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BACTERIA AND FOOD SAFETY
by
D. R. Martin
A Term Paper in Partial Fulfillment
Of the Requirements for
Food & Nutrition 506C
SCHOOL OF HUMAN ECOLOGY
COLLEGE OF APPLIED AND NATURAL SCIENCES
LOUISIANA TECH UNIVERSITY
October 27, 2015
Table of Contents
Page
Introduction..................................................................................................................................................1
Basics of Bacteria........................................................................................................................................1
Adaptation ......................................................................................................................................1
Resistance .......................................................................................................................................2
Identification..................................................................................................................................3
Mechanisms of Damage.............................................................................................................3
Water Activity ..............................................................................................................................................4
Effect During Cooking ................................................................................................................4
Effect on Bacteria Recovery ....................................................................................................5
Thermal Inactivation of Bacteria.........................................................................................................6
Quantification................................................................................................................................6
Parts of Cell Affected by Heat .................................................................................................7
Factors of Effectiveness ............................................................................................................7
Microwave Effects on Bacteria .............................................................................................................8
Cellular Response to Microwaves.........................................................................................9
Parts of Bacterial Cell Affected...............................................................................................9
Recovery of Bacteria from Microwave Damage ..........................................................10
Microwave vs Heat Direct Comparisons .......................................................................................11
Total Inactivation Time Differences .................................................................................11
Comparison of Affected Cell Structures ..........................................................................12
Effects on Proteins....................................................................................................................12
Shock Proteins ..........................................................................................................................................13
Cell Recovery Basics ................................................................................................................13
Effect of Shock Proteins on HACCP Effectiveness.......................................................13
Shared Adaptations..................................................................................................................14
Mechanisms of Adaptation ...................................................................................................15
Adaptations of Bacteria ........................................................................................................................16
Damage Quantified as Stress................................................................................................16
Cellular Structures that Change During Adaptation to Stress...............................16
Environmental Factors .........................................................................................................................17
Fat Content ..................................................................................................................................17
Osmolality ....................................................................................................................................18
pH.....................................................................................................................................................18
Inactivation Curves ..................................................................................................................19
Hurdle Approach.......................................................................................................................19
Summary .....................................................................................................................................................20
References ..................................................................................................................................................21
1
Introduction
The presence of bacteria in the food industry is a major public safety hazard.
Guidelines and critical control points for cooking food have been established with the
goal of reducing the microbial load of food to acceptable levels. In the kitchen, heat is the
primary weapon used against bacteria. The internal temperature achieved in food forms
the basis for food industry safe cooking guidelines.
An emerging issue in food safety is the rise of adaptations in bacteria that can
allow for survival following protocol thought sufficient for inactivation. Research by (De
Jong, Van Asselt, Zwietering, Nauta, & De Jonge, 2012) demonstrates how chicken meat
can reach a safe cooking temperature and still be pathogenic. The authors reason that the
cause must either be re-contamination after cooking or that the bacteria worked down
into the tissues and were more resistant to the heat treatment. Specific adaptations to be
discussed include heat, pH, cold, pressure, antibiotics, and acid resistance.
Recognition of the events that lead to bacterial adaptation will allow for
development of more efficient systems to contain microbial growth along the entire food
production process. New strategies in food safety will need to include factors beyond heat
activation to ensure public safety. Sources of bacterial adaptation, mechanisms of
adaptation, and review of food related anti-pathogenic techniques are the focus of this
research.
Basics of Bacteria
Adaptation
When microbes persist in food cooked to temperatures considered safe, there is
likely to be a subpopulation of the bacteria that naturally express a gene for resistance or
2
has acquired it through adaptation. Bacterial adaptation is not immediately obvious. A
specific test performed on isolated bacteria under strict procedure is required to identify a
trend. Growth curves of laboratory induced high hydrostatic pressure (HHP) resistance
showed no significant difference from that of their parent strains (Vanlint, Rutten,
Michiels, & Aertsen, 2012).
Resistance
When observing bacterial samples exhibiting extreme HHP resistance, it was
observed that most of the cells in culture could not survive modest pressure (Vanlint et
al., 2012). This led researchers to assert that subpopulations of these strains maintain
extreme HHP resistance while the majority of cells are destroyed, a phenomenon known
as persistence. E. coli shows natural HHP resistance, but an isolate has been shown to
possess no resistance at all (Vanlint et al., 2012). This is evidence that there is specific
genetic programming that imparts HHP resistance.
Resistance to various stressors can increase depending on the intensity, duration,
and frequency of the treatment that does not kill the persistent subpopulation. This is
largely due to compositional changes of the cell membranes and induction of regulatory
systems that fortify and repair the cell. As such, the structure of bacteria and its
interaction with the environment are the targets of intervention. Damage to cellular
structures can inactivate bacteria or destroy them completely.
Lysis of the cell occurs when the membranes have been critically compromised
and the cell no longer exists as a coherent structure. Inactivation of bacteria does not
destroy it, but damages it to the extent that it cannot affect other cells or reproduce. The
3
structure of bacteria differs mainly in the structure of the membranes that contain, direct,
and protect the cell.
