CHAPTER 33

Control by Low Temperature

CONTENTS

I. Inroduction ................................................................................................ 467

II. Objectives .................................................................................................. 468
III. Mechanisms of Antimicrobial Action ....................................................... 469
IV. Influencing Factors .................................................................................... 470
A. Nature of Process .............................................................................. 470
B. Nature of Food .................................................................................. 471
C. Nature of Microorganisms ................................................................ 471
V. Methods ..................................................................................................... 472
A. Ice Chilling ....................................................................................... 472
B. Refrigeration ..................................................................................... 472
C. Freezing ............................................................................................. 473
VI. Conclusion ................................................................................................. 473
References ............................................................................................................. 474
Questions ............................................................................................................... 474

I. INTRODUCTION

The effectiveness of low temperature, especially freezing, in food preservation was

probably recognized by our ancestors at least in the last Ice Age, before 40,000 B.C.
Natural freezing and thawing could also have been used to preserve food during the
very early stages of civilization, ca. 10,000 to 12,000 B.C. In the colder regions of
the world, foods (e.g., meat, and fish) are still preserved in natural ice. Ice was used
by the wealthy Romans to reduce the temperature of foods. Until ca. 1800 A.D., ice
blocks from frozen lakes were cut, stored, and used to preserve raw foods (e.g.,
meats, milk, fish, and produce) by lowering the temperature. By 1840, with the help
of ammonia-compressed refrigerator units, ice blocks were produced commercially
and used to reduce the temperature of food for preservation. In 1880, refrigeration

467
468 FUNDAMENTAL FOOD MICROBIOLOGY

was used on ships and trains in Europe to transport meat and fish from other
countries. Linde, in Germany, developed the first domestic refrigerator ca. 1874 and
started commercial production before 1890. The popularity of domestic refrigerators
was initially slow in the U.S.; in 1930, 2 to 3% of the households had the units. In
the U.S., methods to freeze fruits and vegetables were developed and commercially
used around the 1930s. During this time, retail stores also started using cabinets to
display frozen foods.
During World War II (1941 to1944), consumer interest in refrigerated and frozen
foods increased dramatically, which helped develop the technology necessary for
processing, transporting, retailing, and home storage of refrigerated and frozen foods
in the U.S. The popularity of refrigerated and frozen foods has increased steadily
since then. In the 1960s, 1970s, and early 1980s, frozen food consumption increased
sharply, mainly because of the long shelf life. Since the mid-1980s, there has been
increased interest in refrigerated and chilled foods, which consumers view as natural
and healthy. The major drawback of many refrigerated foods is their relatively short
shelf life, ca. 1 to 2 weeks. But in recent years, several technological improvements,
such as oxygen-impermeable packaging materials, good vacuum-packaging equip-
ment, innovative packaging systems, low-temperature refrigeration units (–1rC),
and use of the hurdle concept to preserve food (Chapter 40) have helped increase
the shelf life of many refrigerated foods to 60 d, and for some products, more than
90 d. At present, of the total foods consumed, low-temperature preserved foods
constitute more than 65%, and the trend shows a steady increase in the future. To
suit the taste of consumers, many new products are being developed that are low in
fat (caloric), high in fiber, phosphates, and other additives, and have low amounts
or no preservatives. To achieve the long shelf life and to make these products safe,
extra precautions are being introduced for microbiological control. This has helped
many new or emerging pathogenic and spoilage bacteria, in the absence of compe-
tition from associated microorganisms, to become predominant (Chapter 28). New
designs of processing equipment for high-production efficiency, centralized produc-
tion of large volumes of products, transportation of products for long distances in
regional storage facilities, retailing conditions, and consumers’ handling of the
products have facilitated these so-called new pathogenic and spoilage bacteria in
gaining prominence (Chapter 20). Unless some effective intervention strategies are
developed, new pathogenic and spoilage microorganisms will continue to surface in
refrigerated foods stored for a long time.1–3

II. OBJECTIVES

The main microbiological objective in low-temperature preservation of food is to

prevent or reduce growth of microorganisms. Low temperature also reduces or
prevents catalytic activity of microbial enzymes, especially heat-stable proteinases
and lipases. Germination of spores is also reduced. Low-temperature storage, espe-
cially freezing (and thawing), is also lethal to microbial cells, and, under specific
conditions, 90% or more of the population can die during low-temperature preser-
vation. However, the death rate of microorganisms at low temperature, as compared
CONTROL BY LOW TEMPERATURE 469

with that at heat treatment, cannot be predicted (as D and Z values in heating). Also,
spores are not killed at low temperature. Thus, foods are not preserved at low
temperature in order to kill microbial cells. Freezing is also used to preserve starter
cultures for use in food bioprocessing. This has been discussed in Chapter 13.

