[Advances in Food Research] Advances in Food Research Volume 13 Volume 13 || Fundamentals of Low-Temperature Food Preservation

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  • FUNDAMENTALS OF LO W-TEMPERATU RE FOOD PRESERVATION

    BY 0 . FENNEMA ASD W . D . POWRIE

    . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . Introduction 220 221

    -4 Intermolecular Attractive Force of HOH Molecules 221 B . Proposed Structures for Water and Ice 222 C . Effect of Solutes on the Structure of Water 228 D . Gas Hydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 E . Effect of m-ater on the Structure of Proteins 232 F . Bound Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

    I11 . Phase Diagram of Pure Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 238

    B . Freezing Point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 B . Density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 C . Specific Heat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 D . Latent Heat of Fusion E . Thermal Conductivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Thermal Diffusirity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    V . Crystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 A . Xucleation 244 B . Crystal Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 C . Ice Crystal Size D . Recrystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 E . Location of Ice Crystals in Cellular Suspensions and Tissue . . . . . . . . .

    VI . Freezing Diagrams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 :I . Freezing Diagrams of Watrr and Simple Solutions . . . . . . . . . . . . . . . . B . Ireezing Diagrams of Food Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Structure of kVater and Ice . . . . . . . . . . . . . . . . . .

    . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    . . . . . . . . . . . . . . . . . . . .

    IV . Some Additional Physical Properties of Katcr and Ice . . . . . . . . . . . . . . . . .

    . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 241 243

    . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

    257

    259 261 250 272 274

    B Factors Affecting Rates of Freezing and Thawing 258 C . Comparatire Rates of Freezing and Thawing of Normally Rigid

    VII . Concentration of Nonaqneous Constituents during Freezing . . . . . . . . . . . VIII . Volume Changes during Freezing and Thawing . . . . . . . . . . . . . . . . . . . . . . .

    . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I X . Rate of Frerzing A . Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

    Aqucous Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283 S . A Search for Protective Chemical Additives . . . . . . . . . . . . . . . . . . . . . . 288

    . . . . . . . . . . . . . . . .

    X I . The Effect, of Freezing, Storage, and Tlr:nving on the Physical and Chrmical Prolwrtirs of Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 A . Classification and Characteristics of Food . . . . . . . . . . . . . . . . . . . . . . 289 3 . Changcs in Selected Food Systcnis Resulting from the Freezing Process 292

    219

  • 220 0. FENNEMA A S D W. D. POWRIE

    SII. Conclusions Concerning Possible Causes of Frozen Food Deterioration . 31 1 A. Possible Causes of Damage during Freezing . . . . . . . . . . . . . . . . . . . 311 H. Possible Causes of Damage during Frozen Storage . . . . . . . . . . . . . . 315 C. Possible Causes of Damage during Thawing . . . . . . . . . . . . . . . . . . . . . 317

    SIII. General Aspects of Commercial Freezing Processes . . . . . . . . . . . . . . . . . . . . 317 8. Methods of Freezing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 13. Selection of Fruits and Vegetables for Freezing . . . . . . . . . . . . . . . . . . . 319 C. Prcfrcezing Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 D. The Influencc of Freezing Rate on Quality . . . . . . . . . . . . . . . . . . . . . . . . 322 E. Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 F. Thawing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

    S I V . Closing Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 References . . . . . . . . . . . . . . . . . . . . . . . . . . 330

    I. INTRODUCTION

    The preservation of food by freezing is not new. Early man used ice and snow as one nieans of preserving his food. Even the commercial freez- ing of food by artificial methods had its origin nearly 100 years ago. During the last several decades, the quality of frozen foods has been vastly improved through the use of foods more suitable for freezing and the use of better processing and handling techniques. The widespread present-day acceptance of frozen foods can be attributed largely to these advances.

    Recommended processing procedures for most foods are readily availa- ble in publications serving the food industry. Probably the most useful publications in this regard are Tressler and Eyers excellent two-volumc set, The Freezing Preservation of Foods (1957a, 1957b), and U.S.D.A. Bulletin ARS-74-21, Conference on Frozen Food Quality (Anonymous, 1960a), which partially summarizes an extensive research program on frozen foods conducted a t the Western Regional Research Laboratory, U.S.D.A.

    Tlie value of the above works is indisputable, but a need for a more fundamental approach to the study of frozen foods nevertheless seems evident. As in any field, the technological advances of the future will dc- pend in a large measure on the effectiveness of our present prograni of fundamental research. Yet, anyone seeking knowledge of the fundamental factors involved in the freezing, storing, and thawing of foods will find i t difficult to obtain. A cursory review of the references a t the close of this paper illustrates part of the problem. A great many of these articles appear in publications unavailable to most food scientists. Furthermore, many of the more basic studies inyolve nonfood items such as bull sperma- tozoa, red blood cells, and microorganisms. There is a clear need to evalu- ate these studies, extract the parts pertinent to food materials, and present the results in a conimunication available to food scientists. That is our intent.

  • LOW-TEMPERATURE FOOD PRESERVATION 22 1

    I I . STRUCTURE OF WATER AND ICE

    Kater, like the air we breathe, is so common that i t may hardly semi worthy of consideration. Szent-Gyorgyi (1960), in stressing the important and underemphasized role of water in biological materials, stated that "Biology has forgotten water as a deep-sea fish may forget about it." This is perhaps understandable, since biological materials contain such coni- plex and exciting constituents as proteins, carbohydrates, and lipids. Water appears to be an uninteresting, simple, and inert constituent, but is quite the contrary. Foods intended for freezing invariably contain a large percentage of water; otherwise there would be no need to freeze them. Many cuts of lean meat contain 65-70% water, and as much as 90% water is common in fruits and vegetables. Some water is adsorbed so strongly to food constituents such as proteins and polysaccharides that i t contributes to their native structure and even to the over-all nature of the product. Anything which modifies the normal relationship between water and the other food constituents will likely alter the typical character of the food. A change of state, such as freezing, most assuredly has this effect. Further- more, unfrozen water exists in nearly all frozen foods stored under normal commercial conditions. Although the per cent is small, i t appears to have a much greater influence than would be suspected. There are ample rea- sons, therefore, to undertake a detailed study of both water and ice.

    A. IXTERMOLECULAR ATTRACTIVE FORCE O F HOH AIOLECULES Without prior knowledge, one would expect water and other coni-

    pounds of similar molecular weight and electronic configuration to exhibit similar properties. When the properties of water are compared with those of other hydrides of elements near oxygen in the periodic table (CH,, KH3, HF, PH3, H2S, HCl) , water is found to have unusually high values for melting point, boiling point, heat capacity, latent heat of fusion, latent heat of vaporization, surface tension, and dielectric constant (Edsall and Wynian, 1958; Wells, 1950). To illustrate the striking differences involved,

    TABLE I PHYSIFAL PROPERTIES OF SOME HYDRIDES YEAR OXYGEN I N THE PERIODIC TABLE

    Melting point Boiling point Mold heat of vaporization Substan re ("C) ("C 1 (ral/mole)

    CHI - 183 -161 2200 XH? - 78 - 3 3 5550 HF - n'L +19 7220 H?O 0 +100 9750

    ~ ~ ~~~~~

  • 222 0. FENNESIA A S D \T. D. IO\TRIE

    soiiie of the physical properties of n-atcr antl B fen- of tlic coriipoiintls men- tioned above are listed in Table I.

    These properties indicate the existerice of an unusually strong iittrac- tive force between water molecules. This attractive force arises from n combination of circumstances (Edsall and IYyiiian, 1958; Wells, 1950) :

    1 ) The HOH molecule, by virtue of its covalent hydrogcn-oxygen bonds, is ahlc to undergo intermolecular hydrogcn bonding. This condition results from the greaf, affinity of oxygen for electrons (second only to fluorine as the most, electronegntivc atom), enabling it t o gain electrons at the rxpense of tire nrighhoring hydrogen atoms. The oxygen atom therefore assumes a nct charge of minus two, and each hydrogen atom is left essentially as a positively charged proton. The unsymmetrical distribution of charges in the molecule (callcd a dipole) causes water molecules to orient in a fashion whereby the ncgativc charge of one molecule is associated with the positive charge of a neighboring molcculc. This type of molecular attraction antl orientation is particularly strong when hydrogen and the highly electronegative a t o m F, 0 , or N are involved, and has been given the name hydrogen bonding. The hydrides NHa, H20, and HF exhibit the strongest hydrogen bonding among molecules of low molecular weight.

    The energies of hydrogen bonds are .sniwll (es~tiiniites range from 1.3 to 6.8 kilo- calories per niole ; Kkmethy anti Scheraga, 1962a) compared to covalent bonds but are 1:ti.g~ enough compared to the averagr kineiic encrgics of molecules, i Irat thcy significantly influence the physical 1)ropert ies of biological materials. [For conipai,ison, the enprgies of representative covalent bonds (in kcal/niole) a re : C-C, 80: C-H, 98.2; 0-H, 109.4; C=C, 145.1

    2 ) The atomic composition and groinetry of tlie water molecule facilitatr : a) :I maximum amount of hydrogen bonding (the charged atoms are sterically exposed), antl b) tlircc-diniensionR1 liydrogen bonding. Both factors contribute to the ahnor- i d l y sti.ong association of watcr molecules.

    Tlic forces of attraction are best described by situating the riiolecule in an imaginary tetrahedron as indicated in Fig. 1. With oxygen a t the center of the tetrahedron, the hydrogen atoms may be positioned on any two of the lines originating a t the center of the tetrahedron and passing through any apex. The two lines chosen for the hydrogen at.oins represent tlie di- rection of the two positive forces. HOH molecules tend to orient and associate in accordance with these lines of force.

    lu. PROPOSED STRUCTLRES EOR \VATER ~ N D ICE

    The structure of wttcr and ice lias been inyestigated and discussed by a number of workers (Barnes, 1929; Barrcr and Stuart, 1957; Bernal, 1958; Buswell and Rodcbush, 1956; Claussen, 1951a,b; Edsall and W y - iiiaii, 1958; Eigen and IleMaeyer, 1958; Frank, 1958; Frank and Evans, 1945; Frank and Wen, 1957; Heslop and Robinson, 1960; Klotz, 1958; Lonsdale, 1951, 1958; Luyet and Rapate, 1958; Rlason, 1958a; Mullcr and yon Stackclhcrg, 1952; N h c t h y and Schcraga. 1962a,b,c; Omton, 1958;

  • LO\V-TE1LIPERATURE E 001) PREYERVATIOS 223

    FIG. 1. Orientation of c3liarpt.s arou~id :in HOH molecule.

    Szont-Gyiirgyi, 1957; van P~KIth~h!OIl van Eck e t al.. 1958; Vogcl, 1921 : von Stackelberg and Muller, 1951). Proposed structures are bascd on in- foriiiation provided by X-ray, neutron, and electron diffraction patterns, and by infrared and ranian spcctra.

    r p o n freezing, HOH inolecules associate in an orderly manncr to forin ii rigid structure which is more open (less dense) than the liquid forin. "Rigid," as used here, applies only in a inacro sense, since considerable nioreincnt of individual atoins and iiiolecules has been reported in ice, 1)articularly a t temperatures just below the freezing point (Lonsdale, 1951). At -10"C, for example, an HUH inolccule vibrates with an ainpli- tutle of approximately 0.44 A, which is nearly one-sixth the distance be- tween adjacent HOH niolecules (Owston, 1958). Furthermore, the hydro- gen atoms in ice arc apparently capable of wandering from one oxygen atoiii t o another, lending a certain degree of covalency to thc hydrogen bonds (Lonsdale, 1951; Frank, 1958). These comments should be kept in mind during the following presentation, where bond lengths are gircn as

  • 224 0. FENNEMA AND W. D. POWRIE

    fixed distances and the type of bonding is referred to solely as covalent or hydrogen bonding.

    Each HOH molecule, by virtue of its four tetrahedrally spaced attrac- tive forces, is potentially able to associate, by means of hydrogen bonding, with four other HOH molecules. In this arrangement each oxygen atom is bonded covalently with two hydrogen atoms, each a t a distance of 0.96 A, and hydrogen-bonded with two other hydrogen atoms, each a t a distance of 1.80 A. This results in a quite open, tetrahedral structure, with adjacent oxygen atoms spaced approximately 2.76 A apart and separated by a single hydrogen atom. All bond angles are approximately 109 degrees. This structural arrangement is shown in Fig. 2. Molecules 1 and 2 are

    \!

    i

    0 =oxygen 0 1 - - 1 - - 1 0 = 2.76 0 =Hydrogen = 0.96

    I-+* = Hydrogen bond C-I-I-I~ = 1.80 - =Chemical bond

    FIG. 2. Hydrogen-bonded arrangement of HOH molecules in ice.

    shown as the central oncs, each being surrounded by four equidistant neighbors. Note, however, that every molecule may be considered as a central one since, if the drawing were extended, each would have four tetrahedrally arranged equidistant neighbors.

    Combining the fixed structure shown in Fig. 2 with neutron diffraction patterns and the knowledge of atomic movement in ice gives rise to the more complicated but generally accepted structure known as the statis- tical, or Pauling, half-hydrogen structure (Owston, 1958; Mason, 195%). In this structure each oxygen, in a statistical sense, is surrounded by four

  • LOW-TEMPERATURE FOOD PRESERVATION 225

    half-hydrogen atoms, each a t an 0-I distance of one A (two half-hydro- gen atoms distributed between each two oxygen atoms in the following manner,

    -2.76 a )%X=-x+

    where the half-sphere symbol designates a half-hydrogen atom). This con- figuration merely shows the mean positions of the mobile hydrogen atoms during the course of time. Each half-hydrogen position should be regarded as being occupied by a hydrogen atom approximately half the time and vacant the rest of the time.

    Extension of the model in Fig. 2 draws attention to another charac- teristic of the structure of ice, that is, the hexagonal pattern established when several tetrahedrons are assembled. This arrangement is shown in Fig. 3. When extended to the macroscale, the hexagonal patterns give rise

    A

    C .

    B. * = HOH MOLECULE

    FIG. 3. Structure of ice. A ) Arrangement of HOH molecules in two-dimensional hexagons. The tetrahedral

    substructure can be visualized by considering Molecule 1 and its neighbors, a, b, and c. The fourth neighbor, not shown, would be located either above or below the plane of the paper. The hexagonal grouping represents the top view of a basal plane.

    B) Three-dimensional view of a basal plane. The front edge of basal plane B corresponds to the bottom edge of A.

    C) Thc molecular structure of ice. Obtained by combining three basal planes.

    to the uncountable variations of intricate six-pointed crystals (dendritic) commonly observed in snowflakes and in many aqueous solutions (Luyet and Rapatz, 1958; Mason, 1961 ; Chalmers, 1959).

  • 226 0. FENNEMA AlUD W. D. POWRIE

    Luyet and Rapatz (1958) and Mason (1961) have conducted studies on the basic f o r m of crystals (e.g., hexagonal forms, dendritic, spherulite, needles) which develop under various conditions. Luyet and Rapatz sug- gested that transition from a hexagonal form into a dendritic star form can be explained a t least partly on the basis of geometrical considerations. Since thc heat of crystallization would be dissipated more readily from the edges of a solid polyhedron than from the faces (Vogcl, 1921), i t fol- lows that crystal growth rates would be greater on the edges than on the faces.

    It is apparently necessary to decrease the temperature to -183C or lon-er to achieve maximum hydrogen bonding, where every HOH molecule associates with four others (Heslop and Robinson, 1960). This tempera- ture coincides with the boiling point of oxygen. As the temperature is in- creased above - 183C, molecules possessing fewer hydrogen bonds ap- pear and the structure becomes less ordered, allowing increasing freedom of movement of the H O H molecules. Ice a t 0C still retains a high degree of hydrogen bonding and a structure which is rigid, open, and quite ordered.

    t-pon change of state from ice to water, rigidity is lost, but water still retains a large number of icelike clusters. Use of the term icelike cluster does not imply tha t the clusters existing in water have an arrangement identical to that of ice. Some differences are: 1 ) the HOH-bond angle of water is several degrees less than that of ice (van Panthaleon van Eck e t nl., 1968; Buswell and Rodebush, 1956) ; and 2) the average distance between oxygen atonis is approximately 3.1 A in water, and approximately 2.76 f i in ice (van Panthaleon van Eck et al., 1958). Furtherniorc, there is some question as to whether or not the icelike clusters of water exist in a tetrahedral arrangement, as is characteristic of ice. Since the average intermolecular distance is greater in water than in ice, it follows that thc greater density of water must be achieved by each molecule having more neighbors. A cuhic structure wherein each HOH molecule is surrounded by six others has been suggested (van Panthaleon van Eck e t al . , 1958; Bernal, 1958; Buswell and Rodebush, 1956).

    The highly unstable nature of the icelike clusters in water is another difference worth noting. Frank (1958) pictures water as consisting of flickering clusters of bonded molecules surrounded by, and rapidly al- ternating roles with, nonbonded fluid. He suggests that this pattern may well extend in a reverse fashion to ice, where the continuous ice phase surrounds flickering droplets of water.

    Regardless of thesc differences, hydrogen-bonded clusters of icelike niaterial do exist in water, but inoat of the consequences remain to be discovered. Ni.methy and Schcraga (1962a) have made a theoretical coni-

  • LOM'-TEMPERATURE FOOD PRESERVATlOS 227

    putation of the number and size of clusters existing in water at various temperatures, and found the resulting values to agree well with the ob- served behavior of water. The results are shown in Table 11.

    TAiBLE I1 CALCULATED I\UMBEIt AND SIZE OF ICELIKE CLUbTEItS EXISTING IN WATER

    AT VARIOUS TEMPERATURES"

    Mole fractions of various species for Fraction of

    Tempera- Cluster per molecule : hydrogen ture Cluster concen- - - bonds

    sizeb trationc 0 1 2 3 4 unbrokend ("C,

    the following No. of H bonds

    _________ 0 90.6 0.84 X 0.24 0.21 0.03 0.21 0.30 0.528

    10 71.5 1.02 0.27 0.22 0.04 0.21 0.26 0.493 "0 57.0 1.24 0.29 0.23 0.04 0.20 0.23 0.462 3 0 46.5 1.47 0.32 0.24 0.04 0.19 0.21 0..134 40 38.4 1.72 0.34 0.24 0.05 0.19 0.19 0.409 50 32.3 1.98 0.36 0.25 0.05 0.18 0.17 0.388 60 27.8 2.24 0.38 0.25 0.04 0.17 0.16 0.370 70 24.9 2.43 0.39 0.25 0.04 0.16 0.15 0.356 80 22.9 2.57 0.41 0.25 0.04 0.15 0.15 0.344 90 21.8 2.64 0.42 0.25 0.03 0.15 0.14 0.334

    100 % l . O 2.68 0.44 0.25 0.03 0.14 0.14 0.325

    a From Ndmethy and Scheraga (196%); J. Chem. Phys. 36, 3382. Courtesy of the American Institute of Physics.

    Average number of molecules per cluster. "Moles of clusters" (or number of clusters) per mole of nater. Maximum hydrogen bonding occurs when each HOH molecule associates 1th

    4 others.

    At O"G, water contains icclike clusters averaging 90 molecules per cluster. With increasing temperature, clusters become smaller and more numerous.

    At O"C, approximately one-half the hydrogen bonds originally present a t - 183C remain unbroken, and wen a t 100C approximately one-third arc still present. All hydrogen bonds are broken when water a t 100C changes into vapor a t 100C. The large heat-of-vaporization for water lends support to the contention that considerable hydrogen bonding still exists in water a t 100C (Edsall and Wyman, 1958).

    It is well known that ice, upon melting, undergoes an increase in den- sity, and, as the temperature is raised from O"C, the density continues to increasr until a maximum is obtained a t 4C. Further increases in tem- perature result in progressive decreases in density. The capability of water to retain icelike clusters fa r above the irielting point provides a logical

  • 228 0. FENNEMA AXD W. D. POWRIE

    explanation for this phenomenon (Edsall and Wyman, 1958; Heslop and Robinson, 1960). As the temperature of water is raised above the melting point, two opposing factors apparently influence its density: 1) a decrease in size of the icelike clusters leads to a more dense structure; and 2) thermal expansion causes a decrease in density.

    The effect of cluster breakdown is thought to predominate in the range of W " C , thereby causing an increase in density. At 4"C, the two effects apparently come into balance, and as the temperature is increased above 4C the effect of thermal expansion apparently predominates, causing a decrease in density with further increases in temperature.

    The changes in water with change in temperature are graphically sum- marized in Fig. 4, giving emphasis to the fact that water a t 100C differs

    MAXIMUM

    POINT

    VAPOR NO H-BONDING.

    LIQUID : APPROXIMATELY 30% OF P d E N T J A L H- BONDS REMAIN LN- BROKEN.

    LIQUID : CONTAINS MANY LARGE I C E - L I I ( E CLUSTERS.

    SOLID : R I G I D STRUCTUPJ l44AINTAINED BY H-BONDDIG. L E S S DENSE THAN L I Q U I D .

    COMPLETE H-BONDING. EACH HOH MOLECULE H-BONDED T O 4 6 H E R S .

    FIG. 4. Effect of temperature on the hydrogcn-bonded structure of water and ice.

    from water a t any other temperature and that the extent of hydrogen bonding in icc differs a t all temperatures between 0C and -183C.

    C. EFFECT OF SOLVTES ON THE STRUCTCRE OF WATER Solutes apparently produce considcrablc alteration in the structure of

    pure water. Electrolytes are reported to decrease the formation of icelike clusters in water (Frank and Wen, 1957). The ions of most electrolytes in aqueous solution are highly hydrated, being surrounded by a shell of radially oriented and relatively immobile water molecules. (The ability of HOH molecules to surround and isolate each ion markedly reduces the at- traction between oppositely charged ions and results in the high dielectric constant of water.) The radial orientation of water molecules in the

  • LOW-TEMPERATURE FOOD PRESERVATION 229

    hydration shells is considered detrimental to the formation of hydrogen- bonded HOH clusters. The ions of water itself are an exception to this rule, since they enhance cluster formation (Eigen, 1959; Frank and Wen, 1957).

    Sonpolar solutes such as hydrocarbons, nonpolar gases, and the non- polar side chains of large molecules (proteins being a particularly im- portant example) promote the formation of water-to-water hydrogen bonding (Nkmethy and Scheraga, 1962b; Frank and Evans, 1945; Klotz, 1958). Water molecules in the vicinity of such groups show no inclination to interact with them; instead, they interact more strongly with them- selves, thereby becoming more icelike in character than the remaining water molecules.

    D. GAS HYDRATES The structure of water is undoubtedly affected in the most unique

    fashion by molecules capable of forming clathrate compounds. The term clathrate is from the Latin clathratus, meaning enclosed or protected by crossbars of a grating. Powell (1956) defined clathrate compounds as those in which two or more components are associated without chemical union but through complete enclosure of one set of molecules in a suitable structure formed by another. One can think of the enclosed molecules as the guest (or hydrating agent), and of the molecules that form the enclosure as the host. A clathrate compound in which water serves as the host is often referred to by the more specific term gas hydrate. Gas hydrates are crystalline in nature and consist of small, often gaseous molecules enclosed in an icelike lattice of water molecules. They appear much like ice but are distinguished by their slightly diff went molecular structure and by their ability to exist a t temperatures well above the normal melting point of ice, providing the pressure is sufficient. The freezing of natural-gas pipelincs a t temperatures as high as 20C has been attributed to gas hydrate formation (Hammerschmidt, 1934).

    The two components of a gas hydrate do not interact chemically; in fact, chemical interaction prevents gas hydrate formation (Mandelcorn, 1959). The guest molecules of a gas hydrate are held in place solely by weak van der Waals forces (Bhatnagar, 1962).

    The physical properties of hydrating agents (guests) are quite varied, but some general qualifications have been reported by Towlson (1959) :

    1) Small degree of ionization in water. 2) Low solubility in water. 3) Suitable size and molar volume. Molecules such as He and Hz

    are too small, whereas molecules the size of pentane and hexane are too large.

  • 230 0. FENNEMA A S D W. D. POWRIE

    4) Boiling point usually less than 60C. This limit applies to simple

    Frequent exceptions can be found to these rules (Towlson, 1959). Chlo- rine, bromine and sulfur dioxide all form gas hydrates yet they violate the rules for water solubility and degree of ionization. Values for dipole moment are quite undependable as a means of predicting whether or not a given compound will function as a hydrating agent. For example, gas hydrates can be formed from compounds with large dipole moments (chloromethane and sulfur dioxide) as well as from compounds with zero dipole moment (methane, ethane, carbon dioxide, chlorine).

    The halogenated hydrocarbons (1 or 2 carbons) and propane are some of the more common agents capable of hydrating under low pressure con- ditions (Briggs and Barduhn, 1963).

    Gas hydrates are believed to exist in one of two basic structures (Type I and Type 111, depending on the sizc of the enclosed hydrating agent. Both structures are based on a framework of water molecules coordinated in a tetrahedral fashion (but slightly different from normal ice). When extended to a somewhat larger scale, this results in pentagonal dodeca- hedron units (or other types of polyhedrons with more than 12 faces) arranged in a cubic lattice structure (von Stackelberg, 1949; von Stackel- berg and Muller, 1951, 1954; Muller and von Stackelberg, 1952; Claussen, 1951a,bi.

    The unit cell of the Type I structure consists of 46 water molecules arranged to form six large and two small cavities. Hydrating agents of small molecular size such as methane, carbon dioxide, ethane, ethylene, hydrogen sulfide, nitrous oxide, acetylene, and methyl fluoride promote the Type I structure and are capable of occupying any of the eight cav- ities (von Stackelberg and &fuller, 1954). Hydrating agents of slightly larger molecular sizc, such as ethyl fluoride, chlorine, methyl bromide, sulfur dioxide, methyl mercaptan, difluorochloromethane (Freon-22), and chlorofluoromethane (Freon-31), also promote the Type I structurc but are able to occupy only the larger cavities (Barduhn e t al., 1960).

    The unit cell of the Type I1 structure consists of 136 water niolccules arranged to form sixteen m a l l and eight larger cavities (Claussen, 1951a,b). Hydrating agents such as propane, ethyl bromide, ethyl chlo- ride, and several other halogenated hydrocarbons (1 and 2 carbons) which are too large to be accommodated in a Type I structure, promote the Type I1 structure (von Stackelberg and Muller, 1954; Barduhn e t al., 1960). These hydrating agents, by virtue of their relatively large size, are able to occupy only the eight large cavities of the Type I1 structure. Additional structural characteristics of gas hydrates appear in Table 111.

    gas hydrates (water plus one hydrating agent ) .

  • TABLE I11

    CHARACTERISTI(% OF H o w WA'rElt CAGES INVOLVED I N (:Ah HYmA'rEs" F C 4 4

    molecule accommodated hydrating agent) m

    Diameter lengthc volume cavities cavities m

    E Composition (molecules H20/molecule of 5 Approx. max. size of

    * d

    m

    Kumber of If only larger Length of HOH Nuniher of Molecular Molecular If all

    Structural edge of molecules per cavities designation unit cell unit rell per unit (-ell of ravities" (rr/molej orrupied occupied

    k- ~ _ ~ _ _ _ _ _ _ _ _ . ~- ~ _ _ _ _ _ _ ~- __

    A p p r y . Type I 12 A 4 6

    -~ m !2 136 = 5.65 136 = 17

    Appro?. 16 @ 4.8 4 < * 2

    Type I1 17.3 A 136 24 8 @ 6.9 A 5.5-G.3 K3-92 24 8

    n From Claussen (1951a, b), von Stackelberg and Muller (1!)51, 1954), Mriller ant1 von St:tc.kell)erg (1052), :md B:trduhn et al. (1!)60). 1)iameter which is totally unoccupied even by the periphery of other atoms Measured on Fisher-Hirsc,hfelder-Taylor atomic models.

    Y.

  • 232 0. FENNEMA AND W. D. POWRIE

    In the event that hydrating agents of both large and small molecular size are present, crystals will form in a Type IT structure, and the small cavities will be occupied by the small hydrating agents (von Stackelberg and Meinhold, 1954; von Stackelberg and Fruhbuss, 1954). Pressure will have a considerable influence on the composition of such a hydrate (Bar- duhn et al., 1960).

    Gas hydrates are presently being considered for demineralizing sea water (Barduhn e t al., 1960). The principle involves forming a gas hy- drate in sea water (above the normal freezing point of sea water) under temperature-pressure conditions such that a slush is obtained. The crys- talline gas hydrate, being devoid of foreign solutes (as is any carefully formed crystal), can now be separated, leaving behind the concentrated sea water. Exposure of the crystalline gas hydrate to a higher tempera- ture or a reduced pressure causes decomposition, and removal of the gas leaves only pure water. Table IV gives the properties of several gas hydrates considered promising for the demineralizing of sea water.

    The demineralizing of sea water is unimportant to the frozen-food industry, but the principle involved could be of some value. This tech- nique might be suitable as a partial or complete substitutc for some of the conventional freezing processes, particularly freeze concentration. I n other cases i t might be used as a supplement to the freezing process.

