Vegetable Microflora

The microflora on fresh fruits, grains, and vegetables can range from as low as 102 to 109 colony forming units (CFU) per gram [9,10]. On pickling cucumbers, for example, the aerobic microflora is typically between 104 to 106CFU/ml for fresh fruit, with LAB less than 101CFU/g [11]. In the absence of processing, degradative aerobic spoilage of plant material by mesophylic microorganisms occurs, with Pseudomonas spp., Enterobacter spp., and Erwinia spp. initiating the process [10]. A variety of pathogens, including Salmonella spp., Shigella spp., Aeromonas hydrophylia, Yersinia enterocolitica, Staphylococcus aureus, Campylobacter, Listeria monocytogenes, Escherichia coli, and others, may be present on fresh vegetable products [12-15]. Pathogens on fruits and vegetables may also include enteric, hepatitis, or polio viruses [16]. A variety of sources may contribute to the occurrence of pathogenic bacteria on fruit and vegetable crops, including exposure of plants to untreated manure or contaminated water, the presence of insects or birds, personal hygiene practices of farm workers, postharvest washing or hydrocooling water, and conditions of storage during distribution [12,14]. A study comparing the use of organic fertilizer (composted manure) and inorganic fertilizer from farms in Minnesota showed significantly higher coliform counts on the organically grown vegetables [17]. However, in this and related studies [18,19], pathogens, including E. coli O157:H7, were not detected.

Removal of pathogenic and spoilage bacteria from fruits and vegetables has proved difficult. Surface adherence of bacteria (Figure 14.1) may serve

Coli Fruit Surface

FIGURE 14.1 Attachment of pathogenic bacteria to cucumber fruit. Adhesion of bacteria to the surfaces of pickling cucumbers (Calypso variety) with wax: (A) Staphylococcus aureus; (B) Lactobacillus plantarum; (C) Listeria monocytogenes; (D) Salmonella typhimurium; (E) Enterobacter aerogenes ATCC 13048. Bar 5, 10 mm. (From Reina, L.D., Fleming, H.P., and Breidt, F., J. Food Prot., 65, 1881-1887, 2002.)

FIGURE 14.1 Attachment of pathogenic bacteria to cucumber fruit. Adhesion of bacteria to the surfaces of pickling cucumbers (Calypso variety) with wax: (A) Staphylococcus aureus; (B) Lactobacillus plantarum; (C) Listeria monocytogenes; (D) Salmonella typhimurium; (E) Enterobacter aerogenes ATCC 13048. Bar 5, 10 mm. (From Reina, L.D., Fleming, H.P., and Breidt, F., J. Food Prot., 65, 1881-1887, 2002.)

to enhance survival of bacteria during washing or sanitizing treatments. Bacterial cell surface charge and hydrophobicity measurements have been found to correlate with the attachment of cells to surfaces of cantaloupes and cucumbers [20,21]. Dewaxing cucumber fruit led to increased adhesion of L. monocytogenes and decreased adhesion for other bacteria with higher relative surface hydrophobicity, including salmonella, lactobacilli, and staphylococci [20]. Biofilms of bacteria may be more resistant to sanitizing agents and organic acid treatments than free or planktonic cells [22-24]. It is likely that the vast majority of microorganisms in food processing environments occur in multispecies or multistrain biofilms on food or equipment surfaces [25,26].

14.2.1 Washing Procedures

Washing procedures with water or chemical sanitizers typically result in only a 1 to 2 log10 decrease in bacterial cell numbers [24]. Hydrocooling procedures used for some fruits immediately after harvest may even serve to increase internalization of bacteria due to the vacuum created as internal gases in fruits and vegetables contract with the reduction in temperature [27,28]. Bacteria may be protected in inaccessible locations on fruits and vegetables, such as the cores and calyx of apples [29]. Attachment to wounded regions or entry into the interior of fruits and vegetables through wounded regions or stomata, pores, or channels may occur [20,30-32].

The packaging and storage conditions for minimally processed vegetable products, including the use of modified atmosphere packaging, may significantly alter microbial ecology. The extended shelf life of some minimally processed vegetable products may result in an undesirable "safety index,'' a concept developed to define the risks associated with modified atmosphere packaged foods [33]. This safety index is defined as the ratio of spoilage to pathogenic bacteria in foods, measured as the relative cell concentrations of these organisms. It has been argued, however, that the primary effect of modified atmosphere packaging in extending the sensory quality of vegetable products may be to decrease the metabolic activity of the vegetable material [34]. In a model system, it was found that growth rates for L. monocytogenes, A. hydrophilia, and Bacillus cereus may be reduced by modified atmosphere conditions, but final cell density was not affected [35]. One major source of concern is that Clostridium botulinum spores have been isolated from a variety of vegetables, and this organism may, under the right conditions of temperature, pH, and atmosphere, grow and produce toxin in minimally processed vegetable products if the O2 concentrations drop to 1% or lower [10].

