Glyoxal

The CS-GE hydrogel was prepared by a protocol modified from previous reports [26 (link),54 (link)]. Briefly, 4% chitosan (chitosan glutamate salt, molecular weight: 200–600 kDa, degree of deacetylation: 75–90% w/v, in ddH₂O, NovaMatrix, Sandvika, Norway) and 10% gelatin (w/v, in ddH₂O, St. Louis, MO, USA) were prepared at 60 °C and sterilized by autoclaving. β-GP (28.5% w/v, in ddH₂O, Sigma) was prepared and filter sterilized. After 0.4 mL of 10% gelatin solution was blended with 2 mL of 4% chitosan solution and 0.6 mL ddH₂O homogeneously, 1 mL 28.5% β-GP solution was added drop-wise into the chitosan/gelatin solution with stirring. The CS-GE mixture was composed of 2% chitosan, 1% gelatin and 7.125% β-GP. The glyoxal solution was diluted with ddH₂O until the concentration reached 0.4%. A different volume of 0.4% glyoxal (Sigma) was subsequently added into the CS-GE mixture to reach a final concentration of glyoxal ranging from 0.0025% to 0.04% in the hydrogel.

The MG resins were prepared at M/G molar ratio: 1:6, according to the method of Deng et al. [13 (link)]. Glyoxal (40% water solution, Sigma-Aldrich, St.Louis, MO, USA) was placed in the three-neck flask and the pH was adjusted to 4.5 using 33% NaOH. Subsequently, melamine was added, and the mixture was heated to 60 °C for 1 h. The reaction mixture was cooled to room temperature ready for use.
Under the same reaction conditions, different parts of Glyoxal were substituted with glutaradehyde (50% water solution, Sigma Aldrich, St. Louis, MO, USA) to prepare MGG’ resins. The relative molar proportions of Glyoxal to glutaraldehyde are shown in Table 1 and Table 2.

Reagents of analytical grade were used as received from their commercial sources. Benzyl chloride was obtained from ACROS Organics (Morris Plains, NJ, USA), potassium hydroxide from Riendemann Schmidt (Kajang, Malaysia), carbon disulfide from Fisher Scientific (Chino, CA, USA) and copper (II) acetate from R&M Chemicals (Essex, UK). Hydrazine hydrate, iodomethane, glyoxal, 2,3-hexanedione, 2,3-heptanedione, 2,3-pentanedione, 2,3-butanedione and copper (II) chloride were obtained from Merck (Darmstadt, Germany). Absolute ethanol, 95% ethanol, dimethyl sulfoxide, acetone and dimethylformamide were obtained from RCI Labscan (Bangkok, Thailand). Elemental analysis was conducted via a Vario MACRO CUBE CHNS analyser (Germany), while IR spectra were recorded using a PerkinElmer FTIR spectrophotometer (Waltham, MA, USA). Melting points were measured using an Electrothermal IA9100 melting point apparatus (Essex, UK).
¹H NMR spectra were obtained via a Bruker FT-NMR 300 MHz (Germany). TLC was performed via 60F aluminium silica gel aluminium plates with a 254 nm fluorescent indicator from Merck (Darmstadt, Germany). UV-Vis spectroscopy was performed via a Biochrom UltroSpec 8000 spectrophotometer (Cambridge, UK).

The pH 13 soda bagasse black liquor was obtained from the Pars Company (Haft Tepe, Iran). Lignin extraction from the black liquor was done by acidifying with sulfuric acid. Maleic anhydride, Glyoxal, phenol, 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]), an ionic liquid, and other chemical materials used were acquired from Merck Co. (Kenilworth, NJ, USA).

Commercial samples of
foods (Table 1) were
purchased locally and from the internet and were processed and analyzed
as indicated below. l-Tryptophan, glyoxal (40% in water),
and methylglyoxal (40% in water) were obtained from Sigma-Aldrich
(Saint Louis, MO, USA). d-(+)-Glucose monohydrate was obtained
from Merck (Darmstadt, Germany), d-(−)-fructose from
Sigma-Aldrich, and 3-deoxy-d-glucosone from Biosynth-Carbosynth
(Compton, Newbury, UK). The β-carbolines derived from the reaction
of the α-dicarbonyl compounds glyoxal and methylglyoxal (Figure 1) with tryptophan
were prepared and characterized as follows:

Paraformaldehyde (Merck, #8.18715) was dissolved in PBS (pH 7.4) at a final concentration of 4%. The glyoxal fixative solution was prepared according to published protocol (Richter et al., 2018 (link)). Briefly, 28 ml of ddH₂O, 7.89 ml absolute of ethanol (analysis grade), 3.13 ml of glyoxal (Sigma-Aldrich, #128465), and 0.3 ml of acetic acid (Sigma-Aldrich, #A6283) were mixed well by vortex. After adjusting to pH 4.0 with 1 N of NaOH, the solution was filled up to 40 ml with ddH₂O. The final concentration of glyoxal was 3%. Both fixative solutions were prepared on the day of the experiment and kept cool until use.