CHEMICAL AND PHYSICO-CHEMICAL CHANGES IN
LIPIDS AND OTHER CONSTITUENTS OF
FREEZE-DRIED RAW BEEF DURING STORAGE
UNDER MODIFIED ATMOSPHERES

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
Mohamed Ibrahim EIeGharbawi

I964

THESIS

     
   

  

L 1 B R A R Y
Michigan State
‘ « University

This is to certify that the ,
thesis entitled
CHEMICAL AND PHYSICO-CHEMICAL CHANGES IN

LIPIDS AND OTHER CONSTITUENTS OF FREEZE-DRIED RAW BEEF
DURING STORAGE UNDER MODIFIED ATMOSPHERES

presented by
Mohamed Ibrahim El-Gharbawi

has been accepted towards fulfillment
of the requirements for

PhoDo degree in FOOd SCience

Major professor

 

Date June 10, 1964

 

0-169

" .560 2-0 ;.::. .'.

CHEMICAL AND PINSICO-CHEMICAL CHANGES IN
LIPIDS AND OTHER CONSTITUEN'I‘S OF FREEZE-DRIED RAW BEEF

DURING STORAGE UNDER mDIFIED ADDSPHERES

BY

Mohamed Ibrahim El-Gharbawi

A THESIS

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

DmTOR OF PHILOSOPHY

Department of Food Science

1964

To

My parents who offered me the best in life, education.

My teachers who gave me part of their knowledge and experience.
My wife who was the stimulator to the success.

My children, Eihab. Einaa, Eiman, Haha and Anal.

ABSTRACT
CHEMICAL AND ms ICC-CHEMICAL WES IN

LIPIDS AND CHEER CONSTITUENTS OP FREEZE-DRIED RAW BEEF
mm STORAGE “DER mnnrrn ADDSPEERES

by lbhaned Ibrahim Bl-Gharbawi

The Saninenbranosus. Sanitandinosus and Biceps-femoris muscles of
14-16 month old shorthorn steers were chosen as raw material for this
investigation. The muscles were closely tri-sd of external fat and
were sliced into 1 x l x 1/2 inch cubes. The meat cubes were then quick
frozen to ~40'r. the frozen meat was dried in a Stokes freeze-dryer at
35": and 0.15 ass of Hg for 24 hours. The dried neat containing 2.85%
of its weight water, was packed in lo. 2 lacquered cans. The cans
were hermetically sealed. The and scans of all cans were protected by
a continuous film of Wood's natal to ensure gas tightness of the con-
taineres

the samples were stored for 6 months in nitrogen atmospheres con-
taining 0, 0.1, 0.5 and 2.01 oxygen and at room temperature. At intervals
of 4-6 weeks, samples were removed from storage for analysis.

The solubilities of the nitrogeneous coupoundl in water and in
0.5 3 [Cl solutions were determined by nicro-Kjeldahl method. Generally,
a decrease in total soluble nitrogen, soluble protein nitrogen and
soluble non-protein nitrogen was noted with increasing the storage tins
and the initial concentration of oxygen in can atnosphere. Also. the
free amino groups of freeze-dried raw beef decreased during storage and
the change was dependent on both initial oxygen concentration in storage
smosphere and time.

Dried beef tissue lipids were fractionated into neutral fat and

ii

iii
Mohamed Ibrahim El-Gharbawi

phospholipids by silicic acid. Since onidative degradation of the un-
saturated fatty acids was expected to be the most damaging effect of
storage, the fatty acids composition of each fraction was investigated
by gas- liquid chromatography. The polyunsaturated fatty acids make up
6.12 and 33.811 of the neutral and phospholipid fractions, respectively,
in freeze-dried beef muscles. Oxidation of tissue lipids seems to be
a two-step process; the phospholipids are oxidized first and the neutral
fat autoucidises somewhat later. Also. it was noticed that there was a
very marked loss of the unsaturated fatty acids in the phospholipid
fractions after a short time of storage. whereas those components dis-
appeared more gradually in the neutral lipids of dried beef.
Determination of volatile flavor substances was based on distillation
with steam at ordinary pressure using a nitrogen stream and cooling
traps. An attempt to classify the flavor compounds and to follow the
changes in the flavor spectrum was carried out using both gas-liquid
chromatography and chemical analysis. At least fifteen volatile components
were resolved by gas-liquid chromatography when the steam volatiles of
freeze-dried raw beef were injected directly to the fractionation colt-n.
Acetaldehyde, propanal, hexansl, pentanal. acetone, methyl-mercaptan and
methyl disulfide were tentatively identified. Hydrogen sulfide was
identified in meat volatiles by chemical analysis. It has been found
that volatile compounds increased in both number and concentration in
all samples stored at room temperature and under nitrogen or oxygen-
nitrogen atmospheres. Moreover, the change in the volatile cowounds
was dependent on the initial oxygen concentration in the storage atmos-

phere and on time.

