Preservation of Foods and Drugs Ionizing Radiations

Preservation of Foods and Drugs by Ionizing Radiations

W. DEXTERBELLAMY General Electric Research Laboratory, Scheneclady, New York

............ 49

60

V. VI. VII.

X.

. . . . . . . . . . . . 50 51 54 Dosimetry.. . . . . . . . . . .................................... Physical and Chemical ........................... 54 Microbiological Effects ........................... 60 63 ................. . . . . . . . . . . . . . . 65 A. Insect Eradication., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 65 66 D. Pasteurization. . . . . . .................................... 66 66 .................................... 67 67 Containers.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................... 68 69 70 References. . . . . . . ............................

I. Introduction and Scope Interest and research in irradiation preservation of foods have grown to the point where about 18 tons of food per month will be processed in 1959. This food will be used for a variety of studies ranging from basic chemistry on the isolation and identification of products in irradiated proteins, carbohydrates, and fats to human volunteer feeding experiments. This paper will cover some of the more important aspects of the subject and is not intended to be a comprehensive review. Several hundred technical papers have been published in this field in the past ten years. There have been a number of reviews (Hannan, 1955; Bellamy et al., 1955; Kuprianoff, 1955; Hannan, 1957; Morgan, 1958; Niven, 1958; Siu, 1958) to mention a few. Because of the limited space available only a few references have been given for each subject discussed. A complete listing would leave space for very little else. The interested reader should consult the references and the more comprehensive reviews. Popular interest in the subject has been great, and the number of popular articles probably exceeds that of the technical papers. At first “cold sterili49

50

W. DEXTER BELLAMY

zation” was touted as the panacea for all preservation problems. When some of its limitations were more fully realized, the process was rejected by many as worthless. It is now emerging as a process that will find application in specific areas, depending upon many parameters.

II. History Shortly after the discovery of radioactivity it was found that living cells could be penetrated and killed by this new type of energy. Any practical application had to await the development of sources capable of treating large volumes of material and the accumulation of knowledge of the physical and chemical effects on exposed materials. Coolidge (1925) and Coolidge and Moore (1932) were the first to report on the biological and chemical effects of an intense electron beam. However, little more was done until after World War 11. The present period of activity may be considered to have begun with the work of Brasch and Huber (1947) and that of the M.I.T. group (Dunn et al., 1948; Trump and Van de Graaff, 1948; Proctor and Goldblith, 1948). A large fraction of the present study is supported by the Quartermaster Corps of the United States Armed Forces and by The Office of the Surgeon General. The United States Army Ionizing Radiation Center a t Stockton, California, is expected to be in operation in 1960. The plans call for a 24 m.e.v. linear accelerator as an electron source and two million curies of cobalt60 as a y source. This pilot plant will have a capacity of 1,000 tons per month, and Irradiated Products, Inc. was awarded the production and planning contract .

111. Nature of High-Energy Radiation The term high-energy radiation is applied to types of radiation, both electromagnetic and particulate, in which the energy greatly exceeds that of the chemical bond. Although ultraviolet radiation shorter than 2,000 A should be included, it is of little practical interest because of its limited penetration. Only P- or y-rays from natural sources and X-rays and highvelocity electrons from machines are of interest for sterilization purposes. Other types of high-energy radiation have too little penetration or are likely to produce residual radioactivity. Contrary to visible or ultraviolet light, the absorption of high-energy radiation is dependent upon the atomic number of the target and is almost independent of the chemical structure. The absorbed energy is dissipated by ionization and excitation. The excitation energy may appear as light but will eventually appear as heat. The average ionization energy in air is about 32 electron volts (e.v.). While those occurring in a condensed medium such as biological material are much more difficult to measure,

RADIATION PRESERVATION

51

they are assumed to be about the same.' Thus the available energy is far greater than is necessary for the breaking of any chemical bond. The details of energy absorption are complex and only partially understood. They will be dealt with in later sections. The reader is referred to sections by Lea (1955), Nickson (1952), and Hollaender (1954) for more detailed discussion of this complex subject. Although X-rays and y-rays are electromagnetic rays without mass and p-rays are high-velocity electrons, their effect on biological materials is quite similar. Both interact to a large extent by ejection of an orbital electron, e.g., HIOf

HzO

+e

(1)

The ejected electron may have enough energy to repeat the above process but is eventually captured to form a negative ion, e.g., HzO

+e

+ HzO-

(2)

The excited ions dissociate into radicals, e.g.,

+ OH. HzO- -+H. + OH-

H20+ -+ Hf

(3) (4)

The original molecule may dissociate after excitation rather than eject an electron, e.g., He0 + H -

+ OH.

(5)

The fate of the radicals, ions, and excited molecules formed by the interaction of the high-energy radiation and biological matter depends upon chemical structure of the ionized molecule, although the original interaction was almost independent of chemical structure. The electron because of its mass and charge interacts more strongly with matter than do X- or y-rays. Therefore, the penetration of an electron beam is much less than that of an X- or y-ray of equal energy. Examples of penetration are presented in Fig. 1.

