The control of Listeria innocua and Lactobacillus sakei in broth and meat slurry with the bacteriocinogenic strain Lactobacillus casei CRL705
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.
Section snippets
Bacterial cultures and media
L. casei CRL705, producer of lactocin 705 and lactocin AL705 and L. plantarum CRL691 used as indicator for lactocin 705, were isolated from dry-cured sausages (Vignolo et al., 1993) and were grown in MRS broth at 30°C. L. sakei CRL1424, isolated from vacuum-packaged spoiled raw meat was maintained by growing in MRS broth plus 2 μg/ml of chloramphenicol at 30°C. The antibiotic was purchased from Sigma Chemical Co (St. Louis, Mo). L. innocua 7, used as indicator of lactocin AL705 was obtained from
Results
The control of L. innocua 7 and L. sakei CRL1424 by the bacteriocinogenic L. casei CRL705 in broth and meat system during refrigerated storage under vacuum was studied. A sensitive strain to lactocin 705 selected from the predominant indigenous lactic acid bacterial flora present in spoiled vacuum-packaged raw meat was identified as L. sakei CRL1424 by 16 s rDNA analysis. In this work L. innocua 7 instead of L. monocytogenes FBUNT was used after the growth rate and final population were compared
Discussion
L. sakei CRL1424 was isolated from spoiled vacuum-packaged raw meat as dominant specie of the indigenous microflora considered to be involved in meat deterioration. LAB is the major component of the microbial flora developing on various refrigerated vacuum-packaged meats. In vacuum-packaged cooked meat products Lactobacillus and Leuconostoc have been the main genera associated with the spoilage of these products, L. sakei and Lactobacillus curvatus being isolated commonly (Dykes et al., 1994;
Acknowledgements
The authors are grateful to the Agencia Nacional de Promoción Cientı́fica y Tecnológica (ANPCyT), PICT98 No 09-04632, Argentina, for financial support of this work.
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