iv
Mobs-ed Ibrahim Bl-Gharbawi

A substantial increase in reducing compounds and fluorescing sub-
stances in the freeze-dried raw beef extracts was observed during
storage at room temperature. The water uptake and heat of rehydration
of the stored dried beef was determined. Both characteristics decreased
by increasing storage time and the initial oxygen in the storage atmos-
phere.

All samples. including those stored under purified nitrogen
showed oxidative changes. Loss of the light pink color of oxyhenoglobin
was noticed in the stored meat. Head space gas was analyzed for oxygen-
nitrogen ratio during storage. Results showed that freeze-dried neat
adsorbs oxygen which may be desorbed during storage. Thus. releasing
the vacuum in the freeze-drier with purified nitrogen and improved
packaging Operation are recomended for increasing the shelf-life of

freeze-dried raw beef.

ACKNOWLEDGEMENTS

My sincere gratitude to Dr. L. R. Dugan for suggesting the
problem. effective guidance and for his suggestions in regard to the
inrovelant of the manuscript. I an also indebted to Dr. D. A. Arata.
Dr. 1.. 8. Dawson. Dr. P. liarkakis. Dr. R. J. Evans and Dr. R. w. luecke
who served on the guidance co-ittee and critically reviewed the
manuscript.

The author greatly appreciates the encouragement of
Dr. D. s. Schweigert. Chairman of the tood Science Department.

Thanks also go to Mr. R. Bradley for his help in gas-liquid
chromatography analysis.

The excellent typing and long hours spent carefully preparing the

manuscript by Miss Dee rang is gratefully appreciated.

TABLE OF CONTENTS

Page

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . ii
Acknwledse-ents.....-................ v
Listofl’isuru.......................viii
Listof‘rables-...................... x

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . l
11. LiteratureRevieV...................... 5
m.Experimental........................19
A.Material........................19

B. methods . . . . . . . . . . . . . . . . . . . . . . . . 20
Freeze-dryins.......-...............20
Preparation for storage. , . , . . . . . . . . . . . . . 21
Gas-tightness of containers , , , , , . . . . . . . 21
Replacement of air inside cans‘with desired atmosphere 21

Preparation of nitrogen compound extracts. . 0 , . , , . 22
Waterextract....................22

Soluble nitrogen . . . . . . . . . . . . . . . . . . 23

Soluble protein and non-protein soluble nitrogen . . 23
Aminonitrosen 23

Extraction of lipids . . . . . . . . . . . . . . . . . . 24
Fractionation of crude lipid extract , , , . . . . . . . 25
Preparation of silicic acid column , , . . . . . . . 25

Neutral and phospholipid fraction . . , , . . . . . 25

Fatty acid preparation . . . . . . . . . . . . . . . 25

Methyl ester preparation. . . . . . . . . . . . . . 26

Prep‘r‘tim Of re.1n C O O O O O O O O O O O O O 26

vi

vii

Esterification e e e e

O O O 0 O

Gas-liquid chromatography . . . .

Hematin compounds . . . . . . .
Reducing compounds . . . . . .
Fluorescence determdnation . .
Rehydration . . . . . . . . . .
Best of rehydration . . . . . .
Head space gas composition . .
Flavor volatiles . . . . . . .
IV. Results . . . . . . . . . . . . . .
Freeze-dried raw beef . . . . .
Water extract . . . . . . . . .
Soluble nitrogen . . . . . . .
Soluble protein and non-protein
Free amino nitrogen . . . . . .
Lipids extraction . . . . . . .
Methyl esters . . . . . . . . .
Hematin compounds . . . . . . .
Reducing compounds. . . . . . .
Pluorescing compounds . . . . .
Rehydration . . . . . . . . .
Heat of rehydration . . . . . .
Head space gas analysis . . . .
Flavor volatiles . . . . . . .
V. Discussion and Conclusions . . . .