IV. Sources of High-Energy Radiation Radioactive elements both natural and artificial that emit only 8- or y-rays of suitable energies are potential sources. Of all such elements only cobalt-60 and cesium-137 may become available in sufficient quantities to be considered for commercial use. Mixed radiation from spent fuel plugs from atomic reactors may be used (Ceran et al., 1953). It seems improbable The energy of a C-3 bond is about 4 e.v. (1 e.v. per molecule = 23.05 kg. calories per mole). Because the ionic yields are unknown, the products are frequently reported as G values, i.e., the yield per 100 e.v. absorbed.

52

W. DEXTER BELLAMY

DEPTH OF WATER (cml

FIQ.1A. Distribution of ionization in water by X-rays and cathode rays (Trump and Van de Graaff, 1948).

that the @-radiationwill be of much use because of the low initial energy and the great amount of self absorption in these materials. Some of the pertinent data relating to these sources are the following: Cobalt-60 has a half-life of 5.2 years and average energy of y = 1.2 m.e.v.; cesium-137 has a half-life of 30 years and average energy of y = 0.66 m.e.v.; spent fuel rods’ half-life and average energy is variable, depending upon composition, but may be considered the order of 6 months and 1 m.e.v. All radioactive sources have the disadvantage of continuous decay in activity, thus requiring periodic replacement as well as continuous changes in the dose rate. They cannot be turned off by pushing a button or throwing a switch, thereby necessitating more complex shielding and protection systems. The accelerators can be used for the production of X-rays or electrons. As previously mentioned, X-rays and y-rays are similar and much more penetrating than electrons of the same energy. The accelerators are generally used for the production of electrons because of the much higher efficiency of production and absorption. The types of accelerators now considered for sterilization are: (1) Resonant transformers (Knowlton et al., 1953). (2) Van de Graaff (Foster et al., 1953). (3) Linear accelerator (Dewey et al., 1954). The details of the operation of these machines cannot be included in this article because of lack of space. The interested reader is referred to the

RADIATION PRESERVATION

53

RANGE ( p m l c 3 1

FIG.1B. Distribution of ionization in depth of aluminum produced by cathode rays of different energies (Trump and Van de Graaff, 1948).

specific papers. But, in general, these machines operate by accelerating electrons within a vacuum tube to high velocities. The velocity is directly related to the accelerating voltage. The electron beam can be brought out into the air through a thin window of aluminum, titanium, or stainless steel. If the electron beam is made to impinge on a heavy metal target in place of the thin window at the end of the vacuum tube, X-rays are generated. The conversion of the electron energy into X-rays is inefficient, and the X-rays generated are not as directional as the electron beam. Furthermore, the fraction of the X-ray beam which can be usefully absorbed in the product is much less. Therefore, for those applications where the penetration of the electron beam is sufficient, it is to be preferred. The resonant transformer and the Van de Graaff are direct linear accelerators, and the practical limit is about 5 m.e.v. maximum because the machines become very bulky and unwieldly due to the necessary insulation. A 4-m.e.v. resonant transformer, for instance, is a tank about 9 feet in diameter and 15 feet long with an 18-foot accelerating tube. An indirect accelerator such as the r-f linear type can be built to operate up to several hundred m.e.v. without the dimensional limitations of the direct accelerators. The upper limit is that range which will produce SUEcient nuclear side effects to result in undesirable residual radioactivity. The present evidence seems to indicate that the upper practical limit is

54

W. DEXTER BELLAMY

greater than 25 m.e.v., particularly if there is a storage period of several days following cxposure (Baldwin and Clark, 1953; Hannan, 1955).

V. Dosimetry The control of dosage in radiation processing is a t least as important as the control of time and temperature in thermal processing. The intense beams of high energy have introduced new problems in dosimetry. A search is in progress for a material which can be placed on or in the container of food and which will, with a minimum of processing, indicate the integrated dose received. The indicator should be simple and unaffected by time, temperature, light, etc., and reproducible within a few per cent. Silver phosphate glass fills some of the requirements (Davidson et al., 1956), and a polyvinyl chloride film has been developed as a dosimeter (Anonymous, 1957). Machine sources can be controlled by monitoring the beam current, but radioactive sources with their continuous decay require other methods (e.g., Davidson et al., 1953). The unit of X-radiation was the roentgen which was defined in terms of ionizations in air. The units now used in this work are the roentgen equivalent physical (rep) which is equal to 93 ergs per gram in most biological tissues and the rad which by definition is equal to 100 ergs per gram.

VI. Physical and Chemical Change Knowledge of irradiation chemistry has not developed to the point where it can predict all the changes that will be produced in a material as complex as food. It is still necessary to treat each food as a separate problem. There are, however, a few general observations that can be made which have wide application. Studies on the radiosensitivity of dilute aqueous solutions of an organic molecule may have little relation to its behavior within a living cell or in food products. In dilute solutions most of the radiation is absorbed by the water, resulting in numerous ions and/or radicals which are free to migrate. Because of the low probability of reaction, these ions and radicals may have a relatively long life and migrate correspondingly long distances. Any specific molecule or system in a cell is protected by the high probability that a water radical will react with any of the other more numerous components present. In a cell a large fraction of the water is in an ordered state and is therefore not free to migrate. The probability that a radical will interact with a protein or other large organic molecule is large : E 1 ;therefore, the lifetime of a radical may be only 10-9 seconds. The distance of migration is the order of 30-100 A (Hutchinson, 1957). Estimates of the fraction of indirect

