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. University of Michigan Engineering Research Institute Ann Arbor, Michigan Official Investigator: Lloyd Lo Kempe Collaborators: Jo T. Graikoski Nancy J. Williams Project No. 7-84-01-002 Contract DA 44-109-qm-1826 File No0 S-510 Report Noa 6 (Progress) Period: 6 June 1955-'14 July 1955 Initiation Date: 7 June 1954 Title of Contract: Combined Effects of Heat and Radiation in Food Sterilization SUMMARY Irradiated C. botulinum spores were found to be essentially as sensitive to heat after 3 months storage at 4~C in a refrigerator as they were immediately after irradiation. Irradiated PA 3679 spores are killed quite rapidly by heating at 99 ~C. THIS IS NOT A FINAL.REPORT. CONCLUSIONS STATED ARE SUBJECT TO CHANGE ON THE BASIS OF ADDITIONAL EVIDENCE. THIS INFORMATION IS NOT TO BE REPRINTED OR PLS WITHOUT WRITTEN PERMISSION FROM'HQ, QM ESEARCH AND DEVELOPMENT COMMAND, NATICK, MASSACHUSETTS. 1

I. EFFECT OF STORAGE INTERVAL BETWEEN THE TIME OF IRRADIATION AND THE TIME OF PERFORMANCE OF THERMAL-DEATH TIME STUDIES.OF THE HEAT RESISTANCE OF IRRADIATED BACTERIAL SPORES When planning our studies to be carried out in canned meat during 1955-56 and also in response to a letter from Captain Rueben Pomerantz, it was necessary to answer the following question with experimental data: Is the increased heat sensitivity induced in bacterial spores by irradiation affected by storage? Runs 355 35A, and 35B were conducted by allowing irradiated C. botulinum 62A spores to stand. in the refrigerator overnight before the thermal-death time studies were conducted. The results obtained were normal for C. botulinum spores suspended in 10%l gelatin at pH 7.0 and irradiated before heating (Table I). Runs 36 and 36A were designed to test the effect of a 3-month storage interval at 4~C after irradiation. C. botulinum 213B spores were used in M/15 phosphate buffer for this purpose. The data are presented in Table II and Fig. 1. A comparison with Run 14 (Table III) indicates that storage did not reduce the sensitization effect in this instance, but rather appeared to have accentuated it. A long-time experiment, designed to test this result further has been set up and will be reported at a later date. II. EFFECT OF USING UNHEATED BACTERIAL SPORES THAT HAD BEEN HEATED FOR 15 MINUTES AT 85~C WHEN STUDYING THE PROBLEM OF RADIATION SENSITIZATION OF BACTERIAL SPORES TO HEAT This problem arose as a result of a question in the discussion period following presentation of QMC Paper 544 at the Society of American Bacteriologists Annual Meeting in New York last May. While it is realized that heating for 15 minutes at 85~C is a very mild treatment and is standard procedure among investigators working with anaerobic bacterial spores, it was considered desirable to check briefly this criticism. Run 39 was conducted for 2

TABLE I EFFECT OF PRELIMINARY IRRADIATION BY GAMMA RAYS FROM COBALT-60 ON THE HEAT RESISTANCE OF C. BOTULINUM 62A SPORES Stored Overnight at 4~C Before Heating at 99~C al ___.....Control..._______ Irradiated * *H * V H D0 > r~. o > ~) 1 O. CH U 0.0 O' 0. Suspended in 10% gelatin at pH 7. 0. O O CO 0 Susened n 0~ eltinatpR o~. Run 35 0 o 15 10. 30 24. 45 38. 60 52. 75 66. go 80. (300,000 rep 505 000 8 175,000 6 12,500 5 800 4 lost 3 20 0 7 ) 100.0 34.65 2.48 o.158 2.00 1..540 0.395 -0.801 390,000 23,000 900 40 345 out out 100.0 5.90 0.2305 0.0105 0.000896 2.00 0.771 -0.637 -1.979 -3.047 0.00396 -2.402 0.001385 -2.858 Run 35A (500,000 rep) 0 0 15 10.8 30 24.6 45 38.5 (Same control 60 52.4 as Run 35) 75 66.3 go 80.0 Run 35B (900,000 rep) 0 0 15 10.8 30 24.6 45 38.5 (Same control 60 52.4 as Run 35) 75 66.3 go 80.0 195,000 15,000 250 15 1 out out 85,000 770 1 100.0 7.68 0.128 0.00768 0.000512 100.0 o.905 0.00178 2.00 0.886 -0.892 -2.114 -5.290 2.00 -0.043 -2.749 3

