Ecoli Surrogate K12 in Alfalfa Seed using the Electron Beam Fluidized Bed Process

Lethality Studies for the E. coli 0157:H7 Surrogate K12 in Alfalfa Seed using the Electron Beam Fluidized Bed (ebfb) Process

30 December 1999, Denise Cleghorn and Sam V. Nablo, Electron Processing Systems, Inc., 6 Executive Park Drive, N. Billerica, MA 01862

Treatment of inoculated alfalfa seed with the electron beam fluidized bed process has shown that, using the non-pathogenic surrogate K12 for E. coli 0157:H7, a 5 log reduction is achieved at doses of 10kGy or less. Related germination and yield studies have shown good performance of the treated seed, a prerequisite for its use by the sprout industry.

This work conducted as a member of the NCFST Sprout Task Force team has been pursued, in part, under existing USDA grants through which the eb fluidized bed process has been developed. Our collaboration has been with Peter Slade at NCFST who prepared the inoculated samples, while the assays of treated products were handled under his direction by Nicole Maks, Claudia Rodriguez and Siwalee Supapa. Denise Cleghorn conducted plate preparation and sample handling at EPS. In order to identify individual runs, an alphabetic Greek designation was used. We have just completed “lambda” or eleven trials since this work started in November 1998.

A major goal of this study has been the determination of the treatment level required to achieve a five-log decrease in population as recommended by the USFDA advisory group. We hoped to be able to do this with dry seeds run in the ebfb system, and at levels of 10kGy or under which has been a “generally accepted” level for food products. We realize that a petition is required for practice of the process, if accepted, and expect that this work may only require a modest change of existing petitions for gamma and electron processing of foods already under consideration at USFDA.

This process offers the ability to adjust electron energy so that the depth of penetration into the seed endosperm is controlled. In this manner, the dose delivered to the seed embryo is also affected so that optimization of the process required studies of both energy and dose. Some data on this effect are shown in table 1 from TR23.2 ⁽¹⁾ and table 2 from TR23.4⁽²⁾. Clearly the scarified (shell damaged) seed is much more susceptible to yield loss from treatment while yield loss is “linearly” dependent on depth of penetration. Some reference points for the system used in these studies⁽³⁾ are shown in table 3 for the three different energies (voltages) shown in table 2. Penetration depths are shown where the dose has dropped to 50% of its value at the surface, while the depth assumes a seed shell density of 0.9g.cm^-3.

Table 1
Sprout Yield Data (Germination >95%)
Experiment Beta, 11/9/98 (EPS 98TR23.2)
Reported by Jonathan Sprouts, Inc, Marion Ma

Table 2
Sprout Yield Data (Germination >94%)
Experiment Delta 1/28/99 (EPS 99TR23.4)
Reported by Jonathan Sprouts, Inc, Marion, MA

Treatment	Scarified Seed	Smooth Seed
Control	100%	100%
5 kGy 185 kV	92%	93%
5 kGy 205 kV	90%	96%
5 kGy 225 kV	86%	91%
10 kGy 185 kV	84%	95%
10 kGy 205 kV	82%	90%
10 kGy 225 kV	72%	75%

Detailed studies of seed germination and yield were conducted in this work by Jonathan Sprouts, Inc of Marion, MA (Barbara and Bob Sanderson).

We have continued to be encouraged by the excellent seed embryo vitality retained for this process at elevated surface doses with appropriate adjustment of energy used for treatment. Fett of the USDA ⁽⁵⁾reported on the germination and yield losses in alfalfa with gamma-ray (bulk) treatment of seeds at levels above a few kilograys. The Wyndmoor, PA, USDA team⁽⁶⁾ has concluded that 5D treatment of alfalfa for 0157:H7 is not practicable with gammas, without chemical assistance, (e.g. 30,000ppm calcium hypochlorite). They reported >3D and 2D inactivation of E. coli 0157:H7 and Salmonella respectively on alfalfa at a gamma dose of 2kGy.

Early studies with seeds naturally contaminated with non-pathogenic coliform bacteria (experiments alpha and beta) had shown good ebfb lethality with the expected logarithmic behavior of lethality first studied in November of 1998⁽⁷⁾ on alfalfa seed provided by NCFST. In July of 1999 (Exp’t. Zeta, TR23.6) an experiment was conducted on K12 inoculated seed (considered as a suitable non-pathogenic E. coli 0157:H7 surrogate). The inoculation protocol involved the soaking of the seeds in Phosphate buffer saline for 10 minutes and then adding a sufficient volume of the centrifuged, washed and resuspended (in PBS) culture of K12 to the soak solution to bring the final population to ~10⁶ g^-1 in the dried product. The inoculum was decanted and pipetted off the seed and the seeds dried for 48 hours.

Figure 1 shows the results of this trial, in which it was clear that the lethality asymptote shown was the result of the limited penetration of the electrons into the seed endosperm, which is now thoroughly soaked with the inoculum. The effective penetration depths across the energy range available for this process are presented in table 3. At this point it was decided to continue these studies using a spray technique for introduction of the non-pathogenic surrogate inoculum onto the seeds. Now the challenge would be adequate penetration of the approximately 80gm^-2thickness of the seed shell in order to disinfect the outer depths of the endosperm contaminated via openings (scarified cracks, etc.) in the shell.