55

RADIATION PRESERVATION

effect in living cells have been made by Hutchinson (1958) as not more than 50 %. This will vary, depending upon the system under study. There is ample evidence that energy can be transformed intra- and intermolecularly in solids. (The scintillation counter is based upon intramolecular transfer of excitation.) Under proper circumstances, however, radicals formed in solids by radiation will have a long lifetime (Lawton et al., 1958; Wolfrom et al., 1958). A considerable amount of work on the radiation of synthetic polymers has been reported in the past few years (c.f. Bovey, 1958). Some of these results can be applied to the much more complex biological systems. It was found that all vinyl polymers in which degradation predominates over crosslinking have the structure:

i.e., both the hydrogens on one of the carbons have been replaced. If either R or R' is an H, the polymer will crosslink (Miller et al., 1954). It appears that a simultaneous expulsion of hydrogens from adjacent carbons occurs about as frequently as the formation of a radical by ejection of a single hydrogen [see Eqs. (6a) and (6b)l. -C Hz-CH2-

-

-CH=CH-+Hz -CH2-CH-

(64

+ H.

(6b)

The G value (yield per 100 e.v. absorbed) for each process in high density polyethylene is about 2 (Miller et al., 1956). Crosslinking has been proposed as due to hydrogen abstraction from an adjacent polymer.

-CHz-C-

H

Lauryl methyl methacrylate was found to crosslink (Shultz, 1958). This

56

W. DEXTER BELLAMY

is probably due to the long hydrocarbon side chain. This process can be pictured as shown in Ey. (8) CHa CHs CHZ-C-CH

CHa C-

I *- 1 c=o c=o I I ( C i J L J (C i i H 4 I I CHg

-4

-CH=C

CHs

c=o

I I c=o

CH3

CHs

CH3

C=CH-

I I I (CiIHzi)-(CiiH21) I I

(8)

where the main chain is broken but the long hydrocarbon side chain crosslinks. The backbone of a peptide chain more closely resembles the doubly substituted polyethylene R

H O R

H

N

N

O

\ / It \ / II -C c-c C\ / \ / H

H

and therefore might be expected to degrade upon irradiation. The longer side chains such as in leucine, lysine, etc. would be expected to crosslink because they are aliphatic hydrocarbons. One might predict that proteins would degrade in the backbone but crosslink on the side chains. The over-all result would be a more highly branched polymer. Whether the size went up or down would depend upon the conditions of radiation, as well as the nature of the side chains. Sodium caseinate shows a decrease in particle size as determined by viscosity after the material was irradiated while dry in air with 1.86 X 107 rad. The same material irradiated as a 20% solution set up as a gel which was insoluble in water, 1 N HCl, and 1 N NaOH. The exact nature of the crosslink was not determined (Bellamy et al., 1955). A mechanism for the direct action on peptides and proteins was proposed (Caputo and Dose, 1957; Rajewsky and Dose, 1957) as shown in Eq. (9). Thus the products are an aliphatic imide Hz. The imide is unstable in aqueous solution and decomposes to an a-keto acid a-amino acid iammonia with resulting chain cleavage. The production of carbonyls from amino acids was also reported. This agrees with the earlier work on leucine (Bellamy and Lawton, 1954). Garrison and associates (1958) irradiated dilute solutions of pepsin and carbonyl (imino-intermediate) as shown gelatin. They found an amide in Eq. (10).

+

+

+

57

RADIATION PRESERVATION

H O

O R

I1

RIH 0

\ / 1I I / c-c \ / \ \ /c-c N

N H

\ / NH

\ / NH

0 Rz

II R’ c-c

I

\ /

c-c \ /

H

\ / 4N H

N

0

0

7-

N

H

H

0

II

He

N H H

O

I It R-C-NH-C-C-RI I

1I

Ra

10..

The G value for this reaction was found to be about 1. There is a fundamental difference between radiolysis of a peptide bond and the conventional acid or enzyme hydrolysis in that an imide must be formed between the N and the a-carbon, rather than the carboxyl carbon. Therefore, the subsequent hydrolysis results in an a-keto acid plus an amide or plus ammonia and acid. If this reaction occurs by a molecular process as in Eq. (64, there seems little possibility of a crosslinking in the peptide backbone. It is also very unlikely that any protective agent or inhibitor can be added

