Efecto de la inmovilización en la producción de una bacteriocina por Pediococcus Acidilactici LPS28 aislada de salmón Effect of immobilization on the production of a bacteriocin by the salmon isolate Pediococcus Acidilactici LPS28

Abstract

Lactic acid bacteria (LAB) are use as probiotic microorganism in mammals, fish and fish products, mostly because of production of antagonist substances as lactic acid, acetic acid, oxygen peroxide and bacteriocins. The latter, are known to have a great potential as food bio-preservatives and are used in the production and preservation of food. While immobilization of lactic acid bacteria appears as an alternative to increase both cell concentration and bacteriocin production. In this study, the salmon isolate Pediococcus acidilactici LPS28 was shown to produce an antilisterial bacteriocin. Besides inhibiting the growth of Listeria spp. strains, the antimicrobial compound was also active against several strains of lactic acid bacteria but not against staphylococci. The bacteriocin was purified and its size was estimated by Tris-Tricine SDS-PAGE at about 3 kDa and by LC-MS/MS at 2.25 kDa. It was characterized as a heat-resistant (121°C for 15 min) and pH-tolerant peptide, resistant to treatment with trypsin and α-amylase, but not with proteinase K. Antibacterial activity was related to membrane permeabilization. Also, the relationship between microbial growth, bacteriocin production and the effect of fermentation medium using four different growth media: MRS medium, modified MRS medium (mMRS), whey permeate (WP), and lactase-treated whey permeate (LWP), pH and temperature on the occurrence and the maximum levels, to optimize bacteriocin biosynthesis during growth was studied. Either the medium, pH and/ temperature influenced the growth and bacteriocin production in different levels. The higher bacteriocin titers were obtained in LWP broth, at pH 6.0 at 32°C, and the maximum cell concentration was obtained in the same conditions, but at 37°C. The effect of immobilization of Pediococcus acidilactici LPS28 entrapped in calcium alginate was studied during fermentation, the effect of the medium and alginate percentage, and the reutilization of the beads. Immobilized P. acidilactici LPS28 reached high concentrations in the beads and produced raised bacteriocin titers. Also, better results were obtained in LWP broth at 2% of alginate. The immobilized cells could be reuses up to three times after filtration and resuspension in new medium. P. acidilactici LPS28 production kinetics were studied in both planktonic and alginate-immobilized cultures. In planktonic cultures, bacteriocin production paralleled bacterial growth and was optimal in LWP. Upon immobilization, bacteriocin production was still highest in LWP and also was affected by the percentage of alginate added. In addition, P. acidilactici LPS28 displayed poorer growth in the alginate beads than as planktonic cells, but demonstrated an increased bacteriocin production capacity. In addition, several experiments with immobilized cells indicated a shift in bacteriocin activity towards the stationary phase, which may be related to either altered production kinetics or diffusion effects. Also, a continuous fermentation in a glass column reactor was displayed, obtaining bacteriocin titers similar to those in batch fermentation. Finally pediococci and the listeria were inoculated as mono and co-cultures onto smoked salmon fillets at 4°C and 15°C for 24 h. Enumeration of LAB, L. inoccua ATCC 33090, and bacteriocin production was performed. Overall, results indicate that production of bacteriocins may be affected by the culturing method, which may be important when considering food applications in gel-like environments (e.g., cheeses, fermented meats, fish, etc.). Also, they showed that strain P. acidilactici LPS28 is a good biopreservative candidate for controlling listerial growth in smoked salmon showing a bacteriostatic mode of action against the target bacteria in the inoculated salmon fillets.