Identification
The primary distinction of bacteria is based on Gram staining to identify the
membranes of the cells. Cells are prepped, stained, and rinsed before observation under a
light microscope. Gram-positive bacteria have a cytoplasmic membrane and a thick cell
wall that allows the chemical stain to remain in peptidoglycan. The cell envelope of
gram-negative bacteria consists of the inner cytoplasmic membrane, the periplasm
containing the peptidoglycan network, and a thin outer membrane (Campbell & Reece,
2008). Gram-negative bacteria do not have a cell wall and are not stained during the
treatment, as the outer membrane does not protect the periplasm from the chemical rinse.
Mechanisms of Damage
Chemical damage to the cell occurs in the outer membrane or cell wall first as it
protects the cytoplasmic layer. Gram-positive bacteria exhibit greater chemical resistance
than gram-negative bacteria due to the cell wall (Somolinos, Garcia, Condon, Mackey, &
Pagan, 2009). Research by (Aljarallah & Adams, 2007) demonstrates that heat treatment
damages the outer membrane of Salmonella cells and makes the bacteria more
susceptible to subsequent injury. The outer membrane acts as a selective permeation
barrier (Lefevre, Delepelaire, Delepierre, & Izadi-Pruneyre, 2008), making it integral to
maintaining metabolism and homeostasis of the cell.
Under normal conditions, the human body’s natural serum can disrupt the outer
membrane of gram-negative bacteria and cause the release of lipopolysaccharides (LPS)
as the cell dies. (O’Hara, Moran, Wurzner, & Orren, 2001) LPS release leads to
4
activation of the compliment system, which leads to the formation of the membrane
attack complex. LPS can be used as a measure of cell disruption and give insight to the
initiation of one of the regulatory systems.
Naturally occurring temporins regulate permeability of the cell membrane that
allows for intracellular components to leak from the cytoplasmic membrane (Rinaldi et
al., 2001). The natural cellular property of fluidity and ability to open is exploited and
amplified by application of microwaves to food as will be discussed. Another pore
targeted anti-microbial pathway is the opening of pores in the cytoplasmic membrane by
nisin (Garcera, Elferink, Driessen, & Konings, 1993). It was demonstrated that the outer
membrane of the bacteria must be compromised for nisin action to inactivate bacteria.
Interestingly, acidic pH increased the effectiveness of nisin (Garcera, et al., 1993).
Water Activity
Water activity (aw) is the measure of free water in food. Free water is available for
bacteria to use and is a factor in how foods should be stored to prevent spoilage. Water
activity can also be a factor during cooking as dry heat techniques do not add water and
even reduce the aw while ingredients in soup experience saturation.
Effect During Cooking
The amount of water present during cooking is important, as aw is a variable in the
movement of heat. Transfer of heat is variable among cooking methods and can affect
cooking times and temperatures needed to ensure inactivation of bacteria. Convection
heating methods will raise the temperature of foods more evenly than conduction
methods. Traditional methods of heating food introduce heat to the surface of food and
5
allow it to penetrate for some time. Microwaves vibrate water inside food to generate
heat from within. Foods will heat much more quickly and thoroughly in a microwave
compared to any form of traditional heat.
The evidence on the effect of aw during cooking is consistent. Research by
(McCann, McDowell, & Sheridan, 2009) found that lower aw during cooking increases
heat resistance in beef. Much higher temperatures were needed to inactivate S.
typhimurium on dry meat compared to moist meat (McCann et al., 2009). Researchers
postulate that the proteins damaged by heat are more stable in a dry state. Increased water
activity was found to reduce thermal resistance of Salmonella sp. in peanut butter (He, Li,
Salazar, Yang, Tortorello, & Zhang, 2013).
Effect on Bacterial Recovery
Water activity also has an effect on bacteria recovering from injury. This gives
insight to one of the systems of repair, which can be exploited to increase effectiveness in
reducing pathogens. Researchers found that damaged cells in a high aw test group were
able to repair if no further injury was inflicted. (Aljarallah & Adams, 2007). However,
the low aw group experienced cell death rather than injury (Aljarallah & Adams, 2007).
Heat stress in a low aw kills rather than injure bacteria, presumably from the osmotic
stress and inability to pull substrate back into the cell necessary for repair.
High aw during cooking would provide for faster and more efficient heating. High
aw would subject the microbial load to the heat stress more intensely and for a longer
duration than low aw. Conversely, high aw also allows for the flow of cellular components
back into the cell following sub-lethal injury and may only temporarily reduce the
microbial load to safe levels while the survivors recover.
6
Thermal Inactivation of Bacteria
Regulatory guidelines for food safety are based on thermal inactivation of
bacteria. Much research has confirmed that heat is an effective treatment against most
food borne bacteria. Pertinent variables to bacterial inactivation include: temperature,
time at temperature, the species of bacteria, and the environment the food is cooked in.
Destruction or inactivation is the goal of any antimicrobial measure. Traditionally this
concept has been expressed through first order kinetics.