58

W. DEXTER BELLAMY

which will alter the process. However, if the reaction proceeds by Eq. (Bb), which seems more probable for indirect action, then a radical termination would prevent the abstraction of the second hydrogen. Because aliphatic amides are quite resistant to hydrolysis at ordinary temperatures, the high yield of ammonia from both peptides and amino acids does not support their formation. However, many amides are readily hydrolyzed by enzymes so that the significance of these reactions in foods is in odor and flavor changes because irradiated foods have not been found materially less wholesome (see Section VIII). The production of volatile amines from irradiated beef has been confirmed by Burks et al. (1958). Methyl amine and ethyl amine and a t least four other unidentified amines were found. The largest fraction of the volatile bases was ammonia in both the unirradiated control (E 99.9%) and the 2.3-3.7 megarad irradiated sample (E 93.0%). There was an increase in ammonia as well as volatile amine due to irradiation. The source of these amines is not known. The authors assume that the source of these amines is the nonprotein fraction of the meat because it contains about thirty-eight times as many end amino groups as the protein fraction, but no experiments to check this point have been done. Several papers on the production of carbonyls in irradiated foods have been published. Day et al. (1957a, b) found acetaldehyde, acetone, and butanone in the volatile fraction of irradiated skim milk. They did not search for nonvolatile carbonyls. Batzer et al. (1957) found that the carbonyls produced in irradiated meat could be divided into two groups, depending upon the solubility of their 2,4-dinitrophenyl hydrazine derivatives. The benzene soluble group A was obtained mostly from fat, while the metaphosphoric acid-sodium chloride-soluble group B was obtained chiefly from lean ground beef, The carbonyl compounds in both meat and fat vary directly with dose up to 9.3 megarad, the maximum dose used. The color changes in irradiated meats are largely due to the effect on the heme pigments. Anaerobic irradiation of precooked meats may result in the conversion of the normal brown or grey hematin to red pigments. The red pigment was characterized as denatured globin hemochrome (Tappel, 1957). Fresh meats irradiated aerobically at low doses develop a brownish discoloration due to formation of metmyoglobin. Fresh meats irradiated in nitrogen develop a brighter color due to regeneration of oxymyoglobin. A later study indicates that anaerobic irradiation converts methemoglobin to oxyhemoglobin (Tappel, 1958). Oxygen has a profound effect on both the direct and indirect effects of radiation. In water the H. from Eq. (4)will react with 0 2 to form Hoe. or other oxidizing radical so that instead of an equal number of oxidizing and reducing events, there are chiefly oxidations.

RADIATION PRESERVATION

59

The radicals produced by the direct effect can react with oxygen to produce organic peroxides, carbonyls, or other oxidized products. The radiation-produced radicals may persist for long periods in solids, including foods which have been irradiated and stored frozen. Such stored radicals can react with oxygen or other compounds to cause postirradiation effects. The irradiation of lipids produces crosslinks similar to those in polyethylene and, in addition, if irradiated in air, produces saturated and unsaturated carbonyls and peroxides. The flavor and odor changes could not be correlated with peroxides or carbonyl formation. Ozone and nitrogen oxide may be of importance in flavor and odor change. Carbonyls are also produced under anaerobic irradiation. The radicals produced by irradiation may be involved in autoxidation. The radical concentration is so high, however, that antioxidants are much less effective than in ordinary oxidative changes (Chipault et al., 1957). Transisomerization was found in oleic acid (Pan et ul., 1958). Presumably, transunsaturation occurs as it does in polyethylene, although it was not reported. When irradiated dry, carbohydrates degrade with the production of reducing groups and acid groups. Crosslinking also occurs to some extent (Saeman et al., 1952; Price et al., 1954; Kertesz et ul., 1956). The nature of the crosslink is not known. Carbohydrate degradation is most important in the texture of foods, particularly fruits and vegetables because they do not contribute to off-odors. The products from irradiated mono- and polysaccharides have been implicated in the nonenzymatic “browning” of foods. It appears that mono- and disaccharides are not important in odor or flavor changes at sterilization doses. Sulfur-containing amino acids and proteins have been shown to be a major source of the “irradiated beef” odor, although the exact causes of this odor are not known. Aqueous solutions of cysteine liberate hydrogen sulfide rather than ammonia when irradiated as do other amino acids. No hydrogen sulfide was detected upon irradiation of aqueous solutions of cystine (Swallow, 1952). Irradiation of aqueous solutions of methionine has been found to yield both hydrogen sulfide and methyl mercaptan. Aldehydes and ketones will combine with mercaptans under conditions existing in foods to form mercaptol and mercaptals. Littman et al. (1957) proposed the protection of -SH groups by the addition of carbonyl compounds during irradiation. It also seems possible that some of the irradiation odors and flavors are due to this type of compound formed from condensed carbonyls and mercaptans produced by irradiation of foods. Whether the mercaptal or mercaptol would be volatile and malodorous depends upon its size and other functional groups. Dilute solutions of many enzymes are quite sensitive to ionizing radia-

60

W. DEXTER BELLAMY

tion. However, they are much more resistant in cells, tissues, and in the presence of high concentrations of other organic material. Most natural products retain some enzyme activity after exposure to pasteurization or sterilization doses. The phosphatase of milk was not completely destroyed by ten times the dose necessary to sterilize raw milk (Proctor and Goldblith, 1957). Several workers have found tyrosine crystals in radiationsterilized beef and pork after storage at 72”-100°F. for several months (e.g. Drake et al., 1957) as well as increased amounts of soluble amino acids. From 10 to 90 % of the original activity of other enzyme systems has been found in radiation-sterilized meats. Heating to 160°F. for a few minutes during the processing cycle has been found effective as an enzyme inhibitor. This amount of thermal processing will result in a cooked product with the accompanying physical and chemical changes. It may have beneficial side effects by decreasing the sterilization dose necessary, by inactivation of some viruses, and by inactivation of any toxins (q.v.). Other methods of enzyme inhibition have been sought such as chemical additives, pre- and postirradiation treatment, etc. One method which may have application in irradiation pasteurization and result in a “fresh” meat is that described by Radouco-Thomas et al. (1958)and Zender et al. (1958). These authors found that ante-mortem injection of adrenaline (epinephrine) resulted in muscles with greatly reduced post-mortem autolysis. Because normal muscle is sterile, a combination of epinephrine injection and surface sterilization with electrons resulted in a product that had greatly increased storage tolerance at all temperatures up to 38°C. The process was studied in pork, beef, and rabbit. In addition to greatly decreased autolysis, there was increased fluid retention, better color retention, and increased tenderness. These physical changes in the muscle, as well as the decreased activity of cathepsins, is due to the higher pH of the postmortem meat. The pH of normal muscle drops to about 5.5 a few hours after slaughter, while the adrenaline-injected muscle remains close to 7.0. The injection process does not, however, alter the irradiation odor produced in beef or pork. Some bacterial toxins are molecules about the same size and complexity of enzymes, and one has been found to have the same order of radio resistance. The toxin of Clostridium botulinum was found to have a DWof about 8 megarad (Dack and Wagenaar, 1955).

