ENGINEERING RESEARCH INSTITUTE THE UNIVERSITY OF MICHIGAN ANN ARBOR Final Report COMBINED USE OF HEAT AND RADIATION TREATMENT FOR STERILIZATION OF FOODS June 7, 1955, to July 31, 1957 Lloyd L. Kempe Official Investigator J. T. Graikoski No J. Williams P F, Bonventre Collaborators Project 2391 QUARTERMASTER RESEARCH AND DEVELOPMENT COMMAND NATICK, MASSACHUSETTS CONTRACT NO. DA-19-129-qm-388, PROJECT NO. 7-84-01-002 July 1957

TABLE OF CONTENTS Page LIST OF TABLES iii LIST OF FIGURES v GENERAL SUMMARY vi OBJECTIVE viii PHASE I - EFFECT OF PREIRRADIATION OF INOCULATED PACKS OF CANNED GROUND BEEF ON THE Fo SUBSEQUENTLY REQUIRED FOR STERILIZATION 1 SUMMARY 1 INTRODUCT ION 1 MATERIALS AND METHODS 3 RESULTS 5 DISCUSSION 34 PHASE II - COMPARISON OF Z VALUE OF IRRADIATED AND NONIRRADIATED PA 3679 SPORES 71 ACKNOWLEDGMENT 71 PHASE III - THE EFFECT OF CATALASE ON THE LETHALITY OF Co-60 GAMMA RADIATION FOR CERTAIN ANAEROBIC BACTERIAL SPORES 76 SUMMARY 76 MATERIALS AND METHODS 76 RESULTS AND DISCUSSION 77 PHASE TV - EFFECT OF PREIRRADIATION OF INOCULATED PACKS OF CANNED PEAS ON THE Fo SUBSEQUENTLY REQUIRED FOR STERILIZATION 80 EXPERIMENTAL PROCEDURE 80 REFERENCES 82 ii

LIST OF TABLES Table Page Treatments Required to Sterilize Canned Ground Beef Packed in No. 1 Picnic Tin Cans Using Heat and Gamma Radiation Alone and in Combination 2 I FFo Value Required to Sterilize Ground Beef in No. 1 Picnic Tin Cans, Previously Inoculated with Approximately 10,000 PA-3679 Spores per Can and Then Processed at 230~F. 8 II Summary of Various Combined Irradiation Heat-Processing Treatments Required to Sterilize Cooked Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with 10,000 PA-3679 Spores per Can. 32 III Fo Value Required to Sterilize Cooked Ground Beef in No. 1 Picnic Tin Cans, Previously Inoculated with Approximately 300 PA-3679 Spores per Can and Then Processed at 230~F. 35 IV Summary of Various Combined Irradiation-Heat-Processing Treatments Required to Sterilize Cooked Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with 300 PA-3679 Spores per Can. 43 V Fo Values Required to Sterilize Raw Ground Beef in No. 1 Picnic Tin Cans, Previously Inoculated with 5,000,000 C. botulinum 213B Spores per Can and Then Processed at 230~F. 45 VI Summary of Various Combined Irradiation-Heat Processing Treatments Required to Sterilize Raw Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with 5,000,000 C. botulinum 213B Spores per Can. 58 VII Fo Value Required to Sterilize Raw Ground Beef in No. 1 Picnic Tin Cans, Previously Inoculated with Approximately 300 PA-3679 Spores per Can and Then Processed at 230~F, 60 VIII Summary of Various Combined Irradiation —Heat Processing Treatments Required to Sterilize Raw Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with 300 PA 3679 Spores per Cano 67 iii

LIST OF TABLES (Concluded) Table Page IX Effect of Processing at Various Temperatures on PA 3679 Spores Suspended in Distilled Water Using the Thermoresistometer 72 X Time Required to Kill 99% of the Nonirradiated and Irradiated PA 3679 Spores at the Temperature Indicated 74 XI The Effect of Catalase on the Lethality of Gamma Radiation from Cobalt-60 for Anaerobic Bacterial Spores 78 iv

LIST OF FIGURES Figure Page 1 Fo required to sterilize cooked ground beef packed in No. 1 picnic tin cans, inoculated with approximately 5,000,000 C. botulinum 213B spores per can, and irradiated with gamma rays from cobalt-60 before heat processing. 6 2 Fo required to sterilize cooked ground beef packed in No. 1 picnic tin cans inoculated with approximately 300 C. botulinum 213B spores per can, and irradiated with gamma rays from cobalt-60 before heat processing. 7 3 Fo required to sterilize cooked ground beef packed in No. 1 picnic tin cans, inoculated with 10,000 PA-3679 spores per can, and irradiated with gamma rays from cobalt-60 before heat processing at 230~Fo 33 4 Fo required to sterilize cooked ground beef packed in No. 1 picnic tin cans, inoculated with 300 PA-3679 spores per can, and irradiated with gamma rays from cobalt-60 before heat-processing at 230~F. 44 5 Fo required to sterilize raw ground beef packed in No. 1 picnic tin cans, inoculated with 5,000,000 Co botulinum 213B spores per can, and irradiated with gamma rays from cobalt-60 before heat processing at 230~F. 59 6 Fo required to sterilize raw ground beef packed in No. 1 picnic tin cans, inoculated with 300 PA 3679 spores per can and irradiated with gamma rays from cobalt-60 before heat processing at 230~Fo 68 7 Thermal death time curves for nonirradiated and irradiated PA 3679 spores suspended in distilled water. 75 8 Effect of catalase on the lethality of gamma radiation from cobalt-60 for anaerobic bacterial spores. 79 v

CONTRACT RESEARCH PROJECT REPORT QUARTERMASTER FOOD AND CONTAINER INSTITUTE FOR THE ARMED FORCES, CHICAGO Hq, QM Research and Development Command, QM Research and Development Center, Natick, Mass. The University of Michigan Engineering Research Institute Ann Arbor, Michigan Official Investigator: Lloyd L. Kempe Collaborators: J. To Graikoski Nancy J. Williams Peter F, Bonventre Project No. 7-84-01-002 Contract No. DA-19-129-qm-388 File No. S-510 Final Report Period 7 June 1955 to 31 August 1957 Initiation Date: 7 June 1955 Title of Contract: Combined Use of Heat and Radiation Treatment for Sterilization of Foods GENERAL SUMMARY In order that irradiation processing of foods can be intelligently considered, basic factual information is needed. Such information includes the effects of temperature on the lethality of ionizing radiations for anaerobic bacterial spores that are important in food sterilization, the effects of medium components, as well as the combined effects of temperature and such radiations. Where it may be planned to use irradiation alone for processing foods, it is necessary to know whether or not such a treatment is effective at refrigerating or freezing temperatures which protect the food before irradiation. Further, do chemical additives used in the preparation of the food affect the sterilizing effectiveness of ionizing radiation? Does preirradiation change the Z value of anaerobic bacterial spores still remaining in the food? How is the combined irradiation-heat process affected by the type of food being processed? These and other questions have provided the necessity for and objectives of this work. The work has so far established the following: vi

1o An induction dosage of approximately one megarep of gamma radiation is required before the combined irradiation-heat processing of food shows advantage. For example, using 300 C, botulinum 213B spores in a No. 1 picnic can of ground beef, sterility was attained with an Fo of 0.15 following 1.0 megarep of radiation. But, following 0.5 megarep or less, an Fo of 0.4 was needed to produce sterile cans of meat. 2, A combined process sufficient to sterilize canned ground beef, inoculated with 300 PA 3679 spores per can is much more than adequate to sterilize similar meat containing 5,000,000 C. botulinum 213B spores per can. This provides a large margin of safety against possible botulinus poisoning from underprocessed meat, provided the process used is designed to kill PA 3679 spores. 35 There is little if any difference between the combined irradiation-heat processing treatments required to sterilize canned raw or canned cooked beef. Thus it would be equally acceptable to irradiate raw or cooked beef before applying the reduced heat processing treatment subsequently required for sterilization. 4, Based on one series of experiments the Z values of irradiated and nonirradiated PA 3679 spores appear to be the same when these spores are heat-processed in distilled water. Therefore, the Fo calculations in this report appear to be justifiedo 5. C. botulinum 213B and PA 3679 spores suspended in phosphate buffer (pH 7,02) containing catalase were much less sensitive to the lethal action of gamma radiation than were similarly treated spores in the absence of catalaseo This finding supports the theory that the lethal action of ionizing radiations is at least partially due to secondary effects of the irradiationo Furthermore, such protection of anaerobic bacterial spores is important when sterilization of foods containing catalase is considered since it will likely increase the dosage required. 6. Combined irradiation-heat processing studies of canned frozen peas are incomplete at present. Results will not be available for two or three months due to slow germination of PA 3679 spores in the pea brine. Results will be reported at a later time. 7O Reduced sulfhydryl groups present in anaerobic bacterial spores protect these spores against the lethal activities of gamma radiation, but oxidized sulfhydryl groups are ineffectiveo 80 Different anaerobic bacterial spores have different temperature ranges in which they are less sensitive to the lethal effects of gamma radiation. However, when such radiations are to be used alone for food sterilization, refrigeration or freezing temperatures are best suited for the process since both C. botulinum and PA 3679 spores are more sensitive to gamma radiation at low temperatures than they are at temperatures between 5~C and 95~C. vii

This is compatible with processing schedules designed to protect foods by refrigeration or freezing before and during irradiation. 9. Combined irradiation-heat processing of foods will involve heating the foods to at least 95~C, either subsequent to or during irradiation. This follows from the discovery that a critical temperature exists at about 95~C, below which irradiated anaerobic bacterial spores are no more sensitive to heat than are nonirradiated spores. However, above 95~C, previously irradiated spores are much more easily killed than are nonirradiated spores of C. botulinum or PA 3679. Several papers are in preparation as a result of this work. They will include the following: 1. Combined irradiation-heat sterilization of canned ground beef: by Lloyd L. Kempe, Jo T. Graikoski, and P. F. Bonventre. There will be at least three papers on this subject. 2. The effect of chemicals on the lethality of gamma radiation for anaerobic bacterial spores: by Nancy J. Williams and Lloyd L. Kempe. 35 The effect of temperature during irradiation on the lethality of gamma radiations for anaerobic bacterial spores: by J. T. Graikoski and Lloyd L, Kempe. 4. Sensitization of anaerobic bacterial spores to temperatures above 85~C by gamma radiation: by J. T. Graikoski and Lloyd L. Kempe. OBJECTIVE This work was undertaken to develop combined irradiation —heat processing schedules for canned food and to study the effect of environmental conditions on the lethality of gamma radiation from cobalt-60 for the spores of anaerobic bacteria that are significant in food spoilage. viii