Quantification
First order kinetics reduces the time to thermal inactivation of a bacterium at a
temperature to a straight-line graph. The D-value is the time at a particular temperature
necessary to destroy 90% of the viable cells or spores of a specific organism (Juneja,
Huang, & Yan, 2011). This is a helpful standard to compare rates of inactivation between
pathogens and testing variables. The z-value, an indicator of temperature sensitivity, is
the change in heating temperature needed to change the D-value by 90% or 1 log cycle
(Juneja et al., 2011).
Represented as a line of best fit, the D-value suffers in the ability to fully
represent the inactivation curve of a bacterium at a specific heat. There are two important
deviations in the bacterial thermal inactivation curve not accounted for by first order
kinetics; the shoulder effect and tailing. The shoulder effect is observed when bacteria
remain unaffected by the tested heat for a period before displaying inactivation (Juneja et
al., 2011). Tailing occurs when inactivation slows and an appreciable microbial load
remains viable for a period before becoming inactivated (Juneja et al., 2011). These
7
effects can influence the cooking time necessary to reduce the microbial load and also
help identify subpopulations of bacteria.
Parts of Cell Affected by Heat
The actual cause of cell death or inactivation is difficult to pin down. There is
observable injury to multiple systems following heat stress, which compound to quantify
total damage dealt to an organism during treatment. Membranes can be compromised or
modified, proteins can be denatured or aggregated, and osmotic forces can crush or rip
apart a cell. The total damage to the functional components of the bacteria determines the
effectiveness of a treatment.
Factors of Effectiveness
The higher the initial microbial population in a food, the longer the heating time
at a given temperature required to achieve destruction of the population (Juneja et al.,
2011). Heat required is based on estimated microbial load of ingredients, which can vary
depending on initial growing environment, packaging, and treatment of the food. The
presumed microbial load is one of the keys to how temperature guidelines are supposed
to work. Only when food has been properly handled during the entire process does the
guideline apply. Temperature abuse can increase microbial load to levels that may be
unsafe when cooked to normal guidelines.
Damage to the cell can occur that is not readily visible. Heat can cause
modifications of the cytoplasmic membrane of gram-positive bacteria and the outer
membrane of gram-negative bacteria to allow for greater binding capacity of antibodies
(Kolberg, Hammerschmidt, Frank, Jonak, Sanderova, & Aase, 2008). Heat treatment can
also modify aw that contributes to cell function and repair.
8
Ribosomes are necessary for protein synthesis of the cell and can be damaged by
heat. Research by (Aljarallah & Adams, 2007) shows ribosomes to be stable at lower
inactivation temperatures in both low and high aw tests. Higher temperatures showed
ribosomal inactivation as the major stress causing cellular inactivation (Aljarallah &
Adams, 2007). High heat denatured the 30S ribosomal unit and proteins in the cytoplasm.
Research by (Capozzi, Fiocco, Amodio, Gallone, & Spano, 2009) also demonstrates that
heat can denature proteins and impair enzyme activities in bacteria.
Heat is a proven tool against food borne pathogens; the key is to achieve
sufficient heat for a duration that inactivates the entire microbial load. Shiga toxin
producing E. coli can persist the mozzarella making process by inadequate heating
throughout the entire curd mass (Trevisani, Mancusi, & Valero, 2014). Insufficient
heating is often observed in an attempt to maintain the aesthetic, texture, and flavor of
food.
Boiling Salmonella for three minutes is sufficient to completely inactivate it,
while boiling for two minutes is not (Miaoyun, Zhao, Liu, Gao, & Zhang, 2013). Heat
treatment at 65°C required 20 minutes for complete inactivation during the same trial
(Miaoyn et al., 2013). Visual observation showed darkening, surface irregularity, and
shrinking of heat-treated cells compared to non-treated. It has been observed that brief
steam treatment of lettuce is as effective as chlorine treatment (Capozzi et al., 2009).
Microwave Effects on Bacteria
Microwave ovens have become a part of many kitchens. Expedited reheating of
food is not the only benefit of the home microwave oven; it is also an effective tool
9
against food borne bacteria. Microwaves penetrate the entire food and vibrate water
molecules, increasing the temperature quickly. Pathogens are destroyed or inactivated
from the same thermal processes observed in traditional wet and dry heating methods.
Cellular Response to Microwaves
Microwaves also have the effect of opening large pores in the cellular membranes
of bacteria (Shamis et al., 2013). This compromises osmotic integrity and allows for
substances to leave and enter the cell. Treatment of bacteria in the presence of microwave
radiation, sub-lethal temperature, and fluorescent dextran showed significantly higher
uptake of dextran in the microwave treatment compared to control (Shamis et al., 2011).
Pore formation induced by magnetic radiation allowed dextran to flow into the cell.
