FREEZING OF FOODS

Contents

Damage to Microbial CellsGrowth and Survival of Microorganisms

Damage to Microbial CellsCO 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. 840–845, � 1999, Elsevier Ltd.

(a)

(b)

(c)

(d)

Increasing ice fractionIncreasingly concentrated solution

Maximum ice fractionMaximally concentrated solution

IceGlassy concentrated solution

Tem

pera

ture

(°C

)

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 ofmelting of ice in the maximal ice fraction; (c) temperature of glass tran-sition of the maximally concentrated solution; and (d) �135 �C, the glasstransition temperature of pure water.

Effect of Freezing on the Microbial EnvironmentProvided by Foods

Although some water in foods can be associated with macro-molecules as water of hydration, most of the water in moistfoods usually will be present as a solution of an often complexmixture of solutes. When water contains dissolved solutes, thefreezing point of the water is depressed. Consequently, foodsgenerally commence freezing at temperatures between �1 �Cand�3 �C rather than 0 �C. Freezing occurs with the formationof crystals of pure ice in the solution. If nuclei for ice crystalformation are lacking, the solution may supercool – that is, itmay remain wholly liquid at temperatures below that at whichfreezing can start.

Once ice crystal formation has started, the fraction of waterthat is present as ice will increase with decreasing temperature,and the concentrations of solutes in the remaining liquid waterincrease as well (Figure 1). With decreasing temperature, theamount of ice within a food will reach a maximum value,although the food still may contain unbound water that couldbe frozen. Such water does not freeze because growth of icecrystals is restricted by increased viscosity of the food matrix. Atstill lower temperatures, the concentrated solution will solidifyas a glass.

In any freezing solution, the ice must be in equilibriumwith the remaining liquid water. Consequently, the vaporpressure of the solution is that of ice at the same temperature,which is less than that of liquid water at the same tempera-ture. Therefore, a food that is partially or wholly frozen iseffectively drier than the same food that is not frozen. Theavailability of water can be expressed as its water activity(aw) – that is, the ratio of the water vapor pressure of the foodto the vapor pressure of pure water at the same temperature.The aw of a frozen food is that of pure ice at the sametemperature (Table 1).

All microorganisms in frozen foods must be exposed to lowtemperatures and reduced water activities but may be exposedvariously to other injurious conditions. When a food

964 Encyclopedia of Food Mic

progressively freezes, planktonic microorganisms that are freeto move in the liquid phase will concentrate in the remainingunfrozen solution. Such microorganisms therefore will beexposed to increasing solute concentrations and possibly tolarge pH changes. Organisms that are immobilized withinfoods may escape exposure to concentrated solutions, but theymay be affected by ice crystal formation in their locality or bydesiccation if water sublimes from frozen surfaces to giveregions of freeze-dried food. The various types of

robiology, Volume 1 http://dx.doi.org/10.1016/B978-0-12-384730-0.00130-0

Table 2 Effects on bacteria on turkey carcasses of freezingand frozen storage

Time

Numbers of bacteria (log cfu cm�2)

Aerobes Pseudomonads Coliforms Enterococci

Before freezing 6.5 5.2 4.8 5.0After freezing 4.5 2.7 2.5 3.41-month storage 4.1 2.1 2.7 1.42-month storage 3.1 1.2 2.2 1.14-month storage 2.7 1.0 1.4 .16-month storage 2.7 <.0 .3 .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, 128–137.

Table 1 Effects of freezing temperatures on thewater activities of foods

Temperature (�C) Water activity (aw)

�2 .981�5 .953�10 .907�15 .864�20 .823�30 .746

Source: Leistner, L., Rodel, W. Krispien, K., 1981. Microbi-ology of meat and meat products in high and intermediatemoisture ranges. In: Rockland, L.B., Stewart, B.F. (Eds.), WaterActivity: Influences on Food Quality. Academic Press, NewYork, pp. 885–916.

FREEZING OF FOODS j Damage to Microbial Cells 965

microorganisms can then be affected differently by the inju-rious conditions that develop in frozen foods.

