Antifreeze proteins and their potential use in frozen foods

Pergamon Biot~lmology A d ~ , Vol. 13, No. 3, pp. 375--402, 1995

Colryrisht C 1995 ~ 8,gienoe Inc. Printed in Great Britain. All fishts macrvCd

0734-9750/95 $39.00 + .00

0734-9750(95)02001 -J

ANTIFREEZE PROTEINS AND THEIR POTENTIAL USE IN FROZEN FOODS

MARILYN GRIFFITH* and K. VANYA EWART**

*Department of BioloD, , University of Waterloo, Waterloo, Ontario, Canada N2L 3G I and **Research Inatitute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario,

Canada MSG 1X8

ABSTRACT

Antifreeze proteins (AFPs) are proteins that have the ability to modify the growth of ice,

resulting in the stabilization of ice crystals over a defined temperature range and in the inhibition of the

re.crystallization of ice. AFPs are found in a wide range of organisms, including bacteria, fungi,

plants, invertebrates and fish. Moreover, multiple forms of AFPs are synthesized within each

organism. As a result, it should be possible to select an AFP with appropriate characteristics and a

suitable level of activity for a particular food product. Antifreeze proteins may improve the quality of

foods that are eaten while frozen by inhibiting recrystallization and maintaining a smooth texture. In

foods that are frozen only for preservation, AFPs may inhibit re.crystallization during freezing,

storage, transport and thawing, thus preserving food texture by reducing cellular damage and also

minimizing the loss of nutrients by reducing drip. Antifreeze proteins are naturally present in many

foods consumed as part of the human diet. However, AFPs may be introduced into other food

products either by physical processes, such as mixing and soaking, or by gene transfer.

Keywords: antifreeze glycoprotein, antifreeze protein, cryopreservation, frozen food, freezing

tolerance, ice, ice nucleation, recrystallization inhibition, supercooling, thermal hysteresis

INTRODUCTION

In nature, many organisms are exposed to freezing temperatures. This exposure may be nearly

constant, as occurs over the course of an Arctic winter, for example. However, it can also be brief

and intermittent, as occurs at night at high elevations, even in equatorial regions. An interesting

adaptation to life at subzero temperatures is the elaboration of proteins that modify ice crystal growth.

In this review, we will examine the role of antifreeze proteins in nature and then suggest ways these

proteins may prove useful in improving frozen foods.

375

376 M. GRIFFITH and K. VANYA EWART

PROPERTIES OF ANTIFREEZE PROTEINS

Antifreeze proteins (AFPs), antifreeze glycoproteins (AFGPs) and thermal hysteresis proteins

(THPs) have the ability to bind to ice and modify the normal growth of ice crystals. In this review,

the term AFPs applies to all antifreeze proteins, including the AFGPs and THPs. Ice crystals grown

in the presence of high concentrations of AFPs acquire unusual faceted or needle-like morphologies

(Scholander and Maggert, 1971; Knight et al., 1984). These results suggest that AFPs interact

directly with ice because a facet will only appear on an ice crystal if growth is inhibited in the direction

normal to the facet surface. Evidence for the adsorption of AFPs onto ice crystals has been gathered

from three types of analyses. First, solutions containing ice and AFPs generate second harmonics at

the ice-water interface that are observable by infrared spectroscopy (Brown et al., 1985). This signal

is not generated by pure ice-water interfaces (Brown et al., 1985). Second, unlike most solutes,

AFPs appear to be incorporated into the growing ice crystal during freezing (Raymond and DeVries,

1977; Knight et al., 1991, 1993). Third, the temperature transition during freezing is sudden for

AFP solutions in contrast to the more gradual transition for solutions in which the solute is excluded

from ice (Raymond and DeVries, 1972).

Drawing on current understanding of the effects of impurities on crystal growth, Raymond and

DeVries (1977) proposed that AFPs limit ice crystal growth over a defined temperature range by

adsorbing onto ice crystals and inhibiting the binding of additional water molecules to the crystal

lattice (adsorption-inhibition). Because further ice growth could only occur between adsorbed AFP

molecules, a series of curved ice fronts with higher surface free energies would result and subsequent

growth would not be favored. The model proposed by Raymond and DeVries (1977) also explains

the development of faceted ice crystals in the presence of AFP. In solutions with no AFPs, ice

crystals usually grow along the a-axes (Fig. 1). This causes an increase in the size of the basal plane,

which has a surface parallel to the a-axes. However, in solutions containing AFPs, the direction of

preferred ice growth is along the c-axis with growth occurring parallel to the c-axis along the prism

faces (Raymond and DeVries, 1977) or in other directions that differ from that of the a-axes (Knight

et al., 1991; 1993). This implies that each of the AFPs adsorbs preferentially onto a specific face of

ice and that its adsorption allows facets to develop. Thus, the binding patterns of individual AFPs to

ice appear to be more diverse than initially thought.

There are three known properties of AFPs that all stem from the interaction between AFPs and

ice crystals. They are thermal hysteresis, inhibition of ice recrystallization and interaction with ice

nucleators.

Thermal Hysteresis

Freezing point depression by adsorption-inhibition is the best known property of fish AFPs.

Thermal hysteresis results from a lowering of the apparent freezing temperature of a solution without

affecting the melting temperature because AFPs are 200 to 300 times more effective at freezing point

depression than ideal solutes (DeVries, 1971). Therefore, the supercooled state is stabilized over a

certain temperature interval. Although measurements of thermal hysteresis are routinely used to test

ANTIFREEZE PROTEINS USE IN FROZEN FOODS 377

for the presence of AFPs, this property is only evident when AFPs are present in relatively high

concentrations. In species not thought to have AFPs, these proteins may still be present at low levels,

they may be localized differently within the organism, or they may be present at times when sampling

the organism is not practical.

A a2,

basal prism face plane face

Figure 1. Interaction between antifreeze proteins and ice. A. In a dilute solution of AFPS (nM),

the AFPs (~) adsorb onto the prism faces of the ice crystal and limit crystal growth along the a I -, a 2-

and a3-axes, forming a crystal that is hexagonal in shape. B. In solutions containing higher

concentrations of AFPs (txM), the preferred direction of ice crystal growth is along the c-axis, so that

the crystal forms a hexagonal bipyramid.

Inhibition of Ice Recrystallization

During the recrystallization of ice, larger ice crystals grow at the expense of smaller ones.

Larger ice crystals are more likely to cause physical damage to tissues and cells. Recrystallization

takes place most rapidly at temperatures just below freezing and during warming from the glassy

state. Ice also recrystallizes when environmental temperatures fluctuate within the subzero range.

Extremely low concentrations (i00 l.tg L -I) of AFP are effective in inhibiting ice re, crystallization

(Knight e t a l . , 1984; 1988), even though these concentrations are too low to cause thermal hysteresis.