Parts of Bacterial Cell Affected
Microwave induced modulation of pores in the membrane appears to be short
lived. Bacteria subjected to microwave radiation at sub-lethal temperatures show pore
formation during radiation that was found to be temporary (Shamis et al., 2011). Pores
formed by microwave radiation resolved back to normal within ten minutes of finishing
the treatment. In the experiment, 88% of cells remained viable, indicating that microwave
manipulation of the cell membrane is not enough to inactivate bacteria. The same
radiation treatment performed at 45°C yielded almost complete bacterial inactivation
(Shamis et al., 2011).
The opening of pores in the membranes of bacteria allows for intracellular
substrate to be lost, hindering function and ability to repair. Nucleic acids have been
shown to leak from the cell during treatment, positively relative to the microwave-heated
temperature of the cell suspension (Woo, Rhee, & Park, 2000). When comparing a gram-
10
positive bacteria (B. subtilis) and gram-negative bacteria (E. coli), it was observed that
the gram-positive bacteria leaked more protein from the cytoplasm while the gram-
negative bacteria leaked more nucleic acid (Woo et al., 2000).
Recovery of Bacteria from Damage
Microwave treatment at sub-lethal temperature has been shown to shrink the size
of bacterial cells (Shamis et al., 2011). The shrinkage was reversible as the cells quickly
regained their original size, presumably via dehydration and re-hydration. The extent of
damage determines ability of the cell to recover. Increased time subjected to microwaves
leaves bacteria less able to recover (Benjamin et al., 2009). In regard to the effect of
environmental water, no difference in inactivation times were observed when
microwaving vegetative B. subtilis cells in suspension or dry (Kim, Jo, Kim, Bai, & Park,
2008).
The surface of gram-positive B. subtilis appears undamaged by microwave
irradiation even when the cells are damaged to the point of inactivation (Woo et al.,
2000). This would lead to the conclusion that while surface damage of bacteria occurs
during irradiation, it is not the only or most lethal effect of the process. The surface of
gram-negative E. coli cells changed from smooth to rough, swollen, and damaged
following microwave irradiation (Woo et al., 2000). Microwaves have been shown to
disintegrate cell walls in gram-negative bacteria (Zhou, Shin, Hwang, Ahn, & Hwang,
2010).
The presence of a thick cell wall protects gram-positive bacteria from many
chemical stressors. Microwaves may not visibly damage the cell wall, but are not
inhibited by it. Gram-positive B. subtilis was found to be more sensitive than Gram-
11
negative E. coli during microwave treatment at 60°C (Woo et al., 2000). Both gram-
positive and negative cells exhibit dark spots in the cytoplasm following microwave
irradiation compared to control. This is thought to be protein aggregation and a possible
pathway of bacterial inactivation during irradiation (Woo et al., 2000). Microwave
treatment of foods can also induce creation of hydrogen peroxide molecules that
contribute to destruction of bacteria (Kim et al., 2008).
Both commercial and household microwave ovens generate enough energy to
inactivate bacteria. Inactivation rates of B. subtilis are higher in commercial 2.0W
microwave ovens than in household 0.5W units (Kim et al., 2008). Research by
(Tremonte et al., 2014) also found higher-powered microwaves to be more effective at
inactivating bacteria.
It has been observed that cell density in cell suspensions following microwave
treatment did not decrease despite significant reduction in viable cells (Woo et al., 2000).
Most bacterial cells, both gram- positive and negative, remain osmotically relevant after
irradiation (Woo et al., 2000). This suggests that the cells themselves remain unlysed
while being functionally inactivated by the microwaves.
Microwave vs. Heat Direct Comparisons
As both traditional and microwave heating techniques are proven to inactivate
bacteria, the choice becomes one of convenience, time, and efficiency.
Total Inactivation Time Differences
Complete destruction of B. subtilis has been shown to occur in three minutes of
0.5kW microwave power, two minutes of 2kW microwave power, and 10 minutes of
12
boiling (Kim et al., 2008). Microwaves can disrupt DNA in coliforms at lower
temperatures than external heating (Kim et al., 2008). Coliforms subjected to microwaves
are destroyed at 60°C compared to 100°C with traditional boiling (Kim et al., 2008).
Comparison of Affected Cell Structures
Research on raw milk shows a high microbial load can be completely eliminated
by boiling treatment (Tremonte et al., 2014). Boiling milk also led to a significant
reduction in whey proteins. Microwaving milk for 75s at 900W yielded similar bacterial
inactivation but did not negatively impact the proteins of the milk (Tremonte et al.,
2014). Research by (Kim et al., 2008) verifies that microwaving increases observed
denaturation-induced aggregation and cell envelope damage compared to boiling.
Boiled bacterial cells show degradation of the local cytoplasmic membrane and
several protein aggregations in the cytoplasm (Kim et al., 2008). Significant damage to
the cell membrane of B. subtilis was observed in microwave treatments compared to
boiling treatments (Tremonte et al., 2014). Microwave-irradiated cells have shown
degradation of the cytoplasmic membrane, collapse of the cell wall, and disruption of
cytoplasmic structures (Kim et al., 2008). Research by (Kim et al., 2008) also found that
boiling and microwaving B. subtilis both induced damage to the cytoplasmic membrane,
but only microwaving treatment collapsed the cell wall.
