Table 183

Log Reduction of E. coli O157:H7 Inoculated on Uninjured and Injured Green Pepper Surfaces After ClO2 Gas Treatments for 30 min at 20°C Under 90 to 95% Relative Humidity

Log reduction after different ClO2 gas treatmentsb

Uninjured surface 2.90 ± 0.09Ad 3.99 ± 0.07Ac 7.27 ± 0.68Ab 8.04 ± 0Aac Injured surface 1.67 ± 0.08Bd 1.87 ± 0.03Bc 3.03 ± 0.02Bb 6.45 ± 0.02Ba a The initial populations of E. coli O157:H7 on surface-uninjured and surface-injured green peppers were 7.9 ± 0.29 logCFU/5g.

b Values in the same column with different uppercase subscript letters are significantly different (p < 0.05). Values in the same row with different lower subscript letters are significantly different (p < 0.05).

c No viable E. coli O157:H7 was detected using the end-point method after 1.2mg/l ClO2 gas treatments.

From Han, Y. et al., Food Microbiol., 17, 643, 2000. With permission.

to an uncut or intact surface. Studies [31] have shown that injuries to the wax layer, the cuticle, and underlying tissue layers of green pepper surfaces increased bacterial adhesion, growth, and resistance to washing and ClO2 gas treatments. Han et al. [32] reported that ClO2 gas treatments (0.15 to 1.2mg/l ClO2) had significantly higher reductions for inoculated E. coli O157:H7 on uninjured green pepper surfaces than on injured surfaces (P<0.05) (Table 18.3). Microphotographs obtained using confocal laser scanning microscopy (CLSM) illustrated that bacteria preferentially attached to injured surfaces, where the bacteria could be protected from the treatment. Similar results have been reported for inactivation of Listeria monocytogenes on uninjured and injured green pepper surfaces by aqueous and gaseous ClO2 treatments [33].

Microbial inoculation sites also influenced the efficacy of ClO2 gas treatments. Du et al. [40,41] inoculated L. monocytogenes and E. coli O157:H7 on three sites of an apple: stem cavity, calyx cavity, and pulp skin. After ClO2 gas treatments, bacteria on the pulp skin were less resistant to the treatment compared to the other two sites. At each inoculation site, however, bacterial inoculation levels (at 6, 7, and 8 log CFU/site) did not affect log reductions after treatment [40].

To determine accurately the efficacy of ClO2 gas treatments, appropriate bacterial recovery and enumeration methods should be used. In evaluating the effectiveness of a sanitation treatment on these pathogens in fruits and vegetables, one of the common problems is overestimation of the treatment effectiveness, because sublethally injured cells have not been taken into account since direct selective plating enumeration methods are normally used. Han et al. [35] reported that a membrane-transferring surface-plating method

Polycarbonate membrane

Polycarbonate membrane

Non-selective medium

Selective medium

FIGURE 18.7 Schematic of a membrane-transferring surface-plating method for enumeration of ClO2-treated bacteria.

was better for recovering uninjured and ClO2-injured E. coli O157:H7 and L. monocytogenes on green peppers after ClO2 gas treatments compared to traditional methods using direct surface-plating and overlay surface-plating. In this method (Figure 18.7), a 100 ^ bacterial sample is first spread over a polycarbonate filter membrane (0.4 ^ pore size, 90 mm diameter) (Osmonics Co., Westboro, MA) previously placed on the surface of a tryptic soy agar plate. Plates are incubated at 37°C for 2-4 hours to repair injured cells. Then the membranes are gently transferred onto appropriate selective media using sterile tweezers, followed by further incubation for 20 to 40 hours at 37°C. The membrane-transferring surface-plating method is also able to quantify low levels (<2logCFU/ml) of surviving bacteria by using a filtration procedure to concentrate bacterial populations in test samples [41]. An end-point determination method has been successfully used to detect a complete inactivation of inoculated bacteria on apple surfaces after ClO2 gas treatments [40,41]. This method is useful to validate the efficacy of a ClO2 gas treatment when known levels of bacteria are applied to produce surfaces.

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