Recrystallization inhibition has been shown to occur in the presence of AFGPs (Knight e t a l . , 1984),

type I AFP (MueUer e t a l . , 1991), insect THP (Knight and Duman, 1986) and plant AFPs (Urrutia e t

a l . , 1992; Griffith and Antikainen, 1995). Although it is likely that recrystallization inhibition stems

from the interactions of AFPs and ice crystals, the exact mechanism involved in recrystallization

inhibition at very low concentrations of AFPs is not yet understood (Knight e t a l . , 1988).


Interaction with Ice Nucleators

AFGPs have been shown to inhibit the activity of bacterial ice-nucleating proteins

(Parody-Morreale et al., 1988). Ice-nucleating proteins initiate ice formation at elevated subzero

temperatures. They are thought to act by mimicking the structure of an ice crystal surface and,

consequently, behaving like a seed ice crystal. Thus, the inhibition of nucleation by AFGPs may

result from the ability of antifreezes to interact with ice-nucleating proteins in the same manner as they

do with ice crystals. Apoplastic extracts from cold-acclimated leaves of winter wheat, which contain

several AFPs, also inhibit bacterial ice-nucleation activity (Z~imecnik and J~fnacek, 1992). It is not yet

clear whether this property extends to all types of AFPs or whether it plays a role in freezing

avoidance or tolerance. While it might be assumed that both AFPs and ice-nucleating proteins would

be inactivated if they bound to one another, Duman and coworkers (1993) have shown that this is not

the case. They isolated a 70 kDa protein with ice nucleation activity from the hemolymph of larvae of

the beetle D e n d r o i d e s canadens is . When the protein was added to a D. canadens i s hemolymph

fraction with 1.6°C of thermal hysteresis, the thermal hysteresis of the fraction was increased to 5.2°C

(Duman et al., 1993). In this case, the interaction between an AFP and an ice nucleator resulted in

enhanced antifreeze activity.

DIVERSITY AND DISTRIBUTION OF ANTIFREEZE PROTEINS

Thermal hysteresis has now been observed in extracts obtained from organisms from four of the

five kingdoms: Monera, Fungi, Planta and Animalia (Duman and Olsen, 1993; Duman et al., 1993).

Although AFPs have not been purified from many of these organisms, the fact that treatment with

nonspecific proteases eliminates thermal hysteresis in the extracts suggests that AFPs are present

(Duman and Olsen, 1993; Duman et al., 1993). To our knowledge, there are no reports of thermal

hysteresis in the kingdom Protoctista, although a recent report suggests that a substance which is

capable of binding to the basal plane of ice may be produced by diatoms present in Ant~ctic sea ice

(Raymond et al., 1994).

Antifreeze Proteins in Fish

Although Scholander and coworkers first reported antifreeze activity in marine fish in 1957, it

was 12 years before the causal solutes, AFGPs, were purified (DeVries and Wohlschlag, 1969).

Since that discovery, three additional types of AFPs have been identified and are described in Table 1.

The distribution, structures and functions, regulation and gene organization of the different types of

fish AFPs have been widely studied (see reviews by Davies and Hew, 1990; Cheng and DeVries,

1991; Hew and Yang, 1992). Antifreeze proteins have now been identified in at least 17 species of

North Atlantic fish (Table 2).


Table 1. Characteristics of the four types of AFP present in fish. 1

Characteristic AFGP Type I AFP Type II AFP Type I11 AFP

Molecular mass 2600-33,000 3300-4500 14,000-24,000 6500-7000

Sequence (Ala-Ala-Thr)n Ala-rich, 11 Cys-rich general

residue repeats

Structure unknown 2 single ix-helix 3 similar to 13 sandwich 5

C-type lectin 4

Carbohydrate O-linked none N-linked none

disaccharide (smelt only)

IThis table is updated from the one presented by Hew and Yang, 1992. 2Several different structures

have been proposed for the AFGPs (Berman et al., 1980; Bush and Feeney, 1986; Drewes and

Rowlen, 1993). 3yang et al., 1988. 4Ewart et al., 1992; Ng and Hew, 1992; Ewart and Fletcher,

1993. 5S6nnichsen et al., 1993.

Antifreeze Proteins in Invertebrates

AFPs are present in many invertebrates, including mostly insects, but also terrestrial arthropods

such as spiders and mites (reviewed by Duman et al., 1993). The insect AFPs range in molecular

mass from 14 to 20 kDa, lack carbohydrates and have higher percentages of hydrophilic amino acids

than fish AFPs (Duman et al., 1993). Some of the insect AFPs contain Cys residues that are

important for activity. Immunological cross-reactivity suggests a similarity with a type II fish AFP

(Hew et al., 1983). Although most of the invertebrates do not form part of our diet, an exception is

the blue mussel Myti lus edulis, which is a freezing-tolerant marine mollusk that produces an AFGP

( I 'heede et al., 1976; Guderley et al., 1985).

Antifreeze Proteins in Plants Antifreeze activity has now been reported in more than 27 species of higher plants (Table 3;

Griffith et al., 1992; Urrutia et al., 1992; Z&necnfk and J~acek, 1992; Duman and Olsen, 1993;

Duman et al., 1993), as well as in more primitive plants such as ferns and mosses (Duman and Olsen,

1993). Antifreeze activity is present only when the plants are acclimated to low temperatures (Griffith

et al., 1992; Urrutia et al., 1992; Marentes et al., 1993). Plant AFPs have now been purified from

the woody stems of bittersweet nightshade (Duman, 1994) and from the leaves of winter rye Secale

cereale (Hon et al., 1994a). The AFP from bittersweet nightshade is 67 kDa, has an unusually high

Gly content of 23.7% and may contain galactose. Bittersweet nightshade AFP exhibits about 0.3°C

of thermal hysteresis at a concentration of l0 to 35 mg mL "l (Duman, 1994). This activity is much

lower than that observed for fish AFPs.

Six AFPs, ranging in size from 15 to 38 kDa, have been identified in apoplastic extracts of

winter rye leaves (Hon et al., 1994a). Recent results obtained by N-terminal sequencing,

380 M. (3RIFFITH and K. VANYA EWART

immunoblotting and assays of enzymatic activity revealed that the six winter rye AFPs correspond to

three classes of pathogenesis-related (PR) proteins: two of the AFPs are class I endochitinases, two

are 13-1,3-endoglucanases and two are thaumatin-like (TL) proteins (Hon et al., 1994b). PR proteins

are associated with increased disease resistance in plants because endochitinases and endoglucanases

degrade fungal cell walls and TL proteins inactivate ribosomes in invading organisms (Stintzi et al.,

1993). In contrast to winter rye, pathogen-induced class I endochitinases and ~-l,3-endoglucanases

purified from the leaves of freezing-sensitive tobacco plants do not have antifreeze activity. These

findings suggest that subtle structural changes may have evolved on the surfaces of cold-induced

winter rye proteins and this led to the development of ice-binding properties in these isoforms of PR

proteins (Hon et al., 1994b).