Injury of Microorganisms by Freezing and Thawing

During freezing of foods, microorganisms could be injured bythe low temperatures, by mechanical damage to cell walls ormembranes by ice crystals formed outside or within cells, byincreased concentrations of damaging solutes in the extra-cellular medium, or by dehydration of cells in response toincreased osmotic pressure or drying of the extracellularmedium. During frozen storage, reactions between compo-nents of cells and those of the extracellular medium, orincreasing desiccation of the food may result in cell damage.During thawing, intracellular and extracellular ice crystalsmay enlarge to damage cells, or glassified solutions may meltto expose microorganisms to concentrated solutions. Micro-organisms, however, can be protected from injury duringfreezing and thawing by various solutes that can be present infoods.

Abrupt, relatively large decreases in temperature can resultin injury to growing bacteria, with loss of intracellular metab-olites and proteins and synthesis of novel, cold-shock proteins.Bacteria in foods, however, generally would not experiencerates of cooling sufficiently rapid to induce cold shock. Thus, ingeneral, the simple cooling of microorganisms during freezingis unlikely to be immediately injurious. Microorganisms canprogressively lose viability when their growth is prevented, butsuch loss of viability is generally less at lower than at highertemperatures. Loss of viability during frozen storage may occurat the upper end of the temperature range experienced byfrozen foods, but it may be of little consequence at usual frozenstorage temperatures.

Apparently, damage of cells by extracellular ice is nota major cause of injury to most microorganisms. Formation ofintracellular ice also may be of limited importance for micro-organisms other than multicellular parasites because waterwithin microorganisms tends to supercool and may remainliquid at temperatures below �15 �C. As the water activitywithin supercooled cells must be above that of thesurrounding, frozen medium, the cells lose water to thesurrounding medium and become dehydrated. Ice crystals willform within cells of bacteria and fungi only if the rate of

cooling is such that the temperature limit for cell content’ssupercooling is exceeded before cell dehydration occurs. Themaximum rate of dehydration of a cell will depend on thepermeability to water of the cell membrane and the ratio ofthe cell surface area to its volume. Ice is unlikely to form in thecells of bacteria, yeasts, and molds when cooling rates are�1 �Cmin�1. Ice may form in cells of yeasts and molds, andbacterial cells of larger sizes when rates of cooling are>10 �Cmin�1; however, the formation of ice in the smallestbacterial cells may not occur unless the rate of coolingapproaches 100 �Cmin�1.

If the main cause of lethal injury of microorganisms duringfreezing is dehydration, the rate of survival should decline withdecreasing temperature. This is observed with some parasites,but with bacteria, yeasts, and molds, survival of freezing tendsto increase with decreasing temperature and increasing rates ofcooling up to 10 �Cmin�1. Survival then decreases withincreasing rates of cooling, but it increases again when coolingis rapid. Survival generally is enhanced by rapid rather thanslow thawing. These effects of rates of cooling and thawingindicate that injury at slow rates of freezing probably is due toincreased concentrations of extracellular solutes. At rates ofcooling above 10 �C, ice formed within cells will cause injury.The size of ice crystals will decrease with increased rates ofcooling, however, while the greatest damage is caused by largeice crystals. Slow thawing allows for the enlargement of icecrystals, with greater damage to cells than when thawing israpid.

During frozen storage of foods, the number of viableorganisms can decline (Table 2). Rates of decline are generallyrelatively slow and tend to decrease with time, so after an initialperiod, the numbers of some organisms may be essentiallystable.