    More complete information concerning clathrate compounds and hy- drating agents can be found in articles by Mandelcorn (1959), Bhatnagar (1962), van der Waals and Platteeuw (1959), Swern (1957), Barduhn et al. (1962), Briggs and Barduhn (1963), and Barrer and Stuart (1957).

    E. EFFECT OF WATER OK THE STRUCTURE OF PROTEINS The discussion so far has dealt only with the effect of solutes on the

    structure of water, and not with the effect that water may have on the structure of solutes. Of particular interest in the latter respect arc the proteins. According to Nkmethy and Scheraga (1962~) and Scheraga (1961), the characteristic configuration and reactivity of proteins are gov- erned to a large extent by covalent disulfide bonds and by noncovalcnt interactions between side chains. Included in the latter category are hydrogen bonds, hydrophobic bonds, and ionic interactions.

    Hydrophobic bonds, or the tendency for nonpolar groups to associate in an aqueous environment, are thought to be of considerable importance in stabilizing protein structure. This view can be readily justified by con- sidering the amino acid composition of typical proteins. In general, the amino acids with nonpolar side chains (valine, leucine, phenylalanine, isoleucine, etc.) constitute 35-50% of the total amino acids in proteins (Scheraga e t al., 1962). Since the nonpolar side chains have a low affinity

  • TABLE I V PltoPERTiES OF SOME (+AS HYDRATES A N D HYDI~ATING AGENTS

    Critical Heat of Heat of decomposition conditions vaporization formation

    Hydrating boiling point gas hydrate Temperature Pressure agent in agent (Btu/lb of Normal Structure of Per cent w1w of hydrating of gas hydrate

    agent ( C / O F ) (Type) (C/F) (psig) gas hydrate (Btu/lb) HzO reacted) r 0 P A Methyl Chloride B

    Chlorine - - - (Cl?) - 34/ -29.2 I h 28.7/84 74

    5 Freon-l5Za e

    $ Methyl Bromide m

    8

    - .- - (CHICI) -24.2/ - 11 .(i I b 21/70 (60) est. Freon-31

    (CHzClF) -9.0/+15.8 I b 17.9/64 27 32 150 @ 54F 191 - - - (CHICHF~), -24.7/-12.5 I1 b 15.3/60 51

    2:

    U

    w m m m M a

    - __ - * 0

    (C H3B r ) -3.56/+25.6 I b 14.7/58 8 40 110 @ 48F 170 Freon-142b

    (CH3CClFz) - 9 . G / + 14.i I1 h 13.1 /56 19 Freon-12

    (CC12Fz) Freon-1 2B 1

    (CClBrFz) -4.0/+24.8 I1 b 9.9/50 10 Freon-22B1 <

    2 Freon-21 (CHClzF) +8.!)2/ +48.1 I1 b a.7/4a 0 25 105 (a, 38F I5!) z

    Freon-1 1 (CC1,F) +25/+TS I1 b 8.0/46 -7

    Propane

    - - . __

    - -. - -29.2/-20.6 I1 b 12.1 /54 52 - - -

    (CHBrF2) - 14.5/+5.9 I1 b 9.9/50 24

    - - -

    (C3Hsl -451-49 I1 b 5.7142 65 13 162 @ 32F 150

    a Values for critical decomposition pressures and temperatures are from Briggs and Barduhn, 1963. Adcarms zn Chetnistry &mes 38, 190. Courtesy The American Chemical Society. All other values are from Barduhn rt al , 1060. Research and Dewlopment Proqress Report

    11 See Table 111. Suffix 1) 111 Type designation indicates that the compound fits only in the larger cavities of the struc.ture. N o . 44. Courtesy of the Office of Saline Rater, U. S. Ikpartment of the Interior. t 4

    W W

  • 234 0. FENNE-MA AND W. D. POWRIE

    for water, they tend to approach one another until they touch (within their van der Kaals radii) and thereby minimize their contact with water. Any exposed nonpolar groups will cause the surrounding water molecules to assume a more icelike structure (Nkmethy and Scheraga, 196213). Dispersion of protein molecules in an aqueous environment will therefore encourage configurations with maximum contact among non- polar side chains (hydrophobic bonding) and minimum contact between water and nonpolar side chains. The nonpolar side chains may well asso- ciate in a manner analogous to that of micelles in soap and detergents (Kauzmann, 19591, Regardless of the details, such a configuration will provide maximum protein stability in an aqueous environment.

    Hydrophobic bonding is involved in stabilizing all types of protein structures (alpha-helix, pleated sheets, beta-configuration, and random coil), and, although it is not the only type of bonding involved in protein structure, i t is important and is dependent on the presence of an aqueous environment (Nkmethy and Scheraga, 1 9 6 2 ~ ) .

    During the course of freezing, when water is transferred into purc ice, leaving behind an increasingly concentrated solution of solutes, alteration in hydrophobic, ionic, and hydrogen bonding of proteins would be ex- pected. The extent and mechanisms of such alterations have not, to our knowlcdgc, been investigated.

    Before proceeding into the next section, it would be well to re- emphasize the meaning of icelike as i t is used here. It most assuredly does not mean identical to ice. Rather, i t should be thought of in a comparative sense. A structure is icelike when it has: 1) more water-to- water hydrogen bonds, and 2) a more ordered structure than the remain- ing HOH molecules in the system. This allows one icelike structure to differ widely from ice and from other icelike structures.

    F. BOUND WATER

    Up to this point, emphasis ha5 been given to the ordered, icelike struc- tures that HOH moleculcs assume in pure water and around nonpolar solutes. The water molecules in such cases cooperatively associate to form lattices with a long-range order, i.e., more than several molecules deep. Such water is available as a solvent for any new solutes which may be added to the system.

    In contrast to this arrangement, we have situations where the HOH molecules are strongly attracted to a solute or some part thereof, and are lield in a quite rigid and ordered state. Such an arrangement is especially strong around ions and other charged groups. Water attracted in this manner is known as bound water, and i t is unavailable as a solvent for

  • LOW-TEMPERATURE FOOD PRESERVATIOS 235

    otlicr molecules. Only one or two layers of water iiiolecules are firmly bound, thereby producing an ordered structure over a very limited dib- tance (Szent-Gyorgyi, 1957).

    Use of the term bound water often leads to a great deal of confusion since no one definition has been universally accepted. Definitions for bound water arise from the techniques used for its measurement. The 14 techniques cited by Bull (1943) give rise to several different definitions as well as different results. Bull presents probably the two most common ways of defining bound water:

    1) Bound water can be defined in terms of the water which remains unfrozen a t some prescribed temperature below 0C. The temperature usually chosen is -2OC, although Luyet (1961, page 74) defines it as water which does not crystallize a t any temperature.

    2 ) Bound water can be defined in terms of the amount of water in tlie system which is unavailable as a solvent.

    -4lthough bound water may be difficult to define, i t is nevertheless of great importance. Proteins, by virtue of their many charged groups. are the principal water-binding substance of tissue. Consideration of this fact is essential to understanding protein reactions (Bull, 1943). Borgstrom (1961) pointed out tha t cells containing large amounts of bound water undergo less damage during thawing than cells with smaller amounts. Furthermore, bound water influences reactions during freezing and is an important factor in hindering crystal formation.

    The amount of unfreezable water, based on protein content, appar- ently varies only slightly from one food material to another. Meryman (1960b) estimated that %lo% of the total water in animal tissue is un- available for ice formation. Riedel (1961) found that egg white, egg yolk, meat, and fish all contained approximately 0.4 g of unfreezable water for each gram of dry protein (nitrogen substance). This value corresponds to 11.4% of the total water in lean meat containing 20% protein and 70yG water.

    White bread and other bakery products contain approximately 0.3 g of unfreezable water for each gram of total dry matter (Kuprianoff, 1962).

    Daughters and Glenn (1946) found that most fruits and vegetables contain less than 6% unfreezable water, although they reported a v : d ~ of approximately 34% unfreezable water in whole-grain corn.

    This discussion of the structures of ice and water, along with the I)re*- entation that follows, should help dispel tlie much too common notion that water is simple, uninteresting, and unimportant, and help create a new image in which water is regarded as unique, fascinating, and wortliy of careful considcration when changes in food materials are being eval- uated.

  • 236 0. FENNEMA AND W. D. POWRIE

    Ill. PHASE DIAGRAM OF PURE WATER

    The relationships between the solid, liquid, and vapor states of water are of concern in many food-processing operations, e.g., concentration, dehydration, freezing, freeze-drying, and vacuum cooling. These relations are expressed most easily by a phase diagram.

    A phase can be defined as any homogeneous and physically distinct part of a system which is separated from other parts of the system by definite, usually visible boundaries. The three phases of pure HOH are ice, liquid water, and water vapor.

    A diagram which expresses the conditions for equilibrium between the various phases of a substance is called a phase diagram. Figure 5 is a

    ATM.

    4.579 VAPOR

    I I , 0 00990 1000 3 7 4 -

    TRIPLE POINT CRITICAL TEMP

    TEMPERATURE,C (NOT TO SCALE)

    FIG. 5 . Phase diagram of pure water.

    pressure-temperature phase diagram for water. The various lines repre- sent the temperature-pressure conditions which are necessary for the phases to exist in equilibrium. Equilibrium conditions a t various tem-

  • LOW-TEMPERATCRE FOOD PRESERVATlOK 237

    peratures and pressures are represented for liquid and vapor by curve AO, for vapor and solid by curve BO, and for solid and liquid by curve CO. Curve A 0 is sometimes called a vapor-pressure or boiling-point curve, BO may be thought of as a sublimation curve, and CO as a melting-point curve.

    HOH molecules existing in equilibrium a t some point along curve A 0 can be completely liquefied by raising the pressure or lowering the tem- perature, or completely vaporized by reverse procedures. Point A repre- sents the critical temperature (above which the material cannot be lique- fied, regardless of pressure) of water (374"C), above which the liquid and vapor phases are indistinguishable.

    HOH molecules existing in equilibrium a t some point along curve OB can be completely vaporized by raising the temperature or reducing the pressure, while opposite changes will result in complete solidification. The temperature-pressure conditions needed for sublimation, such as oc- curs in freeze-drying, can be ascertained to a fair degree from this dia- gram. For example, i t is readily apparent that the pressure must be less than 4.579 mm Hg and the temperature less than 0C for rapid sub- limation to occur.

    It is of interest that the melting-point curve, CO, deviates slightly to the left of vertical, indicating that increasing pressure results in a lower melting point. This effect is quite understandable when it is re- called that ice is less dense (more open) than water. Increasing pressure would therefore encourage the open, less dense ice to assume the more coinpact structure of water, this change being permitted a t a progressively lawcr temperature as the pressure is raised.

    Line DO represents the vapor pressure of supercooled water. Of par- ticular significance is the fact that supercooled water has a greater vapor pressure than ice a t the same temperature. Supercooled water, therefore, is thermodynamically unstable and will rapidly transform into ice if a small ice crystal is introduced into the sample.

    All lines intersect a t point 0, a condition occurring a t 0.0099"C and 4.59 mm Hg. This point, known as the triple point, represents the only situation where all three phases can exist in equilibrium. The slightly higher than normal freezing point is the result of the following two effects of low pressure (Dorsey, 1940) :

    1) The open structure of ice is more easily retained a t the low pres- sure (responsible for 0.0075"C elevation).

    2) All dissolved gases are removed (responsible for 0.0024"C eleva- tion).

    Not illustrated in Fig. 5 is the fact that ice can exist in more than one form. Besides ordinary ice (ice I) as shown in Fig. 5, six other distinct

  • 238 0. PENNEMA AND W. 1). POWHlE

    types of icc have bccn reported (Dorsey, 1940). Each has its own definite region of stability. They are not discussed here, since most of them can he produced only under circumstances which are impossible or, at best, iinprobable in foods.

    It is obvious that values obtained for pure water froin Fig. 5 will differ considerably from those observed for aqueous solutions. However, many of the deviations can be easily calculated.

    IV. SOME ADDITIONAL PHYSICAL PROPERTIES OF WATER AND ICE

    A. FREEZING POINT

    The freezing point of pure water a t one atmosphere of pressure is 0C. The addition of nonvolatile solutes lowers the freezing point in accordance with the equation

    1000 AT, = K ~ & = ~ , m

    where A?; = K, =

    G = Y =

    M = m =

    freezing-point depression in "C molal freezing-point constant = 1.86 when water is the sol- vent grams of solvent grams of solute gram molecular weight of the solute molality

    T h e above equation applies to dilute solutions containing solutes which ncitlicr associate nor dissociate. The effect of some selected solutes on the freezing point of water is illustrated in Table V.

    TABLE V IjEPRESSION OF FREEZING P O I N T OF WATEH BY V A R I O U S SOLUTES"

    Substance ~

    sac1 MgC12 . U X 0 3 b

    Acetic. acid Citric acid Dextrose (;lycerol Swrose

    Depression of freezing points ("C) a t molal concentrations of:

    1 .0 2.0 5.0 10.0

    3.37 6.90 - 6.35 17.6

    1.79 - 8.0 11 4 1 .Y4 4.00 1.92 I .w -_ 10.5 2.06 4 (i

    _____---

    ___.__ ________ _ _ _ _ _ ~ - - - -

    - - - 10.6 - -

    - - -

    -

    - -

    Adapted froni "The Handbook of Chemistry and Physics" (Hodgman, 1050). Courtesy of the Chemical Rubber Publishing Co.

    I , Concentration in gram formula weight per liter.

  • LOW-TEMPERATURE FOOD PRESERVATIOS 239

    Particular notice should be given to the greater freezing-point dc- pression achieved by molal quantities of substances which dissociate than by those which do not. This provides proof for the fact that freezing point, like all other colligative properties, is a function of the total number of particles in solution and tha t this number can easily differ, by dissocia- tion or association, from the number of molecules added.

    The fact that solutions prepared in the food industry are generally calculated on the basis of "per cent by weight" also deserves some com- ment. Consider, for example, two 5% aqueous solutions, one prepared froim sodium chloride and the other from sucrose. Addition of 53 g of solute to 1000 ml of water would provide the desired composition. This consti- tutes approximately a 1 inolal solution in the case of sodium chloride (gram inolecular weight 58.45), but only a 0.15 molal solution in the case of sucrose (gram molecular weight 342.3). A 1 molal solution of so- dium chloride would depress the freezing point approximately 3.4"C, whereas the 0.15 inolal solution of sucrose would depress the freezing point approximately 0.28"C. (The molal freezing-point constant for su- crose a t the concentration being considered is approximately 1.86. Since 0.15 mole is involved, the depression would be 1.86 x 0.15, or approxi- mately 0.28OC.) The net result is that a given weight of sodium chloride is twelve times as effective in depressing the freezing point of water as an equal weight of sucrose. The explanation for this difference is quite simple. On an equal-weight basis, six inolecules of sodium chloride are added for every one molecule of sucrose (342/58 = 6 ) , and all of the sodium chloride molecules dissociate. Thus, a given weight of sodium chloride will yield twelve times as many particles as an equal weight of sucrose.

    Thc freezing points of typical food materials will be discussed in S C C - tion VI, dealing with freezing diagrams.

    B. DENSITY Water assumes a maximum density of 1.000 g per ml a t 4C. Raising

    or lowering the temperature from this point decreases density. lTTatcr supercooled to -13C has a density of 0.9969 g per ml, whereas water a t 100C has a density of 0.95838 (Chemical Engineers' Handbook-Perry, 19501. The change in density through the range -13 to +100"C is shown graphically in Fig. 6.

    The density of ice a t 0C is 0.9168 g per ml, compared to 0.9999 g per ml for water a t the same temperature (Chemical Engineers' Handbook- Perry, 1950; International Critical Tables-Stott and Bigg, 1928; Clark, 1928). The expansion resulting when water a t 0C changes into ice a t the same temperature is approximately 9% (the only pure substances

  • 240

    I 00-

    .98--

    2 .96- -

    0. FENNEMA AND W. D. POWRIE

    CHEM ENG. HANDBOOK(HM)GMAN,IOBO) + DORSEY(1940)

    88 -40 -20 0 +20 + 4 0 +60 +80 +I00

    TEMPERATURE OC

    FIG. 6. Density of water and ice at various temperatures.

    known to expand upon freezing are water, gallium, and bismuth; Shortley and Williams, 1961, page 386'1. As the temperature is decreased below O"C, the density of ice increases quite rapidly until, a t -2O"C, it assumes a value of 0.9481 g per ml. Passage through this range revokes approxi- mately three-fifths of the volume increase which occurs during the forma- tion of ice a t 0C. Density changes in ice over the temperature range of 0 to -22C are shown graphically in Fig. 6.

    C. SPECIFIC HEAT The specific heat of water is quite constant throughout the tempera-

    ture range of 0 to +lOO"C, deviating only slightly from a value of 1.00. Upon freezing, the specific heat decreases to a value of 0.492, approxi- mately one-half the value of water a t the same temperature. As the temperature is decreased below O"C, the specific heat of ice steadily de- creases until, a t -250"C, i t assumes a value of approximately 0.1. The change in the specific heat of water and ice in the temperature range of 100" to -250C is shown graphically in Fig. 7.

    D. LATEKT HEAT OF FUSION Changing water a t 0C into ice at the same temperature requires the

    rcmoval of 79.71 gram-calorieslj" per gram of water, or an equivalent

  • LOW-TEMPERATURE FOOD PRESERVATIOS 241

    1 1 4 -250 -200 -150 -100 -50 0 +50 +I00

    TEMPERATURE *C

    FIG. 7 . Specific heat of water and ice a t various temperatures. (Data from "Hand- book of Chemistry and Physics"; Hodgman, 1950.)

    143.5 Btu per pound (Handbook of Cliemistry and Physics-Hodginan, 1950; Dorsey, 1940).

    E. THERMAL CONDUCTIVITY Thermal conductivity, or the coefficient of thermal conductivity, is

    the quantity of heat that is transferred by conduction in unit time, through unit thickness, across unit area, for unit difference in tempera- ture. Two systems of units are in common use:

    thermal conductivity, k'= (Btu)(in.)

    (hr)(sq ft)("P) or

    (cal) (cm) A' = (set> (sq cm) ("C)

    Note that the substance is expressed in terms of volume, not weight. Coefficients of thermal conductivity are determined while the suh-

    stance is in a steady, or stationary, state. A steady state can be achieved by exposing one face of a slab of material to a constant temperature and the other face to a higher constant temperature for a time sufficient to establish a constant temperature gradient within the slab. Under these

  • 242 0. PENNEMA A S D IT. D. PO\VRIE

    p .004-- z 0 0 ,003- -I

    .002-- a w I I- Ool--

    0 .

    conditions the temperature will decrease with dibtance from the hot face to the comparatively cold face. The important feature is that the tein- perature a t any given point within the slab remains constant, giving rise to the name steady state. Coefficients of thermal conductivity indicate the rate a t which heat flows by conduction.

    The thermal conductivity of water is quite constant, increasing only slightly as the temperature is raised from 0 to 100C. Thermal conduc- tivity values for watcr in the range 0 to 80C are shown graphically in Fig. 8.

    *-JACOB a ERK(1929) as ahown in POWELL(IS~I

    POWELL(1958) +- +_+-+-+-+-+-+-+ WTER - VAN DUSENll929) or shown in

    + +POWELL(I956)

    t

    *\ -. \

    %\ cc .\

    Thcrnial conductivity values for ice must be accepted wit11 mine caution since agreement between reported valucs is rather poor. Two sets of values are presented in Fig. 8 (Van Dusen, 1929; Jakob and Erk, 1929). Powell (1958), after carefully evaluating thc experimental techniques, regarded Jakob and Erks data as the more acceptable.

    One of the most important points to gain from Fig. 8 is that the thcr- ma1 conductivity of ice a t 0C is approximately four times that of watcr a t the same temperature, indicating that ice will conduct heat energy much faster than will water. The significance of this fact is deiiionstratcd in Section IX,C, dealing with rate of freezing and thawing.

  • LOW-TEMPERATURE FOOD PRESERVATIOS 243

    F. THERMAL DIFFURIVITY

    During commercial freezing and thawing operations, the temperature and temperature gradient of the product are continually changing. The product is therefore in an unsteady, or variable, state, and the coefficient of thermal conductivity, by itself, is no longer adequate for describing rates of thermal changes. Under these circumstances, a new term: ther- mal diffusivity, becomes appropriate. This term takes into account the heat capacity of the substance.

    The thermal diffusivity of a substance is indicative of the rate a t which i t will undergo a temperature change. Under like conditions, sub- stances with large values of thermal diffusivity will undergo changes in tcmperature more rapidly than substances with small values of thermal diffusivity. Thermal diffusivity, (Y, can he calculated from the following cquation :

    K Sd

    a = -

    where K = the coefficient of thermal conductivity s = specific heat of unit mass d = density (mass per unit volume)

    sd = specific heat of unit volume.

    Thc values of thermal diffusivity are large when a substance conducts heat a t a rapid rate (large coefficient of thermal conductivity) and has a low heat capacity (small values of specific heat and density). Thermal tliffusivity values for water and ice are listed in Table TI.

    TABLE \I TIIEKMAL DIFFUSIVITY VALTES OF ICE AND WYVER

    Calculated

    Sperific heat Density conduct ivty diff usivity S d 01 = K/sd

    Thermal thermal

    Substance ( d / g C) (g/1111)

    Water, 0C 1 .0 1 .0 0.0012 0.0012 Ice, 0C 0.5 0.9 0.0050 0.01 1 I

    Thc most important point t o gain from Table V I is that the tlierinal diffusiyity of ice is iiiore than nine times as great as that of water at the sanie temperature, indicating that ice will undergo a change in tein- pcrature a t a much greater rate than water. The significance of this fact

  • 244 0. FENNEMA AND W. D. POWRIE

    is demonstrated in Section IX,C, dealing with rates of freezing and thawing.

    The present section attempts only a partial coverage of the physical properties of ice and water. For additional information, the reader is referred to Dorsey (1940).

    V. CRYSTALLIZATION

    Knowledge of the principles of crystallization is necessary if food sub- stances are to be frozen, stored, and thawed under optimum circumstances. The crystallization process can be conveniently divided into two parts, nucleation and crystal growth. It may be argued that crystal growth is nothing more than an extension of nucleation. While this is basically true, there are ways of controlling the relative predominance of the two steps and thereby governing the ultimate crystal size. This being the case, a two-step approach seems justified.

    A. SUCLEATION

    Crystallization begins when conditions are appropriate to bring about the aggregation of a group of molecules into a tiny ordered particle known as a crystal nucleus. The establishment of a crystal nucleus, a process known as nucleation, will not occur without some supercooling.

    Two types of nucleation are possible : homogeneous and heterogeneous (or catalytic). m'ater, if exceedingly pure, is limited to nucleation of the homogeneous type. A homogeneous nucleus forms by chance orientation of a suitable number of molecules into an icelike niass. Homogeneous nu- cleation of water is highly improbable a t 0"C, but approaches a proba- bility of 1.0 as thc temperature nears -41C. ,4 temperature of -41C is generally regarded as the limit to which water can be supercoolcd (Mason, 195813).

    The temperature to which ultrapure water can be supercooled is strongly dependent on sample volume. Temperatures of -41C have been obtained only in extremely small droplets measuring a few microns in diameter. Supercooling to about -35C is apparently the limit for water droplets of 1-nim diameter (Langham and Mason, 1958).

    Heterogeneous, or catalytic, nucleation occurs when water molecules aggregate in a crystalline arrangement on a tiny, nonaqueous, solid par- ticle. Mason (1958b) studied the nature of various artificial and natu- rally occurring materials possessing ice-nucleating properties. I n general, he found them to be almost insoluble in water and to have crystalline structures similar to that of ice. Some particularly effective substances arc silver iodide, lead iodide, cupric sulfide, vaterite, P-tridymite, kaolin- ite, and specular hematite.

  • LOW-TE M PERATURE FOOD PRESERVATION 245

    -30

    -25-

    0

    w K 3

    0 -20-

    -15-- a W LL

    I- 3 -lo--

    -5--

    Heterogeneous nucleation is quite unlikely near O"C, but will gen- erally occur a t much higher temperatures than will homogeneous nuclea- tion. Since ice-nucleating particles are present in all aqueous materials except highly purified water, heterogeneous nucleation is the type which occurs in food materials during freezing (Lusena, 1955).

    The addition of solutes will lower the temperature of heterogeneous nucleation. Lusena found that solutes depressed freezing point and nu- cleation temperature to the same extent.

    As was true of homogeneous nucleation, the temperature a t which heterogeneous nucleation occurs is a function of sample volume (Langham and Mason, 1958). Small droplets of water 1 mm in diameter have been supercooled to approximately -25C before the onset of heterogeneous nucleation. On the other hand, large bodies of water such as lakes and ponds rarely supercool more than a few hundredths of a degree. This point may be of considerable importance with regard to the supercooling capacity of certain cellular materials. For example, intracellular freezing has never been observed in bacteria. Considering their extremely small dimensions, supercooling could be a contributing factor.

    The mechanism of nucleation is not well understood, but i t is thought that ordered aggregates of water molecules continually form and disap- pear, and, if the temperature is suitable, some grow large enough to serve as crystallization nuclei. The size a t which a nucleus has an equal oppor-

    -r

    04 I I I I I 1 1 t 0 20 40 60 80 100 120

    CRITICAL RADIUS ( ANGSTROMS 1 Size of critical nuclei in water at various temperaturcs. (From Clialiners,

    1959, Sci. American 200 (2) , 114. Courtesy of Scientific American.) FIG. 9.

  • 246 0. FENNEMA A S D W. D. POWRIE

    tunity of growing or diniinishing is known a5 the critical size. The critical size is strongly temperature-dependent, being extremely large at the melting point and becoming smaller as the temperature is reduced. The effect of temperature on the critical size is shown graphically in Fig. 9.

    The dependence of the size of the critical nuclei on temperature can apparently be explained on the basis of the curvature of the niolecular aggregates. As the size of the aggregate decreases, the radius of curvature decreases, thereby decreasing the area of contact and attraction between the surface molecules and the interior. As the temperature is raised to- ward the melting point, progressively larger aggregates with larger radii of curvature (less curvature) are needed to endure the increased thermal forces.

    Judging from the relative ease with which heterogeneous nucleation occurs, i t would appear that fewer water molecules are required to form a heterogeneous nucleus of critical size than a homogeneous nucleus of critical size. Masons statements (195813) concerning the structural sta- bility of water molecules oriented around ice-nucleating solid particles seem to support this contention.

    When all factors influencing the rate of formation of critical nuclei are considered, i t is found that an almost explosive increase in the rate of nucleation occurs as a certain degree of supercooling (or supersaturation, in the case of solutes) is exceeded. This effect is illustrated graphically in Fig. 10. (The equation for the degree of supercooling is AT = T:, - TI,

    0C - Supercooling (or supersaturation)

    FIG. 10. Hypothetiral nucleation rate of water at. various degrees of supercooling. supercooling increases and tcmperature decreases wi th movrmcnt to the riglit :~lonp the abscissa. (8dapted from van Hook, 1961. Crystallization-Tlleory and Irncl ice. (ourtesy of Reinliold Publishing Corporation.)

  • LOW-TEMPERATURE FOOD PRESERVATIOS 247

    where T P = the actual freezing point of the substance and T I = the tem- perature of the supercooled substance.)

    Information is decidedly lacking on the actual rates of nucleation over broad ranges of supercooling. This is understandable, considering the difficulties of counting minute nuclei accurately and of working with highly supercooled water.

    B. CRYSTAL GROWTH The growth of crystal nuclei is the second step in the crystallization

    process. Crystal growth, in contrast to nucleation, will occur readily a t temperatures very close to the freezing point. This indicates that it is much more difficult to initiate crystallization than it is to continue it. The net crystal growth rate of pure ice is governed by two factors: teni- pcrature and rate of heat removal.

    1. Temperature

    The rate of ice crystal growth decreases with decreasing temperature provided all other factors are held constant, including the degree of super- cooling. This effect is due largely to viscosity, which increases exponen- tially with decreasing temperature (Tipson, 1956). The influence of tem- perature is easily demonstrated when dealing with supersaturated rather than supercooled systems. For example, if crystal growth rates are studied in a series of aqueous sucrose solutions, each held a t a different tempera- ture but supersaturated to the same degree (by adjusting the solute con- centration), it will be found tha t the coldest solution will exhibit the slowest crystal growth (van Hook, 1961, page 183). I n such a case, super- saturation is held constant while crystal growth rate is studied as a function of temperature. An analogous experiment cannot be conducted with watcr, since supercooling and temperature are dependent variables.

    9. Rate of Heat Removal The rate of ice crystal growth increases with an increasing tcmpera-

    ture differential between the surface of the ice crystal and the tempera- ture of the unfrozen medium. Increased supercooling or extremely rapid renioyal of heat energy will tend to increase the rate of ice crystal growth in accordance with this principle.

