**2.5 Extraction of exopolysaccharides from** *L. plantarum*

MRS medium modified with the addition of micronutrients was inoculated with *L. plantarum* bacteria at a concentration of 2% (v/v) and was cultivated at 37°C for 72 hours. Cultures of *L. plantarum* were boiled at 100°C for 15 minutes to inactivate

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

enzymes that degraded exopolysaccharides. The cultures were cooled and centrifuged at 14.534×g, for 15 minutes, at 4°C, to separate the cell biomass and supernatant. The precipitated biomass was then dried and weighed as the weight of *L. plantarum* cell biomass (mg), while the supernatant was extracted to obtain EPSs. An amount of two times of the volume of 96% ethanol was added to the supernatant for precipitation of EPSs. The mixture was stored at 4°C for 24 hours. Then, the EPS was collected by centrifugation (11.772×g, 4°C, 20 minutes). The pellets were dissolved with distilled water. EPSs were purified by adding 15% (w/v) trichloroacetic acid (TCA) [34], then was centrifuged (20,000×g, 10 minutes, 4°C). The pellets were dried at 55°C, weighed as EPS dry weight (mg) [34].

#### **2.6 Total sugar analysis**

Total sugar content was determined by the modified phenol-sulfuric acid method using glucose as a standard [35]. An amount of 1 ml of the sample was mixed with 0.5 ml of 5% phenol solution. Then, 2.5 ml of 95% sulfuric acid was added. The sample were incubated at 25°C for 10 minutes; then stirred for 1 minute. The sample solution was re-incubated for 20 minutes at 25°C. The absorbance of each sample was measured using a spectrophotometer at a wavelength of = 490 nm. Sample control used distilled water (1 ml).

#### **2.7 Protein analysis by the method of Lowry**

The standard solution used in protein analysis was bovine serum albumin (BSA). An amount of 0.5 ml of the sample was added with 0.5 ml of 1 N NaOH, shaken, and boiled at 100°C for 20 minutes, then cooled. An amount of 0.5 ml of the mixed solution (5% Na2CO3, 1% CuSO4.5 H2O and 2% Na-K-tartrate) was added to each sample; and was shaken homogeneously. Add 0.5 ml of Folin-Ciocalteu reagent to each sample; and was shaken homogeneously and let stand for 30 minutes. The absorbance of the BSA standard solution and the test sample was measured with a UV-Vis spectrophotometer at a wavelength of 750 nm. Sample control used distilled water [36].
