Production and Characterization of Exopolysaccharide From Newly Isolated Marine Probiotic Lactiplantibacillus plantarum EI6 With in vitro Wound Healing Activity

Abstract

Because of its safety, biological activities, and unique properties, exopolysaccharide (EPS) from lactic acid bacteria (LAB) has been developed as a potential biopolymer. A few studies have investigated the EPS produced by marine LAB. This study reports the wound healing activity of an EPS produced by a marine isolate identified as Lactiplantibacillus plantarum EI6, in addition to assessing L. plantarum EI6's probiotic properties. EI6 demonstrated promising antimicrobial activity against different pathogenic bacteria, as well as the ability to withstand stomach pH 3, tolerate 0.3% bile salt concentration, and exhibit no signs of hemolysis. Furthermore, EI6 was able to produce 270 mg/L of EPS upon growth for 48 h at 37°C in an MRS medium enriched with 1.0% of sucrose. The chemical features of the novel EI6-EPS were investigated: the UV-vis estimated a high carbohydrate content of ~91.5%, and the FTIR emphasized its polysaccharide nature by the characteristic hydroxyl, amide I, II, & III, and glycosidic linkage regions. The GC-MS and NMR analyses revealed the existence of five monosaccharides, namely, rhamnose, galactose, mannose, glucose, and arabinose, existing mainly in the pyranose form and linked together by α- and β-glycosidic linkages. EI6-EPS was found to be safe (IC50 > 100 μg/ml) and induced human skin fibroblasts (HSF) proliferation and migration. These findings imply that EI6 can be used as a safe source of bioactive polymer in wound care.

Keywords: Lactiplantibacillus; chemical characterization; exopolysaccharide; probiotics; wound healing.

Figure 1

Scanning electron microscope of isolate EI6 (A), and phylogenetic analysis of the marine isolate Lactiplantibacillus plantarum EI6 based on 16s rRNA gene sequence (B).

Figure 2

Effect of different pH values (A), and effect of different bile salts concentrations (0.0, 0.1, and 0.3%) (B) on the growth of L. plantarum EI6 at 37°C for 24 h.

Figure 3

FTIR spectrum (A), GC-MS chromatogram (B), ¹H NMR spectrum recorded at 323 K (2.0% w/v; DMSO-d₆, 500 MHz) (C), and SEM-EDX data of the produced exopolysaccharide (EI6-EPS) (D).

Figure 4

SEM images of the produced exopolysaccharide (EI6-EPS) at 2,000× (A), 9,000× (B), and 15,000× (C).

Figure 5

In vitro cell migration of skin fibroblasts by EI6-EPS scratch was created in monolayer of HSF cells and was treated with EI6-EPS. Control group was without any treatment. (A) Photographs of wound area treated with EI6-EPS at different time intervals were taken using an inverted microscope. (B) Percentage wound closure in control and treated cells at different time intervals.