Two examples of dry seed treatment in the electron beam fluidized bed are shown in figures 2 and 3. The first of these was run on experiment “eta” ⁽⁹⁾on 10/7/99 with seed inoculated with K12 at 10⁵ cfu.g^-1 level. The data shown were taken from plates prepared by P. Slade at EPS and transported back to his laboratory for assay. The actual assay results are the average of duplicates and shown in table 4.

* also treated with hydrogen peroxide before treatment and plotted for comparison on figure 2.

The second experiment (“lambda”) ⁽¹⁰⁾ was conducted on 12/17/99 with seed inoculated at 10⁶ cfu.g^-1 level. Samples were plated by Denise Cleghorn at EPS prior to shipment to NCFST and the results for the assays on both the EPS and NCFST plated samples are compiled in table 5. These data are then plotted in survival form as a function of dose in figure 3.

The lethality data taken on the ebfb pilot at EPS, indicate a compound lethality behavior with an initial D₁₀ value of ~1.0 kGy and a “population” exhibiting a D₁₀ of 2.7kGy. In both of the trials (eta and lambda) reported here, dry treatment of the inoculated seed showed a 5D reduction in the K12 population at 10kGy. The high D value exhibited by these curves after 99% reduction of the inoculum population at 2kGy likely arises from the “protection” offered by the seed shell to the small population which resides in crevices in the seed shell and under the shell adjacent to scarified regions of the endocarp. Of course, one would expect lower D values with bulk treatment of seed with X or gamma rays. Thayer ⁽⁶⁾ has reported gamma-ray D values of 0.60 + 0.01 kGy for E. coli 0157:H7; the ebfb data shows gross D values of 1.6 – 2.0 kGy for the surrogate inoculated alfalfa reported on here.

We believe that these data provide adequate confirmation that the process can achieve the desired 5D disinfection of alfalfa (and other sprout) seeds, without serious penalties in either germination or yield performance. Details of the process needed to support a petition and the industrial application of the process should now be pursued.

EPS has made an effort to commission a facility for the application of the ebfb process on an industrial scale. A 10kGy.ton.h^-1 system is now being completed for this and related applications and we have informed the major seed houses supplying the industry of its features. We believe that this is a well-controlled intervention process⁽¹¹⁾ which can be applied to all seed types of interest to the sprout industry. It can ensure, with good (real time) process documentation, the delivery of “clean” seed to the sprout industry. This is the “sine qua non” for HACCP implementation to an industry seeking a chemical-free intervention manageable through its seed merchants.

The authors wish to express their appreciation to Dr. Charles Cleland of the USDA-SBIR office for the assistance provided by their continuing grants for the development and understanding of this process. The close collaboration with NCFST (Dr. C. Sizer and Dr. P. Slade) has made these studies with the E. coli 0157:H7 surrogate possible.

(1) “E-Beam Irradiation Germination and Yield Tests”, private communications with Jonathan Sprouts Inc. Marion, MA; EPS 98TR23.2.

(2) Nablo, S.V. and Cleghorn, D.A., “Report of Results for Sprout Seed Experiment Delta”, EPS 98TR23.4, Feb 1999.

(3) Nablo, S.V. and Cleghorn, D.A., “ Electron Disinfestation of Powders and Aggregates…”, Final Report for Phase II , Section 2, Penetration Calibration, USDA Grant #96-33610-3115, 30 July, 1999.

(4) Sanderson, R and B, private communication, Jonathan Sprouts Inc, Marion, MA 24 Dec. 1998.

(5) Fett, W.F., “Chemical Treatments and Irradiation to Control Levels of Pathogens on Seeds and Sprouts”, presentation at the Sprout Task Force Meeting, NCFST, April 21, 1999.

(6) Thayer, D.W., Rajkowski, K.T. Fett, W.F. and Boyd, G., “Elimination of E. coli 0157:H7 and control of Salmonella on sprouts and alfalfa seed by gamma irradiation and sanitation”, Proc. Sprout Summit, Best Practices and Recommendations for the Production of Safer Sprouts from Seeds, Chicago, IL, Nov. 15-16, 1999.

(7) Nablo, S.V. and Cleghorn, D.A., “Evaluation of Electron Beam Fluidized Bed Treatment of Alfalfa Sprout Seeds: Test Alpha, EPS 98TR23.1, Nov. 13, 1998.

(8) Cleghorn D.A. and Nablo, S.V., “EBFB Treatment of Alfalfa Seeds Inoculated Using Soaking Technique with K12 E. coli 0157:H7 Surrogate”, EPS 99TR23.6, July 1999.

(9) “Protocol for Sample Preparation and Plating, “Private communication with P. Slade, 2 Nov 1999; Report on Exp’t Eta , EPS 99TR23.7, Oct 23, 1999.

(10) “E. coli 0157:H7 Surrogate K12 Lethality Studies: Dry and with 5% H₂O₂ Treatment”, EPS 99 TR11. 21 December 1999

(11) “Microbiological Safety Evaluations and Recommendations on Sprouted Seeds”, National Advisory Committee on Microbiological Criteria for Food, USFDA, May 28, 1999.