2-2--azino-di-(3-ethylbenzothiazoline)-6-sulfonic-acid and syringaldehyde

The present study aimed at preparing novel free-radical scavenging and water-soluble compounds derived from gelatin. Specifically, gelatin−syringaldehyde, gelatin−anisaldehyde, and gelatin−vanillin were synthesized and thoroughly studied for their physicochemical properties. In particular, the compounds were characterized by UV-Vis spectroscopy, Fourier-transform infrared spectroscopy, and scanning electron microscopy. Notably, as demonstrated by thermogravimetry and differential scanning calorimetry, all three derivatives exhibited higher thermal stability than gelatin itself. Free-radical scavenging activities of the examined compounds were explored by (i) a standard spectrophotometric ABTS assay and (ii) an assay of oxidative degradation of hyaluronic acid monitored by rotational viscometry. We found that gelatin and gelatin−syringaldehyde demonstrated the highest efficacy in scavenging •OH radicals, whereas gelatin−anisaldehyde was the least effective. The efficacy of scavenging alkyloxy- and alkylperoxy-type free radicals via hydrogen-atom-transferring property was in the following order: gelatin > gelatin−vanillin > gelatin−syringaldehyde > gelatin−anisaldehyde. Electron-donor properties determined using the ABTS assay revealed the following order in one-electron reduction of ABTS•+: gelatin > gelatin−anisaldehyde > gelatin−vanillin > gelatin−syringaldehyde.

Topics: Aldehydes; Free Radicals; Gelatin; Hyaluronic Acid; Hydrogen; Spectroscopy, Fourier Transform Infrared; Water

Laccases (LCs) are multicopper oxidases that find application as versatile biocatalysts for the green bioremediation of environmental pollutants and xenobiotics. In this study we elucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxin B₁ (AFB₁) and M₁ (AFM₁). LC enzyme was purified using three chromatographic steps and identified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performed in vitro at 25 °C for 72 h in buffer solution. AFB₁ degradation by Lac2 direct oxidation was 23%. Toxin degradation was also investigated in the presence of three redox mediators, (2,2'-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols, acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediated action of Lac2 with redox mediators univocally proved the correlation between Lac2 activity and aflatoxins degradation. The degradation of AFB₁ was enhanced by the addition of all mediators at 10 mM, with AS being the most effective (90% of degradation). AFM₁ was completely degraded by Lac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pure enzyme as capable of degrading AFB₁ and, for the first time, AFM₁, and on the evidence that the mechanism of an effective degradation occurs via the mediation of natural phenolic compounds. These results opened new perspective for Lac2 application in the food and feed supply chains as a biotransforming agent of AFB₁ and AFM₁.

Topics: Acetophenones; Aflatoxin B1; Aflatoxin M1; Benzaldehydes; Benzothiazoles; Biodegradation, Environmental; Food Microbiology; Fungal Proteins; Laccase; Oxidation-Reduction; Pleurotus; Proteolysis; Substrate Specificity; Sulfonic Acids; Time Factors