ENGINEERING RESEARCH INSTITUTE THE UNIVERSITY OF MICHIGAN ANN ARBOR Progress Report No. 4 COMBINED USE OF HEAT AND RADIATION TREATMENT FOR STERILIZATION OF FOODS Period: 7 December 1955 — 7 February 1956 Lloyd L. Kempe Official Investigator J. T. Graikoski Nancy J. Williams Peter 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 February 1956

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. T. Graikoski Nancy J. Williams Peter F. Bonventre Title of Contract: Combined I Treatment Pr.oject No. 7-84-01-002 Contract DA-19-129-qm-388 File No. S-510 Report No. 4 (Progress) Period 7 December 1955 to 7 February 1956 Initiation Date: 7 June 1955 Jse of Heat and Radiation for Sterilization of Foods The first series of tests to determine the value of the combined use of heat and irradiation treatment for the sterilization of canned meat has been completed. Using an inoculum of approximately 5,000O000 C. botulinum 215B spores per No. 1 picnic can of meat, it was found: 1) Approximately 1,000,000-rep preirradiation is required before a significant effect of irradiation is noted on the Fo value required for subsequent heat sterilization of the canned meat. THIS IS NOT A FINAL REPORT. CONCLUSIONS STATED ARE SUBJECT TO CIHAGE ON THE BASIS OF ADDITIONAL EVIDENCE. THIS INFORMATION IS NOT TO BE PUBLISHED WITHOUT WRITTEN PERMISSION FROM HQ, QM R AIND D COMMA-ND, NATICK, MASS. 1

2) Following irradiation with from 1,200,000 to 1,500,000 rep of gamma radiation, the Fo for sterilizing canned meat under the above conditions is lowered from a value of 1.0 to less than 0.25. Work on the effects of chemicals and temperatures during irradiation on the lethality of gamma radiation for anaerobic bacterial sporeS has continued but has been largely confined to confirming data already reported. Thus it has been shown that glutathione and sodium hydrosulfite protect PA 3679 spores in the same manner as they do C. botulinum spores. And the pH of the medium, in the range of 3.2 to 8.4, does not influence the lethality of gamma radiation for C. botulinum spores. EVALUATION OF CONSECUTIVE IRRADIATION AND HEAT TREATMENT FOR STERILIZING CANNED MEAT The methods and equipment for this study were presented in Progress Report No. 3. Briefly they involve preparation of inoculated packs of lean ground beef in No. 1 picnic tin cans which are closed while hot in order to develop vacua. The cans are then irradiated in the large cobalt60 source at The University of Michigan while held at a temperature of about 4~C. They are next processed in a steam autoclave and finally incubated at 37~C. Sufficient Fo values are used to permit bracketing that Fo value which is required just to sterilize all four cans. Sterility is routinely determined by observation for swelling of the cans during incubation, In Fig. 1 and Table I it is shown that an Fo of 1.0 developed at the coldest spot in the can during processing was required to sterilize ground beef that had been inoculated with approximately 5,000,000 C. botulinum 213B spores per can. This value was reduced to less than 0.25 following irradiation with 1,200,000 rep. However, little reduction of the Fo was noticed until 1,000,000 rep were used. At present, runs are being made with 250 rather than 5,000,000 C. botulinum 213B spores per can, 2

1.0 0.9 0.8 0.7 0.6 0.5 0.4 t I a _ _,-* 7 IT I 0.3 0.2 C.) Z I L_ 0.1.09.08.07.06.05'1 I _______________ I'.,.,,, l X~~~~~~.04.03.02.0 1 0 1.0 2.0 MEGA REP 3.0 Fig. 1. Fo required to sterilize 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. 3

TABLE I - Fo Required to Sterilize Ground Beef Cans, Inoculated with Approximately 5 x 106 C. Then Irradiated with Gamma Rays from Cobalt-5O Packed in No. 1 Picnic Tin botulinum 213B Spores and Before Heat Processing. Run CB-8: Can Size Product Inoculum Preirradiation - No. 1 Picnic - Ground Beef - 5,000,000 C. - 500,000 rep (211 x 400) botulinum 213B Days to Gas Fo Can No. Gas Formation Formation..~~~~~~~~~~~~~omto Uninoculated Control Inoculated Control 1 2 3 4 + 2 1 2 3 4 + + + + 2 2 2 2 Fo Can Can 1, 1.40 2, o.85 Can 1, 0.745 Can 2, 0.639 Can 1, 1.05 Can 2, 0.73 Can 1, 0.881 Can 2, 0.540 Nonirradiated Controls Can 1, 0.86 Can 1, 1.18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Conclusion: None 4

