New Technologies for Decontaminating Sprouting Seed and Produce with Easily Damageable Surfaces

New Chemical, Physical, and Biological Technologies for Decontaminating Sprouting Seed and Produce with Easily Damageable Surfaces

Agricultural Research Service
National Program 108
Food Safety Progress Report 2001 (web version)
Section 10: Produce and Animal Manure,

CRIS: 1935-41420-006

Fett WF, Liao C-H., Ukuku D, Matos A

Food Safety Intervention Technology Research Unit, ERRC, Wyndmoor, PA

Summary Project Aims:

The incidence of foodborne disease due to consumption of contaminated produce appears to be on the rise. Data collected by the CDC indicate that the number of produce-related outbreaks doubled between the periods 1973-1987 and 1988-1992. Fresh produce is often consumed raw and is not subjected to a kill step that would eliminate any pathogens present. Recent outbreaks of foodborne illness due to contaminated produce have shaken consumer confidence in the safety of produce at a time when consumers are being urged to eat more fruits and vegetables.

Since 1995 there have been 18 outbreaks in the US due to consumption of contaminated alfalfa, clover and mung bean sprouts resulting in over 1500 culture-confirmed cases and two deaths. In the year 2001 one outbreak due to alfalfa sprouts and three outbreaks due to mung bean sprouts contaminated with Salmonella were recorded. Most outbreaks have been due to various Salmonella serovars, but two outbreaks were caused by Escherichia coli O157:H7. In almost all outbreaks the original source of the pathogen was determined to be the sprouting seed. The sprout-related outbreaks have resulted in 1) a reduced market for sprouts, 2) the closure of several sprout growing facilities due to outbreak-related litigation and 3) the need for expensive testing of spent irrigation water for pathogens.

Multiple outbreaks of disease due to consumption of fresh-cut cantaloupe contaminated with Salmonella have also been reported, and no nationally distributed fresh-cut melon is available due to safety concerns and quality issues. The most recent outbreak occurred in the spring of 2001 with at least thirty confirmed cases in 14 states and two deaths.

Current methods for controlling human pathogens on sprouting seed as well as fresh or minimally processed produce are not completely effective. New and innovative means of sanitizing seed and produce are required for use by organic and non-organic growers and by processors. In this project, research is being conducted into new and more effective chemical, physical and biological methodologies for pathogen elimination.

In addition, the ecology, detection and isolation of human pathogens on fresh and minimally produce is being studied. Most isolation and detection methods currently available were devised for environmental, clinical and meat samples. Detection methods need to be evaluated and revised, if necessary, for use with produce.

Summary Accomplishments During Entire Project:

Treatment of sprouting seed with 20,000 ppm of free chlorine provided by calcium hypochlorite was shown to result in an approximate 4-log reduction of human bacterial pathogens. This information was used in support of successful petitions to the US EPA and California EPA for temporary approval of the use of this treatment for sanitizing sprouting seed. The method was adopted in FDA guidance to the sprout industry and received a permanent registration from the US EPA. Further research demonstrated that the target 5-log reduction of Salmonella while maintaining seed viability can be achieved by the combination of irradiation with gamma rays (1 kGy) followed by treatment with chlorine (20,000 ppm). This combination treatment of seed can be used to ensure the safety of sprouts consumed in the raw state. The FDA has approved irradiation of sprouting seed up to a level of 8 kGy.

Tests on the efficacy of treating alfalfa seed naturally contaminated with Salmonella Mbandaka and Salmonella Muenchen using 20,000 ppm of free chlorine indicated that this method is effective in sanitizing naturally contaminated seed. As a result of these studies, growers, consumers and regulators will have greater confidence in the safety of sprouts grown from seed treated in this manner.

Addition of antimicrobials to irrigation water was shown to be ineffective in controlling growth of native bacteria on growing sprouts leading to the hypothesis that such bacteria may be present in biofilms. A variety of types of sprouts were examined by scanning electron microscopy. Biofilms consisting of the native microflora were found to be abundant on all types of sprouts. This information will aid in the development of novel antimicrobial treatments for controlling pathogen growth during the sprouting process.

The fate of human pathogens in manure spread on fields used to grow produce is not known. In cooperation with ARS scientists at Orono, Maine, survival of Listeria monocytogenes andE. coli found in liquid dairy manure after application to experimental potato fields was studied. Both bacteria were detected in soil for up to six weeks after addition of manure, but were undetectable at the end of the growing season and in harvested tubers. This work provides producers and regulatory agencies with information useful for developing good agricultural practices.

Summary 2001 Accomplishments:

New methods to eliminate bacterial human pathogens from produce are urgently needed as the incidence of human illness associated with the consumption of fresh produce has increased during the past two decades. The native microflora from green bell peppers, Romaine lettuce and pre-peeled baby carrots was enumerated, characterized and several bacteria and yeasts were isolated. Two microbes were demonstrated to be capable of inhibiting the growth of L. monocytogenes and Salmonella on produce. These microbes represent potential biopreservatives that can be used to suppress the growth of human pathogens on produce.

