FREEZING OF FOODS

Contents
Damage to Microbial Cells
Growth and Survival of Microorganisms

Damage to Microbial Cells

CO Gill, Lacombe Research Centre, Lacombe, AB, Canada
2014 Elsevier Ltd. All rights reserved.
This article is a revision of the previous edition article by Rekha S. Singhal, Pushpa R. Kulkarni, volume 2, pp. 840845, 1999, Elsevier Ltd.

Although some water in foods can be associated with macromolecules as water of hydration, most of the water in moist
foods usually will be present as a solution of an often complex
mixture of solutes. When water contains dissolved solutes, the
freezing point of the water is depressed. Consequently, foods
generally commence freezing at temperatures between
1  C
and
3  C rather than 0  C. Freezing occurs with the formation
of crystals of pure ice in the solution. If nuclei for ice crystal
formation are lacking, the solution may supercool that is, it
may remain wholly liquid at temperatures below that at which
freezing can start.
Once ice crystal formation has started, the fraction of water
that is present as ice will increase with decreasing temperature,
and the concentrations of solutes in the remaining liquid water
increase as well (Figure 1). With decreasing temperature, the
amount of ice within a food will reach a maximum value,
although the food still may contain unbound water that could
be frozen. Such water does not freeze because growth of ice
crystals is restricted by increased viscosity of the food matrix. At
still lower temperatures, the concentrated solution will solidify
as a glass.
In any freezing solution, the ice must be in equilibrium
with the remaining liquid water. Consequently, the vapor
pressure of the solution is that of ice at the same temperature,
which is less than that of liquid water at the same temperature. Therefore, a food that is partially or wholly frozen is
effectively drier than the same food that is not frozen. The
availability of water can be expressed as its water activity
(aw) that is, the ratio of the water vapor pressure of the food
to the vapor pressure of pure water at the same temperature.
The aw of a frozen food is that of pure ice at the same
temperature (Table 1).
All microorganisms in frozen foods must be exposed to low
temperatures and reduced water activities but may be exposed
variously to other injurious conditions. When a food

964

progressively freezes, planktonic microorganisms that are free

to move in the liquid phase will concentrate in the remaining
unfrozen solution. Such microorganisms therefore will be
exposed to increasing solute concentrations and possibly to
large pH changes. Organisms that are immobilized within
foods may escape exposure to concentrated solutions, but they
may be affected by ice crystal formation in their locality or by
desiccation if water sublimes from frozen surfaces to give
regions of freeze-dried food. The various types of

(a)

Increasing ice fraction

Increasingly concentrated solution
(b)

Temperature (C)

Effect of Freezing on the Microbial Environment

Provided by Foods

Maximum ice fraction

Maximally concentrated solution
(c)

Ice
Glassy concentrated solution

(d)

Solids fraction (wt solids/total wt)

Figure 1 Schematic state diagram for food freezing. Temperatures are

(a) 0  C, freezing temperature of pure water; (b) temperature of onset of
melting of ice in the maximal ice fraction; (c) temperature of glass transition of the maximally concentrated solution; and (d)
135  C, the glass
transition temperature of pure water.

Encyclopedia of Food Microbiology, Volume 1

http://dx.doi.org/10.1016/B978-0-12-384730-0.00130-0

FREEZING OF FOODS j Damage to Microbial Cells

Table 1
Effects of freezing temperatures on the
water activities of foods
Temperature ( C)

Water activity (aw)

2

5

10

15

20

30

.981
.953
.907
.864
.823
.746

Source: Leistner, L., Rodel, W. Krispien, K., 1981. Microbiology of meat and meat products in high and intermediate
moisture ranges. In: Rockland, L.B., Stewart, B.F. (Eds.), Water
Activity: Inuences on Food Quality. Academic Press, New
York, pp. 885916.

microorganisms can then be affected differently by the injurious conditions that develop in frozen foods.