TABLE I (Continued) Run CB-9: Can Size Product Inoculum Preirradiation - No. 1 Picnic - Ground Beef - 5,000,000 C. botulinum 213B - 1,500,000 rep spores per can............... Days to Gas Fo Can No. Gas Formation Formation,....,.,,,,...,. Uninoculated Controls Inoculated Controls Can 1, 0.029 Can 1, 0.10 Can 2, 0.11 Can 1, 0.013 Can 2, 0.019 Can 1, 0.062 Can 2, 0.081 1 2 5 1 2 5 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 + + + + 2 2 2 2 + + + + 2 2 2 2 + + + 3 5 3 2 2 3 5 + + + + + 5 + 4 Conclusion: After 1,500,000 Fo of 0.11. rep, sterility dosage is more than an 5

TABLE I (Continued) Run CB-10: Can Size Product Inoculum Preirradiation - No. 1 Picnic - Ground Beef - 6,000,000 C. - 500,000 rep botulinum 213B spores per can Days to Gas Fo Can No. Gas Formation Formation Formation Uninoculated Controls Inoculated Controls Can 1, 0.43 Can 2, 0.26 Can 1, 0.56 Can 2, 0.51 Can, 1.06 Can 1, 0.84 Can 2, 0.77 Nonirradiated Controls 0.81 0.68 0.98 1 2 3 4 17 18 19 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 + 11 + + + + + + + 4+ + + + 2.5 2.5 2.5 2.5 5 6 5 6 6 6 6 9 + - + 20 21 22 25 24 25 26 27 + + + + 7 7 7 6 7 + 8 Conclusion: With 500,000 lies between rep preirradiation the sterility dosage Fo values of 0.77 and 1.06. 6

TABLE I (Continued) Run CB-11: Can Size Product Inoculum Preirradiation - No. 1 Picnic - Ground Beef - 12,000,000 C. botulinum 213B - 1,500,000 rep spores per can Fo Can No. Gas Formation Da to Gas'___ ^__.. ____________Formation Noninoculated Controls 1 2 3 4 + 5* Inoculated Controls Can 1, 0.329 Can 2, 0.206 Can 1, 0.125 Can 2, 0.072 Can 1, 0.047 Can 2, 0.033 Can 1, 0.147 Can 2, 0.087 17 18 19 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 + + + + 2 2 2 2 + 5 -- 3 3 + + + + 3 3 5 5 5 5 + + + 3 6 4 *Toxin present by mouse inoculation test. Conclusion: With preirradiation of 1,500,000 dosage appears to lie between Fo and 0.329. rep the sterility values of 0.087 7

TABLE I (Concluded) Run CB-12: Can Size Product Inoculation Preirradiation - No. 1 Picnic - Ground Beef - 5,000,000 C. - 1,500,000 re] botulinum 213B P spores per can Days to Gas Fo Can No. Gas Formation Formation Noninoculated Controls Inoculated Controls Can 1, 0.15 Can 2, 0.04 Can 1, 0.27 Can 2, 0.26 Can 1, 0.063 Can 1, 0.39 Can 2, 0.25 1 2 3 4 17 18 19 20 1 2 3 4 + + + + 2 2 2 2.+ + + 5 3 3 5 5 5 5 6 7 8 9 10 11 12 + + + + 2 3 2 5 15 14 15 16 Conclusion: With 1,500,000 rep preirradiation the sterility value lies between Fo values of 0.063 and 0.27. 8

