APPTun AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1977, p. 823-831 Copyright © 1977 American Society for Microbiology
Vol. 34, No. 6 Printed in U.S.A.
Cryogenic Gamma Irradiation of Prototype Pork and Chicken and Antagonistic Effect Between Clostridium botulinum Types A and B A. ANELLIS,* E. SHATTUCK, M. MORIN, B. SRISARA,t S. QVALE, D. B. ROWLEY, AND E. W. ROSS, JR. Food Microbiology Group, Food Sciences Laboratory and Office, Technical Director, U.S. Arny Natick Research and Development Command, Natick, Massachusetts 01760
Received for publication 9 May 1977
Inoculated, irradiated pork (2,300 cans) and chicken (2,000 cans) pack studies were performed to establish the 12D dose for these foods. Each can was inoculated with a mixture of 106 spores of each of 10 strains of Clostridium botulinum (five type A and five type B), or a total of 107 spores. The cans received a series of increasing doses of gamma rays (seCo) at -30 + 100C; they were incubated for 6 months at 30 + 2C and examined for swelling, toxicity, and recoverable botulinal cells. The highest rate of swelling for both foods occurred within the first week of incubation, and maximum swelling was observed within 4 to 5 weeks. The minimal experimental sterilizing dose (ESD) based on flat, nontoxic sterile cans was 3.0 < ESD c 3.2 Mrad for pork and 4.0 < ESD c 4.2 Mrad for chicken. An analysis of the partial spoilage data by extreme-value statistics indicated with 90% confidence that the rate of spore death in the two foods was not a normal distribution, but appeared to favor a shifted exponential function. Based on the latter distribution, and assuming one most resistant strain in the mixture of 10 used, the 12D dose computed to 4.37 Mrad, with a shoulder of 0.11 Mrad, for pork and to 4.27 Mrad, with a shoulder of 0.51 Mrad, for chicken. An assumption that there were two or more most resistant strains in the inoculum progressively lowered the 12D dose. There was an apparent antagonism between the irradiated type A and B viable strains in the two foods. Cans with type B cells and toxin predominated over cans with type A cells and toxin, but cans with a mixture of type A and B toxins predominated over cans with a mixture of type A and B cells. At the highest sublethal doses, only type A cells survived in pork, but in chicken there was at least one type B strain that was at least as resistant as type A strains. The principal objective of the U.S. Arny food irradiation program is the long-range preservation, without refrigeration, of prepackaged food products. Prototype radappertization (16) processes, using Clostridium botulinum spores as the indicator organism of safety, have been reported for several foods. Bacon (6), ham (2), and pork loin (3) were irradiated at 2 to 250C before experience indicated that irradiation at cryogenic temperatures produced higher organoleptic acceptance; therefore codfish cake, corned beef, and pork sausage (4) were irradiated at -30 + 100C. It was subsequently determined that four radappertized representative foods (beef, ham, pork, and chicken) would be subjected to a 3-year intensive animal (mice, rats, and dogs) feeding investigation to ascertain their
wholesomeness for unlimited human consumption. Inoculated pack studies, using 'eCo gamma rays at -30 ± 100C, were conducted to determine the prescribed 12D, or equivalent, doses for these four prototype foods. The results for beef have already been presented (7), and those for ham are in press (A. Anellis et al., Spore Research 1976, in press). Uninoculated and inoculated (with C. botulinum spores) irradiation pack studies on pork products have been reviewed previously (3). In addition, Denny et al. (12) irradiated (with spent fuel rods) a mixture of five type A and five type B strains of botulinal spores (10( spores/can) of ground parboiled pork at an initial temperature of -20°C (final temperature not given) and performed most-probable-number (MPN) counts. Interpolation of their survival curve indicated an apparent D value of about 0.35 Mrad, or a
t Present address: Office of Atomic Energy for Peace, Bangkok, Thailand. 823
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ANELLIS ET AL.
