The production of polysaccharides derived from lactic acid bacteria (LAB) can be a valuable alternative to current polysaccharides. In this study, eight samples of Kishk (traditional dairy product) were collected in sterile conditions and directly cultured on MRS agar medium. Following purification and examination of microscopic, macroscopic characteristics and doing biochemical tests, 143 isolates were selected, and the production of exopolysaccharide (EPS) was investigated by the ruthenium red and disc methods. The EPS production of 79 isolates was confirmed. Total carbohydrate of EPS was determined by the phenol sulfuric acid method. Finally, Enterococcus durans K48, Enterococcus faecium R114, Enterococcus faecium T52, strains were selected as the best EPS producers. The optimization of EPS production was then performed using Central Composite Design (CCD) and Response Surface Methodology (RSM). Optimization plots showed the highest EPS yield for E. durans K48, E. faecium R114, E. faecium T52, occurred at 38.4, 37.4 and 36.7 °C and pH of 5.9, 5.6 and 5.8, respectively, cultured in a Sucrose-MRS medium. Under optimal conditions, the maximum predicted and actual production of EPS for the examined isolates were 3.18, 3.21,2.99 and 3.02; 3.15, 3.15; g L−1, respectively. The EPS molecular weight determined by Gel Permeation Chromatography (GPC) was in the range of 2.93×105 to 3.52 × 105 Da. Analysis of the component monosaccharides by HPLC showed that all three tested EPSs were heteropolysaccharides. After appropriate evaluation, the EPS produced by native species of LAB isolated from Iranian Kishk could be of interest for industrial applications or as functional food ingredients.

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