EFFECT OF FREEZENQ TIME ON CELL DESTRUCTION
{N BRCNLER BREAST MUSCLE

Thesis far the Degree :4 M. S.
MICHIGAN STATE UNWERSSTY
50595211 Carter Crig§er
1.966

THESB

   
    
 

  

LIBRARY

Michiga- Sn.

ABSTRACT

EFFECT OF FREEZING TIME ON CELL DESTRUCTION
IN BROILER BREAST MUSCLE

by Joseph Carter Crigler

Broiler breast muscles were frozen at widely varying times (from
0.5 to l8©9 minutes) and thawed under constant conditions (IBOC and
saturated humidity) to study cell destruction. The amount of drip,
rate of drip release and composition of the drip (DNA, total solids and
nitrogen) were determined as measures of cell destruction.

In Part I the amount of drip released from tissue was measured at
specific times to determine the rate at which it was released during.
the thawing process. The largest portion of the drip from the meat
(39.71) was released during the first 9 hours of thawing regardless of
freezing times of 2, 29l and IBb9 minutes. It was also found that the
percent of total drip collected decreased with each successive thawing
period and only a small amount of drip was released during the last
collection period (the l5th to the l8th hours).

The DNA concentration was determined for drip samples collected
during each of the thawing periods. The DNA concentration of the drip
from tissues frozen in 29l and I869 minutes remained relatively con-
stant throughout the thawing process. However, drip from tissues frozen
in 2 minutes had an increase in DNA concentration with each successive
period of collection. The pH of the drip collected from all previously
frozen samples decreased with each successive collection period.

In Part II freezing times varying from 0.5 to l494 minutes were
used to study cell destruction. Excessive cell destruction was found
to occur in tissues frozen in 87, 252. and IDA; minutes. A tendency

I

for increased destruction also occurred in tissues frozen in less than

Joseph Carter Crigler

one minute and in the region from 18 to 35 minutes. Values used to
indicate cell destruction were all lower in drip from unfrozen tissue
that from all frozen samples.

Minimum cell damage as indicated by the amount of drip and concen-
tration of cell components was found in tissues frozen in I to I8, I32
to 223 and longer than IO44 minutes.

The results of this study indicate that the freezing time of
poultry is very important and should be given careful consideration.

A significant linear relationship was not found between apparent cell
destruction and freezing time, thus changing the freezing time will

not assure minimum cell damage.

EFFECT OF FREEZING TIME ON CELL DESTRUCTION

IN BROILER BREAST MbSCLE

By

Joseph Carter Crigler

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science

I966

/’\.CRNOWL EDOEriENT

The author wishes to express his appreciation to Dr. L. E. Dawson
for his guidance. assistance and helpful criticism throughout the
course of study.

He is also grateful to Dr. C. L. Bedford for his helpful dis-
cussions and criticism, and to Dr. R. K. Ringer, Professor of Poultry
Science, for his critical review of the hanuscript.

The author wishes to thank Dr. B. S. Schweigert and the members of
the faculty of the Department of Food Science for making facilities
available and the interest shown during the preparation of this man-
uscript. His sincere thanks go also to Mrs. Maurice Ritchey for her
assistance in typing.

The author is deeply indebted to his wife, Marilyn, for her

encouragement and underSLanding throughout a trying period.

TABLE OF CONTENTS
Page

ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . ii
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . iv
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . v
LIST OF APPENDICES . . . . . . . . . . . . . . . . . . . . . . . vi

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . I

LITERATURE REVIEW . . . . . . . . ..... . .
Factors Influencing Amount of Drip . . . . . . . . . . . . .
Methods of Drip Collection .
Freezing and Thawing of Meat .
Composition of Drip . . . . . . . . . . . . . . . . . . .

\IOKDUJLJ

EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . . . . .
Part I . . . . . . . . . .
Experimental Animals
Preparation . . . . . . . . . . . . . . . . . . . . .
Freezing . .
Drip collection . . . . . . . . . . . . . . . .
Drip rate . . . . .
Chemical Determinations . . . . . . . . . . . . . . .
DNA . . . . . . . . . . . . . . . . . . . . . . . . .
Part II . . . . .
Experimental Animals
Preparation . . . . . . . . .
Freezing . .
Drip collection . . . . . .
Physical and Chemical Determinations . . . . . . . .
Total solids . . . .
Nitrogen . . . . . . . . . . . . . . . . . . .
pH determinations . . . . . .
DNA determinations .

o
T») F) I\.) IQ

\I\I\I\I\J\IU‘U'UIU'IJIQIQ—‘OOQOO

I\J IQ IQ I0 I.) I\) I0 Ix) IO N IQ IQ IQ Ix) IO

N
CD

RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . .
Part I . . . . . . . . . .
Part II . . . . . . . . . . . .

J

I

to
0

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . 48
APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

 

—a
0

Ix.)

LIST OF TABLES

Methods used to obtain different freezing times . . . . . .

Effect of freezing time on rate of drip release from
broiler breast meat during thawing

Effect of freezing time on total and percent drip obtained
from broiler breast meat, and on final pH of the meat . .

pH and DNA concentration of drip from broiler breast meat
frozen at different rates and collected during specific
thawing periods . . . . . . . . .

Freezing times of the meat samples . . . . . . . . . . .

Total solids and nitrogen content of the drip from broiler
breast muscles frozen in different times

Relationship between pH of meat and percentage of drip from
broiler breast muscles frozen at different rates . . . . .

Page

20

29

BI

33

L\
k)

L\
U1

Figure

I.

I0

LIST OF FIGURES

Standard curve for DNA

The effect of freezing time on the amount of drip released
from broiler breast muscles during thawing

The effect of freezing time on amount of total solids in
drip from broiler breast muscles

The effect of freezing time on nitrogen content of drip
from broiler breast muscles .

The effect of freezing time on DNA content of drip from
broiler breast muscles (mg DNA released/I00 g tissue)

The effect of freezing time on DNA content of drip from
broiler breast muscles (mg DNA/ml of drip)

36

37

38

LIST OF APPENDICES

Enzymatic activity during the thawing period

Stability of DNA .

Moisture content of broiler breast muscle
after different freezing treatments

Solids content of drip from broiler breast
muscles which had been previously frozen in
different times . . . . .

Nitrogen content of drip from broiler breast

muscles which had been previously frozen In
different times . .

Aggendix

A. DNA recovery

B.

C.

D. Table I.
Table 2.
Table 3.
Table 4.

DNA content of drip from broiler breast muscles
which had been previously frozen in different
times

Page

60

INTRODUCTION

The utilization of frozen food products has increased steadily
during the past twenty years. Although some poultry products,
especially turkeys, have been successfully marketed in the frozen
form, most young chickens are still merchandized in the ice-packed
condition. Merchandizing of ice-packed fryers (broilers) has con—
tinued as a result of lower retail prices and the availability of
fryers throughout the year.

During the period I960 to I965 production of young chickens
(Inspected and Certified) increased from 3.7 to 5.2 million pounds
(U.S. Department of Agriculture, I962 and I966), and the portion of
this total which was frozen increased from 8.6 to III. The interest
in freezing fryers has increased since freezing eliminates the major
disadvantages of merchandizing ice-packed fryers. These disadvantages
include short shelf-life, repackaging requirements at the retail level,
changes in weight due to water absorption or release, and changes in
appearance due to the presence of melting ice.

Foods expand and ice crystals form during freezing. As the ice
front moves through the product, a concentrated salt solution is formed
in the interstitial spaces. The severity of the damage resulting from
the expansion, the formation of ice crystals and the concentration of
salt in solution affects the quality of the frozen products.

The extent to which the fibers are ruptured or damaged, has been
attributed to the rate of freezing, rate of thawing, and uniformity of
temperature during storage. During and following the thawing process
moisture is released from the tissues, and frequently more moisture is

released than can be reabsorbed by the tissues resulting in drip loss.

I

IQ

Since freezing rate affects both the amount of drip; which con-
tains nutrients, and final meat quality, this study was conducted to
evaluate the effects of different freezing rates of chicken breast

muscles on;

I. Amount of drip loss during thawing

IQ

Rate of drip loss during thawing
3. Composition of the drip
4. Cell destruction as determined by the composition of

the drip.