Table 2. North Atlantic fish species in which AFPs are found.

AFP Type Fish Species Scientific Name Reference

AFGP Atlantic cod Gadus morhua

Greenland cod Gadus ogac

Tomcod Microgadus tomcod

Type I AFP Winter flounder Pleuronectes americanus

Yellowtail flounder Pleuronectesferrugineus

Shorthorn sculpin Myoxocephalus scorpius

Grubby sculpin Myoxocephalus aenaeus

Type II AFP Sea raven Hemitripterus americanus

Smelt Osmerus mordax

Atlantic herring Clupea harengus harengus

Type III AFP Ocean pout Macrozoarces americanus

Atlantic wolffish Anarhichas lupus

Radiated shanny Ulvaria subbifurcata

Rock gunnel Pholis gunnellus

Lavars eelpout Lycodes lavalaei

Unknown l Gaspereau Alosa pseudoharengus

Cunner Tautogolabrus adspersus

Hew et al., 1981

Van Voorhies et al., 1978

Fletcher et al., 1982a

Duman and DeVries, 1976

Scott et al., 1987

Hew etal . , 1980

Chakrabartty et al., 1988

Slaughter et al., 1981

Ewart and Fletcher, 1990

Ewart and Fletcher, 1990

Hew etal . , 1984

Shears et al., 1993

Shears et al., 1993

Shears et al., 1993

Shears et al., 1993

Duman and DeVries, 1975

Valerio et al., 1990

lThermal hysteresis was detected but the AFP has not been identified.

Antifreeze Proteins in Fungi

Thermal hysteresis has been observed in extracts obtained from the oyster mushroom

Pleurotus ostreatus, the winter mushroom Flammulina velupites, and two bracket fungi, Stereurn sp.

and Coriolus versicolor (Duman and Olsen, 1993). The thermal hysteresis disappears upon protease


treaunent of the extracts, which indicates that AFPs are present. Interestingly, an AFP with epitopic

homology to the winter flounder type I AFP and with the same approximate mass was found to be

associated with cell membranes in the snow mold Coprinus psychromorbidus (Newstead et al.,

1994).

T a b l e 3. Higher plant species (angiosperms) in which antifreeze activity is found.

Plant species Common name Reference

Alliara petiolata garlic-mustard

Aster cordifolius blue wood aster

Avena sativa spring oat

Barbarea vulgaris winter cress

Brassica napus winter canola

Brassica oleracea Brussel's sprouts,

cabbage and kale

Oaucus carota carrot

Dicentra cucularia Dutchman's breeches

Euphorbia serpens spurge

Hemerocallis fulva daylily

Hordeum vulgare winter barley

Hydrophyllum virginianum Virginia waterleaf

Plantago lanceolata narrow-leaved plantain

Plantago major plantain

Poa annua speargrass

Poa pratensis Kentucky bluegrass

Populus deltoides Eastern cottonwood

Quercus alba white oak

Secale cereale winter rye

Solanum dulcamara bittersweet nightshade

Solanum tuberosum potato

Stellaria media chickweed

Taraxacum officinale dandelion

Triticum aestivum spring and winter wheat

Triticale sp. triticale

Vinca minor periwinkle

Viola sp. violet

Urrutia et al., 1992


Nantais and Griffith, unpublished



Urrutia et al., 1992; Duman,

personal communication

Duman and Olsen, 1993







Urrutia etal . , 1992

Urrufia etal . , 1992




Griffith et al., 1992





Duman et al., 1993;

7_Amecn~ and Jfmacek,1992;


Zitmecnl"k and J;Inacek 1992




Antifreeze Proteins in Bacteria

Antifreeze activity has been observed in cultures of Micrococcus cryophilus, a well-known

psychrophile, and Rhodococcus erythropolis (Duman and Olsen, 1993) and Pseudomonas put ida

(Sun et al., 1994), which are common soil bacteria. When the bacteria are grown at low temperature

(3 to 5°C), they exhibit thermal hysteresis ranging from 0.10 to 0.35°C (Duman and Olsen, 1993; Sun

et al., 1994). When P. putida is grown at 5°C, antifreeze activity is observed in the growth medium

(Sun et al., 1994). Treatment of the growth medium with either protease or [~-mercaptoethanol

eliminates antifreeze activity (Sun, 1994), which suggests that the bacterial AFP contains

intramolecular disulfide bridges that are important for ice-binding activity, as was previously

observed in type II, insect and plant AFPs.

LOCATION OF ANTIFREEZE PROTEINS IN ORGANISMS

All AFPs described to date in fish, insects and plants are extracellular. In fish, they are

normally purified from blood plasma or serum for ease of collection and purification, hut they are also

present in other tissues. In an Antarctic fish, AFGPs are synthesized predominantly in the liver and

secreted into the blood. From there they are distributed to all interstitial fluids, except brain (O'Grady

et al., 1982a; Alghren et al., 1988), although the possibility of AFGP synthesis in other tissues has

not been excluded (Hudson et al., 1978; Haschemeyer and Mathews, 1980). AFGPs 6, 7, and 8 are

secreted into the intestinal fluids (O'Grady et al., 1982b) and are present in ocular fluids at very low

levels (Turner et al., 1985). The type I AFP of winter flounder is present in approximately equal

amounts in skin and blood (Valerio et al., 1992). AFP was detected in the skin of shorthorn sculpin

and cunner, but not in the blood of either species (Schneppenbeim and Theede, 1982; Valerio et al.,

1990). These findings are consistent with the hypothesis that fish skin is an effective barrier to ice

propagation (Valerio et al., 1992).

Little is known about the location of AFPs in other organisms. In insects, THPs are present

in the circulating hemolymph and are associated with the epidermal layer located just beneath the

cuticle (Duman et al., 1993). In plants, AFPs are secreted into the apoplast, which includes the

xylem, cell walls and intercellular spaces of the tissues (Griffith et al., 1992; Marentes et al., 1993).

Extracts with antifreeze activity have been obtained from the overwintering portions of various plants,

including stems, branches, leaf blades, petioles, berries, buds, flowers, roots, rhizomes and tubers

(Urrutia et al., 1992; Duman and Olsen, 1993). Antifreeze activity may be regulated developmentally

in plants. For example, exudates of white oak acorns germinating early in the winter exhibit thermal

hysteresis even though no antifreeze activity is observed in exudates of the branches of the parent tree

(Duman and Olsen, 1993).