VIe Blbllogtlphy e e e e e e e a e e a

nitrogen

O O O O O

Page
26
27
28
29
29
30
3O
33
36
40

40
40

48
48
52
58
58
59
59
62
65
72

81

Figure

2b

xi

10

11

LIST OF FIGURES

Design of the adiabatic calorimeter used for
determining of heat of rehydration of freeze-
dried t‘" b..f0 e e e e e e e e e e e e e e e e e e e

Design of the parallel column system used for head
.p‘ce 8‘. .n‘ly.1’0 e e e e e e e e e e e e e e e e e a

Design of the head space gas sampling device. . . . . .

The set up used for isolation and trapping flavor
compounds of freeze-dried raw beef. . . . . . . . . . .

Changes in water extract nitrogen during storage
of freeze-dried raw beef at room temperature and
under different oxygen-nitrogen atmospheres. . . . . .

Changes in total soluble nitrogen (0.5 3; KCl) during
storage of freeze-dried raw beef at room teqeratures
and under different oxygen nitrogen atmospheres. . . .

Changes in soluble protein nitrogen (0.5 g 101) during
storage of freeze-dried raw beef at room temperature
and under different oxygen-nitrogen atmospheres. . . . .

Changes in soluble non-protein nitrogen (0.5 3 [(61)
during storage of freeze-dried raw beef at room
temperature and under different oxygen-nitrogen
atmospheres. ......................

Changes in the free amino groups of freeze-dried
raw beef. determined by formal titration. during
storage at room temperature and under different
oxygen-nitrogen atmospheres. . . . . . . . , . . . . .

Gas-liquid chromatographic analysis of methyl esters
from neutral lipids and phospholipid fractions of
freeze-dried raw beef stored at ~40'P. . . . . . . . .

Changes in polyunsaturated fatty acids in the

neutral lipid fractions of freeze-dried raw beef

during storage at room temperature and under

different oxygen-nitrogen atmospheres. . . . . . . . .

Changes in polyunsaturated fatty acids in the
phospholipid fractions of freeze-dried raw beef

during storage at room temperature and under

different oxygen-nitrogen atmospheres. . . . . . . . .

viii

Page

.42

figure
12

13

15

l6

17

18

19

20

The absorption spectra of aqueous extracts of
freeze-driedrawbeef. ,,.,.,.,,,,,,,,

Change in the absorbanca maxima at 5‘0 and 580 m of
water extracts of freeze-dried beef during storage
at room temperature and under different oxygen-
flitt”“.m.’h.t..e eeseeeeeeseeees

Product ion of reducing coqounds during storage of
freeze-dried raw beef at room temperature and under
different oxygen-nitrogen atmospheres. . . . . . .

Change in fluorescence during storage of freeze-
dried raw beef at room temperature and under
different oxygen-nitrogen atmospheres. . . . . . . .

Changes in rehydration capacity of freeze-dried
raw beef during storage at room tsqsrature and
under different oxygen-nitrogen atmospheres. . . . .

Changes in heat of rehydration of freeze-dried
raw beef during storage at room temperature and
under different oxygen-nitrogen atmospheres. . . . .

Changes in head space oxygen during storage of
freeze-dried raw beef at room temperature and
under different oxygen-nitrogen atmospheres. . . . .

Gas liquid chromatograms of flavor volatiles of
freeze-dried raw beef stored at do!) ‘c and room
temperature for six months under purified nitrogen.

Gas liquid chromatograms of flavor volatiles of
freeze-dried raw beef stored at room temperature

and under an atmosphere of 2?. oxygen for four and
sixmthaofstorngt.................

Page

56

57

60

61

63

66

67

69

Table

1.15! OF TABLES

Page

Some chemical and physico-ehsmical characteristics
of freeze-dried raw beef stored at -40’P and under
anatmosphereofnitrogen- .. .............. 41

Analysis of methyl esters of fatty acids of

neutral and phospholipid fractions of freeze-dried

raw beef stored at «40’! and under an atmosphere

OfnitrOSCne seesaesseeeesessesesseSl

INTROWCTION

Since man first killed animals and harvested plants. he has had the
problem of keeping the food from day to day and fru season to season.
For sass thousands of years. therefore. he has had recourse not only to
fresh foods. but has resorted to processing foods to varying degree and
in different ways.