VII. Microbiological Effects The factors affecting the radiosensitivity of microorganisms have been reviewed by Kelner et al. (less), Hollaender and Stapleton (1953), and Bellamy and Lawton (1955).Discussions of the mechanism of action can be found in the above reviews as well as in Lea (1947),Pollard (1954),and

RADIATION PRESERVATION

61

Bacq and Alexander (1955). Inactivation by radiation usually means inability to continue indefinite growth. Inactivated cells may undergo one or more divisions and may have a large fraction of their original enzyme activity immediately following a lethal dose of radiation, but the organism or its progeny cannot reproduce properly. For this reason the lethal effect is thought to be due to genetic damage. It can be easily estimated that within a bacteria such as Escherichia coli less than one bond in lo8is broken by a lethal dose of radiation. Therefore, the damaged structure must be essential. Attempts to identify this structure have led to the target theory. The original target theory has been modified and refined by many, including Lea (1947) and Pollard (1954). Under suitable conditions it has been found that the survival of many bacteria fits a first order decay curve of the type N = N @ - D ‘ D o , where N = number of organisms surviving dose D,N o = original number, and Do = mean lethal dose (dose at which N = Noe-l). This type of survival has been interpreted to mean that a single ionization will be effective if occurring in the proper structure. It is now known that this interpretation is limited to aerobic conditions and within a narrow range of temperature (Howard-Flanders and Alper, 1957). Survival curves of a higher order have been obtained for many organisms and have caused many authors to reject the target theory in favor of the “indirect effect.” Here the lethal action is due to the activated or ionized water, e.g., H., OH., HOz., etc. Because most living systems are largely water, the indirect effect seemed most probable. More recent studies, however, indicate that the indirect effect accounts for not more than one-half of irradiation effect in microorganisms (Hutchinson, 1957). This apparent paradox is probably due to the short life of many of the mobile radicals and also to the fact that much of the water within a cell is not free to migrate but is bound in an ordered system. Most organisms are more radiosensitive under aerobic conditions. Recent work has shown that removal of oxygen 0.02second after aerobic irradiation will not prevent the increased sensitivity. Conversely, addition of oxygen 0.02 second after anaerobic irradiation does not increase the sensitivity (Howard-Flanders and Moore, 1958). Much of the work on the oxygen effect will have to be re-examined in the light of the above findings that very small concentrations are almost immediately effective. Most organisms are more sensitive when irradiated in distilled water than in nutrient broth. The addition of reducing compounds such as ascorbic acid and hydrosulfite, 8-mercaptoethylamine, and other sulfhydryl compounds (Stapleton and Woodbury, 1957; Doudney, 1957) during irradiation decrease sensitivity. In general these compounds do not increase the anaerobic resistance. Pseudomonas geniculata, a very sensitive psychrophilic aerobe, was found to be unaffected by oxygen during irradiation with y-

62

W. DEXTER BELLAMY

rays (Wolin et al., 1957). A threefold increase in resistance was obtained by increasing the number from lo8per milliliter to 8 X los or greater per milliliter. High populations of Micrococcus sp. did not alter the radiation resistance of low numbers of Pseudommas cultures. Recent work (Alper and Gillies, 1958) seems to indicate that many postirradiation restorations of irradiation damage previously reported may act by a common mechanism, viz. the imposition of suboptimal conditions of growth. The type of damage which is susceptible to postirradiation influence is less influenced by oxygen than other forms of damage leading to lethal injury. The whole problem of pre- and postirradiation conditioning is an extremely complex one and undoubtedly differs with different species. Of interest in the field of food preservation are the findings of Morgan and Reed (1954) that the thermal resistance of spores of strain PA 3679 was reduced by prior exposure to sublethal doses of ionizing radiation. The radiation resistance of preheated spores was not changed, however. The spores of C. botulinum were found to respond in a similar manner (Kempe, 1955). Kempe et al. (1957) obtained less spectacular results when they tried pre-irradiation on C. botulinum spores in ground meat. Kan et al. (1957) have confirmed the decreased thermal resistance of pre-irradiated spores of PA 3679 and Bacillus cereus in canned ham. The effect of the temperature during irradiation received considerable attention. In general vegetative cells irradiated aerobically are more resistant when exposed in the frozen state. The sensitivity of anaerobically exposed cells is less altered by freezing. Freezing immobilizes the water radicals and thereby considerably reduces the indirect effect. Most microorganisms are more resistant dry than in aqueous suspension. This again is probably due to the reduction of the indirect effect and decrease of target size. Much less work has been published on the effect of radiation a t elevated temperatures. Denny et al. (1954) reported that spores of C. botulinum suspended in water were only slightly more resistant a t 150°F. (64°C.) than a t 72", 23", and 0°F. when exposed to y-rays from cobalt-60. Kempe et al. (1956) reported that C. botulinum 213B was more resistant at 27'C. by an order of magnitude than a t any other temperature tried between -70" and 95°C. PA 3679 was more resistant by a factor of 25 in the range of 85-95°C. Because of the importance of these observations, for both practical and theoretical reasons further studies on the effect of temperature during irradiation should be undertaken. In 1956 Anderson et al. reported the isolation of a pigmented Micrococcus resembling M. roseus or M . rubens tetragenus from ground beef following 7-doses of 2-3 megarad. Pure cultures on agar slants survived 6 megarad. This non-sporeforming organism is thus the most radioresistant bacteria yet reported. Further studies by Anderson et al. (1956) and Niven (1958)

