Direct Molecular Approach to Monitoring Bacterial Colonization on Vacuum-Packaged Beef

Abstract

Denaturing gradient gel electrophoresis allowed us to monitor total bacterial communities and to establish a pattern of succession between species in vacuum-packaged beef stored at 2 and 8°C for 9 weeks and 14 days. Species-specific PCR was used to confirm the presence of Lactobacillus sakei and Lactobacillus curvatus. Multiplex PCRs using 16S rRNA-specific primers allowed differentiation between Leuconostoc species. These methods provided the desired information about microbial diversity by detecting the main microorganisms capable of colonizing this ecological niche.Vacuum packaging under chilled conditions has proved very effective in extending the shelf life of perishable foods, such as fresh meat and meat products, and preventing the growth of food-borne pathogens (8). The oxygen supply will be restricted, depending on the gas permeability of the packaging film, and thus has a selective effect on the microbial population (22). Lactic acid bacteria (LAB), such as Lactobacillus spp., Leuconostoc spp., Carnobacterium spp., and Brochothrix thermosphacta, are the main spoilage organisms associated with chilled vacuum-packaged fresh-meat products (6,18,21,26,33). Shortly after vacuum packaging of meat, LAB populations are usually below the routine detection limit (Ͻ10 CFU/g), but they increase during storage (19). Although LAB can cause meat spoilage, a selective growth promotion of LAB capitalizing on their ability to control meat-borne pathogens with a preferential growth of benign strains would minimize their spoilage effects (7,25,32,34).Methods in molecular microbiology, especially those including the sequencing of genes coding for 16S rRNA, have become a very important tool in the study of bacterial communities in meat samples. The trend is toward culture-independent methods, because they are believed to overcome problems associated with selective cultivation and isolation of bacteria from natural samples. Genetic-fingerprinting techniques provide a profile representing the genetic diversity of a microbial community from a specific environment. Denaturing gradient gel electrophoresis (DGGE) is usually employed to assess the structure and dynamics of microbial communities in food samples without cultivation in response to environmental variations (13,14,15,27). Species-specific PCR is a rapid and reliable molecular technique for the characterization of bacterial communities, and it can be also applied in situ without colony isolation (2). The variations in length and sequences of the 16S/23S rRNA intergenic spacer regions of the rRNA operon have proved useful for strain and species identification (2,3,17). In this work, we describe the application of cultureindependent methods to the study of the microbial succession dynamics in vacuum-packaged beef stored at 2 and 8°C for 9 weeks and 14 days, respectively.Bacterial control strains and growth conditions. Lactobacillus sakei CRL1463, Lactobacillus curvatus CRL 1465, and Leuconostoc gelidum CRL 1542 (CERELA culture collection) were used as reference strains. L...

Effect of Ultrasound Treatment Prior to Vacuum and Modified Atmosphere Packaging on Microbial and Physical Characteristics of Fresh Beef

“…The initial numbers of LAB at 0 day were 1.768 x 10 3 , 1.831 x 10 3 , 1.818 x 10 3 , 1.845 x 10 3 , 1.836 x 10 3 for control, US-VP, US-MAP, VP and MAP treatments, respectively. The growth LAB increased with an increase in storage time, these results were in good agreement with that observed by [7]. They reported that the LAB counts increased from 10 2 to 10 6 CFU g -1 after 14 days at 8°C and 6 weeks at 2°C.…”

“…They found that the pH reduced in control sample from (5.76 to 5.52). In addition, [7] also found that, the mean of the pH values in fresh beef vacuum package was decreased during storage at 2º and 8°C to final values of 5.17 ± 0.03 and 5.24 ± 0.02, respectively, the drop in pH values observed in meat samples may be attributed to the selective growth of microorganisms especially the lactic acid bacteria, which may also have contributed to the inhibition of gramnegative meat-borne organisms [7].…”

“…Vacuum packaging (VP) under chilled conditions has proved very effective in extending the shelf life of perishable foods, such as fresh meat and meat products, and preventing the growth of food-borne pathogens [7]. Color, lipid oxidation, and microbial criteria are the most important quality criteria for storage of fresh red meat.…”

Characterization of the predominant spoilage bacteria in sliced vacuum-packed cooked ham based on 16S rDNA-DGGE

“…The rapid identification of dominant spoilage microbial which had a strong influence on the final texture and the hygienic quality of the meat products can be used to indicate which preservative methods can be applied most effectively. PCR-DGGE clearly has potential in the analysis of microbial systems from different food in which many microorganisms are difficult to cultivate or are thought to be non-culturable (Fontana et al, 2006). In this study a PCR-DGGE protocol was established for identification and characterization of the dominant spoilage bacteria of cooked meat products.…”

“…In the recent decade, PCR-DGGE has been successfully applied to investigate the microbiology of fermented foods, such as fermented maize dough (Ampe et al, 1999), fermented sausages (Rantsiou et al, 2005), milk products (Temmerman, Masco, Vanhoutte, Huys, & Swings, 2003), etc. In recent years, monitoring the dynamics of microbial populations, and characterizing the dominant spoilage bacteria of beef or chilled pork have been studied using PCR-DGGE (Ercolini, Russo, Torrieri, Masi, & Villani, 2006;Fontana, Cocconcelli, & Vignolo, 2006;Li, Zhou, Xu, Li, & Zhu, 2006a). However, there are few data using PCR-DGGE technique to characterize the dominant spoilage bacteria in cooked meat product.…”