**3. Results and discussion**

#### **3.1 Effect of sodium acetate concentration on the formation of EPS** *L. plantarum*

Exopolysaccharide was extracted from the culture of lactic acid bacteria *Lactobacillus plantarum* fermented in coconut water-MRS medium in a ratio (25:75) at 37°C for 72 hours. The use of coconut water with a percentage of 75% referred to previous studies because it was the best percentage to produce the exopolysaccharide *L. plantarum* [14]. Liquid MRS medium was used because it was a selective medium for the growth of lactic acid bacteria. Seesuriyachan's research used MRS media which was a good growth medium for lactic acid bacteria; the addition of coconut water and sucrose as a carbon source could affect the yield of exopolysaccharides. The research resulted in the production of exopolysaccharides which continued to increase, that is, from a concentration of 0–100% coconut water also contains protein, sugar, amino acids, various vitamins and minerals, so with the relatively complete nutritional content, coconut water is very potential to be used as a basic material for fermenting organic acids and as an alternative carbon source for the production of exopolysaccharides from lactic acid bacteria. Lactic acid-producing bacterium is one of the

*Effect of Sodium Acetate and Trace Element (Se2+, Zn2+) on Exopolysaccharide Production… DOI: http://dx.doi.org/10.5772/intechopen.104547*

#### **Figure 1.**

*(a) Morphology of whole cells of* L. plantarum *visualized by microscop 1000×. (b) EPS crude extracted from*  L. plantarum.

bacteria that tends to be attracted to sugar-containing habitats such as coconut water [43]. **Figure 1** shows the cell morphology of *L. plantarum* with methylene blue dye and crude exopolysaccharide extracted from *L. plantarum* culture.

The fermentation temperature used was 37o C, which was the optimal temperature for the growth of lactic acid bacteria. The fermentation time for 72 hours was the optimal time for harvesting exopolysaccharides for the growth of lactic acid bacteria. The supernatant obtained from the extraction was then added with cold 96% ethanol in a ratio of 1:1; then allowed to stand for 48 hours at 4<sup>o</sup> C. The addition of cold 96% ethanol aimed to precipitate exopolysaccharides [32].

The amount of exopolysaccharide produced by lactic acid bacteria was influenced by several factors such as media composition (source and concentration of carbon and nitrogen), fermentation conditions, effects of growth media (mineral supplementation), interactions between strains (co-culture fermentation), and pharmaceutical technology (fed-batch fermentation), as well as physico-chemical conditions of bacterial growth such as temperature, pH, level of oxygen presence, incubation time, and genetic factors [12].

**Table 1** shows that the production of EPS (g) per dry cell biomass (g) was increasing, in line with the addition of sodium acetate concentration in MRS medium: coconut water (75:25). A significant increase in EPS production against the control (media without sodium acetate) began to be seen at a concentration of 0.75% sodium acetate: increased to 23.81%. At 1% sodium acetate concentration, EPS production increased to 34.94%. The greater the concentration of sodium acetate used the more exopolysaccharide compounds produced. It was influenced by the addition of sodium


#### **Table 1.**

*Dry weight of EPS* L. plantarum *on MRS Media: Coconut water (25:75) with variations in sodium acetate concentration. The numbers followed by the same letter are not significantly different (P < 0.05).*

acetate mineral as an electron acceptor in the metabolism of glucose and other sugars which helped lactic acid bacteria to produce exopolysaccharide compounds [44].

In the exopolysaccharide polymerization reaction, the formation of carbon chains required minerals as electron acceptors binding one monomer to others. Lactic acid bacterium was not limited to oxygen as an electron acceptor. Anaerobically, some organic components could be treated with the same purpose as electron acceptors. This case particularly happened at obligate heterofermentative LAB in the alcohol or acetate formation pathway. However, it turned out that organic electron acceptors could play an important role in homofermentative LAB in anaerobic metabolism on certain substrates [44].

Research by Pham et al. also used several types of minerals in the medium to see the activity of *L. rhamnoses* cells in producing exopolysaccharides. The results showed that during the initial exponential growth phase the biosynthesis of exopolysaccharides did not occur. Production occurred in a stationary phase leading to death and then the exopolysaccharide produced could be reused by microbes as a carbon source due to the presence of enzymes produced by the bacteria themselves that could degrade exopolysaccharides. Consequently, the prolongation of the incubation time decreased the production of exopolysaccharides [45].

The extraction method used could also affect the amount of exopolysaccharide produced. The heating step carried out at the beginning of the extraction was able to increase the recovery of exopolysaccharides, then increased the number of exopolysaccharides obtained. The addition of ammonium sulphate at the time of extraction aimed to precipitate proteins and separate exopolysaccharide compounds from a mixture of other compounds [45].

The production of exopolysaccharides from microbial cultures depended on several different parameters. The formation of polysaccharides was most often associated with the presence of carbohydrates and low or high temperatures. Mineral requirement was also a critical factor. Restriction of nitrogen, phosphorus or sulphur sources increased the production of exopolysaccharides; on the other hand, some researchers reported that phosphate and trace elements were essential elements for the synthesis of exopolysaccharides by *Pseudomonas aeruginosa.* Several types of minerals were needed by microbes as growth factors needed to form energy and compose cell components and the formation of secondary metabolites. Micro elements such as K, Ca, Mg, Cl, Fe, Mn, Co, Cu, Zn, and Mo were needed by almost all microbes. The transfer of nutrients into microbial cells could be in the form of passive diffusion, diffusion with the help of permease, active transfer or through the phosphotransferase system. Minerals were generally transferred by active transfer [44].