Better methods for detecting Salmonella in sprouting seed are needed as foodborne illness due to contaminated alfalfa and other types of sprouts is primarily the result of sprout growers unknowingly sprouting contaminated seed. Various cultural and polymerase chain reaction (PCR) methodologies were compared for their ability to detect Salmonella in naturally contaminated seed lots. An optimized technique for detection of low pathogen levels (one to 10 cells per 25 gram of seed) was developed. This method will be useful for seed distributors, growers and commercial laboratories to help assure that sprouting seed is free of the pathogen.

Effective means of sanitizing sprouting seed are needed due to continuing foodborne outbreaks related to the consumption of contaminated alfalfa and mung bean sprouts. The ability of chlorinated water and citrus-related commercial products to eliminate bacterial human pathogens from mung bean seeds and alfalfa seed, respectively, inoculated in the laboratory was determined. Treatment of seed with high levels of chlorine (16,000 to 18,000 ppm) as well as with three commercial citrus-related products were able to reduce populations of Salmonella and E. coli O157:H7 by up to 99.999% without harming germination of the seed but were not able to completely eliminate the pathogens. Treatment of sprouting seed with chlorinated water or citrus-related commercial products will help to ensure the microbial safety of sprouts.

The organic US sprout industry is in critical need of effective non-chemical interventions that will ensure the microbiological safety of their product. A collection of over 900 resident bacteria were isolated from the surface of a variety of sprouts and over 400 of these were tested in bioassays for their ability to inhibit the growth of Salmonella on alfalfa sprouts. Of these strains, six were found to significantly inhibit pathogen survival and growth. These effective bacteria either singularly or in combination may form the basis of a competitive exclusion product for the sprout industry.

Effective means of sanitizing cantaloupe are required due to continuing foodborne outbreaks caused by bacterial human pathogens. The pathogen L. monocytogenes was demonstrated to survive on the rind of cantaloupe for up to 15 days of storage at 4 C and 20 C. Treating whole inoculated cantaloupe with 1000 ppm free chlorine or 5% hydrogen peroxide for 2 minutes resulted in a 2 to 3.5 log cfu/cm² reduction and negated the transfer of the pathogen to the interior flesh during cutting. In addition, the relationship of bacterial cell surface hydrophobicity and charge with strength of attachment to cantaloupe rinds was investigated for L.monocytogenes, E. coli and Salmonella. Cell surface hydrophobicity, relative negative charge and relative positive charge were all highly correlated with strength of attachment.

QSQR/Impact:

Identify new, more effective chemical treatments to eliminate bacterial human pathogens from sprouting seed and fresh-cut produce. Demonstrated various citrus-related products to be as effective in reducing the populations of bacterial human pathogens on alfalfa seed as is high levels of chlorine. Impact: The citrus-related products may substitute for the very high levels of chlorine currently recommended by the FDA for sanitizing sprouting seed pending EPA approval of these products for seed treatment. Such products would be especially beneficial for use by organic growers. Information on the use of citrus-related products for sanitizing sprouting seed was communicated to the sprouting industry by an oral presentation at the Annual Convention of the International Sprout Growers Association.

Determine the effectiveness of alternative antimicrobial interventions including competitive exclusion and physical treatments. After an exhaustive screening process, identified several highly effective bacterial antagonists that inhibit the growth of Salmonella on alfalfa sprouts. Impact: The antagonistic strains have significant potential for the formulation of a commercial product for ensuring the microbial safety of sprouts. The commercialization of a competitive exclusion product would be highly beneficial for organic growers who are not allowed to use chemical seed treatments.

A CRADA partner will be sought to assist in commercialization.

Investigate endogenous and exogenous factors affecting the survival, growth and interaction of resident microflora including spoilage bacteria and human pathogens on fresh-cut produce and factors affecting the detection of human pathogens. Devised an improved protocol for detecting bacterial human pathogens on sprouting seed. Impact: The new method will allow for increased detection of bacterial human pathogens on seed assisting in both the reduction of sprout-related outbreaks as well as epidemiological studies of outbreaks that do occur. A manuscript describing the technique will be published and the methodology conveyed to the FDA and the International Sprout Growers Association.

Projected Research Accomplishments During the Next 3 Years:

During 2002 we expect to complete studies on the use of citrus-related antimicrobial compounds and initiate studies on various physical methods for decontamination of sprouting seed. Studies on the use of synthetic and natural antimicrobials and various physical methods for ensuring the safety of fresh-cut cantaloupe and other melons will continue. We expect to optimize a PCR-based protocol for use in detection of bacterial human pathogens in sprouting seed. Evaluation of competitive exclusion strategies for inhibiting the growth of human pathogens on sprouts will continue. Highly effective antagonists will be taxonomically identified and studies on the mechanism(s) of action of effective antagonists to be used in a competitive exclusion product will be initiated. A patent on the technology applied for and discussions with an industrial partner concerning development of a CRADA that will lead to commercialization of a competitive exclusion product will be initiated and finalized.

