmon resonance. The understanding of these mechanisms is fundamental for the development of reproducible inorganic bioconjugates with oriented surface as well sensibility of immunoassays.

Controlled biosynthesis of gold nanoparticles with Coffea arabica using factorial design.

Green synthesis of metallic nanoparticles has become incredibly popular, mainly by minimizing problems of environmental contamination and by being able to reduce, stabilize and potentially functionalize nanomaterials. Such compounds have possible applications in various areas, e.g., pharmaceuticals (drug delivery systems, cosmetics), textile industry (clothing with antimicrobial properties), diagnostic medicine (imaging, high efficiency biosensors), energy (solar panels), bioremediation, among others. However, the lack of reproducibility and information on the control mechanisms during synthesis have made the application of green-synthesized nanoparticles unfeasible. Thus, this study proposed the investigation of the main mechanisms affecting synthesis control, using factorial design for the preparation of gold nanoparticles with extract of Coffea arabica. We obtained stable (Zeta Potential, UV-vis and DLS), monodisperse, and quasi-spherical (TEM) nanoparticles, which presented adsorbed aromatic molecules (FTIR and RAMAN) and defined crystal structure (XRD), proving that the plant extract acted as a reducing agent, as well as a stabilizer and functionalizer for the synthesized nanostructures. The factorial design employed here to obtain gold nanoparticles with Coffea arabica extract allowed for a controlled and reproducible synthesis, enabling new possibilities for the application in several fields.

Worsening of Oxidative Stress, DNA Damage, and Atherosclerotic Lesions in Aged LDLr-/- Mice after Consumption of Guarana Soft Drinks.

BACKGROUND: Excessive consumption of soft drinks (SD) has become a health problem worldwide due to its association with related cardiovascular diseases. We investigated the possible impacts associated with the consumption of Brazilian guarana (normal and zero) SD in dyslipidemic mice, thus mitigating potential clinical confounders such as poor-quality diet, lifestyle, body composition, and/or comorbidities. METHODS: Sixteen-month-old LDLr-/- mice were divided into the following groups: (1) control; (2) GSD: normal guarana SD; and (3) Z-GSD: zero guarana SD. All were fed ad libitum, and blood pressure was measured noninvasively. After 8 weeks, aorta, blood, liver, and stomach samples were collected for histological and biochemical analyses. RESULTS: Guarana soft drinks increased atherosclerosis (~60%) and were associated with hypercholesterolemia, hypertension, oxidative stress, DNA fragmentation, and apoptosis (~2-fold) of blood cells, besides presenting an increase in liver and gastric damage even in normoglycemia. Interestingly, Z-GSD did not cause the aforementioned changes, except in hemodynamic and renal parameters. CONCLUSIONS: Chronic administration of GSD is prooxidative, compromising the cardiovascular, gastric, and hepatic systems; the effects are due at least in part to free sugar consumption but not to guarana extract per se.

Virola oleifera-capped gold nanoparticles showing radical-scavenging activity and low cytotoxicity.

The development of effective nanoparticle therapeutics has been hindered by their surface characteristics, such as hydrophobicity and charge. Therefore, the success of biomedical applications with nanoparticles is governed by the control of these characteristics. In this article, we report an efficient green capping method for gold nanoparticles (AuNPs) by a reduction with sodium citrate and capping with Virola oleifera (Vo), which is a green exudate rich in polyphenols and flavonoids. The Vo-capped AuNPs were characterized by UV, DLS, FTIR, Raman, TEM, DPPH, FRAP and their cytotoxicity was evaluated on the viability of Murine macrophage cell. The AuNPs had an average particle size of 15 nm and were stable over a long time, as indicated by their unchanged SPR and zeta potential values. These nanoparticles were assessed for their antioxidant potential using DPPH and FRAP and demonstrated the highest antioxidant activities and low cytotoxicity. We propose that the Virola oleifera-capped AuNPs have potential biomedical applications.

Extracellular biosynthesis of silver nanoparticles using the cell-free filtrate of nematophagous fungus Duddingtonia flagrans.

The biosynthesis of metallic nanoparticles (NPs) using biological systems such as fungi has evolved to become an important area of nanobiotechnology. Herein, we report for the first time the extracellular synthesis of highly stable silver NPs (AgNPs) using the nematophagous fungus Duddingtonia flagrans (AC001). The fungal cell-free filtrate was analyzed by the Bradford method and 3,5-dinitrosalicylic acid assay and used to synthesize the AgNPs in the presence of a 1 mM AgNO3 solution. They have been characterized by UV-Vis spectroscopy, X-ray diffraction, transmission electron microscopy, dynamic light scattering, Zeta potential measurements, Fourier-transform infrared, and Raman spectroscopes. UV-Vis spectroscopy confirmed bioreduction, while X-ray diffractometry established the crystalline nature of the AgNPs. Dynamic light scattering and transmission electron microscopy images showed approximately 11, 38 nm monodisperse and quasispherical AgNPs. Zeta potential analysis was able to show a considerable stability of AgNPs. The N-H stretches in Fourier-transform infrared spectroscopy indicate the presence of protein molecules. The Raman bands suggest that chitinase was involved in the growth and stabilization of AgNPs, through the coating of the particles. Our results show that the NPs we synthesized have good stability, high yield, and monodispersion.

Resin from Virola oleifera Protects Against Radiocontrast-Induced Nephropathy in Mice.

Contrast-induced nephropathy (CIN) is an iatrogenic medical event for which there is not yet a successful therapy. Increasing evidence in rodents has suggested that this disease is associated with renal tubular and vascular injury that is triggered directly by oxidative stress. In the present study, we evaluated whether the antioxidant resin from Virola oleifera (RV) could attenuate renal damage in an experimental mouse model of CIN. Adult male Swiss mice were divided into six groups and pre-treated orally with RV (10, 100 and 300 mg/kg), N-acetylcysteine (200 mg/kg) or vehicle for 5 days before the induction of CIN and Control group. Renal function was assessed by measuring plasma creatinine and urea levels. Additionally, renal oxidative stress and apoptosis/cell viability were determined with flow cytometry. Finally, kidney tissues were sectioned for histopathological examination. In this CIN model, pre-treatment with RV improved renal function, lowered the mortality rate, and reduced oxidative stress and apoptosis in both the medulla and cortex renal cells in a dose-dependent manner. Moreover, the RV treatment had beneficial effects on kidney histopathology that were superior to the standard treatment with N-acetylcysteine. These data suggest that because of its antioxidative and antiapoptotic effects and its ability to preserve renal function, resin from Virola oleifera may have potential as a new therapeutic approach for preventing CIN.