1. Intervention Technologies For Enhancing the Safety of Sprouts W. F. Fett and K. T. Rajkowski Food Safety Intervention Technologies RU wfett@arserrc.gov

2. Overview Interventions for use on sprout seed –Overview of published data –Conclusions Interventions during sprouting and postharvest –Overview of published data –Conclusions Ongoing collaborative study with industry –Sprout seed (Dr. K. T. Rajkowski)

3. Sprout Seed Current guidance (FDA, 1999) – Seeds should be treated with one or more approved antimicrobial treatments such as 20,000 ppm calcium hypochlorite. At least one treatment should be applied immediately before sprouting Challenge is to obtain a 5 log reduction of human pathogens while maintaining seed vigor and sprout appearance and yield at acceptable levels (NACMCF 1999)

4. Sprout Seed Elimination of all pathogens is desirable due to the potential for outgrowth of any surviving pathogens during the sprouting process to levels sufficient to cause illness

5. Published Papers – Development of Interventions (Seed/Sprout)

6. Chemical Interventions - Seed Aqueous sanitizers (along w/ heat, surfactants) –Ca(OCl) 2, NaOCl, ClO 2, acidified ClO 2, acidified NaClO 2, Ca(OH) 2, calcinated calcium, H 2 O 2, acidic electrolyzed water, ethanol, sulfuric acid, lactic acid, citric acid, acetic acid, thyme oil, ozone, trisodium phosphate, colicin type E-2, Tsunami R, Vortexx TM, Vegi-Clean TM, Fit R, Calcifresh-S TM, Citrobio TM, Citricidal TM, Environne TM, Citrex TM Elevated temperature (42 to 50 o C) usually beneficial, but leads to greater loss of seed germination. Effect of addition of surfactants variable.

7. Chemical Interventions - Seed Gas phase treatments –Acetic acid vapor, allyl isothiocyanate, trans- anethole, carvacrol, cinnamic aldehyde, thymol, ammonia

8. Physical Interventions - Seed Dry heat Hot water Irradiation (gamma radiation, pulsed UV) Hydrostatic pressure Radio frequency dielectric heating

9. Biological Interventions - Seed Antagonistic bacteria –Lactic acid bacteria –Fluorescent pseudomonads –Whole bacterial communities Bacteriophage vs. Salmonella

10. Combination Treatments - Seed Combination of aqueous chemicals –Lactic acid followed by chlorine (2000 ppm) –Thyme oil followed by ozonated water and aqueous ClO 2 Combination of physical treatments with aqueous chemicals –Ultrasound and heat in combination with Ca(OH) 2, Tween 80, Tsunami R 200 and Fit R –Ozonated water followed by dry heat (60 o C, 3 h) –Dry heat (50 o C, 1 h) followed by chlorine (200 ppm) or acidic electrolyzed water and sonication Combination of physical treatments –Dry heat (50 o C, 1 h) followed by gamma irradiation

11. Alfalfa Seed - Chlorine (20,000 ppm) Method examined in several laboratories using artificially inoculated seed Data highly variable – from approximately 2 to 7 log reductions reported for Salmonella and E. coli O157:H7 –Differences in inoculation methods and inoculum size, use of seed rinses before and/or after treatment, and treatment methods (e.g., mechanical or hand mixing) Consistent finding - treatment does not consistently eliminate pathogens from inoculated seed Best to evaluate data for alternative chemical treatments with high levels of chlorine where directly compared in the same laboratory in the same set of experiments

12. Chlorine Treatments - Naturally Contaminated Alfalfa Seed 16,000 to 20,000 ppm, with or without buffering to neutral pH, 10-15 min w/ continuous mixing → eliminated Salmonella (Suslow et al. 2002. J. Food Prot. 65: 452-458; Fett 2002. Food Microbiol. 19: 135-149) 20,000 ppm, no buffer, 10 min, intermittent mixing → did not eliminate Salmonella (Stewart et al. 2001. J. Food Prot. 64: 618-621)

13. Potential Alternatives to Chlorine – Alfalfa Seed

14. Chemical Ca(OH) 2, 1%,10 min at RT Fit R, 15 to 30 min at RT Citrex TM, 20,000 ppm, 10 min at RT Pangermex TM (concentrated Citrobio TM ), 20,000 ppm, 10 min at RT All directly compared with high levels of chlorine. All tested in a single laboratory, independent confirmation required, scale up studies; safety/ cost issues?

