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The growing need for Lactobacillus bacteria usage in industry and the expending probiotic market led to a search for new cost-efficient fermentation media from which a high yield of these bacteria could be obtained. The following study aimed to elaborate cultivation medium, for Lactobacillus spp. growth, which main components would be wheat, maize, barley, and rye flours. The optimal temperature for Lactobacillus growth in new semi-solid fermentation (SSF) medium, water content, and pH of the medium were analyzed by the plate count met