    The relative importance of temperature and rate of heat removal can he determined by examining actual rates of ice crystal growth in speci- mens supercooled to various temperatures. If the temperature effect is of primary importance, rates of ice crystal growth will decline as crystalliza- tion is initiated in specimens supercooled to progressively lower tem-

  • 248 0. FENNEMA AND W. I). POWRIE

    peratures. However, if the rate of heat removal is of primary importance, rates of ice crystal growth will increase as crystallization is initiated in specimens supercooled to progressively lower temperatures (rates of heat removal increase as crystallization is initiated a t progressively lower tem- peratures). The information presented in the next few paragraphs appears to show that rate of heat removal has a greater effect than does tem- perature within the temperature range normally encountered during the freezing of food (crystal growth rates increase with increased super- cooling), whereas a t much lower temperatures the temperature effect can predominate (crystal growth actually stops a t very low temperatures).

    As crystallization is initiated in water supercooled to progressively lower temperatures within the range of 0 to -9C, the rate of crystal growth* apart from nucleation is found t o increase rapidly (Hartman, 1914; Walton and Judd, 1914; Lindenmeyer et al., 1957; Hillig and Turn- bull, 1956; Mason, 1961; Lusena and Cook, 1954). Hartmans data are presented in Table VII.

    TABLE VII EFFECT OF TEMPERATURE ON THE LINEAR CRYSTALLIZATION

    VELOCITY OF WATER

    Temperature a t onset of Linear crystallization velocity crystallization (C) (mm/min)

    -0.9 230 -1.9 520 -2.0 580 -2.2 680 -3.5 1220 - 5.0 1750 -7.0 2800

    a From Hartman (1914). 2. Anorg. Chem. 88, 128. Courtesy of Johann Ambrosius Barth, Leipzig.

    Ice crystal growth rates in water supercooled to lower temperatures than shown in Table VII are unavailable because of the difficulty of preventing spontaneous nucleation. It has been established, however, that if the temperature is reduced very rapidly to -160C or lower (by con- densation of moisture vapor on a cold surface), crystallization of watcr will not occur. Water so treated is said to solidify in the glass, or vitreous, state (Pryde and Jones, 1952; Dowel1 and Rinfret, 1960).

    * Refers to the linear velocity of crystallization, in which freezing is initiated at one end of a glass tube filled with supercooled water, while the tube is immersed in n bath maintained at the same temperature as the supercooled water at the mo- ment of freezing.

  • LOW-TEMPERATURE FOOD PRESERVATIOS 249

    The temperature below which the rate of ice crystal growth declines is yet to be determined. However, i t likely lies below -80C (Buckley, 1951, page 265; Luyet, 1960a, page 18).

    A graphical representation of nucleation and crystal growth rates is presented in Fig. 11. The shapes and positions of the curves are somewhat

    Supercooling

    FIG. 11. Hypothctical rates of nucleation and rates of crystal growth of water at various degrees of supercooling. Supercooling increases and temperature decreases with movement to the right along the abscissa. (Adapted from McCabe, 1950. "Chem- icnl Engineers Handbook"; reprinted by permission of McGraw-Hill Book Co.)

    speculative, but nevertheless are of value for explaining the size of crys- tals arising under various circumstances. These curves are similar to those of 1YlcCabe (1950) and agree with principles stated by van Hook (1961, pages 15, 97, 115, 161) and Buckley (1951). Figure 11 is dis- cussed more fully in the next section.

    TABLE T'III

    TJSE kR CRY6TALLIZ.4'I'ION VELOCITY OF iTA'I'EK I N AQUEOUS SOLUTIONS"

    Solution Linear crystallization velocity

    Temperature ("C) (mm/sec)

    Water 0.134 SaCl

    0.l.M Glyrerol 0.1M Sucrose 0.1.V Itaffinose 0.1 .\!I Lactose 0.2% Lactose GelatinP3.i5 g/liter Gelatin-15.0 g/liter Oxygen (saturated)

    0.1M CrH,OH

    -9.1 -9.1 -9.1 -9.1 - 9 . 1 - 9 . 1 -9.1 -9.1 -i.l - i . l -9.1

    61 41 29 29

    6.6 4.6 5.6

    3 8 23 10.6 54

    (1 From Mialton and Urann (1916). J . A m . Cheru. Sor. 38, 31i. Reprinted by per- mission of the American Chemical Society.

  • 230 0. FENNEMA A S D W. D. POWRIE

    Solutes of many types and in quite small amounts will greatly slow ice crystal growth. Table VIII illustrates this effect. More recently, Lu- sena (1955) studied the effect of various inorganic and organic solutes on the rate of ice crystal growth. All samples were supercooled 10C below their respective freezing points before crystallization was initiated. Plots were made of the logarithms of the rate of crystal growth in centi- meters per second versus concentration in per cent (w/w) . I n general, the plots were curvilinear a t low concentrations and linear a t higher concen- trations, indicating that a given increment of solute retards crystal growth inore effectively in a dilute solution than in a concentrated one. A line of best fit was determined for each solute and extrapolated to infinite dilution (zero concentration or y-intercept) . The intercept values and slopes are recorded in Table 1X. The antilogs of the intercept values are also presented in order to provide a convenient means of comparing the mrious crystal growth rates with the value for water.

    It is readily apparent from Table IX that the rate of ice crystal growth was greatly retarded by all solutes tested. The least effective ma- terial a t infinite dilution (sodium chloride) caused a 57% retardation, and the most effective material (ethanol) caused a 94% retardation. Organic substances generally exerted a greater retarding effect on crystal growth rates than did inorganic. The most effective organic substances a t infinite dilution were ethanol, propanol, sucrose, and proteins.

    The effect of increasing solute Concentration can be ascertained from the slope values. All substances caused a reduction in crystal grovth rate with an increase in concentration (negative slopes). The larger the ab- solute slope value, the greater the retardation per unit increase in con- centration. Proteins exerted little additional retarding effect when used in concentrations greater than 10%. This could be anticipated from their molecular weights.

    The mechanisms by which solutes retard ice crystal growth are not known. However, Lusena (1955) postulated that solutes in high concen- trations exert their effect by physically limiting the rate a t which water molecules diffuse to the freezing boundary. He suggested that a different mechanism functions a t low solute concentration, but did not speculate on its nature. Meryman (1956) expressed the belief that the retarding influence of solutes on the rate of crystallization involves more than a simple viscosity effect. He suggested that the solutes may in some way become associated with the growing crystal and thereby create an ob- stacle to further growth.

    The above information appears particularly pertinent considering that solutes become highly concentrated during the latter stages of the freez- ing process.

  • LOLV-TEMPERATURE FOOD PRESERVATIOS 251

    TABLE LX EFFECT OF TYPE AND CONCENTRATION OF SOLUTES ON THE

    LINEAR CRYSTALLIZATION VELOCITY OF WATER"

    Intercept Range of (infinite di1ution)d linearity" Slow

    Solute log (cm/sec) cm/sec log (cm/sec)

    Pure u ater Inorganic salts

    Lithium acetate Lithium chlorideb Sodium acetate Sodium chlorideb Sodium thiocyanate Sodium dihydrogen

    Potassium acetate Potassium chloride Potassium thiocyanate Ammonium acetate Ammonium chloride Ammonium

    phosphate

    thiocyanate

    Organic compound8 Methanolb Ethanolb Propanol Glycerolb Ikxtrose Fructose Sucrose" Acetic acid Glycolic acid Glycine Malic acid Citric acidb Lysosynie Albumin

    -

    5 -15 5 -15 7.5-25 5 -17.5

    10 -25

    10 -25

    10 -15 15 -25

    7.5-17.5

    7.5-25 7.5-12.5

    10 -30

    10 -20 12.5-20 10 -17.5 10 -35 10 -30 10 -25 10 -30 10 3 0 10 -22.5 8 -16

    10 -25 15 -30 11 -16 9 -14

    0.85

    0.15 0.12 0.44 0.49 0.48

    0.24 0.43 0.45 0.45 0.24 0.32

    0.32

    0.08 -0.36 -0.27

    0.14 0.01 0.08

    -0.07 0.33 0.33 0.34 0.22 0.11

    -0.09 -0.12

    7.0

    1.41 1.32 2.75 3.09 3.02

    1.74 2.70 2.82 2.8 1.7 2.1

    2.1

    1.2 0.45 0.54 1.4 1 .0 1.2 0.85 2.1 2.1 2.2 1.7 1.3 0.81 0.76

    -

    - 0.103 -0.114 -0.092 -0.081 -0.055

    -0.050 -0.064 -0.042 - 0.03 1 -0.070 -0.059

    -0.034

    -0.068 -0.070 -0.066 -0.053 -0.013 -0.042 -0.038 -0.045 -0.041 -0.044 -0.033 -0.033 -0.016 -0.016

    a Adapted from Lusena (1955). Arch. Biochem. Biophys. 57, 277. Courtesy of Aca-

    *Values obtained by method of least squares; other values obtained by visual demic Press.

    fit.ting of lines. Concentration of substance (w/w). Obtained by extrapolating linear portion of line to zero concentration.

  • 252 0. F E N N E M A AA-D 11'. D. POWRIE

    The growth of ice crystals is also known to be affected by nienihranes. Studies by Chambers and Hale (1932) and Luyet and Gibbs (1937) in- dicate that cell membranes are quite impermeable to the growth of ice crystals. Lusena and Cook (1953) studied this effect with model iiicin- branes of regenerated cellulose, goldbeater skin, gelatin, and other ma- terials. The permeability of any given membrane to ice crystal growth wab found to increase with porosity and decrease as solutes were added to the medium. The type of membrane also had a great influence on east of permeability.

    l l a z u r (1960b) suggested that tlie permeability of cell menibranes to ice crystal growth may well be governed by the size of the water-filled pores in relation to the radius of curvature of ice crystals a t the tem- perature under consideration. For example, if the pore diameter had a radius of 25 A, a temperature of -15C or lower would be required for an ice crystal to grow into the pore (see Fig. 9) . This may have somc relevance in explaining the preference for extracellular ice formation in ccllular materials, although evidence concerning this possibility is con- tradictory (Salt, 1962; Asahina, 1961).

    C. ICE CRYSTAL SIZE Ice crystal size a t the completion of freezing is related directly to tlie

    nuinher of nuclei. A few nuclei will cause the formation of a few large crystals, whereas numerous nuclei will result in numerous crystals of :t riiialler size (Meryman, 1956). Control of crystal size is therefore tie- pendent on nucleation. The interrelationship between the curves for rate of crystal growth and rate of nucleation (Fig. 11) provides a logical approach to controlling nucleation. Nucleation is not extensive until it moderate degree of supercooling is obtained (Point A, Fig. 111. ( In liquid systems such as sugar solutions and melted chocolate, nuclei in thc form of tiny crystals can be added to rather than developed from the solution. This process is known as seeding.) If the system is main- tained a t a temperature somewhere between the melting point and Point A , only a fcw nuclei will form, and each will grow extensively. The slow removal of heat energy will produce an analogous situation since the heat of crystallization released by the few growing crystals will cause the tcni- peraturc to remain a t or near the melting point, where nucleation is un- likely. I n tissue or unagitated fluid systems, such as water, fruit juices, and niilk, the slow removal of heat energy will result in a continuous icc phase moving slowly inward, with little if any nucleation occurring in advance of the freezing boundary (Lusena and Cook, 1954). Xlildlp agi- tated fluids exposed to a comparable thermal environment will exhibit ice crystals of :in interniediate size.

  • LOW-TEMPERATURE FOOD PRBSERVATIOl- 253

    Conversely, if heat is rapidly removed from the system so that the temperature drops below Point A , then many nuclei will form and each will grow to only a limited extent. In tissue or fluid food materials, the result will be a discontinuous ice phase composed of many tiny crystals arising from numerous nuclei. Any tendency for the growth rate to dimin- ish a t extremely low temperatures will result in ice crystals of an even smaller ultimate size.

    The above principles are in accord with observed facts. It is well established tha t ice crystals are small and numerous in rapidly frozen foods and other biological materials, and large and few in slowly frozen samples (Hanson, 1961; Lusena and Cook, 1954; Woodroof, 1938; Frand- sen and Arbuckle, 1961 ; Tressler and Evers, 1957a).

    The impression should not be left that all tissue samples frozen a t the same rate will contain ice crystals of the same size. Variation is great between different materials, samples of the same material, and even within the same sample (Stephenson, 1960). An illustration of this point is provided by fish frozen in a prcrigor state as compared to a postrigor state. When frozen a t the same rate, the prerigor sample contained smaller and more numerous ice crystals than the postrigor sample (Love and Haraldsson, 1961). The researchers attributed the difference to a greater amount of bound water in the prerigor sample, which could con- ceivably retard the migration of free water and encourage a discontinuous ice phase composed of numerous small crystals.

    Control of crystal size is inherently less difficult in agitated fluid sain- plcs than in unagitated fluid samples and tissue samples. Better control arises mainly from the greater uniformity of temperature during freezing.

    hgitation of the system during freezing is known to encourage the formation of small ice crystals, probably through the creation of more crystal nuclei. Agitation will increase the rate of heat removal, thereby lowering the temperature (if the cooling source is adequate) to the point where nucleation is more likely. I n addition, agitation might conceivably facilitate orientation and aggregation of the molecules, a necessary step in the formation of a nucleus of critical sizc.

    It should not be assumed tha t progressively smaller crystals will be produced as the intensity of the agitation is increased. X study of crystal- lization of inorganic salts from agitated aqueous solutions indicated that ccrtairi intensities of agitation actually slow crystallization, Ixobably by disrupting or impeding the growth of subnuclei (Mullin and Raven, 1961).

    D. RECRYSTALLIZATION Control of crystal size would be quite simple if all crystals, once

    formed, remained unchanged in size during subsequent frozen storage

  • 254 0. FENNEMA A S D W. D. POWRIE

    and thawing. Sucli a static situation is rare. Ice crystals generally have a tendency to enlarge during frozen storage and the early stages of thaw- ing. This phenomenon has been ternied recrystallization.

    Luyet and Gibbs (1937) studied intracellular changes in frozen epi- dermal cells of a yellow onion maintained at -8 to -4C. During a period of 2 hours, they observed an increase in ice crystal transparency which they suggested was due to the property of the crystals to aggrc- gate into larger, more homogeneous masses. Recrystallization was pre- sumably involved in this change. Rapatz and Luyet (1961) observed re- crystallization when frozen red blood cells of the frog were warmed to -10C.

    Lee e t al. (1949) prepared samples of strawberries, raspberries, and sliced peaches; packed them in 50% sucrose syrup; and froze them a t three different rates. One series of samples was slowly frozen in still air at OF, another series was frozen a t an intermediate rate by exposure to a -40F air blast, and a third series was quickly frozen by immersing the open packages in liquid air. The differences in freezing rates were obviously sufficient to produce ice crystals of distinctly different sizes in the three series of samples. Yet, after storage of the samples for six months a t OF, recrystallization had occurred to an extent that all sam- ples contained crystals of essentially the same size.

    Love (1962a) failed to find any evidence of recrystallization in frozen codfish stored for 180 weeks a t a constant -29 or -14C. Recrystalli- zation was observed, however, when the samples were exposed to tempera- tures fluctuating between -14 and -7C. According to Rogers (1958) recrystallization is comnion in stored frozen foods unless precautions are taken to avoid it.

    Examination of the many conditions under which recrystallization has been observed leads to the conclusion that more than one type of recrystallization is possible. Many authors have recognized this fact, but accurate definitions are few, and usage of terms is inconsistent. Thrce distinct types of recrystallization are apparently possible : irruptive, mi- gratory, and a typc that we shall call accretive.

    The term irruptive recrystallization, introduced by Luyet (1960b, page 562), applies to small specimens which have been cooled a t ex- trcmely rapid rates to very low temperatures, so that nucleation occurs but crystal growth is severely retarded. Samples prepared in this fashion are transparent when viewed through a microscope under ordinary light. L-nder polarized light, however, the specimen appears to contain tiny ice crystals known as spherulites. Such a sample, when rewarmed to somc characteristic subfreezing temperature, suddenly becomes opaque when viewctl through a microscope (ordinary light). The opacity arises when

  • LOW-TEMPERATURE FOOD PHEKERVATIOS 255

    the submicroscopic crybtals suddenly grow to a size which interferes with the beam of light. This phenomenon constitutes irruptive recrystal- lization-the resumption of crystallization during the warming of a frozen material previously frozen very rapidly to such a low temperature that crystallization could not be completed.

    Luyet found the temperature of irruptive recrystallization to depend greatly on the molecular weight of the solutes present. Large molecules, as in starch and protein, will delay irruptive recrystallization until the specimen has been warmed to about -5" to -15C. In the presence of smaller molecules, as in sugar and glycerol, however, irruptive recrystal- lization will occur when the specimen is warmed to somewhere in the range -f5" to -25C. Considering the low temperatures and rapid rates of freezing required to produce specimens which are subject to irruptive recrystallization, this type of recrystallization would appear to be of little importance in commercially frozen foods.

    Migratory recrystallization is defined most often as "the growth of large crystals a t the expense of small" (Dorsey, 1940, page 412; Luyet, 196010; Mazur, 1960b; Meryman, 1956). At constant temperature, this occurrence is the result of surface and free-energy differences between large and small crystals (Meryman, 1956; van Hook, 1961). Or, inore simply, the smaller crystals, with their very small radii of curvature, cannot bind their exterior molecules as effectively as larger crystals. Small crystals consequently exhibit greater solubilities and lower melting points than large ones.

    Solubility differences between flat surfaces (infinitely large particles) and extremely small particles can be determined with a fair degree of accuracy from the Ostwald-Freundlich equation,

    L 2 y v RT In - = ~ L, r

    where L = the solubility of a particle of radius r L , = the sohbility of' a plane surface

    y = solid-liquid interfacial tension V = molar volume T = absolute temperature of the system R = gas constant.

    The above equation can be modified to express the lowering of nicking point as a function of particle size (van Hook, 1961, page 79) :

    where X = latent heat of fusion AT = the melting point lowering for a particle of radius T .

  • 2.56 0. FENNEMA A S D W. D. POURIE

    Judging froin studies reported by Buckley (1951), van Hook (1961), and Moran and Hale (1932), the tendency for large crystals to grow a t the expense of sniall ones occurs readily only when small crystals measur- ing less than about 2 p in length are plentiful. Moran and Hale have suggested that many ice crystals of this size would be present in rapidly frozen meat, but later work by Woodroof (1938), with frozen fruits and vegetables, and by Arbuckle (1940), with ice cream, indicates that the average ice crystal size in these products is many times greater than 2 p, even when freezing is accomplished by direct contact with solidified CO,. This of course docs not preclude tlie presence of some ice crystals of a sizc less than 2 p, but i t certainly seems to minimize the possibility that migratory recrystallization occurs in food materials stored a t a constant temperature.

    The possibility of niigratory recrystallization appears much inore likely under fluctuating temperature conditions (Love, 1962a). Any tem- perature gradient would result in vapor-pressure differences and a growth of crystals in the area of low temperature (low vapor pressure) a t tlic expense of crystals in an area of higher temperature (higher vapor pres- sure). Not only would this accelerate the occurrence of migratory re- crystallization of the Ostwald-Freundlich type, it would also cause crys- tals of all sizes to either grow or diminish, depending on their relative temperatures and vapor pressures. Since any temperature gradient would he temporary under a fluctuating temperature environment, the vapor pressure a t various locations would change continually. This being the case, it is logical that sniall crystals, because of their sinall mass, would disappear in areas of high vapor pressure sooner than would crystals of a larger inass. Upon reversal of the vapor-pressure differences, the slightly tliminishcd large crystals would again grow, hut the small crystals which disappeared would not likely reappear, because of the difficulty of nuclea- tion. The result would he a growth of large crystals a t the expense of sniall ones. Regardless of the exact mechanisms, fluctuating teniperaturcs are known to increase the size of ice crystals in frozen food materials, and migratory recrystallization is likely involved.

    Another mechanism leading to an increase in crystal sizc is al>o con- ceivable, i.c., a joining together of adjacent ice crystals. Accretive rc- crystallization appears to be an appropriate designation for this niecha- nisni. Its occurrence has been alluded to by Tressler and Evers (1957a, page 679) and Rogers (1958, page 42) , but its importance remains to be deterniined.

    Rccrystallization, rcgardlcss of the type, is known to be highly tmi- perature-dependent. It occurs a t a decreasing rate as the temperature is lowered below the freezing point (Meryman, 1956; Luyet, 1957a). Low

  • LOW-TEMPERATURE FOOD PRESERVATIOS 257

    and uniform temperatures are the obvious way of niinimizing recrystal- lization during storage of frozen foods. Recrystallization will also occur during thawing, but i t can be minimized by rapid thawing.

    E. LOCATION OF ICE CRYSTALS IN CELLULAR SUSPESSIONS AND TISSCE

    Another interesting and important aspect of crystallization is the dis- tribution of ice crystals in frozen tissue and cellular suspensions. In general, slow freezing will produce large crystals located exclusively in extracellular areas. This statement applies to both plant and animal tis- sue and to suspensions of microorganisms, spermatozoa, and red blood cells (Smith, 1961; Rapatz and Luyet, 1961; Nei, 1954, 1960; Love and Haraldsson, 1961; Hanson, 1961; Clianibers and Hale, 1932; Salt, 1962; Ganc, 1955; Luyet, 1961 ; Meryman, 1960a,b ; Levitt, 1960; Woodroof, 1938; Sherman, 1957; Joslyn and Hohl, 1948; Mazur, 1960a). On the other hand, rapid freezing to a low temperature will generally result in tiny ice crystals, located both extra- and intracellularly.

    The above rules are not free from exceptions. Treatments which severely alter the cells or tissue will apparently overcome the preference for extracellular ice formation, and the specimens will exhibit uniform crystallization throughout even when slowly frozen. -4 treatment known to have this effect is freezing and thawing as a preliminary step to a second freezing treatment (Meryman and Platt, 1955).

    These principles are in accord with the fact that natural cell walls and incnibranes act as barriers to crystal growth, but, once they are altered by some means such as heat or freezing, their barrier properties are con- siderably impaired.

    It is not known why extracellular crystallization occurs in preference to intracellular crystallization during slow freezing. However, Meryiiian (1956) has suggested two logical possibilities: 1 j the freezing point of the extracellular inaterial may be higher than that of the intracellular material, and/or 2 j the intracellular material may be deficient in hetero- geneous nucleation sites.

    Thc sequence of extra- and intraccllular crystallization is still open to question. Smith (1961, page 423) suggested that they may occur simul- taneously in specimens cooled rapidly to low temperatures. Mazur (1960bi expressed the belief that extracellular crystallization always occurs prior to intracellular crystallization, and that the former instigates the latter when conditions are appropriate. According to his theory, this will occur when minute extracellular ice crystals grow through the tiny water-filled pores of the cell membrane and thereby seed the cell con- tents. As previously pointed out, the radius of curvature of an ice crystal

  • 258 0. FENNEMA Ah-D W. D. POWRIE

    of critical size decreases with decreasing temperature. Therefore, as cool- ing progresses, a temperature is eventually reached where tiny, pore-sized crystals can exist and grow through the pores of the cell membrane. Mazur suggested that this temperature is approximately -10C for yeast cells and Aspergillus spores, since they undergo a marked decrease in survival when exposed to -10C or lower. This contention is based on the known association between intracellular ice formation and lethality. Cell mem- branes with larger pores than those just mentioned would acconiniodate larger crystals and would exhibit intracellular ice formation a t corre- bpondingly higher temperatures.

    In the theory suggested by Mazur, i t is assumed that spontaneous heterogeneous nucleation will occur only a t temperatures below that which is necessary for the growth of pore-sized ice crystals. If this assump- tion is invalid, the theory is invalid. I n view of the known inverse rela- tionship between sample size and tendency to supercool, the abo\-e as- sumption appears more valid for small cells such as microorganisms than it does for the large cells of plant and animal tissue. However, the logic of the last statement disappears if pore diameter increases with cell size.

    The fact that slow freezing, even to extremely low temperatures, fails to produce intracellular ice can be explained in a rather logical fashion. Extracellular ice crystals which form during slow freezing presumably have lower vapor pressures than tlie water present a t the cell surfaces. Such a vapor-pressure differential would cause water to migrate out of the cell and deposit on the ice crystals. The slower the freezing, the greater the opportunity for water migration. Cell shrinkage and a decrease in freezing point of the protoplasm will accompany the cellular dehydra- tion, perhaps to such an extent that intracellular freezing never occurs.

    Conversely, rapid cooling to a low temperature apparently provides little opportunity for cellular dehydration, and intracellular crystalliza- tion becomes more probable. Cells frozen in this fashion will exhibit a normal, unshrunken appearance.

    The rate of freezing required to produce intracellular ice formation varies considerably with the material being frozen. Moderate rates, only ,.lightly more rapid than those which occur in a natural environment, are bufficient to produce intracellular ice in plant tissue (Levitt, 1960), whereas in animal tissue a somewhat faster rate is generally required. Yeasts require still faster rates, and intracellular ice has never been ob- ser~-ed in bacteria, regardless of freezing rate. It would appear, then, that the formation of ice crystals is more difficult in small cells than lnrgc. This statement is in accord with the previously cited fact that small hpecimens are more prone to supercool than are large specimens. The large surface-to-volume ratio of small cells would enhance their ability

  • LOW-TEMPERATURE FOOD PRESERVATiOS 259

    to dehydrate, which might also have a bearing on their ability to avoid freezing.

    Even samples of a very similar nature occasionally require markedly different rates of freezing to produce intracellular ice formation. Love and Haraldsson (1961) found that fish frozen in the prerigor state ex- hibited intracellular ice a t slower rates of freezing than fish frozen in the postrigor state. They suggested that the greater amount of bound water in prerigor fish would contribute to this behavior.

    The location of ice crystals is of paramount importance in biological materials which must pass through the freezing and thawing process in a living state, e.g., cultures of microorganisms, spermatozoa, red blood cells, and human tissue. In such specimens, intracellular crystallization is nearly always fatal (Levitt, 1960; Meryman, 1956). It would seem logical that freezing and thawing conditions which are lethal to biological ma- terials might also be detrimental to the quality of food materials. ,4t present there is little evidence to support this possibility, since inany high- quality frozen foods are processed with rates of freezing which produce intracellular ice. This area deserves further investigation, however.

    VI. FREEZING DIAGRAMS

    A. FREEZING DIAGRAMS OF WATER AND SIMPLE SOLUTIONS Plots of product temperature versus heat removal or freezing time can

    provide useful information about the freezing process. Figure 12 presents a freezing curve for pure water and a typical freezing curve for water

    JU

    0

    -10 I \ L -204 I I \i , 0 40 ao 120 160 200

    HEAT REMOVAL (BTUSIPOUND)

    FIG. 12. Freezing c ~ i r w s for water and a simple solution. (Adapted from Somillei,, 1947. Tlieory and Practice of Ice Cream Making.)

  • 260 0. FENNEMA A S D IV. D. POWRIE

    containing one solute. The characteristic shape of the freezing curve for water arises from the following physical properties:

    Specific heat of water at 0C = 1.0 Specific heat of ice at 0C Latent heat of fusion (heat

    = 0.49 = 143.5 Rtu per pound, or

    79.7 calories per gram of crystallization) Water in the range 212-32F requires the removal of one Btu per

    pound for each degree reduction in temperature (50F to Point d in Fig. 12) . Point A represents supercooling, which always occurs to some extent prior to crystallization. Small samples of exceedingly pure water show strong tendencies to supercool, whereas large or impure samples of water generally supercool only slightly.

    With the start of nucleation, thc growth of ice crystals is rapid, with an associated release of lieat of crystallization. As a result of this heat, the temperature rises quickly to the freezing point of pure water and re- mains a t this temperature until all of the water has been solidified iB to C, Fig. 12). Once the water is frozen, further reinoval of heat energy results in a decrease in temperature a t the approximate rate of 1F for each 0.49 Btu removed from each pound of ice (recall that specific lieat varies with tciiiperature) . From comparison of tlic slopes of the cooling curves for ice and water, it is evident that the removal of a given quantity of heat energy will lower the temperature of ice approximately twice as inuch as tha t of water.

    Freezing of a simple solution gives rise to a quite different curve. Kc- nioval of heat from the origin of the curve to Point D results in a steadily decreasing temperature. The solution a t Point D is in a supercooled statc. The sequence of nucleation, ice crystal growth, and release of heat of crystallization causes the temperature to risc to the true freezing point, Point E (this occurs only if supercooling is limited). Additional cooling results in a gradual decrease in temperature, as shown in section EF. During this period, water is gradually transforincd into pure icc crptalb, causing the remaining solution to become progressively more concen- trated. As the concentration increases, the freezing point decreases, ac- counting for the drop in temperature which accompanies crystallization. -4s Point F is approached, the liquid phase becomes saturated with solute, and finally becoincs slightly supersaturated when Point F is reached. Tpon crystallization of the solute, the heat of crystallization cauws tlic tciiiperature to rise to Point G. Point G is known as the cryohydric point or cryohydric tmipcrature, whicli can be defined a3 tlic highest teinpcra- ture a t which iiiaxiniuni crystallization of water and solutes can occur in an aqueous system. (The term cryohydric is applicable only when water is thc solvent. Tlic more gmeral term eutectic is applicable t o anv solvent.)