RADIATION PRESERVATION

63

have shown that the organism is a strict aerobe, sensitive to sodium chloride above 2%, and appears to obtain energy by oxidation of amino acids. Other red pigments were found to be relatively radioresistant, although none approached the resistance of Anderson’s original isolate. Kilburn et al. (1958)were unable to grow this organism without its carotenoid pigment. They were not able to prove conclusively that the great resistance is due to the pigment, although radioresistance varied directly with the pigment content of cells. Most viruses are much more resistant to ionizing radiation than bacteria and would not be inactivated in irradiated foods if present in very large numbers. Because of their smaller size and simpler structure the radiosensitivity of viruses is much more dependent upon their environment than is that of bacteria. In terms of the target theory the increased resistance is due to the smaller size (cf. Pollard, 1954 and Benyesh et al., 1958). Although the radiosensitivity of all viruses is not simply related to their size, it is related to the content and organization of their genetic material. The importance of active virus in preserved foods would have to be evaluated in each product. Polio virus in irradiation-pasteurized milk would not be permitted. However, the presence of virus in fresh meats is not considered a problem now, and their presence in irradiation-pasteurized meats which will be cooked should be no greater problem.

VIII. Wholesomeness The effect of sterilization doses on the vitamin content of foods has been examined. As stated previously, studies on the radiosensitivity of dilute solutions of pure vitamins provide very little information about the same material in foods. They may provide useful information concerning the mechanism of radiolysis. The radiosensitivity varies among the foods and depends upon the conditions during exposure, as well as the pre- and postirradiation treatment. In general, vitamins niacin, riboflavin, folic acid, B12, D, and K are relatively stable to radiation. The stability of vitamins A, E, C, and thiamine is poor while the pyridoximers are intermediate. The vitamin loss in irradiated foods has been found to be about the same as in heat processed foods with the possible exception of thiamine which is more sensitive. In certain long-term animal feeding tests with diets high in individual foods, it has been found desirable to auppIement with vitamins E and K (cf. Groninger et al., 1956). The question of irradiation production of toxic products must be thoroughly investigated before large-scale use is permitted. Experiments have been under way for several years using thousands of animals including mice, rats, chickens, pigs, dogs, monkeys, as well as human volunteers. The

64

W. DEXTER BELLAMY

results of these tests give no reason to believe that irradiation processed food is less safe than food processed by other means. A search for radiation produced carcinogenic materials has been completely negative so far. For a thorough discussion of this problem the reader should consult Siu (1958), Vorhes and Lehman (1956), and Lehman and Laug (1954). Residual radioactivity in irradiated foods and containers is popularly considered to be the most serious problem in irradiated foods. A more thorough examination indicates that the induced radioactivity is low and TABLE I APPROXIMATEDOSERANGE REaUrRED FOR SSVERAL FOOD PRESERVATION PROCESSES Process

Rad

Inhibition of sprouting carrots, onions, potatoes Inactivation of trichina: sterilization of trichina female Insect deinfestatioo of grains and cereals “Irradiation pasteurization” Sterilization of foods Enzyme inactivation

4,000-10,OOO 20,000-50, OOO 100,000-5OO,OOO 1OO,o0o-1,ooo,OOO 2,000,~5,OOO,000 2,000,000-10,OOO, 000

TABLE I1 THETHRESHOLD ENERGY LEVELSFOR THE PRODUCTION OF INDUCED RADIOACTIVITY Element Carbon-12 Oxygen-16 Nitrogen-14 Potassium-39 Sulfer-32 Calcium-40 Iron-54 Magnesium-24 Magnesium-25 Copper-65 Iodine-127 Aluminum-27 Silicon-28 Zinc43 Tin-119 Sodium-22

Threshold (m.e.v.) 18.7 16.3 10.6 13.2 14.8 15.9 13.8 16.2 11.5 10.2 9.3 14.0 16.8 11.6 6.6 12.1

Half-life of product 21 minutes 2.1 minutes 10 minutes 7.5 seconds 3.2 seconds 1 second 8.9 minutes 11.6 seconds 14.8 hours 12.8 hours 13 days 7 seconds 5 seconds 39 minutes 275 days 2.6 years

RADIATION PRESERVATION

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in nearly all cases the half-life so short that there is no hazard. A possible exception might be tin-119 in the containers or sodium-22 in the food if the initial radiation energy is greater than 12 m,e.v, Table I1 gives some of the possible radioactive products, their half-life, and the threshold energy of activation. Measurements on foods exposed to 30 m.e.v. electrons showed that the induced activity was several orders of magnitude less than the established tolerance levels (Skaggs, 1956).

