Z. Ern~hrungswiss.

16, 167-175 (1977)

9 1977 Dr. Dietrich Steinkopff Verlag, Darmstadt
ISSN 0044-264 X

Federal Research Centre ]or Nutrition, Karlsruhe

Some aspects of protein changes in frozen foods

W. P a r t m a n n

With 4 figures

(Received March 26, 1977)

Introduction
For some years the discussion has been going on in G e r m a n y about
the alterations in proteins of foods which are caused by freezing and
frozen storage. Before a well-founded judgement can be made on several
categories of quality changes of such frozen products, particularly f r o m
the standpoint of nutrition physiology, we should know about w h a t
actually happens with the proteins. In this paper therefore the attempt
shall be made to gain, from results hitherto obtained by m a n y researchers,
some insight into the causes underlying these protein changes, and into
the n a t u r e of these phenomena, generally referred to as denaturation
caused by freezing.

Definition and measurement of protein changes

The present conception of protein denaturation is substantially based
on the definitions by N e u r a t h and K a u z m a n n (1). According to a recent
and more comprehensive definition, denaturation is any process that alters
or destroys secondary or tertiary structures in a biopolymer without
breaking covalent linkages to carbon atoms (23). Intermolecular aggre-
gations are not necessarily involved; they may, however, occur at the
same time or subsequently.
Freezing is one of the m a n y influences under which proteins m a y be
changed. Several methods are available to measure the alterations that
proteins undergo in frozen foods; they all comprise the following criteria:
1. Loss of specific biological activities; e.g. changes of enzyme activity
and loss of contractibility of muscle fibres in several kinds of meat.
2. Alterations of physical-chemical properties such as changes in viscosity,
texture, ultrastructure, waterbinding capacity, solubility, isoelectric
point, absorption in the UV range, optical rotation dispersion, sedimen-
tation coefficient in the ultracentrifuge, and electrophoretic properties.
A p a r t from the fact that the physical, chemical and sensorial data,
scatter over wide ranges due to inevitable biological variations, it
is generally impossible to decide by the results of individual determina-
tions w h e t h e r the measured changes are indeed intramolecular in the
sense of true denaturation, or w h e t h e r they are m e r e l y caused by aggre-
gation.
496
168 Zeitschrift fi~r Ernghrungswissenschaft, Band 16, Heft 3 (1977)

Consequences of freezing of water

The disappearance of liquid w a t e r was found to be one of the main
reasons for the alteration of proteins during freezing. P a r t i c u l a r l y the
b r e a k d o w n or change of the structure of the so-called "ordered water" in
the vicinity of hydrophobic groups of protein molecules is supposed to
lead to a change of the native protein structure (2, 3, 4). Such con-
formational changes m a y be supported by the concomitant influence of
various factors such as electrolyte concentrations, semipolaric compounds
like phenylalanine and p h e n y l p y r u v a t e (5), and pH changes (6). In extreme
cases a d e n a t u r a t e d protein m a y exist as a r a n d o m coil with all of its
functional groups exposed. In this situation it is v e r y reactive and can
take part in the formation of new intra- and intermolecular crosslinks.
Certain SH-groups seem to be of special importance for the intramolecular
changes but also for intermoIecular aggregations. According to Connell,
aggregations of this kind can occur without evidence of structural chan-
ges (48). The formation of intramolecular SS-bonds by freezing for steric
reasons seems to be possible only when the molecules have reached a
certain size (7).
Small protein molecules generally found among the albumins are
supposed to also have a small n u m b e r of hydrophobic groups. Hence it is
understandable that m a n y of the proteins resistant to freezing belong to
the albumins (7). M a n y globulins, on the other hand, are h i g h l y susceptible
to freezing or at least to aggregation, as is the case with myosin in
solution (8, 9, 10).
Numerous studies on isolated and purified proteins in solution, e.g.
myosin (8, 9, 10, 11) and actomyosin (12), show that dissolved proteins are
more apt to alterations b y freezing than in situ, w h e r e apparently other
compounds present in the tissue have a protecting effect.