Effects on Proteins
Both treatments damage proteins in the cytoplasm causing aggregation (Kim et
al., 2008). In vegetative cells, no difference in protein leaking was observed but there was
an increase in leaked nucleic acids in the microwave test group cells compared to boiling
13
(Kim et al., 2008). This could be due to the large size of proteins compared to nucleic
acids and inability of proteins to leave the cell.
Shock proteins
When bacteria cells are stressed or damaged, they are able to mobilize shock
proteins that work to repair and fortify the cell.
Cell Recovery Basics
Damage to the cell envelope precludes activation of the extra-cytoplasmic stress
response (Toni, Jovanovic, Huvet, Buck, & Stumph, 2011). Heat shock proteins respond
to increased temperatures by functioning as “chaperones” to direct the repair of damage
and prevention of injury to the cell (Capozzi et al., 2009).
Effect of Shock Proteins on HACCP Effectiveness
This is important due to the fact that accepted cooking guidelines are not based on
stress-shocked bacteria. Any improved resistance, especially thermoresistance, could
result in incomplete inactivation of bacteria in food cooked to the appropriate
temperature. Heat shock has been shown to increase thermotolerance of Salmonella sp.
(Walsh et al., 2005). Heat resistant subpopulations of E. coli have been shown to grow
uninhibited at temperatures greater than the established Tmax point for the strain (Van
Derlinden, Lule, Bernaerts, & Van Impe, 2009). Heat shocked samples of S. typhimurium
were found to be more heat resistant than samples not heat shocked (Walsh et al., 2005).
The SoxRS and MarRA systems recognize stressors of the bacterial cell and
activate stress response pathways including WaaY (Lee et al., 2009). The genes
expressed by the SoxRS pathway are responsible for repairing damaged DNA,
14
maintaining red-ox balance, and defending against cellular radicals (Lee et al., 2009). The
mar regulon confers resistance to many antibiotics while SoxRS targets super oxides and
nitric oxides (Lee et al., 2009). Radical oxygen and nitrogen species are a significant
threat to bacteria as they are capable of damaging proteins, RNA, DNA, and the lipids of
membranes (Capozzi et al., 2009). When one system is activated, there is overlap in
binding of promoters to activators and multiple resistances can be gained from reaction to
a single stressor.
Shared Adaptations
Research by (Vanlint et al., 2012) found that multiple bacterial strains subjected
to heat-shock increased expression of DnaK. The same parent strains subjected to HHP
shock showed no increase in expression of DnaK (Vanlint et al., 2012). Taken together
the findings suggest that different pathways are used to confer resistance to HHP than
heat. Acids can also activate DnaK and GroEL, leading to the conclusion that some
regulatory pathways are redundant and employ the same defense reactions (Capozzi et
al., 2009). Similar heat shock proteins have been identified in non-challenged bacteria.
The proteins DnaK and GroEL have been observed in non heat-challenged E. coli and
Salmonella typhimurium (Capozzi et al., 2009).
Sub-lethal pressure shock can also activate the gene expression of heat shock
proteins (Aertsen et al., 2009) Researchers observed that these genes expressed
themselves more slowly than heat shock activation but that once activated, exhibited
similar levels of resistance.
Multiple rounds of shock and adaptation of the surviving organisms’ compound
and can yield highly resistant bacteria. Research has also shown that multiple HHP
15
shocks yield extremely HHP resistant bacteria when allowed to recover between
treatments (Vanlint et al., 2012). Various abuses along the food processing process could
result in multiple resistances of unknown strength.
Mechanisms of Adaptation
It has been observed that proteins move to the poles of bacterial cells when
subjected to heat and that the relocation is crucial to development of heat tolerance
(Zietkiewicz & Liberek, 2010). The proteins must be unfolded to their original shape for
them to be functional in the cell again. In experiments where the proteins were degraded
rather than disaggregated, thermoresistance was not observed (Zietkiewicz & Liberek,
2010). Membrane damage also dissipates the proton motive force. This force is
responsible for moving proteins to the proper place in the cell membrane to repair
damage (Toni et al., 2011).
It has been observed that Heat Shock Protein sigma32 is transported by signal
recognition particle (SRP) and implanted into the cytoplasmic membrane where it
contributes to regulation of homeostasis in the cell (Lim et al., 2013). Research by
Robinson (2013) confirms that SRP must insert sigma32 into the cytoplasmic membrane
for the heat shock protein to regulate the membrane and cytoplasm. Impairment of SRP
pathway reduces ribosome formation and inhibits protein synthesis within the bacteria
(Burk et al., 2009).
Cold shock proteins are released in an attempt to stabilize the bacteria. One of the
main ways cold shock proteins stabilize the membrane is by altering the composition of
fatty acids to make the membrane more rigid (Capozzi et al., 2009).
