Fermentation conditions optimization, purification, and antioxidant activity of exopolysaccharides obtained from the plant growth-promoting endophytic actinobacterium Glutamicibacter halophytocola KLBMP 5180

Highlights

• The EPS yield of Glutamicibacter halophytocola KLBMP 5180 was increased to 2.89 g/L after RSM.

• Two purified EPSs components were further obtained from G. halophytocola KLBMP 5180.

• Molecular weight of 5180EPS-1 and 5180EPS-2 was found as 58.9 kDa and 10.5 kDa, respectively.

• The two EPSs had certain antioxidant activities.

Abstract

In this study, an endophytic actinobacterium Glutamicibacter halophytocola KLBMP 5180, was investigated for the production and antioxidant activity of exopolysaccharides (EPSs). First, the suitable fermentation time, temperature, inoculation volume, pH value, and the carbon and nitrogen sources for EPSs production were obtained using the one variable at a time method (OVAT). Then, a central composition design was used for fermentation conditions optimization to obtain the maximum EPS yield. The optimal medium and condition were as follows: 100 mL broth in 250 mL Erlenmeyer flasks, including 3.65 g/L maltose, 9.88 g/L malt extract, 3.40 g/L yeast extract, 1.41 g/L MnCl₂, pH 7.5, culture temperature 28 °C, and 200 rpm for 7 days, which increased the yield of EPSs to 2.89 g/L. Two purified EPSs, 5180EPS-1 (MW 58.9 kDa) and 5180EPS-2 (10.5 kDa), comprising rhamnose, galacturonic acid, glucose, glucuronic acid, xylose, and arabinose, were obtained for chemical analysis and antioxidant evaluation. The scavenging ability and reducing power of the superoxide anion and hydroxyl radicals demonstrated the moderate in vitro antioxidant activities of the two EPSs, thus indicating their potential to be a new source of natural antioxidants. However, further structure elucidation and functional studies need to be continued.

Introduction

Actinomycetes are the main producers of bioactive molecules in microorganisms [1]. Plants-associated endophytic actinobacteria have high biodiversity and exhibit many ecological functions. Many biologically active compounds with antimicrobial, antitumor, and immunosuppressive activities have been reported [2], [3]. Recent studies have found that endophytic actinomycetes from medicinal plants also showed the ability to produce the same or similar compounds as their hosts. For example, endophytic Streptomyces sp. F4-20 has been observed to produce wilforgine, a secondary metabolite that was discovered in its host Tripterygium wilfordii Hook.f. [4]. The bioactive metabolites produced by endophytic actinomycetes find important application in the fields of medicine, agriculture, and plant protection [5], [6].

Endophytes have been found to produce structurally different exopolysaccharides (EPSs), which have gained an increased interest in the recent years [7]. However, the production and functional characterization of extracellular polysaccharides from endophytic actinomycetes have been rarely reported. It has been demonstrated that EPSs play an important role in endophyte-host plant interactions [8]. Bacterial EPSs participate in biofilm formation and plant rhizosphere migration. At the same time, EPSs also eliminate the toxicity of reactive oxygen species (ROS) and help bacteria survive under salt stress, drought, and oxidative stress [9], [10], [11]. Furthermore, increased reports demonstrated that EPSs from endophytes exhibit several biological functions, including heavy metal ion removal and absorption [12], [13], and antioxidant [14], [15], antitumor [16], anti-allergic, anti-inflammatory [17], and prebiotic activities [18], meriting their functional application in pharmaceutical and food industries. Culture conditions and medium composition play important roles in the yield of microbial EPSs. The single factor method and response surface methodology (RSM) can be used to optimize the suitable conditions in the production of microbial EPSs [19], [20]. Si et al. [21] demonstrated that the EPS yield obtained from the medicinal mushroom Ganoderma lingzhi was 3.57 ± 0.21 g/L, which was 3.16-fold higher than that produced using the basal medium alone. Wang et al. [15] found that the maximum EPS production of 3.42 mg/mL was obtained from the endophytic fungus Alternaria tenuissima F1 after incubation at 30 °C for 5 d in broth supplemented with 8% mannose, 2% yeast extract, 0.04% MgSO₄·7H₂O, 0.005% Vitamin C at pH 7.0 and an inoculum size of 3%. Thus, it may be concluded that EPS production varies with the microorganisms used and that response surface optimization is a good way to improve the production of microbial EPS.

The Chinese herb Limonium sinense, a halophytic medicinal plant, has a long traditional medicine history in China. It contains flavonoids, polysaccharides, alkaloids, and other such ingredients, which possess antibacterial, antitumor, and immunomodulatory activities, and are, thus, useful in heat reduction, detoxification, and maintenance of hemostasis [22], [23]. At present, there are only a few reports on the endophytes and their bioactive substances obtained from halophytes Limonium [24]. However, in our previous studies, we have reported on G. halophytocola KLBMP 5180, a new endophytic actinobacterium that was obtained from L. sinense [25]. It was found that this halotolerant strain significantly promoted the growth of host plants under salt stress [26], [27]. Besides, we have also reported the complete biosynthetic gene clusters of EPSs found in the genome of the strain [28]. Therefore, this research was undertaken to study the optimization of the production conditions of EPSs and their structure and antioxidant activities from the G. halophytocola KLBMP 5180 strain, which we believe will lay a foundation for the further study on the biological functions of EPSs.