Modeling Bacteriocin Resistance and Inactivation of Listeria innocua LMG 13568 by Lactobacillus sakei CTC 494 under Sausage Fermentation Conditions

Abstract

In mixed cultures, bacteriocin production by the sausage isolate Lactobacillus sakei CTC 494 rapidly inactivated sensitive Listeria innocua LMG 13568 cells, even at low bacteriocin activity levels. A small fraction of the listerial population was bacteriocin resistant. However, sausage fermentation conditions inhibited regrowth of resistant cells.Lactobacillus sakei CTC 494, producer of the bacteriocin sakacin K, inhibits Listeria spp. during sausage fermentation (12). Bacteriocins are small peptides or proteins with antibacterial activity (5). The resistance of the target strain may compromise bacteriocin efficacy (6,7,11,23). This study focuses on in vitro interaction kinetics between Lactobacillus sakei CTC 494 and Listeria innocua LMG 13568 and on listerial sakacin K resistance as a function of key sausage fermentation conditions (temperature, pH, salt, and nitrite) that may affect both bacteriocin production (13,14,17,21,22) and activity (9, 10). Listeria innocua, being more competitive (2) and resistant to sakacin K (12) than Listeria monocytogenes, offered a worst-case approach. An industrial, nonbacteriocinogenic Lactobacillus sakei I strain was used for comparison.Fermentations were done in a Biostat C fermentor (B. Braun Biotech International, Melsungen, Germany) in 10 liters of customized MRS medium (3). Modifications involved the addition of 40 g of sodium chloride per liter (VWR International, Darmstadt, Germany) and 200 ppm of sodium nitrite (VWR International). Some experiments, being highly reproducible (13, 17, 21-23), were repeated. Culture preparation, fermentor setup, and on-line control were as described previously (13). Bacteriocin-free Lactobacillus sakei CTC 494 inocula (10%) were prepared in 100 ml MRS medium (Oxoid, Basingstoke, United Kingdom) at 37°C overnight, since no sakacin K is produced above 33°C (13).Listeria innocua LMG 13568 inocula (1%) were prepared in 100 ml of brain heart infusion (Oxoid) at 30°C overnight. Lactobacilli were enumerated on MRS agar (Oxoid) after incubation at 30°C; Listeria innocua LMG 13568 did not grow on this medium during the first days. Listeriae were enumerated on Palcam agar (Oxoid) after incubation at 30°C. The standard deviation on both measurements equaled 0.07 log CFU ml Ϫ1 . Bacteriocin activity in the cell-free culture supernatant was estimated by a twofold critical dilution method (4, 13). A variation coefficient of 20% on bacteriocin measurement occurs, but activity curves are reproducible (13,14). Salt and nitrite did not interfere with the assay (14).The bacterial population was regarded as an association of subpopulations (in CFU per ml) of Lactobacillus sakei CTC 494 (LAC) and both bacteriocin-sensitive (LIS S ) and bacteriocin-resistant (LIS R ) Listeria innocua LMG 13568. The equations used for the model shown in Table 1 (13-15) were solved by Euler integration in Microsoft Excel by minimizing residual sums of squares. The max , , and K d of LIS R were determined independently; the other parameters were set as for the Listeria monocult...

Bacteriocins from Lactic Acid Bacteria: Production, Purification, and Food Applications

“…In particular, such information is essential when dealing with the potential problem of bacteriocin-resistant target bacteria [44,45] . Application of bacteriocin-producing starter cultures in sourdough (to increase competitiveness and hence establish a desired microbial population), in fermented sausage (anti-listerial effect to meet the zero-tolerance policy in ready-toeat foods), and in cheese (anti-listerial and anti-clostridial effects), have been studied during in vitro laboratory fermentations as well as on pilot-scale level [19,41,[44][45][46][47][48][49] . Results of these studies were highly promising and underline the important role that functional, bacteriocinogenic strains of LAB may play in the food industry as starter cultures, co-cultures, or bioprotective cultures, to improve food quality and safety [2,3,13] .…”

“…In this way, the kinetics of bacteriocin production by LAB strains in foods have been described in detail, amongst others through mathematical modeling and positive predictive microbiology [43] . Insights in the relationship between the food environment and kinetics of the starter culture have yielded valuable information about the in situ production of bacteriocins and its interactions with the target strains, which will be important if bacteriocins or bacteriocinproducing strains are to be increasingly used in food systems [19,44,45] . In particular, such information is essential when dealing with the potential problem of bacteriocin-resistant target bacteria [44,45] .…”

“…Insights in the relationship between the food environment and kinetics of the starter culture have yielded valuable information about the in situ production of bacteriocins and its interactions with the target strains, which will be important if bacteriocins or bacteriocinproducing strains are to be increasingly used in food systems [19,44,45] . In particular, such information is essential when dealing with the potential problem of bacteriocin-resistant target bacteria [44,45] . Application of bacteriocin-producing starter cultures in sourdough (to increase competitiveness and hence establish a desired microbial population), in fermented sausage (anti-listerial effect to meet the zero-tolerance policy in ready-toeat foods), and in cheese (anti-listerial and anti-clostridial effects), have been studied during in vitro laboratory fermentations as well as on pilot-scale level [19,41,[44][45][46][47][48][49] .…”

General Model, Based on Two Mixed Weibull Distributions of Bacterial Resistance, for Describing Various Shapes of Inactivation Curves

“…Among these models are the vitalistic models proposed by Cole et al (13,28,39), models describing both growth and inactivation (26,27,32,37,40,41), the modified Gompertz model (24,32), the exponential model (31), and the log-linear model with latency time (6) and/or with a tail (5). These models cannot deal with all shapes of curves, and most of them are based on log-linear inactivation.…”

Isolation of Highly Heat-Resistant Listeria monocytogenes Variants by Use of a Kinetic Modeling-Based Sampling Scheme

“…The occurrence of stress-resistant variants in monoclonal populations of bacteria remains an important subject of debate, as some research groups have reported the isolation of such variants while others failed to demonstrate their presence and questioned their existence (1,3,5,16,17,19,24,29,31). In this study, we used a new strategy involving a kinetic modeling-based sampling scheme and an optimized stress challenge cycle method for the isolation of stress-resistant variants of L. monocytogenes.…”