16
Adaptations of Bacteria
Damage Quantified as Stress
Stress is used to inactivate and kill bacteria. The subpopulations that survive the
treatment are more capable genetically and likely to have gained new adaptations. As
seen in shock protein activation, survival of one stress can increase the resistance of the
organism to multiple stresses. Many strains of Salmonella sp. are now multi-antibiotic
resistant (Walsh et al., 2009). Research has demonstrated that the stress response can be
shared between heat, cold, acid, and osmotic stress (Capozzi et al., 2009). This cross
protection is worrisome as multiple uncoordinated, sub-lethal measures may increase
resistance to previously effective treatments.
When comparing bacterial survivors heat-challenged at different temperatures,
those challenged at a higher heat exhibited a significantly longer shoulder period than the
lower heat group (Aljarallah & Adams, 2007). Higher growth temperatures of bacteria
have been found to increase heat tolerance (Van Derlinden et al., 2009).). The more
slowly the temperature is increased, the greater the capacities of the bacteria to adapt,
survive, and form resistance (Van Derlinden et al., 2009). Research by (Capozzi et al.,
2009) found that heat adaptation could increase resistance to heat and pH in bacteria.
Cellular Structures that Change During Adaptation to Stress
When stressed, bacterial cells can alter the structure of the LPS in the outer
membrane (Lee, Lee, Yeo, Park, & Roe, 2009). This correlates with increased resistance
to antibiotics, oxidants, and other drugs. Gram-positive Corynebacterineae expresses the
NCgl2775 gene in response to stress, which changes the lipid composition of the outer
membrane (Meniche et al., 2009). Researchers found the gene induced compositional
17
change of the membrane increases the heat resistance of the bacteria. The NCgl2775 gene
has also been observed in mycobacterium under normal conditions (Meniche et al.,
2009). In response to heat, the ratio of saturated mycolic acids to unsaturated mycolic
acids increases as well as the ratio of saturated fatty acids to unsaturated fatty acids
(Meniche et al., 2009). Cold temperatures affect bacteria negatively in multiple ways
such as: decrease in membrane fluidity, impaired protein synthesis, and impaired
ribosome function (Capozzi et al., 2009).
Not all bacterial strains adapt to the same extent. Bacteria subjected to
increasingly severe HHP show vastly different abilities in regard to the pressure
withstood and degree of inactivation at each pressure (Vanlint et al., 2012). When
comparing laboratory induced HHP resistant strains to their parents during successive
generations grown out with no HHP stress it was found that HHP resistance decreased in
both groups (Vanlint et al., 2012). Despite an observable decrease in resistance, it was
observed that after 80 generations with no HHP stress, the HHP resistant strain still
maintained elevated HHP resistance compared to the original.
Environmental Factors
Heat is the primary tool against food borne pathogens, but the environment of the
pathogen must be considered. Just as there are genetic adaptations that can increase
resistance, there are environmental factors that can make complete inactivation more
difficult than expected.
Fat Content
18
Animal type, muscle configuration, pH, fat content and other environmental
factors influence bacterial heat resistance (Juneja, 2007). Fat content can play a
significant role in heat resistance. Higher fat content requires longer heating to inactivate
bacteria, which could be due to lower thermal conductivity or reduced aw (Osalili et al.,
2007). Thick meats take longer for traditional heat to permeate and reach temperatures
lethal to bacteria (Juneja et al., 2011). The longer time necessary to inactivate bacteria in
certain meats and/or cuts represents how environmental factors can induce the shoulder
effect.
Osmolality
Osmotic environment plays a strong role in the ability of bacteria to grow and
reproduce. Bacterial growth is most rapid at high (0.99) aw, while no microbial growth
occurs below 0.6 aw (Capozzi et al., 2009). Bacterial cells must maintain osmotic balance
in the environment and are able to increase uptake of substrate from media to synthesize
solutes that stabilize osmotic pressure (Capozzi et al., 2009). Higher heat resistance to
Campylobacter was seen in pan-fried chicken then in chicken boiled in water (de Jong et
al., 2012).
pH
The pH of food is an important factor influencing microbial heat resistance.
Microorganisms exhibit greatest heat resistance at pH-values close to neutral, with low
and high pH-values generally decreasing heat resistance (Juneja, 2007). Heat is only
effective in destroying the toxin cereulide in pH of over 9 (Rajkovic et al., 2008). At a pH
of 7 the toxin was stable after two hours at 121°C.
19
Acids are effective in damaging bacterial cells. It is hypothesized that in the
presence of acids, protons are driven into the interior of the cell (Capozzi et al., 2009).
The movement of protons inward is damaging to the bacteria in two ways: it dissipates
the proton motive force on the surface while decreasing the pH of the interior (Capozzi et
al., 2009).
Inactivation Curves
A practical representation of the tailing effect can be seen in the mixed culture
model. Assuming that a culture consists of two different strains of bacteria with different
capacities to resist heat, as represented by D-value, the fraction of lower D-value will be
preferentially inactivated by heat, leaving the strain with higher D-value to survive for a
longer time (Juneja et al., 2011). Infected or compromised food is likely to have more
than one pathogen present, which could increase the time at temperature necessary to
completely inactivate the microbial load.
