Generation of BSA-capsaicin Nanoparticles and Their Hormesis Effect on the Rhodotorula mucilaginosa Yeast

Abstract

Capsaicin is a chemical compound found in pungent chili peppers (Capsicum spp.). In biotechnology, capsaicin has been proposed as a pathogen control; however, its low solubility in water and high instability limits its uses. The aim of this work was to study the effect of high concentrations of capsaicin on the synthesis of nanoparticles and to evaluate their inhibitory effect on the growth of Rhodotorula mucilaginosa yeast. Bovine serum albumin (BSA)-capsaicin nanoparticles were formulated at 0, 16.2, 32.5, 48.7 and 65.0 µg of capsaicin per mg of BSA. Nanoparticle properties were evaluated and they were added to cultures of R. mucilaginosa to quantify their effect on cell viability. We found that increased capsaicin levels caused several changes to the physicochemical parameters, probably due to changes in the hydrophobicity sites of the albumin during the nanostructuration. The administration of nanoparticles to cultures of R. mucilaginosa produced a maximal viability with nanoparticles at 16.2 µg/mg; on the contrary, nanoparticles at 65.0 µg/mg caused maximal cell death. R. mucilaginosa cells displayed a hormesis effect in response to the nanoparticle dose concentration. The nanoparticles showed different responses during the uptake process, probably as a consequence of the nanostructural properties of capsaicin in the BSA molecules.

Keywords: Rhodoturola; bovine serum albumin; capsaicin; fungi; hormesis; nanoparticles.

Figure 1

BSA protein transformed into nanoparticles as a function of capsaicin concentration. The quantified protein showed a linear tendency (R = 0.8413) in relation to the concentration. Values are means of three experimental replicates ± standard deviations.

Figure 2

Encapsulated capsaicin in nanoparticles with respect to the concentration. The quantified capsaicin showed a logarithmic tendency (R = 0.9555). Values are means of three experimental replicates ± standard deviations.

Figure 3

FTIR characterization of the BSA–capsaicin pure molecules and the effect of the increment of capsaicin in the nanostructure formation. (a) FTIR spectral characterization of BSA and capsaicin; the scanning spectral range was between 650–4000 cm⁻¹. (b) Spectral graphs of the BSA-capsaicin nanoparticles at 0, 16.4, 32.5, 48.7 and 65.0 µg/mg, (lines in gradient of grey). The treatments showed between 3682 to 3104 cm⁻¹ a deformation of the peak that corresponds to N–H stretching (rectangle blue), and the second region is the amide I and amide II of the BSA with the hydrophobic side chain of the capsaicin, from 1710–1126 cm⁻¹.

Figure 4

TEM micrographs at high magnification show isolated nanoparticles with details in the morphology and nanostructure. TEM micrographs at low magnification show size and shape distribution of BSA-capsaicin nanoparticles at different capsaicin concentrations. All experiments in TEM were carried out at 80 kV high voltage (EHT) at 710,000× to 140,000× (high magnification), and at 8900× to 9000× (low magnification), working pressure 5 × 10⁻³ Pa (5 × 10⁻⁵ Torr).

Figure 5

Effective diameter of the isolated nanoparticles showed an increasing tendency with respect to the concentration of capsaicin. The equation of effective diameter (Ed) is also displayed. Conditions: digital measurement of n = 180 nanoparticle images at 1376 × 1032 pixels and 8 bits of compression; resolution set at 2.1 pixels/nm. Values are the means of three experimental replicates ± standard deviations.

Figure 6

Aspect ratio of the isolated nanoparticles showed a gradual transition from irregular dimension (X-Y axis) to circular shape and irregular aspect at high concentration of capsaicin. The equation of aspect ratio (Ar) is also displayed. Conditions: digital measurement of n = 180 nanoparticle images at 1376 × 1032 pixels and 8 bits of compression; resolution set at 2.1 pixels/nm. Values are the means of three experimental replicates ± standard deviations.

Figure 7

Shape factor of the isolated nanoparticles showed the best circular shape at 32.5 μg/mg of capsaicin concentration. Subsequently, the increase in capsaicin concentration caused a loss of shape. The equation of shape factor (Sf) is also displayed. Conditions: digital measurement of n = 180 nanoparticle images at 1376 × 1032 pixels and 8 bits of compression; resolution set at 2.1 pixels/nm. Values are the means of three experimental replicates ± standard deviations.

Figure 8

The AO/PI staining shows the live and death cells in all treatments. (a) Control treatment with buffer showed a basal growth of R. mucilaginosa. (b) Treatment with 0 µg/mg shows an increment of the cell population. (c) The maximal increment of viable population of R. mucilaginosa was observed at 16.2 µg/mg of capsaicin. (d) Treatment with 32.5 µg/mg causes a decrease in mature viable cells of R. mucilaginosa fluorescent in green channel and an increase in the young death cell in red channel. (e) At 48.7 µg/mg, the growth inhibition was observed in the mature cell. (f) The mature death cells were observed at 65.0 µg/mg and no young cells were observed.

Figure 9

Hormesis response of R. mucilaginosa cells in the presence of BSA-capsaicin nanoparticles administrated directly to the culture. The live cells showed maximal growth at 16.2 µg/mg and growth inhibition at 65.0 µg/mg (the red line shows the positive hormesis behaviour). The death cells showed minimal activity at 0 µg/mg and maximal activity of nanoparticles at 65.0 µg/mg (the blue line shows the negative hormesis behaviour).

Figure 10

Comparative spectra of fluorescence emission of rhodamine B staining. (a) Rhodamine B diluted in water at 1% showed a maximal peak emission at 600 nm. (b) Nanoparticles of BSA-capsaicin stained with rhodamine B showed a maximal peak emission at 575 nm.

Figure 11

BSA-capsaicin nanoparticle uptake by cultures of R. mucilaginosa. (a) Treatment with NaCl buffer did not show rhodamine B signal. (b) Treatment at 0 µg/mg showed nanoparticles aggregated on the surface of cells (see white arrow). (c) Accumulation of the BSA-capsaicin nanoparticles staining with rhodamine B into the R. mucilaginosa cell was observed at 65.0 μg/mg.