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

of exopolysaccharides tended to decrease. The inversely proportional results of cell biomass and exopolysaccharides had been carried out in a study. The yield of exopolysaccharide production was inversely proportional to cell biomass because of the need for energy support in the exopolysaccharide biosynthesis process. The limitation of energy production in cells during the formation of exopolysaccharides caused the amount of biomass to be low, because the formation of the two was interrelated. Higher exopolysaccharides were produced with lower cell growth. It was possible that the cell growth pathway was blocked and converted to an exopolysaccharide synthesis pathway [43].

The production of exopolysaccharides obtained from lactic acid bacteria was influenced by several factors such as fermentation conditions, effects of growth media (mineral supplementation), interactions between strains (co-culture fermentation), and fermentation technology (fed-batch fermentation). Regulation of cell growth at a constant pH resulted in better exopolysaccharide yields. The acidification process occurred because the production of lactate caused the glycohydrolase enzyme to become active (pH range 5). It caused the yield of EPS to decrease due to the enzymatic process. The culture conditions and the composition of the media (not only carbon sources) affect the yield of EPS and the molecular characteristics of the biopolymer [53].

Glucose equivalent EPS levels were determined using the phenol-sulfate method. Glucose did not have a chromophore so it was supposed to be reacted with phenol-sulphate to form an orange-yellow color having a maximum absorption at 490 nm. The addition of concentrated sulfuric acid H2SO4 caused hydrolysis of glycosidic bonds in EPS so that furfural compounds or furfural derivatives were formed which would condense with phenol to form yellow-orange compounds. The results of the measurement of glucose uptake and concentration in EPS can be seen in **Table 5**. The highest average glucose level was obtained in exopolysaccharide samples with the addition of Se2+ 175 mM concentration reaching 55.169% and the addition of 10 mM zinc of 69.20%, while the lowest glucose level was obtained at exopolysaccharide samples in the media without the addition of Se2+ and Zn2+.

Glucose levels in exopolysaccharides would increase because during fermentation, lactose would be broken down into glucose and galactose which became the main carbon sources in increasing the activity of the UDP-glucose pyro phosphorylase and UDP galactose-4-epimerase enzymes. UDP galactose-4-epimerase was a key enzyme in the formation of EPS, and the enzyme would be active when there was a sugar unit in the form of galactose as a precursor in the formation of exopolysaccharides [53].

Analysis of protein content in EPS samples was determined using the Lowry method by visible light spectrophotometry. The protein in the sample would react with copper (II) sulphate under alkaline conditions to form Cu2+ ions and an amino acid radical group which then reacted with Folin-Ciocalteu to produce an unstable product that reduces molybdenum or tungsten blue. The reaction produced a blue-colored complex which gives the maximum absorption at 750 nm. Protein levels would decrease because the longer fermentation means the longer the opportunity for lactic acid bacteria to degrade protein, so it caused protein levels to decrease.

The results of the calculation of the ratio of high levels of glucose to protein provided information on the optimum culture conditions for producing EPS, because glucose was a monomer of EPS. The culture produced EPS with the highest glucose: protein ratio at the concentrations of Se2+ 100 mMol and Zn2+ 12.5 mMol.

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


*\*FRAP Data are means ± S.D. of two replicates.*

*\*\*The ABTS radical is completely reduced to this concentration accompanied by the disappearance of the green color.*

#### **Table 5.**

*Antioxidant activity test of EPS* L. plantarum *using FRAP and ABTS methods.*