III. MECHANISMS OF ANTIMICROBIAL ACTION

The metabolic activities, enzymatic reactions, and growth rates of microorganisms

are maximum at the optimum growth temperature. As the temperature is lowered,
microbial activities associated with growth slow down. Normally, the generation
time, within a certain range, is doubled for every 10rC reduction in temperature.
Thus, a species dividing every 60 min in a food at 22rC will take 120 min to divide
if the temperature is reduced to 12rC. At lower range, generation time can be even
higher than double. For example, if the temperature is reduced from 12rC to 2rC
and the species can grow at 2rC, the generation time for the species could be more
than 240 min. The lag and exponential phases and the germination time (of spores)
for some psychrotrophs (mesophilic types) become increasingly longer as the tem-
perature is reduced to ca. 0rC or even to ca. –1rC. At this temperature, nongrowing
cells of some mesophiles (nonpsychrotrophic) and thermophiles may be injured and
die, especially if they are stored for a long time (weeks) at 2rC or below and the
foods have low Aw, low pH, or preservatives. The rate of catalytic activity of some
enzymes also decreases as the temperature of an environment is reduced. 4–6
Water is present in a food as free water and bound (with the hydrated molecules)
water. As the temperature in a food system drops to ca. –2rC, free water in the food
starts freezing and forming ice crystals (pure water freezes at 0rC, but in a food
with solutes it freezes below 0rC). As the temperature drops further and more ice
crystals form, the solutes get concentrated in the remaining water, which in turn
depresses the freezing point of the water in the solution. The Aw is also reduced.
When the temperature is reduced to ca. –20rC, almost all the free water freezes.
As the temperature of a food is reduced below –2rC, free water inside the
microbial cells also undergoes similar changes. At a slow rate of freezing, as the
water molecules in the food start freezing, water molecules from inside the microbial
cells migrate outside, causing dehydration of cells and concentration of solutes and
ions inside. When the temperature is reduced further (above –20rC) so that the water
in the food has frozen, water inside the cell also freezes. However, before that,
microbial cells are exposed to low pH (due to concentration of ions) and low Aw
(due to concentration of solutes) inside and outside the cells. This can cause dena-
turation and destabilization of the structural and functional macromolecules in the
microbial cells, whose stability and functions depend on their three-dimensional
structures, and can injure the cells. If the freezing is rapid, very small ice crystals
form quickly and the cells are not exposed to the solution effect. This is practiced
in the freeze preservation of starter cultures and frozen stock cultures.
Microbial cells subjected to freezing and thawing suffer sublethal (repairable)
as well as lethal injury. Studies show that different components of the cell wall (or
outer membrane) and cell membrane (or inner membrane) are injured. DNA strand
\
470 FUNDAMENTAL FOOD MICROBIOLOGY

break, ribosomal RNA degradation, and activation and inactivation of some enzymes
have also been reported in some studies. In sublethally injured cells, the structural
and functional injuries are reversible. In lethally injured (or dead) cells, the damages
are irreversible (Chapter 9).4–7

IV. INFLUENCING FACTORS

The effectiveness of low temperature in controlling microbial growth and microbial

enzymatic activity in food depends on many factors. These factors can be arranged
into three groups: those unique to low-temperature, those related to the food envi-
ronment, and those inherent in microorganisms. These factors not only help prevent
or reduce the growth of microorganisms but can also greatly influence the extent of
sublethal and lethal injury that microorganisms incur in food preserved at low
temperatures. An understanding of the influence of these factors and interaction
among them is important to design an efficient and predictable method to preserve
a specific food at low temperature.