15. Biological Whole microbial communities from market sprouts added at seed soak stage → 5 log reduction of Salmonella at day 7 of sprouting for alfalfa Matos and Garland. 2005. J. Food Prot. 68: 40-48.

16. Chlorine – Mung Bean Seed Ca(OCl) 2 (16,000 ppm, pH 6.8, 15 min, constant agitation, rinses both before and after seed treatment) –Salmonella → 5.0 log reduction (survivors) –E. coli O157:H7 → 3.9 log reduction (survivors) –No effect on % seed germination Fett, W.F. 2002. J. Food Prot. 65: 848-852.

17. Potential Alternatives to Chlorine – Mung Bean Seed

18. Gaseous Acetic Acid – Mung Bean Seed Gaseous acetic acid (242 ul per liter of air, 12 h, 45 o C) –Salmonella → 5 log reduction, no survivors –E. coli O157:H7 → 6 log reduction, no survivors –L. monocytogenes → 5 log reduction, survivors –% germination reduced from 96 to 88% (not statistically significant) Note: Similar treatment of alfalfa seed was detrimental to seed germination (Delaquis, personal communication) Delaquis et al. 1999. J. Food Prot. 62: 953-957.

19. Dry Heat – Mung Bean Seed Dry heat (55 o C) –Salmonella → 5 days, 4 log reduction, no survivors –E. coli O157:H7 → 4 days, 6 log reduction, no survivors –No effect on seed germination Note: Similar treatment of alfalfa seed reported to be detrimental to seed germination Hu, et al. 2004. J. Food Prot. 67: 1257-1260.

20. Hot Water – Mung Bean Seed Hot water (5 g/250 ml) (inoc. load >10 8 cfu/g) –55 o C/20 min → 5 log reduction of Salmonella –60 o C/10 min → 5 log reduction of Salmonella –70 o C/5 min → 5 log reduction of Salmonella –80 o C/2 min) → 6 log reduction of Salmonella –None of the treatments reduced seed germination below 95%. Thicker seed coat protects the embryo. Weiss and Hammes. 2003. J. Appl. Bot. 77: 152-155.

21. Ammonia Gas – Mung Bean Seed Ammonia gas (300 mg/L of air), 22 h, RT –5 log reduction of Salmonella (survivors) –6 log reduction of E. coli O157:H7 (survivors) –No effect on germination Note: Reported 2 to 3 log reductions with inoculated alfalfa seed using the same treatment. No effect on germination. Himathongkham et al. 2001. J. Food Prot. 64: 1817-1819.

22. Combinations – Mung Bean Seed Dry heat (50 o C, 1 h) followed by hot (50 o C) acidic electrolyzed water with sonication → 4.6 log reduction of E. coli O157:H7 (survivors), no effect on germination and sprout growth rate Dry heat (50 o C, 1 h) followed by gamma irradiation (2.0 kGy) → 4.6 log reduction of E. coli O157:H7 (no survivors), no effect on germination, reduced sprout growth rate Bari et al. 2003. J. Food Prot. 66: 767-774.

23. Interventions – Other Seed Types Garden cress – High pressure (300 MPa, 15 min, 20 o C) → 6 log reduction of Salmonella, generic E. coli and L. innocua (survivors). Germination delayed by 1 day (sesame, radish and mustard seed more pressure sensitive) Wuytack et al. 2003. J. Food Prot. 66: 918-923. Radish –Dry heat (50 o C, 1 h) followed by gamma irradiation at 2 kGy → 5 log reduction for E. coli O157:H7 (survivors) Bari et al. 2003. J. Food Prot. 66: 767-774

24. Summary – Seed No method is currently available which eliminates pathogens from artificially inoculated alfalfa seed while maintaining seed vigor Laboratory data on the ability of high levels of chlorine to eliminate Salmonella from naturally contaminated seed is variable. Published investigations of outbreaks indicate use of high levels of chlorine may not always be 100% effective under common commercial practice, but may reduce risk.

25. Summary – Seed Several potential alternative treatments (acetic acid vapor, ammonia gas, dry heat, wet heat) reported for mung bean seed High pressure treatment may be an alternative treatment for some seed types (e.g., garden cress) Biological control with whole bacterial communities appears promising, but long-range R&D project

26. Summary – Seed Results need independent confirmation, scale-up studies need to be undertaken and any required regulatory approvals obtained before use of alternatives to chlorine are implemented.