Antifreeze Proteins at Cell Surfaces

The possibility that AFPs may interact with cell membranes is another mechanism by which

AFPs may promote survival at subzero temperatures. The first suggestion that fish AFPs may

interact with membranes was made by DeVries (1980) when he was exploring ways in which AFPs


could prevent ice propagation across the gill epithelia. In insects, AFPs have been immunolocalized

either on the surfaces of plasma membranes or within the membrane bilayer of epidermal cells

(Duman et al., 1993). Moreover, in preliminary studies, anti-insect AFP antiserum cross-reacted

with proteins associated with the plasma membranes of xylem ray parenchyma cells in one plant, the

bittersweet nightshade (Urrutia et al., 1992). These results have been substantiated by the fact that

plasma membrane preparations obtained from cold-acclimated bittersweet nightshade and from D.

canadensis larvae both exhibit thermal hysteresis (Duman et al., 1993). In all the examples presented,

AFPs are located on the surface of cells that provide a barrier to inoculative ice formation in the

particular tissue or organism. Thus, AFPs bound to cell surfaces may promote supercooling of

freezing-sensitive tissues.

Many studies of the interaction of AFPs with cell membranes have dealt with effects at

temperatures above freezing, where interaction of the AFPs with ice is not a possibility. Preliminary

studies using porcine oocytes suggested that all types of AFP substantially increase membrane

integrity and cell survival at hypothermic temperatures. For example, normal membrane potentials

were maintained in oocytes treated with AFGPs at 4°C, while untreated cells exhibited membrane

depolarization at this low temperature (Rubinsky et al., 1990). Voltage clamping revealed that the

type I AFP of winter flounder (0.5 mg mL "1) inhibited Ca 2÷ and K ÷ currents in porcine granulosa

cells (Rubinsky et al., 1992b).

Blockage of Ca 2÷ channels may prevent some forms of damage caused to cells by

hypothermia. Hochachka (1986, 1988) proposed that the reduction in ATP synthesis that occurs in

cells under hypothermic conditions restricts the activity of membrane ATPases, including the

Ca2÷-ATPase, while having no effect on the ATP-independent movement of Ca 2+ and other ions

through membrane channels. This results in an uncontrolled increase in cytosolic Ca 2÷ that, in tom,

sets in motion several signalling pathways leading to cell damage and membrane destruction.

Calcium channel blockers have been shown to protect mammalian cells from hypothermic damage

(Ah'Rajab et al., 1991). Passive Ca 2+ entry was shown to be blocked by 1 mg mL "1 of type III AFP

from ocean pout in rabbit parietal cells loaded with the fluorescent Ca2+-binding dye, fura-2

(Negulescu et al., 1992). Thus, Negulescu and coworkers (1992) suggest that the protective effect of

AFPs may reside in their ability to block the entry of Ca 2÷.

Cell membrane damage may also result from the loss of lipid bilayer fluidity that can occur in

the membranes of cells that are transferred from their normal temperatures to hypothermic conditions

(Mazur, 1984). It is possible that effects of AFPs on cells during hypothermia may result from a

direct involvement with the membrane lipid bilayer. Hayes et al. (1993) found that AFGPs interact with liposomes when added above the phase transition temperature (Tin) of the lipid. Although

liposomes cooled through their T m normally lose 60% of their contents, the addition of 1 mg mL -1

AFGP prevents most of the leakage.

Other studies suggest interactions between AFPs and cell membranes may either cause

damage or show no effect at all. In contrast to the findings of Hayes et al. (1993), Rubinsky et al.

(1992b), and Negulescu et al. (1992), the addition of 20 mg mL 1 AFGP did not influence the

transport of Na + in cultured amphibian renal distal tubule cells (Petzel and DeVries, 1992).


Furthermore, very low (0.1 to 1 ktg mL "1) concentrations of AFGPs and a type I AFP appeared to be

mildly cytotoxic to ovine spermatozoa at 5°C (Payne et al., 1994a) and 1 mg mL "1 of type III AFP

and AFGP increased the leakiness of thylakoid membranes at 0°C (Hincha et al., 1993).

It is evident that AFPs are active at the cell surface. However, the mechanisms by which they

affect ion currents, membrane lipid bilayer integrity and cell survival at hypothermic temperatures are

not yet clear. The possibility of a direct interaction between AFPs and membrane lipids remains to be

investigated. If such an interaction does occur, the divergent membrane lipid compositions of

different cell types used in the various investigations may explain the contrasting results ranging from

hypotherrnic protection to cryotoxicity. While AFGPs have been widely used in these studies, all the

AFP types have been shown to have effects consistent with activities at the cell surface. The different

types of fish AFPs share no obvious similarities aside from their ability to interact with ice crystals,

although it has been suggested that the surfaces of crystals and membranes are similar in some ways

(Knight et al., 1991). Moreover, the concentrations at which the AFPs apparently interact with

membrane components are mostly in the range at which thermal hysteresis occurs. Biochemical

studies of the interactions between AFPs and membranes need to be undertaken in as much detail as

the studies of interactions between AFPs and ice crystals (reviewed by Hew and Yang, 1992) in order

to more clearly understand the activities of these proteins at cell surfaces.

FUNCTION OF ANTIFREEZE PROTEINS IN FREEZING TOLERANCE

Overwintering, terrestrial organisms are subjected to much colder environmental temperatures

and greater temperature fluctuations than found in cold oceans. Rather than risk injury that may occur

if the metastable, supercooled state is disrupted, many of these organisms form ice in a controlled

manner by secreting both ice nucleators and AFPs. The role of ice nucleators is to initiate the

formation of ice at a temperature and a location within the tissues that will minimize physical damage

to the organism. For example, ice nucleation occurs at a threshold temperature of -5°C in winter rye

leaves (Brush et al., 1994), which permits the leaves to supercool during a light frost, but ensures

that they begin to form ice at a high subzero temperature during a hard frost. Observations of frozen

plant tissues using the techniques of cryofixation, freeze substitution and electron microscopy, have

shown that ice does not grow uniformly throughout the tissues, rather it is localized to specific areas

(reviewed by Griffith and Antikainen, 1995). While ice nucleators may start ice formation in specific locations in tissues, they cannot

influence the subsequent growth of the ice crystals. Ice crystal growth must be modified by either

carbohydrates or AFPs. Although plants accumulate AFPs at low temperatures, the low level of

thermal hysteresis of these AFPs (generally 0.2 to 0.5°C; Duman et al., 1993), combined with the

low concentrations of AFPs that can be extracted from the tissues (compared with fish), both suggest

that the primary role of AFPs in freezing-tolerant plants can not be the prevention of the growth of

extraceUular ice. Instead, the function of AFPs in freezing-tolerance may be to limit the extent of ice

formation. An essential role of AFPs may be to bind to extracellular ice to localize its growth and

minimize the ensuing physical damage within the tissues. If AFPs are present on cell surfaces as


well, they could prevent inoculative freezing of the cytoplasm, especially in sensitive tissues that have

high cellular water contents. In addition, AFPs may maintain the small size of extracellular ice

crystals when freezing-tolerant organisms are exposed to conditions that promote recrystallization.

