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_at_uoguelph.ca

2
FDA Review on the Microbiological Safety of
Sprouted Seeds

• Washington DC May 2005

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 O157H7.
• gt6000 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 (gt99 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)

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• 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
TVC
E. coli
Wash
Surface Sterilised 20, 000ppm hypochlorite
22
Apoplastic Fluid from Surface Sterilised Bean
Sprouts
Dead Bacteria
Live Bacteria
23
Sprouts derived from inoculated beans
Control
GUS Assay
24
NACMCF 1999 Sprout White Paper

• Microbial Safety Evaluations and Recommendations
  on Sprouted Seeds - Adopted May 28, 1999
• Basis for FDAs 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

27
Difference in microbial counts between sprouts
and water
Total counts
E. coli O157 counts
mean 0.62 ? 0.67 n 39
mean 0.55 ? 0.47 n 11
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Testing of Water from Each Production Batch
Salmonella
RV / TT
TSB DNP n
BPW n
and
E. coli O157H7
mBPW ACV
29
.Testing of Water from Each Production Batch

• If positive results, then
• discard sprouts and seed disinfect contact
  surfaces
• or
• run confirmatory tests from enrichment

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

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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 O157H7

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
Safer Processing of Sprouts

• Educational video produced and distributed.
• http//www.cfsan.fda.gov/dms/sprouvid.html
• or
• http//www.dhs.ca.gov/fdb/PDF/SproutOrderForm4.PDF

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
FDA advises chlorination of seeds
39
FDA 1998 Field Assignment

• GMP Inspection 83 firms
• 57 performed unsanitary practices
• Samples for microbial analyses
• raw seed ? finished product
• 78 firms

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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

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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

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Seed Decontamination

• Not included in site inspections
• Known to have limited efficacy
• Sprout producers interpretation Seed
  decontamination is not important

43
2004 - Sanitation

• 50 of firms were described as having
  deficiencies including
• unsanitary food contact surfaces (38)
• evidence of pests (33)
• lack of personnel cleanliness (25)
• water quality problems (8)

44
2004 - Seed Treatment

• 70 of sprouts were treated with Ca(OCl)2
• Sodium hypochlorite (25), ozone, and
  peroxyacetic acid were also used.
• only 2 used the recommended
• 20, 000ppm

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2004 - Sampling and Microbial Testing of Spent
Irrigation Water

• 71 of firms collected spent irrigation water for
  microbial testing
• Almost all firms tested for Salmonella spp. and
  E. coli O157H7 but the testing method varied
  greatly.
• Pooling of composite samples

46
2004Testing Spent Irrigation Water

• Record Keeping
• 88 that conduct testing maintain records of test
  results from 48 hour spent irrigation water
  samples.
• 6 maintains a record of 48 hour spent irrigation
  water samples collected but not the results.
• A majority of firms (92) do not have a
  corrective action or product recall plan.

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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

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Biological Interventions - Seed

• Antagonistic bacteria
• Lactic acid bacteria
• Fluorescent pseudomonads
• Whole bacterial communities
• Bacteriophage vs. Salmonella

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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

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Successful Seed Decontamination Methods
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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

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Combinations Mung Bean Seed

• Dry heat (50oC, 1 h) followed by gamma
  irradiation (2.0 kGy)
• ? 4.6 log reduction of E. coli O157H7 (no
  survivors), no effect on germination, reduced
  sprout growth rate
• Bari et al. 2003. J. Food Prot. 66 767-774.

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Ozone Tsunami-100

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

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Integrated Seed Decontamination and Sprouting Unit
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Fatty Acid Based Sanitizer Alfalfa
5 min treatment No survivors L. monocytogenes,
Salmonella, E. coli O157 Seeds tested for
pathogens
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Are any of these treatments practical?
64
Germin-8-or

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

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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

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Methods
Inoculate seeds with suspension of E. coli O157
H7 and Salmonella sp.
Dry seeds for 2 hours
Decontamination Treatment
Sprouting at 30C for 2-3 days (Daily Watering)
Microbiological Analysis
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Effect of Germin-8-or concentration on seed
decontamination efficacy
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Calcium Hypochlorite (20, 000ppm) Vs Germin-8-or
(200ppm)
Initial loading 3-4 log cfu/g ND
lt1 cfu/25g
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Naturally contaminated seeds

• Inoculated flowers with either Salmonella or E.
  coli O157H7
• 10/10 seed batches contaminated with Salmonella
• E. coli O157H7 present on 3/10 seed batches
  tested
• Effectively decontaminated with Germin-8-or.

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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.

Germin-8-or Treated
Control
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Small commercial Trial

• 3 x 25kg batches of mung beans (non-inoculated)
• No difference in sprout development or yield.

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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

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Seed, Spent Irrigation Water Sprout Testing
74

• Screening Seeds Prior to Sprouting
• 1. Seed sampling
• 25 g subsamples from each bag . At least 3 kg per
  seed lot
• 2. Seed inspection
• 3. Sprout seeds (3kg batch)
• 4. Spent irrigation water sampling after 48h
• 5. Enrichment of sampled water
• 6. Pathogen testing

75

• Prevented at least one potential outbreak of
  E.coli O157H7 and prevented shipment of
  contaminated seeds.
• Irrigation water sample 50 100 per test
• 100, 000 per year

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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

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Tangential Flow Filtration System
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Recovery of E. coli O157H7 from the TFF system
79
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.

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Experimental Approach

• Commercial trial
• Generic E. coli
• Fecal Coliforms
• Mesophilic Aeromonas
• Laboratory trial
• 5 strain cocktail of E. coli O157H7 or
  Salmonella.

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Commercial Trial
Sprouting Mung beans
Sampling Point
Centre
Right
Left
Top Middle Base
Collect irrigation water under columns of sprouts
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Generic E. coli
Spent Irrigation Water
Sprouts
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Mesophilic Aeromonas
Spent irrigation water
Sprouts
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Distribution of contamination within sprouting
mung bean beds
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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.

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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.

87
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?

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Introduce Inoculate Seed at Different Locations
3 cm
500g Mung beans
Introduce 1g Inoculated Seed at the top, middle
or base.
15 cm
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Distribution of Pathogens after 48h Sprouting
Top Inoculated
Base Inoculated
Middle Inoculated
Contaminated sprouts
90

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

91

• More homogenous distribution of pathogens when-
• Seeds mixed prior to soaking
• Sprouts and spent irrigation water samples gt72h
  into the sprouting process

92
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)

93
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

94

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

95
Acknowledgements

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

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• 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)