SUMMARY OF TABLE I Run No. CB -3 CB-4 CB-5 CB-6 CB-7 CB-8 CB-9 CB -10 CB-11 CB-12 C-1 C-2 C-3 Number of C. botulinum Spores per Can x 10 2.30 5.20 6.30 6.00 3580 5.00 5.00 6.oo 6.00 12.00 5.00oo 10.70 7.20 10.70 Megarep Fo Required to Sterilize Preirradiation After Irradiation,,..,... 1.350 1.350 0.675 1.000 1.200 0.500 1.500 0.500 1.500 1.500 0 0 0 O O less than 0.23 between 0.11 and 0.21 more than 0.58 between 0.41 and 0.80 between 0.09 and 0.29 more than 0.11 between 0.77 and 1.06 between 0.087 and 0.33 between 0.063 and 0.27 more than but close to 1.0 between 0.75 and 1.30 between 0.36 and 0.93 9

EFFECT OF CHEMICALS IN THE SUSPENDING MEDIUM ON THE LETHALITY OF GAMMA RADIATION FOR ANAEROBIC BACTERIAL SPORES Data presented in Table II and Fig. 2 show that glutathione and sodium hydrosulfite protect PA 3679 spores against the lethal action of gamma radiation to essentially the same degree as was shown for C. botulinum 213B spores in Progress Report No. 2. The effect of pH of the suspending medium during irradiation on the lethality of gamma radiation for C. botulinum 213B spores was also studied over the range of 3.23 to 8.4. O.1N acetate buffer was used in the pH range of 3523 to 5.0, while an M/15 phosphate buffer was used from 6.1 to 8.4. No significant difference was noted in the percent of C. botulinum 213B spores surviving irradiation at any pH in the range of 3.23 to 8.4. This is shown in Table III. Studies have been continued during the past two months to observe the effects of temperature of the suspending medium and the effect of chemicals added to the suspending medium on the lethality of gamma radiation for anaerobic bacterial spores. The work has been confirmatory in nature and so far substantiates the data and results reported in Progress Report No. 2 dated 7 August to 7 October 1955. It is expected that these data presently at hand and data from experiments planned during the next two months will permit publication of this work. EFFECT OF TEMPERATURES OF THE SUSPENDING MEDIUM DURING IRRADIATION ON THE LETHALITY OF GAMMA RADIATION FOR ANAEROBIC BACTERIAL SPORES Table IV and Fig. 3 show that C. botulinum 213B spores, suspended in M/15 phosphate buffer at pH 7.0, are killed somewhat more rapidly at 4~C than at -70~C; similar results for C. parabotulinum 457 and for PA 3679 spores are shown in Tables V and VI, respectively. This confirms similar data previously presented for these and other anaerobic bacterial spores. It has been previously reported in Progress Report No. 2 that irradiation-sensitized Co. botulinum 213B spores suspended in M/15 phosphate buffer at pH 7.10 must be heated to a minimum critical temperature of 80~C before killing occurs. However, once this temperature is reached, the preirradiated spores are killed much more rapidly than are the nonirradiated spores held at the same temperatures. This work has now been extended to PA 3679 spores. 10

TABLE II Effect of Used as a Protective Chemicals Added to the M/15 Phosphate Buffer Suspending Medium for PA 3679 Spores During Irradiation with Gamma Rays from Cobalt-60 Rep Spores per ml- Percent Survivors Log Percent Survivors...~~~~~~~~.'.... a) Control - M/15 Phosphate Buffer at pH 7.0 0 360,000 720,000 900,000 1,080,000 1,260,000 1,440,000 3,130,000 1,120,000 16,200 1,210 129.5 1.5 0.5 100 55.78 0.5176 0.0587 0.00414 0.0000479 0.000016 2.000 1.554 -0.286 -1.412 -2.383 -4.320 -4.796 b) 0.02 M Glutathione Solution in M/15 Phosphate Buffer at pH 7.0 0 360,000 720,000 900,000 1,080,000 1,260,000 1,440,000 3,600,000 1,925,000 720,000 260,000 128,500 36,500 6,250 100 53.47 20.00 7.22 5.57 1.014 0.1756 2.000 1.728 1.301 o.859 0.555 0.0o6 -0.760 c) 0.02 M Sodium Hydrosulfite Solution in M/15 Phosphate Buffer at pH 7.0 0 360,000 720,000 900,000 1,080,000 1,260,000 1,440,000 3,570,000 2,700,000 1,700,000 750,000 440,000 195,000 88,000 100 75.63 47.62 21.01 12.55 5.462 2.465 2.000 1.879 1.678 1.322 1.091 0.737 0.592 11