27. Sprouts Challenge is to significantly reduce or eliminate pathogens on sprout surfaces during growth or postharvest without compromising sprout growth rate, appearance, yield, shelf-life and nutrient content Substantial laboratory evidence for internalization of Salmonella and E. coli O157:H7 during sprouting of inoculated seed (alfalfa, radish and mung bean)

28. Interventions – During Sprouting Antimicrobial additives to the irrigation water –Chlorine (up to 200 ppm) –Chlorine dioxide (100 ppm) –Calcinated calcium (0.4%, w/v) –Ozonated water (9 ppm) –Combination of ozonated water 0.3 to 0.5 pm) and gaseous ozone (0.2 ppm) –Thyme oil alone or with chlorine dioxide and ozonated water –Acidified sodium chlorite (1,200 ppm) → No intervention was highly effective

29. Interventions - Postharvest Acidic electrolyzed water Aqueous ozone (up to 21 ppm) Chlorous acid (268 ppm) Sodium hypochlorite (up to 2,000 ppm) Lactic acid (2%) Acetic acid vapor (up to 500 mg/L) Allyl isothiocyanate (up to 500 mg/L)

30. Interventions - Postharvest RT water rinses Hot water dips (100 o C, 30 sec) Irradiation – gamma and electron beam

31. Effective Postharvest Interventions Irradiation –Gamma: elimination of Salmonella from alfalfa sprouts grown from naturally contaminated seed (0.5 kGy), and > 5 log reduction of Salmonella and generic E. coli from radish and mung bean sprouts (1.5 to 2.0 kGy); no detrimental effects on quality Rajkowski and Thayer. 2000. J. Food Prot. 63: 871-875; Bari et al. 2004. J. Food Prot. 67: 2263-2268. –Electron beam (beta) – elimination (6 logs) of L. monocytogenes from inoculated alfalfa sprouts (3.3 kGy). No change in appearance or odor. Schoeller et al. 2002. J. Food Prot. 65: 1259-1266 Note: Regulatory approvals required

32. Effective Postharvest Interventions Chlorine (2000 ppm, 5 min, RT) → 5 log reduction of Salmonella on alfalfa sprouts Gandhi et al. 2001. J. Food Prot. 64: 1891-1898 Chlorous acid (HClO 2 ) (268 ppm, 10 min) → 5 log reductions of Salmonella and L. monocytogenes on mung bean sprouts, no change in visual quality during storage at 4 o C for 9 days Lee et al. 2002. J. Food Prot. 65: 1088-1092 Note: Regulatory approvals required

33. Summary - Sprouts No effective way of eliminating Salmonella and E. coli O157:H7 during the sprouting process. Addition of antimicrobials to irrigation water would interfere with spent irrigation water testing. After harvest, only gamma or beta irradiation most likely to eliminate both surface-borne and internalized pathogens. Regulatory approval required for this use.

34. ARS/Industry Collaborative Study

35. Table 1. Reduction of Salmonella on inoculated alfalfa seeds after gaseous ozone treatment LOG CFU/G OF SALMONELLA ON TSA* SAMPLE ID** BEFORE AFTER*** REDUCTION A 6.1 4.6 1.5 B 6.7 5.4 1.3 C 5.9 5.1 0.8 D 5.9 5.2 0.7 E 5.9 5.1 0.8 - - - - - - - - - - - - - - - - - - - - - - - - - - - F**** 5.9 5.9 _________________________________________________________________ * Tryptic soy agar – a non selective agar for recovery. ** Samples processed in different packaging material. *** Processing conditions: 30,000 ppm ozone, 65 % RH for 24 hr. **** Processed with little to no RH.

36. Verification of Yield Ratio a.Alfalfa seeds given gaseous ozone treatment of 30,000 ppm ozone, 65 % RH for 24 h. b.Two sprouters given seed to determine germination and yield ratio. c.Both sprout growers reported a 99% germination with good yield ratio. d.One grower now using ozone treated seed for > 3 months.

37. Table 2. Peracetic acid/hydrogen peroxide sanitizing treatment of Salmonella inoculated alfalfa seeds for 20 minutes. log CFU/g Recovered % Sanitizer (v/v) Non-Treated Ozone-Treated 0 6.05 3.64 1 4.30 2.00 2 4.19 1.50 3 3.40 NG* ---------------------------------------------------------- hypochlorite 5.21 ________________________________________ * NG after plating and MPN recovery.