Cryoprotectants

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

Table 4 Time and temperatures for inactivationof Trichinella spiralis in pork specified in USregulations

Temperature (�C) Time (h)

�18 106�23 63�29 35�32 22�37 .5

966 FREEZING OF FOODS j Damage to Microbial Cells

can electrolytes in some instances. Polyols and other low-molecular-weight cryoprotectants are variously synthesizedand accumulated by xerotolerant organisms exposed toosmotic stress. Such compounds readily enter cells, andprobably protect cell components from the injurious effects ofthe dehydration that occurs during freezing. Electrolytessimilarly may stabilize some cell components. In contrast,high-molecular-weight cryoprotectants probably act byinhibiting the formation of ice in the extracellular medium. Asthe complex medium provided for microorganisms by manyfoods are likely to contain a variety of cryoprotectivecompounds, the effects of freezing are generally less delete-rious for microorganisms in foods than for the same organ-isms 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 presentin foods. Viruses, bacteria, yeasts and protozoa, and spores orother resting forms of bacteria, yeasts, protozoa, and moldsmay be planktonic and thus may be affected by increasingsolute concentrations in the remaining liquid water duringfreezing of foods. Mold hyphae are extensive while helminthsand the infective forms of helminthic parasites are relativelylarge, so those types of microorganisms are likely to belocalized within foods and to be damaged by ice crystalformation.

Foodborne enteric viruses are small and of simple struc-ture, being composed of only a nucleic acid core and proteincoat. Spores of bacteria and the sexual spores of molds andyeasts can survive extreme environmental conditions thatinactivate vegetative cells. Thus, in general, these types andforms of microorganisms probably are preserved rather thaninactivated by freezing (Table 3). The extent to which thevegetative cells of bacteria and yeasts are inactivated byfreezing varies greatly between species and strains and isaffected by the physiological state of the organisms as well asthe conditions under which freezing occurs. In general,

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

Organism

Effect of freezingType Form

Viruses – PreservationBacteria Vegetative cells Inactivation/preservation

Spores PreservationYeasts Vegetative cells Inactivation/preservation

Spores, sexual PreservationMolds Hyphae Injury/preservation

Spores, asexual InactivationSpores, sexual Preservation

Protozoa Active forms InactivationSpores, cysts, oocytes Inactivation

Helminths Adult forms InactivationLarvae, metacercariae Inactivation

stationary phase cells and cells exposed to osmotic or someother types of stress before freezing are more resistant tofreezing than are logarithmic phase, unstressed cells. Theasexual spores of molds generally are less resistant to freezingthan are sexual spore, and their resistance may vary with theconditions under which they are formed.

The lethal effects of freezing on filamentous molds aredifficult to quantify and have not been extensively investigated,but damage of hyphae by ice crystals has been reported. Theinfective forms of protozoan parasites (i.e., spores, cysts, andoocysts) generally are inactivated by freezing, with the rate ofinactivation increasing with decreasing temperature. The sameis true of the infective forms of helminthic parasites (i.e., larvaeand metacercariae). Consequently, frozen storage at specifiedtemperatures for specified minimum times is a recognizedmeans 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 freezingof foods can vary greatly with the type of microorganism, itsphysiological state or stage in its life cycle, the composition ofthe 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 largenumbers of any microorganism that may be present in a food.Even so, in some circumstances, freezing will cause extensiveinactivation of some microorganisms in some foods. For suchreductions to be recognized as decontaminating treatment infood production systems, however, they have to be validated forthe microorganisms of concern in each specific process.

See also: Bacterial Endospores; Cryptosporidium; Cyclospora;Freezing of Foods: Growth and Survival of Microorganisms;Fungi: Overview of Classification 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. PoultryScience 42, 128–137.

FREEZING OF FOODS j Damage to Microbial Cells 967

Leistner, L., Rodel, W., Krispien, K., 1981. Microbiology of meat and meat productsin high and intermediate moisture ranges. In: Rockland, L.B., Stewart, B.F.(Eds.), Water Activity: Influences on Food Quality. Academic Press, New York,pp. 885–916.

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. 122–145.

Sablani, S.S., Syamaladevi, R.M., Swanson, B.G., 2010. A review of methods, dataand applications of state diagrams of food systems. Food Engineering Reviews 2,168–203.

Sun, D.W., 2012. Handbook of Frozen Food Processing and Packaging, second ed.CRC Press, Boca Raton, FL.