**Abstract**

*L. plantarum* producing EPS plays an important role as an antioxidant, antiproliferative, and anticancer. This study aims to increase the production of EPS by *L. plantarum* through modification of MRS (de Mann Rogosa Sharpe) media mixed with coconut water, treated with natrium acetate, Se, and Zn at different concentration, as well as understanding its effect on antioxidant activity. The effect of adding sodium acetate with different concentrations of 0.25, 0.50, 0.75, and 1.0% into mixed media MRS coconut water, (1:3) was studied. Fermentation experiments at different of Se2+ concentration (mM): 50; 75; 100; 125; 150; and 175, and addition of variation Zn2+ concentration (mM): 2.5; 5.0; 7.5; 10.0; 12.5; and 15.0), were carried out separately. Antioxidant potential was tested by FRAP (ferric reducing antioxidant power) and ABTS (2.2′-azinobis (3-ethyl benzatiazoline)-6-sulfonate). The results showed that the addition of sodium acetate with different concentrations showed a significant difference to the dry weight of EPS (*P* < 0.05). The increase in sodium acetate concentration was up to 1%, in line with the increase in EPS production by *L. plantarum* (g/g DW biomass). The addition of Se2+ 100 mM increased the ratio of glucose to protein content by 2.121. The value of the antioxidant activity of Fe (II) was 311.54, and the ABTS test obtained IC50 of 83.041. A separate experiment with the addition of Zn2+ in the fermentation medium of *L. plantarum* produced a fluctuating exopolysaccharide. The value of the antioxidant activity of Fe (II) M using the FRAP method was 275.886. The IC50 value with the ABTS method is 73.2942. Characterization of EPS from *L. plantarum* using FTIR (Fourier transforms infrared spectrophotometry) has hydroxyl, carboxylate, and aromatic functional groups.

**Keywords:** exopolysaccharides, *Lactobacillus plantarum*, sodium acetate, Se2+, Zn2+, antioxidant

### **1. Introduction**

Lactic acid bacteria (LAB) are widely used in the manufacture of traditional fermented milk. In the dairy industry, the bacteria are also useful as a culture starter in the fermentation process. Several strains of lactic acid bacteria have important roles in health which are beneficial microflora in the intestinal tract [1–3] and capable of synthesizing exopolysaccharides (EPS) [4–7]. Exopolysaccharides produced by lactic acid bacteria have been given increasing attention in recent years; it is due to their contribution to the rheology and texture of food products. In addition, EPS products, which are GRAS (generally recognized as safe), are declared safe for consumption and stable during storage [4, 5].

EPS is a polymer of high-molecular weight-reducing sugars, which are secreted by microorganisms into their external environment. This polymer has bioactivation, so it can be used for anti-viral and anti-inflammatory treatment. It has an inhibitory effect on tumor growth in vitro or in vivo [8–10]. The structure and composition of EPS is closely related to its anti-tumor biological function. In food industry, exopolysaccharides can function as thickeners, gelling agents, and emulsifiers. EPS from lactic acid bacteria (LAB) can exert functional effects on food, improve the rheology of fermented dairy products, and have beneficial health effects.

Several types of LAB that produce exopolysaccharides are *Lactobacillus acidophillus, L. rhamnosus, L. casei, L. reuteri, Bifidobacterium longum*, and *L. plantarum* [11]. The amount of exopolysaccharide produced by lactic acid bacteria is influenced by several factors, such as media composition (C, N concentration, and mineral supplementation), fermentation conditions, interactions between strains (co-culture fermentation), and fermentation technology (fed-batch fermentation). Other factors include physico-chemical conditions such as temperature, pH, level of oxygen presence, incubation time, and genetic factors [12].

Microbes need minerals to synthesize cellular components, to produce energy, and becomes electron acceptors in the metabolism of glucose and other sugars. Some minerals are enzyme activators for microbial metabolism such as Se2+, Zn2+, Mn2+, Mg2+, Ca2+, and others. The addition of minerals with the right concentration into the growth medium of *L. plantarum* will increase the formation of exopolysaccharides.

Previous research on the effect of mineral species on EPS production and growth characteristics of *L. bulgaricus* strain ropy in milk media showed that the best mineral source was 0.5% sodium acetate, which yielded up to 476.6 mg/L of EPS compared to triammonium citrate, potassium phosphate, and magnesium sulfate at concentrations of 0–0.5% [13]. Referring to the results, this study added the mineral sodium acetate with a concentration variation of 0.25–1.0% into coconut water media to produce EPS by *L. plantarum*. The results of previous studies using coconut water producing the highest EPS of 7.0510 g with a composition of 75% coconut water [14].