LITERATURE REVIEW

Many foods, including poultry, are frozen to preserve quality
during distribution or holding prior to sale and/or consumption.
Freezing is the process of hardening by cold into ice or a like solid.
In biological systems such as food products, freezing has been de-
fined as the solidification or transformation of 90 percent or more of
the free water into ice (Bedford, I966).

After poultry meat has been frozen and thawed. some fluid exudes
from the product and collects as drip (Sair and Cook. l938a). Love
(l955a) referred to fish drip as the cloudy fluid exuding from fillets

1:

which were allowed to stand IOF some time under humid conditions.

According to Cook et al. (I926). drip is the clear, reddish-brown

 

colored fluid which exudes from all cut surfaces of meat that have
been frozen and thawed. They reported that drip from either beef,
lamb or pork contained approximately the same percent (92) protein.
Seagram (I958) reported that drip is primarily of intracellular
origin, and Watson (I965) reported that deoxyribonucleic acid (DNA)
was found only in the nuclei of the cells. Love (l955a) measured the
DNA in the fluid expressed from fish fillets on the assumption that
it would appear only when the muscle fibers had been burst open, and

the amount of DNA would provide an index of cell damage.

Factors Influencing Amount of Drip

 

Koonz and Ramsbottom (l939b) reported that the temperature of
freezing affected the amount of drip obtained from frozen—defrosted
poultry meat. This was based on the fact that the rate of freezing
determined the size, distribution and number of ice nuclei formed.

They reported that white meat frozen at 8°F was much more susceptible

3

43‘

to drip loss than when frozen at —50°F, since with rapid freezing a
maximum fiber to water relationship was created when thawed. Slow
freezing was also shown to result in more drip by Callow (I952) and
Empey and Howard (I954).

The quantities of drip obtained on thawing beef decreased as
freezing temperatures were lowered from l8 to -Il4°F (Hiner _£ _Jx
I945). Increased intrafibrillar freezing and rupturing of the fibers
which permitted the proteins to reabsorb a large proportion of the
water originally frozen in the meat was believed to be the cause of
reduced drip.

Pearson and Miller (I950) studied steaks which had been frozen
at a slow rate (insulated box in a GOP walk-in freezer), intermediate
rate (chest-type freezer, 0°F). and a rapid rate (freezer plate at
-40°F), and then held for varying storage periods (0, 90, l80 days
at OOF). They reported that increased frozen storage time resulted
in increased drip which became apparent after 90 days storage. How-
ever, they found that rate of freezing did not alter the expressible
fluid. Moran (I932) found that drip reached a maximum when frozen
tissues were stored 80 days at -2 to -3°C. He also reported a denatur-
ation effect on the proteins at -l.5°C and at temperatures lower than
-3°C.

Quantitative studies of drip were made by Sair and Cook (l938a)
and they concluded that regardless of rate of freezing, whole birds do
not drip. They noted, however, that removal of skin. cutting the meat,
and particularly mincing the meat increased the probability of drip

from frozen-defrosted tissue. The Quantity of expressible fluid in

frozen fish was also reported by Reay (I934) to be dependent on the

number of cuts made in the flesh. However, he noted that the major
factor in determining the amount of drip from haddock was the time
spent in the temperature range of —I to —5°C (30.2 to 23°F) either
during thawing or freezing.

Nichols and Mackintosh (I952) investigated the structural changes
which occurred in beef and pork muscle during repeated freezing and
thawing. They concluded that both intracellular and intercellular ice
crystal formation contributed to the fragmentation of the fibers, and
that freezing (0 to -5°F freezer) and thawing caused an increased
amount of drip.

Using different methods of freezing including brine, plate, and
moving air (Marion and Stadelman, I958) found that the method of
freezing exerted no significant effect on the amount of drip from
chicken fryers, fowl, turkey fryers and mature tom turkeys. They re-
ported that drip from chicken fryers ranged from 5.3 to 5.8 percent;
fowl, 5.2 to 5.6 percent; turkey fryers, 4.3 to 5.3 percent; and
mature turkeys. 2.3 to 3.0 percent. Hicks _£._l° (I955) studied the
effect of freezing, storage and transportation on frozen meat and
reported that freezing rate did not have a significant influence on
the amount of drip obtained.

Ramsbottom and Roonz (I939) reported that irrespective of
freezing temperature there was little drip from large rib cuts where
the area of the cut surface was small in relation to the volume of
meat. In small cuts where the area of cut surface was large in re-
lation to the volume of meat, the anount of drip was dependent to a
large extent, on the freezing temperature. In a large cut the muscle

tissue had an opportunity to reabsorb the ''frozen out” water while in

small cuts the fluids were more readily lost by the tissue as drip.
During slow freezing, extrafibrillar freezing took place, and druing
defrosting, more of the fluid was lost as drip before it could be
reabsorbed by the partially dehydrated muscle fibers.

Spencer_gt_§l. (I956) reported that the moisture loss during the
defrosting of turkey meat was affected significantly by the cooling
and freezing treatment. They found that losses from carcasses not
cooled in ice-water averaged lower than losses from carcasses cooled
in ice-water. They also found a higher weight loss from turkeys
frozen at 0°F (-I7.8°C) than from turkeys frozen at lower temperatures.

Time of storage, at any one temperature, was shown by Moran and
Hale (I932) to have little effect on the amount of drip, but that
temperature of storage affected the amount of drip. They found no
variation in drip due to thawing rate when using IOOC (50°F) for
rapid thawing and I°C (33.8°F) for slow thawing.

Empey (I933) determined the effect on drip caused by freezing and
thawing rates, age of animal, period in frozen state, breed, sex,
length of time between slaughter and freezing and composition of the
muscle including pH. He concluded that pH was the only factor that
influenced the amount of drip, and lower pH increased the volume of
drip.

According to Bouton__t‘al. (I957) there was a gradual decrease in
the amount of drip from_psgas and_l._dg£si muscles when the pH
increased above 5.8. Maximum drip was obtained from meat in the pH
range between 5.4-5.8. Howard and Laurie (I956) found that the amount
of drip exuded from_l._dg£ii was approximately twice that from_psga§

at the same pH. They concluded that different muscle proteins may be

affected differently by the same pH during thawing.

The quantity of drip obtained from meat frozen at a constant
rate was affected by the pH of the tissue and the period of time
between slaughter and freezing (Sair and Cook, l938b). The amount of
drip obtained was determined primarily by the amount of acid contained
in the tissue; amount of drip increased as pH fell below 6.0. At pH
6.3 or higher there was no decrease in pH of the meat as a result of
freezing rates which required less than 3 days to pass from 0°C to
-3°C. Maximum drip was obtained at pH 5.2-3.3, and increased freezing
rates in this region reduced the amount of drip obtained. The reduced
amount of drip was attributed to the high water-retaining capacity
of tissue proteins at pH 6.4 and was not affected by the size or
number of ice crystals formed during freezing. A decrease in water-
retaining power of the proteins occurred at pH 5.2 which increased
the moisture losses. Rapid freezing reduced these losses, due to the
production of smaller ice crystals and a more uniform distribution of
water during defrosting. At pH 5.2-5.5 the reduced moisture-retaining
capacity of tissue was due to isoelectric conditions rather than to
accelerated denaturation.

Ramsbottom and Koonz (I940) found that thawed beef with pH
values between 6.2 and 6.3 produced only 0.7 percent drip compared
with 4.3 percent drip for beef thawed at pH 5.7-5.9.

According to Koonz and Ramsbottom (l939b) less drip was obtained
from ground dark chicken meat than from ground white meat regardless
of the freezing temperature (~43.5°C, -26.I°C, ~l3.3°£). The pH

value for meat 2-3 hours after slaughter was 5.7 for the white meat

and 6.l for dark meat, and after a 22 day storage period the pH
values were 6.2 and 6.3, respectively. The greater reabsorptive
capacity exhibited by the dark muscle was thought to be due to the
higher pH values.

After examining the effects of freezing on the swelling of
tissues in buffered solution, (Smorodintsev and Bystrov, I937) con-
cluded that the quantity of fluid secreted from the tissue varied
inversely with the degree of swelling. The swelling of frozen tissues
increased with increased acidity of the medium (starting at pH 3.4)
and a decrease was noted at pH's higher than 6.0.