A. Nature of Process

At temperatures above freezing of free water (–2rC), different types of bacteria,

molds, and yeasts can grow in a food. But the lag and exponential phases become
longer as the temperature is reduced. In the low range, even a difference in <1rC
can be highly important. A Pseudomonas fluorescens strain was reported to have a
generation time of ca. 6.7 h at 0.5rC, but 32.2 h at 0rC. Thus, a reduction in 0.5rC
increased the generation time by ca. 4.5-fold. This is much more than the theoretical
estimate, which suggests that the generation time doubles for every 10rC reduction.
Spores of some spoilage Bacillus and Clostridium spp. can germinate even at refrig-
eration temperature (4.5rC or 40rF). Cells of some mesophiles and thermophiles
can be sublethally injured as well as die as the temperature drops below 4.5rC.
As the temperature is reduced enough to cause a large portion of the water to
freeze, the growth of most microorganisms stops except for some psychrophilic
bacteria, yeasts, and molds. Although there are conflicting reports, slow growth
probably can occur up to –10rC, especially by some molds. As the temperature
drops further, to ca. –20rC, and water in a food freezes completely, more cells will
have sublethal and lethal injury. The rate of freezing and the lowest temperature of
freezing dictate the extent of microbial damage from ice crystals. Damage and death
are more extensive at a slower rate of freezing and at –20ºC than at a rapid rate of
freezing and at –78rC or –196rC (temperatures of solid CO2 and liquid N2, respec-
tively). Death and sublethal injury are very high during initial storage (ca. 7 d) but
subsequently slow down.
Fluctuation of temperature of a food during low-temperature storage has great
impact on growth, sublethal injury, and death of microorganisms. This quite readily
happens to foods during storage, transport, retail display, and at home. A fluctuation
of temperature of food from 4.4rC to 10–12rC not only stimulates rapid growth
of psychrotrophic pathogenic and spoilage bacteria, but many mesophilic spoilage
CONTROL BY LOW TEMPERATURE 471

and pathogenic bacteria start to grow and their spores germinate at this range. Just
from the spoilage aspect, a 6- to 8-h temperature abuse (12rC) of a vacuum-
packaged, refrigerated, low-heat-processed meat product can reduce its expected
shelf life of 8 weeks by ca. 7 to 10 d. A fluctuation in temperature of a frozen food
increases microbial death and injury due to repeated damaging solution effect and
mechanical damage from larger ice crystals that form during repeated freezing and
thawing. Dead microbial cells can also lyse, releasing intracellular enzymes, many
of which (e.g., proteinases and lipases) can act on food components and reduce the
acceptance quality of food (see Chapter 21).
The rate of cooling of a food is also very important for effective control of the
growth of pathogenic and spoilage microorganisms. A slow rate of cooling of foods
has been implicated as a major cause of foodborne diseases (Chapter 23). This can
occur by trying to cool a large volume of hot or warm food in a big (deep) container
in a refrigerator or overstuffing refrigerators with hot or warm foods. During thawing
of a frozen food (such as an uncooked chicken), rapid thawing is desirable in order
to control microbial growth, especially growth of pathogens. If the food is thawed
slowly, the temperature on the food surface will soon increase, thereby allowing
microbial growth, even when the inside is still frozen.
Refrigerated foods have limited shelf life, and, with time, microorganisms grow
and spoil the products. In frozen foods, microorganisms (only cells, not spores)
slowly die. However, even after long storage, some survive in frozen foods.

B. Nature of Food

Composition, pH, Aw, and presence of microbial inhibitors (preservatives) in a food

can greatly influence growth, sublethal injury, and viability of microorganisms during
storage at low temperature. A food with higher solid content (especially high pro-
teins, carbohydrates, and lipids, but low ions), pH closer to 7.0, higher Aw, and the
absence of microbial inhibitors facilitate growth and survival of microorganisms at
refrigeration temperature and inflict less injury and cause less death at frozen tem-
perature. Thus, the shelf life of refrigerated foods can be increased by using one or
more of these factors, such as low pH, low Aw, incorporation of suitable microbial
inhibitors, and, when possible, vacuum or modified air packaging (see Chapter 40).
In packaged frozen foods, ice may form in the packages (package ice), especially
if the storage temperature fluctuates. During thawing, the ice melts and is absorbed
by the food, resulting in an increase in the Aw in a localized area (e.g., in a bread)
and making it susceptible to microbial growth after thawing.