  • LOW-TEMPERATURE FOOD PRESERVATIOS 26 1

    Further removal of lieat through section GH results in a change of btate with no change in temperature. During this pcriod, water and solutc crystallize simultaneously in constant proportion, forming an intermingled conglomerate, and the decreasing liquid phase is left unaltered in coin- position. At Point H , the crystallization of water and solute is complete and further removal of lieat merely reduces the temperature.

    The major differences between the two freezing curves should now be apparent. Pure water undergoes coniplete crystallization a t a constant temperature, whereas the simple solution (one solutc) begins to freeze a t a lower temperature than does pure water, and, during solidification, it undergoes solute concentration and a gradual decrease in temperature. The simple solution exhibits a change of state a t constant temperature only after attainment of the cryohydric temperature. Furthermore, com- plete solidification cannot occur a t a temperature above the cryohydric point, which is considerably lower than the initial freezing point.

    I3. FREEZING DIAGRAMS OF FOOD MATERIALS

    All natural food materials and most manufactured ones contain many constituents in true solution. The cryohydric points of the various solutes cover a broad temperature range. Table X lists some representative sub- stances and their cryohydric temperatures.

    Food substances might logically be expected to yield coniplicatcd freezing curves, with a small break appearing as each cryohydric point is passed. This, however, does not generally occur. The reasons are well explained by Sommer (1947, page 264) :

    I n a solution that contains several dissolved substances, the following must he expected. When the saturation point (due to removal of water as ice) has been reached with respect to substance A , further freezing will not increase the concentration of A in the unfrozen portion but will increase the concentrations of B, C, etc. Thcreforc, thr temperature will not be constant on further freezing, as it was in the single conlponcnt solution (Fig. 112 herein], Points G to H ) . Tlirre will merely be a decrease in the rate of temperature change aft,er A has reached its saturation point. The same holds true as the saturation points of R, C, etc., are reached in the remaining unfrozen portion.

    It niust be obvious that the exact, shape of the freezing curve in a mixed solution will be influenced by t,lie amounts of various components present, their ~o l i ih i l i t i~s in the preeence of the others and their effect on the freezing point. Further, it must be expected that if one of the components is present in Inrgc amounts in comparison with the others, it may cause a recognizable break in the freezing curve when the point is reached where i t starts to crystallize out.

    * * *

    li break in the freezing or warming curve of such a mixed solution, however, c.:innol 1 ) ~ rrgiirdcd as tile final cryohydric. point. Thc final wyohydric point is

  • 262 0. FENNEMA A S D W. D. POURIE

    TABLE X CRYOHYDRIC TEMPERATCKES OF VARIOUS SOLUTES

    Cryohydric Solutes temperature (C)

    Ir~oryanzc CaCL HCI

    KCP MgC1,lL Xa2C0p SaCP ?;aK03(L SaH2P04b

    K2C0sn

    Ka2HPOih KH2POAh K2HP0ah KHpPOr, K2HPOab XaH2P04, Na2HPOaf KH2PO L, NaH2POi IiH?POa, Na2HPOAh IGHPOI, Na2HP04b KH2P0,, Na2HP04, T\aHzPOlh KH2P04, Xa2HPOi, K2HPOab

    Sa2HPO4, KClC KH2PO4, NaZHPOI, KCIc KHJOA, NaaHPO4, FaCIc KH2P04, Na2HPOI, SaH,PO?, NaCIc KH2POI, NazHPOd, KCI, NaClC KLHPOi, KH2PO4, halHPO4, KCIc

    Sucrosed Sucrosed Glucosee Lactose, initial Lactose, final for hydrate Lactose, beta anhydrous, initial Lactose. beta anhydrous, final Glyrerolu

    KH2PO4, KClC

    Organzc

    - 55 - 86 -3G.5 - t l . t -33.6

    -2.1 - 2 1.13 - 18.5 -9.7 -0.5 -2.7 - 13.7 - 16.7 -9.9

    -11.2 -4.3 - 14.4 -11.4 - 17.2 -11.5 -11.6 - 12.1 -23.5 -23.6 -23.6 -18.9

    - 13.9 - 14.5 -5 -0.279 -0.65 -2.3 -4.1

    -46.5

    Langes Handbook of Chemistry. !)th ed., 1956. Van den Berg and Rose (1959). Arch. Biochem. Uzophys. 81, 319. Courtesy of

    Van den Berg (1959). Arch. Biochem. Biophys. 84, 305. Courtesy of Acadernic .ic.adernic Press.

    Press. d Washburn, 1927a. International Critical Tables 11, 345. e Washburn, 192ib. International Critical Tables 11, 346. 1 Washburn, 192ic. International Critiral Tables 11, 347. 0 Segur, 1953. Glycerol.

  • LOW-TEMPERATURE FOOD PRESERVATIOS 263

    reached only when tlic last one of thc dissolved substances reachcs its satura- tion point, and in a mixed solution the unfrozen portion a t that point will also be saturated with respect to all the other components. Henre the cryohydric point of a mixed solution must be lower than that of the component with the lowest cryohydric point. . . .

    Tlie cryohydric temperatures also give some indication of how low the temperature must be before a food material will solidify to the maxi- niuiii extent. Sonimer (1947) suggested that the final cryohydric point (highest temperature a t which maximum crystallization can occur) of ice cream lies below -55"C, since calcium chloride is present. Riedel (1961) used a calorimetric method to determine the final cryohydric point of incat and found it to be somewhere between -50" and -60C. Rey (1960h'l employed an electrical resistance technique, and found final cryoliydric points of -50C for horse serum and -56C for embryo ex- tract. Kuprianoff (1962) estimated the final cryohydric point of egg white to be below -55C. Other biological materials would logically have final cryoliydric points of the same general magnitude.

    It follows tha t food materials, when stored under the coldest coni- mercial conditions, still contain a small fraction of highly concentrated, potentially freezable liquid. Present in addition to this liquid would be a quite sizable quantity of bound and unfreezable water.

    It is very likely that the picture of food freezing as presented so far is greatly oversimplified. Rather than one cryohydrate forming for each solute, i t is quite likely that solutes, hydrates of solutes, colloidal con- stituents, or mixtures thereof form not only cryohydric mixtures but solid solutions (mixed crystals) and various other types of physical systems (Luyet, 1960b).

    With the above information, typical freezing curves for food materials can be understood more fully. Figure 13 presents a single freezing curve for slowly frozen strawberries, and Fig. 14 presents several groups of curves representing many different foods. The solid curve in Fig. 14h represents a typical freezing curve for slowly frozen fruits. Freezing curves for apples, figs, grapes, oranges, peaches, and strawberries will fall soine- where between the dotted lines of Fig. 14A. Other fruits would be expected to exhibit similar curves. The solid curve of Fig. 14B represents a typical curve for slowly frozen vegetables. Freezing curves for black-eyed peas, carrots, Swiss chard, lima beans, and potatoes fall within the limits of the dotted lines. Other vegetables would he expected to exhibit similar curves. The solid curve of Fig. 14C represents a curve typical for food materials of animal origin. Freezing curves for beef, eggs, ham, shrimp,

  • 264 0. FENNEMA A S D W. D. POWRIE

    3.3--

    40--

    30--

    20-

    P

    k

    ? 0-

    W m 3 lo--

    f

    -10-

    -30 10 20 30 40 SO 60

    MINUTES

    FIG. 13. Frwzing curve for n strawberry frozen in still air. (Sic.holas arid Perry, 1951. Agr. Eng. 32, 102. Reprinted by permission of the .%incrican Society of Agri- cultural Engineers.)

    and trout fall within the limits of the dotted lines. A separate curve for ice cream is included.

    Supercooling is evident only in Fig. 13 (Point A ) . I n spite of tliis, bupercooling is believed to occur in all biological materials, the extent tle- pending on the particular circumstances (Love and Haraldsson, 1961) . IVhether or not supercooling is detected will depend on the sensitivity, response, and location of the temperature-measuring device. For example, supercooling will probably not be detected if cooling is slow and the junc- tion point of a thermocouple is imbedded deep within a large sample of tissue. Supercooling will occur a t the products surface, but, once crystal- lization begins, the temperature will quickly return to the normal freezing point (if supercooling has been limited). The freezing process will con- tinue with the advance of a continuous freezing boundary toward the center of the specimen. Since the freezing boundary will exist a t the true freezing point of the sample, eventual contact with the thermocouple will give no evidence of supercooling.

  • LOJV-TEMPERATURE FOOD PRESERVATIOS 265

    80 A. FRUITS

    -40A ' i ' 4 ' 6 ' 8 ' 1 0 ' 12' 1 4 ' I6 ' i ' 2 0 22' B. VEGETABLES 80

    60

    4 1 , 2 0 ,\,i --.* 0

    -20 '\ \

    -4 0 0 2 4 6 B 10 12 14 16 18 20 22

    80. C. MATS

    3 -

    0 2 4 6 8 10 12 14 I6 18 20 22 COOLING TIME , HOURS

    FIG. 14. Freezing curves for slowly frozen foods: A ) fruits; B) vegetables; C) meats and ice cream. (Adapted from Short and Bartlett, 1944; courtesy of the Uni- versity of Texas, Bureau of Engineering Rcae:wc.li.)

    As previously mentioned, sinall samples are inore prone to supercool than are large samples. Furthermore, most biological materials are much less inclined to supercool than is pure water. Supercooling of more than 10C is rare in biological materials (Lusena, 1955), whereas pure water can be supercooled to a much greater extent. The composition of the material also influences the extent of supercooling. Colloids and glycols (especially glycerol) are particularly effective promoters of supercooling (Kuprianoff, 1962). Supercooling of fluid materials can be minimized by agitation.

    As would be expected, there is no indication of cryohydric points on any of the curves in Figs. 13 and 14. Section B of all curves represents

  • 266 0. FENNEMA AND W. D. POU'RIE

    the period during which the latent heat of crystallization is being removed and the change of state is occurring. During the initial stages of section B, water separates out in the form of pure ice, whereas during the latter stages of section B, cryohydric mixtures and other types of structures develop. At Point C, crystallization is nearly complete, as evidenced by the rapid drop in temperature as further heat is removed.

    h slow cooling process, as just described, provides little or no oppor- tunity for supercooling and nucleation in areas beyond the advancing ice front. This type of freezing process is characterized by the slow inward advance of a continuous ice front. At inore rapid rates of cooling, supcr- cooling can persist throughout the specimen, causing widespread nuclea- tion and a discontinuous crystallization process. Lusena and Cook (1954) found that freezing following the onset of nucleation could be predicted to be continuous or discontinuous from the ratio of the rate of heat release to tlic rate of heat removal. Thus, when the ratio exceeds 1.0, continuous freezing is probable; when the ratio is less than 1.0, discontinuous freezing is probable. This method of prediction obviously does not apply to fluids that are agitated during freezing.

    Freezing curyes differ in shape, primarily because of differences in product characteristics (composition, freezing point, and physical state) and rate of heat removal. Although manufactured food products often differ greatly in freezing point, natural foods usually freeze within a narrow temperature range. Examination of freezing points compiled by \\-right (1955) reveals tha t 19 common vegetables have freezing points ranging from 26.9" to 30.5"F; 22 coininon fruits have freezing points rang- ing from 27.2" to 30.4"F; fresh beef, pork, and lamb have freezing points in the range of 28 to 29'F; and milk and eggs freeze a t approximately 31F.

    MINUTES

    FIG. 15. Freezing curves for small viinplcs of chick cinbryo ccll suspcnsions frozen a t diffcrent rates. (Adapted from Taylor, 1960. Ann. N . Y . Acad. Sci. 85, 595. Courtesy of the New York Academy of Science.)

  • LOW-TEMPERATCRE FOOD PRESERVATION 267

    2 3 4 5 6 7 8 TIME HOURS

    I. -33OF IN AIR BLAST OF 1400 FT. PER MIN.

    2. -25. F IN NATURAL AIR MOVEMENT

    3. -10. F IN NATURAL AIR MOVEMENT

    4. 0- F IN NATURAL AIR MOVEMENT

    5. +lo' F IN NATURAL AIR MOVEMENT

    6. + P O O F IN NATURAL AIR MOVEMENT

    FIG. 16. Freezing curves for beefsteak frozen at different rates. (Ramsbottom and Coeser, 1955; reprinted by permission from Refrigerating Data Book 1955.)

    Figures 15 and 16 are freezing curves resulting froin different rates of heat removal. Figure 15 represents 0.5-ml samples of chick embryo cell suspensions in thin-walled glass test tubes. The rate of heat removal 11-as governed by changing the coolant temperature (Taylor, 1960). At the slower rates of freezing, supercooling is quite evident and considerable time is required to remove the latent heat of crystallization. As the rate of heat removal is increased, the various stages of freezing become less :,pparent, until, finally, a t very high rates of heat removal, they often become indistinguishable. Figure 16 shows a similar group of curves for beefsteak.

    Mention has been made of the fact that most food materials still con- tain some freezable liquid even under the coldest commercial storage conditions. The amount of freezable liquid decreases as the temperature is lowered. Tables XI and XI1 and Fig. 17 show for a few food materials the relationship between temperature and per cent frozen water (presuma- bly based on total freezable water).

    Before closing the discussion of freezing diagrams, it seems desirable to re-examine the subject of crystal growth, since complex biological materials rather than pure water are being considered. Much of the follow-

  • 268 0. FENNEMA A S D W. D. POWRIE

    TABLE XI PEE C E N T FROZEN &'ATElt IN I C E C R E A M A T V.4IUOU.S T E M P E R A T U I < E S "

    Water frozen Temperature Water frozen Temperature to ice (yo) ("F) 1 to ice (%) ( O F )

    0 5

    10 15 20 25 30 a 5

    27.55 27.35 27.05 26.78 26.40 26.04 25.i0 25.03

    40 45 50 -5 5 60 70 80 n 0 I -

    24.40 23.63 22.@% 21.4% 19.79 14.99 5.14

    22.29

    Composition: fat, 12.5%; seruni solids, 10.5%; sugar, 15%; stabilizer, 0.30%;

    a From Bell (1955). Reprinted by permission from Refrigerating Data Book, 1955. water, 61.7ujo.

    TABLE XI1 AVERAGE P E R CENT ICE I N BEEF, LAMB, A N D P O R K MUSCLES

    AT VARIOUS TEMPERATURES"

    Water frozen to ice (%, Temperature (OF)

    35.5 55.5 60.8 81.6 88.7 04.0 98.2

    29.3 28.4 26.6 23.0 19.4 14.0

    -4.0

    a From Moran (1931). Proc. Roy. SOC. Ser. B. 107, 182. Cour- tesy of The Royal Society, London.

    ing material is takcn froin Luyet's paper, '(An Analysis of the Notions of Cooling and of Freezing Velocity" (195713).

    During thc early stages of icc crystallization in complex systems, small individual crystallization units arise from nuclei. This stage is rc- ferred to by Luyet as "invasion," and its beginning coincides with thc temperature rise following supercooling. During this stage, the rate of crystal growth is determined not only by the rate of heat removal and by temperature, but also by the water content and nature of the system. Crystallization will be rctarded in low-moisture foods since water niole- cules will migrate slowly to the available ice crystal surfaces, and there is a possibility that nonaqueous constituents niay in some way associatc with the ice crystals and interfere with their growth. Some materials

  • LOJV-TEMPERATURE FOOD PRESERVATIOS 269

    I00

    C a, N

    e L

    L a, - p 50 + c a, V

    b

    n

    C

    Temperature (OF)

    FIG. 17. Per cent frozen water in lanib and peaches a t various temperatures. (Desrosier, 1959. The Technology of Food Preservation; courtesy of AVI Publishing Company.)

    known to reduce rates of ice crystal growth are proteins, glycerol, and cell walls. Apparently the two most important factors governing the rate of ice crystallization during the invasion stage are rate of heat removal and the water content of the specimen.

    With tissue, further freezing will eventually give rise to an advanced stage of crystallization, referred to by Luyet as the occupation stage. Crystal growth rates during this stage are governed by a new factor, space limitation. The reason for this is quite apparent, considering that the ever- enlarging ice crystals exist within conipartments of restricted size (cells, intercellular spaces, etc.). During freezing, a point is reached where ice crystals compete with one another for space and for the diminishing supply of freezable water. Rate of growth slows markedly during this btage.

    Transition from the invasion to the occupation stage is not evident froni a freezing curve. It is apparently a gradual and indistinct change, the point of occurrence varying a great deal with the particular circum- stances. The area of transition will occur somewhere along the plateau where latent heat of crystallization is being removed. The end of the occupation stage coincides with the completion of crystallization, recog- nizable on a freezing curve as the final major change in curvature.

  • VII. CONCENTRATION OF NONAQUEOUS CONSTITUENTS DURING FREEZING

    During the freezing of food, water is removed from solution and trans- formed into ice crystals of a variable but rather high degree of purity. The remaining material is left in a concentrated state, somewhat similar to products which have been partially dehydrated by conventional meth- ods. The extent of the concentration is influenced by the product charac- teristics, the rate of freezing, and the ultimate temperature. Highly fluid materials such as fruit juices can be concentrated readily by freezing, and in some cases this process is used commercially (Tucker, 1940; Pederson and Beattie, 1947; Tressler and Joslyn, 1961; Charm, 1963; van den Berg, 1961a). Less obvious is the fact that tissue also undergoes solute concen- tration during freezing (van den Berg, 1961b). Slow freezing will cause a greater degree of solute concentration than rapid freezing (Szent- Gyorgyi, 1957; Borgstrom, 1961 ; Xkiinoto and Ogawa, 1954; Pederson and Beattie, 1947) . The concentration will increase with decreasing tcm- perature in the subfreezing range, until the final cryohydric 1)oint is attained.

    Concentration of solutes during freezing is accompanied by changes of such properties as pH, titratable acidity, ionic strength, viscosity, osmotic pressure, vapor pressure, freezing point, surface and interfacial t en~ion , and oxidation-reduction potential. Except for viscosity, thcse propertics are undoubtedly affected more greatly by electrolytes than by nonclcctro- lytes. Changes in these properties are not difficult to measure in normally fluid materials, since the unfrozen fluid can be separated from the frozen. Changes in the pH, titratable acidity, and viscosity of several fruit juices during freeze concentration were studied by Tucker (1940). I n all cases the liquid collected from the nearly frozen juice had a markedly greater viscosity and titratable acidity than the original unfrozen juice. In most cases a decrease in p H was observed, the greatest being 0.3 p H unit. In ti few samples a slight increase in pH was noted.

    Van den Berg (1959) and van den Berg and Rose (1959) conductctl very interesting and valuable studies on p H and composition changes in buffer solutions during freezing. Sodium and potassium mono- and tliphos- phate buffers, alone and in coiiibination with sodium and potassiuiri chloride, were studied. Buffers consisting of Yarious combinations of sodium and potassium mono- and diphosphate generally exhibitecl largc pH cliangcs upon freezing, sometimes in excebs of one pH unit. I n ooiiie cases the change in p H was caused solely by the removal of water in thc forni of ice, whereas in other cases precipitation of one or niorc of the salts was involved. Except in solutions originally neutral or alkaline, the

  • LOTV-TEMPERATURE FOOD PRESERVATIOS 27 1

    pH decreased during freezing. Precipitation of monobasic salts resulted in an increase in pH, whereas precipitation of dibasic salts was responsi- ble for a decline in pH. Simultaneous precipitation of disodium and mono- potassium phosphates caused the pH to decrease in normally acid solu- tions. The addition of sodium or potassium chloride to the above buffer systems brought about a significant alteration in the pH changes usually observed during freezing. The important point to gain from this work is that even highly buffered systems can and do undergo marked changes in pH during freezing.

    Determination of pH and other properties is a far more difficult task for the liquid portion of partially frozen tissue than i t is for normally fluid materials. Once again, van den Berg has made a valuable contribu- tion to the field, by developing a special glass electrode permitting direct measurement of pH in frozen samples (van den Berg, 1960). With this device he studied pH changes during the freezing and storage of milk, green beans, cauliflower, tomatoes, cod, haddock, and beef (van den Berg, 1961a,b). Changes in pH during the first three months of frozen storage ranged from 0.3 to 2.0 pH units.

    Cauliflower, milk, green beans, and fish all exhibited somewhat similar curves when pH was plotted against duration of frozen storage. These products underwent a marked initial drop in pH, followed by a gradual return to the original pH during two to three nionths of frozen storage. The pH of tomatoes did not change noticeably upon freezing or during t h e e months a t -10C. After 20 days a t -18C, however, the tomatoes suddcnly increased from the normal value of pH 4 to a value in excess of 5.0. This pH was maintained a few weeks, and then abruptly returned t o the original value. The pH of beef increased from 5.6 to 6.0 during freezing and then remained essentially unchanged during frozen storage. KO iiiarked changes in pH occurred in any of the products beyond three months of frozen storage. The great variability noted in the direction of pH change in various products during frozen storage is apparently at- tributable to the type of salt which precipitates.

    Concentration of nonelectrolytes during freezing can also have some noticeable effects. Hydrophilic colloids, for example, can cause marked incrcases in viscosity as water is removed in the form of ice. Dissolved gaseh also become more concentrated during the freezing proccss, fre- quently to the extent that bubbles are formed. Such an occurrence give: rise to ice worms in saniplcs of water frozen a t certain rates (Chaliners 1959). Langhain and Mason (1958) observed a similar occurrence during the freezing of water droplcts 1 nim in diameter. Borgstroin (1961) anc Rinfret (1962) haye suggested that gas concentration during freezing niny have a toxic effect on biological materials.

  • 272 0. FENNEMA ASD W. D. POWRIE

    VIII. VOLUME CHANGES DURING FREEZING AND THAWING

    The alterations that biological tissue undergoes during freezing and thawing have been attributed to a number of factors, one of which is the change in volume associated with the change of state of water. Water a t 0C and one atmosphere of pressure expands nearly 976, transforming into ice. Langham and Mason (1958) effectively demonstrated one con- sequence of the volume change. They suspended millimeter-size droplets of gas-free water from fibers and froze them in cold air. During the initial stages of slow freezing, a clear shell of ice formed around the water drop- let. Continuation of freezing was accompanied by expansion of the interior and a violent rupturing of the shell. Liquid water occasionally spurted from the ruptured spot, solidifying in the form of a large spike.

    I n spite of this example, i t should not be assumed tha t all aqueous substances expand greatly upon freezing. Although some expansion is common, contraction during freezing has been observed in plant materials (Levitt, 1960) and in highly concentrated sucrose solutions (Table XIII) .

    TABLE XI11 C H A N G E S I N V O L V M E OF W A T E R .4ND S U C R O S E S O L U T I O N S ON FREEZIAO"

    Sucrose (54)

    Increase in volume during a decrease in temperature

    from approximately 70" to 0F ( - 17.8"C)

    0 (water) 1 0 20 30 10 50 60 T O

    8.6 8 . i 8.2 6.2 5.2 3.9 Sone 1 .0 (decrease)

    From Joslyn and Marsh (1930). 171d. Erq. Chem. 22, 1192. Courtesr of Industrial and Engineering Chemistry.

    Several reasons can account for the differences in volume changes ob- served in aqueous substances during freezing.

    Water content. The presence of a solute or suspended material has a "substitution" effect. Each volume of aqueous material contains less water as the amount of nonaqueous constituents is increased. Since water alone is responsible for the expansion that occurs during freezing, any decrease in the amount of water per unit volume will decrease the expansion per unit volume that occurs upon freezing.

  • LOJV-TEMPERATURE FOOD PRESERVATIOS 273

    Bound water and supercooling. Any material capable of binding water strongly will decrease the amount of water susceptible to crystallization and thereby decrease the expansion per unit volume upon freezing. Mery- nian (1960b) estimated that between 8 and 10% of the water in animal tissuc is unavailable for ice formation. Riedel (1961) found slightly higher values for fish, meat, egg white, and egg yolk. Daughters and Glenn (1946) found the unfreezable water content of strawberries and rasp- berries to be roughly 57c. Supercooling also decreases the expansion per unit volume tha t occurs upon cooling to a subzero temperature.

    Temperature range. The temperature range over which a volume change is studied will influence the valuc obtained. At temperatures just below the freezing point of a complex aqueous substance, few if any solutes will precipitate, and only a portion of the water will freeze. At somen-hat lower temperatures, more water will freeze and additional solutes will precipitate as they reach their respective cryohydric points. As the temperature is lowered still further, the final cryohydric point i. attained and crystallization is maxiniuni. A further reduction in tempera- ture causes all components to contract.

    The dependence of volume change on temperature range can thus be attributed to the following four events:

    a) Ice formation (expansion) b) Cooling of ice crystals (contraction) c) Solute precipitation and subsequent cooling (contraction) d ) Crystallization and cooling of nonsolutes, such as fats (contraction 1 . I n nearly all cases the predominating event is expansion due to ice

    formation. Air spaces. Air spaces, which arc frequently present in plant tissue, can

    partially accommodate expanding ice crystals, thereby minimizing the ovcr-all expansion. The rate of freezing may conceivably have some bear- ing on the amount of air that is retained in the tissue during freezing. Slow freezing would, logically, facilitate loss of air and tend to minimize ex- pansion during freezing.

    Tlic decrease in expansion that occurs with increasing sucrose conccn- tration (Table X I I I ) is due primarily to the substitution effect. The effect of solute precipitation probably becomes significant in solutions contain- ing high initial concentrations of sucrose, unless supersaturation persists. ;Ilaximum crystallization of water should occur in all samples since the freezing temperature is below the cryohydric temperature of sucrose.

    The action of glycerol on water during freezing offers an excellent example of the water-binding effect. One mole of glycerol, by virtue of its ability to hydrogen-bond, can prevent approximately 3 moles of water from frcczing (Meryman, 1960b). This not only reduces the amount of

  • 274 0. B'ENNEMA AND W. D. POWRIE

    expansion during the freezing process but also decrcases the concentration of solutes that would normally occur.

    Volume changes which occur in some fruit products upon freezing are shown in Table XIV.

    TABIAE XIV EXPANSION OF FRUIT PROOUC~~S DURING FREEZING"

    Av. increase in volume (yo) during a decrease in temperature

    from approximately 70" to 0F Product -

    Apple juice 8.3 Orange juice 8.0 Whole raspberries 4.0 Crushed raspberries 6.3 Whole strawberries 3.0 Crushed strawberries 8.2

    From Joslyn and Hohl (1948). Calif. Uniu. Agr. Ezpt. Sta. Bull. Xo. 703. Courtesy of the University of California Division of Agricultural Sciences.

    Regardless of tlie many complexities involvcd in volume changes, the fact remains that ice crystals tend to form in a pure state, even when they originate from a solution or complex system. As a result, the 9% expan- >ion applies to the freezable-water portion of any aqueous system. The expansion of water as it freezes, coupled with the contraction of most other nonaqueous constituents, results in local stresses, which undoubtedly produce mechanical damage in cellular materials. This statement is true regardless of the over-all volume change which occurs. An indication of the stresscs gcneratcd during freezing is providcd by Callow (1952). H e reported tha t internal pressures of 200 psi have been measured in large pieces of frozen beef.

    During thawing, materials do not necessarily return to their original volume. Most fruits occupy considerably less volume after thawing than they did prior to freezing. This is attributable to structural damage (collapse), loss of intcrcellular gases, and loss of water by diffusion in the presence of added sugar or syrup (Joslyn and Hohl, 1948).

    IX. RATE OF FREEZING

    A. DEFINITIONS Any discussion of freezing processes will eventually neccssitate some

    description of the rate a t which they are conducted. Commonly used terms

  • LOW-TEMPERATURE FOOD PRESERVATIOX 275

    are sharp or slow, rapid or quick, and ultrarapid. AS was true of the term bound water, universally accepted definitions for these terms are nonexistent. Although most investigators generally define their terms, a great deal of variation is evident in the methods of expression and the rate associated with each term. What one author defines as slow freezing, another may define as rapid. Table XV illustrates this point by classifying the methods of expressing freezing rates, along with specific examples found in the literature.

    The variability in definition seems to arise from three basic causes: 1) differences in the size of sample being studied; 2) differences in the facilities available and the accuracy desired; and 3) failure to recognize the relative merits of the various methods for expressing freezing rate. There is some justification for the first two, but none for the last.

    The use of Method I (Table XV), temperature change per unit of time, is beset with an obvious and very serious shortcoming. Application of an average rate of temperature change to the entire freezing process completely ignores the difference between rate of temperature change and rate of freezing. The amount of material that changes state with a one- degree change in temperature is obviously not uniform throughout the entire cooling and freezing curve. This is quite evident from thc shape of a typical cooling and freezing curve, and is further emphasized by thc example given in Table XVI. Use of this quite popular method, therefore, seems highly questionable.

    Method 11, time to traverse a specified temperature range, and Method VIa, rate of heat liberation, are quite similar expressions, with the former being iiiuch easier to determine. Both of these methods overcome the dis- advantage of Mcthod I and appear to be quite suitable for expressing the freezing rate of food materials. The only severe shortcoming of these two methods is found in samples frozen a t extremely rapid rates. I n such cases, the various sections of a normal freezing curve become indistin- guishable, thereby making i t impossible to select a meaningful tempera- ture range for Method 11, or to measure heat liberation accurately with Method VIa. Freezing rates of this magnitude are not. however, gcnerally encountered with commercial food-freezing processes.

