P-Nitrophenol (pNP) is a widely used compound for analytical determinations of several esterases (EC. 3.1.1.X), including lipases (E.C. 3.1.1.3). Most enzymatic measurements employ pNP derivatives such as esters, which are broken down by enzymatic hydrolysis, releasing pNP that is quantified by its absorbance at 410 nm. Although this type of methods was developed a few decades ago, the spectrophotometric analysis of pNP requires analytical measurements of pH and temperature to achieve reliable determinations. The aim of this paper is to offer a graphical update of how pH and temperature affect the p-nitrophenol absorbance at different wavelengths in lipase emulsified media, due to its relevance for the quantitative determination of lipase activity using spectrophotometric methods. To highlight the importance of each variable involved in this analysis, we dissolved pNP in emulsified media (for lipase activity quantification) at several pH values from 4.00 to 11.00, and measured its absorbance in a range of 270 nm – 500 nm and at several temperatures from 25°C to 50°C. The absorption patterns of pNP under the established conditions were graphed in 3D plots. The constructed 3D plots showed that, regardless of the temperature, below pH 6.00, pNP predominantly absorbs at 317 nm, due to the greater abundance of its protonated form, which is completely predominant at pH 3.50 and below. On the other hand, at pH 10.0 and above, the major absorption occurs at about 401 nm, confirming that the equilibrium is completely shifted to the pNP anionic form. These results also indicate that close to neutral pH value pNP, it displays a temperature dependence effect, increasing absorbance to 410 nm at higher temperatures. Due to many analytical determinations of enzymatic activities, the release of pNP is carried around pH 7.00. It is necessary to consider the determinant role of both pH and temperature over these measurements, how these variables must be strictly controlled, and how the calibration curves and blanks should take the reaction media pH and temperature into account.

Coffee wastes have large amounts of by-products rich in phenolic compounds such as chlorogenic and caffeic acid, with potential applications for developing fine chemicals such as caffeic acid phenethyl ester (CAPE). A screening for microorganisms was undertaken in a coffee plantation environment to isolate native tropical species able to modify secondary metabolites present in this kind of biomass enzymatically. From the screening, 130 fungal strains could grow in lipase inducer media. Fungal strains were identified via ITS-based sequencing. Classification based on BLAST assigned 51 isolates to 12 different genera, including Absidia, Aspergillus, Cunninghamella, Fusarium, Metarhizium, Meyerozyma, Mucor, Neocosmospora, Papiliotrema, Penicillium, Rhizopus, and Trichoderma. DNA sequencing identified 14 putative extracellular lipases. According to the extracellular lipase activity, the most promising strain was identified as Fusarium sp. by DNA barcoding. Extracellular lipases from this strain exhibited maximal hydrolytic activity at a temperature of 45 °C, a pH of 7.00, and 200 ppm of NaCl, with an affinity towards substrates having carbon chain lengths of 8 or longer. Under these conditions, lipase instead of esterase activity is the main feature. The Km and Vmax values calculated using p-nitrophenyl palmitate (pNPP) as hydrolysis substrate were 0.003 mM and 299.8 μmol min⁻¹ mg⁻¹, respectively. Fusarium sp. lipases presented high stability during freeze–thawing, allowing the storage of enzyme solutions at −20 °C, but not as a lyophilized powder. According to our kinetic study, these lipases catalyzed CAPE hydrolysis, showing a progressive decrease in the concentration of the CAPE and a correspondent increase in the caffeic acid concentration as a product of this hydrolysis. Being able to carry out this type of reaction under mild conditions shows that Fusarium sp. lipases recognize CAPE as substrate and suggest CAPE synthesis (reverse reaction) and transformation can be engineered, using caffeic acid from coffee biomass, as a potential industrial application for these lipases.

This study reports for the first time the use of chitosan aerogels loaded with gentamicin sulfate as a model drug for biphasic release in wound dressings. Bacterial infections represent one of the main complications following injuries or burns, accounting for 75% of the mortality associated with such conditions. For this reason, the fundamental purpose of dressing is to restore the necessary environment for the healing of the injury. In this study, chitosan aerogels with gentamicin sulfate were developed using the lyophilization technique, incorporating antibiotic concentrations between 0.65 and 1.50 mg/ml. The dressings obtained showed an absorption capacity of 83%–92% of the exudate simulation buffer, which shows their swelling and liquid retention capacity. The drug release profiles showed a biphasic pattern, a rapid initial release, followed by a sustained phase, reaching between 70% and 80% release in 60 min. In vitro antimicrobial assays confirmed the efficacy of the dressings in inhibiting the growth of Staphylococcus aureus and Pseudomonas aeruginosa, with greater effectiveness at higher concentrations of the antibiotic (inhibition zone diameters up to ≈43 mm for S. aureus). Therefore, the developed dressing combines structural properties, fluid absorption, controlled release of a model molecule, and antimicrobial protection, presenting a promising option for wound treatment.

Agricultural by-products are increasingly recognized as sustainable resources for mushroom cultivation and biomass valorization. This study evaluated pineapple stubble and coffee pulp as functional substrate supplements for Cordyceps militaris cultivation. Fruiting bodies were successfully produced on brown rice substrates supplemented with 5–15% pineapple stubble or coffee pulp, either alone or in combination. Substrate composition markedly influenced fungal morphology, biological efficiency (BE), and cordycepin production. Moderate supplementation improved cultivation performance, whereas excessive supplementation reduced BE. Cordycepin accumulation was generally higher in the substrate than in the fruiting bodies. The highest cordycepin content was observed in the substrate supplemented with 15% coffee pulp, reaching 3.93%, approximately twofold higher than that of the brown rice control. Correlation analysis revealed that carbon content and the carbon-to-nitrogen (C/N) ratio were positively associated with BE, while nitrogen content was positively correlated with cordycepin accumulation in the substrate. In addition, coffee pulp extract exhibited notable adenosine deaminase inhibitory activity, suggesting that substrate-derived bioactive compounds may contribute to enhanced cordycepin accumulation during cultivation. After cultivation, the spent mushroom substrate showed reduced C/N ratios and increased nitrogen content, indicating active fungal metabolism-mediated substrate transformation. These findings demonstrate that coffee and pineapple by-products can serve as sustainable supplements to improve cordycepin production in C. militaris. Furthermore, the resulting spent substrate, enriched in nitrogen and bioactive compounds, may represent a valuable secondary resource for agricultural and industrial applications.