Optimization Of Fermentation Conditions For Exopolysaccharide Production From Lactobacillus Plantarum KX041 And The Exopolysaccharide's Physicochemical Properties

Exopolysaccharide is a secondary metabolite,secreted by lactic acid bacteria in order to resist adverse environment such as nutritional deficiencies,toxic substances,phages and osmotic pressure and protect lactic acid bacteria itself.EPS can improve the tast,organizational status and the viscosity of dairy products and is a safe and nontoxic food additives.In this paper,the strain Lactobacillus plantarum KX041 was selected as the research object.Our objectives are to study the optimizing fermentation conditions,isolation and purification,structural analysis and antioxidant activity.The results of our study included the following aspects.1.The preparation and structure characteristics of EPS from Lactobacillus plantarum KX041 were studied.The EPS were prepared from the fermented modified MRS broth by centrifugation,ethanol precipitation,deproteinization by the Sevage method,dialysis and lyophilization.Analysis by phenol sulfuric acid method and Coomassie brilliant blue method showed the EPS consisted of 60.59%of total sugars and 12.88%of protein.Monosaccharide composition of EPS was determined by the pre-column derivatization gas chromatography?GC?method,which was arabinose,mannose,glucose and galactose,the molar ratio was0.95:12.94:7.26:3.31,which implied that the EPS was a hetero-polysaccharide.The Mw was38.67 KDa using high-performance gel permeation chromatography.Fourier transform infrared?FTIR?spectrometry showed that EPS had typical absorption peaks of polysaccharide and did not contain uronic acid.The differential scanning calorimetric?DSC?showed EPS had two distinct melting temperature at 163.93?and 202.16?,a degradation temperature of289.01?,the relative higher degradation temperature suggested that the EPS possessed good thermal property.The EPS was purified by a DEAE-cellulose-52 ion-exchange chromatography column and three fractions were obtained,a neutral polysaccharide EPS-1 and acidic polysaccharide EPS-2 and EPS-3,three fractions were further purified by Sephacryl S-300gel chromatography,elution profile of the EPS-1 andEPS-2 was single symmetrical peak EPS-was separated into EPS-3-1 and EPS-3-2.The Mw of them was 22.52KDa,28.23KDa,798.91KD and 25.88KDa,respectively by gel permeation chromatograph.2.Single factor experiments for maximum EPS yield was carried out by various culture conditions,the results of single factor experiments showed the optimum carbon and nitrogen source were lactose and soybean peptone,respectively,lactose was 3%,soybean peptone was1.5%,MnSO₄ was 0.08%,fermentation time was 28h,fermentation temperature was 30°C,initial pH value was 7 and inoculum concentration was 3%.Moreover,soybean peptone,fermentation temperature and initial pH value screened by the single-factor experiments and was optimized by response surface method.Maximum EPS yield 599.52mg/mL?about 3 folds than the original production?was obtained at optimal conditions of soybean peptone?20 g/L?,fermentation temperature?35??and initial pH value?6.38?.3.Ascorbic acid?Vc?was used as a control to determine the antioxidant activity of EPS on DPPH,ABTS,hydroxyl and superoxide anion radicals and the reducing power.The results showed EPS had a certain scavenging activity on the above four kind of free radicals and reducing power.The scavenging effect of the EPS towards DPPH,ABTS,hydroxyl and superoxide anion radicals had a significant dose dependent relationship in definite concentration range with the EC₅₀ value of 1.46mg/m,0.2mg/mL,1.7mg/mL,5.6mg/mL respectively,therefore EPS has strong comprehensive antioxidant ability in vitro.