    Both Method 111, nature of thc ice front, and Method IV, velocity of the ice front, are valid expressions of freezing rate. Measurement is diffi- cult, however, and Method 111 fails to distinguish adequately between small changes in the rate of freezing.

    Method V, location of ice crystals, appears to be of limited value con- sidering that inany factors other than freezing rate influence the location of ice crystals. Furthermore, this method does not distinguish adequately between small differences in the rate of freezing.

  • TABLE S V METHODS OF EXPRESSING RATE OF FREEZING

    Ilesrription

    Slow (or sharp) Rapid (or quick) UI t rarapid Referenre Appliration"

    I . 7'emperatuie chanp, per umt of tzirir 1C per min

    20C per hr - -

    Approx 50C per min doM n to - . -30C or lower

    5 to 20C per min 1 to 20C per niin 100C per min

    h a l l samples Mazur (l960a)

    Small samples Levitt (1960) Large samples Levitt (1960)

    (natural rondi tions)

    300" to 6000C per niin (5" to Any sample 0.01" to 0 02C per i i i i i i 1" to 100C per t n i n Borgstrom (I!)(il) 100C per sec)

    I I . Tame to traverse a specajied temperature range Approv 1 hr a t tempera- Few ser a t temp. of the freezing Approx 0.01 sec at temp. of Small samples Luyet (1061)

    ture of the freezing plateau the freezing plateau plateau

    ~ Less than 4 hrs in range - 1,:irge s:tuiples Long (1055) 32 "-23 "F

    Less than 30 min in range

    Less than 30 min from start of ice formation to temp. at

    31"-25"F

    which ire format,ion essen- tially c.oniplete

    0" to - i Y D C 10 miti or iiiorc i i i rmgr 2 sec to 5 min in r:trige

    0" t o -70C 2 sc(* or less i i i rntige

    0 t o --I!~O"C

    Large samples Nicholas (1945)

    Large samples Joslyn and Hohl of plant tissue (1918)

    Snidl samples Smith ( l ! ) ( i l )

  • 111 iVatiirr of the we fronf Solid ice front. Single-phase No discrete ph:m surfaw. Ice crystals srn:rll enough to 8in:ill s:~nipIcs Stephenson (l!)AO)

    srirtace separating solid N~mierous ice cryst:Lls be undetec~table M i t h elrv- and liquid tron microscope

    IT-, Vdotzty of ice fronl - a ) Movement of ice front a t -

    least 0.3 rm per sec in a prod- uct possessing a rounded snr- face, such as strawberries, and 11) ire crystals uniformly distributed and about the size of cells

    IT. Locafzon of crystals All crystals lorated extra-

    11 Othcr (not class~jietl as lo m f r ) :I) Rate of heat liberation -~

    Rapid enough to produce some c~ellularly intracrlhilar ire crystals

    h) Quantity of ice formed per unit weight of ma- terial per unit time, preferably with inform:z- tion about the origind moisture content and the

    Large samples Roodroof (1941) Y

    b <

    Animal tissue Meryman (19561 2 w 0 0 0

    General Luyet (1957l)) application

    application General Luyet (1957b)

    amount of freezable water a t lox tempera- tures

    (1 sin:~ll s:ttnple: :I feu gr:iins or less. Imge sample: c.ommerc.ial-si~r retail food packages or larger

  • 278 0. F E N N E M A AND W. D. POWRIE

    TABLE XVI T H E A M O U N T O F \ v A T E R UNDERGOING A C H A N G E O F S T A T E

    AS T H E TEMPERATURE OF I C E CREAM IS L4LTEREDL

    Relative amounts of water undergoing a change of state

    as the temp. is raised 1F Temperature of ice cream ( O F )

    - 5 (hardening room) s i (dispensing cabinet) bY

    23 (manufacturing temperature) 2 5 8

    a From Sommer (1947). Theory and Practice of Ice Cream Making, p. 265.

    AIethod VIb, quantity of ice forined per unit weight of inaterial per unit time, is a very precise method for expressing the rate of freezing but is unsuitable as a routine test.

    B. FACTORS AFFECTING RATES OF FREEZISC AND THAWING The rate a t which a food material freezes or thaws is influenced hy

    1) The temperature differential between the product and the cooling

    2) The means of transferring heat energy to, from, and witliin the

    3) The type, size, and shape of the package 4) The size, shape, and thermal properties of the product. Most of these factors are adequately treated in textbooks dealing with

    heat transfer. However, since such books generally do not contain ade- quate information concerning the thermal properties of food materials, and since such information is necessary for the calculation and under- standing of freezing processes, some space is devoted to i t here.

    The density of food materials generally decreases upon freezing. Den- sity changes vary inversely with changes in volume (discussed in the previous section).

    The specific heat of food materials, when considered apart from the latent heat of fusion, decreases markedly as water changes into ice, and continues to decline gradually with further decreases in temperature. This trend is not evident in the spccific-heat d u e s observed by Short and Bartlett (1944), Table XVII, since tlie effect of latent heat of fusion causes the values to rise above one, just below the freezing point. How- ever, by plotting the values, as sliowii in Fig. 18, the corrected mlues of specific heat can be obtained by extrapolation (dotted line).

    tlie following factors:

    or heating medium

    product (conduction, convection. radiation)

  • TABLE XVII SPECIFIC HEAT OF FOODP

    Product

    Green (English) Lima To- Stram*-

    Temp- peas Carrots beans matoes Apples Oranges Peaches berries Beets erature (80% (88% (68.5% (94.8% (83.77$ (80.7% (89.6% (90.9% (71.50/0 (OF) uater) water) water) water) water) water) water) water) water)

    - 40 -30 - 20 -10

    0 I 0 20

    h

    40 50

    0.45 0.44 0.48 0.47 0.52 0.51 0.51) 0.58 0.70 0.71 0.91 0.98 1.57 2.09 0.82 0.93

    (28.9) (30.4) 0.82 0.93 0.82 0.93

    0.36 0.36 0.37 0.42 0.51 0.68 1.09 0.78

    (28.4) 0.78 0.78

    0.40 0.42 0.44 0.41 0.43 0.43 0.47 0.46 0.47 0.46 0.53 0.53 OX2 0.53 0.62 0.62 O.G2 0.69 0.76 0.77 0.79 1.07 1.07 1.02 1.15 1.85 2.44 1.84 0.88 0.89 0.91 0.91 (30.6) (28.9) (29.3) (29.2) 0.88 0.89 0.91 0.91 0.88 0.89 0.91 0.91

    0.50 0.51 0.53 0.58 0.68 0.92 1.87 0.96

    0.96 0.96

    (30.1)

    0.43 0.44 0.45 0.48 0.50 0.82 1.62 0.95

    (31.3) 0.95 0.95

    F 0 8 Fresh Sea Ice 4

    ham Shrimp trout Eggs cream m (52.0% (78.6% (78.1% (74.2% (70.4% 5 water) water) water) water) water)

    0.39 0.45 0.42 0.44 0.40 2 0.39 0.47 0.43 0.45 0.45 E

    0.45 0.55 0.51 0.49 0.61 tc 0 0.53 0.65 0.61 0.55 0.77 ~

    0.41 0.50 0.46 0.46 0.52

    0.68 0.87 0.80 0.69 1.10 s 1.20 1.56 1.43 1.07 2.40 0.83 0.86 0.88 0.85 0.77 s (31.3) (31.0) (30.9) (31.6) (28.5) 2 0.83 0.86 0.88 0.85 0.77 0 0.83 0.86 0.88 0.85 0.77 '

    ~~

    a Adapted from Short and Bartlett (1944). Courtesy of the University of Texas, Bureau of Engineering Research. Specific-heat values

    b The initial freezing point is shown in parentheses beneath the corresponding specific heat of each product. include latent heat.

  • 280 0. FENNEMA A N D W. D. POWRIE

    -20 -40 -60 -80 -100 -120 -140 -160 Temper at u r e (OC)

    The specific hrat of beef chuck. (Moline ct d., 1961. Food T(,chnol. 15, FIG. 18. 228. Courtesy of the Institute of Food Technologists.)

    Many of the published lists of specific-heat values are calculated rather than observed. An equation with the following general form is fre- quently used for calculating specific heat:

    71

    s = B SIC1 + s2c2 + Sac3 . . s,,c,, 1

    where S = the calculated specific heat of the food s = the specific heat of a food component c = the mass fraction of a food component n = the total number of components being considered in the

    calculation.

    According to illolinc e t al. (19611, calculated values of specific heat are often quite inaccurate, especially those values applying to the change of state temperature range. The accuracy of calculated values is iinproved by considering a large number of food components and using accuratc values of specific heat and mass fraction for each one. If an average error of less than about 5c/( is desired, differcnces in the specific heat of free

  • LOW-TEMPERATURE FOOD PRESERVATION 28 1

    and bound water must be taken into consideration (Moline et al., 1961). The latent heat of fusion of food materials varies directly with their

    water content (Woolrich, 1933). The equation expressing this relationship is :

    Lf = 143.4P

    where Lf = calculated latent heat of fusion in Btu per lb P = the water content of the food material in per cent by weight.

    The presence of appreciable quantities of alcohol invalidates the above relationship. The calculated latent heats of fusion for an extensive num- ber of food materials are summarized in Table XVIII.

    TABLE XVIII CALCULATED LATENT HEATS OF FUSION FOR VARIOUS FOODS'

    Range of latent heat Class of food (Btu/lb)

    Common vegetables (19) Common fruits (22) Eggs Fish, fresh Milk, fluid, whole Meat

    Fresh beef Fresh pork Fresh lamb

    Poultry, fresh

    94 to 137 108 to 132

    96 89 to 122

    125

    89 to 110 50 to 60 86 to 100

    106

    Extracted by permission from ASHRAE Guide and Data Book 1962. Flink (1962). Values calculated by multiplying the water content in per cent by the latent heat of fusion of water (143.4 Btu).

    The thermal conductivities of food materials generally increase mark- edly upon freezing. Changes in the thermal conductivities of meat, fish, and fat during the course of freezing, as determined by Lentz (1961), are shown in Fig. 19. The frozen products have thermal conductivities which are approximately 3 4 times as large as those of the corresponding un- frozen products. Thermal conductivity values somewhat lower than those shown in Fig. 19 were obtained for beef by Miller and Sunderland (1963). Accurate thermal conductivity values for fruits, vegetables, and other food materials over the temperature range +40 to -20F are for the most part unavailable.

    Approximate thermal conductivity values for solid heterogeneous sub-

  • 282 o. FENNEMA A m w. n. POWRIE

    5 . 5 0

    5.00 0

    o_ x 4.5c - z 0 $ 4.0C

    J X 4 0 v w

    2 3 . 5 c

    0

    - N

    z

    3.0C i

    1 a

    I5( W I e

    I01

    0 51

    Lrgend (all percentages given in terms of weight) : ( 1 Indicates test with heat flow parallel to grain of sample I Indicates test with heat flow perpendicular to grain of

    C, Salmon (Gasp-Sallno salar), I, 67y0 water, 12.0%

    0 Salmon (British Columbia-Oncorhynchzts tshazcrg-

    0 Codfish, I, 83% water, 0.1% f a t + Beef (lean sirloin), 1 1 , 75% water, 0.9% f a t X Beef (lean f l a n k ) { , l , 7470 water, 3.4% f a t A Pork (lean, leg), 11, 7270 water, 6.1% f a t

    Pork (lean, leg), I, 72% water, 6.17~ fat Turkey (breast), 1 1 , 74% water, 2.1% f a t

    W Turkey (bieast), I, 74';/u water, 2.1% fut 0 Turkey (leg, I, 7470 water, 3.470 f a t + Butterfat, 0.6qG water X Beef f a t (udder f a t ) , 9% water, 89y0 f a t A Pork f a t (exterior), 6.0% water, 93% f a t 0 Seal blubber, 4.3% water, 95% f a t

    sample

    f,lt

    tscha), I, 7370 water, 5.4% f a t

    FIG. 19. Thermal conductivity of different foods at various temperaturrs (Lent z, 1961. Food Technol. 15, 243. Courtesy of the Instliutc of Food Technologiits.)

  • LOIV-TEMPERATURE FOOD PRESERVATION 283

    stances can be computed from an equation developed by Maxwell (1904) and modified by Eucken (1940) :

    where K = thermal conductivity of the heterogeneous material k , = thermal conductivity of the continuous phase k d = thermal conductivity of the dispersed phase, such as ice

    crystals, fat, etc.

    vd (for accuracy, this term must be small) = v, + V d

    V d = volume of the dispersed phase T;, = volume of the continuous phase.

    Long (1955) measured the thermal conductivity of fish over the range +40 to -20F and found good agreement between measured values and values calculated according to the Maxwell-Eucken equation. Lentz (1961) found that measured and calculated thermal-conductivity values were quite similar for 6 and 12% gelatin gels but differed considerably for 20% gels.

    The authors have been able to locate only a few thermal diffusivity values for food materials ; however, such values can be readily calculated from the equation previously given in Section IV,F.

    c. COMPARATIVE RATES O F FREEZING AND THAWING OF NORMALLY RIGID AQCEOCS MATERIALS

    Food materials, such as plant and animal tissue, gels, and highly con- ccntrated products which transmit heat energy primarily by conduction, exhibit differences in rates of freezing and thawing which often go un- noticed. An example involving like samples of codfish has been reported by Hastings and Butler (1955). When large rounds of cod were frozen and thawed under comparable temperature diffcrcntials, freczing required slightly less than 5 hr whereas thawing required 6 hr. Rinfret (1960), using very small cylindrical specimens, found that freezing to a depth of 5 nim took 60 sec whereas thawing to the same depth took 117 sec. These exainplcs illustrate that aqueous materials, which transmit heat energy priniarily by conduction, freeze more rapidly than they thaw (all condi- tions being comparable but opposite). The reason becomes quite evident when the thermal conductivity and tliernial diffusivity values of the

  • 284 0. FENNEMA AKD W. D. POWRIE

    product are examined. Before proceeding further, it should be mentioned that many of the following thoughts are taken from two of Dr. Meryman's articles (1956, 1957), in which he presents an excellent coverage of this subject.

    The coefficient of thermal conductivity is approximately four times as great for ice as for water, indicating that ice will conduct heat energy four times as fast as water. The coefficient of thermal diffusivity is more than nine times as great for ice as for water, indicating that ice will change temperature nine times as fast as water.

    The significance of these facts is best appreciated by considering an actual experiment conducted each year in a food-science course a t the University of Wisconsin. A nonflowable starch gel is divided among four 303 X 407 cans. Two are equilibrated in a water bath a t +78.5"C, and the other two in a dry ice-acetone bath a t -78.5"C. Four thermocouples are inserted into each can parallel to the major axis and extending to a depth such that the thermocouple junctions reside midway between the top and bottom. One thermocouple is located near the wall of the can, another a t the geometric center, and two others a t intermediate positions. Following equilibration, the two cold cans are transferred to the +78.5"C bath, and the two warm cans are transferred to the -78.5"C bath. Tern- perature and time are recorded until the change of state is complete. In both cases the initial temperature of the specimen is well removed from the freezing point, being as much above the freezing point a t the start of freezing as i t is below the freezing point a t the start of thawing. Further- more, the temperature differential favors neither freezing nor thawing, since the coolant temperature is as much below the product freezing point as the heating medium is above. Figure 20 illustrates the findings.

    The sequence of events is clarified by considering the three layers that exist in any normally solid, aqueous specimen being frozen or thawed: the frozen phase, the unfrozen phase, and the freezing boundary.

    During freezing (Fig. ZO-A,B,C) , the ever-decreasing unfrozen core must lose a considerable quantity of sensible heat in order to reach the freezing point. The rate a t which this occurs depends on the thermal diffusivity of the unfrozen core (low for water) and the temperature and rate of advance of the freezing boundary. The freezing boundary exists at a fixed temperature, characteristic of the initial freezing point of the sample. Regardless of the coolant temperature, thc unfrozen core seeks equilibration at the temperature of the freezing boundary. Whether or not i t attains temperature equilibration prior to the arrival of the bound- ary depends on the temperature of the boundary and the rate of its advance. The higher the coolant temperature, the slower the advance of the freezing boundary and the greater the opportunity for the unfrozen

  • LOW-TEMPERATURE FOOD PRESERVATION 285

    L6:m a ;.1 w n 2 - 6 0 W I- LOCATION

    A. 4 MIN. B. 16 MIN. C. 26 MIN.

    D. 4 MIN. E. 20 MIN. F. 30 MIN.

    FIG. 20. Sequence of freezing and thawing in a cylindrical specimen. A , B, and C = freezing sequence. D , B , and F I t,hawing sequence. Circles represent the central cross section of a 303 x 407 can filled with gelatinized starch. The four thermocouple locations are shown in the rircula; cross section of A . The relationship between the circular cross sections and the temperature profiles immediately beneath them is also shown in A . Arrows indicate the direction of heat flow, and dotted areas represent frozen material.

    core to equilibrate a t the temperature of the freezing boundary prior to its arriral. In the case shown in Fig. 20, the rate of boundary advance is fast enough that temperature equilibration of the unfrozen core does not occur.

    Latent heat of fusion is released as thc freezing boundary progresses inward at a constant temperature. This heat energy, plus the sensible heat released from the unfrozen core, must pass through the frozen ex- terior in order to reach the coolant. The rate a t which this heat energy can be removed governs the rate of boundary advance.

  • 286 0. FENNEMA A S D W. D. POWRIE

    The frozen exterior exists with its outer surface a t or near the tem- perature of the coolant and its inner surface a t the fixed temperature of the freezing boundary. During the course of freezing the ever-enlarging frozen exterior must transmit the heat energy being released from the unfrozen core and from the freezing boundary. This transmission of energy will occur a t a rapid rate because of the high thermal conductivity of ice. I n addition, as the thickness of the frozen exterior increases, the teinpera- ture gradient flattens and the mean temperature of the frozen exterior decreases. This decrease in temperature will be rapid because of the high thermal diffusivity of ice. The rapidity with which the frozen exterior transmits heat energy (high thernial conductivity ) and undergoes tein- peraturc changes (high thermal diffusivity) results in an aliiiost inime- diate establishment of a maximum temperature gradient.

    During thawing (Fig. 20-D,E,F), the phases and the direction of heat flow are reversed. The initial quantities of added heat energy are absorbed rapidly by the high-diffusivity frozen phase, causing the entire splicrc to rise nearly to the melting point before much surface thawing occurs. When surface thawing begins, most of the energy required for the change of state (heat of fusion) has yet to be supplied. Heat of fusion constitutes the majority of the total energy required for the thawing and warming process, and i t must be supplied through an ever-increasing barrier of low- conductivity low-diffusivity water. An analogous point in the freezing sequence occurs when surface freezing just begins and most of the reduc- tion in heat energy required for freceing and cooling has yet to be aceom- plished (Fig. 20-A). I n this case we find a situation which favors a rapid change of state, since further removal of heat energy occurs rapidly through the ever-increasing layer of high-conductivity high-diffusivity ice. Careful comparison of the two situations should adequately illustrate why freezing is inherently faster than thawing in normally rigid materials.

    Additional information concerning the dissimilar nature of freezing and thawing can be gained by plotting the temperature of the geometric center versus time. Figure 21 presents such a plot, based on data from the study just cited. It is readily evident that slightly less than 28 min elapsed before the geometric center froze, whereas 52 min were required for it to thaw. It is further evident that the thawing curve is not merely a reversed freezing curve. Kot only does thawing take longer than freezing, but an additional concern is the temperature a t which it occurs. It is of no small importance tha t all of the additional time required for thawing is spent a t the most damaging subfreezing temperature possible, i.e., just below the melting point.

    The difference in freezing and thawing times observed with simplc

  • LOIV-TEMPERATURE FOOD PRESBRVATIOS 287

    w 20- a

    0 10 20 30 40 50 60

    TIME (MIN.)

    FIG. 21. Comparative freezing and thawing curves for the geometric center of a cylindrical specimen (same data as in Fig. 20).

    system> will generally lie larger than is obscrvcd with tissue. \\.'hercab the simple system used in the above study required nearly twice as long to thaw as to freeze, tissue such as codfish requires something on tlie order of 20F longer to thaw than to freeze. nleryrnan (1960a) reported similar results for rabbit liver.

    The fact remains, however, tha t the thawing of norinally rigid food inaterials is inherently slower than freezing. This leads one to suspect that rate of thawing deserves considerably more attention than i t has received in the past. It seems a bit unreasonable froin a quality standpoint for a manufacturer to utilize ultrafast freezing techniques (liquid nitrogen, etc.) and then expose his product to the extreme daily temperature fluctua- tions encountered in a nornial retail frozen-food cabinet (Peterson, 1961). Moreover, a product may be partially thawed while the consumer is transferring it to her home, refrozen in her storage facility, and, finally, thawcd, often with a total unawareness or a t least a complacency about proper thawing techniquos. Such a sequence of events most assuredly will fail to provide the consumer with foods of maximum quality.

    It should be emphasized that these differences in rates of freezing and thawing apply only to materials which transmit heat energy primarily by conduction and to circunistances involving conventional methods of heat transfer. The principle outlined here would not, for example, apply if dielectric or microwave heating were employed. Likewise, fluid materials can normally be frozen and thawed a t rates fairly well governed by the desire of tlie operator, since agitation and crushing are permissible.

  • 288 0. FENNEMA AND W. D. POWRIE

    X. A SEARCH FOR PROTECTIVE CHEMICAL ADDITIVES

    Chemical additives can in certain instances minimize the deterioration of food materials during freezing, storage, and thawing. The ones in common use are sugar, salt, sulfurous acid and its salts, ascorbic acid, other edible acids, and calcium compounds. The use and function of most of these compounds are discussed in the section on commercial freezing processes (Section XIII).

    Glycerol is somewhat less coininon in the food field and deserves some attention here. Polge e t al. (1949) first discovered that glycerol provides remarkable protection for biological materials which must pass through the freezing, storage, and thawing process in a living state. Of the many substances investigated for this purpose, glycerol is one of the most ef- fective. When used a t a concentration of approximately 5-1570, glycerol permits the successful freezing and revival of nearly every tissue of the body as well as yeast, protozoa, red blood cells, spermatozoa and em- bryonic chick heart (Meryman, 1960b; Smith, 1961). The mechanism of its protective action is still not fully known, but some or all of its prop- erties as listed below are thought to be involved (Luyet and Gonzales, 1952; Meryman, 1960a,b; Rey, 1960a,b; Smith, 1961; Lovelock, 1954a) :

    1) Nontoxic to host cell or tissue a t the concentrations used 2) Highly soluble in aqueous solutions in the range +40 to -40C 3) Ability to penetrate cell membranes rapidly 4) Low cryohydric point 5) Ability to hydrogen-bond with water 6) Strong tendency to form amorphous structures a t low temperatures 7) Ability to decrease the crystallization velocity of ice. The last six properties have the effect of minimizing the amount of

    water that changes into ice, thereby minimizing volume changes and the concentration of solutes. The addition of glycerol also dilutes the solutes, thereby decreasing the maximum concentration attained during freezing.

    There are a few instances where glycerol has been shown to be of some value in food materials. Egg yolk, for example, is frequently blended with glycerol, sodium chloride, sugar, or a combination thereof in order to minimize gelation during freezing. Glycerol has also been investigated as a protective agent for frozen cod (Love, 1962b). Soaking cod fillets in 15% (v/v) glycerol prior to freezing was found to decrease markedly the rate of protein insolubilization during storage a t -14C. Glycerol a t a 10% (v/v) concentration was also effective, but a 5% concentration was of little value. Further investigations seem desirable on the use and protective mechanism of glycerol and other new additives.

    This information is introduced in the hope that glycerol and othcr

  • LOW-TEMPERATURE FOOD PRESERVATION 289

    substances which aid the survival of living biological materials during freezing, storage, and thawing might also have a desirable influence on food quality. This admittedly may be wishful thinking, but the possi- bility should not be overlooked.

    XI. THE EFFECT O F FREEZING, STORAGE, A N D THAWING ON THE PHYSICAL AND CHEMICAL PROPERTIES O F FOOD

    A. CLASSIFICATION AND CHARACTERISTICS OF FOOD Any food may be considered as a whole made up of constituent parts,

    or, more simply, as a system. The specific arrangement of constituents and phases in a food system is responsible for well-defined organoleptic,

    FOOD SYSTEMS

    Gross strawberries, systems, intact 2 i.e. tissue Small-particle systems (particles below 600 p) fish fillets,

    sliced carrots I t

    NondispeWd solids,i.e.

    cocoa powder, flour,

    dry milk powder sugar,

    1 Dispersions

    -colloidal

    -suspension Liquid l---l Solid -colloidal

    a) solid in liquid (sol)

    SOlUtiOn b) liquid in liquid (emulsion) -------suspension

    ~ C O l l O i d a l

    -suspension -colloidal

    \suspension

    solution

    c) gas in liquid (foam)

    d) liquid in solid (solid emulsion)

    ~ C O l l O i d a l

    -suspension e) gzs in solid (solid foam)

    FIG. 22. Classification of food systems

  • 290 0. FENNEMA AND W. D. POWRIE

    physical, and chemical properties. .4ny alteration in thc arrangenient of tlic coiiiponents will lead to changes in the characteristics of the entire bystem. To underbtand how the freezing process brings about changes in food systems, thorough knowledge is needed of the characteristics and interrelationships of food constituents. The following presentation of the general classification and characteristics of food systems will provide a logical basis for evaluating detailed changes in specific food systems.

    -4s shown in Fig. 22, food systems can be divided into two major groups : gross intact tissue systems and small-particle systems. Small- particle systems can be further subdivided into dispersions and undis- persed solids. In general, dry undispersed solids such as sugar granules or flour will not be altered during freezing. Dispersions, particularly those containing water, can be damaged readily during freezing. Dispersions may be simple one-phase systems, such as a liquid sugar solution, or they may consist of several phases. Milk and egg yolk are examples of fluid multiphasc systems.

    1. Food Dispersions

    A dispersion consists of specific ions, molccules, large particles, and/or gas bubbles in a continuous phase. The large particles may be crystals, aniorphous solid matter, cell fragments, cells, or liquid globules. I n most cases, the liquid phase is water. Ostwald (1922) classified dispersions on the basis of particle size. Ostwalds classification can be used for arrang- ing food systems in an orderly nianncr. A molecular dispersion is a solu- tion of ions and molecules whosc sizes are less than 1 inp. Colloidal dis- persions contain particles ranging in size from 0.5 p to 1 mp, and coarse dispersions consist of particles with dimensions greater than 0.5 p. The molecular dispersion or solution is regarded as a one-phase system, whereas the colloidal and coarse dispersions consist of a t least two phases. Diphase food systems may be subdivided into :

    1) Solid in liquid (colloidal solution or suspcnsion) 21 Liquid in liquid (emulsion) 3) Gas in liquid (foam) 4) Gas in solid (solid foam) 5 ) Liquid in solid (solid sol ) .

    A gel is not a dispersion, since it is coinposed of two intermingled, con- tinuous phases. The first three types of diphase systems represent many food systems.

    The uniform distribution of particles in a stable dispersion is dc- pendent on such factors as: electric charges on the particles, viscosity of the continuous phase, and particle hydration. A food system may become unstable or altered if any of the aforementioned stabilizing factors are

  • l,OW-TEMPERATURE FOOD PRESERVATIOS 29 1

    altered. For example, when a food systcni is subjected to freezing tcni- peratures, the physical properties of the particles may be changed to the extent that the over-all physical statc of the thawed food product is much different from that of the original product. The concentration of non- aqueous constituents during freezing can produce alterations in noncellu- lar food systems. The particles in a system may aggregate to form large clumps, or gas bubbles may coalesce to form larger bubbles. The dis- ruption of the clumps in the thawed product will depend on the bond energy. If hydrogen or van der Waals bondings are involved, the clumps may be broken up readily with slight agitation. On the other hand, the dispersion of clumps with high-energy coulombic bonds requires more rigorous treatment. Solutes such as sugars, salts, and glycerol can mini- mize the alteration of some food systems when frozen and thawed. Un- doubtedly the adsorption of sugars and glycerol on the surfaces of par- ticles in a system is responsible for the inhibition of particle aggregation.

    2. Gross Intact Tissue Systems

    Cellular food systenis have much more complex structures than non- cellular systems. I n the first place, the cell wall or the cell membrane encircles a system consisting of hundreds of compounds such as proteins, carbohydrates, pigments, enzymes, and salts. These constituents are not distributed uniformly throughout the protoplasm, but are concentrated in specific cell areas. For instance, sugars and salts are present in the vacuole of some plant cells, and chlorophyll pigments reside in plastids.

    Another outstanding feature of cellular food systems is their resistance t o deformation under sinall pressures. Certainly the rigidity of mcclt is rcsponsible for its delightful textural sensation. With cell walls acting as semipermeable membranes, cells can absorb water to the extent of pro- ducing small turgor pressures. These turgor pressures of cells are essential or obtaining the auditory and textural sensation of lettuce crispness.

