# Presentation: 683567

## Intervention Technologies For Enhancing the Safety of Sprouts

- W. F. Fett and K. T. Rajkowski
- Food Safety Intervention Technologies RU
- wfett@arserrc.gov

## 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)

## 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)

## 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

## Published Papers – Development of Interventions (Seed/Sprout)

## Chemical Interventions - Seed

- Aqueous sanitizers (along w/ heat, surfactants)
- 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
- Elevated temperature (42 to 50oC) usually beneficial, but leads to greater loss of seed germination. Effect of addition of surfactants variable.

## Chemical Interventions - Seed

- Gas phase treatments
- Acetic acid vapor, allyl isothiocyanate, trans-anethole, carvacrol, cinnamic aldehyde, thymol, ammonia

## Physical Interventions - Seed

- Dry heat
- Hot water
- Irradiation (gamma radiation, pulsed UV)
- Hydrostatic pressure
- Radio frequency dielectric heating

## Biological Interventions - Seed

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

## Combination Treatments - Seed

- Combination of aqueous chemicals
- Lactic acid followed by chlorine (2000 ppm)
- Thyme oil followed by ozonated water and aqueous ClO2
- Combination of physical treatments with aqueous chemicals
- Ultrasound and heat in combination with Ca(OH)2, Tween 80, TsunamiR 200 and FitR
- Ozonated water followed by dry heat (60oC, 3 h)
- Dry heat (50oC, 1 h) followed by chlorine (200 ppm) or acidic electrolyzed water and sonication
- Combination of physical treatments
- Dry heat (50oC, 1 h) followed by gamma irradiation

## 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

## 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)

## Potential Alternatives to Chlorine – Alfalfa Seed

## Chemical 

- Ca(OH)2, 1%,10 min at RT
- FitR, 15 to 30 min at RT
- CitrexTM, 20,000 ppm, 10 min at RT
- PangermexTM (concentrated CitrobioTM), 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?

## 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.

## 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.

## Potential Alternatives to Chlorine – Mung Bean Seed

## Gaseous Acetic Acid – Mung Bean Seed

- Gaseous acetic acid (242 ul per liter of air, 12 h, 45oC)
*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.

## Dry Heat – Mung Bean Seed

- Dry heat (55oC)
*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.

## Hot Water – Mung Bean Seed

- Hot water (5 g/250 ml) (inoc. load >108 cfu/g)
- 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*
- 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.

## 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.

## Combinations – Mung Bean Seed

- Dry heat (50oC, 1 h) followed by hot (50oC) 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 (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.

## Interventions – Other Seed Types

- Garden cress
-  High pressure (300 MPa, 15 min, 20oC) → 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 (50oC, 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

## 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.

## 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

## 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.

## 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)

## 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

## 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)

**Notes:**
Chlorous acid (HClO2) is the antimicrobial species for acidified sodium chlorite formed by the disassociation of sodium chlorite in the presence of acid

## Interventions - Postharvest 

- RT water rinses
- Hot water dips (100oC, 30 sec)
- Irradiation – gamma and electron beam

## 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

## 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 (HClO2) (268 ppm, 10 min)
- → 5 log reductions of *Salmonella* and *L*. *monocytogenes* on mung bean sprouts, no change in visual quality during storage at 4oC for 9 days
**    **
**     ****Lee et al. 2002. J. Food Prot. 65: 1088-1092**
- Note: Regulatory approvals required

## 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.

## ARS/Industry Collaborative Study

## 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.**

## Verification of Yield Ratio

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

## 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.