### **3.2 Effect of sodium acetate concentration on total sugar content and protein in EPS**

Determination of glucose levels in exopolysaccharide samples was carried out using the phenol-sulphate method using visible light spectrophotometry. This method was chosen to determine the total sugar content in exopolysaccharides because it was commonly used to determine the total carbohydrate content of bacterial polysaccharides. The advantage of this method was that the reagents used were cheap and easy to obtain, little equipment was needed and the analysis was simple [46]. The principle of this method was that simple sugar and oligosaccharide could react with phenol in concentrated sulfuric acid to produce a stable yellowish orange color. The addition of phenol and concentrated sulfuric acid aimed to form a color complex in the sample so

*Effect of Sodium Acetate and Trace Element (Se2+, Zn2+) on Exopolysaccharide Production… DOI: http://dx.doi.org/10.5772/intechopen.104547*

#### **Figure 2.**

*Schematic overview of the phenol sulfuric method for the total carbohydrate analysis [47].*

that it could be detected by UV-VIS spectrophotometry. The addition of concentrated sulfuric acid would produce a yellowish orange hydroxymethyl furfural compound absorbing at a wavelength of 490 nm [47]. The mechanism of the dehydration reaction of glucose to hydroxy methyl furfural can be seen in **Figure 2**.

**Figure 3a**: the correlation coefficient value was 0.9924 showing that there was a linear relationship between the concentration of the standard glucose comparison solution and the absorption at 490 nm; the increase in concentration was proportional to the increase in absorption. **Figure 3b**: analysis of variance (ANOVA) and DMRT test (Duncan multiple range test) showed that there was a significant difference in the addition of sodium acetate with a concentration of 0–0.75% with an increase in glucose levels at that concentration. Meanwhile, at the concentration of sodium acetate concentration of 0.75% with 1% there was no significant difference even though glucose levels in the 1% sodium acetate treatment increased. **Figure 3b** the highest glucose level was obtained in 1% sodium acetate sample (75.54%) and the lowest was found in 0% sodium acetate sample (29.05%).

The absorption of BSA standard solution with concentrations of 20, 40, 60, 80, and 100 ppm was measured by a spectrophotometer at a wavelength of 750 nm (39)

#### **Figure 3.**

*(a) Relationship of standard glucose concentration to absorption at λ490 nm and (b) effect of Na acetate concentration on glucose levels in EPS (%). The numbers followed by the same letter are not significantly different (P < 0.05).*

**Figure 4.**

*(a) Relationship of protein standard concentration (BSA) to absorption at λ750 nm and (b) effect of Na acetate concentration on protein content in EPS (%). The numbers followed by the same letter are not significantly different (P < 0.05).*

resulting in a calibration curve with the regression line equation y = 0.0034x + 0.1492 with the correlation coefficient value obtained 0.9909 (**Figure 4a**).

Determination of protein content using the Lowry method based on two different reactions. The first reaction was the formation of copper Cu+. Under alkaline conditions formed by a solution of Na2CO3 in NaOH, Cu2+ ions formed a complex with peptide bonds reducing Cu2+ to Cu+. The second reaction was a reduction reaction by Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate). The Cu+ ion and the radical groups of tyrosine and tryptophan reacted with Folin's reagent to produce an unstable product that reduces molybdenum or tungsten blue. Protein would react with Folin-Ciocalteu reagent to form a complex compound giving a blue color [48].

The results of analysis of variance (ANOVA) and DMRT showed a significant increase in protein content due to treatment with sodium acetate concentration (0–0.75%). However, the results did not show a significant difference between 0.75 and 1% sodium acetate concentrations. **Figure 4b** shows that the highest protein content was obtained as a result of 1% sodium acetate treatment (9.70%) and the lowest was found in 0% sodium acetate treatment (7.24%).

#### **3.3 Interpretation of IR spectrum of EPS and glucose samples**

The functional groups of exopolysaccharide samples produced from *L. plantarum* isolates with comparison, namely glucose, were determined using an FT-IR (Fourier transform infra-red) spectrophotometer [49].

The results of the interpretation of the infrared spectrum of the exopolysaccharide sample of *L. plantarum* and the reference standard (glucose) are presented in **Table 2**. Glucose was used as a comparison because glucose is the main compound contained in the exopolysaccharide.

The results of the FT-IR analysis in **Table 2** show that the exopolysaccharide and glucose samples contained IR spectra similarities indicating a typical absorption of polysaccharide compounds. Glucose showed the presence of hydroxyl groups (-OH), alkane groups (–CH), and C–C vibrations. Meanwhile, the exopolysaccharide isolated from *Lactobacillus plantarum* showed the presence of a hydroxyl group (-OH), a carbonyl group (–C=O) in the carboxylate and a C–C vibration. These results indicated that the extract obtained was a carbohydrate compound.

*Effect of Sodium Acetate and Trace Element (Se2+, Zn2+) on Exopolysaccharide Production… DOI: http://dx.doi.org/10.5772/intechopen.104547*


#### **Table 2.**

*Interpretation of IR Spectrum of* Lactobacillus plantarum *EPS samples and glucose standard.*

The area of 200–900 cm−1 was the fingerprint area for carbohydrates and could be used for carbohydrate identification. Exopolysaccharides from *L. plantarum* with the addition of sodium acetate had an absorption band of 1085 cm−1. Absorption bands in the area around 1080 cm−1 were characteristic for carbohydrates derived from microbial biomass glucose, galactose, and mannose had absorption bands in the region of 983–1200 cm−1 [50]. The sample and glucose had an absorption band in that area. Specifically, glucose was in the wave number of 1100–1124 cm−1 and the exopolysaccharide sample was in the wave number of 1000–1150 cm−1. It indicated the presence of sugar monomers such as glucose, galactose and mannose in the sample. Thus, the sample can be said to be a polysaccharide based on the resulting IR spectra and its similarity to glucose.
