Selenium-Fortified Kombucha-Pollen Beverage by In Situ Biosynthesized Selenium Nanoparticles with High Biocompatibility and Antioxidant Activity

Abstract

Biogenic selenium nanoparticles (SeNPs) have been shown to exhibit increased bioavailability. Fermentation of pollen by a symbiotic culture of bacteria and yeasts (SCOBY/Kombucha) leads to the release of pollen content and enhances the prebiotic and probiotic effects of Kombucha. The aim of this study was to fortify Kombucha beverage with SeNPs formed in situ by Kombucha fermentation with pollen. Response Surface Methodology (RSM) was used to optimize the biosynthesis of SeNPs and the pollen-fermented Kombucha beverage. SeNPs were characterized by Transmission electron microscopy energy-dispersive X-ray spectroscopy (TEM-EDX), Fourier-transform infrared spectroscopy (FTIR), Dynamic light scattering (DLS), and Zeta potential. The pollen-fermented Kombucha beverage enriched with SeNPs was characterized by measuring the total phenolic content, antioxidant activity, soluble silicon, saccharides, lactic acid, and the total content of Se⁰. The polyphenols were identified by liquid chromatography-mass spectrometry (LC-MS). The pollen and the bacterial (nano)cellulose were characterized by scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX), FTIR, and X-Ray diffraction (XRD). We also assessed the in vitro biocompatibility in terms of gingival fibroblast viability and proliferation, as well as the antioxidant activity of SeNPs and the pollen-fermented Kombucha beverage enriched with SeNPs. The results highlight their increased biological performance in this regard.

Keywords: antioxidant; biocompatible; biogenic nanoselenium; kombucha fermentation; response surface methodology; selenium-nanoparticles-enriched bee bread.

Figure 1

Three-dimensional representations of the influence of the three variables on the properties of pollen-fermented Kombucha beverage, as well as on maximizing the production of biogenic SeNPs: (a) TPC; (b) TFC; (c) HAT; (d) DPPH; (e) FRAP; (f) CUPRAC; (g) Silicon content; (h) Se⁰ biosynthesis yield; and (i) Se⁰ content. The color gradient corresponds to the range of the response variables (min to max), which are presented in Table S1. The dots highlight the experimental points.

Figure 2

Pearson correlation plot for the responses of the RSM experimental design.

Figure 3

Desirability: (a) 3D representation of the desirability function for maximizing Se⁰ yield and (b) 3D representation of the desirability function for maximizing Se⁰ yield and Se⁰ content. The color gradient corresponds to the range of the desirability function (min = 0 to max = 1). The dots highlight the predicted points.

Figure 4

Soluble silicon content; K—Kombucha beverage with 30 mL SCOBY; KPol5—Kombucha beverage with 30 mL SCOBY, 5 g pollen, and 190 mg sodium selenite; KPol15—Kombucha beverage with 30 mL SCOBY, 15 g pollen, and 190 mg sodium selenite; KPol25—Kombucha beverage with 30 mL SCOBY, 25 g pollen, and 190 mg sodium selenite (±error bars, α < 0.05, n = 3 for K, n = 2 for Kpol5, KPol25, and n = 5 for KPol15, different letters indicate statistically significant differences between samples).

Figure 5

Influence of Kombucha modulation on total phenolic content and antioxidant activity: (a) total phenolic content; (b) antioxidant activity. K—Kombucha beverage with 30 mL SCOBY; KPol—Kombucha beverage with 30 mL SCOBY, 15 g pollen, and 10 mg sodium selenite; KPol5—Kombucha beverage with 30 mL SCOBY, 5 g pollen, and 190 mg sodium selenite; KPol15—Kombucha beverage with 30 mL SCOBY, 15 g pollen, and 190 mg sodium selenite, KPol25—Kombucha beverage with 30 mL SCOBY, 25 g pollen, and 190 mg sodium selenite (±error bars, α < 0.05, n = 3 for K, n = 2 for KPol, Kpol5, KPol25, and n = 5 for KPol15, different letters indicate statistically significant differences between samples).

Figure 6

ATR-FTIR spectra of freeze-dried samples of SCOBY-Kombucha beverage and SeNPs: K—Kombucha beverage with 30 mL SCOBY; KPol—Kombucha beverage with 30 mL SCOBY, 15 g pollen, and 10 mg sodium selenite; KPol5—Kombucha beverage with 30 mL SCOBY, 5 g pollen, and 190 mg sodium selenite; KPol15—Kombucha beverage with 30 mL SCOBY, 15 g pollen, and 190 mg sodium selenite; KPol25—Kombucha beverage with 30 mL SCOBY, 25 g pollen, and 190 mg sodium selenite; SeNPs—SeNPs Kombucha.