The addition of selenium (Se2+) micronutrients to MRS media was intended to review its effect on increasing antioxidant potential. Selenium as a microelement acted as a component of the enzyme glutathione peroxidase (GPX) which had antioxidant activity by reducing peroxide compounds, so it reduced free radicals in the body. Selenium is an essential function in the biological system [15]. The results of previous studies reported that the amount of selenium that could be added to bacterial culture was around 100–150 mM [16]. Micronutrient selenium became an *Effect of Sodium Acetate and Trace Element (Se2+, Zn2+) on Exopolysaccharide Production… DOI: http://dx.doi.org/10.5772/intechopen.104547*

important nutrient for cell proliferation that played its role in increasing cell growth [17]. Some LAB strains have been reported to be capable of resisting selenium oxyanions at high concentrations during cultivation. Especially, *L. plantarum* has been suggested as Se-enriched lactobacilli for food applications.

Research on micronutrients Zn and Cu added to goat's milk production showed an effect of increasing antioxidant activity. *L. casei* KCTC 3260, was found to possess a high antioxidant ability by chelating Fe2+ or Cu2+, although no detectable SOD activity was observed [18]. This paper reports the results of research on the effect of adding selenium to the growth medium of *L. plantarum* bacteria for the production of exopolysaccharides that have potential as antioxidants.

Another micromineral is zinc (Zn) which is important for health. Zn is needed by various organs of the body, such as the skin of the gastrointestinal mucosa. Zn increased the antioxidant capacity of SOD which played its role in protecting pancreatic beta cells from damage caused by reactive oxygen species (ROS). Superoxide is one of the most abundant ROS produced by the mitochondria, while SOD catalyzes the breakdown of superoxide into hydrogen peroxide and water and is therefore a central regulator of ROS levels [19]. The complex form of copper zinc SOD (Cu Zn-SOD) compound was able to increase the activity of SOD. Study on the *L. fermentum* E-3 and E-18 were able to express Mn-SOD to resist oxidative stress [20]. Increased antioxidant activity of EPS from *L. plantarum* culture with the addition of zinc (Zn) with various concentrations of 2.5–15 mM will be reported here. Previous researchers reported that the uptake of zinc (Zn) in lactic acid bacteria (LAB) was 10 mM [13]. The results were used as a reference. Few studies about Zn-enriched LAB have been conducted, but it has been found that the bacterial growth and probiotic effect of *L. plantarum* can be enhanced by zinc in the gut [21].

In the past few years, intensive research of EPS from microbes has developed and widely applied as ingredients of functional foods, pharmaceuticals, nutraceuticals, cosmetics, and insecticides. EPS from microbes are predicted to quickly develop as fast as EPS from plants and microalga which now dominate the market. This is due to their role as an immunostimulatory [22, 23], antivirus activity, antibacterial, and anticancer [23–26]. The potential of EPS as antioxidant becomes crucial in the field of medicine and food industry due to their action as scavengers of reactive oxygen species (ROS). ROS are a diverse group of unstable and highly reactive oxygen-derived molecules, such as hydrogen peroxide (H2O2), hydroxyl radical (•OH), singlet oxygen ( 1 O2), and superoxide (O2−). These oxidants are produced under stress conditions that cause destruction of macromolecules (lipids, proteins, and DNA) and disrupt various redox signalling pathways in eukaryotic cells. The oxidative stress condition caused by the abnormally high levels of ROS triggers cardiovascular disease, and has also been implicated in diabetes, various types of cancer, neurologic and inflammatory diseases, ageing [27–29], and autoimmunological disorders [30].

Sustainable Development Goal 3 of the 2030 Agenda for Sustainable Development is to "ensure healthy lives and promoting well-being for all at all ages" [31]. One of the objectives that must be achieved is to reduce the mortality rate caused by noncommunicable disease such as cardiovascular, cancer, diabetes, or chronic respiratory disease. In 2019, it was reported that globally, 74% of all mortality were caused by non-communicable diseases. In this regard, this study aims to produce EPS from *L. plantarum* bacteria as raw material for drugs that are useful for human health. The study focused on increasing the production of EPS through modification of MRS media mixed with coconut water by adding natrium acetate, Se, and Zn2+ and evaluating the potential of EPS as an antioxidant.