Kaloyereas (I947) found that rapid freezing was not as beneficial
for decreasing drip loss from products with high boundwater content
as from products with low boundwater content.

The amount of drip obtained was closely related to the water-
holding capacity (WHC) of meat, Hamm (I953, I958). When a high WHC
was observed in meat before freezing, a comparable WHC was noted after
defrosting. Whitaker (I959) found that pH affects the water-holding
capacity of the meat by modifying the charges of the protein. Meat
with a pH in the range of its isoelectric point (5.0-5.5) shows a
greater drip loss after freezing and thawing than meat with a higher
pH (Kuprianoff, I952). He also found that there may be no drip on
defrosting under certain conditions (in the pH range of 6.3-6.4).

Wierbicki__t.al. (I957) studied the effects of added cations on
meat shrinkage at 70°C, and found that the cations sodium, calcium,
potassium, and magnesium, increased the water-holding capacity of meat.
NaCl added to the meat prior to freezing decreased the amount of drip

on thawing. They also stated that the pH shifts toward alkalinity

produced by the addition of MaCl decreased after freezing and thawing,

indicating possible protein modification due to freezing.

Methods of Drip Collection

 

Numerous methods or variations in methods have been devised to
collect drip, some considerably more elaborate than others. Cook
.33 .l' (I926) measured drip quantitatively by using absorption on
blotting paper, and then determined the loss in weight of the samples.

<oonz and Ramsbottom (l939b) used frozen molds of ground dark
and white chicken meat for collection of drip. They placed the molds
in containers on elevated wire meshing and defrosted for two days at
I0°C (50°F).

A unique method of determining the amount of drip from frozen
products was developed by Kaloyereas (I947) using Skellysolve B or
ether, which was previously saturated with water, as the thawing medium.
He placed the frozen sample in a suitable container, and covered it
with petroleum ether allowed to stand for 24 hours. The liquid was
drained off the sample and the volume of the aqueous phase was read
directly from a graduated cylinder.

Pearson_gt_al. (I95I) analyzed drip from steaks that were wrapped

in cellophane, and overwrapped with butchers paper, frozen and stored

in a home freezer at 0°F. The Iongjssimus dorsi muscle was dissected

 

from the remainder of the steak and thawed for l4 to I5 hours at
approximately 26°C in a large funnel which drained into a graduated
cylinder. They weighed the samples before and after defrosting, and
measured the drip to the nearest 0.l ml.

WIadyka (I965) collected drip from poultry meat by placing it on

ID

a wire grid inside a glass funnel supported by a ring stand. The
apparatus was placed in a container with a layer of water on the
bottom and sealed to provide a saturated humidity. Meat was thawed
in a 60°F cooler for I8 hours. and the drip was collected in a
graduated cylinder and measured to the nearest 0.l ml.

When measuring the amount of fluid lost from thawed fish, Banks
(I955) employed a method in which samples were submitted to pressure.
Pressure was used since much of the free liquid in fish thawed after
storage at relatively high temperatures was retained by simple physical
forces. He applied a pressure of 7 p.s.i. to the fillets for 6 hours
under relatively humid conditions (O-2°C).

Howard _t _1. (I960) placed meat samples on a glass grid inside
air-tight dishes and thawed them for 48 hours at l0°C. Drip fluid

was collected under the samples and measured as the percentage loss

in weight of the samples.

Freeziflg and Thawing of Meat

 

The histological structure of frozen poultry was inveStigated by
Koonz and Ramsbottom (l939a). In small pieces of meat which were
frozen almost instantaneously, the water frozen within the fibers
appeared as minute evenly distributed ice columns. In tissues frozen
somewhat slower, fewer ice columns were present within the fibers,
but they were somewhat larger in diameter and had a peripheral dis-
tribution. With further decrease in freezing temperature a single
centrally located ice column resulted. However, when the freezing
process was sufficiently prolonged, a temperature was reached at which

water was lost from the fibers; freezing externally to the fibers.

ll

Pennington (I94l) reported the size of the ice crystals formed
are determined to a large extent by the speed with which the product
passes through the temperature range of plus 32°F to plus 25°F. The
faster the freezing rate, the smaller and more numerous the ice
crystals. The temperature range was defined as the l‘zone of maximum
crystallization.H

Bystrov (I938) reported that quick-frozen meat swells more than
slowly frozen meat. He deduced that quick-frozen meat undergoes less
change than meat frozen in buffer solutions when an equilibrium was
reached in 24 hours. Quick frozen foods also retained more natural
juices since there was little or no rupture of the cells by large ice
crystals (Anonymous, I964).

According to Ramsbottom and Koonz (I939) in small steaks which
were rapidly frozen, intrafiber freezing occurred, and the drip was
retained on defrosting. However, with steaks which were slowly
frozen, extrafiber freezing took place, and upon defrosting more of
the fluid was lost as drip before it could be reabsorbed by the
partially dehydrated fibers.

Chambers and Hale (I932) found that the fluids outside muscle
fibers had a lower osmotic pressure than the fluid within the fibers.
Thus, ice formation tends to start outside the fibers. and it is only
with rapid rates of freezing which give rise to a large number of
crystallization centers that freezing occurs within the fibers.

Hiner t l. (I945) froze the longissimus dorsi muscle of beef

 

at l8, 0, IO, -40, and -lI4°F in air without forced circulation. He
found that at l8°F large interfibrillar ice areas formed which pushed

the fibers into groups (no interfibrillar ice crystals were observed).

l2

It was also noted that the size of the ice crystals decreased as
freezing temperatures were lowered, with some intrafibrillia freezing
and fiber-wall damage visible at 0°F. At the lowest temperature
(—Il4°F) the fibers were found to be Split longitudinally into several
sections containing very extensive and small ice crystals. As the
freezing temperatures were lowered, precipitated proteins and nuclear
fragments outside the fibers became more extensive.

The explanation of drip formation during thawing of fish or meat
included more than the rupturing of cells by the expansion of ice
crystals (Taylor, I930). He stated that there were certain physico—
chemical changes related to the conditions under which colloidal
protein systems were converted from liquid to gel or gel to liquid
and form heterogeneous systems that have a role in the formation of
drip. Three means by which the cell jelly could be converted to a
liquid were suggested by Taylor: I) the salting out of protein by
strong brine in the frozen cells, 2) the contraction of the jelly and
squeezing out of the then dilute liquid, and 3) autolysis of the cell
membrane.

Reay (I933, I934) emphasized the denaturation phenomenon by in-
vestigation of the influence of freezing temperature on haddock. He
found that muscle protein solubility in salt solutions decreased after
frozen storage of the fish. The globulin (actomyosin) fraction was
most affected and the change was greatest in the range -2 to -6°C.

He also concluded that the denaturation and deterioration which occurred
during thawing was similar to that which occurred during freezing.

Drip composition may vary, but its nitrogen content seems to be

l3

derived entirely from the myogen of the fish muscle cells (Dyer gt 3!.
I956). Dyer interpreted drip as a result of cell damage and con-
sidered the exudate resulting from freezing and thawing to be valid as
an indication of cell damage. Further release of free water was

taken as a sign of dehydration of protein thus protein denaturation.

Plank (I925) emphasized the importance of two effects of freez-
ing: I) the damage to the protOplasmic structure by formation of ice
crystals, and 2) the consequent dehydration of the colloid. It was
pointed out by Callow (I952) that the salts in the muscle juice become
more concentrated during the formation of ice in muscle and (Callow,
I955) at high concentrations salt may be capable of denaturing some
cellular proteins.

Smorodintsev and Bystrov (I937) reported that low temperature
freezing (-25°C) resulted in no denaturation of muscle proteins as
determined by ATP-ase activity or by the solubility and swelling of
tissue in salt solution. Finn (I932) reported that rapid freezing and
thawing together with storage at low temperatures may be important
in denaturation, since these conditions minimize changes due to
denaturation of the proteins by strong salt solution.

It was found by Deatherage and Hamm (I960) that quick freezing
and thawing of beef caused no significant decrease in hydration and
no change in the isoelectric point of the meat; however, small but
significant changes in acidic and basic groups of muscle proteins by
quick freezing were noted. Marsh and Thompson (I958) reported that
the shortening and dehydration of muscle during thawing did not occur
in slowly thawed muscles. The melting was sufficient to allow the

czhemical changes, yet enough ice remained to maintain shape and

rigidity.