Figure 7

ATR-FTIR spectrum of SeNPs compared with (a) Na₂SeO₃; (b) comparison of SeNPs spectra with those of gallic acid, catechin hydrate, and BSA.

Figure 8

TEM-EDX analysis of SeNPs: (a) TEM analysis; (b) EDX analysis.

Figure 9

Antioxidant activity of SeNPs by DPPH, FRAP, and CUPRAC assays (±error bars, α < 0.05, and n = 3, different letters indicate statistically significant differences between samples). The antioxidant activity was expressed as µM Trolox Equivalents (TE).

Figure 10

Dynamics of bacteria and yeast populations: (a) D-glucose and D-fructose content; (b) D-lactic acid and L-lactic acid content. K—Kombucha beverage with 30 mL SCOBY; KPol5—Kombucha beverage with 30 mL SCOBY, 5 g pollen, and 190 mg sodium selenite; KPol15—Kombucha beverage with 30 mL SCOBY, 15 g pollen, and 190 mg sodium selenite; KPol25—Kombucha beverage with 30 mL SCOBY, 25 g pollen, and 190 mg sodium selenite (±error bars, α < 0.05, n = 3 for K, n = 2 for Kpol5, KPol25, and n = 5 for KPol15, different letters indicate statistically significant differences between samples).

Figure 11

Biocompatibility of SeNPs: (a) CCK-8 assay (± error bars, α < 0.05, and n = 3, different letters indicate statistically significant differences between samples); (b–g) LIVE/DEAD assay (live cells—green fluorescence, dead cells—red fluorescence): (b) untreated cells; (C−; negative cytotoxicity control); (c) cells treated with 7.5% DMSO (C+; positive cytotoxicity control); (d) cells treated with 0.1 µg/mL SeNPs Kombucha; (e) cells treated with 0.5 µg/mL SeNPs Kombucha; (f) cells treated with 2.5 µg/mL SeNPs Kombucha; and (g) cells treated with 10 µg/mL SeNPs Kombucha.

Figure 12

Cell morphology–Fluorescence microscopy images after actin cytoskeleton labeling with Alexa Fluor 488-coupled phalloidin (green fluorescence) and the nuclei with DAPI (blue fluorescence): (a) untreated cells and (b) cells treated with 2.5 µg/mL SeNPs.

Figure 13

In vitro antioxidant activity of SeNPs: (a) Labeling and quantifying total ROS with H₂DCFDA(±error bars, α < 0.05, and n = 3, different letters indicate statistically significant differences between samples); (b–e) Fluorescence microscopy images after labeling total ROS with H₂DCFDA (green fluorescence): (b) untreated cells (C−; negative control); (c) cells treated with 37 µM H₂O₂ (C+; positive control; ROS inducer); (d) cells treated with 0.5 µg/mL SeNPs Kombucha in the presence of ROS inducer; and (e) cells treated with 2.5 µg/mL SeNPs Kombucha in the presence of ROS inducer.

Figure 14

Biocompatibility of Kombucha beverage: (a) CCK-8 assay (±error bars, α < 0.05, n = 3, *—σ between 0.05 and 0.01, **—σ between 0.01 and 0.001, and ***—σ < 0.001; black stars indicate statistically significant values that exceed C−; and red stars indicate statistically significant values that are below C−); (b–m) LIVE/DEAD assay (live cells—green fluorescence, dead cells—red fluorescence): (b) untreated cells (C−; negative cytotoxicity control); (c) cells treated with 7.5% DMSO (C+; positive cytotoxicity control); HGF-1 cells treated with: (d) 0.1 mg/mL K; (e) 1 mg/mL K; (f) 3 mg/mL K; (g) 5 mg/mL K; (h) 7 mg/mL K; (i) 0.1 mg/mL KPol25; (j) 1 mg/mL KPol25; (k) 3 mg/mL KPol25; (l) 5 mg/mL KPol25; (m) 7 mg/mL KPol25. K—Kombucha beverage with 30 mL SCOBY; KPol5—Kombucha beverage with 30 mL SCOBY, 5 g pollen, and 190 mg sodium selenite; KPol15—Kombucha beverage with 30 mL SCOBY,15 g pollen, and 190 mg sodium selenite; and KPol25—Kombucha beverage with 30 mL SCOBY, 25 g pollen, and 190 mg sodium selenite.