12D dose of 4.2 Mrad as a minimal radiation dose (MRD). Aneilis et al. (3) irradiated (with 'Co gamma rays at 2 to 25°C) a series of enzyme-inactivated (center temperature, 71 to 740C) pork loin packs, each pack inoculated with an individual strain (five type A and five type B) of botulinal spores. Partial spoilage data obtained for swelling, toxicity, and recoverable cells after 6 months of incubation at 30 ± 2°C yielded a computed (reference 8, equations 3 and 4) MRD of 4.33 Mrad based on the one most resistant strain (62A) as determined by recoverable botulinal cells. The available literature on high-dose irradiation of chicken is scanty. Schmidt and Nank (38) and Schmidt (37) prepared a chicken pack containing a combination of light and dark meat and skin, inoculated the contents with a spore mixture (108/can) of five C. botulinum strains (three type A and two type B), and irradiated the cans with spent fuel rods at about 27°C. After incubation at 29.4°C for 12 to 15 months, they obtained partial spoilage data for swelling, toxicity, and viable spores, and computed a D value of 0.32 to 0.35 Mrad (12D of 3.84 to 4.20 Mrad) on the basis of the spore viability of one most resistant strain. Wheaton et al. (43) irradiated (0.85 or 1.7 Mrad), with spent fuel rods at a freezing temperature (latter not given), enzyme-inactivated (790C) deboned ground chicken inoculated with five individual strains (three type A and two type B) of botulinal spores and performed MPN survival counts. The most resistant strain (12885A) was reduced to 34% survivors by the lower dose and to 0.31% by the higher dose. Obviously, this study did not provide a reliable estimate of a D value. [Using the equation D = dose/log (No/n), where No is the initial spore density and n is the surviving number, one obtains 1.82 and 0.64 Mrad for the lower and higher doses, respectively.] Proctor et al. (31) inoculated sterile boneless chicken (with skin) with C. sporogenes (390 spores/sample), irradiated the food with electrons at -29°C, and then assayed for survivors and computed D37 values. As in Wheaton's study (43), the D values (D = 2.303D37) of the organism decreased (2.32, 0.66, and 0.48 Mrad) with increasing doses (0.23,0.47, and 0.70 Mrad, respectively), although sterility was achieved with a dose of 1.86 Mrad. Yet when they irradiated uninoculated nonsterile controls with 1.86 Mrad, they found indigenous viable anaerobic bacteria (43/g) but not the putrefactive variety; standard plate counts were
Vol. 34, No. 6 Printed in U.S.A.
Cryogenic Gamma Irradiation of Prototype Pork and Chicken and Antagonistic Effect Between Clostridium botulinum Types A and B A. ANELLIS,* E. SHATTUCK, M. MORIN, B. SRISARA,t S. QVALE, D. B. ROWLEY, AND E. W. ROSS, JR. Food Microbiology Group, Food Sciences Laboratory and Office, Technical Director, U.S. Arny Natick Research and Development Command, Natick, Massachusetts 01760
Received for publication 9 May 1977
Inoculated, irradiated pork (2,300 cans) and chicken (2,000 cans) pack studies were performed to establish the 12D dose for these foods. Each can was inoculated with a mixture of 106 spores of each of 10 strains of Clostridium botulinum (five type A and five type B), or a total of 107 spores. The cans received a series of increasing doses of gamma rays (seCo) at -30 + 100C; they were incubated for 6 months at 30 + 2C and examined for swelling, toxicity, and recoverable botulinal cells. The highest rate of swelling for both foods occurred within the first week of incubation, and maximum swelling was observed within 4 to 5 weeks. The minimal experimental sterilizing dose (ESD) based on flat, nontoxic sterile cans was 3.0 < ESD c 3.2 Mrad for pork and 4.0 < ESD c 4.2 Mrad for chicken. An analysis of the partial spoilage data by extreme-value statistics indicated with 90% confidence that the rate of spore death in the two foods was not a normal distribution, but appeared to favor a shifted exponential function. Based on the latter distribution, and assuming one most resistant strain in the mixture of 10 used, the 12D dose computed to 4.37 Mrad, with a shoulder of 0.11 Mrad, for pork and to 4.27 Mrad, with a shoulder of 0.51 Mrad, for chicken. An assumption that there were two or more most resistant strains in the inoculum progressively lowered the 12D dose. There was an apparent antagonism between the irradiated type A and B viable strains in the two foods. Cans with type B cells and toxin predominated over cans with type A cells and toxin, but cans