Although it is difficult to measure concentrations of AFPs in situ, recrystallization inhibition has been

observed at concentrations as low as 25 lag protein L -I in solutions containing all six winter rye AFPs

(C.A. Knight, W.C. Hon and M. Griffith, unpublished results). Therefore, it is very likely that

AFPs are present in the tissues of freezing-tolerant plants in sufficient quantity to inhibit

recrystallization.

AFPs may also slow the rate of growth of extracellular ice crystals during freezing. When

type I AFP from winter flounder was added at a concentration of 20 mg mL -I to bromegrass (Bromis

inermis) cell suspension cultures, the rate of freezing at a given temperature was slower than observed

in untreated cell suspension cultures (Cutler et al., 1989). The amount of ice that formed at a given

temperature was also lower in the presence of the fish AFP (Cutler et al., 1989). However, these

effects of AFP on freezing were observed at AFP concentrations that are much higher than the

concentration likely to be present in freezing-tolerant plants.

Resistance to low temperature diseases, such as snow molds and powdery mildew, is an

important factor determining winter survival in winter cereals. The fact that winter rye AFPs are also

PR proteins suggests that these proteins may play a role in disease resistance as well as in freezing

tolerance. For instance, winter cereals are more resistant to fungal diseases after the plants are cold-

acclimated (Tronsmo, 1984; 1985). In winter barley, increased disease resistance is correlated with

the appearance of PR proteins in plants exposed to low temperature as well as in plants inoculated

with mildew (Tronsmo et al., 1993). The fact that cold-induced endochitinases and endoglucanases

from winter rye exhibit both enzymatic activity and antifreeze activity suggests that these proteins

could play a dual role in disease resistance and freezing tolerance (Hon et al., 1994b).

USE OF ANTIFREEZE PROTEINS IN CRYOPRESERVATION

The interaction of AFPs with ice crystals and cell surface components makes them interesting

candidates for use in enhancing the cold storage and cryopreservation of cells and tissues for many

different purposes. However, results to date suggest that AFPs may either protect or harm cells,

depending on the AFP used, the concentration of AFP and whether the particular AFP also interacts

with membranes.

Protective Effects of Antifreeze Proteins The presence of AFPs may enhance the preservation of tissue integrity and cell survival

during freezing and thawing. In one study, the expression of a synthetic type I AFP-protein A gene

in yeast (Saccharomyces cerevisiae) increased cell survival to about 2.5% after rapid freezing to

-196°C (McKown and Warren, 1991). Although extracts from the transformed yeast cells showed

marked recrystallization inhibition, intracellular targeting of the synthetic AFP may have limited its

effectiveness in promoting cell survival. Porcine oocytes, treated with 20 mg mL -I of each of the


four fish AFPs, exhibited increased cell survival following apparent vitrification and subsequent

warming to above 0°C. Although oocytes did not survive freezing in the absence of AFPs, survival

in the presence of these proteins was nearly 75% of controls held at ambient temperature (Arav et al.,

1993).

The addition of AFPs may also improve the functions of cells and organs subjected to

freezing. Ovine spermatozoa showed significantly more motility after freezing in the presence of 0.1

to 10 gg mL "1 of type I AFP or AFGP, but higher AFP concentrations were less effective (Payne et

al. , 1994a). Adding 1 mg mL -1 AFGP and 0.5 M glycerol resulted in post-freeze/thaw bile

production in mammalian liver that was 2 to 5 times that obtained in the absence of additives

(Rubinsky et al., 1994).

The concentrations of AFPs that are protective to cells are too low to cause thermal hysteresis

or the stabilization of membranes reported to be conferred by the AFPs. The only known property of

AFPs at these very low concentrations is recrystallization inhibition. Carpenter and Hansen (1992)

compared the effects of type I AFP on rapid and slow warming of erythrocytes in the presence of

hydroxyethyl starch to determine whether AFPs protect cells by recrystallization inhibition. The

AFP, at approximately 40 gg mL 1, reduced the normally extensive hemolysis that results from

recrystallization in slowly warmed samples, while having no effect on the low levels of hemolysis

that occur when recrystallization is minimized by rapid warming. Thus, inhibition of re, crystallization may be the mechanism mediating some part of the observed protective effects of AFPs on cells during

freezing and thawing.

Injurious effects of AFPs Although reports of beneficial effects of AFPs during freeze/thaw events are accumulating,

several studies describe toxicity and physical damage to membranes, cells and organs frozen in the

presence of AFGPs. For example, AFGPs and AFP types I and III increase freeze-thaw damage to

photosynthetic membranes isolated from spinach (Spinacia oleracea) leaves (Hincha et al., 1993).

Disruption of the chloroplast membranes was determined by the release of plastocyanin from the

lumen of thylakoid vesicles and appeared to occur through direct interaction between the AFPs and

the membranes because increased leakage was also observed in the absence of freezing (Hincha et al.,

1993). At the cellular level, red blood cells frozen and thawed in the presence of glycerol containing

40 mg mL "l of the smaller AFGPs or 5 mg mL -1 of the larger AFPs exhibited elevated levels of

hemolysis compared with controls to which no protein was added (Petzel and DeVries, 1979).

Cryotoxicity was also observed in organs when rat hearts were flushed with solutions containing

AFGPs and frozen to -1.4°C for several hours (Wang et al., 1994). Hearts treated with 10 mg mL -1

AFGP failed to beat after thawing, those treated with 5 mg m L 1 AFGP had less damage, and those

treated with 10 gg mL 1 still showed reduced recovery compared with unfrozen controls (Wang et al.,

1994).

Carpenter and Hansen (1992) suggest that a delicate balance exists between cell preservation

and cell damage resulting from the presence of AFPs because effects of type I AFP on red blood cell

survival during freezing and thawing are concentration-dependent. During freezing, high (>1 mg


mL "1) concentrations of most AFPs cause ice crystals to grow as bipyramids or needles that may

cause damage to cells if the direction of growth is towards the surface of a cell. During rapid

warming, low concentrations of AFPs decrease cell damage, whereas higher concentrations (0.16 -

1.5 mg mL "l) of AFPs increase cellular injury. Microscopic evaluation revealed that higher

concentrations of AFPs resulted in the formation of bipyramidal ice crystals and greatly increased ice

growth around cells. In a subsequent study, Hansen et aL (1993) examined the effects of type I AFP

addition on damage to KG-1A (human leukemia) cells during slow freezing and rapid thawing in

dimethylsulfoxide. The addition of AFP did not improve cell recoveries at any concentration and

AFP concentrations greater than 100 gg mL "1 reduced cell viability. In agreement, Wang et al.

