Production of melanin from the larvae of the Black Soldier Fly
The study of melanin
The study of melanin: a comparison of the content of paramagnetic centers, antioxidant activity and sorption properties of between a dry sample of melanin “Bioprogress” (hereinafter BP) and a solution of melanin-substance (hereinafter SUB)
The melanin has been compared in its properties with the well-known commercial melanin obtained from Antarctic yeast (hereinafter astromelanin). We studied absorption spectra, fluorescence spectra of degradation products by potassium superoxide, electron paramagnetic resonance (EPR) spectra, antiradical activity in terms of the effect on the chemiluminescence amplitude of luminol, and sorption activity in binding the organic dye methylene blue (MB). The absorption spectra, fluorescence, and chemiluminescence were measured on a Shimadzu UV-1601 PC spectrophotometer (Japan) and a Shimadzu-5301 PC spectrofluorimeter (Japan). EPR spectra were recorded on a Bruker EMX EPR spectrometer (Germany).
BP quickly and almost completely dissolved in potassium phosphate buffer (concentration 0.1 M, pH 7.4). The maximum concentration of the MN used in the experiments was 8 mg / ml. Unfortunately, the exact concentration of the dissolved SUB is not known, so some data for this melanin have not been calculated.
The absorption spectrum of the BP in the buffer is a line almost identical to the spectrum of astromelanin with the same absorption maxima in the region of 280 nm, which is characteristic of protein impurities (Fig. 1). At the same time, the SUB melanin spectrum has very low absorption in the long-wavelength region and significant absorption in the short-wavelength region with a maximum at about 265 nm. Thus, it differs significantly from the spectra of astromelanin and BP and is not typical for polymer melanins.
Comparison of the fluorescence characteristics of the two melanin showed the following. SUB exhibited initially high fluorescence intensity at the excitation wavelength of 470 nm, which did not increase upon oxidation of the melanin with potassium superoxide (Fig. 2, curves 1 and 2). The BP initially exhibited a significantly lower fluorescence intensity, which strongly increased upon oxidation with potassium superoxide (Fig. 2, curves 3 and 4). Since the observed fluorescence spectrum is characteristic of low molecular weight products of oxidative decomposition of melanin, it can be assumed that their content is significant in comparison with polymer melanin in SUB.
Fig. 2. Comparison of the fluorescence of two melanin before and after the reaction with potassium superoxide. 1 – fluorescence of the initial solution of melanin substance (SUB), 2 – also after its oxidation with superoxide, 3 – fluorescence of the initial melanin solution “Bioprogress” (BP), and 4 – also after reaction with superoxide
EPR studies have shown that BP is identical in its main parameters to astromelanin, but the concentration of paramagnetic centers is approximately three times lower (Fig. 3 and table).
Fig. 3. EPR spectra of BP melanin and astromelanin
EPR parameters table
SUB, measured in liquid form in a capillary, did not give a reliable EPR signal.
A study of the antiradical activity of BP and SUB showed that they exhibit a fairly good ability to quench the chemiluminescence of luminol induced by free radicals. The study of the dependence of the quenching of luminol chemiluminescence on the concentration of melanin confirms that astromelanin is a somewhat more effective antioxidant. This follows from the dependence of the decrease in the amplitude of chemiluminescence on the concentration of melanin (Fig. 5), which shows a comparison of the antioxidant activity of BP and astromelanin. According to these results, astromelanin is about 4 times more effective than BP melanin at quenching luminol chemiluminescence per unit weight of the drug.
Concentration, μg / ml
Fig. 4. Comparison of the AOA activity of astromelanin and melanin BP.
The BP adsorbed methylene blue (MG) dye, and the sorption efficiency was high. 1 gram of BP was maximally able to bind about 300 µg of the dye (Fig. 5). True, astromelanin was also slightly better in terms of sorption and could bind to a maximum of about 550 μg of the dye.
1 / MB added x 10-3 , ml / mk
Fig. 5. Binding of the dye methylene blue by melanin BP versus astromelanin
Summary. Melanin sample BP is readily soluble in buffer solutions, exhibits good antiradical activity and sorption capacity, and is typical melanin in its EPR and spectral characteristics. Melanin sample SUB does not exhibit an EPR signal and appears to contain a large amount of low molecular weight products.