14.2.2 Biocontrol in Minimally Processed Vegetable Products

The survival and growth of bacteria on vegetable products can depend on the competitive microflora present and the environmental conditions and processing treatments [15,36]. The use of competitive microflora to enhance the safety of minimally processed foods, including vegetable products, has been proposed by a number of authors [5,37-39]. LAB have been nominated for this role, partly because of their GRAS (generally regarded as safe) status and their common usage in food fermentations. Application of this approach for minimally processed fruit and vegetable products has led to mixed results. Vescovo and co-workers isolated LAB from salad vegetables and, subsequently, re-inoculated the vegetables with both the biocontrol cultures and selected food pathogens, including aeromonas, salmonella, staphylococcus, and listeria species [6,40]. The added LAB cultures were found to reduce or prevent the growth of microbial pathogens. Conversely, a Lactobacillus delbruckii lactis strain, known to inhibit E. coli on chicken skin due to the production of hydrogen peroxide, did not alter the survival of E. coli O157:H7 on fresh-cut vegetables, possibly due to the presence of catalase on the plant surfaces [8].

Competition from aerobic microflora isolated from fresh vegetables, other than LAB, including yeasts, Bacillus spp. and Pseudomonas spp., can influence the survival and growth of microbial food pathogens. Pseudomonas spp. have been shown to enhance [41], inhibit [42-44], or have no effect [45] on the growth of L. monocytogenes in fruits and vegetables. A variety of pseudomonas and aeromonas isolates from fresh vegetables were found to confer inhibitory activity against E. coli, salmonella, listeria, and staphylococcus strains using an agar diffusion assay [46]. Competition studies have shown iron sequestration by siderophores may influence the competition between pseudomonads and L. monocytogenes [42,47], although some Listeria spp. may be able to use exogenous siderophores as an iron source [48]. Buchanan and Bagi [49] demonstrated that the effects of salt and temperature can control the outcome of competitive growth of a L. monocytogenes Scott A and a Pseudomonas fluorescens culture that was screened for the inability to produce siderophores or bacteriocins. In a study by Del Campo et al. [45], competition for nutrients between a Scott A strain of L. monocytogenes and saprophytic bacteria from green endive was investigated. Enterobacteriaceae and pseudomonas were grown in competition with L. monocytogenes in minimal media and media supplemented with yeast extract. In this case, enterobacteriaceae but not pseudomonads species were effective in reducing the growth of the L. monocytogenes culture. Because culture filtrates from enterobacteriaceae were found to have no inhibitory effects in broth supplemented with yeast extract, the data indicated that competition for nutrients (not end product inhibition) was responsible for the inhibitory effect [45].

These studies illustrate the complexity of microbial interactions in and on fruit and vegetable products. Varying environmental conditions may include changes in the availability of nutrients, salt concentration, temperature, atmosphere, pH, and others. While further research is clearly needed, the use of protective cultures should only be considered as a supplement to good manufacturing practice, not as a substitute for the proper handling and packaging of vegetable products [5]. The use of biocontrol cultures may, therefore, be considered to enhance existing hurdle technology to prevent the growth of pathogens in foods. The hurdle concept [50] advocates the use of multiple preservative factors to prevent the growth of pathogens. In fresh fruit and vegetable products, the main factors affecting the growth of the indigenous bacterial populations are sanitation, modified atmosphere packaging, and refrigeration, as well as the competitive interactions of bacteria.

Bacteria cultures selected for use in biocontrol applications should ideally be isolated from the products for which they are intended to be used [39]. Development of successful biocontrol strategies for fresh fruit and vegetable products may include the following steps: (1) isolation of potential biocontrol LAB from the product for which they are intended to be used; (2) reduction of the total microflora in and on the vegetable product by one of a variety of procedures, including heat, washing using chemical sanitizers, irradiation, or others; (3) addition of the biocontrol culture to achieve an appropriate initial population, as determined experimentally; (4) storage of the product under refrigeration temperatures [39]. The shelf life of the product would then be dictated by the growth of the biocontrol culture, but, to be successful, the growth rates of a biocontrol culture presumably should be faster than that of the target pathogens. While rapid growth and production of inhibitory metabolites may be desirable from a safety standpoint, this may be a liability as far as the quality of the product is concerned. Breidt and Fleming [7] investigated the kinetics of acid production and inhibition of L. monocytogenes by L. lactis using a mathematical modeling approach [7]. It was observed that the growth and death of the L. monocytogenes culture could only be accurately predicted by the model if pH was assumed to be the limiting variable, rather than acid concentration, with cessation of growth around pH 4.6. Further studies to characterize the kinetics of bacterial competition are needed to aid in the development of biocontrol strategies.

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