TABLE II EFFECT OF STORAGE AT 4~C BETWEEN TIME OF IRRADIATION AND HEATING ON THE SENSITIVITY TO HEAT INDUCED BY PREIRRADIATION ON C. SUSPENDED IN M/15 PHOSPHATE BOTULINUM 213B SPORES BUFFER AT pH 7.0 Control Irradiated *~r dH 0)H p.' U 11 3?o1 UC P P- U 0 0 1,050,000 100 2..00 130,000: 100.0 2 00 5 3.3 690,000 65.7 1.818 110,000 84.5 1.927 10 8.3 460,000 43.8 1.642 27,000 20.8 1.318 15 13.3 290,9000 27.6 1.441 8,700 6.69 0.825 20 18.3 350,000 33.3 1 -523 2,700 2.o8 0.318 30 28.3 150,000 14.3 1 J156 250 0.192 -0o716 40 38.3 30. 023 -1.638 Run 36A (540,000 rep) and storage from Jan. 31, 1955, to May 1, 1955, at 4~C 0 0 64,0000 100.0 2.00 5 3.3 26,000 40.6 1.961 10 8.3 3 7,00 5.46 1.738 15 13.3 (Same control 19,00 2.97 0.473 20 18.3 as Run 36) 320 0.150 -0.301 30 28.3 8 0.125 -1 903 40 38.3 out

2.00 U) 0 c 1.00 D 03 " -2.00 -3.00 RUN 36 -4.00 0 20 40 60 80 EFFECTIVE TIME AT 99~C Fig. 1. Effect of storage at 4~C, between the times of irradiation and heating, on the sensitivity to heat induced by preirradiation of C. botulinum 213B spores suspended in M/15 phosphate buffer at pH 7.0. 5

TABLE III EFFECT OF PRELIMINARY IRRADIATION BY GAMMA RAYS FROM COBALT-60 ON THE HEAT RESISTANCE OF C. BOTULINUM 213B SPORES ____. Control ___. ___ Irradiated -'1: U 0 0 0 Or-I 0 0 Run 14 (500j CD rep) 0 0 369,.0 100.0O0 2.000 35.000 100.0 2.0000 15 10.8.106-000 2870 1.4579 1.4.0 0 4.M 0.6021 co p ^a CO o O o o A) Suspended in M/15 phosphate buffer-at pH 7.0 O 0 569,000 L00.O0 2.000 353,000 100.0 2.0000 45 37.5 25,900 7.03 0.8470 20 0.0572'-1.2426 60 52.4 11, 900 3.23 0.5092 2 0.00572 -22426 75 66.3 5 100 1.382 0.1405 0 -- this purpose. In this experiment previously unheated C. botulinum 213B spores were grown and harvested. They were diluted into M/15 phosphate buffer at pH 7.0 and then four portions were withdrawn. Two of these portions were heated at 85~C for 15 minutes. Then, one portion of the heated and one portion of the unheated samples were set aside as controls and the other two were irradiated at 250,000 rep in the cobalt-60 gamma-ray field. Thermal-death time studies were then made of all four samples. The results are presented in Table IV and Fig. 2. It will be observed that there is no essential difference, for our purposjes, between the unheated spores and similar spores that have received: a preliminary heat treatment at 85~C for 15 minutes. 6

