Novel Nanomaterials Made of Humic Substances from Green Composts and Chitosan Exerting Antibacterial Activity

Green compost represents a sustainable source of humic substances (HS), which are supramolecular associations of heterogeneous natural compounds bearing a variety of functional groups. Novel humic-chitosan nanoconjugates (NC) were synthesized by using HS from three green composts [coffee husks (HS-COF), artichoke residues (HS-CYN), and fennel residues (HS-FEN)]. Solid-state NMR spectra showed that HS-CYN and HS-FEN contained the largest relative amount of polysaccharidic and phenolic carbons, whereas HS-COF had the greatest relative content of alkyl and carboxyl groups. While size and z-potential of NC depended upon the HS origin, NC-HS-FEN revealed the greatest thermal stability, followed by NC-HS-CYN and NC-HS-COF. Scanning electron microscopy and transmission electron microscopy analyses suggested the formation of spherical nanoparticles only for NC-HS-FEN and NC-HS-CYN. This was explained by more stable conformations of HS-FEN and HS-CYN that enabled regular spherical nanoparticles, as assessed by high-pressure size-exclusion chromatography measurements. NC bioactivity against bacterial human pathogens was strain-specific, and the inhibition of bacterial development was attributed to the positive z-potentials that facilitated NC adhesion on bacterial cell walls, smaller nanosizes favoring cell penetration, and subsequent release in the cell of NC toxic components that altered microbial biochemical functions. This study indicates that not only novel NCs can be obtained by humic matter derived from recycled biomass, such as green compost, but they can also be employed as effective and sustainable antimicrobial agents

Additional file 1 of Antibacterial and antioxidant properties of humic substances from composted agricultural biomasses

Additional file 1. Table S1. List of the main products released by the thermochemolysis from humic substances from artichoke (HS-CYN), coffee grounds (HS-COF) and pepper (HS-PEP). Fig. S1. Total ion chromatograms of thermochemolysis products of humic substances from artichoke (HS-CYN), coffee grounds (HS-COF) and pepper (HS-PEP)

Antioxidant activity of HS-FEN at different concentration (50, 30, 25 μg mL^-1).

Vertical bars represent the standard deviation (s.d.). Different capital letters indicate significant differences according to Tukey test (p ≤ 0.05).</p

Relative distribution (%) of signal areas over chemical shift regions (ppm) and structural indexes^a in ¹³C CPMAS-NMR spectrum of HS.

Relative distribution (%) of signal areas over chemical shift regions (ppm) and structural indexesa in 13C CPMAS-NMR spectrum of HS.</p

S1 Dataset -

Helicobacter pylori (H. pylori) is a common human pathogen causing inflammation. Recent studies have suggested a sophisticated interplay between mitochondria, innate immunity and inflammatory response, thus proposing mitochondrial disfunction as the hallmark of severe inflammatory disorders. In this study, humic substances isolated from composted fennel residues (HS-FEN) were tested as potential therapeutical strategy to restore the mitochondrial physiology and control the inflammation associated with H. pylori infection. The molecular features of HS-FEN were characterized by infrared spectrometry, thermochemolysis-GC/MS, NMR spectroscopy, and high-performance size-exclusion chromatography (HPSEC), which revealed the presence of aromatic polyphenolic components arranged in a rather stable conformation. In vitro results showed antioxidant and anti-inflammatory properties of HS-FEN, that was found to increase the expression level of OPA-1 and SOD-2 genes and in AGS cells stimulated with H. pylori culture filtrate (Hpcf) and concomitantly decrease the expression level of Drp-1 gene and IL-12, IL-17 and G-CSF proteins. The hydrophobic features of HS, their conformational arrangement and large content of bioactive molecules may explain the beneficial effects of HS-FEN, that may potentially become an interesting source of anti-inflammatory agents capable to counteract or prevent the H. pylori-related inflammatory disorders.</div

Relative yield (%) of main thermochemolysis products a released from HS-FEN.

Relative yield (%) of main thermochemolysis products a released from HS-FEN.</p

Total ion chromatograms of thermochemolysis products of HS-FEN.

Total ion chromatograms of thermochemolysis products of HS-FEN.</p

¹³C-CPMAS-NMR spectrum of HS-FEN.

Helicobacter pylori (H. pylori) is a common human pathogen causing inflammation. Recent studies have suggested a sophisticated interplay between mitochondria, innate immunity and inflammatory response, thus proposing mitochondrial disfunction as the hallmark of severe inflammatory disorders. In this study, humic substances isolated from composted fennel residues (HS-FEN) were tested as potential therapeutical strategy to restore the mitochondrial physiology and control the inflammation associated with H. pylori infection. The molecular features of HS-FEN were characterized by infrared spectrometry, thermochemolysis-GC/MS, NMR spectroscopy, and high-performance size-exclusion chromatography (HPSEC), which revealed the presence of aromatic polyphenolic components arranged in a rather stable conformation. In vitro results showed antioxidant and anti-inflammatory properties of HS-FEN, that was found to increase the expression level of OPA-1 and SOD-2 genes and in AGS cells stimulated with H. pylori culture filtrate (Hpcf) and concomitantly decrease the expression level of Drp-1 gene and IL-12, IL-17 and G-CSF proteins. The hydrophobic features of HS, their conformational arrangement and large content of bioactive molecules may explain the beneficial effects of HS-FEN, that may potentially become an interesting source of anti-inflammatory agents capable to counteract or prevent the H. pylori-related inflammatory disorders.</div

HS-FEN subverts detrimental effects elicited by Hpcf.

Cellular expression of OPA-1 (a), Drp-1 (b) and SOD2 (c) genes detected by quantitative PCR performed on RNA extracted from AGS cells cultured for 3 hours with HS-FEN 25 μg mL-1 in presence or absence of Hpcf (1:2) stimulation. GAPDH was used as housekeeping gene to normalize all samples. Data were represented as means ± s.d. of three independent experiments, each performed in triplicate. One-way ANOVA followed by Bonferroni post hoc correction was used to determinate statistically significant differences (*** p <0.0001).</p

Weight average (Mw) and number average (Mn) molecular weights, and polydispersity (P), as calculated from UV-detected HPSEC chromatograms for HS-FEN, before and after addition of acetic acid (AcOH).

Weight average (Mw) and number average (Mn) molecular weights, and polydispersity (P), as calculated from UV-detected HPSEC chromatograms for HS-FEN, before and after addition of acetic acid (AcOH).</p