Hurdle Approach
One of the growing strategies in the food service industry to reduce pathogens is
known as the “hurdle” approach. This approach is based on the observation that
antimicrobial factors can act synergistically, their combined effect being greater than the
sum of the individual factors (Aljarallah & Adams, 2007). The hurdle principle was
demonstrated by (Somolinos et al., 2009) in combining mild heat treatment with citral
essential oil application to achieve inactivation of E. coli. Microwave heating is an
example of the hurdle effect in that the cell membranes are compromised in the presence
of heat. Microwaves are more efficient at inactivating bacteria under the same conditions,
lower heat, and shorter times than traditional heat.
20
Summary
Bacteria in food are able to adapt to stress and gain multiple types of resistance.
The method of heating food determines the primary way the bacteria are thermally
stressed. Increased water activity enhances heating speed and thoroughness, but can also
facilitate bacterial recovery. Proteins are more resistant to heat in a low water activity
environment.
Microwaves generate heat from within while traditional methods apply heat
externally. Microwaves show great potential for inactivating bacteria during cooking as
they heat more efficiently and provide a hurdle effect of enlarging pores in the bacterial
cell. Damage to bacterial cells must be comprehensive to ensure food safety.
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References
Aertsen, A., Vanoirbeek, K., De Spiegeleer, P., Sermon, J., Hauben, K., Farewell, A., Nystrom, T., & Michiels, C. W. (2004). Heat shock protein-mediated resistance to high hydrostatic pressure in Escherichia coli. Applied and Environmental Microbiology, 70, 2660-2666.
Aljarallah, K. M., & Adams, M. R. (2007). Mechanisms of heat inactivation in Salmonella serotype typhimurium as affected by low water activity at different temperatures. Journal of Applied Microbiology, 102, 153-160.
Benjamin, E., Reznik, A., Benjamin, E., Pramanik, S. K., Sowers, L., & Williams A. L. (2009). Mathematical models for Enterococcus faecalis recovery after microwave water disinfection. Journal of Water and Health, 7, 699-706.
Burk, J., Weiche, B., Wenk, M., Boy, D., Nestel, S., Heimrich, B., & Koch, H. (2009). Depletion of the Signal Recognition Particle Receptor inactivates ribosomes in Escherichia coli︎. Journal of Bacteriology, 191, 7017-7026.
Campbell, N. A., & Reece, J. B. (Eds.). (2008). Biology, 557. San Francisco: Pearson Benjamin Cummings.
Capozzi, V., Fiocco, D., Amodio, M. L., Gallone, A., & Spano, G. (2009). Bacterial stressors in minimally processed food. International Journal of Molecular Sciences, 10, 3076-3105.
De Jong, A. E. I., van Asselt, E. D., Zwietering, M. H., Nauta, M. J., & de Jonge, R. (2012) Extreme heat resistance of food borne pathogens Campylobacter jejuni, Escherichia coli, And Salmonella typhimurium on chicken breast fillet during cooking. International Journal of Microbiology, 2012, 1-10. doi:10.1155/2012/196841
Garcera, M. J. G., Elferink, M. G. L., Driessen, A. J. M., & Konings, W. N. (1993). In vitro pore-forming activity of the lantiviotic nisin – role of protonmotive force and lipid composition. European Journal of Biochemistry, 212, 417-422.
He, Y., Li, Y., Salazar, J. K., Yang, J., Tortorello, M. L., & Zhang, W. (2013). Increased water activity reduces the thermal resistance of Salmonella enterica in peanut butter. Applied and Environmental Microbiology, 79, 4763-4767.
Juneja, V. K. (2007). Thermal inactivation of Salmonella spp. in ground chicken breast or thigh meat. International Journal of Food Science and Technology, 42, 1443-1448.
Juneja, V. K., Huang, L., & Yan., X. (2011). Thermal inactivation of foodborne pathogens and the USDA pathogen modeling program. Journal of Thermal Analysis & Calorimetry, 106, 191-198.
22
Kim, S. Jo, E., Kim, H., Bai, K., & Park, J. (2008). The effects of high-power microwaves on the ultrastructure of Bacillus subtilis. Letters in Applied Microbiology, 47, 35-40. **68,
Kolberg, J., Hammerschmidt, S., Frank, R., Jonak, J., Sanderova, H., & Aase, A. (2008). The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococci. Immunological Medical Microbiology, 53, 222-230.
Lee, J., Lee, K., Yeo, W., Park, S., & Roe, J. (2009). SoxRS-mediated lipopolysaccharide modification enhances resistance against multiple drugs in Escherichia coli. Journal of Bacteriology, 191, 4441-4450.
Lefevre, J., Delepelaire, P., Delepierre, M., & Izadi-Pruneyre, N. (2008). Modulation by substrates of the interaction between the HasR outer membrane receptor and its specific TonB-like protein, HasB. Journal of Molecular Biology, 378, 840-851.
