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Department of Microbiology, Iowa State University, Ames, Iowa
2 . 1 . INTRODUCTION
The ability of radiation to inactivate microorganisms has been the main rationale
for the use of food irradiation. Radiation has been demonstrated to be an effective
means of destroying both pathogenic and nonpathogenic bacteria, as well as para-
sites and, to a lesser degree, viruses. In this context, radiation can be seen as
analogous to various other food processes used to inactivate microorganisms, such
as the various forms of heating.
2 .2 . MECHANISMS OF INACTIVATION
Radiation, whether ionizing or nonionizing (i.e., a photon of energy or an electron),inactivates microorganisms by damaging a critical element in the cell, most often
the genetic material. This damage prevents multiplication and also randomly ter-
minates most cell functions. Damage to the genetic material occurs as a result of a
direct collision between the radiation energy and the genetic material, or as a result
of the radiation ionizing an adjacent molecule, which in turn reacts with the genetic
material. In most cells, the adjacent molecule is usually water (Grecz et al. 1983).
In the first instance, the effects are straightforward. A photon of energy or an
electron randomly strikes the genetic material of the cell and causes a lesion in theDNA. The lesion can be a break in a single strand of the DNA or, if the orientation
of the DNA is appropriate, the energy or electron can break both strands on the
DNA. Single-strand lesions may not be lethal in and of themselves, and may in fact
result in mutations. However, large numbers of single-strand lesions may exceed
the bacterium's repair capability, which ultimately results in the death of the cell.
Food Irradiation: Principles and Applications, Edited by R. A. Molins
A doub le-strand lesion occurs w hen the photon or electron strikes adjacent areas
on both strands of the DN A. This in effect severs the DNA into two pieces. D ouble
strand lesions are almost invariably lethal, as the mechanism necessary to repair a
double-strand lesion is beyond the ability of virtually all biological systems. How-
ever, because of the necessary orientation of the DNA in relation to the irradiationsource, double-strand lesions occur much less frequently than do single-strand
lesions.
The interactions of radiation with molecules adjacent to the genetic material are
more complex. The chemistry of the irradiation of water is well known. Radiation
causes water molecules to lose an electron, producing H 2 O+ and e~. These pro-
ducts react with other water molecules to produce a number of compounds, includ-
ing hydrogen and hydroxyl radicals, molecular hydrogen and oxygen, as well as
hydrogen peroxide (Arena 1971). The reactive components of these equations,
which are generally believed to be most significant, are the hydroxyl radicals
(OH~) and hydrogen peroxide (H 2O 2). These m olecules react with the nucleic acids
and the chemical bonds that bind one nucleic acid to another in a single strand, as
well as with the bonds that link the adjacent base pair in the opposite strand. Since
the location of the ionization of the water molecules is random, the subsequent
reactions with the nucleic acids are random. As with the direct interaction of
radiation with DN A, the indirect action can result in both single- and double-strand
lesions, with the same overall effects.
In add ition to effects on the genetic m aterial, radiation has a variety of effects on
the other components of the cell. Applying radiation to a cell results in the direct
and indirect interaction with cell components such as membranes, enzymes, and
plasmids. These interactions m ay h ave the potential to be lethal to the cell, in and of
themselves but in most cases would not be so unless there were also damage to the
genetic material. These interactions may have a role in the survival of sublethally
injured bacteria, in that a cell that has not sustained lethal genetic damage may bedamaged in other ways that complicate or impede survival of the injured cell.
The radiation sensitivity of various organic compounds is proportional to their
mo lecular w eight. On the basis of this assumption, it has been e stimated that a dose
of 0.1kGy would damage 0.005% of the amino acids, 0.14% of the enzymes, and
2.8% of the DNA within a given cell (Pollard 1966). It is difficult to separate the
effects of genetic damage from the nongenetic damage of irradiation, and the
differentiation may not be of any practical value. However, one important aspect
of this point is that the damage is random and not related to a specific genetic locusor cell component. This is a significant factor in the elucidation of radiation resis-
tance of bacteria, especially in relation to the ability of microorganisms to develop
or acquire radiation resistance.
2.3. MECHANISMS OF MICROBIAL SURVIVAL AND REPAIR
Since the primary means of inactivation of microorganisms by radiation is damageto DNA, the mechanisms of survival and repair center on the repair of DNA. The
sensitivity of a microorganism to irradiation is often based on the efficiency of its
repair m echanisms for DN A, and organisms that have a mo re efficient DNA repair
mechanism are more resistant to irradiation. An extreme example of this is the
bacterium Deinococcus radiodurans, which was first identified as Micrococcus
radiodurans in foods that were thought to be sterilized by radiation (Brooks andMurray 1981). This bacterium is exceptionally resistant to radiation, as it has been
isolated from foods exposed to doses in the 35-4OkGy range. The enzymatic DNA
repair system within D. radiodurans is very efficient (Moseley 1976), while other
radiation resistant bacteria possess efficient excision mechanisms (Lavin et al.
1976), to remove damaged portions of the DNA.
In addition to the efficiency of DNA repair, another mechanism of survival for
microorganisms relates to the number of copies of a given gene within the DNA.
2 .4. RAD IATION SENSITIVITY OF SPECIFIC MICROO RGA NISMS
Bacterial populations increase in numbers by doubling; that is, one bacterium
reproduces by growing and dividing, forming two bacteria. On a population basis,
this becomes
b = (l x 2") (2.1)
where b is the bacterial population after n generations, beginning with a single cell.
In most cases, the growth from a single cell is limited to laboratory experimenta-
tion. Therefore
b = (B x 2") (2.2)
where b is the bacterial population after n generations, beginning with an initial
population of B cells. When the numbers of bacterial cells are converted to Iogi 0
values and plotted during the active phase of the growth curve (logarithmic growth),
the results form a straight line.