According to Dyer (I95l) the formation of ice crystal fragments
in frozen fish was due to the dehydration of tissue cells. During
the freezing process proteins present in the sol form in the fresh
tissue were converted to the gel form which was denatured by salts in
the fresh tissue at the eutectic point.

While the overall extent of denaturation depended on the length
of storage, Love (l955a) showed that large differences in denaturation
existed between the freezing rates of fish. Love (I956) concluded
that an important factor which affected denaturation was the mode of
ice formation, distribution and the resulting concentration patterns
of tissue components rather than actual mechanical damage.

Love (l955a) in re-examining the question of tissue damage re-
ported that the appearance of deoxypentosenucleic acid (DNA) in the
expressible fluid from the muscle was taken to indicate rupture of the
sarcolema with liberation of nucleic materials. It seemed to provide
a more meaningful method for determining the degree of cell damage.
Considerable cell damage was shown to result with a freezing rate of
less than 28.2 minutes. The freezing rate (time required to go from
0 to -5°C) which was found to cause the greatest damage was about I30
minutes after which there was a decrease in cell destruction. This
peak of major destruction was thought to correspond with the change
over from intracellular to intercellular ice crystal formation, with
a decrease in destruction at slower freezing rates. The great damage
reported in the ultra rapid freezing range was thought to be due to
a different type of cell damage, an expansion of the interior during

cooling, and subsequent cracking of the hard outer shell which had

I5

formed earlier.

Using different freezing methods (solid CO2 and plate at -78°C)
Love (I958a) further examined the action of intercellular ice on
muscle cells. He showed that the DNA content in the drip corresponded
with actual rupture of the sarcolemmas, both from histological evi-
dence (Love, l958a) and from the presence of cell particles in the
expressible fluid (Love. l958b)- A zone of minimum damage was found
with a freezing rate of about II5 minutes, (Love, l958a) the freezing
time at which all ice was able to form in the intracellular spaces.

At slower freezing rates (200 to 500 minutes) cell damage was found to
be at a maximum value and then decreased. With very slow freezing,
the strong salt solutions, which were created by the freezing out of
water from the weakly saline interstitial fluids, exercised a con-
siderable solvent effect on the fibers without actually rupturing the
cells. An increase in the protein concentration in the interstitial
fluid tended to increase during very slow freezing (approximately

750 minutes).

Love (I962a) found that denaturation was less when the quantity
of ice present at a given temperature was reduced, since a reduction
in the amount of ice reduced the concentration of tissue salts. These
results further supported the theory (Love, l958c) that denaturation
results directly or indirectly from the action of concentrated salts
on the proteins.

Chicken meat muscle proteins undergo both denaturation and pro-
teolysis during frozen storage according to Khan _t‘_l. (I963).
Stepwise denaturation of actomyosin decreased ATP-ase activity and

protein solubility. Proteolysis also occurred in both leg and breast

lb

muscle at -l8, -l0, and -4°C, and the rate of change depended directly
on storage temperature and time. Bandack-Yuri and Rose (I96l) re-
ported that muscle proteases have a pH optimum of 4 to 7 and tempera-
ture optimum of 37°C. Since beef contains a protease active at
freezing temperatures (Balls, I938), it has been concluded that a
similar enzyme system could possibly be present in chicken muscle
(Khan _£‘al., I963).

Tarr (I947) found that undesirable changes in fish (formation of
large ice crystals ruptured the cells during freezing) took place
during freezing, with much of the deterioration resulting from a
change (denaturation) in the myosin. the most important single protein
present. This denaturation was caused by chemical changes, probably
irreversible dehydration. which rendered the myosin insoluble. De-
naturation occurred most rapidly between the temperatures of 30°F and
23”F though denaturation occurred slowly at much lower tempera-
tures. Tarr concluded that ”the only practical method of retarding
protein denaturation is by very rapid freezing and storage at a con—
stant low temperature. preferably below —4°F.”

Sawant and Hagar (l96l) studied the denaturation of proteins in
frozen fish and concluded that denaturation was restricted to the
actomyosin fraction of protein while the sarcoplasmic fraction re-
mained unchanged. Dyer (l95l) observed that the albumen fraction was
not denatured during frozen storage, while Seagram (I958, I939)
suggested that sarcoplasmic fraction was not associated with the for-
mation of drip.

Denaturation and autolytic changes in compounds of muscle tissue

during cooling and freezing of meat were studied by Pavlovskii (I963).

l7

Autolysis of muscle was characterized by a degradation as a result of
the activity of ATP-ase and glycolytic conversion and resynthesis of
adenosine triphosphate. Since, freezing increased NaCl, myosin and
ATP-ase concentration, he concluded that during refrigeration pro-
cessing the denaturation of muscle was largely characteristic of meat
autolyzed before freezing.

Proteolysis in poultry was indicated during frozen storage by an
increase in non-protein and soluble nitrogen of both light and dark
meat (Swanson and Sloan, I953). They also noted a decrease in amino
acid nitrogen which suggested that some metabolic processes were being
continued in the frozen state resulting in additional degradation of
amino acids which were formed during proteolysis.

The protein components of autolyzing muscle tissue were studied
during cooling and freezing of meat by Pavlovskii and Grigor'eva (I963).
The amount of readily extractible protein of the tissues was decreased
during cooling and freezing when compared to that of unfrozen meat.
However, during the initial storage of frozen meat the soluble protein

increased, but after three months this fraction decreased.

Composition of Drip

 

Khan and Lentz (I965) reported on the influence of prerigor,
rigor and postrigor freezing on drip losses and protein changes in
chicken meat. They found an average of 0.8 ml drip released per IOO
grams carcass and 5.9 mg total nitrogen released per IOO grams of car-
cass in postrigor chicken muscle. Nonprotein nitrogen accounted for
35 percent and protein nitrogen 65 percent of the total nitrogen.

According to NIadyka (I965) the drip from the white meat con-

tained larger quantities of protein than drip from dark meat, at

I8

storage periods of 30 or 90 days. He reported that the drip from
white meat held for 90 days contained l0.92 percent protein nitrogen
and 9.l7 percent when stored for 30 days. Similarly, the protein
nitrogen content of drip from dark meat stored for 90 days (6.26 per-
cent) was larger than the protein content of drip from dark meat stored
for 30 days (5.I6 percent).

The deoxypentosenucleic acid (DNA) concentration of expressible
fluid has been shown to vary with different freezing rates (Love,
l955a, l955b, I958a, and l958c). Love (I955a) found that the DHAP
(deoxypentosnucleic acid phosphorus) concentration varied from 0.2l mg
per IOO ml expressible fluid (freezing time 28.2 minutes) to a maximum
value of 0.6 mg per IOO ml expressible fluid (freezing time of I3I.O
minutes). He found that the drip from an unfrozen control contained
O.l56 mg DNAP per IOO ml. The appearance of DNA in the drip from an
unfrozen control contained 0.I56 mg DNAP per IOO ml. The appearance
of DNA in the drip from unfrozen muscle was unexpected, but was
probably due to cutting the fibers when removing the fillets.

Love (l958a) investigated cell damage in fish muscle during slow
freezing and reported solids and ash content. Solid matter was found
to vary from 7.29 to 8.47 grams per IOO grams of expressible fluid
with freezing rates of 2 and 47 minutes, respectively. Ash content
in the expressible fluid did not vary to any great extent regardless
of the freezing rate (I.27 to I.34 grams per IOO grams of expressible
fluid with freezing rates of 2 and 42 minutes, respectively). Love
(I955a) reported the solids and nitrogen content of expressible
fluid from unfrozen fillets which were used as controls. Solids and

nitrogen were found to be 8.20 and l.l50 grams per IOO ml of

expressible fluid,

respectively.

l9

EXPERIMENTAL PROCEDURE

PART I

Experimental Animals:

Forty-five (45) 9 week old, ready—to-cook, broilers were obtained
from a commercial source. The broilers were ice packed for transpor-
tation to the laboratory, and then removed from the ice pack and
placed in slush ice for a total chilling time of eight hours.