    In many instances, the consumer assesses the acceptability of a prod- uct on the basis of the fresh product. For example, the consumer con- sidcrs fresh strawberries as the most acceptable from the standpoint of flavor, color, and texture. Any alteration in a quality attribute will usu- ally reduce the acccptability of the product. When fresh fruit or meat i* frozen, altcrations occur, particularly in the organized structure, and the consumer can usually detect a textural change. The niode and degree of structural alteration are dependent on the typcs of constituents in and among the cells, the rigidity of the cell walls, the concentration of water in the tissue, and the organization of constituents in the cells. Certainly one ~ o u l d expect that the structural alterations in plant tissue would differ from those in animal tissue. Levitt (1960) pointed out that the

  • 292 0. FENNEMA AND W. D. POWRIE

    fieezing injury of plant cells is probably dissimilar to that for animal cells, because: 1) the plant cell has a large vacuole, which is responsible for the high moisture content of plant tissue; 2) plant cells have thicker cell walls than do animal cells; 3) plant cells possess intercellular spaces with entrapped air whereas animal cells are surrounded entirely by fluid.

    B. CHANGES IN SELECTED FOOD SYSTEMS RESULTING FROM THE FREEZING PROCESS

    The purpose of this section is to illustrate briefly some changes in selected biological systems.

    1. Milk and Milk Products

    JThole milk consists essentially of a continuous phase, called serum, within which fat globules and calcium caseinate-phosphate particles are suspended. The size of the globules varies from about 0.1 to 10 p (Camp- bell, 1932). The average fat content of milk has been reported to vary from 3.55c/o, for Holstein, to 5.197., for Guernsey (Jenness and Patton, 1959). When whole milk stands, the fat globules gradually rise, but the globules do not coalesce, since a protective film, called a fat-globule membrane, is present on the surface of each globule (King, 1955).

    The calcium caseinate-phosphate particles are made up of as-, p-, 7- , and K-caseins as well as bound ions such as phosphate, citrate, calcium, and magnesium. These particles in serum are stabilized for the most part by charges (Jenness and Patton, 1959). Thus, any change in the ionic environment may lead to destabilization of the caseinate particles. Sitsch- mann (1949) stated that the somewhat spherical caseinate particles vary in diameter from about 30 to 300 p.

    The serum contains soluble materials such as p-lactoglobulin, a-lactal- bumin, globulins, lactose, and ions of salts (Jenness and Patton, 19591.

    When fluid milk, concentrated niilk, or cream is frozen, stored in the frozen state, and thawed, the physical properties are altered from those of the corresponding unfrozen samples. The freezing-storage-thawing process causes a disruption of the fat emulsion and a destabilization of milk proteins.

    Disruption of the fat emulsion by the freezing of a fat-containing niilk product leads to the formation of a surface layer of fa t aggregates in the thawed material (Webb and Hall, 1935; Doan and Baldwin, 1936; Dahlc, 1941). According to Doan and Baldwin (1936), the fat aggregates in thawed cream appear to be identical to the fat aggregates brought about by churning. Webb and Hall (1935) found that freezing of cream or whole milk without any frozen storage was capable of destabilizing the fat emulsion. The amount of aggregated or agglomerated fat in a thawed

  • LOW-TEMPERATURE FOOD PRESERVATION 293

    product increases with an increase in fat content and percentage of frozen water (Doan and Baldwin, 1936; Webb and Hall, 1935). Doan and Bald- win (1936) suggested that the cmulsion breakdown during freezing is due to pressures within the frozen mass. However, this supposition has not been thoroughly investigated. Undoubtedly the disruption of lipid-protein complexes in the fat globule membrane is involved in the mechanism of de-emulsification. Certainly lipoproteins in other biological systems are altered by freezing and thawing (Lovelock, 1957; Powrie e t al., 1963).

    Several methods have been proposed for minimizing the disruption of the fat emulsion in whole-fluid and condensed milk. Both Webb and Hall (1935) and Doan and Baldwin (1936) found that sucrose in milk products retarded the separation of fat. High levels of sucrose, however, cause the product to be excessively sweet. An increase in the solids-not-fat of milk products will inhibit emulsion disruption (Webb and Hall, 1935). Homogenization of whole milk, concentrated milks, and creams with fat contents up to 2070 can prevent freezing damage to the emulsion (Webb and Hall, 1935; Bell, 1939; Webb, 1951). Homogenization pressures up to 3000 psi have been recommended.

    Destabilization of proteins in fluid milk and concentrates by storage in the frozen state is reflected by the flocculation or precipitation of pro- tein masses in the thawed materials. Anderson and Pierce (1929) reported that frozen-storage periods of 1-2 months wcre required to obtain sig- nificant precipitates in thawed raw and sterilized skim milk previously frozen and held a t -12.2"C. The amount of precipitate in thawed samples of raw and sterile milk increased progressively with storage time (-12.2OC) up to the fifth month. In concentrated milks, the flocculated particles in the thawed product may be so numerous that a gel structure is formed (Corley and Doan, 1940; Doan and Featherman, 1937). Early in frozen storage, the flocculated masses in thawed samples can be redis- persed by agitation or heat. As storage time progresses, the flocs become more resistant to redispersement (Doan and Featherman, 1937; Doan and Warren, 1947). Thcb coagulated particles, or flow, have been shown by Doan and Warren (1947) and Wildasin and Doan (1951) to be cal- cium caseinate, which has properties similar to those for salted-out cal- cium caseinate from concentrated skim milk.

    Although the mechanism of flocculation has not been explored exten- sively, some reports have provided valuable clues. Doan and Warren (1947) suggested that the flocculation of calcium caseinate micelles is due to the salting-out effect in the presence of a high concentration of salts in the unfrozen liquid of the frozen product. Wildasin and Doan (1951) indicated that a 10% reduction in total calcium content of milk can retard the flocculation of caseinate in concentrated milk. Conse-

  • 294 0. FENNEMA AND W. D. POWRIE

    quently, those investigators postulated that soluble calcium is a require- ment for caseinate flocculation. The possibility of pH change as a factor in protein destabilization led Tessier and Rose (1956) and van den Berg (1961a) to investigate pH changes during freezing and frozen storage. Van den Berg (1961a) found no relation between pH change and protein flocculation. Desai e t al. (19611 noted a gradual migration of casein phosphorus into the serum during the frozen storage of condensed milk. They suggested that the loss of phosphorus from casein may be a result of the protein destabilization. They also found that proteins became destabilized when the a-lactose content reached 80% of the total lactose.

    Many factors have been reported to influence thc destabilization of proteins in frozen milk and concentrates. I n the first place, the degree of milk concentration has a marked effect on the degree of flocculation. Bell and illuclia (1952) showed that, as the solids in milk are increased, the amount of precipitate increases in thawed products previously frozen and stored a t +lo or -8F. Doan and Warren (1947) and Wildasin and Doan (1951) found similar results.

    The rate of freezing and the storage temperature influence the sta- bility of proteins in milk products. Rose and Tessier (1954) reported that proteins in whole milk and concentrated milk held a t -12C were not destabilized as extensively during 5 1 0 ~ freezing as during very rapid freezing (liquid nitrogen, -196C). They assumed that slow freezing pro- vided timc for the establishment of new equilibriums. The advantage of slow freezing was not apparent when milk was stored at -18C.

    Many reports h a w been publislied to substantiate the initial observa- tion by Anderson and Pierce (1929) that, as the frozcn-storage tempera- ture is dccreascd, the destabilization of calcium caseinate is diminished (Bell and Mucha, 1952; Doan and Warren, 1947; Babcock et al., 1946; Rose and Tessier, 1954). Babcock et al. (1946) indicated that a rise in storage temperature could increase the rate of protein destabilization.

    Heat treatment and homogenization have been reported to influence the stability of proteins in frozen milk products. According to Corky and Doan (1940), tlie formation of irreversible flocs in frozen-thawed concen- trated milk can bc prevented by heating tlie original fluid milk 15 min a t 82.2"C. Forewarming skim milk to above 765C was found by Doan and Warren (1947) to lower the stability of the protein in frozen stored concentrate. A more recent report (Webb, 1951) indicates that heat treat- inents of milk in excess of 155'F for 30 min can cause a decrease in pro- tein stability. Homogenization of milk or concentrate prior to freezing increases the rate of caseinate destabilization during frozen storage.

    Sugars and glycerol added to milk prior to freezing h a w been reported to inhibit protein destabilization (Wildasin and Doan, 1951 ; Babcock

  • LOW-TEMPERATURE FOOD PRESERVATIOY- 295

    e t al., 1952; Rose, 1956). Tuniernian e t al. (1954) reported that enzyme- hydrolyzed lactose in concentrated milk inhibited protein destabilization. Loss of soluble lactose through crystallization during frozen storage of milk products undoubtedly enhances destabilization (Desai e t al., 1961 ; Braatz, 1961). Rose (1956) theorized that the protective effect of sucrose can bc attributed to an increase in the Yiscosity of the unfrozen portion and to a freezing-point depression.

    The rcmoval of inorganic ions from milk products by dialysis and ion- exchange methods has extended the storage life of frozen concentrates (Christianson et al., 1952; Haller and Bell, 1950; Wildasin and Doan, 1951). Doan and Warren (1947) and Wallgren (1961) found tha t poly- phosphates markedly retarded the rate of protein flocculation from frozen storage of concentrated milk.

    Excellent reviews on the commercial aspects of frozen milks have been written by Doan (1952) and Winder (1962).

    2. Egg Yolk

    Yolk is a complex Iiological iiiixturc consisting of microscopically dctcctahle granules and a continuous fluid phase, called the plasma (Schmidt e t al., 1956). On a dry-weight basis, granules represent about 23% of the total yolk solids (Burley and Cook, 1961). According to Burley and Cook (1961), granules are 70% 01- and p-lipovitellins, 16% phosvitin, and 1276 low-density lipoprotein. The plasma, with 7770 lipid (on a dry-weight basis), contains 0 1 - , p-, and y-livetins as well as low- density lipoproteins (McCulley e t al., 1962). Martin e t al. (1959) and Sugano and Watanabe (1961) reported that the plasma low-density lipo- protein fraction consists of a t least two lipoproteins. Saari e t al. (1964) have developed a technique for the separation of two low-density frac- tions, LPLl and LPL2, from yolk plasma. LPL1 and LPLa contained 89 and 8676 lipid, respectively.

    The structure of low-density lipoproteins has been discussed in sev- eral recent review articles (Cook and Martin, 1962; Vandenheuvel, 1962). Low-density lipoprotein niicelles consist of triglycerides, phospholipid, cholesterol, carbohydrates, and proteins. Most investigators agree tha t each micelle consists of a lipid core surrounded by a protein film. Since the concentration of protein in low-density fractions is rather low, cov- erage of the entire micelle surface is impossible (Cook and Martin, 1962).

    When fluid yolk is frozen a t a temperature below -6"C, the mass becomes pasty upon thawing (Moran, 1925). This viscosity change is called gelation. Moran (1924) noted that supercooled yolk did not be- come pasty when warmed to room temperature. Apparently, ice crystal formation in yolk is a requisite for gelation. As the temperature of yolk

  • 296 0. FENNEMA AND W. D. POWRIE

    is lowered below -6"C, the rate of viscosity change is increased (Powrie e t al., 1963). The influence of storage temperature on yolk viscosity is shown in Fig. 23.

    -792 .

    - ._ 400

    a a +"-- 0 200 400 600 800

    Storage time, min.

    FIG. 23. Influence of frozen-storage temperature on the viscosity of thawed yolk. (Powrie et al., 1963. J. Food Sci. 28, 38; courtesy of the Institute of Food Tech- nologists.)

    The alteration of yolk stored a t -14C is presumably most extensive during the first few hours of storage. Moran (1924) stated that the pastiness of thawed yolk in whole eggs a t -11C increased in proportion to storage time up to about 24 hr. According to Pearce and Lavers (1949), alteration in the viscosity of thawed yolk (frozen and stored a t -18C) could be detected up to the eighth month of frozen storage. According to some investigators (Moran, 1924; Pearce and Lavers, 1949), rapid thaw- ing inhibits the increase in yolk viscosity.

    Mechanical and chemical treatments have been reported to inhibit yolk gelation. Pearce and Lavers (1949) and Lopez e t al. (1954) found that homogenization and colloid milling of native yolk minimized gela- tion. Moran (1925) reported that sucrose prevented a change in the fluidity of frozen-thawed yolk. Other sugars, sodium chloride, and glycerol have been reported as inhibitors of gelation (Thomas and Bailey, 1933; Lesser, 1948; Lopez e t al., 1954). Proteolytic enzymes have been reported to be effective agents for inhibiting the viscosity alteration of frozen- thawed yolk (Tressler, 1932; Lopez et al., 1955).

    So far, the mechanism of yolk gelation has not been elucidated. Moran (1925) suggested that the salts of yolk are concentrated a t temperatures below -6"C, whereupon irreversible precipitation of lecitho-vitellin oc- curs. Urbain and Miller (1930) suggested that the coagulation of lecithin was responsible for the gelation of yolk. However, recent studies have indicated that no free lipid matter is present in yolk (Evans and Ban- demer, 1957; Turner and Cook, 1958). The implication of lipoproteins in the gelation of yolk has been suggested by Fevold and Lausten (1946),

  • LOW-TEMPERATURE FOOD PRESERVATION 297

    Feeney e t al. (1954), and Powrie e t al. (1963). Powrie e t al. (1963) re- ported that the electrophoretic properties of a lipoprotein, L2, which is the major lipoprotein fraction in the plasma, were altered when yolk was frozen and thawed. Paper electrophoresis of frozen yolk displayed the ab- sence of the lipoprotein L3 band, but a new lipoprotein band, with a much lower mobility, was apparent. Saari and Powrie (1961) noted that the plasma fraction of yolk became pasty when the fluid mass was frozen below -6C, stored, and thawed. From this observation, it can be con- cluded that the particulate yolk granules do not play a significant role in the gelation of yolk.

    3. Meat

    Meat is a food product derived mainly from domesticatcd mammals, fowl, and fish. Although meat is composed of several types of tissue, the major portion is usually skeletal muscle tissue.

    Meat is generally regarded as a very perishable food commodity (Ayres, 1955; Hastings and Butler, 1955; Evans and Niven, 1960). The meat of fish, poultry, and domesticated animals is susceptible to rapid microbial decomposition a t ambient temperatures. Freezing temperatures retard microbial growth and metabolic proccsses, and inhibit enzymic and chemical reactions which are responsible for deleterious changes in the meat quality. However, under certain conditions the freezing process can cause detrimental alterations in meat. Typical consequences of muscle changes from freezing are an exudation of fluid and a tough texture when meat is thawed. In most instances the deterioration is related to alteration of the native fibrillar structure of muscle. Consequently, the following knowledge of the structural characteristics of the native muscle should be helpful in explaining or hypothesizing the mechanisms of the freezing damage.

    The unit cell of a skeletal muscle is a long, cylindrical element, termed a fiber. The fibers of bovine muscle have diameters ranging from about 40 to 100 p (Tuma e t al., 1962). The fiber length has not been estimated for domestic animals, but studies on human muscles indicate that fibers can extend to a t least 34 cm long (Lockhart and Brandt, 1938). Accord- ing to Huber (1916) and Van Harreveld (1947), the muscle fibers of the rabbit are long, though few of them extend from the origin to the inser- tion. Love (1958a) found that the fiber diameters and lengths of cod myotonies were related to the body length of the intact fish. Each fiber possesses a thin membrane called a sarcolenima. According to Wall (1960), this elastic membrane has a thickness of 0.1 p and acts as a sheath for the protoplasm.

    The protoplasm of the fiber is composed of contractile fibrils imbedded

  • 298 0. FENNEMA AND W. D. POWRIE

    in a liquid cytoplasm, called the sarcoplasm. The sarcoplasm consists of water-soluble albumins along with other soluble constituents. Sarcoplas- mic proteins are the major nitrogenous constituents in the drip of thawed mcat. Many nuclei and inclusions, such as mitochondria and fat globules, are scattered throughout the sarcoplasm. The fibrils have diameters be- tween 1 and 3 p and are aligned parallel to the long axis (Wall, 1960; Huxley and Hanson, 1960). The cross striations which can be detected in the muscle fiber with a light microscope can be shown in a fibril by electron microscopy. This indicates that the fibrils are oriented in a defi- nite manner. Since fibrils do not have membranes around them, exchange of thc constituents between the fibrils and the sarcoplasm is unrestricted. Wit11 regard to the freezing of muscle, such a structural fcature should not be overlookcd.

    The cross striations of a fibril are caused by variation in light trans- parency. Thc pattern of striations repeated along each fibril depends on the condition of the muscle (i.e., prerigor or postrigor) and the spccies of animal. The length of the repeat pattern (sarcomere) is commonly be- tween 2 and 3 p for the vertebrate animals (Huxley and Hanson, 1960). The typical A , H , and I bands in a sarcomere have been attributed to the organization of fibrillar protein subunits called filaments. Recent studies with the electron microscope have shown that two types of filaments arc present in the fibril (Huxley and Hanson, 1960). The diameters of the two kinds of filaments have been estimated to be about 110 and 50 A

    (b) - - - I

    ~ ; I G . 24. Structure of a muscle fibril: a) bands in an intact sarcomere; b) filaments in an intact sarcomere. (Huxley and Hanson, 1960. I n The Structure and Function of Musrle, Vol. 1 ; courtesy of Academic Press 1

  • LOW-TEMPERATCRE FOOD PRESERVATIOK 299

    (Huxley and Hanson, 1957). As shown in Fig. 24, the filaments are ar- ranged in a parallel form, with each type of filament in an alternate position (Huxley and Hanson, 1960). Evidence indicates that the thick filaments consist of myosin, and the thin filaments of actin and tropo- myosin. Myosin is the major protein constituent in the fibril (about 54% of the total protein). Although actin is an important muscle constituent* i t is present in the fibril only to the extent of about 25%.

    According to Huxley and Hanson (1960), the thick myosin filaments are situated about 450 A apart. Crystals of ice could undoubtedly fornl, particularly in the H-band areas, and disrupt the filament organization. Even more plausible is the growth of ice crystals in the sarcoplasm between fibrils, which are normally spread 0.5 p apart. When intracellular crystal formation occurs during the rapid freezing of muscle tissue, me- chanical damage of the cell, as well as chemical alteration of proteins from the increase in ionic strength of salts, is certainly possible.

    Muscle fibers are separated from each other by thin connective tissue called endomysium (mammals, fowl) or myocommata (fish). The endo- mysium, as well as other types of connective tissue, consists of collagenous fibers, elastin fibers, and reticulin fibers. The endomysium is elastic, and consequently may withstand small forces. Thus, i t is not surprising that extracellular ice crystal formation per se causes only minor damage to the cellular structure. The elastic nature of the sarcolemma and endo- mysium apparently prevents mechanical rupture of cells that are distorted by large extracellular ice crystals.

    In mammalian muscle, fibers are grouped into bundles (fasciculi) sur- rounded by connective tissue called periniysium. The perimysium is gen- erally thicker than the endomysium. The perimysium also surrounds the secondary and tertiary bundles, but a much thicker connective-tissue sheath, called the epimysium, surrounds the entire muscle. Undoubtedly, the elastic properties of the perimysium and epimysium are responsible for the retention of the integral muscle structure when extracellular ice crystals are present, and for reversion of the tissue to near-normal ar- rangement during thawing.

    In the above discussion, no mention was made of the fact that, imme- diately after the slaughter of an animal (including mammalian, fowl, and fish muscle), physical and chemical changes of the fibers are initiated. The most outstanding post-mortem physical alteration is the stiffening of extensible muscles. The phenomenon is termed rigor mortis. From the onset of rigor, the strength of the bondings between myosin and actin filaments increases constantly, and the extensibility of fibers is reduced (Bendall, 1960). During this period the texture of a muscle is changed from a soft tissue to a hard mass. Along with the physical changes the

  • 300 0. FENNEMA A S D W. D. POWRIE

    lactic acid content increases, and, under normal circiiinstances, the ATP concentration as well as the pH value decreases. After a period of post- rigor, exudation of fluid may occur, particularly if the p H of the muscle is much below pH 6.0. Factors influencing the amount and type of muscle alteration during freezing and thawing are the degree of rigor and the p H of the muscle.

    a. Fish. The edible portions of fish are located in the dorsal position, where large skeletal muscles occur. The musculature of fish is not as com- plicated as tha t of higher vertebrates. The lateral trunk muscles of fish consist of flake-shaped segments (myomeres) held together by connective tissue called myosepta or myocommata (Eton, 1960; Lagler e t al., 1962). Each myomere contains striated fibers with features similar to those of mammals. Some fibers are red because myoglobin is present. The fibers which terminate at the myosepta are arranged parallel to the axis of the muscle. The lengths of the longest fibers have been shown by Love (1958a) to be proportional to the length of the intact fish.

    Other than water, the major constituent of skeletal muscle in most fish is protein. I n some high-fat fish, lipids may be the predominant com- ponent. Significant amounts of nonprotein nitrogenous constituents are present in fish flesh (Simidu, 1963). Hamoir (1955) compiled the corn- positions of flesh from several types of fish. The water content of most fish varies between 75 and 80%. The majority of common fish have 15-20% protein in the flesh portions. The lipid concentration of fish flesh depends on the type of fish and the position of the flesh. Lipid values are less than 1% for cod, haddock, and whiting, but in herring, sardines, and salmon may exceed 10%.

    Fish may be frozen in the form of fillets, steaks, eviscerated products, or whole fish. A protective ice coating, called a glaze, has been advocated for prevention of moisture loss and inhibition of lipid autoxidation. Frozen fish may be glazed by dipping the frozen product into water or into water containing specific inorganic salts and gums (Pottinger and Aliyauchi, 1956).

    During the freezing of fish, chemical and physical alterations may occur in muscle. For instance, thawed muscle previously held in frozen storage for long periods will consist of two major portions: intact inuscle tissue and a fluid called drip (Stansby, 1956; Seagran, 1956; Love, 1958b; Sawant and Magar, 1961). When fish previously held in frozen storage is cooked, a taste panel can detect toughness and dryness, and in some cases off flavors. The loss of tenderness and development of off flavors in fish apparently occur during frozen storage rather than during freezing. According to Stansby (1956), cooked fish previously frozen and immediately thawed cannot bc clibtinguislied from cooked unfrozen fish

  • LOW-TEMPERATURE FOOD PRESERVATION 30 1

    by the average consumer. Love (196213) observed tha t the toughness of cod supercooled to - 15C did not increase significantly during storage for 12 days. Dyer (1951) demonstrated that, as the frozen storage period increased, the quality of cod and halibut declined. Furthermore, the qual- ity deterioration was faster a t higher storage temperatures. The cause of off-flavor development in frozen fish has been attributed to lipid oxidation. The increase in toughness of fish Acsh, paralleling the decrease in quality, has been related to a loss in protein solubility by Dyer (1951). More recent investigations (Dyer e t al., 1956; Luijpen, 1957), however, have indicated tha t soluble-protein values do not correlate with toughness a t low storage temperatures. Love (1958d, 1960) suggested that changes in soluble protein and toughness are brought about by two conipletely inde- pendent mechanisms. This investigator has suggested that the damage to cell walls may be involved in the increase in toughness during frozen storage. However, the possibility should not be overlookcd that fibrillar protein alteration could be responsible for some aspect of the quality deterioration of fish.

    Interest in the alteration of proteins in fish during the freezing process was initiated by Reay (1933). He reported that the protein in haddock muscle became less solublc during frozen storage. In frozen-fish research, all protein alterations responsible for the decrease in the solubility of proteins are designated as denaturation. The denaturation of proteins has been assessed by most researchcrs by determining the amount of fish protein which can be extracted from homogenized tissue with 5% sodium chloride. Actomyosin has been found to be the protein fraction which is denatured during frozen storage (Dyer e t al., 1950; Dyer, 1951; Sawant and Magar, 1961).

    Many investigators have reported that the solubility of fish proteins in salt solution decreases as the frozen storage period increases (Dyer, 1951; Husaini and Alm, 1955; Love and Ironside, 1958). For example, Love and Ironside (1958), using techniques to limit biological variation, found that the per cent soluble-protein values for cod fillets declined pro- gressively up to a 20-week storage period a t -14C and remained con- stant thereafter (Fig. 25).

    The rate of freezing was shown by Love (1958d) to be related to the extent of denaturation. Xikkila and Linko (1954) studied the effect of thawing rate on the protein denaturation of herring in frozen storage for 12 days a t -20C. During defrosting a t 18C, the soluble protein in fish frozen in rigor-mortis state did not decrease until after 17 hr, whereas fish frozen after the resolution of rigor niortis had a depression in soluble protein after a defrosting time of 10 hr. Love (1962b) found tha t protein was denatured less during freezing in prerigor cod than in postrigor cod.

  • 302

    40

    STORAGE TIME AT *14,wcckr

    FIG. 25. Influence of frozen-storage time on the solubility of cod muscle proteins in 5% aqueous sodium chloride. Freezing times: 0 15 hr; 48 min. (Love and Iron- side, 1958. J. Sci. Food Agr. 9, 604; courtesy of the Society of Chemical Industry, London.)

    The complete mechanism of fish protein denaturation is not yet under- stood. According to Connell (1959), myosin is a very reactive protein and can undergo aggregation during the freezing and thawing of the solution. Apparently no chemical changes occur in myosin during freezing, and Connell consequently postulated that unfolding of the protein molecules is not involved in the myosin alteration. This supposition may be extended to proteins in intact fish tissue, particularly since Seagran (1956) found no bignificant changes in the sulfhydryl content in rockfish during freezing. Connell (1960) reported that actin, isolated from actomyosin in frozen cod, had properties similar to those of actin from unfrozen cod. Thus, he deduced that the actin portion of the actomyosin complex is not impli- cated in the aggregation phenomenon. The reason for aggregation and decreased solubility of actomyosin in frozen fish has been proposed by Love (1958d, 196213). He suggested that the formation of intracellular ice crystals creates localities with high concentrations of salts and thereby denatures the proteins.

    Protein denaturation in frozen fish muscle can be inhibited by sugars, glycerol, and glaze application. Tamoto e t al. (1961) found that mono- and disaccharides are capable of inhibiting protein denaturation in frozen Alaska pollack fish. Love (1962bl found slower protein denaturation in

  • LOW-TEMPERATURE FOOD PRESERVATION 303

    frozen glycerol-treated cod muscle. According to Sawant and Magar (1961) , the loss of protein solubility was slower wlien fish muscle was coated with various types of glazes.

    An increase in concentration of free fatty acids during frozen storage has been reported in a large variety of fish by Dyer and Morton (1956), Dyer and Fraser (1959'1, and Wood and Haqq (1962). Free fatty acids were formed from the breakdown of phospholipids (Olley and Lovern, 1960) , particularly phosphatidylethanolamine and phosphatidylcholine (Bligh, 1961). Apparently, enzymic hydrolysis of lipids is responsible for the production of free fatty acids in fish tissue (Olley e t al., 1962).

    Protein denaturation in frozen cod is accompanied by a rise in the free fatty acid content of the muscle (Dyer and Morton, 1956; Dyer et al., 1956; Dyer and Fraser, 1959). Dyer and Dingle (1962) indicated that the lipid is bound to actomyosin and thus stabilizes the protein. Upon lipid hydrolysis, however, the protein becomes unstable and denaturation takes place. Further, King e t aZ. (1962) have shown that free fatty acids in the presence of myosin will form complexes and bring about protein denaturation.

    Several reports have been presented to substantiate the theory that the freezing damage to tissue can be caused by mechanical rupture of cell walls and internal cell structure during the formation of intracellular ice crystals. In particular, Love (1955) noted that fluid expressed from rapidly frozen cod fillet contained more desoxypentose nucleic acid than fluid expressed from unfrozen fish. Apparently, the sarcolemmas were ruptured by large masses of ice formed intracellularly during rapid freez- ing. Consequently, upon thawing, the D S A could escape from the broken fibers and mix with intercellular fluid.

    Love (1957, 195813, 1958c) reported that cell damage (measured by DNA technique) was extensive when cod fillets were frozen a t one of three different rates. The critical rates were expressed as the times (about 25, about 75, and 200-500 min) required for the temperature a t the center of the fillets to decrease from 0 to -5C. With the 25-min freezing rate, the cell damage was apparently due to mechanical forces developed dur- ing ice crystal expansion (Love, 1958~) . Love presumed that the lack of sufficient organic constituents on the inside cell walls was responsible for the bursting of the sarcolemmas by ice crystals. A very large ice crystal column was formed in each fiber during freezing a t the 75-min rate, and broke the cell wall because of cryohydric expansion (Love, 195813). At the very slow freezing rates (200-500 min) , cell damage, Love suggested, was probably caused by the movement of both fibers and intercellular ice crystals during temperature changes.

    b. MmzmaZs. At present, uncooked mammalian meat, such as beef,

  • 304 0. FENNEMA AND W. D. POWRIE

    pork, and lamb, is not frozen to any great extent for the retail trade. A small amount of frozen meat is marketed in such forms as ground-meat patties, steaks, and chops.

    The freezing process produces less change in mammalian meat than in fish. The primary defect of frozen mammalian meat is the exudation of drip from tissue during thawing.