PHASE I EFFECT OF PREIRRADIATION OF INOCULATED PACKS OF CANNED GROUND BEEF ON THE FO SUBSEQUENTLY REQUIRED FOR STERILIZATION SUMMARY Data are presented to show that the Fo required to sterilize inoculated packs of either raw or precooked ground beef can be considerably reduced by preirradiation with gamma rays from cobalt-60o Ground beef packed in No. 1 picnic tin cans was sterilized by conbined irradiation-heat treatments, as well as by heat and by irradiation processing alone. Results of these studies are summarized in the following table (on page 2). It will be noted that a combined process, sufficient to sterilize precooked ground beef containing 300 PA 3679 spores per can, is much more than adequate to sterilize similar meat containing 5,000,000 C. botulinum 213B spores per cano This provides a large margin of safety against possible botulinus poisoning from underprocessed meat if the process is originally designed to kill PA 3679 spores. The data adequately demonstrate little if any difference between cooked and naw meat with respect to the severity of irradiation-heat processing treatments required to produce sterile products, INTRODUCTION Combined irradiation-heat processing of canned, cooked, ground beef inoculated with C. botulinum 213B spores was previously shown to require less of these forms of energy when they were used together than when either form was used alone.3 However, it was necessary to extend the studies to include bacterial spores of greater heat resistance than those of C. botulinumo Therefore the work was continued using PA 3679 spores. It was also considered desirable to determine whether the results obtained with cooked ground beef would be applicable to such meat preirradiated in this raw state. For this purpose, raw meat inoculated with CO botulinum and like packs inoculated with PA 3679 spores were tested in a manner similar to that reported for cooked ground beef. This work, then, was designed to establish combined irradiationheat processing treatments that would sterilize canned ground meat. From these treatments, it should be possible to select the most desirable combined 1

TREATMENTS REQUIRED TO STERILIZE CANNED GROUND BEEF PACKED IN NO. 1 PICNIC TIN CANS USING HEAT AND GAMMA RADIATION ALONE AND IN COMBINATION Sterilization Conditions Condition of ~Condit~i~on of~ Number of Irradiation Only Heat A Combined Process Ground Beef Kinid of Spores Ground Beef Kind of Spores Sp Approx Approx. Only Preirradiation Heat During Irradiation |Megarep Megarad F Megarep IMagari F Precooked C. botulinum 213B 5,000,000 354-3.9 3.2-3.6 1.0 1.2 1.12 0.20 Precooked C. botulinum 213B 00 1.6-1.8 1.5-1.7 0.4 1.0 0.93 0.15 Precooked PA 3679 10,000 2.1 2.0 7.0 1.9 1.77 1.0 Precooked PA 3679 300 1.8-2.0 1.7-1.9 6.0 1.3 1.21 1.0 Raw C. botulinum 5,000,000 2.8-3.0 2.6-2.8 1.1 1.2 1.12 0.2 Raw PA 3679 300 2.0-2.3 1.9-2.1 6.0 1.3 1.21 1.0

process for future organoleptic and similar studies. MATERIALS AND METHODS a Packing lo Cooked Meat Packs.-Lean ground beef is purchased locally from The University of Michigan food stores. The meat is placed in shallow pans and autoclaved at 15 psig steam pressure for one-half hour. Excess liquid is poured off, and the hot meat is packed into No. 1 picnic tin cans, some of which have previously been fitted with 0. F. Ecklund thermocouples. Covers are set loosely on the cans of meat which are then placed in an autocalve where they are sterilized at 17 psig steam pressure for one hour. Next, each can is removed individually from the hot autoclave and the meat is inoculated with 1 ml of a spore suspension. The cans are then sealed in a commercialtype closing machine, immersed in cold tap water for about 20 minutes, and placed in ice water for an hour. 2o Raw Meat Packs. —The cold, lean ground beef is spread out in shallow pans and then placed in an evacuation chamber. Here the dissolved metabolic gases are removed by evacuating to about 25 inches of mercury, after which the vacuum is releasedo This is repeated three times while the meat is in the panso Next the ground beef is packed into No. 1 picnic tin cans, being careful to avoid air pockets. Some of the cans are equipped with Ecklund thermocouples. Now the cans are placed in the evacuation chamber and the gas exhaustion procedure previously described is again carried out three timesO The meat remains very cold throughout this procedure. Following degassing, the meat is inoculated at approximately the geometrical center of the can with 1 ml of a spore suspension that has been prepared in distilled watero Finally the cans are sealed in a commercial-type vacuum closing machine at a vacuum of about 26 inches of mercury. Experimental cans are then either irradiated or temporarily stored in a refrigerator, as required; controls are placed in an 85~F incubator immediately; processed cans are quickly cooled by immersion in cold water before incubation. b. Irradiation The canned meat is irradiated in the "center well" of the large cobalt-60 source here at The University of Michigan. The temperature of the meat is kept below 4~C during irradiation. Actual dosage delivered at the center of the can is measured by ferrous-feric sulfate dosimetry as previously described.1 c. Heat Processing This process is carried out as follows: 5

1o A 3-gallon pail is positioned in the upper part of a steam-heated autoclave, and the pail is half filled with water at 180~F. 2. Six cans of meat, two of which contain thermocouples, are removed from the refrigerator, thermocouples leads are attached to the two control cans, and then all six cans are placed in the 1800F water. Temperature measurements of the water in the pail and in the center of each can containing thermocouples are begun immediately. 35 The autoclave cover is clamped shut, and steam is introduced at such a rate that the water surrounding the cans is maintained at 1800F until the two thermocouples in the cans of meat show identical temperatures of 1700F or more. A temperature of 180~F was selected because (a) it represents "hot-filling" temperatures of industrial practice, (b) it is not sufficient to cause appreciable killing of spores anywhere in the can during temperature equilibration, and (c) it is high enough to make "come-up" rates to the processing temperature essentially uniform in all the cans. 4o When the cans have equilibrated at some temperature between 170~F and 180~F, the water-bath temperature is quickly brought to 250~F by introducing steam into the autoclave. Processing time to attain the desired Fo value, less the cooling increment, is now providedo Note:e A processing temperature of 2500F was first used (Runs PA-2 to PA-9) but this was found to be too high and 230~F was adopted instead. 5. At the proper time, the autoclave is quickly opened and the cans are plunged into ice water. Temperature measurements are continued until the temperature at the center of the cans reaches 180~Fo 6. Experimental cans of meat are incubated at 85~F; the cans containing thermocouples are again refrigerated; they are used a second time only. 7. Four sets of cans are autoclaved for each run, using arbitrarily selected processing times designed to provide suitable Fo increments. After the runs are completed, the actual Fo accomplished for each set of four cans is computed. This, together with the incubation results, constitutes the basic data acquiredo 8. Fo values are calculated by 0. To Schultz's graphical modification of C. 00 Ball's General Methodo In these calculations the Z value of both irradiated and nonirradiated Co botulinum 215B spores is assumed to be 18. do Spores The spores of anaerobic bacteria used in these studies are prepared and used according to techniques described in previously published work from this laboratory 1 4

RESULTS Canned ground beef packed in No. 1 picnic tin cans was sterilized by combined irradiation-heat processingo Data from the previous year's work, showing suitable combined processes of this type for cooked ground beef inoculated with C. botulinum 213B spores, are included here for reference and for purposes of comparison. These data, shown in Figs. 1 and 2, indicate the following: a. A preirradiation "induction" dosage was apparent, before sensitization of the spores to heat became significantly important, b. The data in Fig. 1 show that when 5,000,000 C. botulinum 213B spores were used per can of meat, heat processing alone required an Fo of approximately l.0 for sterilizationo Similarly, irradiation alone required between 3.4 and 3.9 megarep to produce sterility. But when preirradiation with 1.2 megarep of gamma radiation was used, sterility was attained with an Fo of approximately 002. c. From Figo 2 it will be observed that when using 300 C. botulinum 213B spores per can of meat, sterilization was attained with heat alone using an F0 of approximately 0.4, while with irradiation alone approximately 1.7 megarep of gamma radiation from cobalt-60 were required. On the other hand, when irradiation and heat processing were combined, sterility was attained with an F0 of approximately 0.15 following 1.0 megarep of radiation. This year the combined irradiation-heat processing treatments required to sterilize cooked ground beef inoculated with PA 3679 spores have been determined. Studies were also carried out with raw meat pack. Table I includes data from runs PA-2 through PA-25, all of which were carried out under similar conditions as indicated. Results from these runs, are summarized in Table I1o Data from Table II, plotted in Fig. 3, indicate the following for inoculated packs treated as designated: Can size: Noo 1 picnic (211 x 400) Product: Cooked ground beef Inoculum: 10,000 PA 3679 spores per can Preirradiation: As indicated Processing temperature: 230~F (NOTE: 250~F for Runs PA-2 to PA-9) Incubation temperatures 85~F Without irradiation, an Fo of more than 7 was required to sterilize the canned beefs with irradiation alone, approximately 2.1 megarep were required for this purpose. Preirradiation with 1.0 megarep of gamma radiation reduced the F0 required to produce sterility to 4o4; further reduction to an Fo of 1.0 was obtained by preirradiation with approximately 1.9 megarep. 5

I.C 0.8 0.6 0.5 0.4 0.3 11 I I I D:: LL 0.1.08 I ____ ______ I..I........ 5,000,000 SPORES C BOTULINUM 213B COOKED BEEF IRRADIATION ALONE 3.4- 3.9 MR.06.05.04.03.02.01 0 I.C 2.0 MEGAREP 3.0. Fig. lo Fo required to sterilize cooked ground beef packed in No. 1 picnic tin cans, inoculated with approximately 5,000,000 C. botulinum 213B spores per can, and irradiated with gamma rays from cobalt-60 before heat processing. 6

1.0 0.8 0 0 0 0 0 LLJ Cl) ILi?'.6.5 i.4 13.3'I.2 18 I )6 5 --- I. 14 300 SPORES )3 ___3 C. BOTULINUM 213B - COOKED BEEF 10 -I 0.0 j-.0.0.0.0 nr A. C IRRADIATION ALONE 1.64 -a 1.80 MR.01 0 1.0 2.0 3.0 MEGAREP Fig. 2. Fo required to sterilize cooked ground beef packed in No. 1 picnic tin cans inoculated with approximately 300 C. botulinum 213B spores per can, and irradiated with gamma rays from cobalt-60 before heat processing. 7

TABLE I, Fo Value Required to Sterilize Ground Beef in No. 1 Picnic Tin Cans, Previously Inoculated with Approximately 10,000 PA-3679 Spores per Can and Then Processed at 250~F. Run No. PA-2-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-3679 spores per can - none - 250~F - 85oF F0 Can No. Days to Gas Formation 0o Noninoculated Controls Inoculated Controls NI 1 NI 2 NI 3 NI 4 26 27 28 29 3 5 3 3 Can 1, 2.5 Can 2, 2.5 1 3 4 7 5 4 5 Can 1, 4.1 Can 2, 4.1 Can 3, 3.7 Can 1, 1.1 Can 2, 1.1 Can 3, 1.3 2 5 6 8 6 9 10 11 12 4 4 4 4 Can 1, 3.7 Can 2, 3.7 Can 3, 2.8 13 14 15 16 4 5 5 4 Can 1, 6.2 Can 2, 3.3 18 19 20 21 Can 1, 8.8 Can 2, 8.8 22 23 24 25 Conclusion: Ground beef, packed in No. 1 picnic tin cans, and inoculated with 10,000 PA-3679 spores per can, required an Fo value between 2.8 and 8.8 for sterilization when processed at 250~F. 8