C. Nature of Microorganisms

Although some microorganisms can grow at as low as –10rC, many mesophilic and
thermophilic bacterial cells can be sublethally injured and may die with time at low
temperatures above freezing. At temperatures below –10rC, vegetative cells of
microorganisms can sustain sublethal injury and die. In general, Gram-negative or
rod-shaped bacteria are more susceptible to the damaging effect of freezing than
Gram-positive or spherical-shaped bacteria. Also, cells from the early exponential
\
472 FUNDAMENTAL FOOD MICROBIOLOGY

phase of growth are more susceptible to freezing than those from the early stationary
phase. Species and strains of microorganisms also differ greatly in sensitivity and
resistance to freezing damage. Germination and outgrowth of spores of some
Clostridium spp. can occur at as low as 2rC and maybe at a slightly higher temper-
ature for some Bacillus spp. spores. Spores do not lose their viability in frozen foods.
Some microbial enzymes, either heat stable or released by the dead and lysed cells,
can catalyze reactions at temperatures above –20rC, but at a slow rate, and can
reduce the acceptance quality of a food.1–7

V. METHODS

Foods are stored at low temperature in different ways to extend their shelf life. Many
fresh fruits and vegetables are kept at temperatures between 10 and 20rC or lower,
mainly to reduce their metabolic rate. Microorganisms to which these products are
susceptible, namely yeasts and molds (and some bacteria), can grow at this temper-
ature. Maintaining a low relative humidity to prevent moisture build-up on the food
surface is very important to reduce their growth. Highly perishable products are
generally stored at a low temperature, below 7ºC, often in combination with other
preservation methods. The importance of rapid cooling of a food for microbiological
safety has been mentioned before. The methods used for low-temperature preserva-
tion of foods and in food safety are briefly discussed.

A. Ice Chilling

This is used in retail stores where the foods are kept over ice; the surface in contact
with the ice can reach between 0 and 1rC. Fresh fish, seafood, meats, cut fruits,
vegetable salads (in bags), different types of ready-to-eat salads (prepared at the
retail store), salad dressing (high pH, low caloric), sous vide, and some ethnic foods
(e.g., tofu) are stored by this method. The trend is increasing.
Temperature fluctuation (due to the size of the container or melting of ice),
duration of storage (fresh or several days), and cross-contamination (raw fish, shrimp,
oysters, and ready-to-eat fish salads in an open container in the same display case
at the retail store) can cause microbiological problems, especially from foodborne
pathogens.

B. Refrigeration

The temperature specification for refrigeration of foods has changed over time
(Chapter 20). Previously, 7rC (45rF) was considered a desirable temperature.
However, technological improvements have made it economical to have domestic
refrigeration units at 4 to 5rC (40 to 41rF). For perishable products, 4.4rC (40rF)
is considered a desirable refrigeration temperature. Commercial food processors may
use as low as 1rC for refrigeration of perishable foods (such as fresh meat and
fish). For optimum refrigeration in commercial facilities along with low temperature,
the relative humidity and proper spacing of the products are also controlled.
CONTROL BY LOW TEMPERATURE 473

Raw and processed foods of plant and animal origin, as well as many prepared
and ready-to-eat foods, are now preserved by refrigeration. Their volume is increas-
ing rapidly because consumers prefer such foods. Some of these foods are expected
to have a storage life of 60 d or more.
For refrigerated products expected to have a long shelf life, additional preserva-
tion methods are combined with the lowest possible temperature that can be used
(close to –1rC). However, as the products are nonsterile, even a very low initial
microbial population (e.g., 10 cells or spores per 10 g), capable of growing (or
germinating) under the storage condition, can multiply to reach hazard (for pathogen)
or spoilage levels, thereby reducing the safety and stability of the product. Any
fluctuation in temperature or other abuse (e.g., a leak in a vacuum or modified
atmosphere package, or oxygen permeation through the packaging materials) can
greatly accelerate their growth. The processing and storage conditions may provide
environments in which different types of spoilage and pathogenic microorganisms
grow advantageously. This may increase spoilage and wastage of foods unless
appropriate control measures are installed quickly.

