microbiological safety of sprouted seeds: current status and future directions

1. Microbiological Safety of Sprouted Seeds: Current Status and Future Directions  Keith Warriner Department of Food Science University of Guelph kwarrine@uoguelph.ca

3. Sprouted Seeds Mung bean sprouts Alfalfa Soy bean Anti-oxidants Anti-carcinogens Anti-cholesterol

4. Sprouts United States Alfalfa Canada and Europe Mung bean sprouts: lightly cooked (stir fry) Soy bean sprouts More exotic sprouts appearing (broccoli, buckwheat, onion, cabbage, rice)

5. Organic Sector & 5-a-Day Campaign USA $200-250m (300, 000 tons per year) Ontario $4m Expanding market Small/domestic producers Health benefits

6. February 2006 Aquafuchsia Quebec Product Recall Presumptive Salmonella on alfalfa Previous recall 2003 Follow good practice

7. Ontario 2005 October 2005 648 cases of salmonellosis traced to contaminated mung bean sprouts Product Recall 24th Nov 2005 Production re-started Dec 14th 2005 Product Recall 24th Dec 2005

8. Update on Investigation CFIA: No Comment Could the outbreak have been prevented if current guidelines had been followed? Not necessarily

9. Foodborne illness Outbreaks Linked to Sprouts

10. Pathogens Associated with Sprouts

11. Sakai City, Japan, in 1996 Radish sprouts contaminated with E. coli O157:H7. >6000 cases 13 deaths Further 4000 cases reported in other cities

12. Sprout Outbreaks in United States

13. Contaminated Seed Australia China Mongolia Burma United States

14. Sources of Seed Decontamination Contaminated irrigation water Grazing animals Manure Equipment

15. Mung Beans Seeds specifically produced for sprout production Australia: On-farm HACCP in mung bean production Traceability

16. Alfalfa Major alfalfa seed produced in the heart of cattle country Seeds not specifically produced for sprout production No motivation for on-farm HACCP

17. Sprout Production Pre-soak 3-16h Trays, drums or bins 25-30?C (>99% relative humidity) for 4-5 days Irrigation Alfalfa: 15 second spray every 2h (4 liters per min) Mung beans: Shower every 3-4h (40 liters per min)

20. 25-70kg mung bean batches 24-30?C for 4-5 days Irrigated every 3 h

21. Growth of E. coli during mung bean sprouting

22. Apoplastic Fluid from Surface Sterilised Bean Sprouts

24. NACMCF 1999 Sprout White Paper Microbial Safety Evaluations and Recommendations on Sprouted Seeds - Adopted May 28, 1999 Basis for FDA’s sprout guide http:/vm.cfsan.fda.gov/~mow/sprouts2.html International Journal of Food Microbiology, November, 1999

25. Seed Decontamination 5 log reduction required 20, 000 ppm Calcium hypochlorite Problems Not totally effective Worker safety Incompatible with organic production No other sanitizer listed

26. Spent Irrigation Water Testing Guidelines based on studies with alfalfa seed. Growth of pathogens during sprouting: 48h Screen water as opposed to sprouts directly

30. Federal Register Notice: Guidance Failure to adopt effective preventive controls can be considered insanitary conditions - FDA will consider enforcement actions against any party who does not have effective preventive controls in place, in particular, microbial testing

31. Broad Sprout Guide Everyone has a responsibility: 1. Seed production - Good Agricultural Practices (GAPs) 2. Seed conditioning, storage, and transportation - minimize contamination 3. Sprout production - Good Manufacturing Practices (GMPs) should be standard operating procedure

32. Broad Sprout Guide Seed treatment - applying one or more approved treatments shown to reduce pathogens prior to sprouting (SUCH AS 20,000 ppm calcium hypochlorite) 5. Microbial testing – testing spent irrigation water from each batch of sprouts for pathogens before sprouts enter the food supply - Salmonella - E. coli O157:H7

33. FDA Guidelines Guidelines not regulation Does not provide detailed information on all individual steps that should be followed to produce seeds and sprouts Resources and references CDHS/FDA sprout video

34. Educational video produced and distributed. http://www.cfsan.fda.gov/~dms/sprouvid.html or http://www.dhs.ca.gov/fdb/PDF/SproutOrderForm4.PDF “Safer Processing of Sprouts”

35. 1999 Consumer Advisory Advised all persons to be aware of the risks associated with eating [all] raw sprouts. People in high risk categories should not eat raw sprouts. Persons wishing to reduce the risk of foodborne illness from sprouts should not eat raw sprouts.