Changes of enzyme activity

A m o n g the proteins, considerable interest was devoted to enzymes in
frozen foods. Whereas some enzymes retain their full activity, some show
a decrease and others a striking increase (13).
As an example for such an "activating effect" of freezing the enzymatic
b r e a k d o w n of the acid-labile energy-rich phosphate (mainly ATP) in thin
muscle strips of carp,, w i t h d r a w n immediately after death shall be pre-
sented in dependence upon t e m p e r a t u r e in the range from 9 20 ~ to
- 25 ~ The phosphates were split more quickly at - 2 ~ than in unfrozen
muscle at W 10 ~ (Fig. 1). It is assumed that such increases of activity as
found for other systems, too (14, 15, 16), are generally due to removal of
the natural b a r r i e r between enzyme, substrate or activator. This means
that the separating m e m b r a n e s are affected by freezing. In the molecular
domain these changes take place mainly in the lipoproteins of the
m e m b r a n e o u s system.
The difficulties arising in the freezing of fresh milk and eggs are also
attributable mainly to the lability of lipoproteins. In fish meat and meat
from w a r m b l o o d e d animals the m e m b r a n e o u s systems are changed b y
 Partmann, Some aspects ol protein changes in ]rozen 1oods 169

% initiot - p
I00
/J
r~

i
60 i

,~0-

20

0
920 .70 0 - lO - 20 -30
Temperoture =C

Fig. 1. Contents in acid-labile energy-rich phosphate (Np) of two carps (X andO)

at various times post mortem (. 18 hours, 24 hours, and
...... 48 hours) in dependence on temperature.

f r e e z i n g ; t h e i r d a m a g e is of less immediate i m p o r t a n c e , h o w e v e r , for t h e

s e n s o r i a l q u a l i t y of t h e f r o z e n m e a t .

T h e s o - c a l l e d " d e n a t u r a t i o n " of f i s h m u s c l e p r o t e i n s

General viewpoints:
A g r e a t m a n y scientific s t u d i e s o n p r o t e i n c h a n g e s of f r o z e n foods w e r e
c o n c e r n e d w i t h fish. T h e m e r e a d e q u a t e f r e e z i n g of fish m u s c l e s e e m s to
b e of n e g l i g i b l e i m p o r t a n c e for t h e s o l u b i l i t y (20) a n d w a t e r s o r p t i o n
a b i l i t y (21) of fish m u s c l e p r o t e i n s . W h e n t h e l i m i t s to s t o r a g e t i m e s

100 ~O

_.q

9O 30
Z
~ 1 7 6~
oo ~

10 2

Texlurescore
Fig. 2. Relation between consistency and extractability of the myosin fraction D,
and between consistency and contractibility of the muscle fibres . obtained
from slices of coalfish (Gadus virens L.) stored for different times at - 8 ~
- 18 ~ and - 30 ~
170 Zeitschri$t ]i~r Ern~ihrungswissenscha]t, Band 16, Heft 3 (1977)

determined in time-temperature--tolerance studies have been exceeded,

v e r y undesirable textural changes appear in m a n y fish species which m a y
lead to complete toughening. At least in the t e m p e r a t u r e range above
- 2 9 ~ a fairly reliable correlation between protein extractability and
toughness of the meat seems to exist (17, 18, 19) (Fig. 2).
During frozen storage the alterations of proteins generally increase
with higher temperatures and extended storage times. For m a n y proteins
the t e m p e r a t u r e range between - 2 ~ and - 10 ~ is particularly critical.
Several m a x i m u m reaction rates for the formation of undesirable com-
pounds related to protein changes, like f o r m a l d e h y d e (Alaska pollack
around - 10 o) fall in this range (22).
Research over the past t h i r t y years has been able to show that the
decrease of protein extractability is due to alterations of the myofibrillar
proteins. Until now the role o f the individual proteins has not been
completely elucidated. Cross-links between elements of the sarcoplasmic
reticulum and fibrillar proteins m a y also occur. It is generally held that
the sarcoplasmic proteins do not undergo significant alterations which
might be responsible for textural changes.
It is obvious that in fish meat, like in other tissues, the freezing of
w a t e r and the correlated concentration of polar and semi-polar com-
pounds m a y change the conformation in certain areas of fibrillar protein
molecules, w h e r e b y particular aggregation reactions m a y be initiated (23).
This alone does not explain yet the different rates of protein changes in
frozen fish of related fish species with similar gross chemical composi-
tion (24),