TABLE IV EFFECT OF PREIRRADIATION WITH SUBSEQUENT HEATING AT 99~C ON UNHEATED C. BOTULINUM 213B SPORES AND ON SIMILAR SPORES THAT HAD BEEN HEATED AT 85~C FOR 15 MINUTES TO KILL VEGETATIVE CELLS Control.._ Irradiated, [ o. o 0 0 01)PA[ 0 C' (UrrA CCC CO Heated but not irradiated Ieated -nd'-;irradiated (250 000 rep) 0 0 1,260,000 100.0 2.00 1,730,000 100.0 2.00 15 10.8 725,000 57.5 1.76 178,000 10.3 1.01 30 24.6 140,500 11.16.0o48 3,750 0.2165 -0.664 45 38.5 30,500 2.42 0.384 150 00oo865 — 2.063 60 52.4 3,200 0.254 -0.595 25. 001445 -2.837 75 66.3 9O00'0.0715 -1o346 4 0.000231 -5.636 Not heated, not irradiated Not heated but irradiated (250,000 rep) 0 0 *12,100,000 100.0 2.00 8,300,000 100.0 2.00 5 8,.550, 000 - 5,350 000 64.5 1.81 10 4,450 000.- 1,350,000 16,3 1.212 15 10.8 2,380,000 57 5 1.760 550,000 6.59 0.807 30 24.6 425,000 10.27 1. 012 6,05 0. 0729 -1.137 45 58.5 85,000 2.00 0.01 1,000 0.0121 -1.917 60 52.4 9,900 0.259 -0.621 175 0.00211 -2.676 75 66.5 2,150 0.052 -1.284 50 0.000605 -5.219 *Contains vegetative cells; a calculation was made indicating 4,140,000 spores were present in this sample at 0 minutes. 7

2.00 I.OC 0 C) cr 0 > U) 0 - 0 -.OC - 2.00 * HEATED-NOT IRRAD. E\ HEATED-IRRAD. (250,000 rep) * NOT HEATED-NOT IRRAD. ) \ S~ 0 NOT HEATED- IRRAD. (250,000 rep) NOTE: ~\ \. ~ USED CALCULATED VALUE FOR iNITIAL SPORE COUNT FOR NOT HEATED NOT IRRADIATED VALUES 30 RUN 39 -3.00 -4.OC -5.00 0 20 40 60 EFFECTIVE TIME AT 99~C 80 Fig. 2. Effect of preirradiation with 250,000 rep of gamma radiation followed by heating at 99~C on unheated C. botulinum 213B spores and on similar spores that had been heated at 85~C for 15 minutes to kill vegetative cells. 8

III. EFFECT OF TEMPERATURE ON THE SURVIVAL OF BACTERIAL SPORES A. THE EFFECT OF TEMPERATURE DURING IRRADIATION ON THE SURVIVAL OF BACTERIAL SPORES This study involves the irradiation of spore suspensions contained in small, heat-sealed glass vials. Irradiation is carried out principally in the center well of the cobalt-60 gamma radiation source. During irradiation the vials are fixed in an especially designed container which permits immersion of the vials in a fluid bath whose temperature can be controlled. After irradiation, the surviving spores are counted and the numbers found are compared with those surviving in suitable controls. Counting is carried out according to standard techniques similar to those described by Reed, Bohrer, and Cameron.* B. THE EFFECT OF TEMPERATURE DURING IRRADIATION ON THE SENSITIZATION OF BACTERIAL SPORES TO THE SUBSEQUENT LETHAL ACTION OF HEAT Other workers have suggested that irradiation of food at low temperatures may reduce off-flavor development due to the irradiation treatment. The question naturally arises then as to whether irradiation at low temperatures will still sensitize bacterial spores to the subsequent lethal action of heat. Also, how will high temperatures during irradiation influence this phenomenon? The following experiments have been carried out to answer these questions: 1. PA 3679. —PA 3679 spores were suspended in M/15 phosphate buffer at pH 7.0 and then placed in several glass ampoules which were sealed. The spores were then irradiated at 5 and 95~C, ampoules being withdrawn from the irradiation chamber at specified intervals. Following irradiation, all the vials containing spores were heated at 99~C for 1 hour. An unirradiated control was also included. Data in Table V and Figs. 3 and 4 indicate that irradiation sensitizes PA 3679 spores in some manner that causes a portion of them to be killed by 1 hour of heating at 99~C. Unirradiated *J. M. Reed, C. W. Bohrer, and E. J. Cameron, "Spore Destruction Rate Studies on Organisms of Significance in the Processing of Canned Foods," Food Research, 16, 383-408 (1951). 9