TABLE I (Continued) Run No. PA-3-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 600,000 PA-3679 spores per can - none - 250~F (except cans 1, 2, 3, and 4 - 85~F at 2300F) Fo Can No. Days to Gas Formation Noninoculated Controls NI 1 NI 2 Inoculated Controls I 1 3 12 +* Can 1, 2.3 1 4 Can 2, 2.3 2 4 Can 3, 2.3 3 4 4 4 Can 1, 6.1 5 4 Can 2, 6.1 6 6 Can 3, 3.8 7 4 8 5 Can 1, 9.4 9 9 Can 2, 8.3 10 8 Can 3, 8.7 11 6 12 9 Can 1, 12.4 13 9 Can 2, 12.4 14 13 Can 5, 11.6 15 15 16 16 *Date not recorded. Conclusion: With 600,000 PA-3679 spores ture of 250~F, the Fo value required for exceeds 12.4. per can and a processing temperasterilization of the canned meat 9

TABLE I (Continued) Run No. PA-4- Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 200 PA-3679 spores per can - as indicated - not heat processed - 85~F Irradiation Dosage, megarep Can No. Days to Gas Formation Noninoculated Controls See Run 5 Inoculated Controls See Run 5 3.550 1 2 3 4 3.200 5 - 6 7 8 1.383 9 10 11 6 12 1.816 13 14 15 16 Conclusion: Under the above conditions canned ground beef was sterilized with between 1.383 and 1.816 megarep of gamma irradiation. 10

TABLE I (Continued) Run No. PA-5-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 200 PA-5679 spores per can - none - 250~F - 85~F Fo Noninoculated Controls Can No. NI 1 NI 2 NI 3 NI 4 Days to Gas Formation....... Inoculated Controls Can 1, 2.7 Can 2, 2.7 Can 1, 3.6 Can 2, 6.8 Can 3, 8.3 INC 1 INC 2 INC 5 INC 4 3 3 3 3 1 2 3 4 5 6 8 9 Can 1, 12.3 Can 2, 12.3 Can 3, 15.3 (disregard) Can 1, 9.0 Can 2, 8.0 Can 3, 8.1 (disregard) 10 11 12 13 14 15 16 17 18 19 20 21 23 24 25 26 Can 1, 4.9 Can 2, 2.7 Can 3, 6.8 Can 1, 4.4 Can 2, 4.4 Can 1, 14.1 Can 2, 12.6 7 22 27 28 value less than 2.7 was __ Conclusion: Under the above conditions needed to sterilize ground beef. 11 an Fo

TABLE I (Continued) Run No. PA-6-Can Size Product Inoculum Preirraidation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 54,000 spores per can - 1.060 megarep - 250~F - 85~F Fo Can No. Days to Gas Formation Noninoculated Controls NI 1 NI 2 Inoculated Controls 17 4 18 4 19 4 20 4 Can 1, 3.1 1 Can 2, 5.1 2 10 Can 3, 2.7 (disregard) 3 - 4 Can 1, 5.8 5 8 Can 2, 3.5 6 Can 3, 4.1 7 8 Can 1, 1.0 9 5 Can 2, 1.2 10 7 Can 5, 1.7 11 9 12 7 Can 1, 8.6 13 - Can 2, 6.2 14 - Can 3, 5.5 15 - 16 Conclusion: Under these conditions sterilization of canned ground beef is obtained by irradiation with 1.060 megarep of gamma rays followed by an Fo value between 3.5 and 8.6. 12

TABLE I (Continued) Run No. PA-7-Can Size Product Inoculum Irradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground, Beef - 13,500 PA-3679 spores per can - as indicated - not-processed - 85~F Megarep Can No. Days to Gas Formation Noninoculated Controls See Run 8 Inoculated Controls 35 36 37 38 3 3 3 3 0.8553 1 2 3 4 4 4 4 4 1.878 13 14 15 16 6 4 1.620 31 32 33 34 4 4 4 4 2.560 9 10 11 12 3.330 5 6 7 8 Conclusion: Irradiation-sterilization dosage for canned ground beef under these conditions lies between 1.878 and 2.560 megarep. 13

TABLE I (Continued) Run No. PA-8-Can Size Product Inoculum Pre irradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 13,500 PA-3679 spores per can - none - 250~F - 85~F Fo Can No. Days to Gas Formation Noninoculated Controls NI 1 NI 2 NI 3 NI 4 Inoculated Controls 35 36 37 38 4 4 4 4 13 6 Can 1, 3.1 Can 2,, 2.5 1 2 3 5 4 Can 1, 9.6 Can 2, 9.6 Can 1, 5.6 Can 2, 5.6 5 6 7 I 8 9 10 11 12 Can 1, 3.8 Can 2, 2.9 13 14 15 16 9 6 Can 1, 7.9 Can 2, 10.7 Can 3, 12.8 17 18 19 20 Can 1, 7.0 Can 2, 7.0 21 22 25 24 Can 1, 11.6 25 Can 2, 9.2 26 27 28 Conclusion: Under these conditions canned ground beef was sterilized with heat processing at 250~F, having an Fo value between 2.5 and 5.6. 14

TABLE I (Continued) Run No. PA-9 —Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 16,000 PA-3679 spores per can - 0.8553 megarep - 250~F - 85~F Fo Can No. Days to Gas Formation Inoculated Controls 21 3 23 3 24 3 Irradiation Controls, 17 3 1.340 megarep 18 3 19 3 20 3 Can 1, 2.3 1 4 Can 2, 2.3 2 5 Can 3, 0.6 (disregard) 3 4 4 5 Can 1, 6.3 5 6 Can 2, 3.8 6 Can 3, 4.2 7 8 7 Can 1, 0.5 9 5 Can 2, 0.5 10 4 11 4 12 4 Can 1, 6.5 13 Can 2, 9.0 14 Can 3, 11.7 15 16 Conclusion: Under these conditions canned ground beef was sterilized by irradiation with 0.853 megarep of gamma irradiation followed by heat processing to an F. value of between 3.8 and 11.7. 15

TABLE I (Continued) - Fo Value Required to Sterilize Ground Beef in No. 1 Picnic Tin Cans, Previously Inoculated with Approximately 10,000 PA-3679 Spores per Can and Then Processed at 230~F Run No. PA-10 —Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-3679 spores per can - none - 230~F - 85~F Fo Can No. Days to Gas Formation Noninoculated Controls NI 1 NI 2 NI 3 NI 4 Inoculated Controls Can 1, 6.0 Can 2, 6.0 Can 3, 5.2 Can 1, 3.8 Can 2, 3.8 Can 3, 3.8 Can 1, 3.0 Can 2, 3.0 Can 3, 3.0 17 18 19 20 3 3 3 3 1 2 3 4 9 11 8 11 5 6 7 8 5 6 5 5 9 10 11 12 6 5 5 5 Can 1, 2.0 Can 2, 2.0 Can 3, 2.0 13 14 15 16 value 4 4 4 4 sterilize Conclusion: Under these conditions the Fo canned ground beef exceeds 6.0. required to

TABLE I (Continued) Run No. PA-l —Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-3679 spores per can - 1.261 megarep - 230~F. 85~F Fo Can No. Days to Gas Formation Noninoculated Controls NI 1 NI 2 NI 3 NI 4 Inoculated Controls 17 18 19 20 3 3 3 3 Can 1, 1.0 Can 2, 0.8 Can 3, 0.4 1 2 3 4 5 5 5 5 Can 1, 1.6 Can 2, 1.0 Can 3, 0.8 5 6 7 8 6 5 5 6 Can 1, 2.0 Can 2, 2.0 Can 3, 3.0 9 10 11 12 9 6 9 Can 1, 3.9 13 Can 2, 3.9 14 Can 3, 4.9 15 16 Conclusion: Canned ground beef was sterilized under these conditions by 1.500 megarep of preirradiation followed by heat processing with an Fo value between 2.0 and 4.9. 17

TABLE I (Continued) Run No. PA-12 —Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-3679 spores per can - None - 230~F - 85~ F Fo Can No. Days to Gas Formation Noninoculated Controls NI 1 NI 2 NI 3 Inoculated Controls INOC 3 Can 1, 2.9 1 4 Can 2, 2.9 2 4 3 3 4 5 Can 1, 5.8 5 14 Can 2, 5.8 6 7 11 8 14 Can 1, 4.3 9 6 Can 2, 5.5 10 7 11 6 12 6 Conclusion: Canned ground beef requires an Fo value of more than 5.8 for sterilization under these conditions.

TABLE I (Continued) Run No. PA-13-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-3679 spores per can - 0.841 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation Can 1, 4.4* 1 Can 2, 4.4 2 7 5 4 Can 1, 0.7 5 4 Can 2, 0.5 6 3 7 4 8 4 Can 1, 2.3 9 7 Can 2, 2.3 10 4 11 5 12 5 Can 1, 5.7 13 Can 2, 5.7 14 15 16 Conclusion: Canned ground beef was sterilized under these conditions by 0.841 megarep preirradiation followed by heat processing with an F value between 4.4 and 5.7. *Same controls as used for Run 12. 19

TABLE I (Continued) Run No. PA-14-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-3679 spores per can - 1.420 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation Noninoculated Controls NI 1 NI 2 NI 3 NI 4 Inoculated Controls Can 1, 5.6 Can 2, 4.9 Can 1, 6.5 Can 2, 5.6 Can 1, 1.1 Can 2, 1.1 17 18 19 20 3 3 3 5 1 2 3 4 5 6 7 8 9 10 11 12 9 9 Can 1, 3.2 13 Can 2, 3.2 14 Can 3, 3.2 15 16 Conclusion: Canned ground beef was sterilized 1.420 megarep of gamma irradiation followed by value between 1.1 and 3.2. under these conditions by heat processing with an F0 - c 20

TABLE I (Continued) Run No. PA-15-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-5679 spores per can - None - 250~F - 85~F Fo Can No. Days to Gas Formation Noninoculated Controls NI 1 NI 2 NI 3 NI 4 Inoculated Controls 17 3 18 5 19 3 20 3 Can 1, 6.5 1 21 Can 2, 7.0 2 17 Can 3, 7.0 3 54 4 13 Can 1, 8.4 5 Can 2, 7.3 6 7 8 Can 1, 6.0 9 8 Can 2, 6.0 10 8 Can 5, 6.0 11 7 12 7 Can 1, 5.0 13 11 Can 2, 5.0 14 11 Can 3, 5.0 15 11 16 8 Conclusion: Under these conditions, canned ground beef was sterilized with an Fo value between 6.5 and 8.4. 21

TABLE I (Continued) Run No. PA-16-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-5679 spores per can - None - 230~F - 85~F Fo Can No. Days to Gas Formation Noninoculated Controls NI 1 II 2 II 5 II 4 Inoculated Controls 13 3 14 3 15 3:NOC 4 Can 1, 7.1 1 L33 Can 2, 7.1 2 59 Can 3, 7.1 3 47 4 63 Can 1, 8.7 5 Can 2, 8.7 6 7 8 Can 1, 4.1 9 7 Can 2, 4.6 10 7 11 7 12 7 Conclusion: an Fo value Under these conditions canned ground beef was sterilized with between 7.1 and 8.7. 22

TABLE I (Concluded) Run No. PA-17-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-3679 spores per can - 0.708 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation Noninoculated Controls NI 1 NI 2 NI 3 NI 4 Inoculated Controls 13 14 15 INOC 3 3 3 4 Can 1, 10.2 Can 2, 10.2 Can 3, 10.2 Can 1, 5.5 Can 2, 4.9 1 2 3 4 5 6 7 8 Can 1, 6.9 Can 2, 6.6 9 10 11 12 Can 1, 3.7 13 Can 2, 3.1 14 17 15 12 16 12 Conclusion: Canned ground beef was sterilized under these conditions by 0.708 megarep irradiation followed by heat processing with an Fo value between 3.1 and 5.5.