Free l a i t y acids:
Dyer and his groups concluded from their first studies on frozen fish
that fish species with lower fat contents had a higher reaction rate of
protein changes than species with higher fat content (25). F u r t h e r experi-
ments gave rise to the assumption that free f a t t y acids support protein
denaturation, whereas n o n - h y d r o l y z e d fats or lipoprotein complexes have
a stabilizing effect on proteins (26).
Olley et al. (27, 28) found that the main source of free f a t t y acids in
m a n y fish species were phospholipids which, so the authors assumed,
particularly originated from the membraneous system of the muscle
fibre. The results of this group and of other researchers (18, 29, 30, 31,
32, 33) suggest that mainly verY specific free f a t t y a c i d s and above all the
oxidation products of u n s a t u r a t e d f a t t y acids react with the fibrillar
muscle proteins r a t h e r than the total of all free f a t t y acids.
Recently Sikorski et al. (24) emphasized that free fatty acids m a y bind
polypeptide side chains under formation of intermolecular hydrophobic-
hydrophilic or hydrophobic-ionic linkages. This process is favoured by
appropriate concentrations of inorganic ions. That hydrophobic adherences
are mainly involved in the aggregation of flish proteins during frozen
storage follows from the observation that solubility in sodium dodecyl
sulphate (SDS) not breaking covalent cross-links decreases only slightly
with the time of storage (34).
 Partmann, Some aspects of protein changes in frozen foods 171

Oxidation products of lipids:

The products of lipid oxidation have been well k n o w n to produce
aggregation of proteins, hence causing a decrease of solubility. According
to recent ultrastructural and chemical studies, linoleic acid hydroperoxides
were about 10 times more effective in decreasing the protein solubility in
KC1 solutions of incubated cod myofibrils than linoleic acid (33). This
explains w h y fibrillar proteins in the surface layers of frozen blocks of
cod fillets deteriorated much more rapidly during frozen storage of four
years than those in deeper layers. This became manifest not only in the
loss of extractability, but also in the decrease of contractibility of the
muscle fibres (55).
Formaldehyde:
A m o n g the substances contributing to protein changes of fish meat
during frozen storage, certain aldehydes like malonaldehyde, which are
formed during the autoxidation of fats, are of importance (35, 36). For
some years formaldehyde originating, besides dimethylamine, from the
enzymatic b r e a k d o w n of trimethylamine oxide in certain fish species, has
been the subject of intensive studies (54). Castell et al. found that the
enzyme capable of formaldehyde formation and largely concentrated in
the red lateral body muscle and blood (56) was highly active in gadoid
species, but not in non-gadoid ones. In such species like halibut, wolffish
and ocean perch the solubility changes of the proteins during frozen
storage were considerably slower, and the values of the extractable
proteins became not as low as in the gadoid fish (37).
Model experiments showed that among the proteins of cod muscle
homogenates h e a v y meromyosin and tropomyosin were completely
inextractable in buffered 5 % NaC1 solution after reaction with formal-
dehyde of 0.04% concentration. Actin, on the other hand, was least
reactive (38). It is not surprising that formaldehyde concentrations of this
order of magnitude lead to a time-dependent inhibition of the mainly

t
o 0.05 % HCHO
l I I/ x ~02
oH,2% HCHO
1

~
Q

! 2c
~'~

0.1 0.2 0.4 0.60.81 4. 6 810 20 40 6080"I00

Time of action of HCHOin hrs.
Fig. 3. ATP-splitting in ground muscle tissue of carp in dependence on reaction
time with formaldehyde.
172 Zeitschrift 5fir Erndhrungswissenscha~t, Band 16, He]t 3 (1977)

50

4 0 84

2~2e3 0
a

o 20

I I I
I0~" 1 2 3 ~ 5 6 7 8
Storage time [veeks]

Fig. 4. Changes in the extractable myosin fraction in comparable parts of

5 cods (Gadus morrhua L.) stored in evacuated bags at - 8 ~ ~ minced slices;
O intact slices.