After chilling, uniform sized birds were divided into treatments

of eight birds each. The breast muscles (Pectoralis major and minor)

 

were carefully separated from the birds and the right and left muscles
were individually packaged in 6 x IO inch Cryovac bags. Each treat-
ment was divided into two groups of four birds each to obtain dupli-
cate samples for each freezing rate. To minimize variation between
groups, the left breast muscles of one group of birds were exchanged
with the left breast muscles of the other group in each treatment.
Breast muscles were identified, and placed one layer thick on a fiber

board tray for freezing.

Preparation:

Freezing:

Samples were frozen at three different rates identified as fast,
intermediate, and slow. Freezing time is defined as the time elapsed
during which the temperature of meat changed from O to -5°C. Tempera-
ture was measured with a Honeywell-Brown Automatic recording potentio-
meter with attached thermocouples. These were inserted through small
openings in the Cryovac bags, positioned in the center of the largest

two muscles in each sample, and held in position by a clamp fastened

2O

7i

s.

to the tray. Temperatures of the meat frozen at the slowest rates
were recorded every four minutes and that of the fastest rate every
l5 seconds. The freezing tine used for each treatment was the longest
time recorded among individual samples in each treatment.

Variation in freezing time was accomplished by using different

freezing methods as follows:

 

Treatment: Freezing Method:
A Dry ice and acetone
B Walk-in freezer, -l8°C, still air,

slight protection, placed in
cardboard box

C Walk-in freezer. -l8°C, three inch
thick polystyrene box filled with
expanded mica, samples placed in
center of box

Drip Collection;

 

All samples were thawed at I6°C for l8 hours. Drip was collected
using a method similar to that reported by Pearson _£.El' (I959). The
apparatus consisted of a glass funnel supported by a ring stand over a
graduated cylinder. The entire apparatus was sealed in a metal
cylinder, with a layer of water on the bottom to provide a saturated
atmosphere.

After the packaging material was removed from the frozen meat
samples, they were suspended above the funnel by a clamp with the
anterior end of the muscles down. A few drops of chloroform were put
into the graduated cylinder to reduce microbial spoilage of the drip.
At the end of the thawing period, the meat samples were reweighed, pH

readings of the drip and meat were taken. and the drip measured to the

nearest O.I ml. The drip was stored at -23°C until the DNA

[\3
I‘d

determinations were made.

Drip Rate:

To determine the thawing time, the thermocouples were left in the
breast muscle and the temperature rise was determined by the use of a
Honeywell-Brown Automatic recording potentiometer.

Drip rate was determined by measuring the amount of drip released
after 9, ll, I5 and I8 hours. These particular times were determined
in preliminary studies, since they resulted in sufficient drip to be
collected for analysis. The temperatures, pH readings and DNA concen-

trations of the drip samples were determined at each of these times.

Chemical Determinations;

Each sample of meat was ground through a l/8II plate with a grind-
ing attachment on a Kitchen-Aid mixer and mixed in a stainless steel
pan. After mixing, each sample was reground and mixed again to insure
a uniform mixture. A l0 gram sample of ground meat was mixed with 30 ml
of deionized water in a Waring Blender and blended to a liquid con-
sistency. The pH of the meat was determined by placing the electrodes
in a beaker containing the blended meat samples. and pH of drip by
placing electrodes directly into the drip collection container. A
Beckman Zeromatic pH meter was used, and all pH measurements recorded

to the nearest O.I of a unit.

DNA:

DNA (deoxyribonucleic acid) was extracted from the drip samples
using a modified procedure suggested by Ogur and Rosen (I950). The
protein was precipitated from the drip with 951 ethanol (2:l volume

ethanol to drip). After centrifugation (3000 rpm——l5 min) the super—

23

natnat was decanted off, and the residue was suspended and washed in

5 ml of 70% ethanol plus O.IZ perchloric acid (HCIOA) and again centri-
fuged. The residue was then washed in 5 ml of 3:l mixture of ethanol:
ethyl ether. and heated in a water bath (80°C) for 3 minutes. After
centrifugation. the supernatant was decanted off and discarded, and the
ethanol:ethyl ether washing step repeated.

The precipitate was broken up with a glass rod and stirred with
0.5 ml of l N HCIOQ. After stirring, approximately 3 ml of l N HCIO4
were added and the suspension heated in an 80°C water bath, and stirred
occasionally for 30 minutes to extract the DNA (DNA is soluble at 80°C
in l N HCIOA). The heated mixture was centrifuged and the total volume
of the precipitate and supernatant read from a l5 ml graduated centri-
fuge tube. An aliquot (I or 2 ml, as convenient) of the supernatant
was used for the diphenylamine reaction.

A blue color was developed by the reaction of the diphenylamine
reagent with deoxyribose according to the procedure of Burton (I956).
The diphenylamine reagent has been found to be very specific for
deoxyribose.

Absorbency of the diphenylamine deoxyribose mixture was read at
600 mu on a Beckman DB Spectrophotometer and recorded. These readings
were then used to quantitate the DNA concentrations from a standard
curve (Figure l).

The accuracy of the method for recovery of DNA was evaluated by
adding known amounts of DNA to drip samples containing predetermined
amounts of DNA and determing the amount recovered. Stability of DNA
during drip collection was evaluated by collecting drip in the normal

manner and comparing its content with that of DNA in drip that had

24

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been collected in a slush ice bath. The DNA stability was also
evaluated by determining DNA content of drip before and after l8
additional hours of holding at 16°C. Results of the above determina-

tions are reported in the appendix.

PART II

Experimental Animals;

Nine-week old White Rock broilers from the same brood were ob-
tained from the University Farm. All of these birds were raised on
the same diet.

A total of 96 birds were slaughtered, scalded in a Rotomatic
scalder for 25 seconds at 53°C (l28°F) and picked in a Cyclomatic
rubber fingered picker. After the birds were picked, eviscerated and
washed, they were placed in slush ice for 8 hours. At the end of the
chilling period, the birds were removed from the slush ice and divided

into treatments of four birds each. The breast muscles (Pectoralis

 

_mai9£ and_migg£ muscles) were carefully separated from the birds and
each breast packed individually in a 6 x l0 inch Cryovac bag. The
breast muscles were marked and placed one layer thick on a fiber board
tray for freezing. To insure two uniform samples of two birds each at
each freezing rate, the same procedure used in Part I of exchanging

left breasts was used.

Preparation;

Freezing:

Seventeen different freezing procedures were used to obtain differ-

ent freezint times as presented in Table l.

Table l.

Methods used to obtain different freezing times

 

 

 

Treatment Freezing method

I Liquid nitrogen

2 Dry ice and acetone

3 Dry ice and acetone, two thicknesses of
Cryovac around the muscle

4 Walk-in freezer, -34°C, moving air

5 Walk-in freezer, -l8°C, moving air

6 Walk-in freezer, -29°C, still air

7 Walk-in freezer, -23°C, still air

8 Walk-in freezer, -l8°C, still air

9 Walk-in freezer, —l8°C. still air,
double layer of Cryovac

l0 Chest freezer, -l5°C, still air

ll Walk-in freezer, —l8°C, tray and samples
enclosed in large unsealed paper bag

l2 Walk—in freezer, —l8°C, tray and samples
enclosed in large sealed paper bag

l3 Walk-in freezer. -l8°C, samples placed
in 5 gallon can and sealed

l4 Walk—in freezer, -l8°C, samples sealed
in heavy cardboard box

l5 Chest freezer, -l5°C, samples placed in
aluminum foil lined cardboard box

l6 Walk-in freezer, -l8°C, samples packed in
expanded mica and sealed in a cardboard
box

I7 Walk-in freezer, -l8°C, three inch thick

polystyrene box 3/4 filled with expanded
mica samples placed in center of
insulating material

 

Drip Collection;

 

The thawing of meat samples and collection of drip was the same

as described in Part I.
Physical and Chemical Determinations:

Total Solids: .

 

Five ml of the drip were pipetted into a volumetric flask and made
up to a volume of 25 ml with deionized water. Ten ml of the diluted
drip were pipetted into a tarred evaporating dish and an equal volume
of 95% ethanol was added. The samples were dried to a constant weight
in a drying oven at l00 : 2°C. Total solids of the drip were reported

as the difference in weights.