IX. Applications Table I presents the dose range required for some preservation processes which are discussed in more detail below.

A. INSECT ERADICATION The control of such insects as Sitophilus granum'us (granary weevil) and Tribolium confusum (confused flour beetle) in grains and cereal products has been suggested. The dose necessary to bring about rapid death of the insects and larvae was found to be about 300,000 rad, while that necessary to prevent reproduction was the order of 15,000 to 30,000 rad (Hassett and Jenkins, 1952). Similar results were reported by Proctor et a,?. (1954) for five species of insects normally found in infected cereal and fruit products. Plans have been suggested for the design of facilities for deinfestation of bulk grain (Hassett and Jenkins, 1952; Brownell et al., 1955, 1957), but its use in prepackaged cereal and cereal products seems more feasible because of the possibility of recontamination of bulk materials. B. SPROUTINHIBITION Because of the low dose required (4,00040,000 rad) sprout inhibition of vegetables such as potatoes, onions, carrots, etc. has been examined (Sparrow and Christensen, 1954; Brownell et al., 1955; Brownell and Nehemias, 1955). The increased storage life without sprouting and the accompanying softening and loss of water is very clear-cut and may increase the usable fraction from 0 to over 90% after prolonged storage. It was found that doses higher than 14 kilorad caused a permanent increase in respiration during storage, while doses of 4.7 and 14.0 kilorad caused a temporary increase in respiration that dropped to approximateIy normal after the seventh week of postirradiation storage (Gustafson et al., 1957). Irradiated potatoes have been found more sensitive to bruises and less resistant to rot. Potatoes exposed to 2.3 kilorep or more did not exhibit periderm formation after wounding, although there was distinct soberization (Brownell el at., 1957; Burton and Hannan, 1957).

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W. DEXTER BELLAMY

C. TRICHINA The use of ionizing radiation to break the trichinosis cycle has been suggested (Gomberg et al., 1954), e.g., because of the relatively low dose (20,000-50,000 rad). It has been pointed out, however, that in this country other approaches to this problem appear more practical (Urbain, 1958). Its application in other countries with different technical and economical parameters merits study.

D. PASTEURIZATION (‘Irradiationpasteurization” has been suggested as a means of increasing storage l i e at low temperatures. Wolin et al. (1957) reported that the psychrophilic pseudomads, the principal cause of spoilage of fresh meats at low temperature (2”C.), were very sensitive to radiation, and doses of lob rad or less were found to extend the low temperature life severalfold. However, the meats eventually spoil due to growth of more radio resistant microorganisms such as Microbacterium thermosphactum. This increased low-temperature life may make prepackaging of fresh meat a commercial possibility. Physical changes due to nonmicrobial spoilage in certain meats may be a serious problem (Urbain, 1958). Minced chicken meat refrigerated anaerobically was wholesome after 80 days if pre-irradiated with 250,000 rad. Spoilage was due to M . thermosphactum and Streptococcus faecium (Thornley, 1957). Irradiation at 1 megarad was found to inactivate all microorganisms except endospores and has been proposed as a “pasteurization dose.” Brownell and Purohit (1956), Brownell et al. (1955), and Proctor et al. (1955) found that certain meats and vegetables had longer low temperature storage life after doses ranging from 250 to 1 X lo6 rad. Beef products developed undesirable off flavors at the higher doses, however.

E. STERILIZATION The fact that C. botulinum spores and toxin will not be destroyed by these pasteurization doses means that these foods will have t o be treated as possibly contaminated and uncooked. As previously mentioned the spores of C . botulinum are among the most radiation-resistant of microorganisms and any radiation-sterilized product must be free of this organism. If the criterion used in thermal processing is used, i.e., the dose necessary to reduce 1 X 10l2 spores to less than one survivor, then the sterilization dose has been calculated to be 4.5 megarad (Niven, 1958). Much of the earlier work based on a sterilization dose of 2 megarad must be re-examined in the light of the increased dose.

RADIATION PRESERVATION

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F. PHARMACEUTICALS Studies on the properties of irradiated pharmaceuticals have been reported by several workers. Colovos and Churchill (1957) examined a series of parenteral products that had been sterilized by cathode rays. Stability and toxicity were used to determine the influence of exposure to 2 megarep (1.86 megarad). The pharmaceuticals fell into the following categories: antibiotics, hormones and steroids, multivitamin preparations, anticoagulants, proteins, alkaloids, and sulfonamides. Storage times up to 4 years a t 4",2 5 O , and 40°F. were reported, They concluded that irradiated products used clinically showed no unfavorable reactions and that with few exceptions drugs under the proper conditions are stable to sterilizing doses of cathode rays. However, each product must be treated as an individual problem. Ethicon has announced the commercial sterilization of sutures by a 7 m.e.v. linear acceIerator (Anonymous, 1957). Some of the advantages claimed are: (1) increased tensile strength, (2) sterilization in their final sealed containers, and (3) substantial increase in the safety margin. The sterilization by ionizing radiation of human blood vessels and bones for transplants has proved successful and is being used in several places (e.g. DeVries et aE., 1955). The use of ionizing radiation to prepare vaccines has been suggested (Traub et al., 1951) because of the differential sensitivity of the Virus components. The properties of irradiated virus have been examined (Bellamy et al., 1957; Benyesh et al., 1958), but further knowledge of the factors involved is needed.

