D

CD era

Sis'

FIGURE 8.2 Scanning electron micrograph of alfalfa seeds showing extensive cracking of a seed coat and natural openings: C, crack in the seed coat; H, hilum; M, micropyle.

MN), Tween 80, Vegi-cleanTM (Microcide, Inc., Detroit, MI), and Vortex® (Ecolab). Treating with aqueous chemicals at elevated temperatures can lead to greater reductions of pathogen populations on seed, but is often detrimental to seed germination [83]. Addition of high levels of the surfactant Tween 80 (1%, w/v) to 1% Ca(OH)2 led to only an additional 1 log10 reduction or less in the population of salmonella on alfalfa seed [62,63]. Sonication of seed during treatment with aqueous antimicrobial compounds also did not have a significant effect, only slightly increasing the log10 kill obtained [68,83].

Treatment with gaseous acetic acid was reported to eliminate both salmonella and E. coli O157:H7, but not Listeria monocytogenes, from artificially inoculated mung bean seed without reducing seed germination [55]. Similar treatments of inoculated alfalfa seed led to either unacceptable reductions of seed germination [84] or were not effective [56]. Hot water treatments of alfalfa seed inoculated with generic E. coli were reported to eliminate the bacterium [79], but results both with alfalfa seed artificially inoculated with human pathogens as well as naturally contaminated seed have not been as promising due to lowered effectiveness and/or detrimental effects on seed germination [34,85]. Under commercial practice, the ability of hot water treatments to ensure consistent elimination of bacterial human pathogens from alfalfa seed was put into question by a recent multistate outbreak of salmonellosis due to contaminated alfalfa sprouts grown from seed treated with hot water followed by a soak in low levels (2000 ppm) of chlorine [20]. However, a recent laboratory study indicates that treatment of mung bean seed with hot water may be an effective seed-sanitizing step. Treatment of seed inoculated with salmonella at 55°C for 20 minutes, 60°C for 10 minutes, or 70°C for 5 minutes led to an approximate 5 log10 reduction [80]. Treating seed at 80°C for 2 minutes was even more effective resulting in an over 6 log10 reduction. None of these temperature/time treatments led to a decrease in germination of the treated seed.

There have been a very limited number of studies on seed sanitization using naturally contaminated rather than artificially inoculated seed and these studies have evaluated the efficacy of hot water and chlorine treatments only [44,65, 85]. The use of naturally contaminated seed rather than artificially inoculated seed may give a more accurate prediction of the efficacy of seed treatments for eliminating bacterial human pathogens in commercial practice. This may be due to differences in bacterial populations per gram of seed (normally much lower on naturally contaminated seed than on artificially contaminated seed used for laboratory studies), possible differences in the location and physiological status of the pathogens and the potential presence of pathogens in biofilms. In contrast to studies with artificially inoculated seed treated with high levels of chlorine, research conducted independently in two laboratories using alfalfa seed lots naturally contaminated with salmonella indicated that treatment with chlorine (unbuffered and buffered to neutral pH, from 2,000 to 20,000 ppm) completely eliminated the pathogen [65,85]. However, a third laboratory published contrasting results using 20,000 ppm of unbuffered active chlorine also using seed naturally contaminated with salmonella [44]. The reasons for the differing results between laboratories may include differences in the degree of mixing during seed treatment as well as differences in the population and location of the pathogen on the particular naturally contaminated seed tested even if originating from the same seed lot.

Several physical treatments have also been tested for sanitizing sprout seed (Table 8.2). In 2000 the FDA approved exposure of sprout seed to ionizing radiation at doses up to 8 kGy [86]. Treatment with ionizing radiation can significantly reduce bacterial pathogens on sprout seed. Exposure of inoculated alfalfa seed to a 2 kGy dose of gamma irradiation led to a 3.3 and 2.0 log10 reduction in E. coli O157:H7 and salmonella populations, respectively, while still maintaining commercially acceptable yields as well as nutritive values of sprouts grown from the treated seed [71,87,88]. Higher dosages led to unacceptable reductions in yields. For alfalfa seed naturally contaminated with salmonella and treated with gamma radiation, Thayer et al. [89] reported a D-value of 0.81 kGy. An absorbed dose of 4 kGy was required to eliminate the pathogen, a dosage that results in significant reductions in yield. A required dosage of 4 kGy for pathogen elimination along with a D-value of 0.81 kGy indicates that individual naturally contaminated seeds may harbor pathogen populations in excess of 4 log10 CFU. Electron beam radiation or use of so-called soft electrons (low-energy electron beam, energies <300 kV) may also be useful for reducing pathogen populations on the surface of seed [90], but both have lowered penetration ability compared to gamma radiation.

Various treatment combinations (hurdle concept) for reducing contaminants on sprout seed have also been tested. Bari et al. [68] reported that the combination of dry heat (50° C, 1 hour) followed by treatment with hot acidic electrolyzed oxidizing (EO) water and sonication was able to reduce populations of E. coli O157:H7 on artificially inoculated mung bean seed by 4.6 log10, but the combination treatment was less effective when tested against inoculated radish and alfalfa seed. Seed germination and subsequent sprout growth were not adversely affected. In the same study, a dry heat (50° C, 1 hour) seed treatment in combination with exposure to 2 to 2.5 kGy of gamma radiation led to the elimination of the pathogen on mung bean, radish, and alfalfa seed, but resulted in decreases in yield, most significantly for mung bean and radish. Lang et al. [52] found that successive treatments of alfalfa seed artificially inoculated with E. coli O157:H7 with lactic acid and chlorine (2000 ppm) were slightly more effective than lactic acid treatments alone, but were less effective than high levels of chlorine (20,000 ppm). Sharma et al. [91] found that treating alfalfa seed inoculated with E. coli O157:H7 first with ozone (continuous sparging in water) followed by a dry heat treatment (60°C, 3 hours) led to a greater than 4 log10 reduction of the pathogen population, but survivors were detected by enrichment. A sequential washing treatment with thyme oil (5 ml/l) followed by ozonated water (14.3 mg/l) and aqueous ClO2 (25 mg/l) led to a 3.3 log10 reduction of E. coli O157:H7 on inoculated alfalfa seed [92].

The large body of research reported subsequent to the release of the FDA guidance documents [53] indicates that several alternative chemical and physical treatments may be similar or greater in efficacy to high levels of chlorine for reducing pathogen populations on sprout seed. For sanitizing alfalfa seed such treatments include seed soaks in 1% Ca(OH)2, 1% calcinated calcium, FIT®, 8% H2O2, or 2% CITREX™ [62,63,67,69,83,93]. For sanitizing mung bean seed exposure to gaseous acetic acid or soaking seed in hot water appear especially promising [55,80]. The efficacy of these alternative chemical and physical treatments needs to be confirmed by other researchers ideally using naturally contaminated seed. In contrast to high levels of chlorine, several of these alternative methods of sanitizing seed may be acceptable for use by organic growers as well as conventional growers pending any required regulatory approvals. The cost of some of these alternative methods to the commercial grower may be prohibitive, however. Cost may not be as much of an issue for home growers.

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