Bacterial populations also decline in a similar fashion after being subjected to
an environmental stress, such as heat or radiation. The kinetics of bacterial death
follows a first-order reaction, with the same proportion "or percentage killed over
time. To allow com parisons between different microorganism s and the same micro-organism under different conditions, a decimal reduction value is calculated.
This value is the amount of radiation required to reduce the population of a
specific bacterium by 90% (Ilog 1 0 cycle) under the stated conditions. The
bacteria that survive radiation doses at a higher-than-expected level. Although this
survival phenomenon is less well understood than the shoulder, it has been well
documented with radiation and with other environmental stresses. Although the
explanations for this phenomenon are mostly unsatisfactory, it is clear that this is
a subset of the population that exhibits this characteristic in response to environ-mental stress. This characteristic is not heritable, in that subcultures from the
survival tail do not exhibit higher radiation resistance than the homologous parent
population, which suggests that this is a response to environmental stress.
2.4 .1 . Bacteria of Public Health Significance
The application of any nonchemical antimicrobial process to foods can be regardedin terms of the number of logio reductions (D 1 0 values) required to achieve a
predetermined level of safety. A common target level of reduction in the United
States has been 5 Iog10 cycles (5Z)). Although there are limitations to the use of the
D 1 0 value (shoulder and tail effects) as described previously, it still provides a
standard point of reference for process evaluation and control. In addition to these
limitations, the effect of irradiation on the death of microorganisms can also be
significantly affected by environmental conditions and the nature of the food ma-
trix. This is discussed in more detail in Section 2.5. Tables 2.1 and 2.2 present D 1 0
values for selected bacteria of public health significance.
As can be seen from a review of the information in Tables 2.1 and 2.2, there is
wide variation in microbial sensitivity to irradiation. However, the greatest resis-
tance to radiation is seen with spore forming bacteria. Bacterial spores are more
resistant to radiation than vegetative cells, in part because of their extremely low
moisture content. A "typical" vegetative cell may be composed of as much as 70%
water, while the moisture content of a "typical" spore is less than 10%. Thereduced levels of moisture in spores minimize the secondary effects of irradiation,
with a net result of an increase in resistance to radiation.
2.4.2. Viruses
Although not as extensively researched as bacteria, there are data available on the
sensitivity of pathogenic viruses to radiation. Because of the biology of viruses,most notably the small size of their genetic material and a very low moisture
content, human viruses are even more resistant to radiation than bacterial spores.
Table 2.3 presents D 1 0 values for some viruses of public health significance. Food-
borne viruses account for a significant portion of foodborne disease in the United
States (Mead et al. 1999), but typically enter the food chain during preparation. A
typical viral outbreak would occur if a food preparation employee, ill with the
virus, were to subsequently contaminate food that was served to many people.
Irradiated foods would be equally susceptible to contamination at this point inthe food chain, with irradiation offering neither an advantage or disadvantage to
contamination. The resistance of viruses to radiation would only be a factor in
processing shellfish that would be consumed raw.
2.4.3. Parasites
Parasites of public health significance are far more sensitive to radiation than
are either bacteria or viruses. The parasite Trichinella spiralis has been the most
extensively studied in regard to radiation, with a report from 1921 demonstrating
the ability to control this parasite with radiation (Schwartz 1921). Further studies
have shown that a dose of 0.3 kGy is sufficient to eliminate the public health
concern regarding this parasite in pork (Brake et al. 1985). Other parasites, such
as Taeniarhynchus sag inatus (known as Cysticercus bovis in cattle), exhibit a
relatively high resistance to radiation [3 kGy, (Van Kooy and Robjins 1968)], but
are rendered noninfective at lower doses [0.4 kGy, (Tolgay et al. 1972)].
2.5. ENVIRONMENTAL FACTORS AFFECTING
RADIATION SENSITIVITY
The lethal effect of radiation on biological hazards is in part affected by theenvironmental conditions under which the organism is irradiated. The most sig-
nificant environmental factor is the temperature at which irradiation occurs. The
effect of temperature on the lethality of a given radiation dose is seen clearly during
irradiation at freezing and above-freezing temperatures. As an example, the D i 0
value for Clostridium botulinum type A is almost 1 kGy greater when the bacterium
is irradiated at freezing temperatures in comparison to refrigeration temperatures
[Table 2.1 (Anellis et al. (1977)]. Perhaps one of the best illustrations of this effect
has been reported with Escherichia coli O157: H7, where the reported D i 0 valuealmost doubled between + 5 0 C (0.2SkGy) and - 5 0 C [0.44 kGy (Thayer and Boyd
1993)]. This research clearly shows the biphasic response of the bacterium to
temperature, as the D 1 0 values were relatively constant at temperatures above
O0C, and were likewise relatively constant at irradiation temperatures below O0C.
The cause of this change in sensitivity to radiation is due to the change of state of
the water molecules in the cell. When the water is no longer in a liquid form, the
radiation chemistry of the water is changed, so that the secondary or indirect effects
of irradiation are minimized.Other environmental factors may also affect the sensitivity of microorganisms to
radiation. The composition of the medium in which the microorganism is sus-
pended may have a profound effect on radiation sensitivity. In one study, the
reported D i 0 value for Listeria monocytogenes in nutrient broth was 0.35 kGy,
but the D I O value in ground chicken was 0.77 kGy (Huhtanen et al. 1989). Another
study reported that the D 1 0 values for Sa lmonella senftenberg were 0.13 kGy (buf-
fer) and 0.56 kGy [bone meal (Ley et al. 1963)]. Many of these effects attributable
to media may, at a very basic level, also be attributable to the availability of waterin the medium.
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