36. Impact of Guidelines Year 2000 No outbreaks linked to alfalfa or clover Outbreak linked to mung bean sprouts contaminated with Salmonella enteritidis Guidelines working?

37. Sprout Outbreaks 1996-2004

38. Alfalfa Sprout Outbreaks and Compliance with FDA Guidelines by Year

39. FDA 1998 Field Assignment GMP Inspection 83 firms 57% performed unsanitary practices Samples for microbial analyses raw seed ? finished product 78 firms

40. FDA 1998 Microbial Analyses Firm Stage of growth A RAW PRE GERM ----- FIN* B RAW PRE GERM ----- FIN* C RAW PRE* GERM* WW* FIN* * (+) Salmonella sp., alfalfa

41. 2000 Field Assignment 1 Year Post-Guidelines Target: 150 firms Limited Inspections (focus on practices in guidance) 72% of firms failed to follow guidelines in full - Warning letters 65 firms (47%; 10% ) Unsanitary conditions Failure to implement effective controls, emphasis on microbial testing

42. Seed Decontamination Not included in site inspections Known to have limited efficacy Sprout producers interpretation: Seed decontamination is not important

43. 2004 - Sanitation

44. 2004 - Seed Treatment

45. 2004 - Sampling and Microbial Testing of Spent Irrigation Water

46. 2004Testing Spent Irrigation Water

47. Future Regulation? Ineffective seed decontamination method Unreliable screening method Stakeholder motivation

48. How can safety be improved Guidelines vs Regulation Seed decontamination interventions Spent irrigation water

49. Seed Decontamination Eliminate pathogens Maintain seed viability Low cost and practical

50. Chemical Interventions - Seed Ca(OCl)2, NaOCl, ClO2, acidified ClO2, acidified NaClO2, Ca(OH)2, calcinated calcium, H2O2, acidic electrolyzed water, ethanol, sulfuric acid, lactic acid, citric acid, acetic acid, thyme oil, ozone, trisodium phosphate, colicin type E-2, TsunamiR, VortexxTM, Vegi-CleanTM, FitR, Calcifresh-STM, CitrobioTM, CitricidalTM, EnvironneTM, CitrexTM Gas phase treatments Acetic acid vapor, allyl isothiocyanate, trans-anethole, carvacrol, cinnamic aldehyde, thymol, ammonia

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

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

53. Seed Decontamination Alfalfa seeds more difficult to decontaminate than mung beans 5 log reduction vs complete elimination Majority of methods reduce but do not eliminate pathogens

54. Successful Seed Decontamination Methods

55. Gaseous Acetic Acid – Mung Bean Seed Gaseous acetic acid (2500ppm, 12 h, 45oC) Inactivates Salmonella and E. coli O157 on mung beans. Reduces but does not eliminate L. monocytogenes % germination reduced from 96 to 88% Reduces viability of alfalfa seed Delaquis et al. 1999. J. Food Prot. 62: 953-957.

56. Dry Heat – Mung Bean Seed Dry heat (55oC 4-7 days) Eliminates Salmonella and E. coli O157 No effect on mung bean germination Alfalfa viability reduced Hu, et al. 2004. J. Food Prot. 67: 1257-1260.

57. Hot Water – Mung Bean Seed Hot water (5g seed/250 ml) 55oC/20 min ? 5 log reduction of Salmonella 60oC/10 min ? 5 log reduction of Salmonella 70oC/5 min ? 5 log reduction of Salmonella 80oC/2 min) ? 6 log reduction of Salmonella No effect on seed germination

58. Daisy Company Japan Hot water pasteurization Unreliable Reduced seed viability

59. Combinations – Mung Bean Seed Dry heat (50oC, 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.

60. Ozone Tsunami-100 30, 000ppm Ozone RH 65% 24h 3% Tsunami-100 20 mins No survivors

61. Fatty Acid Based Sanitizer: Alfalfa

62. Are any of these treatments practical?

63. Germin-8-or Germin-8-or is Phyto-compatible and can be introduced into steep water used for germinating seeds.

64. Evaluation of SDH Seed Decontamination Efficacy Escherichia coli O157 E. coli O157: H7-C1033 E. coli O157: H7- C1032 E. coli O157-C652 E. coli O157-C476 E. coli O157-C477 Salmonella. Sal Meleagridis E1 Sal Oranienburg C1 Sal Newport C2 Sal Seftenberg Sal Montevideo

