Immunodetection of the Bacteriocin Lacticin RM: Analysis of the Influence of Temperature and Tween 80 on Its Expression and Activity

Abstract

Immunoassays with specific antibodies offer higher sensitivity than do bioassays with indicator strains in the detection and quantification of several bacteriocins. Here we present the purification of lacticin RM and the production of specific polyclonal antibodies to a synthetic peptide resembling an internal fragment of the mature bacteriocin. The specificity and sensitivity of the generated polyclonal antibodies were evaluated in various immunoassays. The detection limits of lacticin RM were found to be 1.9, 0.16, and 0.18 g ml ؊1 for Western blot, immuno-dot blot, and noncompetitive indirect enzyme-linked immunosorbent assays, respectively. Immunoassay sensitivities were 12.5-fold higher than that of the agar diffusion test (ADT). The production of lacticin RM showed temperature dependency, with 3, 4.2, 12.7, 28.9, 37.8, and 12 g ml ؊1 at 37, 30, 20, 15, 10, and 4°C, respectively. Temperature-stability analysis demonstrated that lacticin RM is sensitive to mild temperature, but the loss of activity does not seem to result from protein degradation. Tween 80 increased the concentration of lacticin RM eightfold and probably affected the results of the ADT either by enhancing the activity of lacticin RM or by increasing the sensitivity of the indicator strain. The use of antibodies for the specific detection and quantification of lacticin RM can expand our knowledge of its production and stability, with important implications for further investigation and future application.Bacteriocins are antibacterial proteins produced by bacteria that kill or inhibit the growth of closely related bacteria. Many lactic acid bacteria (LAB) produce a high diversity of bacteriocins, with a fairly broad inhibitory spectrum (19). In recent years, many bacteriocins from LAB belonging to different groups have been described, characterized, and purified. Nisin, produced by several strains of Lactococcus lactis, is the most studied bacteriocin, and it is used as a commercial food preservative (6,11,16). The role of LAB and their bacteriocins as food biopreservatives is expected to grow in the future as a result of consumer awareness of the potential risks derived from food-borne pathogens as well as from the artificial chemical preservatives currently used to control them (14).Lacticin RM is a bacteriocin produced by Lactococcus lactis subsp. lactis EZ26 (DSM ID-95-131), isolated from goat's milk. It displays a wide range of inhibition, including Listeria monocytogenes and Staphylococcus aureus (36). The operon encoding lacticin RM is located on a 2.5-kb fragment in the plasmid pHU1, and it is rather simple in comparison to other bacteriocin systems, including only four genes. lacA is the bacteriocin structural gene, while lacF, lacG, and lacI participate in immunity against lacticin RM (36). Preliminary results indicate that lacticin RM is optimally produced at low growth temperatures, with maximal inhibition zones against an indicator strain at 10 and 15°C. Although most of the characterized bacteriocins are produced at the producer...

Immunochemical Characterization of Temperature-Regulated Production of Enterocin L50 (EntL50A and EntL50B), Enterocin P, and Enterocin Q byEnterococcus faeciumL50

“…Of interest is the high specificity ( Table 2) and sensitivity of the antibodies generated in the present work, which show a detection limit by an NCI-ELISA of 1 ng/ml (for EntL50A and EntL50B) and 15 ng/ml (for EntQ). These values are similar to the best reported data for nisin A and nisin Z (7,23) but are considerably lower than most data described for other bacteriocins (16,29,38,(45)(46)(47)(48)(49)61).…”

“…Similar to results observed for EntL50A and EntL50B, production of mesenterocin 52A by Leuconostoc mesenteroides subsp. mesenteroides FR52 (40), amylovorin L471 by Lactobacillus amylovorus DCE471 (19,41), curvacin A by Lactobacillus curvatus LTH1174 (52,65), sakacin K by L. sakei CTC494 (53), sakacin A by L. sakei Lb706 (20), sakacin P by L. sakei CCUG 42687 (1), lacticin RM by Lactococcus lactis EZ26 (38), and bacteriocin by Lactobacillus pentosus B96 (17) was shown to be stimulated by temperatures unfavorable for growth, particularly low temperatures. Moreover, production of EntL50A and EntL50B is restricted to temperatures below 42°C, although the strain grows at up to 47°C, as observed for mundticin KS production by Enterococcus mundtii NFRI7393 (37).…”

“…Moreover, production of EntL50A and EntL50B is restricted to temperatures below 42°C, although the strain grows at up to 47°C, as observed for mundticin KS production by Enterococcus mundtii NFRI7393 (37). Optimal bacteriocin production at suboptimal temperatures for growth could be ascribed to one or more of the following phenomena: (i) decreased degradation/inactivation of the bacteriocin, (ii) lower rate of enzymatic reactions resulting in reduced growth rate and increased pools of metabolites essential for bacteriocin synthesis, (iii) decreased adsorption of the bacteriocin to the producer cells, (iv) higher gene expression at the transcription and/or the translation level, and (v) higher level of production of the inducing peptide (1,20,38,42,53). Maximum volumetric EntQ production by E. faecium L50 was observed at 42 and 47°C.…”

“…On the other hand, before approval of the use of bacteriocins in the food industry, analytical methods to determine their presence, activity, and stability in foods should be available (31). In this respect, specific antibodies against bacteriocins can be successfully used for bacteriocin identification and detection by immunochemical assays, such as immunoblotting and enzyme-linked immunosorbent assay (ELISA) (5,29,38,45,61).…”

Caracterización de cepas del género Bifidobacterium con carácter probiótico.

“…Uno de los factores que modula positivamente la actividad antimicrobiana detectada en los sobrenadantes neutralizados de bifidobacterias fue la adición de Tween 80 (polisorbato) al medio de cultivo de acuerdo con otros estudios (Huot et al, 1996;Keren et al, 2004). Este surfactante puede incrementar los niveles de bacteriocines en los sobrenadantes mediante la estimulación de crecimiento o de la producción de bacteriocinas o mediante mecanismos que facilitan su secreción o deadsorción, aunque también podría actuar aumentando la sensibilidad del microorganismo empleado como indicador (Degnan et al, 1993;Aymerich et al, 2000;Keren et al, 2004).…”