The control of Listeria innocua and Lactobacillus sakei in broth and meat slurry with the bacteriocinogenic strain Lactobacillus casei CRL705

Abstract

The effect of the bacteriocin-producing strain, Lactobacillus casei CRL705, in the control of Listeria innocua 7 and Lactobacillus sakei CRL1424 in MRS medium and meat slurry during the storage under vacuum at chill temperatures was evaluated. L. sakei CRL 1424 isolated from vacuum-packaged contaminated raw meat was identified as the predominant indigenous lactic acid bacterial flora. Co-inoculation of MRS broth at 8°C with L. casei CRL705 caused growth inhibition of L. sakei CRL1424 and L. innocua 7 after 10 and 4 days of storage, respectively. At 4°C the complete inhibition of both strains occurred within 14 and 8 days for L. sakei CRL1424 and L. innocua 7, respectively. Bacteriocin activity in broth was observed to be maximal at 8°C reaching 2130 AU ml⁻¹ after10 days and 400 AU m1⁻¹ after 8 days of storage, while at 4°C maximal activities, 690 and 30 AU ml⁻¹ were obtained at 14 and 10 days, respectively. Addition of bacteriocinogenic strain to the meat slurry did not allow the growth of L. sakei and L. innocua, showing a bacteriostatic effect during 21 days of storage at 4°C. In addition, L. casei CRL705, as a protective culture did not change significantly the pH of the meat slurry in the assayed storage conditions. The results presented here confirm that the bacteriocinogenic strain L. casei CRL705 can be used as a useful tool to improve the microbial stability and safety in the commercial meat preservation.

Introduction

Bacteriocinogenic strains of lactic acid bacteria (LAB) have been isolated from dry cured sausages and considerable research have been done on their ability to inhibit growth of spoilage and pathogenic micro-organisms (Vignolo et al (1993), Vignolo et al (1996a), Vignolo et al (1996b)). Recent approaches in the preservation of cooked meats and minimally processed refrigerated foods are increasingly directed towards biocontrol using a protective microflora, usually LAB, to inhibit growth of Listeria monocytogenes and other undesired micro-organisms (Schillinger et al., 1996; Leisner et al., 1996; Duffes et al., 1999; Scannell et al., 2000; Katla et al., 2001; Bredholt et al., 2001).

LAB form the dominant population in fresh meat after 3 weeks of chill storage under vacuum or in a modified gas atmosphere. Under anaerobic conditions these organisms have a considerable advantage in growth rate over competing species such as Enterobacteriaceae and Brochothrix thermosphacta (Venugopal et al., 1993). A widely used practice in meat marketing is the vacuum packaging of primal cuts for distribution and extension of storage life followed by removal of the meat from the package for preparation of retail cuts. As a result of these practices meat can become contaminated with pathogens such as L. monocytogenes. A method for preventing L. monocytogenes growth on refrigerated raw or cooked meat would help reduce this potential hazard to public health.

Bacteriocin-producing LAB cultures have been successfully used to control natural LAB microflora in Spanish-style green olive fermentations with Lactobacillus plantarum producing plantaricins S and T (Ruiz-Barba et al., 1994); in sausage fermentations with Lactobacillus curvatus producing curvacin A (Vogel et al., 1993); in fresh pork sausages using a lacticin 3147-producing strain of Lactococcus lactis (Scannell et al., 2000); in the control of beef spoilage with bacteriocinogenic Leuconostoc gelidum UAL187 producer of leucocin A (Leisner et al., 1996) and during commercial production of cooked meat products using Lactobacillus sakei TH1 (Bredholt et al., 2001) among others. In general, LAB do not grow at refrigeration temperatures, but if the product is temperature abused, LAB cell can serve as indicators of the abuse by spoiling the product (Holzapfel et al., 1995; Jay, 1996; Hugas, 1998). To be successful in biopreservation, a bacteriocinogenic LAB culture must survive during storage at refrigeration temperatures, compete with the relatively high indigenous microbial loads of raw meat, actively inhibit pathogenic and spoilage bacteria, and do not alter the sensory properties of meat except under temperature-abuse conditions. In previous works, the synthesis and mode of action of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL705 isolated from dry-cured sausage were studied (Cuozzo et al (2000), Cuozzo et al (2003)). However, lactocin 705 was not active against L. monocytogenes, but evidence about the production of a second inhibitory substance, an anti-Listeria bacteriocin, is well documented (Vignolo et al (1996a), Vignolo et al (1996b)) whose characterization is currently in progress.

The objective of this study was to determine if the bacteriocinogenic L. casei CRL705 could produce an antagonistic action toward an indigenous Lactobacillus strain (identified as L. sakei CRL1424) and L. monocytogenes surrogate Listeria innocua on vacuum-packaged meat slurry during storage at refrigeration temperatures. L. innocua was used instead of L. monocytogenes for safety considerations.