Optimization and Characterization of a Novel Exopolysaccharide from Bacillus haynesii CamB6 for Food Applications

Abstract

Extremophilic microorganisms often produce novel bioactive compounds to survive under harsh environmental conditions. Exopolysaccharides (EPSs), a constitutive part of bacterial biofilm, are functional biopolymers that act as a protecting sheath to the extremophilic bacteria and are of high industrial value. In this study, we elucidate a new EPS produced by thermophilic Bacillus haynesii CamB6 from a slightly acidic (pH 5.82) Campanario hot spring (56.4 °C) located in the Central Andean Mountains of Chile. Physicochemical properties of the EPS were characterized by different techniques: Scanning electron microscopy- energy dispersive X-ray spectroscopy (SEM-EDS), Atomic Force Microscopy (AFM), High-Performance Liquid Chromatography (HPLC), Gel permeation chromatography (GPC), Fourier Transform Infrared Spectroscopy (FTIR), 1D and 2D Nuclear Magnetic Resonance (NMR), and Thermogravimetric analysis (TGA). The EPS demonstrated amorphous surface roughness composed of evenly distributed macromolecular lumps. GPC and HPLC analysis showed that the EPS is a low molecular weight heteropolymer composed of mannose (66%), glucose (20%), and galactose (14%). FTIR analysis demonstrated the polysaccharide nature (-OH groups, Acetyl groups, and pyranosic ring structure) and the presence of different glycosidic linkages among sugar residues, which was further confirmed by NMR spectroscopic analyses. Moreover, D-mannose α-(1→2) and α-(1→4) linkages prevail in the CamB6 EPS structure. TGA revealed the high thermal stability (240 °C) of the polysaccharide. The functional properties of the EPS were evaluated for food industry applications, specifically as an antioxidant and for its emulsification, water-holding (WHC), oil-holding (OHC), and flocculation capacities. The results suggest that the study EPS can be a useful additive for the food-processing industry.

Keywords: Bacillus; additive; antioxidants; emulsification; exopolysaccharide; flocculation; food industry; oil holding; water holding.

Figure 1

Map image showing the location of the Campanario hot spring located in the Andean Mountains in the Maule region, Chile, and photographs of the study site. On the right, the maximum likelihood phylogeny of isolate CamB6 (filled red circle) shows similarity with B. haynesii (outlined red circle).

Figure 2

(A) Normal plot of residuals; (B) Predicted versus actual plot for EPS production.

Figure 3

Response surface plot (2-D) interaction effect expressed, respectively, in X1 and X2 axes. Here, inputs are 30 experimental runs carried out under conditions established by CCD matrix. EPS production as function with (a) Glucose (g L⁻¹) and yeast extract (g L⁻¹) (Actual factors C. pH = 7.00, D. Inoculum size = 30.00 mL L⁻¹); (b) Glucose (g L⁻¹) and pH (B. Yeast extract = 30.00 g L⁻¹, D. Inoculum size = 30.00 mL L⁻¹); (c) Glucose (g L⁻¹) and inoculum size (mL L⁻¹) (B. Yeast extract = 30.00 g L⁻¹, C. pH = 7.00); (d) Yeast extract (g L⁻¹) and pH (A. Glucose = 30.00 g L⁻¹, D. Inoculum size = 30.00 mL L⁻¹); (e) Yeast extract (g L⁻¹) and inoculum size (mL L⁻¹) (A. Glucose = 30.00 g L⁻¹, C. pH = 7.00), and (f) pH and inoculum size (mL L⁻¹) (A. Glucose = 30.00 g L⁻¹, B. Yeast extract = 30.00 g L⁻¹).

Figure 4

Surface morphology of the EPS produced by B. haynesii CamB6, where (A) ×3000 (B) ×5000; and (C) ×10,000, respectively; (D) 2D; and (E) 3D AFM images, and (F) size distribution patterns.

Figure 5

(A) HPLC analysis of the constituting monosaccharides, and (B) GPC analysis of the EPS.

Figure 6

FTIR-ATR spectra of CamB6 isolated polysaccharide.

Figure 7

(A) 1H NMR spectra (B) 13C NMR spectrum (106-58 ppm amplified zone plus carbonyl signal) of EPS, (C) HSQC spectrum (anomeric zone) of EPS obtained at 60 °C in 2H₂O, and (D) Edited gradient-enhanced HSQC spectrum of ESP, showing A: anomeric region, B: CH groups, C: CH position 2 (linkage position) on α-D-Manp and β-D-Manp, D: CH2 groups in position 6.

Scheme 1

Predicted glycosidic bonds in the EPS structure obtained from the heteronuclear 1H-13C HSQC spectrum.

Figure 8

Thermogravimetric curves of Camb6 exopolysaccharide.

Figure 9

In vitro antioxidant capability of the EPS produced by B. haynesii CamB6; where (A) DPPH radical scavenging activity; (B) ABTS radical scavenging activity; (C) H₂O₂ radical scavenging activity; and (D) Bioflocculation activity of the EPS.