(1994) report unpublished observations describing the destruction of isolated cardiomyocytes in the

presence of high concentrations of AFGPs when spicular ice touched the cells during freezing.

Another factor that may contribute to damage during freezing in the presence of AFPs is the

increased speed of ice crystal growth. When a solution containing AFGPs is cooled to the

temperature at which ice crystals do begin to grow (the hysteresis freezing point), the crystals grow at

a rate up to five times greater than in water with no AFP (Harrison et al., 1987). The bipyramidal or

needle-like shapes of the crystals and their rapid growth during cooling may both contribute to cell

damage at high concentrations of AFPs. In contrast, cell protection may occur at lower AFP

concentrations through inhibition of recrystallization.

USE OF ANTFREEZE PROTEINS IN FROZEN FOODS

The ability of AFPs to depress solution freezing points, inhibit recrystallization during

freezing and thawing, and neutralize the effects of ice nucleators suggests possibilities for the use of

AFPs as natural ice modulators in the cold storage of food.

Recrystallization Inhibition Ice crystals may grow in frozen foods due to temperature gradients formed within the

products during freezing or thawing, or to the fluctuating temperatures that occur during defrost

cycles in storage or when products are in transit. The inhibition of ice recrystallization may be an

important factor determining the texture of frozen foods, especially foods such as ice cream and

popsicles that are eaten while frozen. The presence of AFPs in these products may inhibit ice crystal

growth and preserve the smooth, creamy texture of a high quality product.

The inhibition of recrystaUization may also be important in foods that are eaten after they have

thawed. When large ice crystals form intracellularly in tissues such as meat and fish, they can

damage membranes and cause increased drip during thawing. This may result in a lower quality

frozen product due to reduced water-holding capacity and loss of nutrients from the tissue. Meat

(bovine and ovine muscle) soaked in solutions of up to 1 mg mL -1 type I AFP or AFGP prior to

freezing at -20°C showed evidence of reduced ice crystal size when examined by light and scanning

electron microscopy following thawing (Payne et al., 1994b). It is not yet known whether the

reduced ice crystal size improved the quality of the meat.


It is possible to reduce the growth rate of ice and alter the shape of ice crystals using polymers

that form gels (Holt, 1991). Smith and Schwartzberg (1985) have shown that gelatin at

concentrations up to 0.5 mg mL -1 causes a reduction in ice recrystallization during freezing and

thawing. However, the mechanism of action of the AFPs appears to be distinct from those of other

inhibitors known to date. Therefore, AFPs may represent an interesting and versatile alternative for

use in inhibiting ice recrystallization in frozen food.

Preservation of Cellular Integrity

There are many foods, such as strawberries, raspberries and tomatoes, that cannot be frozen

without a reduction in quality. The cellular structure of these foods is damaged by freezing and leads

to the changes in both texture and flavor. There are two possible roles for AFPs in preserving this

type of food. One possible role of the AFPs at low concentrations is to promote and maintain the

formation of smaller ice crystals during freezing, which may preserve a somewhat higher percentage

of cellular integrity. The use of AFPs rather than sugars to control ice formation may lower the

extracellular solution concentration, so that less water is withdrawn from the ceils before freezing and

water can then be more readily absorbed by intact cells after freezing. The other possibility is to

introduce AFPs at a high concentration and to use higher storage temperatures (e.g. -5°C) to promote

supercooling rather than freezing of the tissues.

Reduction of Microbial Growth

When food is stored at temperatures below the minimum for microbial growth, spoilage is

generally very slow. However, there are two major sources of microbial contamination of frozen

foods (Robinson, 1985). The first is the microflora present in the food before freezing. The second

source of spoilage occurs during thawing when microbial growth accelerates. Although blanching,

cooking, pasteurization or other treatments may be used to reduce microbial contamination of foods

before freezing, these treatments are not always practicable. For example, in the case of ice cream,

the addition of fresh fruit or other flavorings may introduce yeasts and molds into the final product

(Rothwell, 1985).

In the U.S.A., approximately 24% of fruits and vegetables are lost to postharvest diseases

before processing (Wilson et aL, 1994). One strategy to reduce spoilage now under study is to use

postharvest treatments to induce disease resistance within the harvested fruits and vegetables.

Treatments with physical and biological agents such as heat, UV-C radiation and application of

chitosan have all proven effective in inducing the accumulation of PR proteins, such as endochitinases

and endoglucanases, within harvested fruits and vegetables (Wilson et al., 1994). As a result,

harvested crops, such as apples, carrots, citrus fruit, green peppers, peaches and tomatoes, all exhibit

reduced spoilage following treatment to induce resistance (Wilson et al., 1994). PR proteins induced

by low temperature in winter cereals also enhance disease resistance by inhibiting the growth of fungi

within the plant tissues (Tronsmo, 1985; Tronsmo et al., 1993). Thus, the presence of cold-induced

PR proteins in foods at harvest may act to reduce microbial contamination of the food products before

freezing, as well as inhibit microbial growth during thawing.


SELECTION OF ANTIFREEZE PROTEINS FOR USE IN FOODS

All of the AFPs described to date cause thermal hysteresis and many AFPs have been shown

to inhibit the recrystallization of ice. The exact relationship between recrystallization inhibition and

thermal hysteresis activities of the AFPs are not completely understood. However, it is reasonable to

suggest that all of the AFPs have both activities. The membrane-related properties of the fish AFPs

are less clear. Although different types of AFPs affected photosynthetic membranes differently

(Hincha et al., 1993), the effects on oocytes were reported to be quite similar for all AFP types

(Rubinsky et al., 1992a). A closer examination of the mechanism of AFP interaction with membrane

components will be necessary in order to determine the precise activities of the diverse AFPs and their

response to different membrane lipid compositions, membrane proteins and thermal regimes.

Four different AFP types have been isolated from fish to date (Davies and Hew, 1990).

Additional AFPs have been isolated from winter rye (Hon et al., 1994a; 1994b, bittersweet

nightshade and four insects (Duman et al., 1993). This alone presents a substantial choice for use in

applications. In addition, all the organisms produce multiple isoforms of their respective AFPs. For

example, Atlantic cod produces many isoforms of AFGPs which vary in both protein size and

specific activity (Hew et al., 1981; Kao et al., 1986). Moreover, AFPs have been engineered that are

more active than their counterparts isolated from fish. Synthetic forms of type I AFP containing four

and five helical repeats, instead of the three repeats normally found in winter flounder AFP, and fused

to another protein have been shown to be progressively more effective in recrystallization inhibition

than their 3-repeat relative fused to the same protein (Mueller et al., 1991). In addition, a recombinant

type III AFP with two additional amino acids expressed in Escher ich ia col i was found to have a

stronger effect on ice crystal growth and enhanced resistance to heat denaturation when compared

with the native ocean pout AFP (Li et al., 1991). Finally, the discovery that cold-induced winter rye

enzymes such as glucanase and chitinase exhibit both antifreeze and enzymatic activity (Hon et al.,

1994) raises the possibility of engineering antifreeze activity into other proteins naturally present in

food products.