TABLE V EFFECT OF A COMBINED TREATMENT CONSISTING OF IRRADIATION WITH GAMMA RAYS FROM COBALT-60 FOLLOWED BY HEATING FOR 1 HOUR AT 99'~C ON THE SURVIVAL' OF. PA 3679 SPORES SUSPENDED IN M/15 PHOSPHATE BUFFER AT pH 7.0 Dosage, Spores per Log % rep ml Survivors Survivors la Irradiated at 5~C 0' 570,000 550,000 650,000 833,000 1,100,000 1, 100,000 2,700,000 460,000 95,000 32,000 14,000 3,200 340 54 100. 17.0 3.51 1.18 0.519 0.118 0.0126 0.002 2.00 1.250 0.545 0.0719 -0.2848 -0.9281 -1.8996 -2.699 lb Irradiated at 5~C and Heated for 1 Hour at 99~C 0 370,000 550,000 852,000 1,000,000 2,300,000 450,000 14,000 700 2 92.0 19.5 0.61 0.0504 o.000oooo8 1.964 1.29 -0.2147 -2.5171 -4.0605 2a Irradiated at 95~C 0 370,000 550,000 740,000 852,000 925,000 1,017,000 1,100,000 1,200,000 900,000 570,000 140,000 51,000 30,000 10,400 5,100 100.0 75.0 47.5 11.7 4.25 2.5 0. 866 0.258 2.00 1.875 1.676 1. 068 0.628 0. 398 -0.063 -0.588 2b Irradiated at 95~C and heated 1 hour at 99~C 0 570,000 550,000 740,000 885,000 925, 000 1,000,000 1,100,000 1,100,000 600,000 120,000 8,700 1,200 260 36 8 100.0 54.5 10.9 0.791 0.109 0.0236 0.00528 0. 000726 2.00 1.7564 1.0374 -0. 1018 -0. 9626 -1.6271 -2.4840 -3.139.1 Control Held at 95~C for 5 hours (the 1,200,000 spores per ml remain, originally present. time required for 925,000 rep); which is the same number as was 10

PA 3679 spores show little if any decrease in numbers under like conditions. This finding will be investigated further since it could be significant if a process of irradiation of food followed by heat treatment should be applied in food preservation. 11

20C CI) 0 U) I.OC 0 -1.00 -2.0C O0-HEATED AT 99 C I hr AFTER IRRADIATION \-\ IRRADIATED (ONLY) AT 5~C.:)~zz 0 0"l (. 0 0 -J -3.0C -4.0C 0 0.4 0.8 MEGAREP 1.2 14 Fig. 35 Effect of heating 1 hour at 99~C on PA 3679 spores that had been previously irradiated with gamma rays from cobalt-60 while held at a temperature of 5~C. Note: Unboiled control, 2,700,000 spores per ml; boiled 1 hour at 99~C, 2,300,000 spores per ml. 12

2.0C I.OC 0 oo CKl n0 -OC (9 0 -J -2.0C -3.OC -4.0C ) - 0 O-HEATED at 990C I hr AFTER IRRADIATION I-IRRADIATED (only) AT TEMP OF 95~ C (SEE TABLE VII) ) - ---------------- 0 0.4 0.8 MEGAREP 1.2 1.4 Fig. 4. Effect of heating 1 hour at 99~C on PA 3679 spores that had been previously irradiated with gamma rays from cobalt-60 while held at a temperature of 950C. Note: Unboiled control, 2,500,000 spores per ml; boiled 1 hour at 99~C, 2,300,000 spores per ml. 13