    The rate of freezing mammalian meat has been found by many in- vestigators to be related to the amount of drip. Cook et al. (1926) indi- cated that the drip from thawed beef could be decreased by increasing the freezing rate. Moran (1932) found that very little drip followed when small pieces of beef were frozen in liquid air. Callow (1952) and Hiner e t al. (1945) recently provided further evidence supporting the relation- ship of freezing rate and amount of drip. According to Ramsbottom and Koonz (1939), the rate of freezing is related to the per cent drip only if the volume of meat is small in comparison with the cut surface area.

    The location and size of ice crystals have been found to be dependent on the freezing rate of meat. When beefsteaks were frozen a t -45.6OC, Ramsbottom and Koonz (1939) noted that intrafiber ice was formed. With a freezing temperature of -23.3"C, however, the ice crystals formed ex- tracellularly. Those investigators suggested that small intrafiber ice crystals, which fornied during rapid freezing, would facilitate the reten- tion of cellular fluids. Histological studies by Hiner et al. (1945) have shown that intracellular ice formation in frozen beef tissue (stored for about 24 hr) ruptures celI walls. With a freezing temperature of -17.8"C, some intrafiber ice was formed and a few fiber walls were damaged. As the freezing temperature was lowered to -40C, the rupturing of cell walls was more extensive. Those researchers suggested that an increase in fiber- wall damage reduces the amount of drip from thawed meat.

    Deatherage and Hanim (1960) found that rapid freezing (-55C) of ground and cut beef gave a small increase in water-binding capacity, whereas slow freezing ( - 15C) decreascd water-holding capacity. Fur- ther, those researchers observed that rapid freezing of beef caused a slight increase in the number of charged groups. They therefore suggested that a loosening of the protein structure might be responsible for the increase in water-binding capacity. Undoubtedly the water-binding capacity of thawed meat is intimately connected with the exudation of fluid.

    &loran and Hale (1932) presented data showing that the amount of drip increases as the temperature of frozen storage is raised. Moreover, ice crystals in frozen meat stored a t -3.1"C increased in size during a storage period of 180 days. Ranisbottom and Koonz (1941), in contrast, indicated that the amount of drip in thawed beefsteak v7as not influenced

  • LOW-TEMPERATURE FOOD PRESERVATIOX 305

    by storage temperature and that no crystal growth was noted in beef tissue stored 1 year a t -12.2" and -34.4"C.

    Several investigators have shown that the per cent drip in thawed mammalian meat depends on the pH of the tissue and the time between slaughter and freezing. Empey (1933) indicated that thawed meat with pH values above 6.3 did not exude very much fluid. Data of Sair and Cook (1938b) indicated tha t drip in thawed beef, pork, and mutton was maximum when the pH was between 5.0 and 5.2. As pH values increased, the amount of drip decreased progressively until no drip was obtained with thawed tissue a t pH 6.4. Then drip was negligible, regardless of whether the meat had been frozen rapidly or slowly. Sair and Cook con- cluded that crystal size alone cannot be related to formation of drip. I n accord with the above observations, Ramsbottom and Koonz (1940) noted that thawed beef with pH values between 6.2 and 6.5 produced only 0.7% drip, compared with 4.3% drip for thawed beef of pH 5.7-5.9.

    Sair and Cook (1938b) suggested that, although pH and the period between slaughter and freezing influenced drip, their actions were prob- ably independent of each other. They found that the per cent drip in thawed beef and pork decreased as the t h e of storage (a t 0-10C) before freezing was extended. Ramsbottom and Koonz (1940) reached a similar conclusion with beef cuts.

    According to Hankins and Hiner (1940) and Hiner et al. (1945), the tenderness of beef can be increased by freezing. They found that the ten- derness of the thawed beef was increased as the freezing temperature was lowered. Hiner e t al. (1945) concluded that the greater tenderness of beef frozen a t low temperatures was caused by the rupture of cell walls by intracellular freezing. Lee et al. (1950) indicated no significant differences in tenderness and juiciness between samples frozen a t -18" and -46C.

    c. Poultry. Several studies have shown that cooked frozen poultry meat is less palatable than cooked unfrozen meat (Hanson et al., 1942; Stewart e t al., 1943). Frozen poultry meat generally possesses less flavor and juiciness than unfrozen meat. There is no significant drip from thawed whole birds, regardless of freezing rate (Koonz and Ramsbottom, 1939; Sair and Cook, 193%). A discoloration of meat adjacent to the bones has been noted in chickens after freezing and thawing (Woodroof and Shelor, 1948). Apparently, the heme compounds in the bone migrate from the spongy bone areas when the chickens are frozen and thawed.

    The rate a t which juiciness is lost depends on freezing temperature and the duration of frozen storage (Stewart et al., 1943; Stewart et al.. 1945). Lowe (1948) presented an excellent review of the factors influcnc- ing the juiciness of poultry.

  • 306 0. FENKEMA A S D W. D. PO\VRIE

    Khan e t al. (1963) recently found that, as the time of storage a t -10 and -18C increased, the amount of soluble protein in chicken breast and leg muscles decreased. The loss of protein solubility was attributed to a change in the actomyosin fraction. Of particular interest is the fact that the number of sulfhydryl groups in the chicken meat decreased dur- ing frozen storage.

    Histological studies by several researchers have shown that the factors that influence the size and locality of ice crystals are similar in poultry meat to those in fish and mammalian meat (Koonz, 1955; Lowe, 1948).

    4, Plant Products Immediately after harvest, all fruits and vegetables deteriorate pro-

    gressively a t ambient temperatures to the point where they are incdiblc. Freezing plant tissue can inhibit deterioration by microorganisms and enzymes and help retain natural flavor. I n many instances, however, the normal structure of fruits and vegetabIes is damaged appreciably by freezing them. I n particular, the delicate cells in many fruits are sus- ceptible to mechanical rupture by ice crystals. The freezing damage to tissue may be so severe that the resulting soft, limp thawed product, wit11 excessive drip, would be unacceptable to the consumer.

    Very few research reports arc available on the influence of the freezing process on the structural disruption of edible plant products. The pioneer- ing work of Woodroof (1938) is still recognized as invaluable for under- standing freezing injury to fruits and vegetables from the standpoint of histology. Joslyn and Diehl (1952), in their review on the preservation of plant products by freezing, pointed out the lack of extensive informa- tion on histological changes. At present, the mechanism of freezing dam- age to fruits and vegetables is in a hypothetical state. With these facts in mind, i t was thought that a presentation of the general anatomy of plant tissue in relation to freezing would provide a stimulus for further scientific research in this area of food science.

    Most edible plant tissues consist of parenchyma cells, which are clas- d i e d as storage cells, along with some interlacing conducting cells. The exact shape of a parenchyma cell depends on intercellular contact anti pressures. During the growth of plant organs, parenchyma cell walls are flattened by internal forces, and the cells are closely packed. Some inter- cellular areas in parenchyma tissue are filled with gases. These areas have been termed intercellular spaces. Fleshy fruit tissue contains many large intercellular spaces, whereas seed endosperm and cotyledons have small spaces or none a t all. For example, mature apple tissue contains many largc intercellular gas spaces (Reeve, 1953), whereas the spaces in

  • LOW-TEMPERATURE FOOD PRESERVATIOS 307

    parenchyma tissue of the mature Nary bean cotyledons are relatively small (Powrie et al., 1960).

    Intercellular spaces undoubtedly are locations where ice-crystal nu- cleation and growth progress during the freezing of native parenchyma tissue. The water vapor in the large intercellular regions may condense as water on the adjacent cell walls, and, after a period, the water may be transformed into microscopic ice crystals (Tetley, 1931 ; Woodroof, 1938). Chandler (1913) indicated that the mechanism of plant death by freezing may involve the transfer of water from the protoplasm to the intercel- lular spaces where ice crystals are formed. Woodroof (1938) stated that flabbiness of thawed young stems may be due to lack of complete reab- sorption of the water which migrated from the cells to the intercellular spaces during slow freezing. Since blanched tissue generally contains intercellular spaces with cell sap (Crafts, 1944), large ice crystals would be expected in these areas of frozen tissue. According to Woodroof (1938), blanched asparagus contained much larger ice crystals than did un- blanched asparagus. As ice crystals grow in the intercellular spaces dur- ing slow freezing, the cells may become distorted and cell walls may be ruptured (Woodroof, 1938 ; MacArthur, 1948).

    Plant cells are surrounded by cellulosic protective coatings called cell walls. All parenchyma cells have a primary wall on the exterior, and some of them have an additional layer, called the secondary wall. Dif- ferentiation between the middle lamella and primary wall is sometimes impossible, so the term compound middle lamella has been used to include both the middle lamella and primary walls of two cells (Esau, 1953). The secondary wall is formed on the inner portion of the primary wall after the volume of each cell has reached a maximum. The function of the secondary wall is to strengthen the primary wall and provide re- sistance to injury. The parenchyma cell walls in fleshy fruit and vegetable tissue are generally thin and quite susceptible to disruption by small forces of ice crystals (Woodroof, 1938; TVeier and Stocking, 1949). In many seed cotyledons, in contrast, parenchyma cells possess thick cell walls, breakable only by large forces.

    Cavities in primary and secondary walls are frequently observed in parenchyma tissue (Esau, 1953). Primary walls may have definite de- pressions, which have been named primary pit fields. When a cell wall is thick, the entire cavity is called a pit. il pit in one cell wall is usually opposite to another pit in the adjacent cell wall. A pit membrane, com- posed of two primary walls and the middle lamella, closes the opening betwcen the pit pair. Sometimes a pit may be opposite to an intercellular space, with a pit membrane as the barrier. Presumably, the pit membrane

  • 308 0. FENNEMA AND W. D. POWRIE

    is semipermeable and allows low-molecular-weight cornponents and ions to migrate between cells. Pi t meiiibranes are very thin and, obviously, could be broken very easily by a slight pressure. Thus, if ice crystals are formed in the pits, they conceivably would rupture pit membranes during freezing. As a consequence, solutes in the cells could diffuse out readily, reducing turgor.

    The most common nonaqueous constituent of the parenchyma cell wall is cellulose, while other carbohydrates such as hemicellulose and pectic substances occur in smaller quantities. I n sonic areas of a cell wall, cellulose molecules are aligned in a parallel direction to the extent that these regions (micelles) have crystalline properties. On the other hand, some regions of a cell wall may have unoriented cellulose molecules, and the cellulose thus has amorphous properties (Eeau, 1953). The propor- tion of crystalline and amorphous cellulose depends on the type and ma- turity of plant cell. As the cell matures, the amount of crystalline mi- celles increases. Meyer and Anderson (1952) have indicated that small spaces separate micelles and are filled with polysaccharide. I n the primary wall of any cell, pectic substances are present in these spaces.

    From the microscopic viewpoint, cell walls consist of microfibrils which are aggregates of cellulose molecules. I n the primary wall, the fine fibrils are orientated in such a way as to form a network, and the spaces are filled with pectic compounds. Fibrils are often thicker in the secondary wall than in the primary wall.

    Water, both bound and free, is abundant in cell walls (Crafts e t al., 1949). The polysaccharides, such as pectic substances and cellulose, of the cell walls are highly hydrophilic and can bind large amounts of water. The possibility of ice crystal formation in cell walls should not be over- looked. Very small ice crystals could easily distort the well organized cell- wall fibrils, and the thawed walls may consequently lack rigidity. More- over, cold injury to the cell walls may be partially responsiblc for the migration of water from the cells into the intercellular spaces during the slow freezing of tissue.

    Simpson and Halliday (1941) reported that the cell walls of carrot tissue were thinned by steaming. Apparently the pectic substances were solubilized and migrated out of the interfibrillar spaces, since their data indicated a decrease in total pectic substances when carrot tissue was steamed. Without pectic substances as physical barriers in the inter- fibrillar spaces of steamed or blanched tissue, the development of ice nuclei during freezing would be expected, with ultimate seeding of the protoplasmic water phase to form intracellular ice crystals.

    The cell contents of a parenchyma cell, as well as of other types of cells, can be classified as protoplasiiiic and nonprotoplasmic. Protoplasinic

  • LOW-TEMPERATCRE FOOD PRESERVATIOP; 309

    components include the cytoplasm, nucleus, plastids, and mitochondria ; the nonprotoplasmic constituents are the vacuoles, crystals, starch gran- ules, and oil droplets. From the physical or textural standpoint, the food scientist is not concerned with the nucleus or the mitochondria. The cyto- plasm of a cell is the continuous ground mass which is the matrix for granules, plastids, oil droplets, and other particulate matter. Cytoplasm, a transparent semifluid, is regarded as a mass consisting of organic and inorganic constituents in solution. Proteins and polysaccharides may be constituents of the cytoplasm of some parenchyma cells such as lettuce or onion tissue. The water content of active cytoplasm has been reported to be 85-9070 (Crafts e t al., 1949). Undoubtedly, the cytoplasmic water concentration of parenchyma cells is governed by the maturity of the cells. The formation of ice crystals in the cytoplasm is dependent on the concentration of sugars and salts, the viscosity, the moisture content, and the temperature. During rapid freezing of plant tissue, small crystals de- velop within the cells (Chambers and Hale, 1932; Woodroof, 1938), and a large amount of cytoplasmic water is undoubtedly converted to ice. The destruction of the so-called colloidal complex in the plant cells, due to freezing, is probably the salting out of proteins in the cytoplasm in the presence of high salt concentrations, brought about by intracellular ice formation or withdrawal of water during slow freezing. Newton and Brown (1931) found that protein coagula of plant juice could not be dispersed after thawing.

    Plastids are protoplasmic bodies imbedded in the cytoplasm of a cell. I n most cases, plastids are located in the parenchyma cells of edible tis- sues. A thin membrane presumably envelops each plastid. Plastids are classified on the basis of the presence or absence of pigments. The color- less plastids, called leucoplasts, are the primary bodies which synthesize starch molecules t o form granules. Chronioplasts are plastids which con- tain pigments such as carotenoids and chlorophylls. The pigments may occur in a variety of shapes. The leaves of spinach and cabbage contain disc-shaped plastids, called chloroplasts. A chloroplast is composed of granules, or grana, which are organized in an orderly pattern (Granick, 1949). The granules containing chlorophylls a and b are imbedded in a proteinaceous matrix called a stroma (Esau, 1953). Many edible plant tissues contain carotenoids such as a- and p-carotene and lycopene. The parenchyma cells of the carrot root and the tomato fruit contain plastids with carotenoids in the form of crystal-like solids. Weier (1942, 1944) and Reeve (1943) reported that carotenoid-containing plastids in the carrot root are disrupted during blanching, and that the pigments beconic dissolved in the cellular oil droplets. During freezing, the carotenoid- containing oil droplets in blanched tissue may agglomerate to some extent.

  • 310 0. FENNEMA AND W. D. POWRIE

    However, no significant amounts of pigment are lost in the drip upon thawing, since all of the carotenoids are insoluble in water. Woodroof (1938) reported that very rapid freezing (at dry-ice temperature) of blanched asparagus decreased green color. The reason is unknown, but it is possible that the chloroplasts are disrupted to such an extent by intra- cellular ice crystals that the chlorophylls become susceptible to degrada- tion by heat-resistant enzymes.

    Cavities filled with a liquid cell sap have been termed vacuoles. The vacuoles are separated from the cytoplasm by a vacuolar membrane, or tonoplast. The immature parenchyma cell may have numerous small vacuoles, whereas a mature parenchyma cell usually has one large vacuole. The major constituent of the vacuole is water, and the minor constituents are, for the most part, molecularly dispersed. Sugars, organic acids, pro- teins, tannins, and anthocyanins have been found in vacuoles of paren- chyma cells (Esau, 1953). In parenchyma cells of beet root, potato tuber, and the onion bulb, the vacuoles contain such nonaqueous mate- rials as amides, proteins, and sugars. The anthocyanins of strawberries are located in the vacuoles of parenchyma cells (Blank, 1947).

    Disruption of the intact vacuoles in fresh tissue during the freezing process has been noted (Woodroof, 1938), but the nature of thc vacuole damage is not known. Apparently, not only water is removed from the vacuoles, but also the cell sap constituents. For example, the leakage from thawed strawberries contains anthocyanins and sugars (Woodroof, 1938) which occur naturally in the vacuoles of the tissue.

    Starch granules are abundant in the parenchyma cells of the potato tuber, pea seed, and corn endosperm. The rigidity of a native plant cell is dependent to a large extent on the amount of starch granules present. Actually, native starch does not bind a large amount of water, but, when plant tissues are blanched, large amounts of water are imbibed by the gelatinized granules. When blanched starch-containing tissue is frozen, even a t a slow rate, the resulting thawed product is generally firm and palatable (Woodroof, 1938; Lee e t al., 1946). It is possible that the closely packed, swollen granules inhibit excessive nieclianical damage to tissue by ice crystals.

    Within pea and bean cotyledon cells, starch granules are imbedded in protein matrices (Powrie e t al., 1960). The granular protein particlcs should be considered as nonprotoplasmic matter distributed throughout tlie cytoplasm. Protein matrices, being strong barriers, may inhibit ex- tensive gelatinization of the starch granules during heating (Veiss and Powrie, 1959). These protein matrices probably contribute to the rc- sistance to cell damage in blanched or cooked tissue during freezing and iliawing.

  • LOW-TEMPERATURE FOOD PRESERVATIOS 311

    The intercellular matter, which binds the two primary walls of ad- jacent cells, consists of pectic substances and minor quantities of other polymers (Bonner, 1950; Kertesz, 1951; Joslyn, 1962). The amount of water-insoluble protopectin and water-soluble pectic substances depends on the cell type, maturity of the cell, and storage conditions. The con- tribution of pectic substances to the firmness of tissue is not well known. Van Buren et a2. (1962) and Kaczmarzyk e t al. (1963) have shown that the firmness of canned green beans is dependent on the nature of the pectic substances.

    During the freezing of blanched and unblanched tissue, cells can be separated along the middle lamella, which is probably the area of least resistance to mechanical forces from growing ice crystals (Woodroof, 1938). According to Weier and Stocking (1949), any weakening of the adhesive forces within the middle lamella will bring about textural ch an ges.

    XII. CONCLUSIONS CONCERNING POSSIBLE CAUSES OF FROZEN FOOD DETERIORATION

    At this point an attempt will be made to draw some general conclu- sions concerning the possible means by which food deteriorates during the entire freezing process (freezing, frozen storage, thawing). For con- venience, each phase of the freezing process is considered separately.

    ,4. POSSIBLE CAUSES OF DAMAGE DURING FREEZING The removal of heat energy from foods being frozen has two basic

    effects: a decrease in temperature and a change of state from water to ice. During the freezing of any complex material, these two consequences of heat removal are inseparable. However, it is of more than theoretical interest to determine how the consequences of a decrease in temperature with a change of state of water to ice compare with the consequences of the same decrease in temperature with no change of state. This approach will enable the causes of freezing damage to be determined more accu- ratrly.

    1. T h e Effect of a Temperature Decrease lcith No Change of State

    Supercooling provides us with one means of studying the effects of a temperature decrease in the subzero (below O O C ) range without the com- plication of a water-to-ice transformation. Several studies have been made of supercooled biological materials. A study by Mazur (1960b) compared freezing with supercooling in terms of survival of microorganisms. He found near-complete survival of microorganisms supercooled to tempera-

  • 312 0. FENNEMA AND W. D. POWRIE

    tures well below their freezing point and then warmed. When, however, like specimens were exposed to the same temperature treatment but al- lowed to undergo a change of state, survival upon thawing was markedly less.

    Brown and Dolev (1963) studied the autoxidation of aqueous solu- tions of oxymyoglobin which had been cooled to -10"C, stored for various periods, and then warmed. Some samples froze a t -10C whereas others remained in a supercooled state. In all cases, autoxidation of oxymyo- globin was much less in supercooled samples than in frozen samples.

    Shell eggs which had been supercooled for several days a t -11C and warmed to room temperature were compared with eggs which had been frozen and warmed under an identical temperature program (Moran, 1924). When examined a t room temperature, the previously frozen eggs were far more viscous than normal, while the previously supercooled eggs had a normal viscosity.

    Love (1962a) measured the insoluble protein content of cod muscle which had been cooled to -1.5"C and then warmed. Some samples froze when cooled to -1.5"C, whereas others remained in a supercooled state. \Then examined following warming, the previously frozen samples con- tained larger amounts of insoluble protein than the samples which had been supercooled.

    The above studies indicate that damage to biological materials in the subzero temperature range is due to the formation of ice rather than to the temperature decrease per se. However, this does not mean that tem- perature decreases in the subzero range are devoid of any consequence other than ice formation. A subzero decrease in temperature (no change of state) decreases the kinetic energy of all molecules in the system and causes many alterations in physical, chemical, and biological properties.

    Values for surface tension and viscosity will increase with decreasing temperature. Moreover, the amount of hydrogen bonding and protein aggregation will also increase with a temperature depression. Values for vapor pressure and solubility will decrease with decreasing temperature, and so will the rates of cheinical and enzymic reactions such as acid hydrolysis, enzymic hydrolysis, metabolism, microbial growth, and oxida- tion. Rates of diffusion will also decrease with decreasing temperature. As a consequence, materials such as sugar, ascorbic acid, and glycerol will penetrate cellular structures quite slowly a t low temperatures. How- ever, if penetration is desired, it can normally be accomplished under re- frigerated conditions prior to freezing. I n any of the above cases, the ex- tent to which a property or rate is altered will be related to the magni- tude of the temperature decrease.

    Two other phenomena should be mentioned with regard to dccreases

  • LOW-TEMPERATURE FOOD PRESERVATlOS 313

    in tcinperature (no change of state) : 1) chilling injury (also called phys- iological cold injury) ; and 2 ) thcriiial shock (also called temperature shock and cold shock). Chilling injury consists of damage that occurs in certain living materials when they are stored a t relatively low but nonfreezing temperatures. For example, pineapples, avocados, bananas, citrus fruit, and many other plants of tropical or subtropical origin have optimum storage temperatures tha t are well above freezing, and will undergo chilling injury if stored a t temperatures close to freezing (Wright e t al., 1954). The low temperature apparently upsets the normal meta- bolic processes in some way, thereby causing a variety of disorders (Meyer and Anderson, 1952). Since chilling injury is confined to living materials stored above freezing, i t apparently has no bearing on the qual- i ty of food exposed to temperature decreascs in the subzero range.

    Sudden decreascs in temperature, both above and below freezing, can have a lethal effect on some types of cells. This effect is known as thermal shock. The fact that slow cooling through identical temperature ranges is innocuous lends support to the Contention tha t the lethal effect is due to sudden cooling. Thermal shock has been observed in spermatozoa, red blood cells, and bacteria (Chang and lJ7alton, 1940; Lovelock, 1954b; Sherman and Cameron, 1934). The rate of decrease in temperature neces- sary to produce thermal shock is generally much greater than is possible in most cellular food materials. Damage to the quality of frozen foods hy this iiicans is, therefore, improbable.

    In general, it can be said tha t subzero temperatures (no change of state) are responsible for little if any damage to food quality during freezing. In fact, all the benefits of preservation a t subzero tempcratures accrue froin low temperature per se. Quality darnage which does occur during freezing must therefore be caused hy the transformation of water to ice.

    2. Effects of Change of State

    The consequences of a change of state can be classified in two major categories: a ) mechanical damage arising froin changes in volume; and b) darnage caused by concentration of nonaqucous constituents.

    a. Mechanical Damage. Mechanical damage can arise from changes in volume during freezing. The nct voluine change occurring in any coin- plex system is equal to the volunie changes of all the individual con- stituents (see Section VII I ) . Water, generally the most prevalent constit- uent, expands on freezing whereas most other constituents contract. Materials containing large amounts of water and few intercellular air spaces will likely show a net expansion upon freezing-and a greater like- lihood of darnage (MacArthur, 1948; Dietrich e t nl., 1957; Borgstrom,

  • 314 0. FENNEMA AND W. D. POWRIE

    1961 ; Joslyn and Diehl, 1952). Materials containing lesser amounts of water and many air spaces will likely show little or no expansion upon freezing, and may even contract.

    Solutes can limit the expansion during a given freezing process-by hydrogen-bonding with water, by promoting prolonged supercooling, or by lowering the cryohydric point. Each of these occurrences decreases the amount of water undergoing a change of state during a normal freezing process. Nevertheless, the system will contain areas of expansion and con- traction, frequently resulting in mechanical damage.

    The extent and type of mechanical damage will depend on the nature of the product and the rate a t which the change of state occurs. Plant and animal tissues, for example, react quite differently. Plant tissue, consist- ing of rigid, unaligned cells, is subject to cell separation, cell breakage, and damage to cellular contents (Woodroof, 1938, 1944; MacArthur, 1948; Levitt, 1960). Animal tissue, consisting largely of long, pliable fibers (cells) arranged in a parallel fashion, is subject primarily to separation of fibers or intrafiber filaments, and to damage to cellular contents (Rapatz and Luyet, 1959; Meryman, 1960b). Furthermore, there is a great variation in the resistance to freezing damage of different species, types, and maturities within the plant and animal kingdonis (Cruess, 1958; Tressler and Evers, 1957a; Joslyn and Hohl, 1948). In general, plant tissue suffers greater damage to texture during the freezing-thawing process than does animal tissue.

    Rapid freezing (rapid change of state), with the formation of small, uniformly dispersed ice crystals, followed by minimum frozen storage and rapid thawing, is thought to rnininiize cell breakage and changes in tissue structure (Deatherage and Hamni, 1960; Davis et al., 1952; Fellers, 1955; Webstcr e t al., 1962; MacArthur, 1948; Tressler and Evers, 1957a; Wood- roof, 1938). However, factors other than mechanical damage may be in- volved.

    b. Concentration of ATonaqueous Constituents. The concentration of solutes and suspended materials which occurs during the transforniation of water to ice, is another factor which can lead to damage during the freezing process. Changes in such properties as pH, titratable acidity, and ionic strength are known to result from this occurrence and undoubtedly have some effect on the stability of hydrophilic colloids, emulsions, and perhaps even larger cellular and intercellular structures. The extent of these changes will vary with the product and the rate of freezing. Slow freezing allows a greater opportunity for concentration damage than will rapid freezing.

    Little is known concerning the influence of freeze concentration on the quality of frozen foods. The effects of freeze concentration on the viability

  • LOW-TEMPERATURE FOOD PRESERVATION 315

    of biological materials have been studied extensively, but divergent view- points still exist (Smith, 1961, page 425; Lovelock, 1957; Borgstrom, 1961, page 221; Levitt, 1960; Mazur, 1960b; Sloviter, 1962).

    B. POSSIBLE C.4CSES O F DAMAGE DURING FROZEN STORAGE Undoubtedly the single most important point to remember about foods

    in the frozen state is that they are not inert. Foods deteriorate during frozen storage a t a rate which depends on the temperature. The rate of deterioration will generally decrease as the temperature is decreased. De- terioration may occur through chemical or physical means but not by microorganisms, since they normally decrease in number during frozen storage (Guerrant e t al., 1953; Bennett e t al., 1954; Elliott and Michcner, 1960).

    Some typical chemical changes which occur during frozen storage of food products are degradation of chlorophyll and ascorbic acid, denatura- tion of animal-tissue proteins and lipoproteins, hydrolysis of phospho- lipids, oxidation of lipids, browning, loss of cloud in juices, and flavor deterioration (Dietrich e t al., 1960; Boggs e t al., 1960; Pierce e t al., 1955; Guadagni, e t al., 1957a,b,c; Love and Ironside, 1958; Lovern and Olley, 1962; Anonymous, 1960a; Lee et al., 1955; Saari, 1963).

    Some typical physical changes that occur during frozen storage are gelation, diffusion, and recrystallization (Moran and Hale, 1932 ; Mac- Arthur, 1948; Guadagni and Nimmo, 1957b; Saari, 1963; Powrie e t al., 1963).

    Quite a few studies have been conducted to dctcrmine the rate of food deterioration under fluctuating subfreezing storage temperatures as com- pared to comparable constant-temperature conditions (for example, a temperature cycling from -5" t o +5"F on a sine wave pattern would be considered approximately comparable to a constant temperature of 0F) . I n nearly all studies involving fruits, vegetables, poultry, fish, and meat, changes in over-all food quality or various aspects thereof were found to occur a t approximately the same rate under both storage conditions (Hus- trulid and Winter, 1943; Gortner e t al., 1948; Winter e t al., 1952; Klose e t al., 1955, 1959; Guadagni e t al., 1957a, 1958; Guadagni and Nimmo, 1958; Hanson and Fletcher, 1958; Dietrich e t al., 1959, 1960, 1962; Boggs e t al., 1960). A study by Woodroof and Shelor (1947) involving frozen fruits apparently led to conclusions contrary to the above.

    Fluctuating storage temperatures apparently cause sizable quantities of frost to accumulate on the surface of products packaged in moisture- impermeable materials, but this is relatively unimportant (Wcisman, 1956; Klose e t al., 1955).

