Optimization Of Fermentation Conditions,Structure Analysis And Antioxidant Activity Of Exopolysaccharide Produced By Lactobacillus Fermentum CECT 5716

Lactic acid bacteria exopolysaccharides are high molecular weight carbohydrate polymers secreted by lactic acid bacteria to the outside of cells during growth and metabolism.Because of its good physical and chemical properties and biological properties,it has received extensive attention from domestic and foreign researchers.In this study,the fermentation conditions of Lactobacillus fermentum CECT 5716（isolated from healthy human breast milk）strain to produce exopolysaccharide（EPS）were optimized,and the EPS obtained under the optimal conditions were isolated,purified and analyzed.Finally,the antioxidant activities of EPS in vitro were preliminarily studied.The main conclusions of this paper are summarized as follows:Firstly,using EPS yield as an indicator,the medium composition of Lactobacillus fermentum CECT 5716 was optimized by single factor and orthogonal experiments.The optimal medium composition was:maltose 2.00%,sucrose 1.00%,yeast extract 2.50%,L-Cysteine hydrochloride 0.20%,sodium acetate 0.50%,dipotassium hydrogen phosphate0.20%,magnesium sulfate 0.02%,manganese sulfate 0.005%,hydrogen citrate diamine0.20%,Tween 80 0.10%.After Lactobacillus fermentum CECT 5716 was inoculated in an optimized medium with an initial p H of 6.20,and cultured at 37℃for 16 h,the EPS yield reached 1575.22 mg/L,which was 6.38 times that before the optimization.Secondly,EPS was isolated,purified and structurally elucidated.EPS was obtained by removing protein,dialysis and freeze-drying.The EPS was purified by Sepharose CL-6B column to obtain a main polysaccharide component,which was named p EPS.Its purity measured by phenol sulfuric acid method was 98.20±1.31%.p EPS was analyzed by high performance liquid chromatography（HPLC）,ion chromatography,Fourier transform infrared spectroscopy（FT-IR）,methylation and Nuclear magnetic resonance spectroscopy（NMR）to structural identification.The results showed that p EPS was a glucan with a molecular weight of 3.76×10⁴ Da.Scanning electron microscopy（SEM）analysis showed that p EPS was porous and highly branched structure.Finally,a preliminary study on the antioxidant activity of EPS was carried out.The antioxidant activity of the EPS was also studied by evaluating the indexes of the scavenging activity of EPS for DPPH and ABTS free radicals,the total antioxidant capacity and the total reducing power of Fe³⁺.The antioxidant activity of EPS was dose-dependent.As the EPS concentration was 8 mg/m L,the scavenging activities of EPS for the DPPH and ABTS free radical were（84.17±1.30）%and（35.37±1.24）%,respectively.Moreover,the total antioxidant capacity was 0.31±0.01 and the total reducing power of Fe³⁺was 0.55±0.01.