2.00 1.00 l -I 0 03 I) 0 -2.00 (,9 o 0 Control -l \0 A Glutathione 0 Sodium Hydrosulfite - 3.00 Control- 4.00 _5.00.2.4.6.8 1.0 1.2 1.4 1.6 Fig. 2. Effect of protective chemicals at 0.02 M concentration when present in the M/15 phosphate buffer (pH 7.0) used as a suspending medium for PA 3679 spores during irradiation with gamma rays from cobalt-60. 12

TABLE III Effect of the pH of the Medium in Suspended During Irradiation from Cobalt-60 Which C. botulinum 213B Spores Were on the Lethality of Gamma Rays for These Spores Rep Spores per ml Percent Survivors Log Percent Survivors pH 3.4 0 10,900,000 100 2.000 360,000 8,400,000 77.06 1.887 720,000 1,910,000 17.52 1.244 900,000 725,000 6.651 0.823 1,080,000 195,500 1.794 0.254 pH 6.75 0 13,000,000 100 2.000 360,000 8,650,000 66.54 1.823 720,000 5,300,000 40.77 1.610 900,000 2,050,000 15.77 1.198 1,080,000 945,000 7.27 0.862 pH 7-01 0 10,800,000 100 2.000 360,000 8,200,000 75.93 1.880 720,000 3,065,000 28.38 1.453 900,000 1,940,000 17.96 1.254 1,080,000 665,000 6.157 0.789 pH 8.40 0 360,000 540,000 720,000 900,000 pH 7.17 0 560,000 540,000 720,000 900,000 pH 6.10 0 360,000 540,000 720,000 900,000 6,000,000 2,230,000 330,000 42,000 3,100 5,800,000 2,155,000 240,000 69,500 5,650 4,870,000 1,910,000 255,000 56,000 3,740 100 37.17 5.50 0.70 0.0517 100 37.16 4.158 1.200 0.0974 100 39.52 5.236 1.150 0.0768 2.000 1.570 0.740 -0.155 -1.287 2.000 1.570 0.617 0.079 -1.011 2.000 1.594 0.719 0.061 -1.115 15

TABLE III (Concluded) Rep Spores per ml Percent Survivors Log Percent Survivors pH 5.00 0 4,730,000 100 2.000 360,000 1,180,000 24.95 1.397 540,000 174,000 3.679 0.566 720,000 20,400 0.4313 -0.365 900,000 1,625 0.0344 -1.463 pH 4.00 0 3,900,000 100 2.000 360,000 845,000 21.67 1.336 540,000 139,500 35577 0.554 720,000 8,650 0.2218 -0.654 900,000 176 0.04513 -1.346 pH 3.23 0 1,553,000 100 2.000 360,000 420,000 27.04 1.432 540,000 75,000 4.829 0.684 720,000 5,500 0.354 -0.451 900,000 850 0.05473 -1.262 Data in Table VII and Fig. 4 show that PA 3679 spores in M/15 phosphate buffer at pH 7.0 are not killed in any significant number by heat until a temperature of 105~C is reached. Although such spores that have previously been irradiated with 400,000 or 800,000 rep die in significant numbers when heated to 100~C, it is apparent that a critical temperature of about 1000C is still necessary before irradiated PA 35679 spores become susceptible to killing with heat. The data in Table VII also indicate that although the heat resistance of irradiated PA 5679 spores is reduced considerably above 100~C, until this critical temperature range is reached, there is no significant difference between the heat resistances of irradiated and nonirradiated spores.