Lim, B., Miyazaki, R., Neher, S., Siegele, D. A., Ito, K., Walter, P., Akiyama, Y., Yura, T., & Gross, C. A. (2013). Heat shock transcription factor s32 co-opts the Signal Recognition Particle to regulate protein homeostasis in E. coli. PLoS Biology, 11, e1001735. doi:10.1371/journal.pbio.1001735
McCann, M. S., McDowell, D. A., & Sheridan, J. J. (2009). Effects of reduction in beef surface water activity on the survival of Salmonella typhimurium DT104 during heating. Journal of Applied Microbiology, 106, 1901-1907.
Meniche, X., Labarre, C., de Sousa-d’Auria, C., Huc, E., Laval, F., Tropis, M., Bayan, N., Portevin, D., Guilhot, C., Daffe, M., & Houssin, C. (2009). Identification of a stress-induced factor of Corynebacterineae that is involved in the regulation of the outer membrane lipid composition. Journal of Bacteriology, 191, 7323-7332.
Miaoyun, L., Zhao, G., Liu, J., Gao, X., & Zhang, Q. (2013). Effect of different heat treatments on the degradation of Salmonella nucleic acid. Journal of Food Safety, 33, 536-544.
O’Hara, A. M., Moran, A. P., Wurzner, R., & Orren, A. (2001). Complement-mediated lipopolysaccharide release and outer membrane damage in Escherichia coli J5: requirement for C9. Immunology, 102, 365-372.
Osalili, T. M., Griffis, C. L., Martin, E. M., Beard, B. L., Keener, A. E., & Marcy, J. A. (2007). Thermal inactivation of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in breaded pork patties. Journal of Food Science, 72, 56-61.
Rajkovic, A., Uyttendaele, M., Vermeulen, A., Andjelkovic, M., Fitz-James, I., in ‘t Veld, P., Denon, Q., Verhe, R., & Debevere J. (2008) Heat resistance of Bacillus cereus emetic toxin, cereulide. Letters in Applied Microbiology, 46, 536-541.
23
Rinaldi, A. C., Di Giulio, A., Liberi, M., Gualtieri, G., Oratore, A., Schinina, M. E., Simmaco, M., & Bozzi, A. (2001). Effects of temporins on molecular dynamics and membrane permeabilization in lipid vesicles. Journal of Peptide Research, 58, 213-220.
Robinson, R. (2013). Heat shock response regulator is pinned to the membrane. PLoS Biology, 11, e1001736. doi:10.1371/journal.pbio.1001736
Shamis, Y., Taube, A., Mitik-Dineva, N., Croft, R., Crawford, R. J., & Ivanova, E. P. (2011). Specific electromagnetic effects of microwave radiation on Escherichia coli. Applied Environmental Microbiology, 77, 3017-3022.
Somolinos, M., Garcia, D., Condon, S., Mackey, B., & Pagan, R. (2009). Inactivation of Escherichia coli by citral. Journal of Applied Microbiology, 108, 1928-1939.
Toni, T., Jovanovic, G., Huvet, M., Buck, M., & Stumpf, M. P. H. (2011). From qualitative data to quantitative models: analysis of the phage shock protein stress response in Escherichia coli. BioMed Central Systems Biology, 5, 1-15. doi:10.1186/1752-0509-5-69
Tremonte, P., Tipaldi, L., Succi, M., Pannella, G., Falasca, L., Capilongo, V., Coppola, R., & Sorrentino, E. (2014). Raw milk from vending machines: effects of boiling, microwave treatment, and refrigeration on microbiological quality. American Dairy Science Association, 97, 3314-3320.
Trevisani, M., Mancusi, R., & Valero, A. (2014). Thermal inactivation kinetics of shiga toxin-producing Escherichia coli in buffalo mozzarella curd. Journal of Dairy Science, 97, 642-650.
Van Derlinden, E., Lule, I., Bernaerts, K., & Van Impe, J. F. (2009). Quantifying the heterogeneous heat response of Escherichia coli under dynamic temperatures. Journal of Applied Microbiology, 108, 1123-1135.
Vanlint, D., Rutten, N., Michiels, C. W., & Aertsen, A. (2012). Emergence and stability of high-pressure resistance in different food-borne pathogens. Applied and Environmental Microbiology, 12, 3234-3241.
Walsh, C., Duffy, G., Sheridan, J. J., Fanning, S., Blair, I. S., & McDowell, D. A. (2005). Thermal resistance of antibiotic-resistant and antibiotic-sensitive salmonella spp. on chicken meat. Journal of Food Safety, 25, 288-302.
Woo, I., Rhee, I., & Park, H. (2000). Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure. Applied and Environmental Microbiology, 66, 2243-2247.
Woo, I., Rhee, I., & Park, H. (2000). Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure. Applied and Environmental Microbiology, 66, 2243-2247.
24
Zhou, B. W., Shin, S. G., Hwang, K. H., Ahn, J., & Hwang, S. (2010). Effect of microwave irradiation on cellular disintegration of Gram positive and negative cells. Applied Microbiological Biotechnology, 87, 765-770.
Zietkiewicz, S., & Liberek, K. (2010). Dispose to the pole – protein aggregation control in bacteria. The EMBO Journal, 29, 869-870.