During 2003 we expect to optimize effective chemical and physical antimicrobial treatments for sprouting seed and fresh-cut cantaloupe and transfer new technologies to end-users by presentations to growers and trade associations and by publications. Multiple hurdle approaches combining natural antimicrobials and physical methods for decontaminating seeds, cantaloupe and other melons will be tested. Complete studies on the effect of the natural microflora on produce on the detection of bacterial human pathogens and continue working with an industrial partner towards commercialization of a competitive exclusion product for use on sprouts.

In 2004 we expect to complete the studies on the detection of human pathogens on produce, work with a CRADA partner to obtain FDA approval of a competitive exclusion product for use on sprouts and commercialize a first-generation competitive exclusion product. The efficacy of the most effective intervention technologies developed for fresh-cut cantaloupe and sprouting seed will be optimized. The transfer of new technologies to the produce and sprout industries will continue.

Technology Transfer:

Information on methods for decontaminating produce and sprouting seed has been disseminated to growers, processors, trade organizations such as the International Sprout Growers Association, the International Fresh-cut Produce Association, regulators and other scientists by means of presentations at meetings, visits to growers and processors, telephone and e-mail correspondence and through peer-reviewed publication. Significant outreach activities with small, rural sprout growers were undertaken. Many questions concerning the sanitizing of seed and sprouts received from growers from around the US and representatives of the International Sprout Growers Association (ISGA) via the telephone or electronic mail were responded to. As chair of the technical session for the ISGA Annual Convention held in August of 2001, selected and introduced speakers on the topic of microbial food safety and presented an update of research activities at ERRC. The use of high levels of chlorine was shown to be effective for sanitizing alfalfa seed naturally contaminated with Salmonella, a bacterial human pathogen, giving growers greater confidence in the efficacy of this treatment for helping to ensure the microbial safety of their sprouts. Invited oral and poster presentations of research results were given at several US and international meetings including two oral presentations at the 14^th Annual Conference of the International Fresh-Cut Produce Association, Phoenix, AZ, April 5-7, 2001 and two posters for a poster symposium at the 88^th Annual Meeting of the International Association for Food Protection, Minneapolis, MN, August 5-8, 2001. Coordinated research efforts under a grant funded by the National Alliance for Food Safety in support of a collaborative research effort between ARS, Pennsylvania State University and The University of Georgia to examine the use of ozone for sanitizing seeds and sprouts.

PUBLICATIONS:

Liao, C-H., Cooke, P. H. Response to trisodium phosphate treatment of Salmonella Chester attached to fresh-cut green pepper disks. Canadian Journal of Microbiology. 2001. v. 47, p. 25-32.

Liao, C-H., Fett, W.F. Analysis of native microflora and selection of strains antagonistic to human pathogens on fresh produce. Journal of Food Protection. 2001. v. 64. p. 32-37.

Fett, W.F. Bacterial extracellular polysaccharides. Maloy, O.C., Murray, T. D., editors. John Wiley & Sons, Inc., New York, NY. Encyclopedia of Plant Pathology. 2001. p. 78-79.

Fett, W.F. Amylovoran. Maloy, O. C., Murray, T. D., editors. John Wiley & Sons, Inc., New York, NY. Encyclopedia of Plant Pathology. 2001. p. 37-38.

Fett, W.F. Bacterial ooze/exudates. O. C., Murray, T. D., editors. John Wiley & Sons, Inc., New York, NY. Encyclopedia of Plant Pathology. 2001. p. 81.

Fett, W.F. Xanthan. O. C., Murray, T. D., editors. John Wiley & Sons, Inc., New York, NY. Encyclopedia of Plant Pathology. 2001. p. 1207.

Fett, W.F. Alginate. O. C., Murray, T. D., editors. John Wiley & Sons, Inc., New York, NY. Encyclopedia of Plant Pathology. 2001. p. 32-33.

PROCEEDINGS/ABSTRACTS:

Liao, C-H., Ukuku, D., Fett, W.F. Factors limiting the efficacy of sanitizer treatments for fresh produce. Proceedings of the 29^th United States-Japan Cooperative Program in Natural Resources – Protein Resources Panel Meeting. Honolulu, Hawaii. November 19-25, 2000.

Fett, W.F. Sanitizing sprouting seed with chlorine and citrus extracts. Proceedings of the 12^th Annual Convention of the International Sprout Growers Association Annual Convention. Nashville, TN. July 12-14, 2001.

Fett, W.F. Research on ensuring the microbial safety of sprouts. Pages 77-81. Proceedings of the 30^th UJNR Meeting. Tsukuba, Japan. October 14-16, 2001.

Matos, A. Microbial composition and physiological profiling of sprout-associated microbial communities. Abstract no. FR.048. Proceedings of the 9^th International Symposium on Microbial Ecology. Amsterdam, The Netherlands. August 26-31, 2001.