Development of Biological Coating from Novel Halophilic Exopolysaccharide Exerting Shelf-Life-Prolonging and Biocontrol Actions for Post-Harvest Applications

Abstract

The literature presents the preserving effect of biological coatings developed from various microbial sources. However, the presented work exhibits its uniqueness in the utilization of halophilic exopolysaccharides as food coating material. Moreover, such extremophilic exopolysaccharides are more stable and economical production is possible. Consequently, the aim of the presented research was to develop a coating material from marine exopolysaccharide (EPS). The significant EPS producers having antagonistic attributes against selected phytopathogens were screened from different marine water and soil samples. TSIS01 isolate revealed the maximum antagonism well and EPS production was selected further and characterized as Bacillus tequilensis MS01 by 16S rRNA analysis. EPS production was optimized and deproteinized EPS was assessed for biophysical properties. High performance thin layer chromatography (HPTLC) analysis revealed that EPS was a heteropolymer of glucose, galactose, mannose, and glucuronic acid. Fourier transform infrared spectroscopy, X-ray diffraction, and UV-visible spectra validated the presence of determined sugars. It showed high stability at a wide range of temperatures, pH and incubation time, ≈1.63 × 10⁶ Da molecular weight, intermediate solubility index (48.2 ± 3.12%), low water holding capacity (12.4 ± 1.93%), and pseudoplastic rheologic shear-thinning comparable to xanthan gum. It revealed antimicrobial potential against human pathogens and antioxidants as well as anti-inflammatory potential. The biocontrol assay of EPS against phytopathogens revealed the highest activity against Alternaria solani. The EPS-coated and control tomato fruits were treated with A. solani suspension to check the % disease incidence, which revealed a significant (p < 0.001) decline compared to uncoated controls. Moreover, it revealed shelf-life prolonging action on tomatoes comparable to xanthan gum and higher than chitosan. Consequently, the presented marine EPS was elucidated as a potent coating material to mitigate post-harvest losses.

Keywords: % disease incidence; antagonistic attributes; coating material; marine exopolysaccharide.

Figure 11

Analysis of the composition of EPS by (A) biochemical and spectral study, (B) HPTLC assay, and (C) FTIR analysis.

Figure 1

EPS producing halophiles selected after primary screening.

Figure 2

Antagonistic activity of EPS of TSIS01 by Agar diffusion assay and % growth inhibition by EPS against fungal phytopathogens. Five replicates were observed, and results were represented as mean ± SD which showed comparable outcome with positive control with no significant variation.

Figure 3

Characterization of TSIS01 by (A) microscopic (B) 16S rRNA sequence alignment, and (C) Phylogenetic tree revealed that the identified strain was Bacillus tequilensis MS01 that was highlighted.

Figure 4

EPS production optimization by analysis of (A) different media, (B) carbon sources and (C) nitrogen sources (for incubation time 48 h). Each experimentation was performed in triplicate and results were represented as mean ± SD.

Figure 5

Optimization of concentration of minimal media (A) starch (C-source), (B) beef extract (N-source), (C) K₂HPO₄, (D) MgSO₄ and (E) NaCl (%) for better EPS production at incubation time 48 h. Each experiment was performed in triplicate and results were represented as mean ± SD.

Figure 5

Optimization of concentration of minimal media (A) starch (C-source), (B) beef extract (N-source), (C) K₂HPO₄, (D) MgSO₄ and (E) NaCl (%) for better EPS production at incubation time 48 h. Each experiment was performed in triplicate and results were represented as mean ± SD.

Figure 6

Effect of water (D/W and seawater) on EPS production. Each experiment was performed in triplicate and results were represented as mean ± SD.

Figure 7

Optimization of physical parameters (A)Temperature, (B) pH, (C) Incubation time, and (D) shaking and static incubation conditions for higher EPS production. Each experiment was performed in triplicate and results were represented as mean ± SD.

Figure 8

Comparison between optimized and unoptimized culture conditions of EPS production for checking the effectiveness of optimization. The significant variation in O.D. at 600 nm and EPS production is denoted as ** for p < 0.01 and *** for p < 0.001.

Figure 9

Determination of phase of growth cycle ofTSIS01 for EPS production.

Figure 10

Analysis of EPS stability at different (A) Temperatures, (B) pH, and (C) Incubation time. The significant reduction in carbohydrate content was recorded at 200 °C (** p < 0.01) temperature as compared to room temperature.

Figure 12

Analysis of EPS properties viz. (A) XRD analysis, (B) rheological behavior.

Figure 13

Analysis of biological activities viz. (A) antimicrobial activity against (a) E.coli, (b) S. aureus, (c) P. valgaris, (d) B. Cereus, (e) S. typhi, (f) Nocardia sp., (B) antioxidant, and (C) anti-inflammatory actions of EPS (D) Cytotoxic activity assay. % viable cells are higher in EPS inoculated tubes in concentration dependent manner compared to control tubes and extent of variation is denoted as * for p < 0.05, ** for p < 0.01, *** for p < 0.001.

Figure 14

Effect of EPS coating on (A) firmness and (B) weight loss (%) of tomato fruits. Comparative statistical analysis revealed significant variation (p < 0.05) in chosen parameters for periodic incubation in case of control or uncoated fruits and marked as * superscript. * p < 0.05, ** p < 0.01.

Figure 15

Analysis of % disease incidence in uncoated (UC), EPS-coated (EC), chitosan (CC) and xanthan gum (XC)-coated tomato fruits after treatment of phytopathogenic fungus A. solani. The highest variation reported in uncoated (UC) tomatoes than coated fruits (p < 0.001) and denoted as *** superscript.