Nitrogen:

All nitrogen determinations were conducted by the micro-Kjeldahl
method as outlined by the American Instrument Company (l96l). One ml
of the diluted drip used for the total solids content was used to
determine the nitrogen content of the drip, and 0.2 gram sample of the
ground and mixed tissue was used to determine the nitrogen content of
the meat. The nitrogen content was reported as mg nitrogen per ml of
drip, or as per gram of tissue. All nitrogen determinations were run

in triplicate.

pH Determinations:

 

The pH determinations were conducted in the same manner as in

Part I.

DNA Determinations:

 

DNA determinations were made using the same procedure as in Part I.

RESULTS AND DISCUSSION
PART I

Drip was collected from each sample as it was released during and
following the thawing process. The frozen meat was thawed at a temper-
ature of l6°C with the first drip released after about 5-l/2 hours
(temperature of the meat 0°C). Thawing (time to pass from -5 to 0°C)
time was approximately 230 minutes.

The amount of drip collected was recorded after 9, ll, l5 and I8
hours, respectively, since all of these time periods, except the last
collection period, resulted in sufficient fluid for later analysis.
Table 2 shows the percentage of the total drip collected during each
period from samples that had been frozen at times of 2, 29l, and I,869
minutes. Freezing time had little effect on the rate of drip released
since approximately equal percentages were obtained from the samples
during each collection period.

The maximum amount of drip was collected during the first collec-
tion period (0-9 hours) with each successive collection period yielding
a smaller amount of fluid (Table 2). This may have been the result of
a longer period for collection, or that proteins had not reached a
temperature or obtained a structural condition at which they were able
to reabsorb the free liquid released.

Table 3 shows the total percent of drip obtained from meat and
the final pH of the tissue which had been frozen at different freezing
times. As freezing time increased the amount of drip from the meat
increased. Since the final pH of the tissue was approximately the same

for each treatment (5.80 to 5.87), the differences among treatments in

28

Table 2. Effect of freezing time on rate of drip release from
broiler breast meat during thawing

 

 

 

 

 

1/ Freezing Thawing time periods (hrs)
Treatment— times (min) 0-9 9-ll ll-I5 l5—l8
Percentz/

A 2 39.8 29.8 2l.2 9.2

B 29l 42.0 33.7 l7.5 6.8

C I869 37.3 33.9 2l.3 7.5

Ave. 39.7 32.5 20.0 . 7.8

l—Eight birds per treatment - two replicate samples of four

birds each.

7
5Average of duplicate samples.

Table 3. Effect of freezing time on total and percent drip obtained
from broiler breast meat, and on final pH of the meat

 

 

 

 

 

 

Freezing Chicken meat Total drip
time Weight Final 1/
(min) (9) pH ml Percent-

2 750 5.80 34.7 4.6
29] 685 5.87 50.3 7.3
I869 725 5.83 56.0 7.7

l/

— Based on original weight of the meat.

30
amount of drip obtained was a result of freezing rate. These results
agree with those of Koonz and Ramsbottom (I939b), Callow (I952), and
Empey and Howard (I954), who found that rapid freezing rates decreased
the amount of drip obtained.

The DNA concentration and the pH of the drip samples collected
during each successive thawing period are reported in Table 4. The DNA
concentrations of the drip obtained from samples frozen at times of
I869 and 29I minutes remained relatively constant for all thawing
periods. However, DNA concentrations of drip from meat frozen very
fast (freezing time of 2 minutes) increased with each successive thaw-
ing period. This effect could have been caused by minimum cell damage
during rapid freezing, thus a reduction in the amount of DNA in the
interstitial spaces for removal at the beginning of the drip collection.
Cell damage during thawing, similar to damage during freezing, could
have been responsible for the increased DNA concentration of the drip
at later collection periods (Reay, I933, I934).

The pH of the drip decreased slightly from samples collected at
each successive thawing period (Table 4). This decrease in pH could be
due to increased mobility of organic acids at higher temperatures or to

a change in the charge on the protein (Whitaker, I959).

PART II

Since re5ults shown in Part I indicated variability in DNA con-
centrations of drip due to freezing time, additional studies were
planned using l7 different freezing procedures. Cell damage was eval-
uated by determining the amount of drip released and the concentration

of total nitrogen, total solids, and deoxyribonucleic acid (DNA) in the

Table 4.

pH and DNA concentrations of drip from broiler breast meat

frozen at different rates and collected during specific

thawing periods

 

 

 

 

FrffELHg Time_periods (hrs)l/
(min) 0-9 -ll ll-I5
Temperature of
ineat °C Z‘i l 7_f l l3.: l
. .2/
I869 DNA conc.(mg/mIi—- 0.0l34 0.0l29 0.0l32
pH of dripi/ 5.94 5 88 5.86
Z9I DNA ConC.(_mg/i‘.il) 0.0I42 0.0llli/ 0.0l45
pH of drip 6.0l 5.96 5.95
2 DNA conc.(mg/ml) 0.0l06 0.0ll4 0.0l37
pH of drip 5.95 5.95 5.87

 

l . . . . . .
-—/lnsuffICient drip to make determinations for the period from

l5-I8 hours.
9
-:/Average of four samples.

3 .
-/Average of two readings.

4/ .. . .
-— Tnis value represents only one determination.

3l

32

drip from samples frozen at different rates. Freezing times obtained
in this study are presented in Table 5.

Maximum percent drip was collected from meat which had been frozen
at times of 87, 252, and l044 minutes (Figure 2), indicating either
maximum cell rupture (Ramsbottom and Koonz, I939) or a solvent effect
by a high salt concentration without actual cell rupture (Callow, I952,
I955). Dyer El 21' (I956) reported the amount of drip released to be
a result of cell damage.

The amount of solids released in the drip per I00 grams of tissue
is shown in Figure 3. These data, when compared with Figure 2, indi-
cate a direct relationship between the percentage of drip and total
solids released per IOO grams of tissue.

As determined by the amount of drip released and grams of solids
released per l00 grams of tissue, freezing times resulting in excessive
cell destruction were found to cause excessive cell damage when eval-
uated by the mg of nitrogen released per IOO grams of tissue (Figure 4).
In addition to the cell destruction noted above, excessive cell des-
truction was also noted in meat frozen in 35 minutes. This was indi-
cated by the increased level of nitrogen found in the drip. The
increased amount of nitrogen released at approximately 35, 87, 252, and
1044 minute freezing times could be the result of either release of
intracellular fluids which accounts for most of the nitrogen present
in drip (Seagram, I958) or by the presence of cell fragments which
were the result of ice particles breaking the cell walls.

The quantity of DNA released per IOO grams of tissue from each
sample is reported in Figure 5, and mg DNA per ml of drip collected is

reported in Figure 6. At a freezing rate of IS minutes, there was an

Table 5. Freezing times of the meat samples

 

 

 

Treatmentl/ Freezing time

number minutes;
1 0.5
2 l
3 18
s, 35
5 65
6 74
7 87
8 I32
9 I66
10 225
II 252
i2 260
13 321
I4 441
15 552
16 1,044
l7 1.494

 

l .
—/Each treatment reported in
procedure, Table l.

2 . - .
—/Time reqUired for the center of the
tissue to go from 0 to -5°C.

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increase in destruction of the cells indicated by an increase in the
DNA released per ml (peak A). however, when DNA was reported as mg
released per 100 grams of tissue, peak A did not occur, but a steady
increase in amount of DNA released to a peak value at a freezing rate
of 74 minutes (peak 8), followed by a decrease in DNA released. A
possible explanation for the lack of peak A when DNA is reported as mg
DNA released per 100 grams of tissue is the release of a smaller amount
of drip at a freezing time of 18 minutes than at 35 minutes, resulting
in a diluting effect with the longer freezing time.

A small shift in the position of peak 8 (freezing time approxi-
mately 80 minutes) was noted when evaluating the effect of freezing
time on cell destruction using DNA as the criteria. With percent drip
and total solids or nitrogen released per 100 grams of tissue, peak B
was located at a freezing time of 87 minutes, whereas with DNA reported
either as concentration per ml or amount released per 10D grams of
tissue, peak B was found at freezing time of 74 minutes. The shift
in position of peak 8 could be the result of the concentrated salts
having an increased solvent effect on the cell walls of tissues
frozen the slowest. thus increasing the amount of total solids and
nitrogen available for removal by the drip. Also, the tissue fluids
have more time to migrate, therefore there can be an increase in the
concentration of total solids and nitrogen, and the amount of drip
without an increase in the DNA released. The above is based on the
theory that DNA is found solely within the cell (Watson, 1965), and
cell rupture must occur if the DNA is to escape in the drip (Love,
1955a). An increase in amount of drip could be the result of the con-

centrated salt solutions raking the cell wall more porous allowing the

40
fluid to escape from the cell (Love, 1958a).