Drying. probably the oldest form of preservation, developed as a
practical art which in fact deprived the microbial population of the
water necessary for growth and production. In practice. fish have been
split and slowly dried in the wind; grain has been dried; and products
such as panican has been made fra meat. cut thin and dried in the sun.
for many thousands of years. British-Peed Imnufacturing Industries
Research Association has four thousand year old suples of dried foods
from Jericho. Seven hundred years ago liarco Polo found the Tartars
drying milk in goatskin bottles in the smoke of their cmspfires. The
process used by the seventeenth century American Indians for making
pal-iota by drying lean mat in the sun. powdering it and mixing it
with melted fat was adopted by the early settlers and later by Arctic
explorers.

a greater utilization of dehydrated meats would be desirable for a
mater of reasons. The msonal variations in supply and price of
fresh meat. which are interrelated. could be leveled out to a great
extent if a portion of the production could be dried during the time of
large supply. that storage costs would be decreased by dehydration since
the need for expensive refrigeration would be decreased. Purther
savings would be made in the cost of transportation by elimination of

l

2

50 to 752 of water found in fresh mat. Opportunities also exist for
the use of dehydrated meat in isolated areas or in tines of emergency
when normal supply is limited.

The dried neat which is available in the meat nrkets today is a
salted. usually snoked. air dried product. It bears little flavor or
textural resemblance to fresh meat.

The techniques of freeze-drying have been developed over the past
half century for the purpose of preserving certain biological materials
which are costly to produce and which are highly unstable. Perhaps its
nest noticeable success has been in the preservation of hunn plane
for transfusion purposes and in the preservation of early samples of
penicillin produced during World war II. Today. it has nuaerous
applications in the pharnaceutical industry. but though the product
of the various plants is adequate for these particular purposes. it falls
far short of requirements for the food industry.

The cost of the process is still too high to make freeze-drying a
practical proposition for many of the cheaper foods. Nevertheless.
with the advances of vacuun techniques and increasing knowledge of the
process of freeze-drying. it should be possible to design and build
apparatus of any desired output at a coqetitive price. though, as yet.
the truly continuous process. as distinct free a continuous batch
process, has not been suitably developed.

No special difficulty is found in freeze-drying raw beef steaks.
chops or stew neat (236). The meat has a light pink color and essentially
the sane volune as when fresh. The structure of the dry meat is some-

what similar to that of balsa wood. although more friable. (h

3

rehydration. by i-ersion in water. freese-dried meat takes up to 901 of
the original weight of water removed. After cooking. the flavor of the
froeae-driod product is practically ‘ indistinguishable from fresh frosen
neat. but the texture sons to be slightly drier. Thus the rehydratod
frosts-dried product has most of the desired characteristics of a fresh
nest in terns of acceptability and versatility.

Freeze-dried. raw and precookod beef undergoes oxidation very
rapidly when stored in air (237). However. enidative deterioration nay
be largely eliminated by packaging in an inert gas or under a good vacuum.
Non-enidative deteriorative reactions then become the important practical
factors in storage stability. Studies on precoeked dehydrated most
have indicated that active carbonyl-nine browning is probably the nest
inertant factor in the storage stability of that product (219).

The main advantage to be sought from freeze-drying of foodstuffs
is inproved preservation of the product over an indefinite period. even
at tropical tenperatures. This roquirenent arises from the current
foroeoable need of an 'instant nsal' made up of procooked freeze-dried
food. in light weight plastic. to which hot water nay be added to pro-
duce a full and attractive noel. This appears to have advantages for
space flights. the highly mobile field forces of modern arnies. and nay
as well also have an impact on civilian life.

The purpose of this study is four-fold: To characterise sons of
the axidative and non-oxidative deteriorative changes in the major
coqonents of frosts-dried raw beef. to ascertain the effect of oxygen
on the quality of the product by increasing the initial level of oxygen
in the headopace. to correlate different changes and products. and to

4

develop ideas for possible control of the deterioration to give a

product of increased storage stability.

LITERATURE

Freeze-Dug. Sir John Leslie. best known for his use of the
"Leslie cube" scans to have been the first to make a scientific study of
freezing by evaporation and of the subsequent sublimation of the ice.
This was in 1811 (161). He described the disappearance of ice. without
melting. in an evacuated vessel in which the water vapor pressure was
kept low by sulfuric acid.

According to Flosdorf (60) freeze-drying was first used by
Schackell in 1909. The reduction of the technique to a practical process
did not come until 1935 when llosdorf and Md modified the procedure to
operate with the tenperature of the heating surface above freezing (S9).

