Biological

In contrast to the voluminous literature concerning biological control of plant pathogens [94] as well as numerous studies on the biological control (competitive exclusion) of pathogens in poultry, meat, and dairy products [95,96], there is little published information on the use of antagonistic microorganisms to control human pathogens on produce. The ideal biocontrol product for use on sprout seed and sprouts would contain a nonpathogenic microorganism(s) that is genetically stable, easily cultured and formulated using low-cost substrates and materials, has a long shelf life, is easily applied to seeds and/or sprouts, is highly effective on a variety of sprout types and against several human pathogens, and is affordable for the grower. For control of pathogens in poultry, meat, and dairy products, single microbial strains or defined or undefined consortia of microbes have been tested as antagonists.

Most of the studies on biological control of bacterial human pathogens on produce have examined the use of lactic acid bacteria (LAB) as antagonists [95]. LAB are attractive candidates for commercial biological control agents due to their common occurrence on sprout surfaces [41,97], their ability to produce multiple antimicrobial agents including bacteriocins, hydrogen peroxide, and organic acids in vitro, their extensive use in the food industry for fermentation, and their lack of known pathogenicity [95]. A strain of Lactococcus lactis inhibitory in vitro against Listeria monocytogenes due to acid production was tested for control of the pathogen when the two bacteria were co-inoculated onto alfalfa seed before sprouting [98]. Results indicated that the strain was much less inhibitory towards the pathogen in situ than in vitro, reducing pathogen populations on the sprouts by only 1 log10. In a second study on LAB, Wilderdyke et al. [99] found that of 58 isolates of LAB isolated from alfalfa seeds and sprouts, 32 were inhibitory towards the three pathogens salmonella, E. coli O157:H7, and Listeria monocytogenes in agar spot tests. One strain of Lactococcus lactis subsp. lactis was particularly inhibitory towards all three pathogens on agar media and in broth culture. The same group reported a significant reduction in populations of Listeria monocytogenes on alfalfa sprouts after application of a strain of LAB in the seed soak solution [100]. A commercial product containing a lactic acid bacterium is available in Japan for controlling E. coli O157:H7 on Daikon radish sprouts [101]. This product is to be sprayed onto seeds and sprouts several times during the sprouting process. In our laboratory, we have tested hundreds of plant-associated bacteria, primarily isolated from sprout surfaces, for their ability to inhibit growth of salmonella inoculated to alfalfa seed in small-scale laboratory bioassays [102]. Of these, a few isolates (none are LAB) have been identified that consistently reduce growth by several log10 units in small-scale laboratory bioassays. Currently, the effectiveness of these antagonists is being evaluated in larger scale experiments and studies on their mode of action are also underway.

More than a single antagonist may be required for controlling pathogens on germinating seeds of various sprout types due to compositional differences in the native microflora [103]. Treating artificially contaminated alfalfa seed with a novel purified bacteriocin, colicin HU194, led to reductions ranging from 3 log10 CFU/g to complete elimination of E. coli O157:H7. Efficacy was dependent on the particular strain of E. coli O157:H7 used for seed inoculation [104]. Bacteriophages are also being researched as a possible antimicrobial intervention for application to sprout seed [105]. Biological control agents may also be useful for reducing spoilage caused by soft-rotting bacteria [106].

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