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coumestrol

Tomato

Lycopersicon esculentum

Alkaloids

Yam

Discorea rotundata

Hicicol, isobatasin

Adapted from Walker, J.R.L., Antimicrobial compounds in food plants, in Natural Antimicrobial Systems and Food Preservation, Dillon, V.M. and Board, R.G., Eds., CAB International, Wallingford, U.K., 1994, p. 181; Whitehead, I.M. and Threlfall, D.R., J. Biotechnol, 26, 63, 1992.

of salmonella recovered. While blending, homogenizing, or macerating may be acceptable in preparing samples of some types of fruits and vegetables, a simple surface washing without rupturing of plant cells may be required for other types.

The presence of inhibitory or protective residues from crop management practices and variations in surface morphology unique to specific fruits or vegetables should also be a consideration when selecting a procedure for preparing samples for analysis. Microorganisms may be most effectively retrieved by washing the surface of fruits and vegetables such as tomatoes, mangoes, avocados, watermelons, oranges, and other produce with a relatively smooth, rigid surface. Even produce with hard, apparently blemish-free surfaces, however, can harbor microorganisms in areas that are not easily accessible by washing or homogenization [53]. Infiltration of microbial cells into stomata, lenticels, broken trichomes, and cracks in the skin surface can occur. The porous stem scar tissue of tomatoes, for example, offers a relatively easy port of entry for microorganisms compared to intact skin [27,54]. Microorganisms are also known to partition into the cut tissues of produce. Infiltration of E. coli O157:H7 into cut tissue of lettuce is affected by temperature [55]. Microorganisms harbored in subsurface and other protected areas should be considered when selecting a method to retrieve them from produce tissues for the purpose of detection and enumeration.

Sonication of samples may be an alternative method for removal of microorganisms from the surface of produce with minimal tissue disruption, although this approach has not been thoroughly researched. Seymour et al. [20] evaluated the use of ultrasound to promote decontamination of raw vegetables. Cavitation caused by treatment appeared to enhance the release of Salmonella Typhimurium. Release of salmonellae and E. coli O157:H7 from inoculated alfalfa seeds is enhanced by ultrasound treatment [56]. These observations suggest that ultrasound treatment, perhaps in combination with other methods for removing microorganisms from produce tissues, may result in a more accurate assessment of populations. Tissues of leafy and floret vegetables, strawberries, raspberries, blackberries, and other produce with complex surface tissues are easily ruptured by rubbing, thus exposing surface microflora to stress conditions imposed by reduced pH or other factors associated with tissue juice. Agitation using a mechanical shaker or by manually shaking in a wash fluid with standard composition and volume for a set period of time may be the most suitable method for removing microbial cells from these produce items.

To avoid too many modifications of a standard sample preparation protocol, the sample weight or number of pieces and the volume of wash solution or diluent should be standardized for each type or group of produce. Results of analysis can be reported as CFU/g of sample or be converted to CFU/cm2 using a conversion table listing estimated values for specific fruits and vegetables categorized as spheres, cylinders, two-sided planes, or perhaps other geometric shapes. An alternative would be to calculate microbial populations on the basis of CFU/piece of fruit or vegetable, although this method has little meaning if the weight of each produce piece is not reported.

Removal and disposal of microbial cells from surface tissues of fruits and vegetables that have been mechanically cleaned by brushing or that have been waxed or oiled may be more difficult, compared with retrieval from untreated produce. Microorganisms entrapped in bruised tissue, waxes, and oils may be more difficult to remove and disperse in homogenates or wash fluids, resulting in an underestimation of populations. Dip inoculation of bruised, unwaxed apples in a suspension of E. coli O157:H7 is known to result in lodging and infiltration of cells in broken tissues, the waxy cutin layer, and lenticels (Figure 24.1). Cells can be harbored in lenticels at depths up to 24 making their retrieval difficult [57]. It may be necessary to modify the sample preparation protocol to maximize release of cells from tissues, as well as from

FIGURE 24.1 (Color insert follows page 594) Confocal laser scanning microscopy (CLSM) images showing attachment of E. coli O157:H7 to various sites on the surface of apples. (A) Bruised tissue of unwashed, unrubbed, bruised apple at a junction (4.8 p,m depth) between wax platelets: edge of wax platelets (open arrow); most cells attached to the edge of the wax platelets (filled arrow). (B) Bruised tissue of unwashed, unrubbed, bruised apple at a junction (6.6 p,m depth) between wax platelets: heavy colonization of junctions between wax platelets (arrow). (C) Bruised tissue of unwashed, rubbed, bruised apple showing a cuticular crack on surface of apple (16.6 p,m depth): cells are trapped within the cuticular crack (arrow). (D) Bruised tissue of unwashed, unrubbed, bruised apple showing a lenticel (9.2 p,m depth): cells are within lenticel (arrow) at a depth of 20.6 p,m below the surface of the apple. (From Kenney, S.J., Burnett, S.L., and Beuchat, L.R., J. Food Prot., 64, 132, 2001. With permission. Copyright International Association for Food Protection, Des Moines, IA.)

FIGURE 24.1 (Color insert follows page 594) Confocal laser scanning microscopy (CLSM) images showing attachment of E. coli O157:H7 to various sites on the surface of apples. (A) Bruised tissue of unwashed, unrubbed, bruised apple at a junction (4.8 p,m depth) between wax platelets: edge of wax platelets (open arrow); most cells attached to the edge of the wax platelets (filled arrow). (B) Bruised tissue of unwashed, unrubbed, bruised apple at a junction (6.6 p,m depth) between wax platelets: heavy colonization of junctions between wax platelets (arrow). (C) Bruised tissue of unwashed, rubbed, bruised apple showing a cuticular crack on surface of apple (16.6 p,m depth): cells are trapped within the cuticular crack (arrow). (D) Bruised tissue of unwashed, unrubbed, bruised apple showing a lenticel (9.2 p,m depth): cells are within lenticel (arrow) at a depth of 20.6 p,m below the surface of the apple. (From Kenney, S.J., Burnett, S.L., and Beuchat, L.R., J. Food Prot., 64, 132, 2001. With permission. Copyright International Association for Food Protection, Des Moines, IA.)

the naturally occurring waxy cutin layer and waxes or oils that may be applied to enhance appearance or extend the shelf life of some fruits and vegetables.

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