The possible consrcial application of freeze-drying of food was
described by Gene (74).

The histological characteristics of freeze-dried meat have been
extensively studied by many investigators (252, 253, 254). A process
for the preparation and some of the properties of freeze-dried raw beef
have been described by Tappel 3_t_ :1. (239). The freeze-drying of cooked
meat has also been studied to some extent (10). Harper and Tappel (96)
have extensively reviewed the freeze-drying of foods. Recently. many
scientists (88, 93, 96, 97, 105. 128. 146. 165) discussed the various
aspects of freeze-drying technology.

Deterioration of Dehydrated Food Products Burg Storage. Con-
taninstion. damage. or infestation by rodents or insects could be

 

serious problens with dehydrated foods. The causes in these cases are
relatively obvious and will not be dwelt upon further.

Deterioration of dehydrated food products due to the growth of

5

6

nicroorgenions is not col-non. The critical level of noisture content
for growth of nicroorganinss is that which is equivalent to about 651
relative humidity (167).

Extensive work has been done on the deterioration of dehydrated
fruits and vegetables (69. 232). Nearly all of this work has been di-
rected at elucidation of the mechanina of. and nethods for, the control
of the browning reaction.

The studies on the deterioration of milk have shown that even in a
relatively dilute system such as evaporated milk. the browning apparently
proceeds at a significant rate at 90’! (226). It has also been known
that there is a decrease in the biological value of non-fat dry nilk
powder during storage (103. 136).

Probably the greatest mount of work on the storage stability of
dehydrated foods has been on eggs. attensive study has been node of
the relationship between palatability and the fluorescent material which
is forned during storage (18,156). It was found in these studies that
the salt water soluble fluorescent material was produced by the brownim
reaction (54). A comprehensive investigation of the effects of a nuber
of variables established the deterioration nechanisn as an' active aldehyde
anine browning reaction (132, 133).

No detectable change wns found in the biological value of air
dried neat during storage (107. 139). The thianine of dried pork was
found to be sensitive to oxidation during storage. The losses of
thiamine were extensive at temperatures above 70’! in eight weeks. The
other 3 vitanins were apparently quite stable under the same conditions
(188).

7

It has been stated that the initial brown color that develops during
storage of dried nest is usually nasked by the natural pigments of the
nest during the early stages of browning (81). This browning was found
in dehydrated light colored pork. When the sugar was fermented out of
the neat juice. this color development was reduced (81).

The removal of the free sugar (glucose) by yeast or by an enzyme
system was shown to decrease the rate of off-flavor development in pre-
cooked dehydrated pork (101. 102). The yeast when used at levels of
lore than 101 inarted a characteristic flavor of its own to the product.
The nest effective removal of the reducing sugar was found to occur when
the neat was precooked and finely ground.

The effects of several storage atnospheres on the storage stability
of dehydrated precooked beef and pork were satanined by Whitmore gt _a_l_.
(256). They found no significant difference in free fatty acids.
peroxide value, or palatability between material stored in air, vacuua,
or nitrogen. It is probable that those precooked meats dehydrated at
relatively high temperatures have high free fatty acids. some peroxides
and a strong flavor due to browning. Therefore. oxidative and other
deteriorative reactions caused by storage in air are not readily
apparent. 0n the basis of spectrophotonstric reflection curves, they
concluded that the red-brown color that developed during storage was
not due to hone pignsnt, or bone pig-ant products. The storage toner-
ature was found to be the most important factor in the rate of deterior-
ation of dehydrated neat (204. 245).

Terr has studied the browning of white fish during heating (242.
243). He concluded that the cause of browning was a free ribose-protein

8

reaction. The free ribose apparently comes from the enzymatic hydrolysis
of nucleic acids during storage at o'c prior to heat processing.

The browning of freeze-dried extractives fru fish were studied by
Jones (124). He found traces of glucose and galactose. but no free
ribose. The maximal: rate of browning was at pH 7.0. A change of 10°C
in the reaction conditions caused about a five-fold change in the rate
of browning.

The storage of precooked freeze-dried pork has been extensively
studied by Sharp (219,220). He found that the deterioration of his
product. in the absence of oxygen. appeared to be a carbonylamine
browning reaction. He studied its rate of formation as a function of
moisture. pa. tenperature. and concentration of reactive sugar. As
measured by the brown color formed. 57! relative lumidity (a treasure
of the activity of water) caused the maximal rate of deterioration.