TABLE IV Effect of Temperature During Irradiation with Gamma Rays from Cobalt-60 on the Survival of C. botulinum 213 B Spores Suspended in M/15 Phosphate Buffer at pH 7.0 Dose Rep Spores per ml Percent Survivors Log Percent Survivors a) Irradiated at 5~C 0 630,000 100 2.000 340,000 172,000 27.3.438 510,000 72,000 11.4 1.057 595,000 11,500 1.83 0.263 680,000 6,100 0.968 -0.014 765,000 760 0.121 -0.917 850,000 370 0.0587 -1.231 b) Irradiated at -70~C 0 7,500,000 100 2.000 Frozen 9,700,000 129.0 2.076 340,000 5,600,000 57.7 1.761 510,00 12000 250000 12.9 1110 595,000 710,000 7.33 o.865 680,000 270,000 2.78 0.440 765,000 186,000 1.92 0.283 850,000 35,000 0.361 -0.443 15

(o) 100 0 0 0 (n Fg Effect of temperature dring irradiation on the survival of C. botulinum 21-B spores suspended in M/15 phosphate buffer at pH 7.0\ 0 -3 5OC 1.00 - 0 Irrad at 5~C * Irrad at-700C 2.00 0 0.2 0.4 0.6 0.8 1.0 1.2 REP x 106 Fig. 3* Effect of temperature during irradiation on the survival of C. botulinum 213B spores suspended in M/15 phosphate buffer at pH 7.0. 16

TABLE V Effect of Temperatures from Cobalt-60 on the Spores Suspended in During Irradiation with Gamma Rays Survival of C. parabotulinum 457A M/15 Phosphate Buffer at pH 7.0 Dose Rep Spores per -ml Percent Survivors Log Percent Survivors a) Irradiated at 5~C 0 490,000 100 2.000 340,000 145,000 36.6 1.593 510,000 29,000 5.92 0.772 595,000 9,250 1.89 0.277 680,000 2,550 0.52 -0.284 765,000 950 0.193 -0.714 850,000 145 0.0296 -1.535 b) Irradiated at -70~C 0 1,900,000 100 2.000 Frozen 2,300,000 121.0 2.083 340,000 1,030,000 44.8 1.651 510,000 385,000 16.7 1*223 595,000 123,000 5.4 0,732 680,000 58,000 2.52 0.401 765,000 17,100 0.774 -0.111 850,000 7,700 0.334 -0.476,,,,..,,,,.,,.,...,,,,.. 17

TABLE VI Effect of Temperatures During Irradiation with Gamma Rays from Cobalt-60 on the Survival of PA 3679 Spores Suspended in M/15 Phosphate Buffer at pH 7.0 Dose Rep Spores per ml Percent Survivors Log Percent Survivors a) Irradiated at 50~ 0 700,000 100 2.000 340,000 230,000 32.8 1.516 510,000 30,000 4.28 0.631 595,000 9,850 1.41 0.149 680,000oo 2,950 0.237 -0.625 765,000 890 0.127 -0.896 850,000 175 0.025 -1.602 b) Irradiated at -70~C 0 590,000 100 2.000 Frozen 650,000 110 2.014 340,000 270,000 41.5 1.618 510,000 46,000 7.07 0.849 595,000 46,000 7.07 0.849 680,000 8,600 1.32 0.121 765,000 3,500 0.508 -0.294 850,000 1,200 0.185 -0.735 18

TABLE VII Effect of Postirradiation Heating for One Hour at Various Temperatures on Previously Irradiated PA 3679 Spores Temperature, ~C Spores per ml Percent Survivors Log Percent Survivors a) Nonirradiated Control 70 80 90 95 100 105 110 860,000 650,000 660,000 640,000 500,000 580,000 310,000 0 100 75.5 76.7 74.5 58.2 67.5 34.9 o 2.000 1.878 1.885 1.872 1.765 1.829 1.543 b) Irradiated with 400,000 rep Control 70 80 90 95 100 105 110 150,000 170,000 160,000 162,000 175,000 134,000 3,600 0 100 113 106 108 116 89.5 2.40 0 2.00 2.055 2.025 2.033 2.o65 1.952 0.58o m c) Irradiated with 800,000 rep Control 70 90 95 100 105 110 1,340 200 305 560 65 0 0 100 14.9 22.8 41.7 4.85 0 0 2.000 1.1752 1.558 1.620 0.686 19

0 o 1 oA 400,000 Rep, 0>> Q 800,000 Rep C!) I 8 -1.00 I I -2.00 70 80 90 100 110 TEMPERATURE CENTIGRADE Fig. 4. Effect of postirradiation heating for one hour at various temperatures on previously irradiated PA 5679 spores. 20