Injury of Microorganisms by Freezing and Thawing

During freezing of foods, microorganisms could be injured by
the low temperatures, by mechanical damage to cell walls or
membranes by ice crystals formed outside or within cells, by
increased concentrations of damaging solutes in the extracellular medium, or by dehydration of cells in response to
increased osmotic pressure or drying of the extracellular
medium. During frozen storage, reactions between components of cells and those of the extracellular medium, or
increasing desiccation of the food may result in cell damage.
During thawing, intracellular and extracellular ice crystals
may enlarge to damage cells, or glassied solutions may melt
to expose microorganisms to concentrated solutions. Microorganisms, however, can be protected from injury during
freezing and thawing by various solutes that can be present in
foods.
Abrupt, relatively large decreases in temperature can result
in injury to growing bacteria, with loss of intracellular metabolites and proteins and synthesis of novel, cold-shock proteins.
Bacteria in foods, however, generally would not experience
rates of cooling sufciently rapid to induce cold shock. Thus, in
general, the simple cooling of microorganisms during freezing
is unlikely to be immediately injurious. Microorganisms can
progressively lose viability when their growth is prevented, but
such loss of viability is generally less at lower than at higher
temperatures. Loss of viability during frozen storage may occur
at the upper end of the temperature range experienced by
frozen foods, but it may be of little consequence at usual frozen
storage temperatures.
Apparently, damage of cells by extracellular ice is not
a major cause of injury to most microorganisms. Formation of
intracellular ice also may be of limited importance for microorganisms other than multicellular parasites because water
within microorganisms tends to supercool and may remain
liquid at temperatures below
15  C. As the water activity
within supercooled cells must be above that of the
surrounding, frozen medium, the cells lose water to the
surrounding medium and become dehydrated. Ice crystals will
form within cells of bacteria and fungi only if the rate of

965

Table 2
Effects on bacteria on turkey carcasses of freezing
and frozen storage
Numbers of bacteria (log cfu cm
2)
Time

Aerobes

Pseudomonads

Coliforms

Enterococci

Before freezing
After freezing
1-month storage
2-month storage
4-month storage
6-month storage

6.5
4.5
4.1
3.1
2.7
2.7

5.2
2.7
2.1
1.2
1.0
<.0

4.8
2.5
2.7
2.2
1.4
.3

5.0
3.4
1.4
1.1
.1
.1

Source: Kraft, A.A., Ayres, J.C., Weiss, K.F., Marion, W.W., Balloun, S.L. Forsythe,
R.H. 1963. Effect of method of freezing on survival of microorganisms on turkey.
Poultry Science 42, 128137.

cooling is such that the temperature limit for cell contents

supercooling is exceeded before cell dehydration occurs. The
maximum rate of dehydration of a cell will depend on the
permeability to water of the cell membrane and the ratio of
the cell surface area to its volume. Ice is unlikely to form in the
cells of bacteria, yeasts, and molds when cooling rates are
1  C min
1. Ice may form in cells of yeasts and molds, and
bacterial cells of larger sizes when rates of cooling are
>10  C min
1; however, the formation of ice in the smallest
bacterial cells may not occur unless the rate of cooling
approaches 100  C min
1.
If the main cause of lethal injury of microorganisms during
freezing is dehydration, the rate of survival should decline with
decreasing temperature. This is observed with some parasites,
but with bacteria, yeasts, and molds, survival of freezing tends
to increase with decreasing temperature and increasing rates of
cooling up to 10  C min
1. Survival then decreases with
increasing rates of cooling, but it increases again when cooling
is rapid. Survival generally is enhanced by rapid rather than
slow thawing. These effects of rates of cooling and thawing
indicate that injury at slow rates of freezing probably is due to
increased concentrations of extracellular solutes. At rates of
cooling above 10  C, ice formed within cells will cause injury.
The size of ice crystals will decrease with increased rates of
cooling, however, while the greatest damage is caused by large
ice crystals. Slow thawing allows for the enlargement of ice
crystals, with greater damage to cells than when thawing is
rapid.
During frozen storage of foods, the number of viable
organisms can decline (Table 2). Rates of decline are generally
relatively slow and tend to decrease with time, so after an initial
period, the numbers of some organisms may be essentially
stable.

Cryoprotectants
Although some extracellular solutes can be injurious, others
can protect microorganisms against freezing damage. These
cryoprotectants include glycerol and other polyols, glycine,
sugars, and various other low-molecular-weight organic
compounds. Soluble, high-molecular-weight compounds,
such as starch and proteins, can have cryoprotective effects, as

966

FREEZING OF FOODS j Damage to Microbial Cells

can electrolytes in some instances. Polyols and other lowmolecular-weight cryoprotectants are variously synthesized
and accumulated by xerotolerant organisms exposed to
osmotic stress. Such compounds readily enter cells, and
probably protect cell components from the injurious effects of
the dehydration that occurs during freezing. Electrolytes
similarly may stabilize some cell components. In contrast,
high-molecular-weight cryoprotectants probably act by
inhibiting the formation of ice in the extracellular medium. As
the complex medium provided for microorganisms by many
foods are likely to contain a variety of cryoprotective
compounds, the effects of freezing are generally less deleterious for microorganisms in foods than for the same organisms in simple media.