TABLE I (Continued) - Fo Value Required to Sterilize Ground Beef in No. 1 Picnic Tin Cans, Previously Inoculated with 10,000 PA-2679 Spores per Can and Irradiated with Gamma Rays from Cobalt-60 Before Processing at 230~F. Run No. PA-18-Can size Product Inoculum Preirradiation Processing temperature Incubation temperature - No. 1 picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-3679 spores per can - 1.652 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation Inoculated Controls 1 2 3 4 4 4 4 4 Can 1, 1.1 Can 2, 1.1 Can 3, 0.68 1 2 3 4 Can 1, 1.8 Can 2, 1.8 5 6 7 8 13 Can 1, 0.16 Can 2, 0.16 Can 3, 0.10 9 10 11 12 5 5 6 Can 1, 0.57 Can 2, 0.40 Can 3, 0.25 13 14 15 16 5 5 Can 1, 0.27 17 6 Can 2, 0.27 18 Can 3, 0.27 19 8 20 4 Conclusion: Under these conditions canned ground beef was not sterilized by 1.652 megarep of gamma radiation followed by heat processing to an Fo of 1.8. 24

TABLE I (Continued) Run No. PA-19-Can size - No. 1 picnic (211 x 400) Product - Cooked Ground Beef Inoculum - 10,000 PA-3679 spores per can Irradiation only Incubation temperature - 85~F Megarep Can No. Days to Gas Formation Inoculated Controls 1 4 2 4 Noninoculated controls See run PA-20 1.560 13 4 14 4 15 4 16 5 1.970 9 10 7 11 12 2.625 1 2 3 4 3.040 5 6 7 8 Conclusion: Canned ground beef was sterilized under these conditions by 1.970 to 2.625 megarep of gamma radiation. 25

TABLE I (Continued) Run No. PA-20-Can size Product Inoculum Preirradiation Processing temperature Incubation temperature - No. 1 picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-3679 spores per can - 0.410 megarep - 230~F - 85OF Fo Can No. Days to Gas Formation Noninoculated Controls 1 2 3 4 Inoculated Controls See run PA-19 Can 1, 7.9 1 Can 2, 7.9 2 3 4 Can 1, 4o5 5 40 Can 2, 4.5 6 7 23 8 13 Can 1, 6.1 9 6 Can 2, 6.1 10 17 Can 3, 5.7 11 12 6 Can 1, 351 13 6 Can 2, 3.1 14 7 Can 35 3.1 15 - 16 5 Conclusion: Under these conditions, canned ground beef was sterilized by 0.410 megarep of gamma radiation followed by heat processing with an Fo between 5.7 and 7.9. 26

TABLE I (Continued) Run No. PA-21-Can size Product Inoculum Preirradiation Processing temperature Incubation temperature - No. 1 picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-3679 spores per can - 1.313 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation Controls See run PA-19 Can 1, 0.44 1 4 Can 2, 0.34 2 3 Can 35 0.29 4 3 8 3 Can 1, 6.6 5 Can 2, 6.6 6 Can 3, 6.0 3 7 Can 1, 4,0 9 Can 2, 4.0 10 Can 3, 4.0 11 12 Conclusion: Under these conditions, canned ground beef was sterilized by 1.313 megarep of gamma radiation followed by an Fo between 0.29 and 4.0. 27

TABLE I (Continued) Run No, PA-22-Can size - No. 1 picnic (211 x 400) Product - Cooked Ground Beef Inoculum - 10,000 PA-3679 spores per can Preirradiation - 1.780 megarep Processing temperature- 230~F Incubation temperature- 85~F Fo Can No. Days to Gas Formation Noninoculated Controls Inoculated Controls Can 1, 2.4 Can 2, 2.1 Can 3, 1.9 Can 1, 1.5 Can 2, 0.97 Can 3, 0.78 Can 1, o056 Can 2, 0.56 1 2 3 4 1 2 3 4 3 3 3 3 5 1 2 3 4 5 6 7 8 9 10 11 12 8 4 4 4 6 3 4 4 5 Can 1, Can 2, Can 3, 0.23 0.20 0 20 13 14 15 16 Conclusion: Under these conditions, canned ground beef was sterilized by 1.780 megarep of gamma radiation followed by an Fo between 0.78 and 2.4. 28

TABLE I (Continued) Run No, PA-23-Can size Product Inoculum Preirradiation Processing temperature Incubation temperature - No. 1 picnic (211 x 400) - Cooked Ground Beef - 10,000 PA-5679 spores per can - 1.538 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation Controls See run PA-22 Can 1, 2.3 1 4 Can 2, 2.3 2 Can 3, 2.3 3 4 Can 1, 1.1 5 4 Can 2, 0.97 6 4 Can 3, 0.97 7 8 Can 1, 0.14 9 5 Can 2, 0.14 10 3 Can 3, 0.16 11 3 12 3 Can 1, 3.1 13 Can 2, 3.7 14 Can 3, 3.4 15 16 Can 1, 4.2 17 Can 2, 4.1 18 Can 3, 3.8 19 20 Conclusion: Under these conditions, canned ground beef was sterilized by 1.538 megarep of gamma radiation followed by an Fo between 2.3 and 3.7. 29

TABLE I (Continued) Run No. PA-24-Can size - No. 1 picnic (211 x 400) Product - Cooked Ground Beef Inoculum - 10,000 PA-5679 spores per can Preirradiation - 1.985 megarep Processing temperature - 230~F Incubation temperature - 85~F Fo Can No. Days to Gas Formation Noninoculated Controls Inoculated Controls Can 1, 0.25 Can 2, 0.23 Can 3, 0.22 Can 1, 0.061 Can 2, 0.055 Can 3, 0.068 Can 1, 1.61 Can 2, 1.40 Can 3, 1.32 Can 1, 0.63 Can 2, 0.49 Can 3, 0.41 1 2 1 2 3 4 3 3 5 3 5 1 2 3 4 5 6 7 8 4 w 9 10 11 12 13 14 15 16 Conclusion: Under these conditions, canned ground beef was sterilized by 1.985 megarep of gamma radiation followed by an Fo between 0.055 and 0.25. 30

TABLE I (Concluded) Run No. PA-25 —Can size - No. 1 picnic (211 x 400) Product - Cooked Ground Beef Inoculum - 10,000 PA-3679 spores per can Irradiation only Incubation temperature - 85~F Megarep Can No. Days to Gas Formation Controls See run PA-24 1.620 1 4 4 3 5 3 8 3 2.160 2 3 6 7 2.440 9 10 11 12 13 14 Conclusion: Under these conditions, canned ground beef was sterilized by 1.620 to 2.160 megarep of gamma radiation. 31

TABLE II. Summary of Various Combined Irradiation Heat-Processing Treatments Required to Sterilize Cooked Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with 10,000 PA-3679 Spores per Can. Run No. Preirradiation, megarep* Fo Range, minute PA-10 PA-11 PA-12 PA-13 PA-14 PA-15 PA-16 PA-17 PA-18 PA-19 PA-20 PA-21 PA-22 PA-25 PA-24 PA-25 0 1.261 0 0.841 1.420 0 0 0.708 1.652 1.970-2.625 0.410 1.513 1.780 1.538 1.985 1.620-2.160 > 6.0 2.0-4.9 > 5.8 4.4-5.7 1.1-3.2 6.5-8.4 7.1-8.7 3.1-5.5 > 1.80 0 5.7-7.9 0.29-4.0 0.78-2.4 2.3-3.7 0..055-0.25 0 *Note: corrected on basis of source recalibration, 1-7-57 52

Irnn 8.0 go I. ---- 6.0 5.0 4.0 \ 3.0 2.0 1.0 0.8 0.6 0.5 0.4 0.3 0.2 0.1.08 _ 10,000 SPORES _ 06 PA 3679 COOKED BEEF.05.04 l - u) w z I.03.02.01 I I I I I I IRRADIATION ONLY 1.97 — 2.16,, ________ \ ___, i 1.0 2.0 MEGAREP 3.0 Fig. 3. Fo required to sterilize cooked ground beef packed in No. 1 picnic tin cans, inoculated with 10,000 PA 3679 spores per can, and irradiated with gamma rays from cobalt-60 before heat processing at 230~F. 33

Table III includes data from runs PA26 through PA-33. These runs are similar to those previously described in Table I except that only 300 PA 3679 spores were inoculated into each can. The results for these runs are summarized in Table IV and plotted in Fig. 4. They show that, under these conditions, without irradiation an Fo of approximately 6 was required to sterilize the precooked ground beef; with irradiation alone, between 1.750 and 2.020 megarep were needed; but when irradiation preceded heat processing, those cans of cooked ground beef receiving more than 1.3 megarep were subsequently sterilized with F0 values less than 1.0. Table V includes data from runs RCB-1 through RCB-11 which are similar to those previously described in Table I except that the meat was packed into the cans while still in the raw condition, and each can was inoculated with approximately 5,000,000 C. botulinum 213B spores. The results for these runs are summarized in Table VI and plotted in Fig. 5. The data show that, under these conditions, sterilization was accomplished by heat processing alone with an F0 between 1.O and 1.1; with irradiation only, between 2.75 and 35.00 megarep were needed; but when irradiation preceded heat processing, 1.2 megarep of gamma radiation reduced the Fo subsequently required for sterilization to approximately 0 2. Table VII includes data from runs RPA-1 through RPA-7. These runs are similar to those tabulated in Table V except that 300 PA 3679 spores were used for the inoculum in each can of raw meat. The results for these runs are summarized in Table VIII and are plotted in Fig. 6. The data show that raw ground beef, packed in No. 1 picnic tin cans and inoculated with 300 PA 35679 spores per can, was sterilized by heat processing alone with an Fo of approximately 6, and by irradition alone, with between 2.000 and 2.300 megarep, but when irradiation preceded heat processing, 1.3 megarep reduced the Fo subsequently required to less than 1.0. DISCUSSION The spores of C. botulinum were chosen for the initial portion of this work because of their importance in food poisoning. Also, the toxin developed by their growth permitted testing of the processed cans to insure that spoilage was caused by growth of the injected spores and not by contaminants. This was useful for development of the techniques later used for PA 5679 spore packs, The first part of the work was also carried out with cooked, sterile, ground beef. This was done for several reasons, viz: 1. Cooked meat is a classical medium for growing anaerobic bacterial spores to produce gas. 2. A sterile medium permits the usual methods of pure culture bacteriological 34

TABLE III. Picnic Tin Spores per Fo Value Required to Sterilize Cooked Ground Beef in No. 1 Cans, Previously Inoculated with Approximately 500 PA-3679 Can and Then Processed at 230~F Run No. PA-26-Can Size Product Inoculum Preirradiat ion Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 300 PA-3679 spores per can - 0.500 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation -- - I! I I I I,l...Il I I III! 1 - J m,.. Noninoculated Controls Inoculated Controls Can 1, 1.97 Can 2, 1.97 Can 3, 1.97 1 2 3 4 1 2 3 4 3 3 3 3 1 2 3 4 5 5 4 5 6 9 4 45 Can 1, Can 2, Can 35 2.87 2.83 2.49 5 6 7 8 Can 1, 7.42 Can 2, 7.43 Can 3, 6.51 9 10 11 12 Can 1, Can 2, Can 3, 4.50 4.81 4.85 15 14 15 16 - - Conclusion: Sterilization was accomplished followed by an Fo between 2.5 and 4.9. with 0.500 megarep irradiation 35

TABLE III (Continued) Run No. PA-27-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 300 PA-3679 spores per can - none - 230~F - 85 F Fo Can No. Days to Gas Formation Controls - See Run No. PA-26 Can 1, 0.63 Can 2, 0.55 Can 3, 0.62 Can 1, 0.36 Can 2, 0.28 Can 3, 0.29 Can 1, 8.10 Can 2, 8.46 Can 39 7.77 Can 1, 1.91 Can 2, 1.76 Can 1, 1.02 Can 2, 0.59 Can 1, 5.01 Can 2, 5.01 Can 1, 3.14 Can 2, 3.14 Can 3, 3.14 Can 1, 4.07 Can 2, 4.31 Can 3, 3.53 Can 1, 7.05 Can 2, 7.05 Can 3, 7.05 1 2 3 4 4 4 4 4 5 6 7 8 3 3 3 3 9 10 11 12 13 14 15 16 5 5 4 4 17 18 19 20 4 3 4 4 21 22 23 24 25 26 27 28 17 7 5 6 7 8 6 6 5 29 30 31 32 33 34 35 36 Conclusion: Sterilization was accomplished 36 with an Fo between 5.0 and 7.1.