m y o s i n - b o u n d ATPase system as observed in own e x p e r i m e n t s w i t h

ground muscle tissue of carp (Fig. 3) (39). During frozen storage of corn-
m i n u t e d fish m e a t f o r m a l d e h y d e and DMA are produced f a s t e r t h a n in in-
tact muscle tissue, leading to a higher r a t e of protein changes, as observed b y
several authors (40, 41, 42). The higher losses in e x t r a c t a b i l i t y of fibrillar
muscle proteins in minced fillets of cod t h a n in corresponding intact fillets
during frozen storage at - 8 ~ m a y serve, as an e x a m p l e (Fig. 4). In
coalfish the differences in solubility are even m o r e pronounced; the same
applies to the contractability of muscle fibres (43). Recently, the causes of
these findings and their o w n convincing results w e r e reason enough for
Canadian scientists to advise the fishing i n d u s t r y not to m i x minced fish
m e a t of the Gadidae f a m i l y w i t h t h a t of other scarcely f o r m a l d e h y d e
producing fish species like flatfish (56).
This conception of f o r m a l d e h y d e playing an essential role in protein
alterations of certain fish species during frozen storage was doubted b y
Conne~l (34). His a r g u m e n t a t i o n is based on the assumption t h a t the
p o l y m e r i z i n g activity of f o r m a l d e h y d e leading to toughness is m a i n l y
caused b y the f o r m a t i o n of covalent m e t h y l e n e cross-links b e t w e e n protein
molecules. In this case the f o r m e d aggregates should be insoluble in
h y d r o g e n - b o n d b r e a k i n g solvents like sodium dodecyl sulphate. The
a u t h o r found, however, t h a t m y o f i b r i l l a r proteins of cod stored for
29 weeks at - 1 4 ~ in contrast to fresh cod t r e a t e d w i t h formaldehyde,
can be dissolved in 1 ~ SDS. C o n n e l l t h e r e f o r e regards the role of f o r m a l -
dehyde as a m e t h y l e n e cross-linking agent acting during frozen storage of
Gadus species as questionable. He admits, however, t h a t these e x p e r i m e n t s
do not exclude the possibility t h a t t h e r e m a y still be other w a y s of
 Partmann, Some aspects o] protein changes in $rozen /oods 173

irreversible reactions between formaldehyde and proteins during frozen

storage, not necessarily producing covalent cross-links.
The changes leading to inextractability of fibrillar proteins of fish
obviously are not initiated by only one of the factors mentioned. The
effects of w a t e r freezing m a y support the reactions caused by certain free
f a t t y acids and their oxidation products. In species of the family Gadidae
the formation of formaldehyde m a y be the predominant factor for protein
deterioration.

The molecular range:

There seems to be general agreement in the pertinent literature that
the insolubilization of fibrillar proteins correlated with the toughening of
the muscle results from aggregation reaction. Connell (44) and Anderson
and Ravesi (45, 46) believe that most of the cross-links formed between
the proteins during frozen storage result from formation of intermolecular
h y d r o g e n or hydrophobic bonds. Sikorski, Olley and Kostuch are of the
opinion that lipid autoxidation products and formaldehyde lead at least to
a limited n u m b e r of covalent cross-links, although no direct experimental
evidence has been furnished (24).
Connell found no significant changes for the highly reactive or total
SH groups in frozen fish or frozen protein solutions (8), and no reduction
of the n u m b e r of acidic and basic groups in myofibrils of cod stored at
- 1 4 ~ (46). He therefore concluded that disulfides do not contribute to
the aggregation of myosin (44), and that, in fact, the bulk of the protein in
c o d - u n t i l it is extremely t o u g h - i s not covalently cross-linked to a large
extent (47, 48).
Buttkus, however, proposed a mechanism for the aggregation of myosin
which involves disulfide-sulfhydryl exchange reactions between activated
myosin molecules without a net change of existing SH groups (10). Neither
can it be completely excluded that only a few scarcely detectable cross-
links of this kind contribute considerably to toughening.
There has apparently been no answer to the question yet w h e t h e r the
single molecules of the fibrillar proteins which aggregate during frozen
storage are still native or denaturated. Connell showed that, w h e n the
actomyosin fraction in the cod muscle became completely insoluble at a
storage t e m p e r a t u r e of - 14 ~ there was only a v e r y small change in the
properties of the actin (49). Genuine myosin capable of combining with
actin to an ATP-sensitive complex was extractable from cod muscle stored
at - 1 4 ~ for up to 52 weeks (50). Non-extractability hence cannot be
regarded as a reliable indicator for denaturation in the classic sense of the
aggregated fibrillar proteins of frozen fish muscle. All possibilities con-
ceivable seem to exist: the molecules, or part of them, m a y still be native
or be more or less changed in their conformation before or during
aggregation.