65. Methods

66. Effect of Germin-8-or concentration on seed decontamination efficacy

67. Calcium Hypochlorite (20, 000ppm) Vs Germin-8-or (200ppm)

68. Naturally contaminated seeds Inoculated flowers with either Salmonella or E. coli O157:H7 10/10 seed batches contaminated with Salmonella E. coli O157:H7 present on 3/10 seed batches tested Effectively decontaminated with Germin-8-or.

69. Microbial Populations Associated with Sprouted Seeds DGGE analysis of 16S rRNA. Herbaspirillium spp and Klebsiella spp missing in sprouts derived from SDH treated seeds. No new populations introduced via SDH treatment.

70. “Small” commercial Trial 3 x 25kg batches of mung beans (non-inoculated) No difference in sprout development or yield.

71. SDH decontamination of other seed types Successfully Decontaminated Mung bean Alfalfa Cress Soy bean Flax Clover Mustard (V) Failed to Decontaminate Radish Broccoli Sesame Chickpeas Sunflower Onion seed Buckwheat

72. Seed, Spent Irrigation Water & Sprout Testing

74. Prevented at least one potential outbreak of E.coli O157:H7 and prevented shipment of contaminated seeds. Irrigation water sample: $50 – 100 per test $100, 000 per year

75. Developments in Spent Irrigation Water Testing Test at 24h as opposed to 48h (early product release) Pooling of samples from different beds to reduce costs Pre-concentration in combination with rapid detection methods

78. Distribution of Contamination within Sprouting Mung Bean beds Mung bean sprouted in bins (25kg lots) Spent irrigation water testing based on alfalfa (sprouted as a monolayer) Spent irrigation water testing assumes homogenous distribution of microflora within seed bed.

79. Experimental Approach Commercial trial Generic E. coli Fecal Coliforms Mesophilic Aeromonas Laboratory trial 5 strain cocktail of E. coli O157:H7 or Salmonella.

80. Commercial Trial

81. Generic E. coli

82. Mesophilic Aeromonas

84. Commercial Trial Conclusions Spent irrigation water testing more reliable than sprouts. Contamination heterogeneously distributed within seed bed. Variation in contamination levels in sprouts beds produced in the same growth room and from the same batch of seed.

85. Significance Individual bins should be sampled Single spent irrigation water samples do not provide an assessment on the microbiological status of the seed bed. Multiple spent irrigation water samples need to be collected.

86. Laboratory Studies Naturally contaminated seed harbors low levels of pathogens. Reduced but not eliminated by seed decontamination treatment. Only a small proportion of the seed contaminated within a sprouting seed bed How does contamination spread?

87. Introduce Inoculate Seed at Different Locations

88. Distribution of Pathogens after 48h Sprouting

89. Gradient of contaminated sprouts Highest density of contamination at the point of inoculation. High proportion of false negative results from screening spent irrigation water.

90. More homogenous distribution of pathogens when:- Seeds mixed prior to soaking Sprouts and spent irrigation water samples >72h into the sprouting process

91. Spent Irrigation Water Testing in Bean Sprout Production Tangential Flow Filtration System Electrochemical Immuno-sensor In-house testing Sampling at the latter stages of sprout production OMAFRA Food Safety Program Collaboration with T. J. Fu (USDA)

92. Research Needs Standardized methods for validating and verifying seed decontamination methods. Introduction of alternative seed decontamination methods Seedborne vs environmental contamination Rapid and reliable screening methods Seeds produced specifically for sprout production

93. Detailed food safety guidelines Consider different seed types Organic sector Regulation? Focus on health benefits of sprouted seeds

94. Acknowledgements OMAFRA Innovation and Risk Management Program. Vernagene Ltd (UK) Bob Rust Bob & Barbra Sanderson

95. Rajneesh Hora Manoj Kumar Dr M. Kostrzynska (AAFC) Dr M. Griffiths (CRIFS, UoG) Prof M. Dixon (Department of Environmental Biology, UoG) Luis Garcia (OMAFRA) Bengt Schumacher (OMAFRA)