TABLE III (Continued) Run No. PA-28-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 300 PA-3679 spores per can - 1.000 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation, ~, ii, I.'.... Noninoculated Controls Inoculated Controls 1 2 3 4 1 2 3 4 3 3 3 3 Can Can Can 1, 0.90 2, 0.90 3, 0.46 1 2 3 4 5 10 8 Can 1, 1.8 Can 2, 1.1 5 6 7 8 7 Can 1, Can 2, Can 3, 5.50 4.68 4.95 9 10 11 12 Can Can Can 1. 2.0 2, 2.6 3, 2.9 13 14 15 16 Conclusion: Sterilization was accomplished with 1.000 megarep of gamma radiation followed by an Fo between 2.0 and 5.0. 37

TABLE III (Continued) Run No. PA-29 —Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 300 PA-3679 spores per can - 0.750 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation Controls: - See Run No. PA-28 Can 1, 1.84 1 6 Can 2, 1.43 2 6 Can 3, 1.65 3 5 4 5 Can 1, 2.56 5 Can 2, 2.56 6 10 Can 3, 2.56 7 11 8 Can 1, 0.74 9 5 Can 2, 1.02 10 5 Can 3, 0.53 11 5 12 5 Can 1, 3.48 13 Can 2, 3.48 14 Can 3, 3.68 15 16 Conclusion: Sterilization was accomplished by 0.750 megarep of gamma radiation followed by an Fo between 2.6 and 3.7.

TABLE III (Continued) Run No. PA-30-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 300 PA-3679 spores per can - 1.250 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation Noninoculated Controls Inoculated Controls 1 2 3 4 1 2 3 4 3 3 3 3 Can Can Can 1, 2.32 2, 1.47 3, 1.70 9 10 11 12 Can 1, Can 2, Can 3, 0.38 0.24 0.12 15 14 15 16 5 4 4 4 Can Can Can Can Can Can 1, 0.98 2, 0.98 3, 0.48 1, 1.29 2, 1.29 3, 1.50 17 18 19 20 5 21 22 25 24 Conclusion: Sterilization was accomplished by 1.250 megarep of gamma radiation followed with an Fo between 0.48 and 1.5. 39

TABLE III (Continued) Run No. PA-31-Can Size Product Inoculum Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 300 PA-3679 spores per can - 85 F Radiation Dosage (megarep) Can No. Days to Gas Formation,~ ~~~~~. 1. ~. l.,-i Controls - See Run No. PA-30 1.750 2 160 1.250 1.390 2.020 1 2 3 4 5 5 17 18 19 20 9 15 12 13 3 5 4 3 10 11 14 16 5 4 5 4 5 6 7 8 Conclusion: Sterilization was accomplished megarep of gamma radiation. with between 1.750 and 2.020 40

TABLE III (Continued) Run No. PA-32-Can Size - No. 1 Picnic (211 x 400) Product - Cooked Ground Beef Inoculum - 300 PA-3679 spores per can Preirradiation - 0.250 megarep Processing Temperature - 230~F Incubation Temperature - 850F Fo Can No. Days to Gas Formation --. i i i 1 l i Noninoculated Controls Inoculated Controls Can 1, 3.28 Can 2, 3.49 Can 1, 4.83 Can 2, 4.83 Can 39 4.52 1 2 3 4 1 2 3 4 1 2 3 4 3 3 3 3 6 5 6 5 7 7 11 9 5 6 7 8 Can 1, Can 2, Can 3, Can 1, Can 2, Can 3, 6.93 6 61 6.66 7.65 8.09 8.09 9 10 11 12 13 14 15 16 Conclusion: Sterilization was accomplished radiation followed by an Fo between 4.5 and with 0.250 megarep of gamma 6.9. 41

TABLE III (Concluded) Run No. PA-33-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Cooked Ground Beef - 300 PA-3679 spores per can - 1.350 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation --,, i, i, ~ ~~~ ~ ~ ~ ~ ~ ~ ~~~~~~~~~~~~~~~~~~~~~~~. —,i,, i! I I Noninoculated Controls Can 1, 0.31 Can 2, 0.31 Can 1, 1.46 Can 2, 1.46 Can 1, 0.77 Can 2, 1.13 Can 3, 1.13 1 2 3 4 1 2 3 4 5 7 5 6 7 8 9 10 11 12 7 5 Can 1, Can 2, Can 3, 0.58 0,42 0.54 13 14 15 16 Conclusion: Sterilization was accomplished by radiation followed with an Fo between 0.77 and 1.350 megarep of gamma 1.5. 42

TABLE IV - Summary of Various Combined Irradiation-Heat-Processing Treatments Required to Sterilize Cooked Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with 300 PA-3679 Spores per Can. Run No.00 _Preirradiation, megarep.. Fn Range, minute PA-26 0.500 2.5-4.9 PA-27 none 5.0-7.1 PA-28 lo 000 2.0-5.0 PA-29 0.750 2.6-3.7 PA-30 1.250 0.48-1.5 PA-31 1.750-2.020 none PA-32 0.250 4.5-6.9 PA-53 1.350 0.77-1.5

10 8.0 6.0 50 4.0 3.0 2.0 1.0 0.8 0.6 0.5 0.4 0.3 in LIJ pD I 10 LLo 300 SPORES il- I PA 3679 -i Ix _COOKED BEEF _ I I IRRADIATION ONLY 1. 75 -- 2.02 0.2 0.1.08.06.05.04.03.02.01 0 1.0 2.0 3.0 MEGAREP Figo 4~ Fo required to sterilize cooked ground beef packed in No. 1 picnic tin cans, inoculated with 300 PA 3679 spores per can,: and irradiated with garna rays from cobalt-60 before heat processing at 2300F. 44

TABLE V. Fo Values Required to Sterilize Raw Ground Beef in No. 1 Picnic Tin Cans, Previously Inoculated with 5,000,000 C. botulinum 213B Spores per Can and Then Processed at 230~F. Run No. RCB-1-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Raw Ground Beef - 2,500,000 C. botulinum 213B spores - None per can - 230~F - 85OF Fo Can No. Days to Gas.Formation Can 1, 0~25 1 8 Can 2, 019 2 7 3 7 4 8 Can 1, 0.74 5 9 Can 2, 0.74 6 11 Can 3, 0.74 7 8 10 Conclusion: Under these conditions, raw ground beef required an Fo in excess of 0.74 for sterilization. 45

TABLE V (Continued) Run No. RCB-2-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Raw Ground Beef - 2,500,000 C. botulinum 213B spores None per can - 230~F - 85~F Fo Can No. Days to Gas Formation Can 1, 0.58 1 4 Can 2, 0.59 2 4 Can 5, 0.61 3 4 4 4 Can 1, 0.76 5 4 Can 2, 0.76 6 4 Can 5, 0.76 7 4 8 4 Can 1, 1l13 9 7 Can 2, 0.93 10 7 11 7 12 7 Can 1, 2.08 13 Can 2, 2.08 14 Can 3, 2.08 15 16 Can 1, 1.49 17 Can 2, 1L49 18 Can 5, 1.49 19 20 Conclusion' Under these conditions, an Fo between 0.95 and 1.5. raw ground beef was sterilized with 46

TABLE V (Continued) Run No. RCB-3-Can Size Product Inoculum Preirradiation Processing Temperature - Incubation Temperature - No. 1 Picnic (211 x 400) Raw Ground Beef 5,000,000 C. botulinum 2 1.400 megarep 230~F 85~F 15B spores per can Fo Can No. Days to Gas Formation Inoculated Control 17 1 18 1 Irradiated and Inoculated 27 4 28 5 No Heat Process 29 5 1.4 Megarep 30 4 Can 1, 0.13 1 4 Can 2, 0.16 2 3 6 4 Can 1, 0.34 5 Can 2, 0.29 6 Can 3, 0.29 7 8 Can 1, 009 9 4 Can 2, 0.06 10 6 Can 3, 0.06 11 4 12 4 Can 1, 0.22 13 Can 2, 0.22 14 Can 3, 0.28 15 16 Conclusionl Under these conditions, raw ground beef was sterilized by 1.400 megarep preirradiation plus an Fo between 0.13 and 0.28. 47

TABLE V (Continued) Run No. RCB-4-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Raw Ground Beef - 5,000,000 C. botulinum 213B spores - 2.000 megarep per can - 230~F - 85~F Fo Can No. Days to Gas Formation Inoculated and Irradiated No Heat Process 2o130 Megarap Inoculated and Nonirradiated Heat-processed Can 1, 1.03 Can 2, 1.03 Can 3, 1.03 Inoculated and Nonirradiated Heat-processed Can 1, 1.67 Can 2, 1.67 Can 3 Noninoculated and Nonirradiated Can 1, 0.17 Can 2, 0.17 Can 35 0.20 Noninoculated No heat Process 2.000 Megarep Can 1, 0.033 Can 2, 0.033 Can 3, 0.033 Can 1, 0.28 Can 2, 0.28 25 26 27 28 29 30 31 32 37 38 39 40 1 2 3 4 11 11 5 6 17 18 19 20 21 22 23 24 48

TABLE V (Continued) Run No. RCB-4 (Concluded) Fo Can No. Days to Gas Formation Can 1, 0.05 13 Can 2, 0.04 14 Can 3, 0.04 15 16 Can 1, 0.12 41 Can 2, 0.12 42 43 Conclusion~ 1) Under these conditions, raw ground beef was sterilized by 2.000 megarep preirradiation plus an Fo less than 0.033. 2) An Fo of 0.20 was not quite enough to sterilize the noninoculated raw meat. 49

