Semi-preparative scale purification of enterococcal bacteriocin enterocin EJ97, and evaluation of substrates for its production

Abstract

The influence of substrate composition on the production of enterocin EJ97 and the conditions for semi-preparative bacteriocin recovery have been studied. Final bacteriocin concentrations of 12.5 or 15.6 mg/l were obtained in the commercial media brain heart infusion broth (BHI) and tryptic soya broth, respectively. The bacteriocin was also produced in the complex medium CM (8.75 mg/l), in which the vitamin supplement was essential for production. Some combinations of meat peptone and yeast extract plus either soy peptone or BHI also supported bacteriocin production, at concentrations of 6.25-7.5 mg/l. In cow milk (whole, half-skimmed, and skimmed), the final bacteriocin concentrations obtained ranged from 7.5 to 11.25 mg/l. Highest bacteriocin activity was obtained by using pasteurised milk whey as growth substrate (up to 25 mg/l), suggesting that this bacteriocin can be obtained on a large scale by using this cheap food-grade industrial by-product. Highest bacteriocin titres were always obtained after 8 h of incubation at 37 degrees C. Semi-preparative concentration and purification of enterocin EJ97 produced in a complex medium was achieved by bulk cation exchange chromatography without previous cell separation, followed by reversed-phase chromatography. This two-step procedure allowed preparation of milligram quantities of purified bacteriocin, which is an improvement compared to purification procedures established for most other bacteriocins (35). The availability of purified enterocin EJ97 will facilitate other studies such as the elucidation of its molecular structure and its interaction with target bacteria.

Antimicrobial compounds produced by Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian meat product

“…Many other strategies have been reported in the literature for the purification of antimicrobial peptides and proteins produced and secreted by bacteria [1,3,9,21,34,40,41,44,47]. Generally, the first step is a precipitation with ammonium sulphate [29] or an aqueous extraction of secreted compounds by medium acidification to release cationic compounds bound to the bacteria cell wall [27].…”

Lactobacillus sakei 1 and its bacteriocin influence adhesion of Listeria monocytogenes on stainless steel surface

“…In general, the metabolism of LAB leads to an improvement of sensorial attributes of dairy, vegetable, fish and meat products and some strains may even inhibit growth of spoilage and pathogenic microorganisms, contributing for safety and shelf-life extension of those products (Castellano, Belfiore, Fadda, & Vignolo, 2008;Castellano et al, 2004;Guerrieri et al, 2009;Hammami, Rhouma, Jaouadi, Rebai, & Nesme, 2009;Lopez et al, 2007;Minei, Gomes, Ratti, D'Angelis, & De Martinis, 2008). Specifically, Lactobacillus sakei presents tolerance to low temperatures and high salt concentrations, playing important role in fermentation and/or preservation of fresh meat and fish products mainly due to its ability to produce acid, which prevent the growth of a great variety of pathogens (Champomier-Verges, Chaillou, Cornet, & Zagorec, 2001;McLeod et al, 2008;Najjari, Ouzari, Boudabous, & Zagorec, 2008;Ringo & Gatesoupe, 1998).…”

Culture medium of diluted skimmed milk for the production of nisin in batch cultivations

“…In this case, under microaerophilic conditions, the cells had only the residual oxygen contained inside of bioreactor at their disposal. López et al (2007) studied enterocin EJ97 bacteriocin production in bovine milk (whole, half-skimmed, and skimmed) and obtained the highest titers (18 AU/mL or 11.25 mg/L) after 8 h of incubation in half-skimmed milk; this level is fourfold lower than that obtained in assay 3 (Table 1). These authors also verified that bacteriocin activity was much lower in whole milk than in half-skimmed or skimmed milk, suggesting that Ej97 may interact with milk fat.…”