Polyphasic characterization of bacterial community in fresh cut salads

Abstract

In the present work we describe a polyphasic study of bacterial community in fresh cut salads packaged under ordinary and modified atmospheres. Samples of fresh cut salads were aseptically collected at 0, 3, and 6 days of storage and analysed both by culture-dependent and -independent methods. DNA extracted from fresh cut salad samples was used as a template for PCR amplification of 16 S rRNA gene; the PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE); finally, clone libraries of 16 S rRNA gene from the fresh cut salad was constructed. Results of plating count revealed a significant increase of all microbial loads in fresh-cut salad samples packaged in OA and that the microbial growth of the different groups was significantly affected by the conditions applied for MA packaging. A constant presence, throughout storage, of the pathogenic bacteria in all the fresh-cut salads samples was highlighted by PCR-DGGE analysis. Therefore, the polyphasic approach used in the present study allowed us to characterize the main species involved in the fresh cut salad products and to better understand their dynamics throughout storage.

Introduction

Fresh cut salad (FCS) sold in ready-to-eat or ready-to use form have become a very important area of potential economic growth for fresh cut industry. Today these products have gained popularity mainly because consumers perceive such products, besides their well known nutritional qualities, as fresh, healthy, convenient, tasty and easy to use (Garret et al., 2003).

Fresh cut processing includes unit operations such as peeling, trimming or cutting that alter the integrity of the commodity's tissues and induce wounding stress. For these reasons, these products have a limited shelf-life (≤ 12 days), which is one of the greatest problems faced by commercial marketers (Soliva-Fortuny and Martín-Belloso, 2003). Although FCS products are lightly acidic foods (pH ≤ 6.0), their high humidity and the large number of cut surfaces can create ideal conditions for several microrganisms growth consequently leading to shelf-life reduction (Willocx et al., 1993). Bacterial species on the outer plant surface are often related to soil bacteria, which are members of Pseudomonas, Enterobacter or Erwinia genera, lactic acid bacteria (LAB) and yeast species (Nguyen-The and Carlin, 1994, Bennik et al., 1998, Beuchat, 2002, Tournas, 2005). Moulds are less important in FCS products due to the intrinsic properties such as slightly acid to neutral pH favouring bacteria and yeasts which will overgrow moulds (Lund, 1992, Moss, 1999, Giménez et al., 2003, Tournas, 2005). Among spoilage microrganisms, Pseudomonas fluorescens, Pantoea agglomerans, and Rahnella aquatilis, have frequently been isolated from minimally processed lettuce. These microrganisms have been also found to dominate the microbial population of FCS products at the end of shelf-life under refrigerated conditions (Zagory, 1999, Ragaert et al., 2007). Moreover, several opportunistic pathogens, such as Escherichia coli, Salmonella and Shigella spp., Aeromonas hydrophila, Yersinia enterocolitica, Listeria monocytogenes and Campylobacter spp. may also grow in FCS products, leading to a safety problem (Gleeson and O, 2005, Abadias et al., 2008). Preservation techniques (e.g. chlorine-containing compounds, irradiation, light or electric pulses, ClO₂ with cysteine) alone or in combination with active packaging and low temperature both during production and storage could minimise microbial development ensuring the shelf-life of the products (Everis, 2004, Gomez-Lopez et al., 2005, Gomez-Lopez et al., 2008). Nevertheless, microbial load reduction depends on the sensitivity of the microrganisms and the accessibility of the agent or the treatment to the microbial population (Ragaert et al., 2007). Hence, a better understanding of microbial ecosystems of the FCS products would be extremely useful in order to prevent the growth of pathogens, and to reduce spoilage microrganisms at the various stages of processing and storage.

Present knowledge of microbial population in FCS products is based mainly on bacterial cultivation, and on the identification of the dominant microorganisms; both methods are time-consuming and poorly reproducible. Moreover, most microrganisms widespread in nature are not cultivable under conditions typically used; microbial diversity can be over- or underestimated with media that are not sufficiently selective (Randazzo et al., 2002). Thus, molecular approaches, especially those based on the use of rRNA genes (DNA), like denaturing gradient gel electrophoresis (DGGE), and related technique, have provided the opportunity to analyze complex communities on the basis of sequence diversity (Muyzer et al., 1993). Bacterial species can be identified by generating clone libraries of the 16S rDNA followed by sequencing and comparison with databases containing thousands of ribosomal sequences to allow a phylogenetic affiliation to cultured, as well as uncultured microorganisms. DGGE is now frequently applied in food microbial ecology to compare the compositions of complex microbial communities and to study their dynamics (Randazzo et al., 2002, Ercolini, 2004).

To obtain knowledge on the bacterial communities of fresh cut salad, packaged under ordinary and modified atmosphere, in the present study a polyphasic characterization mainly based on molecular approaches, was applied. Whole bacterial community diversity was detected using PCR-DGGE analysis whereas the dominant bacteria were identified throughout the clone libraries of the 16 S rRNA gene amplified fragments.