TABLE V (Continued) Run No, RCB-6-Can Size - No. 1 Picnic (211 x 400) Product - Raw Ground Beef Inoculum - 5,000,000 C. botulinum 215B spores Preirradiation - 0.600 megarep per can Processing Temperature - 230~F Incubation Temperature - 85~F Fo Can No. Days to Gas Formation Inoculated Not Can Can Can irradiated 1, 1.84 2, 1.75 3, 1.75 1 2 3 4 Inoculated Not Can Can Can irradiated 1, 0.99 2, 0.99 3, 0.99 5 6 7 8 Noninoculated Nonirradiated Can 1, 0.23 Can 2, 0.23 Can 3, 0.26 Inoculated Irradiated only 0.600 megarep Can 1, 0.59 Can 2. 0.59 Can 3, 0.59 Can 1, 0.21 Can 2, 0.21 Can 3, o.18 Can 1, 1.62 Can 2, 1.60 Can 3, 1.60 NI-5 NI-6 NI-7 NI -8 8 62 3 3 24 28 9 10 13 14 21 22 26 27 4 4 4 5 11 12 16 17 50

TABLE V (Continued) Run No. RCB-6 (Concluded) Fo Can No. Days to Gas Formation Can 1, 0.94 25 Can 2, 0.94 29 Can 3, 0.94 30 34 Can 1, 0.38 15 Can 2, 0.38 18 Can 5, 0.37 19 20 7 Conclusion: Under these conditions, raw ground beef was sterilized by 0.600 megarep preirradiation plus an Fo between 0.57 and 0.59. 51

TABLE V (Continued) Run No. RCB-7-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Raw Ground Beef - 5,000,000 C.botulinum 215B spores - 1.000 megarep per can - 250~F - 85~F Fo Can No. Days to Gas Formation Can 1, 0.33 Can 2, 0.55 Can 5, 0.55 Can 1, 0.10 Can 2, 0.10 Can 3, 0.10 Can 1, 0.69 Can 2, 0.66 Can 3, 0.66 13 14 15 16 9 10 11 12 7 8 6 17 18 19 20 Can 1, Can 2, Can 3, 0.16 0.13 0.13 21 22 27 28 Can 1, 1.25 Can 2, 1.25 Can 1, 0o14 Can 2, 0.14 Can 3, 0.14 Inoculated Nonirradiated Cani, 0o88 Can 2, 0.88 Inoculated Nonirradiated Can 1, 0.213 Can 2, 0.213 29 30 35 36 5 6 7 8 8 6 6 37 38 39 40 7 8 8 8 1 2 3 4 7 7 7 52

TABLE V (Continued) Run No. RCB-7 (Concluded) Fo Can No. Days to Gas Formation Inoculated 23 Nonirradiated 24 16 Can 1, 0.761 31 32 Noninoculated NI 7 Nonirradiated NI 47 Can 1, 0.17 NI Can 2, 0O17 NI Can 3, 0.17 Conclusion~ Under these conditions, raw ground beef was sterilized by 1.000 megarep followed by an Fo between 0.14 and 0.33. 53

TABLE V (Continued) Run No. RCB-8-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Raw Ground Beef - 5,000,000 C. botulinum 213B spores - 1.700 megarep per can - 230~F - 85~F Fo Can No. Days to Gas Formation Noninoculated Nonirradiated Can 1, 0.21 Can 2, 0.21 Can 3, 0.19 Inoculated Nonirradiated Can 1, 1.20 Can 2, 1.20 Can 3, 1.20 NI NI NI NI 25 26 33 34 Can 1, Can 2, Can 3, 0.02 0.02 0.02 9 10 14 17 7 7 7 7 Can 1, 0.03 Can 2, 0.03 Can 1, 1.54 Can 2, 1.54 Can 3, 1.54 Can 1, 0.025 Can 2, 0.025 Can 3, 0.025 13 15 16 18 6 7 6 45 46 47 48 41 42 43 44 Conclusion: Under these conditions, raw ground beef was sterilized by 1.700 megarep followed with an Fo between 0.03 and 1.5. Note: Cans 41 through 48 were run with a different batch of meat and 3 weeks later. 54

TABLE V (Continued) Run No. RCB-9-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Raw Ground Beef - 5,000,000 C. botulinum 215B spores - 0.700 megarep per can - 230~F - 85~F Fo Can No. Days to Gas Formation Can 1, 1.09 1 Can 2, 1.09 2 Can 3, 1.09 3 4 Can 1, 0.31 11 10 Can 2, 0.31 12 Can 3, 0.31 21 15 22 Can 1, 0.43 24 Can 2, 0.43 26 Can 3, 0.39 27 28 8 Can 1, 0.15 23 5 Can 2, 0.15 25 5 19 5 20 5 Conclusion: Under these conditions, raw ground beef was sterilized by 0.700 megarep followed by an Fo between 0.39 and 1.1 55

TABLE V (Continued) Run No. RCB-10-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Raw Ground Beef - 5,000,000 C. botulinum 213B spores - None per can - 230~F - 85~F Fo Can No. Days to Gas Formation Noninoculated NI Nonirradiated NI Can 1, 0.12 NI Can 2, 0.12 NI Can 3, 0.12 Can 1, 0.53 1 7 Can 2, 0.53 2 7 Can 3, 0.55 3 7 4 7 Can 1, 1.02 5 Can 2, 1.02 6 Can 3, 1.09 7 12 8 Can 1, 0.79 9 21 Can 2, 0.72 10 12 Can 3, 0.75 11 12 12 12 Can 1, 1.12 13 Can 2, 1.12 14 Can 3, 1.12 15 16 Conclusion: Under this condition, raw ground beef was sterilized by an Fo between 1.0 and 1.1. 56

TABLE V (Concluded) Run No. RCB-11 —Can Size - No. Product - Raw ( Inoculum - 5,00( Preirradiation - as ii Processing Temperature - Incubation Temperature - 85~F 1 Picnic (211 x 400) Ground Beef 0,000 C. botulinum 2: ndicated 13B spores per can Megarep Can No. Days to Gas Formation 5.000 1 2 5 4 5.500 5 7 8 2.250 9 4 10 4 11 4 12 4 2.750 15 14 4 15 16 Conclusion~ Under these conditions, raw ground beef was sterilized by between 2.75 and 3.000 megarep of gamma radiation. 57

TABLE VI - Summary of Various Combined Irradiation-Heat Processing Treatments Required to Sterilize Raw Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with 5,000,000 Co botulinum 213B Spores per Can. Run No _Preirradiation, megarep Fo Range, minutes RCB-1 none > 0.74 RCB-2 none o0.93-15 RCB-3 1o400 0o13-0o28 RCB-4 2,000 <.033 RCB-6 0.600 0.37-0,59 RCB-7 1.000 0o14-0o33 RCB-8 lo 700 0o03-1.5 RCB-9 0700 0,39-1.1 RCB-10 none 1.0-1.1 RCB-11 2.750-53 000 none 58

2 0. 0. 0. 0. O U) Ld I-:D z 2 Li 0..0 5,000,000 SPORES C BOTULINUM 213B RAW BEEF.0 8.6 _ _ 2.I 6 - 5 5 - _- - 2_ IRRADIATION ONLY 2.75 - 3.00 0.0.0.0.0.0.0.0 5 1.0 2.0 MEGAREP 3.0 Fig. 5. Fo required to sterilize raw ground beef packed in No. 1 picnic tin cans, inoculated with 5,000,000 C. botulinum 213B spores per can, and irradiated with garmma rays from cobalt-60 before heat processing at 230~F. 59

TABLE VII. Fo Value Required to Sterilize Raw Ground Beef in No. 1 Picnic Tin Cans, Previously Inoculated with Approximately 300 PA-3679 Spores per Can and Then Processed at 230~F. Run No. RPA-1-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Raw Ground Beef - 300 PA-3679 spores - None - 230~F - 85~F Fo Can No. Days to Gas Formation Can 1, 5o25 1 33 Can 2, 5o25 2 9 Can 3, 5.25 3 4 Can 1, 7~84 5 Can 2, 7~84 6 Can 3, 7~84 7 8 Conclusion: Under these conditions, raw ground beef was sterilized by an Fo between 5,3 and 7,8, 60

TABLE VII (Continued) Run No. RPA-3-Can Size Product Inoculum Preirradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Raw Ground Beef - 300 PA-3679 spores per can - 0.400 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation Not Can Can Can Not Can Can Can Can Can Can irradiated 1, 4o35 2, 4.35 3, 4355 irradiated 1, 6.63 2, 6.63 3, 6.63 1 2 3 5 4 6 5 4 5 6 7 8 9 10 11 12 1, 35, 6.54 6054 6.54 Can 1, 4.45 Can 2, 4.45 Can 3, 4.45 Can 1, 3506 Can 2, 3506 Can 3, 3.06 13 14 15 16 17 18 19 20 7 Can 1, Can 2, Can 35 2o10 2o10. 2 10 21 22 23 24 5 4 5 5 Conclusion: Under these conditions, raw ground beef was sterilized by 0o400 megarep followed by an Fo between 3.1 and 4.5; without irradiation, an Fo between 4~4 and 606 was required.

TABLE VII (Continued) Run No. RPA-4 —Can Size - No. 1 Picnic (211 x 400) Product - Raw Ground Beef Inoculum - 300 PA-3679 spores per can Irradiation - As indicated Processing Temperature - Incubation Temperature - 85~F Megarep Can No. Days to Gas Formation 2.000 1 4 2 3 4 2.300 5 6 7 8 1.400 9 discarded 10 discarded 11 discarded 12 discarded 1o4o0 13 discarded 14 discarded 15 discarded 16 discarded Discussion: This was an unusual run for a number of reasons, viz.: Meat was ground on June 11 and received that day. Due to changes in persoaer.e at this time, it was placed in the refrigerator and not packed until the morning of June 135. On June 14, the first 8 cans were irradiated while the second 8 were kept in the refrigerator. Two r.efrigerators were usedo'both kept the meat at 40 to 50~Fo During irradiation, the cans were cold, being packed with dry ice, which, however, evaporated before termination of irradiation. Cans 1, 2, 3, and 5 were slightly "soft" when removed from the centerwell of the irradiation source on the morning of June 14. However, cans 9 through 16 were soft at this time, which was the time when they were placed in the center well. By the morning of June 15, cans 9 through 16 were slightly swollen and when warmed to incubator temperature, they were soft springers. These phenomena have been noted in lesser degree before with raw meat. These results were interpreted to indicate growth of psychrophile bacteria,before irradiation,with gas production. By packing the meat as 62

TABLE VII (Continued) Run No. RPA-4 (Concluded) soon as it was ground, and then storing the 8 cans awaiting irradiation at 370F, this problem has been completely circumvented for runs RPA-5 through RPA-7o This is important when considering possible commercial use of the combined irradiation procedure, and will be considered in the report under "Discussiono. Conclusione Under these conditions, between 2.000 and 2.300 megarep were required to sterilize the raw ground beef. 63

TABLE VII (Continued) Run No. RPA-5-Can Size Product Inoculum Irradiation Processing Temperature Incubation Temperature - No. Picnic (211 x 400) - Raw Ground Beef - 500 PA-3679 spores per can - 0.800 megarep - 250~F - 85~F Fo Can No. Days to Gas Formation Noninoculated 1 4 Nonirradiated 2 5 Can 1, 0.24 3 4 Can 2, 0.24 Can 1, 1.47 4 6 Can 2, 1.47 5 5 Can 5, 1.47 6 4 7 Can 1, 2.14 8 4 Can 2, 2.14 9 4 10 4 11 Can 1, 2.86 12 11 Can 2, 2.86 15 Can 3, 2.86 14 15 Can 1, 4.13 16 Can 2, 4.13 17 18 19 Conclusion~ Under these conditions, raw ground beef was sterilized by 0.800 megarep followed with an Fo between 2.9 and 4.1.