with a mixture of type A and B toxins predominated over cans with a mixture of type A and B cells. At the highest sublethal doses, only type A cells survived in pork, but in chicken there was at least one type B strain that was at least as resistant as type A strains. The principal objective of the U.S. Arny food irradiation program is the long-range preservation, without refrigeration, of prepackaged food products. Prototype radappertization (16) processes, using Clostridium botulinum spores as the indicator organism of safety, have been reported for several foods. Bacon (6), ham (2), and pork loin (3) were irradiated at 2 to 250C before experience indicated that irradiation at cryogenic temperatures produced higher organoleptic acceptance; therefore codfish cake, corned beef, and pork sausage (4) were irradiated at -30 + 100C. It was subsequently determined that four radappertized representative foods (beef, ham, pork, and chicken) would be subjected to a 3-year intensive animal (mice, rats, and dogs) feeding investigation to ascertain their
wholesomeness for unlimited human consumption. Inoculated pack studies, using 'eCo gamma rays at -30 ± 100C, were conducted to determine the prescribed 12D, or equivalent, doses for these four prototype foods. The results for beef have already been presented (7), and those for ham are in press (A. Anellis et al., Spore Research 1976, in press). Uninoculated and inoculated (with C. botulinum spores) irradiation pack studies on pork products have been reviewed previously (3). In addition, Denny et al. (12) irradiated (with spent fuel rods) a mixture of five type A and five type B strains of botulinal spores (10( spores/can) of ground parboiled pork at an initial temperature of -20°C (final temperature not given) and performed most-probable-number (MPN) counts. Interpolation of their survival curve indicated an apparent D value of about 0.35 Mrad, or a
t Present address: Office of Atomic Energy for Peace, Bangkok, Thailand. 823
824
ANELLIS ET AL.
12D dose of 4.2 Mrad as a minimal radiation dose (MRD). Aneilis et al. (3) irradiated (with 'Co gamma rays at 2 to 25°C) a series of enzyme-inactivated (center temperature, 71 to 740C) pork loin packs, each pack inoculated with an individual strain (five type A and five type B) of botulinal spores. Partial spoilage data obtained for swelling, toxicity, and recoverable cells after 6 months of incubation at 30 ± 2°C yielded a computed (reference 8, equations 3 and 4) MRD of 4.33 Mrad based on the one most resistant strain (62A) as determined by recoverable botulinal cells. The available literature on high-dose irradiation of chicken is scanty. Schmidt and Nank (38) and Schmidt (37) prepared a chicken pack containing a combination of light and dark meat and skin, inoculated the contents with a spore mixture (108/can) of five C. botulinum strains (three type A and two type B), and irradiated the cans with spent fuel rods at about 27°C. After incubation at 29.4°C for 12 to 15 months, they obtained partial spoilage data for swelling, toxicity, and viable spores, and computed a D value of 0.32 to 0.35 Mrad (12D of 3.84 to 4.20 Mrad) on the basis of the spore viability of one most resistant strain. Wheaton et al. (43) irradiated (0.85 or 1.7 Mrad), with spent fuel rods at a freezing temperature (latter not given), enzyme-inactivated (790C) deboned ground chicken inoculated with five individual strains (three type A and two type B) of botulinal spores and performed MPN survival counts. The most resistant strain (12885A) was reduced to 34% survivors by the lower dose and to 0.31% by the higher dose. Obviously, this study did not provide a reliable estimate of a D value. [Using the equation D = dose/log (No/n), where No is the initial spore density and n is the surviving number, one obtains 1.82 and 0.64 Mrad for the lower and higher doses, respectively.] Proctor et al. (31) inoculated sterile boneless chicken (with skin) with C. sporogenes (390 spores/sample), irradiated the food with electrons at -29°C, and then assayed for survivors and computed D37 values. As in Wheaton's study (43), the D values (D = 2.303D37) of the organism decreased (2.32, 0.66, and 0.48 Mrad) with increasing doses (0.23,0.47, and 0.70 Mrad, respectively), although sterility was achieved with a dose of 1.86 Mrad. Yet when they irradiated uninoculated nonsterile controls with 1.86 Mrad, they found indigenous viable anaerobic bacteria (43/g) but not the putrefactive variety; standard plate counts were