Sharp (220) found that glucose-é-phosphate is responsible for a
large share of the browning that occurred in his material during
storage. In his system. fermentation by yeast. or additim of a fairly
large amount of sulfur dioxide (500 ppm) coupletely inhibited the forma-
tion of the brown color. The Q10 of the rate of loss of free fer-ent-
able sugar and the development of brown discoloration. over the range
of 15’ to 250‘c were fond to lie between 3.2 and 4.3.

Nearly all of Sharp's work was with pork which had been ground as
well as precooked before drying. The cellular material was thus brought
into intimate nixture before dehydration. and sons of the possible
chemical reactions not have been started. if not conpleted. before the

meat was dry.

9

Stor_a_ge of Freeze-Dried new leaf. Lee (147) made one limited

observations on the storage characteristics of freeze-dried beef in 1943.
He noted the development of a paints-like odor on storage in atmosphere
containing air. In the absence of air a fishy odor developed which was
mostly removed on rehydration and cooking. When a very low moisture con-
tent material was stored. the intensity of the fishy odor was much less.
His work pointed up the need for storage of freeze-dried meat in an
inert atmosphere in containers which are impermeable to aygen and water.
Tappel (237) has shown that the oxidation of the extractable lipid
material can ‘ account for only about one-half of the oxygen uptake of the
freeze-dried meat. A major portion of the oxygen uptake was ascribed to
the oxidation of the protein. The hemetin compounds were shown to be
deoxygenated during the freeze-drying process. During storage these
piments undergo oxidation to "mot" forms which are brown in color.
Oxidative carbonyl-mine browning was shown not to be responsible for
the oxygen absorption. Freeze-dried purified proteins as well as
freeze-dried most were found to show significant oxygen absorption.
Regier and Tappel (203) studied the non-oxidative deterioration of
freeze-dried raw beef stored in nitrogen atmosphere at controlled tenor-
ature and water content. . The effect of towerature. p11. various
additives. water content and the volatile materials on the rate of
deterioration were also investigated. The apparent activation energy
for the deterioration during storage was found to be 25 Keel/mole. The
caer cmnpounds were found to be the apparently limiting reactants
in the deterioration. Lowering the p11 causes a decrease in the rate of

deterioration.

10

It is concluded by Regier and Tappel (203) that active carbonyl-
amins browning is the major non-oxidative deterioration mechanism. The
volatile compounds produced in the deterioration of freeze-dried beef
are probably end or side products of the deterioration and apparently
do not enter into further reactions.

The effect of water and tqerature on the deterioration of freeze-
dried beef during storage was studied by Thomson. Fox and Landmenn (246).
A study of the effect of 1.7-5: moisture content in freeze-dried beef
stored at 0. 68. and 90’!I showod appreciable deterioration with both in-
creasing moisture and teensrature of storage. They concluded that
freeze-dried meat must contain less than 21 moisture to keep well. This
conclusion holds when the effect of temperature is considered. Moisture-
temperature interact ions were found to be significant in most of the
factors analyzed statistically. and the sample at 1.72 moisture stored
at 68 and 90‘! showed mch loss deterioration than those at 3.9 or 5.1%
moisture. The estimated muount of oxygen taken up at the various
moisture and tenerature levels was only 1/6-113 the total reducing
capacity of the eagles. They concluded that oxygen taken up could be
escalated for by oxidation of browning reaction roductants.‘ Such a
reaction may be of benefit in the storage of freeze-dried meat. for
previous studies by Kline. g_t_ 3;. (133) and Tarr (263) have shown that
in some cases browning deterioration is greater in nitrogen or in
vacuo than in air.

EM Reactions in Simplified glotems. The effect of amorous
factors and possible reaction mechanimss for browning in model systems
were thoroughly reviewed by Hodge (109) .

11

The process of browning in siqle systems such as amino acids and
aldoses forms reductones. Schiff bases. and other intermediate coqoundo
which are more readily oxidised than the aldoses (107.151.207.259).

This effect has also been observed in slightly more complicated systems
such as isolated proteins and sugars (154, 255). Those reducing com-
pounds are generally considered to be reductones which are intermediates
in a sequence of react ions eventually leading to brown insoluble poly-
noric materials.