TABLE VII (Continued) Run No. RPA-6-Can Size - No. 1 Picnic (211 x 400) Product - Raw Ground Beef Inoculum - 300 PA-3679 spores per can Irradiation - 1.200 megarep Processing Temperature - 230~F Incubation Temperature - 85~F Fo Can No. Days to Gas Formation Noninoculated 1 Nonirradiated 2 Can 1, 0.38 3 3 Can 2, 0.38 4 4 Can 3, 0.38 Can 1, 0.54 5 6 Can 2, 0.54 6 3 7 4 8 Can 1, 0.86 9 4 Can 2, 0.86 10 11 12 5 Can 1, 1.12 13 5 Can 2, 1.12 14 5 Can 3, 1.12 15 16 6 Can 1, 1.66 17 Can 2, 1.66 18 Can 5, 1.66 19 20 Conclusion: Under these conditions, raw ground beef was sterilized by 1.200 megarep followed by an Fo between 1.1 and 1.7. 65

TABLE VII (Concluded) Run No. RPA-7-Can Size Product Inoculum Irradiation Processing Temperature Incubation Temperature - No. 1 Picnic (211 x 400) - Raw Ground Beef - 300 PA-3679 spores per can - 1.500 megarep - 230~F - 85~F Fo Can No. Days to Gas Formation Noninoculated 1 Nonirradiated 2 3 Can 1, 0.33 3 4 Can 2, 0.33 4 4 Can 3, 0.33 Can 1, 0.71 5 Can 2, 0.71 6 Can 3, 0.71 7 8 Can 1, 0.42 9 4 Can 2, 0.42 10 4 Can 3, 0.42 11 12 7 Can 1, 0.23 13 Can 2, 0.23 14 Can 3,0.23 15 16 Can 1, 0.13 17 Can 2, 0.13 18 4 Can 3, 0.13 19 5 20 Conclusion: Under these conditions, raw ground beef was sterilized by 1.500 megarep followed with an Fo between 0.42 and 0.71. 66

TABLE VIII - Summary of Various Combined Irradiation —Heat Processing Treatments Required to Sterilize Raw Ground Beef in No. 1 Picnic Tin Cans Previously Inoculated with 300 PA 3679 Spores per Can. Run No. Preirradiation, megarep Fo Range, minutes RPA-1 none 5.3-7.8 RPA-3 0.400 3.1-4,5 RPA-3 none 4.4-6.6 RPA-4 2.000-2.500 none RPA-5 0.800 2.9-4.1 RPA-6 1.200 1.1-1.7 RPA-7 1.500 0.42-0.71 67

10. ra 6.( 5.( 4. 0_ 300 SPORES PA 3679 RAW BEEF 0: -- I 3.i w \ O n C,) en 0 IL o 1.1_ 1.0 0.8 0.6 _ r)-..==M~i= 0.4 0.3 I 0.2 0.1 ~ I \ IRRADIATION ONLY 2.00 - 2.30 ____ I___I. ) 1.0 2.0 MEGAREP 3.0 Fig. 6. Fo required to sterilize raw ground beef packed in No. 1 picnic tin cans, inoculated with 300 PA 3679 spores per can and irradiated with gamma rays from cobalt-60 before heat processing at 230~Fo 68

techniques to be used for interpretation of the results. 53 Experience was already at hand for dealing with irradiations of cooked, canned ground beefo.It was realized, however, that commercial adaptation of any process developed would likely involve preirradiation of raw meat. Since it had already been established3 that chemicals could alter the lethal character of ionizing radiations, it was desirable to learn whether the substances present in raw meat could also act in this manner. The results indicated that, for raw beef at least, there is no difference between combined irradiations —heat processing schedules developed for precooked and for raw meat. It was necessary to develop techniques for the heat-processing phase of this work that would produce consistent results. The method described under "Materials and Methods" provided reasonable certainty in the reported results, but ultimately any process used commercially should be tested with a larger number -of canso Data presented in the 1956 annual report show that irradiation sensitization of spores to heat occurred only if temperatures above 85~C were used. For this reason it was possible to preheat the cans to 180~F in the autoclave before heat processing without affecting the results. The preheating permitted the development of much more uniform Fo values among the cans being processed than when they were processed with lower starting temperatures. It will be noted from the results with C. botulinum 213B spores that the difference between 5,000,000 and 300 spores per can did not appreciably change the combined processing treatment required. On the other hand, where either of these treatments was used alone, spore concentration was an extremely significant factor.15 Actually more difference was observed between 10,000 and 300 PA 3679 spores than between 5,000,000 and 300 C. botulinum 213B spores in this regard. Some interesting considerations are developing from this project. Sterilization of foods with ionizing radiations alone will likely require very high dosages because C. botulinum spores are very resistant to radiationo This contrasts with their relatively low heat resistance. Combined irradiation-heat processing of canned foods restores the desirable processing resistance-relationship between C. botulinum and PA 3679 spores, viz.: the latter are more resistant to combined processing than the former.Therefore, when a combined processing treatment is designed to sterilize canned meat containing PA 3679 spores, it will provide an inherent safety factor insofar as Co botulinum spores are concerned. A question developed concerning the finding of botulinus toxin in raw meat. Part of the routine testing involves establishment of the presence 69

of botulinus toxin in cans of meat that develop gas upon incubation following insufficient processing when C. botulinum spores are being used for the inoculum.. We have observed that botulinus toxin is often difficult to demonstrate in raw meat under these conditions. A few preliminary tests were therefore made to determine whether or not botulinus toxin developed in canned raw meat that had been previously sterilized with gamma radiation and then inoculated with C. botulinum spores. Similar results were obtained. Some of these cans of meat contained no demonstrable toxin, others contained a small amount as determined by mouse inoculation tests, Parallel, control cans of cooked meat developed large amounts of botulinus toxin. It is possible that botulinus toxin does not develop in raw meat, but more likely that it forms and then disappears. This observation should be investigated in some detail as a separate project since, if the observation is substantiated, it has considerable theoretical and practical significance. It is of interest in this connection that we cannot find any published instance of botulism poisoning resulting from raw meat, this in spite of the probable opportunity for C. botulinum organizms to grow in improperly refrigerated raw meat. Besides the previously observed24 synergistic action of gamma radiation and heat for killing anaerobic bacterial spores, other considerations indicate the desirability of a combined irradiation.-heat processing treatment for sterilizing canned foods. For example, the dosage of 3.0 megarep that is often suggested for sterilizing foods will not completely inactivate enzymes, pathogenic viruses, certain micrococci, or botulinus toxin. On the other hand, viruses, micrococci, botulinus toxin, and all but a few enzymes are quickly destroyed by moist heat at 212~F. Sinc;e all these should be inactive in canned foods, a combined irradiation-heat processing treatment that is designed to kill PA 3679 spores should produce safe canned foods because any such process would involve heating the food to temperatures of 230~F or above; and, as has been'shown in this report, PA 5679 spores are more resistant to combined irradiation-heat processing than C. botulinuw spores. 70

PHASE II COMPARISON OF Z VALUE OF IRRADIATED AND NONIRRADIATED PA 35679 SPORES In order that Fo calculations can be intelligently carried out, it is necessary that the Z value of the spores of test bacteria be known. There are many techniques for carrying out Z-value determinations. In general they are of two varieties, end-point analysis and decimal reduction determinations. We have previously reportedl preliminary Z-value determinations based on end-point analysis. This time we used the survivorcurve technique, using the thermoresistometer at the American Can Co. Research Laboratories in Barrington, Illinois. The PA 3679 spores used for this study were taken from the same stock supply as those used for the work discussed in Phase I of this report. A portion of these were irradiated with 0.500 megarep in the large cobalt-60 gamma radiation source at The University of Michigan. They were suspended in distilled water during storage and irradiation, and were refrigerated at all timeso The spore suspensions were pipetted into each of three cups of the thermoresistometer in 0,01 ml portions. These were thermally processed at the temperatures indicated, and then dropped into 0o99-ml of distilled water. The three l.OO-ml samples were then pooled and titrated as previously described. The titrations and calculated surviror data are shown in Table IXo The times required to kill 99% of the spores at the temperatures indicated are listed in Table X, The Z-value curves are plotted in Fig. 7. It will be noted that the Z-value lines for irradiated and for nonirradiated PA 3679 spores have the same slope; this slope corresponds to a Z value of 22.3. The previous work using end-point analysis6 developed a Z value of 23 for irradiated, and 21.3 for nonirradiated PA 3679 spores suspended in M/15 phosphate buffer at pH 7.0o The data for this phase of the work are based on one series of experiments, however, so the results must still be considered preliminaryo Nevertheless, two experiments using different techniques indicate no significant difference between the Z values of irradiated and nonirradiated PA 3679 spores. ACKNOWLEDGMENT We wish to express our sincere appreciation to the American Can Co. for permission to use their thermoresistometer for this work. In particular, we wish to thank Dr. E. Wheaton for his advice and assistance. 71

TABLE IX - Effect of Processing at Various Temperatures on Suspended in Distilled Water Using the Thermnoresistometer PA 3679 Spores Time Number of Soores % SlIrvi vnrs LoT.no Survivors 110C - Nonirrad.ated llO~ C - Nonirradiated I 0 10 50 50 59,000 550 7 o 100 0.93 0.o1185 _ _. _om. 2.000 -0. 031 -1.926 ___ 110~C - Irradiated 0 10 30 50 69,000 20 100 0.029 _W"S 2:. 000 -1o537 _._"M 115~C - Nonirradiated 0 265 5oO 500 10.0 1500 59,000 500 192 8 4 100 0.847 0.326 0.01358 0.00678 2.000 -0,072 -0.486 -1.867 -2. 168 115~C - Irradiated 0 2.5 5.0 10 o.O 10.0 15.0 69,000 64 4 100 0.0926 o0 o58 2.000 -1.033 -2.2537 117.5~C - Nonirradiated 0 350 5.0 10.0 59,000 79 22 100 0.134 0.0373 2.000 -0.873 -1.427 _.,, 117.5~C - Irradiated 0 5.0 5.0 69,0oo 2 100 0.0029 2.000 -2.538 0m__ 72

TABLE IX (Concluded) Time. Number of Spores % Survivors Log C% Survivors 120~C - Nonirradiated 0 1.0 2.0 4.0 59,000 105 25 100 00178 0.0425 2.000 -0.749 -1.370;. ". 120~C - Irradiated 0 10o 2.0 69,000 12 __.W 100 0o0174 2.000 -1.785 _ -._w 130~C - Nonirradiated 0 0.2 0.4 59,000 61 100 0.103 -2.000 -0.986 A_ _ 130~C - Irradiated 0 0-.2 0.4 69,000 4 100 0.0057 ___. 2.000 — 2.244 _... 73