X. Containers Radiation does not impart some miraculous self-sterilizing property to foods; therefore, treated foods must be protected against recontamination. Several studies are under way to determine the effect of sterilization doses on containers, of the interaction between irradiated foods and irradiated containers, and of storage of irradiated foods in irradiated containers. Both conventional rigid metal cans and Aexible plastic films are under consideration. Most films have been found to be permeable to oxygen and/or water. Some irradiated films have been found to interact with the foods to produce off odors or flavors. Mylar, nylon, polystyrene, and polyethylene alone or in combination with each other or several mills of metal film have shown promise. Plastic film is more permeable to electrons than metal cans, but so far problems of oxidation, dehydration, and light destruction have not been satisfactorily solved. Some of the standard can enamels appear to be satisfactory for irradiation processing of foods (cf. Morgan, 1958; Siu, 1958).

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XI. Economic Considerations Many papers written on this subject have not considered many of the factors that a business must deal with such as taxes, capital write-off, and profit on the investment. Cook (1958a, b) has examined some of these parameters. For instance, much has been made of the fact that cesium-137 has a half-life of 30 years and is essentially free as a by-product of reactors; however, there are few companies that do not expect to liquidate their investment in a much shorter time, usually in not more than 10 years. A medium-risk business requires 10-15 % net earnings before taxes to attract capital. Such considerations make cesium-137 an expensive source. Estimates ranging from $ 3 4 per curie to a “rock bottom” of $0.30 per curie have been made. At $1.00 per curie cesium-137 costs $240,000 per initial kilowatt of 7-radiation. Cobalt-60 is made by absorption of neutrons in a pile. The neutrons thus used are not available for other uses. Therefore, the minimum price for

I I I I l l

O.\

I

I 1 1 1 1 1 1

1.0 COST OF IRRADIATION

I

4

I IIllll1

10.0

I

I 1 1 1 1 1

PER POUND

FIG.2. The relatioiiship between the cost of radiation production (kilowatt hours absorbed) and the cost per pound of irradiated product; e.g., if radiation costing $0.60 per kilowatt hour is used for sterilizing meat at 4 megarep, the cost per pound will be $0.028.

RADIATION PRESERVATION

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cobalt-60 cannot be less than the neutrons could bring in other uses, e.g., production of power. Neutron cost is estimated a t about $58,000 per mole (Cook, 1958b). Because 1 mole of neutrons is required to make 1 mole of cobalt-60, $58,000 per mole must be a minimum amount. This is equivalent to $60,000 per initial kilowatt of y-radiation. Present costs of cobalt-60 are two t o ten times this number with a “rock bottom” of $4,000 per initial kilowatt suggested by W. F. Libby of the Atomic Energy Commission. Linear accelerators now cost $35,000 to $40,000 per kilowatt of electron beam. A sufficiently large market to allow quantity production could reduce this amount to $10,000 or even $5,000 per kilowatt of electron beam. Resonant transformers now cost $10,000-$12,000 per kilowatt, and this cost might drop to $5,000 per kilowatt of electron beam. Machines will have servicing and replacement of parts costs but can be shut off when not in use, whereas isotope sources radiate continuously whether used or not. It is obvious that only that radiation which is absorbed in the food is useful. That part lost in the air, in the container, or in the shielding is wasted. The fraction of the beam used in overdosing part of the product in order that all the product receives a minimum dose is also wasted. Utilization may vary from 10 to 90 %, depending upon the product and the engineering. In general, electron beams can be utilized more completely than the undirected radiation from radioactive sources. Figure 2 presents the relation between the cost of radiation absorbed and the cost per pound of irradiated product. These numbers were calculated from the relationship: cost of radiation (cents per pound) = cost of beam absorbed (dollars per kilowatt hour) X dose in megarad X 1.18.2 Products which are highly unique can absorb a higher cost per pound than irradiated products which are little different from materials produced by other methods. High-cost items such as drugs can absorb a few cents per pound, whereas low-cost large-volume materials such as fruits and vegetables may be overpriced by addition of a fraction of a cent per pound, These and other problems make the financial considerations as important as the technical studies.

XII. Summary and Conclusions Irradiation preservation offers a new and different process to the food and drug industry. While many of the problems are formidable, there is no evidence at present which definitely precludes their solution. The production of irradiation odors and flavors is probably the major obstacle to its use in some meat products. Research now in progress may lead to methods 2 1 kw.-hr. = 3.6 X 101s ergs; 1 megarad = 1 X 108 ergs gram-’; 1 kw.-hr. 106 gram megarad, or % 840 lb. megarad.

3.6 X

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W. DEXTER BELLAMY

of preventing or masking them. It is clear that radiation will not replace the conventional methods of thermal processing, freezing, and drying but will supplement them in restricted areas, depending upon many parameters. Sterilization of sutures, of heat-sensitive medicinals, and of army field rations are examples of special applications which are now in use. It appears unlikely that irradiation costs can compete with present methods where the conventional product is satisfactory. Entirely new products with new properties seem more probable. Irradiation pasteurization in combination with enzyme inhibition may have wide application in countries where household refrigerators are not common. The potentials of irradiation preservation of foods and drugs cannot be overlooked by a world beset by an inadequate food supply for an exploding population.

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