Conclusive remarks
In other meats, e.g. p o u l t r y (51, 52) and probably in m a n y other foods
similar alterations of proteins during freezing and frozen storage m a y
occur, f r e q u e n t l y without aggregation. The single molecules m a y retain
174 Zeitschrift fiJr Erniihrungswissenschaft, Band 16, Heft 3 (1977)

t h e i r n a t i v e s t a t e o r u n d e r g o c o n f o r m a t i o n a l c h a n g e s to d i f f e r e n t e x t e n t .
T h i s is in a g r e e m e n t w i t h t h e w e l l - f o u n d e d v i e w of L e w i n on p r o t e i n
d e n a t u r a t i o n in g e n e r a l (23). T h e l a r g e v a r i e t y of p o s s i b l e a l t e r a t i o n s , so
I b e l i e v e , does n o t a l l o w a g e n e r a l c o n c l u s i o n r e g a r d i n g t h e p r o p e r t i e s of
t h e p r o t e i n s c o n c e r n e d of f r o z e n foods f r o m t h e v i e w p o i n t of n u t r i t i o n
p h y s i o l o g y , u n l e s s s u b s t a n t i a l e v i d e n c e is f u r n i s h e d b y e x p e r i m e n t s , f o r
i n s t a n c e , b y f e e d i n g tests in h u m a n v o l u n t e e r s . C o l v i n has a l r e a d y u n d e r -
l i n e d t h a t t h e d i f f e r e n t t r e a t m e n t s of p r o t e i n s l e a d i n g to " d e n a t u r a t i o n "
do n o t p r o d u c e i d e n t i c a l c h a n g e s of t h e m o l e c u l e s a n d h e n c e no i d e n t i c a l
a l t e r a t i o n s of t h e i r p r o p e r t i e s (53).

Summary
In the past decades m a n y results on changes of food proteins caused by
freezing and their relation to quality changes were found. Recently especially
the nutrition physiological viewpoint of these alterations has aroused consid-
erable interest. In the present paper the a tt e m p t is made to outline some
aspects of these changes which are usually r e f e r r e d to as "denaturation" caused
by freezing. Af t er a description of the phenomena observed and their m e a s u r e -
ment, a survey of the m a i n causes presumed for the "denaturation" of fish
muscle proteins by freezing is given. With special consideration of the molecular
range it can be said that generally aggregation p h en o m en a prevail and that the
participating molecules, or part of them, m a y still be native or more or less
changed in their conformation before or during aggregation. Definite j u d g e m e n t
of the nutritional properties of the altered proteins in frozen foods is possible
only when convincing results of feeding experiments will be available.

Zusammenfassung
In den v e r g a n g e n e n J a h r z e h n t e n w u r d e n viele Ergebnisse fiber Ver~nderun-
gen yon P r o t ei n en in Lebensmitteln durch Gefrieren und ihre Beziehungen zu
Qualit~tsver~nderungen erhalten. Neuerdings hat speziell der ern~hrungsphy-
siologische Gesichtspunkt dieser Ver~nderungen besonderes Interesse gefunden.
Im vorliegenden Bericht wird der Versuch gemacht, einige Aspekte dieser Ver-
~nderungen zu umreii~en, die gewShnlich als ,,Gefrierdenaturierung" bezeichnet
werden.
Nach einer Beschreibung der beobachteten K r i t er i en und ihrer Messung w i r d
auf die Ursachen eingegangen, die v e r m u t l i c h ffir die ,,Denaturierung" der
Fischmuskelproteine durch Gefrieren v e r a n t w o r t l i c h sind.
U n t e r besonderer Berficksichtigung des molekularen Bereichs kann gesagt
werden, dab im allgerneinen Aggregationsph~nomene vorherrschen und dab
die beteiligten Molekfile oder ein Teil yon ihnen noch n at i v sein k~nnen oder
vor oder w~hrend der Aggregation in ih r e r Konformation m e h r oder w en i g er
ver~ndert sind. Eine verl~{31iche Beurteilung der ern~hrungsphysiologischen
Eigenschaften der Proteine in gefrorenen Lebensmitteln wird erst m6glich sein,
w e n n iiberzeugende Ergebnisse von Fiitterungsversuchen vorliegen.