A slight shift in the position of peak C (freezing time approxi-
mately 252) was noted when DNA was reported as mg DNA released per 100
grams of tissue. Since the shift in position of peak C is over a
narrow range in freezing rates and the difference in DNA concentration
per ml is snall, it does not seem significant.

Love (1957), l958a, 19586), from studies using fish muscles,
found similar critical rates of destruction. With a 25 minute freezing
rate, the cell damage was apparently due to mechanical forces developed
during ice crystal expansion (Love, l958b). He presumed that the lack
of sufficient organic constituents on the inside cell wall was responsi-
ble for the bursting of the sarcolemmas by ice crystals. A very large
ice column was formed in each fiber during freezing at the 75 minute
rate. and broke the cell wall because of cryohydric expansion (Love,
l958a). At the slow freezing rates (200-500 minutes) cell damage,

Love (1957) suggested, was probably caused by the movement of both
fibers and intercellular ice crystals during temperature changes.

Peak D (freezing time of 1044 minutes) was observed when DNA was
evaluated as mg DNA released per 100 grams of tissue (Figure 5);
however, peak D was not observed when DNA was reported as mg DNA per
ml of drip (Figure 6). The absence of peak D with a corresponding
decrease in the amount of DNA with increased freezing time agrees with
work reported by Love (l955a). The conflicting results can be
explained in part by the large amount of drip (Figure 2) obtained,
resulting in a diluting of the DNA extracted by the fluid.

The occurrence of peak E (freezing time of 552 minutes) was

observed when DNA was reported as mg DNA per ml of drip (Figure 6).

41

The absence of peak E in all the other determinations was the result

of the occurrence of peak D which masked the presence of peak E.
However, the amount of the other materials determined for a freezing
time of 552 minutes was approximately equal or slightly higher than the
previous freezing time and would have resulted in a peak E in the
absence of peak D.

A tendency for increased cell damage was shown with a freezing
time of 0.5 minutes (Figures 2 through 6). The results for total
solids and nitrogen per ml of drip (Table 6) indicated that when the
tissue was frozen in liquid nitrogen, cell destruction was only exceeded
in tissue frozen in times longer than 1000 minutes. Love (l955a) in-
dicated that the destruction at this ”ultrarapid” freezing rate was
the result of a different type of cell destruction. An outside layer
was frozen to a very hard shell before the interior had cooled below
the freezing point. When the inside commenced to freeze, it expanded
and set up great internal pressure. The outer shell being rigid was
cracked by this pressure in many places and the muscle fibers were cut
across. Thus, on thawing, the cell contents were able to escape, and
caused a high concentration of solids, DNA, and nitrogen in the drip.
Faint crackling sounds were observed by Love and the experimenter
conducting this study when the tissue was removed from the freezing
media.

Deatherage and Hamm (1960) reported that the amount of drip was
closely related to the water—holding capacity (WHC) of the meat. Rapid
freezing resulted in a small but significant increase in meat hydration,
probably by mechanical loosening of tissue by the formation of tiny ice

crystals inside the cell. Histological studies (Love, 1955b) showed

Ta

ble 6.

Total solids and nitrogen content of the drip from

broiler breast muscles frozen

in different times

 

 

 

Freezing time Solids contentl Nitrogen contentg/

(min) (gr/m1) (mg/m1)
Unfrozen 0.1028 1.47
0.5 0.1418 2.06

1 0.1338 2.02

18 0.1298 1.93

35 0.1122 1.97

65 0.1173 1.73

74 0.1215 1.84

87 0.1151 1.66

132 0.1286 1.89

166 0.1276 1.79

225 0.1285 1.88

252 0.1325 1.92

260 0.1320 1.85

321 0.1348 1.91

441 0.1308 1.80

552 0.1316 1.77
1,044 0.1535 2.22
1,494 0.1458 2.09

 

1 . .
—/Average of four determinations.

-_2/

Average of six determinations.

if).

1“.)

that with a freezing rate of 86 minutes, maximum cell rupture occurred
(changeover from intracellular to intercellular freezing). Since this
was done by a single ice column, considerable disruption of the cell
constituents occurred reducing the ability of the fibers to reabsorb
the fluid lost to the ice formation.

Considerable cell destruction was found with a freezing time of
252 minutes (peak C); however, there was a Small decrease in destruc-
tion after this point, with freezing times of the tissue between 260
to 552 resulting in relatively high tissue damage. An increase in des-
truction of the tissues was found to occur at peak D (freezing time
1044 minutes). Love (l958a) found similar results and indicated a
tendency for increased destruction to a freezing rate of 400 minutes
and to 500 minutes (Love, 1958c). Since Love did not have freezing
times in the range of 1,000 minutes, peak D could have been missed.

He suggested the damage was caused by the movement of both fibers and
intercellular ice crystals during temperature changes.

The decrease in destruction of the tissue with freezing times in
excess of those causing peak D was believed to be the result of the
fibers forming clumps with very slow freezing. Thus, the fibers in the
middle of the clumps were protected from ice damage by the surrounding
fibers (Love, 1958a). The larger fiber clumps, formed during increased
freezing times, protected the inner fibers from damage.

The occurrence and dominance of the extensive cell damage occurring
at peak 0 seemed to be of considerable importance. Peak 0 was believed
to be the result of a severe solvent effect on the cell walls by the
resulting concentrated salt solutions. The fiber clumping suggested

by Love (1955a) for very long freezing rates was not sufficient to

IL\
‘L'\

offer protection of enough individual fibers, allowing considerable
protein denaturation. The protein denaturation reduces the ability
of the proteins to reabsorb the fluid, increasing the amount of drip
resulting in excessive leaching of the tissue materials.

According to Bouton _t 21. (1957), Sair and Cook (l938b), and
Ramsbottom and Koonz (1940), the amount of drip was affected by the pH
of the meat. In this study. average pH values varied from 5.6 to 6.0
(Table 7). No significant correlation coefficients were found between
pH and the amount of drip among treatments.

The amount and composition of drip were determined to estimate
cell destruction, and results indicate that all frozen tissues were
damaged during the freezing and/or thawing process. These results
agree with those reported by Love (1955a), who found that DNA concen-
tration of expressible fluid from fish was lower from unfrozen tissue

than from tissues frozen at different rates.

Table 7. Relationship between pH of meat and percentage of drip
from broiler breast muscles frozen at different rates

 

 

 

 

 

Freezing
time Drip (L) EH of meat

(min) Average Range Average Range
Unfrozen 2.8 2.7-2.9 5.8 5.8-5.9
0.5 3.1 2.9-3.2 5.9 5.805.9

1 2.9 2.7-3.1 5.8 .5.6-5.9

18 3 1 2 5-3.6 5 6 5 6-5 7

35 5 0 4 6-5.3 5 6 5 5-5 8

65 5 0 4 8-5.2 5 6 5 5-’ 8
74 6 1 5 3-6.8 5 8 5 7-5 9
87 8 0 .5 6-10.3 5 8 .5 7-5 9
132 5 1 4 7-5.6 5 7 5 6-5 7
166 4 1 4 1-4.l 6 0 5 8-6 1
225 3 O 2 2-3.7 5 7 5.7-5 8
252 6 2 4 7-7.6 6 0 6 0-6 1
260 5 4 4.6-6.1 5 9 5 8-5 9
321 5 2 4 0-6.4 5 8 5 7-5 9
441 4 5 4 2-4.8 6 0 5 9-6 1
552 4 7 3 2-6.2 6 0 5 9-6 0

l 044 7 5 6 3-8.6 5 6 5 5-5 8
1,494 4 6 2 5-6.7 6 0 6 0-6 1

 

45

 

SUMMARY

Cell destruction, resulting from different freezing times, (time
required for the tissue to pass from 0 to -5°C) was evaluated by
studying broiler breast muscles and the drip obtained after freezing
and thawing. The degree of cell destruction was determined by the
amount of drip released and by total solids, nitrogen and DNA concen-
tration of the drip.