    With bread (Walker, 1956), foods thickened with eggs and starch

  • 316 0. FENNEMA AND W. D. PO\\RIE

    (Hanson e t al., 1957), and ice cream (Somnier, 1947; Frandsen and Ar- buckle, 1961), fluctuating subfreezing storage temperatures are reportedly fa r more detrimental to quality, primarily texture, than comparable con- stant-temperature conditions.

    Returning to the natural foods originally mentioned, it is evident that the majority of investigators regard fluctuating temperatures as having approximately the same effect on food quality as comparable constant temperatures. It should be pointed out, however, tha t some investigators apply this conclusion to food quality or to the rate of food deterioration whereas other investigators are more cautious, applying it only to the specific quality attributes they measured, such as color, flavor, reduced ascorbic acid, ete.

    Considering the entire collection of articles, i t seeitis quite safe to con- clude that most quality attributes of natural foods will not deteriorate more rapidly under fluctuating subfreezing storage temperatures than they will under comparable constant-temperature conditions. However, the authors of this paper feel that there is insufficient evidence as yet to apply this conclusion to the texture of fruits. Xone of the articles prc- viously cited in regard to the effect of storage conditions on natural foods pravitles the necessary evidence, because of one or inore of the following reasons :

    1) The texture of fruit was not investigated. 2 ) The texture of fruit was investigated but only as one component

    of an over-a11 evaluation of quality. 31 The texture of fruit was investigated, but the evaluation technique

    n-as poor and/or the results were not statistically analyzed. This criticisin applies particularly to those investigations conducted in the 1940s and early 1950s.

    41 The frozen fruit was stored for tirnes insufficient to produce pig- nificant changes in quality.

    5 1 The constant temperature employcd was not comparable or nearly coinpirable to the fluctuating tcniperaturcs.

    Considering that texture is an important aspect of food quality (Szczcsniak and Kleyn, 1963) and that the texture of fruits generally u:-tl-.rgoes considerahlci damage during the freezing-thawing process (.Tor- lyn and Hohl, 1948), it is surprising that this point has not received iiiore attcntion. Until further invcstigations prove otherwise, it seems advisablc t o assume that constant subfreezing storage temperatures are best for preserving the texture of frozen fruits.

    The optimum storage temperature will depcnd on the product, how i t i> packaged, and how long it is to he stored. Most products generally requirc 0F or lon-er.

  • LOW-TEMPERATURE FOOD PRESERVATlOS 317

    C. POSSIBLE CAUSES OF DAMAGE DURING THAWING

    During thawing, food materials are subject to all the possible sources of darnage previously mentioned: exposure to concentrated liquids, re- crystallization, niechanical damage, and growth of microorganisms.

    Faster thawing reduces the opportunity for product damage. Achieving rapid thawing poses a difficult problem since: 1) thawing of normally rigid materials is inherently slowcr than freezing; 2) temperature diff crentials are frequently less during thawing than during freezing; and 3) thawing is frequently conducted by persons unaware of or unconcerned with proper thawing procedures.

    The fact that normally rigid food materials thaw more slowly than they frveze is more serious than the relative times involved would indicate. During thawing, the temperature of the product rises very rapidly to the melting plateau and remains near the frcezing point for the remainder of the prolonged thawing time (see Fig. 21) . Of all teniperatures in the subfreezing range, those near the freezing point permit undesirable rcac- tions to occur a t the niaxiniurn rate (recrystallization, niicrobial growth, and other physical and chemical changes).

    The temperature differentials during thawing are frequently subject to limitations which are not present during freezing. For example, materials which are consumed in an uncooked state, such as fruits, must be thawed a t reasonably low temperatures; otherwise, heat damage or, in some cases, excessive growth of microorganisms will occur. Recommendations for thawing whole poultry and large chunks of meat generally call for tem- peratures below 50F, since more elevated temperatures might result in surface spoilage by microorganisms.

    Education of the consumer would undoubtedly result in better thawing techniques and better-quality foods.

    Restoration of the original properties upon thawing is never complete, but foods differ in this respect. Plant tissue, with its normally rigid char- acter, is frequently somewhat flabby when thawed. Thawed animal tissue, in contrast, usually has propcrties quite similar to those of the original product.

    Fluid is quite commonly lost during the thawing of plant and animal tissue. This occurrence is known as drip or leakage. The severity of fluid loss depends on the type of product, the surface area exposed, and the conditions of freezing, storage, and thawing.

    XIII. GENERAL ASPECTS OF COMMERCIAL FREEZING PROCESSES

    This section briefly reviews the technological aspects of the frozen- food industry. A more complete discussion is given in any of several

  • 318 0. FENNEMA AND W. D. POWRIE

    books on this subject (Tressler and Evers, 1957a,b; Joslyn and Hohl, 1948 ; Rogers, 1958).

    A. METHODS OF FREEZING The freezing of a food material involves the transfer of heat energy

    froin the product to a coolant or refrigerant. The coolant may exist in one or more of three physical states: solid, liquid, or gas. Examples of solid coolants are cold metal plates (indirect freezers, such as plate freezers), solid carbon dioxide, and ice. Some liquid coolants are ice water, cold aqueous solutions, and liquefied gases (carbon dioxide, nitrogen, air) , and air is the common gaseous coolant. Combinations of the above are used sometimes.

    The three most common types of freezing systems are air blast (gase- ous coolant), indirect contact (solid coolant) , and direct immersion (liquid coolant). Each type possesses certain advantages and disad- vantages.

    1. Air Blast

    Freezing in air is economical, but it is generally slower than the other methods and will generally result in dehydration of an unpackaged food material. Moisture lost from the food necessitates frequent defrosting of the equipment. Packaging of the product will overcome the last two dis- advantages but will also slow the freezing.

    Commonly used air temperatures range from 0" to -40"F, and air velocities in the freezing chamber range from 100 to 3,500 lineal feet per minute. Tressler and Evers (1957a) have suggested that an air tempera- ture of -20F and a velocity of 2500 lineal feet per minute are practical and economical.

    2. Indirect Contact

    Indirect contact involves placing the food product against a cold metal surface. The metal surface is generally cooled by circulating a liquid coolant through the hollow cores of freezing plates or, in ice cream freez- crs, through a jacket. Packaged food materials may rest on, or slide against, a cold plate or be pressed between two plates. Liquid products such as ice cream mix arc frozen under agitation, with the cooling surface continually scraped to keep it free of frozen material. Most equipment for indirect-contact freezing is quite expensive but is capable of freezing foods rapidly.

    3. Immersion Freezing

    Immersion freezing involves immersing (or spraying) the packaged or unpackaged product in a cold liquid medium, such as liquid nitrogen,

  • LOW-TEMPERATURE FOOD PRESERVATIOX 319

    liquid nitrous oxide, liquid air, or aqueous solutions of sugar, salt, or glycerol. Heat transfer is faster than in any other freezing method availa- ble. Some properties of suitable liquefied gases are shown in Table XIY.

    TABLE XIX

    PROPERTIES OF SOME LIQUEFIED GASES SUITABLE FOR IXMERSION FREEZING OF FOOD"

    Gas

    Property Nitrous oxide Nitrogen ( O F ) ( O F )

    Boiling point (1 atm.) - 127.2 -320.5 Latent heat of vaporization a t boiling point (Btu/lb) 161.8 85.7 Sensible heat (gas to 70F in Btu/lb) 40.0 97.0

    a From Webster (1961). Technical and Process Bull. ADE 895. Courtesy of Air Reduction Sales Co.

    Since the coolant conies into direct contact with the product, the con- sequences of the interact,ion must be considered. The coolant must be nontoxic, and, ideally, should impart only those organoleptic alterations which are compatible with the food being frozen. For example, salt brine would be entirely Unsuitable for fruits but would be quite acceptable for fish. The reverse would be true of sugar solutions. Furthermore, since the product and coolant do interact, care must be taken to maintain a uni- form coolant composition (in t#he case of aqueous coolants) and to main- tain the coolant in a sanitary state.

    B. SELECTION OF FRUITS AND VEGETABLES FOR FREEZING The suitability of fruits and vegetables for freezing varies greatly

    with the type and variety. Some types of fruits and vegetables, such as watermelon and celery, are totally unsuitable for freezing, regardless of variety. However, most types can be frozen successfully if the proper variety is selected. The conditions under which varieties are evaluated for freezing quality should be carefully conttrolled. A variety which is desira- ble under one set of growing and processing conditions may not be de- sirable under a different set of conditions. Care should therefore be taken to grow and process all test varieties under the commercial conditions anticipated.

    The optimum maturity for freezing fruits and vegetables is generally the same as for table use.

  • 320 0. FENNEMA A S D W . I). POWHIE

    C. PREFREEZING TREATMENTS

    All food materials require some sort of processing prior to freezing. I n addition to such coinmon operations as washing, grading, cutting, and packaging, most major classes of food materials require special prefreezing treatments if the original quality is to be retained to a maximum extent during freezing, frozen storage, and thawing. The more important special treatments for each class of food material will be discussed.

    1. T'egetables

    Most vegetables should be heated for a few ininutes in hot water or steam prior to freezing. This process is comnionly referred to as blanching. Blanching serves primarily to inactivate enzymes which would otherwise cause undesirable alterations in color, flavor, texture, and nutritive value (loss of vitamins A, B,, B2, C) during subsequent freezing, storage, and thawing. Blanching has no detrimental effect on vegetables, since nearly all vegetables are cooked before being eaten.

    Good sanitary practices should be observed throughout tlie cntire process, particularly with regard to maintaining tlie blancli water and postblanching equipment in a satisfactory bacteriological state. The product should be cooled promptly after blanching. Failure to observe these precautions can result in extremely high bacteria counts and poor product quality (Splittstoesser e t nl., 1961a,b).

    2 . Fruits

    Fruits contain enzynics, which will causc undesirable eff ccts if not controlled. Of particular concern are tlic polyphenoloxidases, which cata- lyze the reaction betwecn atmospheric oxygen and phenolic substanccs. Thib reaction, because of its enzymic nature and the brown color pro- duced, is commonly referred to as enzymic browning.

    Since nearly all fruits are consunied uncooked, control of enzymic hrowning by blanching is generally unacceptable. However, a few fruits, such as apples, apricots, and peaches, can be carefully blanched without undergoing objectionable changes (Tressler and Evers, 1957a ; Guadagni and Nimmo, 1957a). In fruits which cannot be blanched, enzymic brown- ing is controlled with additives which either inhibit thc activity of tlie enzymes or minimize contact of oxygen with the phenolic substances.

    Sulfur dioxide and acids bucli as citric and malic all act to inhibit tlic activity of the polyphenoloxidases. Sulfur dioxide apparently functions by reacting with the enzyme, whereas the acids function by lowering the pH to a value which is less suitable for the activity of the polyphenoloxi-

  • LOW-TEMPERATURE FOOD PRESERVATIOS 32 1

    dascs (Ponting, 1960). Use of sulfur dioxide is frequently avoided in fruits which are not cooked prior to consumption, since a chemical flavor can sometimes be detected.

    Ascorbic acid is an extremely effective agent for controlling enzymic browning, as well as being a valuable nutrient. The antibrowning capa- bility of ascorbic acid apparently arises from: 1) its role as a reducing agent: and 2) its ability to inhibit polyphenoloxidases when conditions are suitable (Ponting, 1960). It has only a slight influence on pH. As a reducing agent, ascorbic acid helps maintain the phenolic substances in a reduced and colorless state. If present in an adequate concentration, the oxidized ascorbic acid will inhibit polyphenoloxidases. For extended effectiveness against browning, it is essential tha t ascorbic acid function in both capacities. Usually adequate is a fraction of one per cent of as- corbic acid, based on the weight of the syrup.

    Sugar or sugar syrups serve several valuable functions: 1) they con- tribute a desirable sweetness; 2) they help retain volatile aromas; 3) they lower the cryohydric point and thereby minimize volume changes during freezing; and 4) they inhibit enzymic browning. The decrease in enzymic browning results primarily from the action of sugar syrup as a barrier to oxygen transmission (Ponting, 1960).

    3. Meats

    Beef, veal, pork, and lamb require no special treatments prior to freez- ing other than assuring that cooling takes place rapidly following slaugh- ter. Aging of beef prior to freezing is considered desirable. For special uses, beef frozen in a prerigor state is soinetimes desired.

    4. Fish

    Fish is one of the most perishable classes of food materials, being highly susceptible to spoilage by psychrophilic bacteria and by its own enzyiiie system (Nikkilii and Linko, 1954). Good sanitary practices and prompt freezing are especially important if the initial fresh quality is to be retained to a maxinium extent. Prior to freezing, fish are sometimcs immersed for a few seconds in a 67. sodium chloride solution. This helps reduce drip during thawing (Stanshy, 1956).

    5. Poultry and Eggs

    Toughness of poultry which has passed through a freezing, thawing, and cooking sequence can be minimized by aging in ice slush for several hours prior to freezing (Winter and Funk, 1960; May e t al., 1962; Rogers, 1958; Klose e t al., 1956). However, the same effect can be achieved by

  • 322 0. FENNEMA AND W. D. POWRIE

    holding the poultry a t chilling temperatures following thawing (Klose e t al., 1956). If chilling following thawing is assured, then prefreeze aging can be shortened or eliminated, depending on the duration of post-thaw chilling.

    Freezing of untreated whole eggs or egg yolk causes the thawed product to be undesirably gelatinous. Sodium chloride or sugar (5-10%) is usually added to overcome this defect.

    D. THE INFLUENCE OF FREEZING RATE ON QUALITY When examined immediately after freezing, food tissue which has

    been frozen very rapidly will generally appear more like the original un- frozen commodity than food tissue which has been frozen quite slowly (Dyer, 1951; Dykstra, 1956; MacArthur, 1948; Joslyn and Hohl, 1948). If thawed following minimal frozen storage, rapidly frozen samples will generally exhibit less loss of fluid (leakage or drip) than slowly frozen samples (Deatherage and Hamm, 1960; Webster et al., 1962; Woodroof, 1938), and the superiority of the rapidly frozen sample will usually still be evident. As the time of normal frozen storage is extended, the differ- ences due to rate of freezing gradually disappear, and if the samples are cooked prior to examination the differences may no longer be detectable (Dyer, 1951 ; Dykstra, 1956; Tressler and Evers, 1957a; MacArthur, 1948; Cruess, 1958; Lee e t al., 1946, 1949, 1950; Rogers, 1958).

    In frozen desserts such as ice cream, very rapid freezing is essential in order to produce an acceptable texture (small ice crystals). Frozen poultry is improved in appearance if freezing is rapid (Loy, 1956). In any case, all products should be frozen a t a rate rapid enough to inhibit microbial growth and enzymic and other chemical reactions to the point that no noticeable changes in quality occur from these causes during freezing.

    The question inevitably arises as to what constitutes the optimum commercial rate of freezing. A satisfactory answer to such a question is difficult since many complicating factors are involved. Freezing is always followed by frozen storage, which is usually followed by thawing and often by cooking. As a consequence, the benefits of a given freezing tech- nique should be evaluated following the sequence of events which normally prevail between freezing and final consumption. The benefits of ultra- rapid freezing are frequently dissipated during normal commercial storage and thawing. On the other hand, damage inflicted during freezing can- not be undonc, regardless of how well the remaining operations are con- ducted. The rate of freezing should therefore be compatible with the product, the anticipated time and temperature of storage, and other conditions which precede final consumption.

  • LOW-TEMPERATURE FOOD PRESERVATIOS 323

    E. STORAGE

    Food materials arc never completely frozcn during normal coin- mercial storage, nor are they inert. Deteriorative chemical and physical changes occur continually, a t a rate governed by the storage temperature and the type of product. Generally, as the storage temperature of the product is raised the rate of deterioration increases. However, a t any given storage temperature, large differences are found in the rate a t which various products deteriorate. An indication of these differences can be gained from Fig. 26. The average time that a product remains in good condition a t various storage temperatures is of niore direct value in establishing commercial storage conditions. Tressler (1960a,b) has coin- piled this information for an extensive number of products, and the re- sults are presented in Table XX.

    Foods are not subject to deterioration by microorganisms during proper frozen storage, since cven psychrophilic organisms cease growing a t temperatures lower than approximately 14F (Elliott and Michener, 1960). Pathogenic food organisms arc of even less concern, since tempera- tures of 38F or higher are necessary for their growth (Schmidt e t al . , 1961 ; Elliott, 1963). These temperature-growth relationships are shown in Fig. 27.

    During frozen storage, the viability of mixed cultures of microorgan- isms such as found on natural foods will either remain constant or decline slightly. ,4s the storag- temperature is lowered from 20F, a larger per cent of the organisms will usually survive frozen storage of a given dura- tion (Elliott and Alichener, 1960).

    Ideally, the desired storage time should be dcterniined for each product and the storage temperature adjusted accordingly. This is obviously im- practical, since an excessive number of separate storage and marketing facilities would be required. The problem has been met by selecting a storage temperature which is reasonable for all products and adjusting the storage time of each product accordingly. A temperature of 0F or less is generally selected, since i t allows a reasonable storage time for most products. Temperatures well below 0F arc generally recommended for the more perishable or unstable products, such as fish and ice cream, or if long storage times are anticipated.

    Some of the more recent references dealing with storage times and temperatures for frozen fruits are Kulp and Bechtel (1962), Winter et nl. (19521, Chadagni et al . (1957b, 1958, 19601, and Guadagni and Niniiiio (1957a,b, 1958). Siniilar references for frozen vcgetables are Lindquist et al. (19501, Boggs et trl. (1960), and Dietrich e t al. (1959, 1960, 1962 1 . Similar rtiferenccs for frozen meats are Weisman (1956) and Anonynious

  • 324

    --

    10-

    0. FENNEMA AND W. D. POWRIE

    \

    0 I.

    (well packaged)

    \\\

    , 1 I I ,

    TEMPERATURE O F

    30

    0.3-

    Strawberries

    0 5 10 15 20 25 TEMPERATURE O F

    FIG. 26. Relative stabilities of frozen foods. The lines for poultry, vegetables, and intact fruits represent the average times a t various temperatures to develop the first detectable change in quality. Thc data for beef, pork, fish, and heat-treated orange juice concentrate represent the work of several investigators using somewhat different evaluation techniques. These lines generally depict changes in quality which are somewhat greater than the first detectable change. (Shepherd, 1960; courtesy of the Agricultural Research Service, U. S. Department of Agriculture.)

  • LOW-TEMPERATURE FOOD PRESERVATION 325

    TABLE XX STORAGE LIFE OF FROZEN FOODP

    AVERAGE LIFE IN "GOOD CONDITION" FOR PRODUCTS PI~OCESSED A N D PACKAGED IS A NORMAL MANNEK

    Product - 10F

    Apricotsh Asparagus Beans, green Beans, Lima Broccoli Brussels sprouts Cauliflower Corn, on coh Corn, rut Carrots Fish, fatty Fish, lean Lobsters Peaches * Peas Raspberries, sugared Spinach Strawberries, sliced Beef

    Roasts, steaks Cubed, small pieces Ground

    Roasts, chops Thin cutlets, ruhes Ground

    Veal

    Lamb Roasts, chops Cubed Ground

    Roasts, chops Ground, sausage Pork or ham, smoked Bacon

    Unsalted Salted

    Variety meats Beef or lamb liver, heart Veal liver, heart Pork liver, heart Tongue Kidneys Sweet breads Brains Oxtails Tripe Spiced sausage or

    Pork

    Lard

    deliratessen meats

    Months at :

    0F + 5F +lO"F 24

    16-18 lfi-18

    2'4 34 6-1 8 24 2- I4 36 36 0-12 4-16

    10-12 24 24 24 24 24

    18-24 8-12 8-12

    14-16 14-16 8-12

    14-16 8-10 24 24 6-8

    10-12 8-10

    18-24 14-16

    18 14-16

    18

    12-14 10-12

    8

    10-12 8-1 0

    6

    12-14 10-12

    8

    6-12 4

    5-7 3

    3-6 1-3

    4 3 2 4 3 1 1 4 1

    2-3

    " From Tressler (l960a,b). Frozen Food Almanac., Quick Frozen Foods. Courtesy of F. Mi. Williams Publications.

    Contains ascorbic acid.

  • 326 0. FENNEMA A S D W. D. POWRIE

    FOOD POISONING

    ? RAPID GROWTH RATE i SLOW GROWTH OF SOME TYPES

    TiYCHROPHILIC ORGANISMS

    I

    RAPID GROWTH RATE 1 SLOW GROWTH

    OF SOME TYPES

    -SLOW DEATH, RARELY COMPLETE-

    ZONE OF SLOW SPOILAGE WTHOUT

    DANGER TO HEALTH

    FIG. 27. Effect of low temperature on the growth of inicroorganisms important in frozen foods. (Elliott and Michener, 1960; Elliott, 1963; courtesy of thc Agricultural Research Service, U. S. Department of Agriculture.)

    (1960b). Similar references for frozen fish are Dyer (1951), Butler (1956), Dassow (1956), Holston (1956), Pottinger (1956), and Borgstroni (1961- 1962). Similar references for poultry are Loy (1956), Hanson et al. (1957, 1959), and Klose e t al. (1959). Similar references for cereal products are Kalker (1956), Pence and Heid (1960), and Bechtel (1963). Recommen- dations for storing frozen orange juice concentrate are presented by Mc- Collocli e t al. (1957). Some additional general references are Tressler and Evers (1957a,b), Dykstra (1956), Anonymous (1960a), and Jul (1963).

    Fluctuating storage temperatures should generally be avoided. Al- though the chemical properties of food materials are usually not damaged by fluctuating storage temperatures, the physical properties may be. This is particularly true of products with unstable textures, such as ice cream, gels, and many plant tissues. Attainment of a uniform temperature poses no particular problem in a well-opcrated warehouse. However, the same cannot be said for the conditions which prevail during shipment and dur- ing storage in retail cabinets and home freezers. The remarkable non- uniformity of storage temperatures in properly operated retail cabinets was well established by Peterson (1961). He studied the temperature history of a series of small cans of water properly stored in a connner-

  • LOW-TEMPERATURE FOOD PRESERVATION 327

    cially available retail cabinet, operating a t thc lowest possible tempera- ture setting. The cabinet was equipped with an automatic defrost system, preset by the manufacturer to operate for a 1-hr period once every 24 hr. The results are shown in Fig. 28. These data should leave no doubt as to

    FIG. 28. Temperature history of small cans of water stored in a frozen food retail cabinet. Photographs show cabinet, row of cans which contained thermocouples (ar- row), and the location numbering system. (Peterson, 1961 ; Z n Proceedings Low- Temperature Microbiology Symposium; courtesy of Campbell Soup Company.)

    the variable tcmperatures that most frozen foods experience during marketing.

    According to Kaess (1961), most natural foods and many processed ones can develop a defect known as freezer burn during frozen storage. The prirnary causc has been shown to be sublimation of ice from the product surface (Tressler, 1935). Freezer burn is particularly noticeablc on the surface of poultry, where i t appears as unattractive dry, brown spots. Prevention is usually achieved by packaging the product in a ma- terial which is quite impermeable to water vapor.

    F. THATVIKG It is to the food processors benefit to have frozen foods thawed in a

    manner that provides maximum retention of quality. This not only re- quires explicit instructions on the retail package but also a general edu-

  • 328 0. FENNEMA AKD W. D. POWRIE

    cational effort to inform consuiners of the need for good thawing tech- niques.

    Spoilage of food by microorganisms, particularly psychrophiles, can occur during and after thawing if proper precautions are not observed. It is true that freezing kills a great many microorganisms, but in no case does i t come close to having a sterilizing effect. Quite variable values have been reported for the survival of psychrophilic microorganisms fol- lowing freezing and immediate thawing, but a value of approximately 50% can be regarded as typical (Elliott and Michener, 1960). Additional organisms often die during frozen storage, but a sufficient number always remain to promote spoilage during and after thawing if the time-tempera- ture conditions are suitable.

    Deteriorative chemical and physical changes will also occur during and after thawing. Higher tcinperatures will normally be accompanied by faster rates of change, but some exceptions to this rule occur in the temperature range involving the change of statc.

    Most vegetables can generally be thawed and cooked by placing tlic frozen material directly in boiling water. Since high temperatures arc detrimental to the quality of most fruits, thawing must be carried out a t moderate temperatures (room temperature is common) . Quality is inaxi- muni when thawing is just complete. If consumption is delayed much bc- yond this point, a decrease in quality will frequently he evident. Small cuts of frozen meat, fish, or poultry can be cooked without a separate thawing step. Larger pieces cannot be treated in this nianncr, since the outer surface will usually become overcooked before the interior thaws. It is best to thaw such pieces in a refrigerator a t temperatures below 50"F, or in cold water. Thawing at temperatures ranging from room temperature to 120F should be avoided since surface spoilage may occur before thawing is complete.

    Dielectric and microwave heating have received some attention as possible commercial means of thawing foods. Providcd the food niaterial is reasonably hoiiiogenc~ous, dielectric or microwave heating will enable more rapid and more uniform heating than is possible by thermal con- duction (Copson, 1962 ; Cable, 1954). Unfortunately, foods being tliawed are not reasonably homogeneous, since frozen and unfrozen phases exist simultaneously. These phases heat a t markedly different rates when placed in a dielectric field, which often leads to localized overheating before all areas have thawed. Proper adjustment of operating conditions and careful selectioii of product geometry and product orientation help minimize this difficulty.

    Jason and Sanders (1962a,b'l have thawed fish satisfactorily by di-

  • LOW-TEMPERATCRE FOOD PREHERVATIOS 329

    electric means. Srveral applications for microwave thawing have been cited by Copson (1962).

    XIV. CLOSING COMMENTS

    A great many occurrences associated with the freezing, storage, and thawing of foods have been cited. However, relatively few comments have been made concerning the effect of these occurrences on the quality of food. The reason for this shortcoming is quite simple: in most cases the specific effects and mechanisms are unknown. Even such common and widely known rules as lowering the temperature decreases the rate of reaction, must be re-examined in the case of frozen foods, since we have seen that sonic reactions will occur more rapidly just below the freezing point than they will just above the freezing point. Furthermore, such an important question as HOW are foods damaged during freezing, storage and thaw- ing? cannot be answered other than by listing factors which arc likely to be involved. Obviously, if we do not know how foods arc damaged, we are in a poor position to combat the damage. Research efforts must pro- vide answers to these problcms if the general quality of frozen foods is to be improved a t the fastest and most economical rate.

    The first step in solving these complex proBlems involves farniliariza- tion with the fundamental factors involved. That has been the primary objective of this paper, and the authors earnestly hope that it has been adequately accomplished. The next step involves determination of specific problem areas. These are easily discovered by starting with a few broad generalities and gradually considering more detailed aspects until state- mcnts can no longer be made with reasonable accuracy. Deterioration of foods during freezing, storage, and thawing can be treated in this manner.

    Even though proper freezing and frozen storage will preserve foods better than any other cornrnercial method, some damage does occur. It is highly unlikely that low temperature per se has anything but a desirable effect. Damage, thcrefore, must result directly or indirectly from the change of state. Direct consequences of the change of state involve volume changes and mechanical damage. Indirect consequences of the change of state involve concentration of all nonaqueous constituents, associated with alteration of many physical and chemical properties. At this point we are left with a long series of partially or totally unanswera- ble questions :

    1) To what extent is food quality affected by the change in volume which accompanies the change of state? T o what degree does damage from this source vary with the type of product, rate and direction of

  • 330 0. FENNEMA AND W. D. POWRIE

    change of state, and the temperature range involved? How can volume changes be minimized?

    2) To what extent is food quality affected by the concentration of nonaqueous constituents which accompany the change of state? To what degree does damage from this source vary with the type of product, rate of change of state, and the temperature range involved? How can damage from this source be minimized?

    3) How do various product characteristics influence susceptibility to damage during freezing, storage, and thawing, and how can these char- acteristics be favorably modified? Some factors to consider under this heading would be the amount and nature of constituents such as water, carbohydrates, proteins, and gases; bound (unfreezable) water and the means for increasing i t ; the type of physical systems present; structural characteristics, such as cell size, shape, strength, pliability, nature of the cell wall and contents, nature and amount of intercellular substances, arrangement of cells, space between cells, etc. ; other physical and chemi- cal properties such as pH, ionic strength, type and character of buffer systems, etc.

    41 To what extent is fruit quality affected by ice crystal size, shape, location, purity, and enlargement and dislocation during storage?

    51 What relationship, if any, exists between conditions affecting sur- vival of biological materials during freezing, storage, and thawing as com- pared to conditions affecting the quality of food materials during the same process?

    6) Do gas hydrates have any useful applications in the frozen-food field? For example, can they be employed advantageously for commercial freeze concentration of fruit juice, or can they be used to supplement or replace traditional freezing processes?

    7) What additional prefreezing treatments can be employed to reduce quality deterioration during freezing, storage, and thawing?

    8) What are the mechanisms and best means of controlling some of the typical changes which occur in frozen foods, e.g., gelation of egg yolk and insolubilization of proteins?

    hfany more additions could be made to the above list, but these illus- trate the abundance of unsolved problems. Some of the questions posed above have received the attention of a few investigators, and this list is not intended to minimize the importance of their contributions. However, in every case the answers are incomplete and in many cases totally un- known.

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  • LOW-TEMPERATCRE FOOD PRESERVATION 33 1

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