TABLE X - Time Required to Kill 99% of the Nonirradiated and Irradiated PA 3679 Spores at the Temperature Indicated Tempe rat......... oTime to Kill 99% Temperature, C Nonirradiated I Irradiated (0.500 megarep) 110 9 5.6 115 2.7 1.8 117.5 2.1 1.3 120 0.7 0.5 130 0.2 0.13 74

10 8.0 g 1, K I 0 IRRADIATED 0 NONIRRADIATED 6.0 5.0 4.0 3.0 2.0 ] UJ ID z w I 1.0 0.8 0.6 0.5 i ~\ \o \ NONIRRADIATED \ X z- z 22.3 \ \ _ IRRADIATED --- - \ Z =22.3\ I R\ \' ____i_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 0.4 0.3 0.2 0.1 110 120 130 TEMPERATURE —C Fig, 7 Thermal death time PA 3679 spores suspended in curves for nonirradiated and irradiated distilled water, 75

PEASE III THE EFFECT OF.CATALASE ON THE LETHALITY OF Co-60 GAMMA RADIATION FOR CERTAIN ANAEROBIC BACTERIAL SPORES SUMMARY Catalase present in the suspending medium during irradiation significantly reduced the lethal action of gamma radiation from cobalt-60 for anaerobic bacterial sporeso This finding supports the theory that the lethal action of ionizing radiations is at least partially due to secondary effects of the irradiation~ Furthermore, such protection of anaerobic bacterial spores is important when sterilization of foods containing catalase is considered since it will likely increase the dosage required, Two explanations are offered for the lethal action of ionizing radiations on living cells. The first postulates a direct action of the radiations on genetic material, the other assumes that an initial change takes place in the medium or at some nongenetic locus and that this change brings about lethal effects in the cell. The work of Hollander et al. (1951)7 and Burnett et alo (1951)8 support the latter theory. Similarly, the work presented in this paper further supports the indirect action theory by describing a protective effect of catalase for anaerobic bacterial spores when catalase is present in the suspending medium during irradiation. Obviously, such protection of anaerobic bacterial spores will probably increase the amount of ionizing radiations required to sterilize foods containing catalase. MATERIALS AND METHODS Spores used in this work were grown, harvested, and suspended in distilled water as described by Kempe et al. (1954).l Immediately prior to use in an experiment, the stock spore suspensions were shaken with glass beads for five minutes to disperse the spore clumpso The desired quantity of spores was next pipetted into a sterile test tube and heated at 85~C for 15 minutes to kill the vegetative cells. The spore suspension was then diluted into the final solutions to be irradiated. The control for these experiments was sterile phosphate buffer to which only the spores of either Clostridium botulinum 213B or Putrefactive anaerobe No. 3679 were added. Purified crystalline catalase for this work was obtained from General Biochemicals, Inc., Chagrin Falls, Ohio. For use in an experiment, 76

phosphate buffer (pH 7.02) was sterilized by autoclavingo Following this, 60 mgm of catalase was added to 14.5 ml of the cooled experimental solution and then 0.5 ml of a spore suspension was added to both the control and the catalase solutions. Four-ml quantities of these preparations were next aseptically pipetted into sterile 5-nl glass vials which were finally sealed in an oxygen flameo Irradiation was carried out in an ice water bath in the center well of the large cobalt-60 source in the Fission Products Laboratory at The University of Michigan. After completion of the irradiation, a sample from the irradiated or control (0-hours radiation) vial was withdrawn, diluted to the proper spore concentration, and counted using techniques previously described by Reed et al. (1951)o9 RESULTS AND DISCUSSION The survival of spores of Clostridium botulinum 213B after varying exposures to Co-60 gamma radiation either in a phosphate buffer solution or in a phosphate buffer solution containing catalase is shown in Table XI and in Fig. 80 In the control solution, only 55 spores per ml remained viable after 8 hours radiation from an original population of 15,000,000 per ml. On the other hand, in the solution containing catalase, 59,000 spores per ml were viable after 8 hours irradiation from an original population of 16,900,000 per ml. This represents a 1500-fold increase in survival caused by the addition of catalase. Similarly, the effect of catalase on the lethality of Co-60 gamma radiation for the spores of Putrefactive anaerobe No. 3679 is shown in Table XI and in Fig. 8. Here only 145 spores per ml were viable after 7 hours irradiation in the control solution as compared to 179,500 spores per ml after a similar dosage of gamma radiation in the solution containing catalase. This represents a 1250-fold increase in the survival ratio. Since many investigators, including Allen (1954),1 have reported that hydrogen peroxide is developed during irradiation of water by gamma rays, and since Curran and Evans (1940)11 reported that the sporicidal action of hydrogen peroxide could be dissipated by catalase, the inference is drawn that catalase protected the anaerobic spores tested in this work by destroying hydrogen peroxide that was produced in situ by the irradiation treatment. This is evidence for the indirect lethal action of radiation on anaerobic bacterial sporeso The magnitude of the observed effect suggests that the sporicidal action of gamma radiation may be largely indirect in natureo In any event, the protection of anaerobic bacterial spores against the lethal action of ionizing radiations by catalase must be considered when the sterilization of raw foods by such radiations is contemplated. 77

TABLE XI THE EFFECT OF CATALASE ON THE LETHALITY OF GAMMA RADIATION FROM COBALT-60 FOR ANAEROBIC BACTERIAL SPORES No. of Hours R, No of Organisnms Log -Radiation Survivirng per ml % Survivors a) C. botulinum 213B ~ _....J...u. Control Catalase 0 3 5 6 7 8 0 3 5 6 7 8 0 465,000 775,000 930,000 1,085, 000 1,240,000 0 465,000 775,000 930,000 1,085,000 1,240,000 15,000,000 3,500,000 136,000 3,330 380 *35 16,900,000 6,800,000 1,690,C00 710,000 233,000 59,000 2.000 1.5368 -0o043 -1.654 -2.596 -5.632 2.000 1.605 1.000 0.623 0.139 -0.456 b) PA 3679 Control Catalase 0 5 6 7 0 5 6 7 0 775,000 930,000 1,085,000 0 775,000 930,000 1,085,000 10,700,000 61,500 8,100 145 11,600 000 1,460,000 410,000 179,500 2. 000 -0..241 -1o121 -2.868 2.000 1.100,. 548 0.1895 *One rep unit is a dose of ionizing radiation capable of producing energy absorption of 93 ergs per gram of tissue. 78

2 \ -Control \A -Catolose o 0 —- \ 0 >) 0 -I -2 -3 -4 | ] 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2 MEGAREP Fig. 8. Effect of catalase on the lethality of gamma radiation from cobalt:-60 for anaerobic bacterial spores. 0 79

PHASE IV EFFECT OF PREIRRADIATION OF INOCULATED PACKS -OF CANNED PEAS ON THE FO SUBSEQUENTLY REQUIRED FOR STERILIZATION All the work under this contract on the combined irradiation —heat processing of canned foods has been based on ground beef. Canned vegetables also might be improved if reduced heat processing is required following irradiation. For convenience in testing,,it is desirable to use gas formation as a criterion of growth of anaerobic bacteria. Therefore, peas were selected for study from among the Several vegetables that are commercially preserved by canning. Frozen peas were used because fresh peas are only seasonably available Although this is a final report, the results from these studies cannot be available for some time-perhaps three months or more —because anaerobic bacterial spores appear to germinate slowly in the canned peas. Therefore, onLy the method presently in use will be described here. When incubation results are available they will be reported in progress reports of the next project. EXPERIMENTAL PROCEDURE Frozen peas are purchased from The University of Michigan Food Service. These are thawed by being placed in a refrigerator over night..They are then dumped into a stock pot, covered with a 1l8% salt, 2.2% sucrose brine, and heated to 200~Fo The preliminary heating serves to drive out gases and to pasteurize the peas. Following this, the peas and brine are packed into No. 1 picnic tin cans, and more brine is added if necessary. Some of the cans contain Ecklund thermocouples. Lids are then set on the cans and the cans are placed in an autoclave where they are kept in flowing steam at atmospheric pressure for a few minutes while individual cans are removed, inoculated, and closed. As soon as a can is closed, it is placed in cold, running tap water where it remains for about 20 minutes. Then the experimental cans are either irradiated, or placed in a refrigerator at 35~F. The controls are placed in an 85~F incubator. Irradiation is carried out in the center well of the large cobalt-60 gamma radiation source here at The University of Michigan. The cans are cooled with dry ice during this irradiation. Following irradiation, the cans are heat processed and incubated in the same manner as has been previously described for ground beef (see Phase I)o 8o

At present, an inoculum of 300 PA 3679 spores per can is being usedO With this inoculum, ground beef control cans usually develops hard swells overnight-when incubated at 85~F; the canned peas, under these contions require two or three dayso It can be expected that the experimental cans will take proportionately longer to develop sufficient gas to indicate positive anaerobic bacterial growth0 As previously stated, no quotable results are.available as this is written (middle of July, 1957), but the results will be reported in about three months or more depending upon the incubation time required.

REFERENCES 16 Kempe, Lo Lo, Graikoski, Jo To, and Gillies, Ro Ao, "Gamma Ray Sterilization of Canned Meat Previously vInoculated with Anaerobic Bacterial Spores," Applo Microbiolo, 2, 330-332 (1954). 2o Kempe, Lo Lo,."Combined Effect of Heat and Radiation in Food Sterilization," Appl. Microbiolo. 3, 346-3552 (1955). 3. Kempe, Lo Lo,.et alo Combined Use of Heat and Radiation Treatment for Sterilization of Foods, The University of Michigan Engineering Research Institute Report Noo 2391-8-F, Ann Arbor, Michigan, September, 1956. 4. Morgan, B. H., and Reed, J. Mo, "Resistance of Bacterial Spores to Gamma Radiation," Food Research, 19, 357-366 (1954). 5. Stumbo, C. Ro, "Thermobacteriology as Applied to Food Processing,." Advances in Food Research, Volo 2, Academic Press, Inc., N. Y,, 1949, p. 73 ff. 60 Kempe, L. L., et alo, Combined Use of Heat and Radiation Treatment for Sterilization of Foods, The University of Michigan Engineering Research Institute Report No. 2391-2-P, Ann Arbor, Michigan, October, 1955. 7o Hollaender, Ao, Stapleton, Go Eo, and Martin, F. Lo, "X-Ray Sensitivity of E. coli as Modified by Oxygen Tension," Nature, 167, 103-104 (1951). 80 Burnett, Wo To, et alo,. "Reduction of X-Ray Sensitivity of Escherichia colil B/r by Sulfhydryl Compounds, Alcohols, Glycols, and Sodium Hydrosulflte," Proco Soco Expo Biol. and Med., 77, 636-638 (1951). 9. Reed, J. Mo, Bohrer, Co Wo, and Cameron, Eo Jo, "Spore Destruction Rate Studies on Organisms of Significance in the Processing of Canned Foods," Food Research,.16, 385-408 (1951). 10 o Allen, Ao 0, "The Yields of Free H-and OH in the Irradiation of Waters" Radiation Research, 1, 85-96 (1954). 11o Curran, H. Ro, Evans, Fo R., and Leviton, Ao, "The Sporicidal Action of Hydrogen Peroxide and the Use of Crystalline Catalase to Dissipate Residual Peroxide,". Bact,.40, 423-434 (1940). Peroxide~ _ ------..__ 82