References
1. Kauzmann, W., J. Cell. Comp. Physiol. 47, Suppl. 1, 113 (1956). - 2. Baust,
J. G., Cryobiology 10, 197 (1973). - 3. Mazur, P., Science N.Y. 168, 939 (1970). -
4. Meryman, H. T., Cryobiology 8, 489, (1971). - 5. Heber, U., L. Tyankova, K. A.
Santarius, Biochim. biophys. Acta 291, 23 (1973). - 6. Van denBerg, L., Cryobiology
3, 236 (1966). - 7. Levitt, J., Interactions. Cryobiology 3, 243(1966). - 8. Connell, J. J.,
Biochem. J. 75, 530 (1960). - 9. Connell, J. J., Nature 183, 664 (1959). - 10. Buttkus,
 Partmann, Some aspects of protein changes in ]rozen ]oods 175

H., J. F d . Sci. 35, 558 (1970). - 11. Hanalusa, N., C o n t r i b . I n s t . L o w T e m p . Sci. J.

S e r . B 1-20 ( 1 9 7 2 ) . - 12. Matsumoto, J. J., S. Noguchi, P r o c . X I I I I n t e r n . C o n g r .
R e f r i g . 3, 237 (1973). - 13. Partmann, W., T h e e f f e c t s o f f r e e z i n g a n d t h a w i n g
o n f o o d q u a l i t y . I n : W a t e r r e l a t i o n s o f f o o d s . E d i t . R. B. Duckworth, p. 505-537
( L o n d o n 1975). - 14. Hamm, R., L. K6rmendy, F d . Sci. 34, 452 (1969). - 15. Harem,
R., A. EI-Badawi, Z. L e b e n s m . - U n t e r s . u. F o r s c h g . 150, 12 (1972). - 16. Gould, E.,
J. A s s o c . O f f i c . A n a l y t . C h e m . 56, 541 (1973). - 17. Dyer, W. J., Fdu R e s . 16, 522
(1951). - 18. Connell, J. J., F i s h m u s c l e p r o t e i n s a n d s o m e e f f e c t s o n t h e m o f
p r o c e s s i n g . I n : P r o t e i n s a n d t h e i r r e a c t i o n s . E d i t . H. W. Schultz, a n d A. F. Angle-
ruler, p. 255-293, A V I W e s t p o r t - C o n n . (1964). - 19. Partmann, W., Q u a l i t y c h a n g e s
o f c o a l f i s h ( G a d u s V i r e n s , L.) d u r i n g f r o z e n s t o r a g e . P r o c . X I V t h I n t e r n . C o n g r .
R e f r i g . i n p r e s s . - 20. Love, R. M., J. I. M. Ironside, J. Sci. F d . A g r i c . 9, 604
(1958). - 21. Mao, W. W., C. Sterling, J . T e x t u r e S t u d . 1, 338 (1970). - 22. Tokunaga,
T., B u l l . J a p . Soc. Sci. F i s h . 40, 167 (1940). - 23. Lewin, S., D i s p l a c e m e n t o f w a t e r
a n d i t s c o n t r o l o f b i o c h e m i c a l r e a c t i o n s , 367 S. ( L o n d o n a n d N e w Y o r k 1974). -
24. Sikorski, S., J. Olley, S. Kostuch, C r i t i c a l R e v . F d . Sci. a n d N u t r i t i o n 7, 97 (1976). -
25. Dyer, W. J., M. L. Morton, J. F i s h . R e s . B d . C a n . 13, 129 (1956). - 26. Dyer,
W. J., J. R. Dingle, F i s h p r o t e i n s w i t h s p e c i a l r e f e r e n c e to F r e e z i n g . I n : F i s h a s
f o o d . E d i t . G. Borgstr6m. Vol. 1 p. 275 (1961). - 27. Olley, J., J. A. Lovern, J. Sci.
F d . A g r i c . 11, 644 (1960). - 28. Olley, J., R. Pirie, H. Watson, J. Sci. F d . A g r i c . 13,
501 (1962). - 29. King, F. J., M. L. Anderson, M. A. Steinberg, J. F d . S c L 27, 363
(1962). - 30. Olley, J., W. R. H Duncan, J. S c L F d . A g r i c . 16, 99 (1965). - 81. Butt-
