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 Seedusing the Electron Beam Fluidized Bed (ebfb) Process

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

SUMMARY

Treatment of inoculated alfalfa seed with the electron beam fluidized bedprocess has shown that, using the non-pathogenic surrogate K12 for E. coli0157:H7, a 5 log reduction is achieved at doses of 10kGy or less.  Related germination and yield studies have shown goodperformance of the treated seed, a prerequisite for its use by the sproutindustry.

INTRODUCTION

This work conducted as a member of the NCFST Sprout Task Force team hasbeen pursued, in part, under existing USDA grants through which the eb fluidizedbed process has been developed.  Ourcollaboration has been with Peter Slade at NCFST who prepared the inoculatedsamples, while the assays of treated products were handled under his directionby Nicole Maks, Claudia Rodriguez and Siwalee Supapa.  Denise Cleghorn conducted plate preparation and samplehandling at EPS.  In order toidentify individual runs, an alphabetic Greek designation was used.  We have just completed “lambda” or eleven trials sincethis work started in November 1998.

A major goal of this studyhas been the determination of the treatment level required to achieve a five-logdecrease 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, andat levels of 10kGy or under which has been a “generally accepted” level forfood products.  We realize that apetition is required for practice of the process, if accepted, and expect thatthis work may only require a modest change of existing petitions for gamma andelectron processing of foods already under consideration at USFDA.

This process offers the ability to adjust electron energy so that thedepth of penetration into the seed endosperm is controlled.  In this manner, the dose delivered to the seed embryo is alsoaffected so that optimization of the process required studies of bothenergy and dose.  Some data on thiseffect 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 toyield loss from treatment while yield loss is “linearly” dependent on depthof penetration.  Some referencepoints for the system used in these studies⁽³⁾ are shown in table 3for the three different energies (voltages) shown in table 2. Penetration depths are shown where the dose has dropped to 50% of itsvalue 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

Table 3
Effective ebfb Penetration Depth in Alfalfa Seed

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

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

EXPERIMENTAL

Early studies with seeds naturally contaminated with non-pathogeniccoliform bacteria (experiments alpha and beta) had shown good ebfb lethalitywith the expected logarithmic behavior of lethality first studied in November of1998⁽⁷⁾ on alfalfa seed provided by NCFST. In July of 1999 (Exp’t. Zeta, TR23.6) an experiment was conducted onK12 inoculated seed (considered as a suitable non-pathogenic E. coli 0157:H7 surrogate).  Theinoculation protocol involved the soaking of the seeds in Phosphate buffersaline 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 thefinal population to ~10⁶ g^-1 in the dried product. The inoculum was decanted and pipetted off the seed and the seeds driedfor 48 hours.

Figure 1 shows the results of this trial, in which it was clear that thelethality asymptote shown was the result of the limited penetration of theelectrons into the seed endosperm, which is now thoroughly soaked with theinoculum. The effective penetration depths across the energy range available forthis process are presented in table 3.  Atthis point it was decided to continue these studies using a spray technique forintroduction 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 theendosperm contaminated via openings (scarified cracks, etc.) in the shell.

Two examples of dry seed treatment in the electron beam fluidized bed areshown in figures 2 and 3.  The firstof these was run on experiment “eta” ⁽⁹⁾ on 10/7/99 with seedinoculated with K12 at 10⁵ cfu.g^-1 level. The data shown were taken from plates prepared by P. Slade at EPS andtransported back to his laboratory for assay. The actual assay results are the average of duplicates and shown in table4.

Table 4
K12 Lethality Data for Exp’t Eta (10/7/99)

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

The second experiment (“lambda”) ⁽¹⁰⁾ was conducted on12/17/99 with seed inoculated at 10⁶ cfu.g^-1 level. Samples were plated by Denise Cleghorn at EPS prior to shipment to NCFSTand the results for the assays on both the EPS and NCFST plated samples arecompiled in table 5.  These data are then plotted in survival form as a function ofdose in figure 3.

Table 5
K12 Lethality Data for Exp’t Lambda 12/17/99

DISCUSSION

The lethality data taken on the ebfb pilot at EPS, indicate a compoundlethality 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 ofthe inoculated seed showed a 5D reduction in the K12 population at 10kGy. The high D value exhibited by these curves after 99% reduction of theinoculum population at 2kGy likely arises from the “protection” offered bythe seed shell to the small population which resides in crevices in the seedshell and under the shell adjacent to scarified regions of the endocarp. Of course, one would expect lower D values with bulk treatment ofseed 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 thesurrogate inoculated alfalfa reported on here.

We believe that these data provide adequate confirmation that the processcan 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 industrialapplication of the process should now be pursued.

EPS has made an effort to commission a facility for the application ofthe ebfb process on an industrial scale.  A10kGy.ton.h^-1 system is now being completed for this and relatedapplications and we have informed the major seed houses supplying the industryof its features.  We believe thatthis is a well-controlled intervention process⁽¹¹⁾ which can beapplied to all seed types of interest to the sprout industry. It can ensure, with good (real time) process documentation, the deliveryof “clean” seed to the sprout industry. This is the “sine qua non” for HACCP implementation to an industryseeking a chemical-free intervention manageable through its seed merchants.<imgwidth=695 height=”911″ src=”/sprout-images/Lethality-Image002.gif”></imgwidth=695>

Figure 1

Figure 2

Figure 3

ACKNOWLEDGEMENT

The authors wish to express their appreciation to Dr. Charles Cleland ofthe USDA-SBIR office for the assistance provided by their continuing grants forthe development and understanding of this process. The close collaboration with NCFST (Dr. C. Sizer and Dr. P. Slade) hasmade these studies with the E. coli0157:H7 surrogate possible.

REFERENCES

(1)              “E-Beam Irradiation Germination and YieldTests”, private communications with Jonathan Sprouts Inc. Marion, MA; EPS98TR23.2.

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

(3)              Nablo, S.V. and Cleghorn, D.A., ” ElectronDisinfestation 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 andIrradiation to Control Levels of Pathogens on Seeds and Sprouts”, presentationat the Sprout Task Force Meeting, NCFST, April 21, 1999.

(6)              Thayer, D.W., Rajkowski, K.T. Fett, W.F. andBoyd, G., “Elimination of E. coli0157:H7 and control of Salmonella onsprouts and alfalfa seed by gamma irradiation and sanitation”, Proc. SproutSummit, Best Practices and Recommendations for the Production of Safer Sproutsfrom Seeds, Chicago, IL, Nov. 15-16, 1999.

(7)              Nablo, S.V. and Cleghorn, D.A., “Evaluationof 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., “EBFBTreatment 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 , EPS99TR23.7, Oct 23, 1999.

(10)         “E.coli 0157:H7 Surrogate K12 Lethality Studies: Dry and with 5% H₂O₂Treatment”, EPS 99 TR11. 21 December 1999

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