Structural elucidation and cytotoxic analysis of a fructan based biopolymer produced extracellularly by Zymomonas mobilis KIBGE-IB14

Highlights

• Levan was produced and characterized in detail using indigenous isolate.

• This biopolymer possesses high water solubility and low water holding capacity.

• It exhibited non-cytotoxic effect against murine cell line.

Abstract

Fructan based biopolymers have been extensively characterized and explored for their potential applications. Linear chained biopolymers, like levan-type fructan, have gained attention because they have exhibited unconventional stretchable and unbendable properties along with biodegradable and biocompatible nature. Current study deals with the chemical characterization and cytotoxic analysis of fructose based exopolysaccharide that was extracellularly produced by an indigenously isolated bacterial species (Zymomonas mobilis KIBGE-IB14). Maximum yield of exopolysaccharide (44.7 gL^-1) was attained after 72 h of incubation at 30 °C under shaking conditions (180 rpm) when the culture medium was supplemented with 150.0 gL^-1 of sucrose as a sole carbon source. This exopolysaccharide displayed high water solubility index (96.0%) with low water holding capacity (17.0%) and an intrinsic viscosity of about 0.447 dL g⁻¹. This biopolymer exhibited a characteristic linear homopolysaccharide structure of levan when characterized using Fourier Transform Infrared (FTIR), Nuclear Magnetic Resonance (NMR) spectroscopy (¹H, ¹³C, TOCSY and NOESY) while, Atomic Force Microscopy (AFM) revealed its pointed and thorny structure. The decomposition temperature of levan was approximately 245 °C as revealed by Thermal Gravimetric Analysis (TGA). X-Ray Diffraction (XRD) results revealed its amorphous nature with crystalline phase. Cytotoxicity of different concentrations of levan was investigated against mouse fibroblast cell lines by measuring their cellular metabolic activity and it was noticed that a higher concentration of levan (2.0 mg ml⁻¹) permitted the normal cell growth of NIH/3T3 cell lines. This non-cytotoxic and biocompatible nature suggests that this levan has the capability to be utilized in food and drug-based formulations as it exhibited biomedical potential.

Introduction

A wide variety of polysaccharides are biosynthesized by plants, bacteria, algae and fungi. Among them, biopolymers of microbial origin have been the major focus of research in the last few decades because of their unique physicochemical nature, structural properties, and bioactive functionalities. In recent years, demand for biodegradable exopolysaccharide (EPS), which have exhibited multiple industrial applications, have increased. Microbial EPSs are usually long-chain polymers and are consist of a variety of sugar moieties with branched and sometimes with repeating units of sugars [1]. Among different types of EPSs, fructan based biopolymers such as levan and inulin have been comprehensively studied in the last few decades. A wide range of bacterial species including Bacillus subtilis, Zymomonas mobilis, Paenibacillus bovis, Erwinia herbicola and Leuconostoc citreum are reported for the synthesis of a fructan based levan using sucrose as a substrate [[2], [3], [4], [5], [6]]. Levansucrase [E.C. 2.4.1.10] catalyzes tranfructosylation reaction in the presence of sucrose or a relevant substrate to synthesize a unique biopolymer of β-(2,6) fructosyl-fructose molecules commonly known as levan. Microbial levan has exhibited high water solubility, low viscosity, and elevated thermal stability along with antioxidant, anti-tumor, immunostimulatory and anti-inflammatory properties [5,[7], [8], [9], [10], [11]] that enforced its applications in food, pharmaceutical and cosmetic industries. This unique exopolysaccharide has also been studied for the formation of thin films, drug delivery carrier system, plasma substituter and as a radio protector [[12], [13], [14], [15]]. Levan has also been tested for transient electronic system to be used in bioresorbable electronic implants and drug release systems [16].

Zymomonas mobilis is considered as one of the exceptional bacteria in microbial domain that can excessively produce ethanol and have also demonstrated the capability to synthesize an exopolysaccharide [17,18]. This organism is a natural ethanologen that have exhibited high tolerance level against higher concentrations of alcohol and sugar molecules therefore, it can be cultivated at a broad range of pH (pH 3.5 to 7.5) and is generally regarded as safe to be used for fermentation purpose [18]. Z. mobilis also exhibits lower biomass generation as compared to other microorganisms because it uses Entner-Doudoroff metabolic pathway for the metabolism of carbon source regardless of the carbon source consumption during fermentation [19]. The levansucrase produce by this bacterium can synthesize levan as a biopolymer along with other metabolites such as sorbitol, gluconic acid or fructooligosaccharides (FOS) in the presence of sucrose as a substrate [20]. The transfructosylation reaction is highly dependent on the fermentation conditions which are used to produce exclusive byproducts. Bekers et al. [20] have reported that high fermentation temperature along with higher concentrations of sucrose could leads towards the formation of FOS whereas, the lower values of these fermentation parameters could increase the production of ethanol. Therefore, culture cultivation conditions should be strictly monitored during the production of levan. Various applications of levan, in numerous industries, have already been explored but its utilization specially in medical and food sectors is exclusively dependent on its biocompatible and non-cytotoxic nature.

Keeping the above available information in view, the current study was designed to produce and purify levan from and indigenously isolated bacterial isolate. The purified exopolysaccharide was structurally characterized for the first time that was produced by Z. mobilis KIBGE-IB14. To determine the application of this exopolysaccharide, different concentrations of levan was tested against mouse fibroblast cell lines to evaluate its cytotoxicity.