A non-proteinaceous antimicrobial substance (nonbacteriocin) produced by Weissella paramesenteroides DFR-8. an isolate from Cucumber (Cucumis sativus), was purified using cell adsorption-desorption and gel permeation chromatography methods. A single peak observed in the purity check by analytical RP-HPLC strongly indicates the homogeneity of the nonbacteriocin preparation. The active substance is insensitive to proteolytic enzymes, lipase. amylase and catalase and shows a broad spectrum of activity towards food-borne/spoilage pathogens including Gram-negative organisms. The non-proteinaceous antimicrobial molecule has a molecular mass similar to 2.5 kDa and is thermostable up to 121 degrees C at pH similar to 4.0. A central composite rotatable design (CCRD) was employed to Study the interactive effect of temperature and pH on antimicrobial activity and an equation was developed to deduce the residual activity of inhibitory substance under any conditions of temperature and pH within the experimental domain. The broad inhibitory spectrum and heat stability of the antimicrobial substance advocates its application as food-biopreservative. (C) 2009 Elsevier Ltd. All rights reserved.