Purification and characterization of an exopolysaccharide produced by Lactobacillus plantarum HY isolated from home-made Sichuan Pickle

https://doi.org/10.1016/j.ijbiomac.2019.05.010Get rights and content

Highlights

  • A high yield of exopolysaccharide was obtained from Lactobacillus plantarum HY.

  • The purified HY EPS has an average molecular weight of 9.549 × 104 Da.

  • The HY EPS is mainly composed of mannose, galactose, glucuronic acid and glucose.

  • The HY EPS has fibrous tubular microstructure and many irregular cylindrical protrusions.

  • The HY EPS has excellent thermal stability, antioxidant properties and also has the ability to inhibit α-amylase.

Abstract

In the present study, the exopolysaccharide (EPS) producing strain HY was isolated from home-made Sichuan Pickle and identified as Lactobacillus plantarum. The EPS was purified by ethanol precipitation, and fractionation by column chromatography, and the yield of purified HY EPS was 1.423 g/L. The molecular weight of the purified HY EPS was 9.549 × 104 Da and composed of mannose, galactose, glucuronic acid and glucose in a mass percentage of 72.99%, 17.27%, 6.99% and 2.75%, respectively. FT-IR spectrum exhibited the existence of carboxyl and hydroxyl group. Partial sugar residues and glycosidic bond present in HY EPS could be identified by NMR spectroscopy, i.e. α-d-glucose and α-d-mannose residues. The degradation temperature (320 °C) and melting point (122.78 °C) of the HY EPS were determined by thermogravimetric analysis (TGA) and differential scanning calorimeter analysis (DSC). A highly porous structure of EPS was observed by SEM, and the AFM analysis of the EPS revealed presence of spherical lumps. Meanwhile, the in vitro biological activity assays of HY EPS showed considerable antioxidant and α-amylase inhibitory activities, suggesting its promising potential to be applied in the food and pharmaceuticals industry.

Introduction

Polysaccharides produced by plant and microorganisms, are regularly used as thickeners, stabilizers and gelling agents due to their unique physicochemical properties in food industry [1]. Many microorganisms such as bacteria, fungi, and algae, possessed the capability of producing exopolysaccharides (EPSs). EPSs have been classified into two groups according to their different production locations: capsular (attached to the surface of the cell) and slime (released into the external environment). The EPSs produced by bacteria have received increasing attention, in particular by lactic acid bacteria (LAB) with GRAS (Generally Regarded as Safe) status which were reported to show several health beneficial activities, e.g. antioxidation [2], lowering cholesterol level [3], and antimicrobial capability [4], etc. Meanwhile, LABs have a long history as fermentation starter [5], contributing to the improvement of rheology, texture and taste of dairy products [[6], [7], [8]].

In recent years, increasing evidences have demonstrated that EPSs synthesized by LAB could maintain stability in the gastrointestinal environment and enhance the colonization of other probiotics [9]. Many reports have indicated that LAB EPSs also exhibited various physiological activities, such as antitumor [10], immunomodulation [11], antioxidant activity [12,13], antibiofilm formation [14], antidiabetic [15]. Thus, EPSs from LAB not only can be utilized in food production as functional additives, but also has great potential for application in pharmaceutical industry due to their biological activities [16].

In past decades, a number of EPSs producing LAB have been isolated from Chinese traditional fermented foods, such as kefir [17], soybean paste [18], acid beans [19], pickle [20]. Pickle or Paocai, as a Chinese traditional fermented vegetable, is rich in LAB [21,22], especially, Lactobacillus plantarum is a quite common LAB species found in pickle. EPSs produced by L. plantarum in the fermented products have received considerable attention due to their biological activity and health benefits. However, up to date, most of the studies focused on the functional characteristics, with less knowledge about the relationship between structure and function yet.

We all know that the chemical structure of EPS serves as the basis for its biological function. Therefore, structure illumination of EPS is indispensable for exploring its biological activity. However, the structure analysis is quite challenging due to the diversity and complexity of EPSs, particularly, to determine its three dimensional structure.

Therefore, in the present study, an EPS-producing strain Lactobacillus plantarum HY from home-made Sichuan Pickle was isolated and purification of crude EPS was conducted by column chromatography fractionation. Then, the chemical structure and physicochemical property of the purified EPS were characterized by High Performance Liquid Chromatography (HPLC), ultraviolet (UV), fourier transform infrared spectroscopy (FT-IR), one dimensional 1H and 13C nuclear magnetic resonance (NMR), thermogram analysis (TGA), and differential scanning calorimeter (DSC), scanning electron microscopy (SEM), atomic force micrograph (AFM), aiming at providing evidence for its potential application in food industry and study about the relationship between its structure and biological activity. In addition, the antioxidant activities were investigated using various in vitro assays including 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity, reducing power assay and total antioxidant activity, and the antidiabetic activity of HY EPS was studied using alpha amylase enzyme inhibition assay [23].

Section snippets

Isolation and identification of EPS- producing LAB

The EPS-producing strain HY was isolated from home-made Sichuan pickle collected from Nanchong city of Sichuan province, China. The strain HY was stored as frozen (−80 °C) stocks in De Man Rogosa Sharpe (MRS) broth supplemented with 25% (v/v) glycerol. EPS production was carried out in semi-defined medium (SDM) containing (per 100 mL): 4.0 g of lactose, 4.0 g of yeast nitrogen base, 0.5 g of sodium acetate, 0.1 g of ammonium sulfate and 0.1 mL of Tween 80 [24].

The strain HY was primarily

Screening and identification of HY strain

As Fig. 1A and B showed, HY strain showed a smooth and hydrated colony appearance on SDM agar plate and a round shape and medium-sized cell morphology. A phylogenetic tree was constructed by comparing with 16S rDNA sequences of the other strains from NCBI (Fig. 1C). The results on this basis of morphological characteristics and 16S rDNA sequence analysis identified HY strain as Lactobacillus plantarum.

Isolation and purification of HY EPS

Crude EPS produced by strain HY was obtained from SDM liquid medium through a series of steps

Conclusion

In the present study, a EPS-producing strain HY was isolated from home-made Sichuan pickle and identified as Lactobacillus plantarum. The purified HY EPS was obtained by column chromatography fractionation, and further subjected to structure characterization, whose yield was 1.423 g/L. The purified HY EPS is a heteropolysaccharide and composed of mannose (72.99%), galactose (17.27%), glucuronic acid (6.99%) and fucose (2.75%) with a Mw of 9.549 × 104 Da. The FT-IR spectrum exhibited the

Acknowledgement

This work was supported by the fund of the Beijing Laboratory for Food Quality and Safety, Beijing Technology and Business University, National Natural Science Foundation of China (31701518), Science & Technology Foundation of Sichuan Province (Grant No. 2019YFN0110).

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