The rate at which drip was released from the tissues during thaw-
ing (16°C) was not affected by freezing times of 2, 291 and 1869
minutes (Part 1). The maximum percent (average of 39.77) of the total
drip was noted approximately 5-1/2 hours after the thawing process was
initiated. A smaller percentage of the total drip was released during
each of the subsequent thawing periods regardless of the rate at which
the meat was frozen.

The pH of the drip declined from all samples collected during each
successive thawing period. The DNA concentration of successive
collections of drip for meat frozen in 291 and 1869 minutes remained
relatively constant. However, the DNA concentration of the drip from
meat frozen in 2 minutes increased with each successive collection.

It was found that cell destruction was not uniformly related to a
change in freezing times from 0.5 to 1494 minutes (Part 11). In
general, increased freezing times resulted in greater cell destruction,
up to 1044 minutes, after which a decrease in cell destruction was
found to occur. However, several exceptions were noted. Cell des-
truction was relatively severe in tissues frozen in 87, 252 and 18—35
minutes, and relatively low for tissues frozen in times of 1 to 18

minutes, 132 to 225 minutes and longer than 1044 minutes. The unfrozen

46

1.x
\1

muscles had less cell damage than did the frozen tissues.

Results of this study indicate that more attention should be given
to freezing rates for poultry meat. Loss of nutrients, cell des-
truction, and amount of drip released are all affected by specific
freezing rates, and neither increasing nor decreasing the freezing rate

will assure minimum cell damage.

L\

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APPENDIX A
DNA RECOVERY

To determine the percentage of DNA recovered from drip, a known
amount of DNA was added to drip containing a predetermined amount.

The following results were obtained:

 

 

Initial DNA DNA
concentration added Total DNA DNA recovered
(mg/m1) (mg/m1) (WE/W12 (mg/m1) Percent
0.0232 0.0081 0.0313 0.0281 89.78
0.0265 ' 0.0081 0.0346 0.0315 91.04
0.0265 0.0081 0.0346 0.0322 93.06
0.0232 0.0081 0.0313 0.0282 90.09
0.0242 0.0081 0.0323 0.0291 90.09
0.0242 0.0081 0.0323 0.0289 89.47

Av. percent recovery = 90.61
The average recover of DNA from the drip was 90.6 percent with

a range from 89.5 to 93.1 percent.

U1
45

APPENDIX B
ENZYMATIC ACTIVITY DURING THE THAWING PERIOD

Drip was collected from two groups of birds in the normal manner,
and in a container in an ice water bath for the other two groups. The
ice water bath was used to minimize enzymatic activity in the drip

during the long collection period. The following results were

 

 

obtained:
DNA content

Treatment Drip collection method (mg/m1)

2A In ice-water bath 0.0134

28 In ice-water bath 0.0131

Av. 0.0133

3A Collection in normal manner 0.0132

38 Collection in normal manner 0.0138

Av. 0.0135

‘C

No DNA destruction was observed during thawing; however, destruc-
tion could have occurred in the meat, or in the drip before it reached

the collection container.

APPENDIX C.

STABILITY OF DNA

Drip, collected in the normal manner, has allowed to stand an
additional 18 hours at 16°C and was then analyzed for DNA content. The

following results were obtained:

 

 

 

 

 

Initial DNA DNA DNA recovered after

concentration added Total DNA an additional 18 hrs at 165C
(mg/ell (mg/n1i_ (mg/n1) (mg/ml) Percent
0.0243 -- 0.0243 0.0220 90.53
0.0243 0.0081 0.0324 0.0301 92.90
0.0234 -- 0.0234 0.0209 89.32
0.0234 0.0081 0.0315 0.0291 92.38
0.0196 -- 0.0196 0.0178 89.79
0.0196 0.0081 0.0277 0.0259 93.50
Av. DNA recovered with no DNA added = 89.9L
Av. DNA recovered with 0.0081 mg added per m1. = 92.9}

pH of drip before adding DNA = 5.78

pH of drip plus 0.0081 mg per m1 DNA added = 5.81

 

These results indicate that approximately 10 percent of the DNA was

lost during the additional 18 hour standing period.

APPENDIX D

Table 1. Moisture content of broiler breast muscles after different
freezing treatments

 

 

Freezing time Percent moisture
01101 of meat
unfrozen 75.5
0.5 75.1
1 75.0
18 74.3
35 76 2

0‘
U1
\J
b'l
KC

74 75 5
87 75 3
132 75 3
106 74 4
225 74 6
252 75 1

[Q
C‘
O
\1
U‘I
O O

321 75

441 7a 5

552 75.8
1.044 74.5
1,494 — 75.4

Table 2. Solids content of drip from broiler breast muscles which
had been previously frozen in different tines

 

. Total solids
FreezIng .

 

time Averagei/ Range 9 Released

(min) (g/ml) per 100g tissue
unfrozen 0.1028 0.1004-0.1047 0.237
0.5 0.1418 0.1387-0.1449 0.438
1 0.1338 0.1298-0.1377 0.385
18 0.1298 0.1215-0.1381 0.396
35 0.1122 0.1083-0.1163 0.552
65 0.1173 0.1152-0.1194 0.585
74 0.1215 0.1148—0.1282 0.739
87 0.1151 0.1121-0.1176 0.913
132 0.1286 0.1267-0.1306 0.672
166 0.1276 0.1202-0.1348 0.529
225 0.1285 0.1254-0.1314 0.380
252 0.1325 0.1302-0.1348 0.816
260 0.1320 0.1287-0.1351 0.714
321 0.1348 0.1284-0.1412 0.699
441 0.1308 0.1131-0.1487 0.596
552 0.1316 0.1306-0.1322 0.603
1,044 0.1535 0.1509-0.1559 1.132
1,494 0.1458 0.1381-0.1553 0.657

 

l/Average of 4 determinations

58

Table 3. Nitrogen content of drip from breast muscles which had
been previously frozen in different times

 

Freezing

 

time AverageL/ Range rg Released

(mini (mg/m1} ,LDg/hlt Aper 100 g tissue

unfrozen 1.47 1.44—1.48 3.39

0.5 2.06 2.04-2.13 6.34

1 _ 02 I 92-2 17 7 82

18 1 93 1.81-2 10 5 92

35 1 97 1 75-2.38 9 69

65 1 73 1.65-1./6 8 60

74 1 84 1 76—1 87 11 18

87 1.66 1.63—1.68 13.18

132 1.89 1.80-1.98 9.85

166 1.79 1.66—1.92 7.40

225 1.88 1.78-1.98 5.56

252 1.92 1.86-2.01 11.81

260 1.85 1.80-1.91 9.99

321 1.91 1.84-1.94 9.91

441 1.80 1.52-2.04 8.19

552 1 77 1 71—1.87 8.13

1 044 2 22 2.16—2 33 16.38

1,494 2.09 l 95-2.23 9.44

 

I ,. . .
—/Average of 6 determInatIons

Table 4. DNA content of drip from broiler breast muscles which had
been previously frozen in different times

DNA content

 

Freezing

 

time Averagel/ Range mg Released

(min1 [mg/ml1 ,ng/ml) per 100 9 tissue

unfrozen 0.0110 0.0105—0.0115 0.024

0.5 0.0160 0.0159-0.016l 0.049

1 0.0144 0.0135-0.0152 0.043

18 0.0173 0.0148-0.0198 0.051

35 0.0118 0.0108-0.0130 0.059

65 0.0128 0.0118-0.0137 0.064

74 0.0180 0.0156-0.0202 0.108

87 0.0120 0.0105-0.0135 0.095

132 0.0125 0.0120-0.0137 0.065

166 0.0129 0.0119-0.0136 0.053

225 0.0132 0.0130-0.0135 0.039

252 0.0201 0.0186-0.0216 0.124

260 0.0204 0.0164—0.0235 0.110

321 0.0178 0.0168-0.0192 0.092

441 0.0175 0.0143-0.0208 0.080

552 0.0182 0.0176-0.0190 0.083

1.044 0.0162 0.0128—0.0183 0.122

1.494 0.0121 0.0111-0.0139 0.051

 

I . . .
—/Average of 4 determInatIons

60

‘-\“"‘_ v.“

 

 

 

II
'1'
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