The diversity of AFPs found among different organisms and their demonstrated possibifities

for useful modification through protein engineering make it feasible to choose the most appropriate

AFP for addition to a particular product or for expression in a particular plant or animal. Individual

AFPs have characteristics that could make them less appropriate for certain uses. For example, the

type I AFP, which is o~-helical at O°C, is largely unfolded at temperatures above 20°C and may not be

suitable for use in products that are heated before freezing. Other AFPs behave in ways that might

pose problems in certain foods. For example, the similarity of the type II AFPs to the C-type lectins

suggests that the AFPs might bind carbohydrates, although no such interaction has been detected

(K.V. Ewart, unpublished observations). Thus, if type II AFP were chosen for use in a food

product, the possibility of interaction with an abundant carbohydrate in the food would have to be

ruled out. Another such example involves the two winter rye AFPs that are similar to thaumatin (Hon

et al., 1994b). Thaumatin was originally isolated because it is an intensely sweet-tasting protein

(reviewed in Cornelissen et al., 1986). If the thaumatin-like proteins also taste sweet, their use may

be limited to food products that are normally sweetened during preparation.


~In all organisms where antifreeze activity has been observed, multiple AFPs are present

(DeVries, 1988; Duman et al., 1991: Hew and Yang, 1992). The distinct AFPs from different fish

have been shown to adsorb onto specific planes on the surface of ice (Knight et al., 1991: 1993).

Although binding to a single plane by an individual AFP may completely inhibit ice crystal growth

(the mattress felting hypothesis, Knight et al., 1991), the cooperative effect of multiple AFPs may

prove more effective (Hon et al., 1994a). This is an important point to consider when adding AFPs

to food. The addition of a single AFP at low concentration may prove effective in inhibiting the

recrystallization of ice. However, in some cases, the addition of several AFPs may be necessary to

lower the freezing temperature of an organism or to reduce the size of ice crystals formed in tissues

subjected to freezing.

PRESENCE OF ANTIFREEZE PROTEINS IN FOODS

Antifreeze proteins are present in a wide variety of organisms normally consumed as part of

the human diet. Because AFPs known to date are secreted and are normally active outside the cell, it

may be possible to introduce AFPs into other foods by physical methods such as soaking or vacuum

infiltration. It may also be possible to introduce AFPs into organisms or modify antifreeze activity in existing proteins by genetic engineering.

Antifreeze Proteins in the Human Diet

In nature, AFPs are synthesized by many organisms that are used for food. Examples include

winter flounder, Atlantic cod, blue mussels, carrots, cabbage and Brussel's sprouts. In most cases,

the levels of AFPs are higher in the tissues of organisms exposed to cold temperatures.

Differences in AFP levels in fish occur among species with different distributions, between

distinct populations within species, and within individuals according to the stage of development and

time of the year. AFP levels appear to be closely coupled to the extent of freezing risk, which

suggests that AFPs play a key role in the survival of fish in icy seawater. For example, fish residing

in permanently cold Arctic waters may have high AFP levels throughout the year. The Arctic

shorthorn sculpin has AFP levels in August that are equal to the maximum winter levels of AFP in

Newfoundland populations (Fletcher et al., 1982b). In contrast, the AFP levels in fish inhabiting

north temperate environments follow seasonal cycles with maximal levels occurring in the plasma in

winter. In winter flounder, AFP production follows a seasonal cycle in response to photoperiod

(Davies et al., 1988), reaching maximal levels in January and disappearing in May (Fletcher, 1977),

whereas AFGP production in Atlantic cod is responsive to temperature (Fletcher et al., 1987).

Timing of AFP production appears to closely precede the beginning of ice accumulation in the natural

habitat (Fletcher et al., 1985). In addition to the seasonal cycles of AFP expression, plasma AFP

levels can change over the course of development in some fish species, such as Atlantic cod. AFGP

levels in juvenile Atlantic cod are nearly twice the levels found in adults at the same time of time of the

year (Kao and Fletcher, 1988). Juvenile cod are believed to overwinter near shore (Keats et al.,

1987) where ice is prevalent, whereas adults are thought to move offshore (Templeman, 1979) where

ice is less of a risk.


Vegetable crops that are harvested after the occurrence of fall frosts also have accumulated

AFPs. Examples of such crops include carrots, cabbage and Brussers sprouts (Duman et al., 1993).

No antifreeze activity has been observed in plants from temperate climates during the summer (Urrutia

et al., 1992; Duman and Olsen, 1993; Duman et al., 1993). However, AFPs are induced by low

temperature (Griffith et al., 1992; Urrutia et al., 1992; Marentes et al., 1993), and so it is entirely

possible that crops such as carrots and cabbage may synthesize AFPs when they are placed in cold

storage for eventual sale on the fresh market. At this time, there is no information regarding the post-

harvest low-temperature physiology of vegetable crops. Data are also unavailable for comparing

seasonal differences in the quality of frozen vegetables that could be attributed to cold acclimation in

crops harvested late in the growing season.

Addition of Antifreeze Proteins Directly to Foods

The isolation, characterization and cloning of a number of distinct AFPs from fish makes it

possible to obtain large amounts of the AFPs and to introduce these proteins into frozen foods.

Products such as ice cream that are not composed of cells may be frozen with higher concentrations of

AFPs to minimize the size of ice crystals. The fact that AFPs are located extracellularly in freezing-

tolerant organisms also indicates that these proteins can be added to foods by physical means such as

mixing, injection, soaking or vacuum-infiltration. A promising example is the report discussed earlier

that demonstrated that meat soaked in a solution of AFGPs or type I AFP exhibits reduced ice crystal

size and tissue damage after freezing (Payne et aL, 1994b).

In several cases, the addition of AFPs directly into plants and animals influences their freezing

characteristics. The lethal freezing temperature of rainbow trout is lowered in direct proportion to the

amount of type I AFP injected into the fish (Fletcher et al., 1986). The response is somewhat more

variable in plant tissues. When leaves were vacuum-infiltrated with 1 mg mL l type I AFP from

winter flounder, the threshold nucleation temperature was lowered by 1.8°C for canola and by 4.0°C

for Arabidopsis thaliana (Cutler et al., 1989). In contrast, there was an increase from -7.2°C to

-0.8°C in the threshold ice nucleation temperature for potato leaves (Solanum tuberosum) vacuum-

infiltrated with AFP, a result that suggests that not all plant tissues respond in the same way to the

addition of AFPs (Cutler et al., 1989). Cutler and co-workers (1989) also examined the freezing

characteristics of bromegrass cell cultures suspended in 20 mg mL "l type I AFP by NMR and

determined that there was a decrease in the amount of freezable water that froze at a given

temperature, as well as a decrease in the rate at which water froze.