Effects of Freezing on Microorganisms in Foods

Microorganisms of all types (i.e., viruses, bacteria, yeasts,
molds, protozoa, and multicellular parasites) can be present
in foods. Viruses, bacteria, yeasts and protozoa, and spores or
other resting forms of bacteria, yeasts, protozoa, and molds
may be planktonic and thus may be affected by increasing
solute concentrations in the remaining liquid water during
freezing of foods. Mold hyphae are extensive while helminths
and the infective forms of helminthic parasites are relatively
large, so those types of microorganisms are likely to be
localized within foods and to be damaged by ice crystal
formation.
Foodborne enteric viruses are small and of simple structure, being composed of only a nucleic acid core and protein
coat. Spores of bacteria and the sexual spores of molds and
yeasts can survive extreme environmental conditions that
inactivate vegetative cells. Thus, in general, these types and
forms of microorganisms probably are preserved rather than
inactivated by freezing (Table 3). The extent to which the
vegetative cells of bacteria and yeasts are inactivated by
freezing varies greatly between species and strains and is
affected by the physiological state of the organisms as well as
the conditions under which freezing occurs. In general,

Table 3
Predominant effects of freezing on the various types
and forms of organisms that may be present on foods

Table 4
Time and temperatures for inactivation
of Trichinella spiralis in pork specied in US
regulations
Temperature ( C)

Time (h)

18

23

29

32

37

106
63
35
22
.5

stationary phase cells and cells exposed to osmotic or some

other types of stress before freezing are more resistant to
freezing than are logarithmic phase, unstressed cells. The
asexual spores of molds generally are less resistant to freezing
than are sexual spore, and their resistance may vary with the
conditions under which they are formed.
The lethal effects of freezing on lamentous molds are
difcult to quantify and have not been extensively investigated,
but damage of hyphae by ice crystals has been reported. The
infective forms of protozoan parasites (i.e., spores, cysts, and
oocysts) generally are inactivated by freezing, with the rate of
inactivation increasing with decreasing temperature. The same
is true of the infective forms of helminthic parasites (i.e., larvae
and metacercariae). Consequently, frozen storage at specied
temperatures for specied minimum times is a recognized
means of inactivating some parasites in foods for example,
larvae of Trichinella in meat (Table 4).

Conclusion
The extent to which the microorganisms are injured by freezing
of foods can vary greatly with the type of microorganism, its
physiological state or stage in its life cycle, the composition of
the food, and the rates at which freezing and thawing occur.
Except with large larval or adult forms of multicellular parasites,
it cannot be safely assumed that freezing will inactivate large
numbers of any microorganism that may be present in a food.
Even so, in some circumstances, freezing will cause extensive
inactivation of some microorganisms in some foods. For such
reductions to be recognized as decontaminating treatment in
food production systems, however, they have to be validated for
the microorganisms of concern in each specic process.

Organism
Type

Form

Effect of freezing

Viruses
Bacteria

Vegetative cells
Spores
Vegetative cells
Spores, sexual
Hyphae
Spores, asexual
Spores, sexual
Active forms
Spores, cysts, oocytes
Adult forms
Larvae, metacercariae

Preservation
Inactivation/preservation
Preservation
Inactivation/preservation
Preservation
Injury/preservation
Inactivation
Preservation
Inactivation
Inactivation
Inactivation
Inactivation

Yeasts
Molds

Protozoa
Helminths

See also: Bacterial Endospores; Cryptosporidium; Cyclospora;

Freezing of Foods: Growth and Survival of Microorganisms;
Fungi: Overview of Classication of the Fungi; Helminths;
Trichinella; Virology: Introduction; Injured and Stressed Cells;
Water Activity.

Further Reading
Evans, J.A., 2008. Frozen Food Science and Technology. Wiley-Blackwell, Oxford.
Kraft, A.A., Ayres, J.C., Weiss, K.F., Marion, W.W., Balloun, S.L., Forsythe, R.H.,
1963. Effect of method of freezing on survival of microorganisms on turkey. Poultry
Science 42, 128137.

FREEZING OF FOODS j Damage to Microbial Cells

Leistner, L., Rodel, W., Krispien, K., 1981. Microbiology of meat and meat products
in high and intermediate moisture ranges. In: Rockland, L.B., Stewart, B.F.
(Eds.), Water Activity: Inuences on Food Quality. Academic Press, New York,
pp. 885916.
Lund, B.M., 2000. Freezing. In: Lund, B.M., Baird-Parker, T.C., Gould, G.W. (Eds.),
The Microbiological Safety and Quality of Food. Aspen Publishers, Gaithersburg,
pp. 122145.

967

Sablani, S.S., Syamaladevi, R.M., Swanson, B.G., 2010. A review of methods, data
and applications of state diagrams of food systems. Food Engineering Reviews 2,
168203.
Sun, D.W., 2012. Handbook of Frozen Food Processing and Packaging, second ed.
CRC Press, Boca Raton, FL.