Background and ObjectivesLactic acid bacteria (L.A.B.) can produce exopolysaccharides (EPSs) using agricultural and industrial waste materials. This approach can prevent the harmful disposal and buildup of these wastes in the environment in addition to producing valuable products. Thirteen LAB-producing EPS isolates were selected, and the similarity and distance indices were determined between them through Rep-PCR DNA fingerprinting, and molecularly identified LAB from silage samples. Evaluation of the ability of the isolated strains to produce exopolysaccharides was carried out, in addition to the optimization of the polysaccharides from renewable resources.Materials and methodsLAB-producing EPS isolates were molecularly identified by the 16S rRNA gene sequencing and deposited their DNA sequences to NCBI. EPS production using the examined 13 strains was carried out on MRS as a standard production medium and ranged between 1.53 and 7.53 g/l. Then, the highest significant EPS-producing strains i.e., Lacticaseibacillus paracasei strain LAB 64, Lacticaseibacillus rhamnosus strain LAB 160, and Lacticaseibacillus rhamnosus strain LAB 192 were further examined for EPS production from the agro-industrial wastes sugarcane molasses, salted cheese whey, and their mixture.Results and conclusionThe maximum EPS production by the three strains was obtained in a mixture of molasses: whey (1/1 v/v). Calcium carbonate addition to the production mixture significantly improved EPS production in almost all cases and it is important to neutralize the media. Moreover, increasing the mixture sugar concentration of the fermentation mixture from 2\% to 5\% enhanced EPS production by all strains. In this regard, a 2-fold increment in EPS production was achieved by the Lactic. rhamnosus strain LAB 160 22.39 g/l. The extraction and analysis of the EPS product were carried out using both FT-IR and HPLC compared to an EPS standard. FTIR and HPLC analysis confirmed the polymer as an alpha-glucan, which was identified as dextran through a comparison between its retention time and the retention time of the dextran standard.