kus, H., J. F d . Sci. 32, 432 (1967). - 32. Childs, E. A., J. F i s h . R e s . B d . C a n . 31, 111
(1974). - 33. Jarenbtick, L., A. Liljemark, J. F d . T e c h n o l . 10, 437 (1975). - 34.
Connell, J. J., J. Sci. F d . A g r i c . 26, 1925 (1975). - 35. Kuusi, T., O. E. Nikkilti,
K. Savolainen, Z. L e b e n s m . U n t e r s . - F o r s c h g . 159, 285 (1975). - 36. Babbitt, J. K.,
D. K. Law, D. L. Crawford, J. F d . Sci. 41, 35 (1976). - 37. Castell, C. H., B.
Smith, W. J. Dyer, J . F i s h . R e a . B d . C a n . 30, 1205-1213 (1973). - 38. Childs, E. A.,
J. F d . S e i 38, 1009 (1973). - 39. Partmann, W., F d . R e s e a r c h 22, 51 (1957). - 40.
Babbitt, J. K., D. L. Crawford, D. K. Law, J. A g r i c . F d . C h e m . 20, 1052 (1972). -
41. Sorenson, T., S e p a r a t e d c o d m i n c e . D e t e r i o r a t i o n o f f u n c t i o n a l p r o p e r t i e s
d u r i n g f r o z e n s t o r a g e . O r s b e r e t n i n g 1975 F i s k e r i m i n i s t e r i e t s F o r s ~ g s l a b o r a t o r i u m
L y n g b y , D e n m a r k , p. 28-38. - 42. Hiltz, D. F., B. Smith Lall, D. W. Lemon, W. J.
Dyer, J. F i s h . R e s . B d . C a n . 33, 2560 (1976). - 43. Partmann, W., L e b e n s m . W i s s .
u. - T e c h n o l . 7, 186 (1974). - 44. Connell, J. J., J. Sci. F d . A g r i c . 16, 769 (1965). -
45. Anderson, M. L., E. M. Ravesi, J. F d . S c L 35, 139 (1970). - 46. Anderson, M. L.,
E. M. Ravesi, J. F d . Sci. 35, 551 (1970). - 47. Connell, J. J., P. F. Howgate, J. F d .
S c L 29, 717 (1964). - 48. Connell, J. J., T h e e f f e c t o f f r e e z i n g a n d f r o z e n s t o r a g e
on the proteins of fish muscle. In: Low temperature biology of foodstuffs. Edit.
J. Hawthorn, a n d E. J. Rolfe, p. 333 ( O x f o r d 1968). - 49. Connell, J. J., J. Sci.
F d . A g r i c . U , 515 (1960). - 50. Connell, J. J., J. Sc. F d . A g r i c . 13, 607 (1962). - 51.
Kahn, A. W., L. van den Berg, C. P. Lentz, J. F d . S c i 28, 425 (1963). - 52. Kahn,
A. W., C r y o b i o l o g y 3, 224 (1966). - 53. Colvin, J. R., D e n a t u r a t i o n : A r e q u i e m .
I n : S y m p o s i u m o n f o o d s : P r o t e i n s a n d t h e i r r e a c t i o n s . E d i t . H. W. Schultz a n d
A. F. Anglemier, p. 69-83, A V I W e s t p o r t . C o n n . (1964). - 54. Amano, K., K.
u The biological formation of formaldehyde in cod flesh. In: The
t e c h n o l o g y o f f i s h u t i l i z a t i o n . E d i t . R. Kreuzer, p. 7 3 - 7 8 ( L o n d o n 1965). - 55.
Partmann, W., J. Gutschmidt, K ~ l t e t e c h n i k 24, 327, 328, 331, 332, 343, 344, (1972). -
56. Dingle, J. R., B. Lall, R. A. Keith, E n v i r o n m e n t Canada, Fish and Marine
S e r v i c e , H a l i f a x L a b o r a t o r y N e w c i r c u l a r N o 59 (1976).

Author's address:
W. Partmann, F e d e r a l R e s e a r c h C e n t r e f o r N u t r i t i o n ,
E n g e s s e r s t r a l 3 e 20, D - 7 5 0 0 K a r l s r u h e 1