Waste to Wealth Approach: Improved Antimicrobial Properties in Bioactive Hydrogels through Humic Substance-Gelatin Chemical Conjugation

Abstract

Exploring opportunities for biowaste valorization, herein, humic substances (HS) were combined with gelatin, a hydrophilic biocompatible and bioavailable polymer, to obtain 3D hydrogels. Hybrid gels (Gel HS) were prepared at different HS contents, exploiting physical or chemical cross-linking, through 1-ethyl-(3-3-dimethylaminopropyl)carbodiimide (EDC) chemistry, between HS and gelatin. Physicochemical features were assessed through rheological measurements, X-ray diffraction, attenuated total reflectance (ATR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and scanning electron microscopy (SEM). ATR and NMR spectroscopies suggested the formation of an amide bond between HS and Gel via EDC chemistry. In addition, antioxidant and antimicrobial features toward both Gram(-) and Gram(+) strains were evaluated. HS confers great antioxidant and widespread antibiotic performance to the whole gel. Furthermore, the chemical cross-linking affects the viscoelastic behavior, crystalline structures, water uptake, and functional performance and produces a marked improvement of biocide action.

Figure 1

Torque as a function of time during a DTST at 30 °C by fixing ω = 10 rad/s and γ = 5% for gelatin samples (see legend for details). Symbols are experimental data, and lines are fit obtained with eq 4.

Figure 2

Viscous and elastic moduli as a function of frequency at 30 °C for (a) Gel, (b) Gel HS 3-EDC, (c) Gel HS 8-EDC, and (d) Gel HS 16-EDC. Error bars are shown along with data at different frequencies, as a result of the standard error of multiple measurements (often smaller than data symbols).

Figure 3

Gel strength (measured as the G′ value evaluated at 30 °C at a fixed frequency of 1 rad/s) as a function of relative percentage of HS in solution. Error bars are shown along with the data, as a result of the standard error of multiple measurements.

Figure 4

XRD analysis on physical gels (panel A) and EDC-cross-linked gels (panel B).

Figure 5

FTIR analysis on physical gels and EDC-cross-linked gels.

Figure 6

¹³C NMR solid-state spectra of various composite components and gelatin-cross-linked composite blends.

Figure 7

Relaxation times (t1ρH) of Gel HS and Gel HS-cross-linked blends determined by VSL 13C NMR experiments.

Figure 8

SEM images of (A) HS; (B) HS EDC; (C) Gel EDC; (D) Gel HS 3-EDC; (E) Gel HS 8-EDC; and (F) Gel HS 16-EDC.

Scheme 1. Chemical Cross-Linking between Activated Carboxyl Groups of HS and Gelatin by Means of the EDC/NHS Complex

Figure 9

Antibacterial activity of gelatin-based physical and chemical gels combined with HS against Gram-negative and Gram-positive bacterial strains. The error bars indicate the standard error (n = 3); the standard deviation was less than 5%.

Figure 10

Swelling analysis on physical gels (A) and chemical gels (B).