C. Freezing
The minimum temperature used in home freezers (in the refrigerator) is –20rC, a
temperature at which most of the free water in a food remains in a frozen state. Dry
ice (–78rC) and liquid nitrogen (–196rC) can also be used for freezing; they are
used for rapid freezing (instant freezing) and not for only freezing a food to that
low temperature. Following freezing, the temperature of the foods is maintained ca.
–20 to –30rC. Depending on the type, foods can be stored at refrigerated temperature
for months or even more than a year. Raw produce (vegetables, fruits), meat, fish,
processed products, and cooked products (ready-to-eat after thawing and warming)
are preserved by freezing.
Microorganisms do not grow at –20rC in frozen foods. Instead, microbial cells
die during frozen storage. However, the survivors can multiply in the unfrozen foods.
Accidental thawing or slow thawing can facilitate growth of survivors (spoilage and
pathogenic microorganisms). Spores can also germinate and outgrow, depending on
the temperature and time following thawing. Enzymes, released by the dead micro-
bial cells can reduce the acceptance quality of the food.1–5

VI. CONCLUSION

Microbial growth in a food depends on the storage temperature and the microbial
types. Based on these two factors and the specific need, foods are stored below room
temperature, refrigerated or frozen. Although microorganisms cannot grow in frozen
foods, some can grow in refrigerated foods. In addition to preventing and retarding
growth, microbial cells are also injured and killed at frozen and refrigerated tem-
peratures. Because refrigeration maintains the acceptance quality of foods, lower
temperature (= –1r to 5rC) is used in combination with other factors (low Aw, pH,
etc.) to extend the shelf life of foods for 60 d or more. Like low temperature, reduced
Aw is also used to control microbial growth in food and is discussed in Chapter 34.
\
474 FUNDAMENTAL FOOD MICROBIOLOGY

REFERENCES

1. Kraft, A.A., Refrigeration and freezing, in Psychrotrophic Bacteria in Foods, CRC

Press, Boca Raton, FL, 1992, p. 241.
2. Ray, B., Kalchayanand, N., and Field, R.A., Meat spoilage bacteria: are we prepared
to control them?, Natl. Provision., 206(2), 22, 1992.
3. Ray, B., Kalchayanand, N., Means, W., and Field, R.A., The spoiler: Clostridium
laramie, Meat Poult., 41(7), 12, 1995.
4. Ray, B., Enumeration of injured indicator bacteria from foods, in Injured Index and
Pathogenic Bacteria, Ray, B.. Ed., CRC Press, Boca Raton, FL, 1989, p. 10.
5. Olson, J.C. and Nottingham, P.M., Temperature, in Microbial Ecology, Vol. 1, Silliker,
J.H., Ed., Academic Press, New York, 1980, p. 1.
6. Speck, M.L. and Ray, B., Effects of freezing and storage of microorganisms from
frozen foods: a review, J. Food Prot. 40, 333, 1977.
7. Kalchayanand, N., Ray, B., and Field, R.A., Characteristics of psychrotrophic
Clostridium laramie causing spoilage of vacuum-packaged refrigerated fresh and
roasted beef, J. Food Prot. 56, 13, 1993.

QUESTIONS

1. List the microbiological objectives of low-temperature preservation of food. How

do these objectives differ from those for food preserved by heat?
2. Briefly discuss the mechanisms of microbial control by reducing the temperature
of a food to 10ºC, to –1ºC, to –10ºC, and to –20ºC.
3. List the major factors that need to be considered for effective control of microor-
ganisms in a food at low temperature, and briefly discuss the importance of each.
4. Discuss the microbial implications of the following in low-temperature preserva-
tion of foods: (a) fluctuation of storage (refrigerated and frozen) temperature, (b)
slow cooling of a warm food, and (c) slow thawing of a frozen food.
5. Briefly discuss the microbiological problems of foods stored by chilling, refrig-
eration, and freezing.
6. List the psychrotrophic pathogens that can cause food hazard in refrigerated ready-
to-eat foods and suggest a method that can be used to overcome the problem
(consult Chapter 40).