Figure 15

In vitro antioxidant activity of Kombucha beverage: (a) labeling and quantifying total ROS with H₂DCFDA (±error bars, α < 0.05, and n = 3, different letters indicate statistically significant differences between samples); (b–i) fluorescence microscopy images after labeling total intracellular ROS with H₂DCFDA (green fluorescence): (b) untreated cells (C−; negative control); (c) cells treated with 37 µM H₂O₂ (C+; positive control; ROS inducer); HGF-1 cells incubated in the presence of ROS inducer and (d) 3 mg/mL K; (e) 5 mg/mL K; (f) 7 mg/mL K; (g) 3 mg/mL KPol25; (h) 5 mg/mL KPol25; and (i) 7 mg/mL KPol25. K—Kombucha beverage with 30 mL SCOBY; KPol5—Kombucha beverage with 30 mL SCOBY, 5 g pollen, and 190 mg sodium selenite; KPol15—Kombucha beverage with 30 mL SCOBY, 15 g pollen, and 190 mg sodium selenite; KPol25—Kombucha beverage with 30 mL SCOBY, 25 g pollen, and 190 mg sodium selenite.

Figure 16

SEM-EDX analysis of fresh polyfloral pollen, and fermented pollen: (a) P; (b) EDX spectrum of P; (c) PK; (d) EDX spectrum of PK; (e) PK25; (f) EDX spectrum of PK25. P—fresh polyfloral pollen; PK—fermented pollen from KPol (30 mL SCOBY, 15 g pollen, and 10 mg sodium selenite); PK25—fermented pollen from KPol25 (30 mL SCOBY, 15 g pollen, and 190 mg sodium selenite).

Figure 17

ATR-FTIR spectra of main pollen samples and SeNPs: P is raw pollen; PL is lyophilized pollen; PK is fermented pollen with SCOBY and 10 mg Na₂SeO₃; PK25 is fermented pollen with 190 mg Na₂SeO₃; and SeNPs are the selenium nanoparticles.

Figure 18

Analysis of never-dried bacterial cellulose in terms of weight and topography: (a) Never-dried bacterial cellulose weight (±error bars, α < 0.05, n = 3, different letters indicate statistically significant differences between samples). (b) Topographical analysis of BCK, and BCKPol25; BCK—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY; BCKPol5—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY, 5 g pollen, and 190 mg sodium selenite; BCKPol15—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY, 15 g pollen, and 190 mg sodium selenite; and BCKPol25—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY, 25 g pollen, and 190 mg sodium selenite.

Figure 19

SEM-EDX analysis of BC: (a) BCK 100×; (b) BCK 1000×; (c) EDX spectrum of BCK; (d) BCKPol5 100×; (e) BCKPol5 1000×; (f) EDX spectrum of BCKPol5; (g) BCKPol15 100×; (h) BCKPol15 1000×; (i) EDX spectrum of BCKPol15; (j) BCKPol25 100×; (k) BCKPol25 1000×; (l) EDX spectrum of BCKPol25. BCK—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY; BCKPol5—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY, 5 g pollen, and 190 mg sodium selenite; BCKPol15—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY, 15 g pollen, and 190 mg sodium selenite; and BCKPol25—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY, 25 g pollen, and 190 mg sodium selenite.

Figure 20

XRD analyses of bacterial celluloses membranes (BC) compared with the corresponding bacterial nanocelluloses (BNC) obtained by purification and microfluidization: (a) Control samples (without pollen and sodium selenite): BCK—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY; BCKw—purified/washed bacterial cellulose; BNC1p—bacterial nanocellulose after 1 pass of microfluidization; BNC10pK—bacterial nanocellulose after 10 passes of microfluidization; BNC20pK—bacterial nanocellulose after 20 passes of microfluidization; (b) Samples from Kombucha modulation with pollen, and sodium selenite: BCPol25—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY, 25 g pollen, and 190 mg of sodium selenite; BCKPol25w—purified/washed bacterial cellulose; BNC1pKPol25—bacterial nanocellulose after 1 pass of microfluidization; BNC10pKPol25—bacterial nanocellulose after 10 passes of microfluidization; and BNC20pKPol25—bacterial nanocellulose after 20 passes of microfluidization. The red circle filled with yellow in Figure 20b highlights the necessary scale break due to the high intensity of 22.66° peak.

Figure 21

ATR-FTIR spectra: BCK—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY (control); BNC20pK—bacterial nanocellulose (control) after 20 passes of microfluidization; BCKPol25—bacterial cellulose from Kombucha beverage prepared with 30 mL SCOBY, 25 g pollen, and 190 mg of sodium selenite; and BNC20pKPol25—bacterial nanocellulose after 20 passes of microfluidization.