Introduction of Antifreeze Proteins to Foods by Gene Transfer

The introduction of an AFP into plants and animals through AFP gene transfer has resulted in

organisms that exhibit lower freezing temperatures and increased inhibition of ice recrystallization.

AFP gene transfer has also resulted in expression of AFPs in several food organisms, including

salmon and tomato. Atlantic salmon (Salmo salar) do not normally produce their own AFP; however,

transgenic salmon produce type I AFP from introduced flounder genes (Hew et al., 1992). The

expression of AFP in the transgenic salmon is intended to protect salmon from freezing when they are


raised in coastal enclosures where sea ice may form, but expression levels are, so far, too low to be

protective (Hew et al., 1992). The goal of the transgenic tomato project is to express a protein A-type

I AFP gene within the tomato fruit in order to improve the quality of the fruit after freezing.

Transgenic tomato plants have been obtained that accumulate the protein A-type I AFP fusion protein

in the leaves in sufficient quantities to inhibit ice recrystallization (Hightower et al., 1991), but

expression of the gene in fruit has not been demonstrated.

The use of gene transfer to generate food organisms that produce AFPs needs to be carried nut

with careful consideration of the concentrations sought and the type of AFP most appropriate for the

organism used. As observed in polar fish, high concentrations of AFPs are required to promote

supercooling in the presence of ice. In fish, these high concentrations of AFPs are obtained by the

expression of multiple copies of the genes and by the expression of genes encoding different isoforms

of the AFPs (Fourney et al., 1984; Scott et al., 1985; Davies et al., 1984; Hew et al., 1988; Scott et

al., 1988; Hayes et al., 1989; 1991; Hsiao et al., 1990). This level of AFP gene expression may be

difficult to obtain by gene transfer. Moreover, work discussed earlier suggests that the interaction of

some AFPs with cell surfaces may be detrimental to plants and animals if the proteins are sufficiently

abundant and they are present under the proper conditions. Thus, the most promising use of AFP

genes in foods at this time is to obtain low levels of AFP gene expression in order to inhibit the

recrystallization of ice. One point to consider, however, is that our understanding of the effects of

AFPs above the freezing point is rudimentary at present and more research into activities of the AFPs

will be necessary in order to predict their behaviour in different systems. Another important

consideration is that of consumer acceptance of transgenic products. For example, the transformation

of tomatoes with an AFP gene from fish has proven to be unacceptable to some consumers.

Consumer acceptance may depend on the transfer of genes from closely related organisms, and the

recent identification of AFPs in a wide range of overwintering organisms may facilitate selection of

the most appropriate AFPs for specific gene transfers. Thus, it may be more acceptable to consumers

to transfer an AFP gene from P seud om on as put ida to Lactobaci l lus sp. for use in the production of

frozen yogurt, or an AFP gene from winter rye to raspberries for production of a more disease and

freezing resistant fruit, than to use fish AFPs in all applications. In very closely related species, it

may be possible to obtain AFP gene transfer by normal breeding and selection.

The challenges of introducing AFPs into organisms to enhance the freezing properties of their

tissues when processed as foods are best illustrated using the type I AFP as an example. The type I

AFP, while an obvious choice for transformation based on our understanding of its activities, may be

difficult to synthesize in some systems and may not be stable, particularly at the temperatures of

mammals. Transgenic fruit flies harbouring the type I AFP gene did not appear to translate the

mRNA efficiently, possibly because of a codon bias, and resulting protein levels were very low

(Rancourt et al., 1992). This problem can be obviated by designing a synthetic gene with a codon

composition appropriate to the animal or plant to be used as done by Georges et al. (1990). Yet, even

if translation is efficient, the type I AFP may have a short half life in some systems because it unfolds

progressively as the temperature is raised above 0°C (Anantharayanan and Hew, 1977) and it may

become more susceptible to protease digestion. In transgenic tobacco transformed with the gene


encoding the winter flounder type I AFP, AFP mRNA was detected in plants grown at 25°C, but the

protein was not (Kenward et al., 1993). The AFP was detected in leaf tissue only after the plants had

been transferred to 4°C, which suggests that either translation was less efficient or the protein was

degraded at warmer temperatures. However, the protein that accumulated at 4°C co-migrated with

proAFP, which suggests that the preproAFP was targeted correctly into the endoplasmic reticulum for

eventual secretion, but was not processed further (Davies et al., 1982; Kenward et al., 1993).

ProAFP has 70% of the activity of the mature protein, so the final processing step may not be

essential for successful transformation (Hew etal . , 1986). In summary, an appropriate choice of an

AFP gene for expression in a dissimilar species depends in part on the ability of the host to produce

the protein, to process and target it correctly and to maintain it at stable levels.

Gene transfer may be more appropriately used in an overexpression system to produce large

amounts of AFPs in order to harvest them for subsequent addition to food products. However, no

attempts to use this method have been reported.

C O N C L U S I O N S

AFPs are natural products that form part of a normal diet through their occurrence at high

concentrations in food fishes and vegetables. Their diversity and their various activities show that the

proteins are intriguing candidates for use in preserving the quality of foods during chilling and frozen

storage. This may make them ideal for addition to other foods either directly or by gene transfer, but

the concentrations and types of AFPs used will have to be selected carefully. For example, it will be

essential to choose AFPs that are stable throughout the range of temperatures used in processing. At

high (>1 mg/mL) concentrations, AFPs depress the freezing temperature non-colligatively.

However, even the presence of high concentrations of fish AFPs does not depress the freezing point

by more than about 2°C and the amount of protein required may lead to unreasonable costs. At

elevated concentrations, the use of a single AFP promotes rapid, spicular ice formation that may

increase cellular injury. However, these high concentrations of AFP appear to interact with some

membrane components to protect ceils from chilling damage. This property requires further study in

order to understand the mechanism by which it occurs and its potential usefulness. At low

concentrations, the AFPs can be employed very effectively to inhibit recrystallization during freezing

and thawing to maintain the texture and flavor of frozen foods. The use of AFPs from overwintering

cereals may have the additional effect of reducing spoilage before freezing or during thawing.


Acknowledgements We thank Dr. Choy L. Hew for his generous support and encouragement and we thank

Shashikant Joshi for many helpful discussionS. -K-.V..Ewart i.s a post-doctoral fellow in the

laboratory of Dr. C.L. Hew and she is supported by a fellowship from the Medical Research Council

of Canada. M. Griffith's research is supported by the Natural Science and Engineering Research

Council of Canada. This review was presented at the Second European Congress on Food Freezing

held at the University of York, York, U.K., April 6 and 7, 1995.


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Antifreeze proteins and their potential use in frozen foods