As browning proceeds in model systems. there is a loss in free
amino groups due to the formation of l-glycesylamines. The latter then
apparently undergo further transformations which irreversibly tie up
the nitrogen in a non-amino form (35.161.151.216).

It has been shown that when the broning of a mixture in proteins
and aldoses occurs. there is a decrease in the solubility of the pro-
teins (153.156). This could be due in part to the decrease of the
amber of free amino groups which are strongly polar. or to the in-
crease in molecular sise caused by the formation of polymers character-
istic of the browning reaction.

The effect of temperature on the rate of browsing has been studied
in a amber of different systems. naugaard. Tumsrnnn and Silvestri
(98) found the activation energy for the developent of the brown color
to be 19.7 Kcal. for o leucine-glucooe system. In a casein-glucose
system. Lea and assess (153) found the loss of nine-nitrogen to have an
apparent activation energy of 29,000 Cal. Similarly. Pearce (195) found
the development of fluorescence in glucose-glycine and in egg powder to
have an apparent activation energy of 26.0 Kcal. With bovine sort-

12
albuain in glucose. unbound. Traakel-Conrat and Olcott (181) cal-
culated the activation energy for browning to be 30,300 Cal. Wolfrom.
Kolb ad banger (260) found the activation energy to be 20,000 Cal. in
a glycine or alanine with D-xylooo system.

When free sugars and amino groups are present in food products.
additives such as bisulfite. which combined with the carbonyl group.
has been shown to have an inhibitory effect on browning. Triodman and
Kline (72) found that sodium bisulfite prevented color formation in
mixtures of 12 different amino acids with glucose. Acetylation of
casein before drying and storing it with glucose was found by Lea.
llannan and Rhodes (156) to cause a significant decrease in the darken-
ing of the system. Kretovich and Tohareva (140) found no color develop-
ment in sugar-amino systems when the aldehydes were tied up by the
addition of dimedone.

The dependence of the rate of browning on the p11 of the system is
well known (1”). In a casein-glucose system. Lea and Batman (150)
found a nearly linear relationship between the rate of browning (loss
of free amino groups) and the pi over the range of pH 3 to pH 8.
W. Praenkel-Conrat ad Olcott (181) found a linear increase in
the log of the initial reaction rate with an increase in p11 in the
browning of a bovine serum albumin-glucose syston. Wolfrom's group
(260) found that the browning of amino acid-eldose mixtures exhibited
strong base catalysis at pH 6.5 to pH 8.5 or weak base catalysis
between pH 3 and pa 5. and acid inhibition in the pa 1 to 3 range.
Robert and Ponaranda (208) noted that the reaction between glycine.
hiotidino. lysine or serine and acetaldehyde at 37 '0 in th. “1‘“ 20 ‘0

13
to 60 minutes appears to be bimolecular at pl 7. At other pn's. the
reaction appeared to be of an order higher than 3. This is attributed
to the appearance of several reaction products which are capable of
reacting with acetaldehydo. They conclude that the effect of pa shows
that the anionic form of the amino acid reacts with acetaldehydo.
Kretovich and Toharova (140.) noted that the browning of amino acid-
aldose mixture is accmspanied by formation of furfural and other vola-
tile aldehydes. Speck (231) identified pyruvaldehyde ad diacetyl in
the distillatos from solutions of sugars and amino acids. Other
volatiles were indicated (acotal-glycolaldohyde) from the periodate
oxidation sum... of the distillates.

W. The bright red color of fresh meat is due to the
chemical state of pigment called myoglobin. which represents 902 of the
total pigsonts in muscles. moglobin differs in chaical structure and
reactivity from blood pipent. hemoglobin. It is more difficult to
obtain this pigment in sufficient state of purity comparable to hemoglobin
although both have been crystallized (77). liyoglobin canbinos reversibly
with oxygen. to form bright red oxymoglobin. In this coqoumd. the
iron rennins in the ferrous form. Also. it may lose an electron be-
coming oxidisod to the corresponding brown "met" pigments. Moreover.
the pig-ant may undergo changes of the porphyrin ring to give green or
gray deconposition products.

Two types of oxidativo changes are chiefly responsible for the
abnormal brow. gray and green discoloration. One involves the oxida-
tion of the ferrous ion in the has coaound to ferric state; the

second is the direct attack by oxygen on the porphyrin ring. The most

16
co-only encountered type of discoloration is that of the brown oxida-
tion products. methomoglobin and mstmyoglobin. formed from the noral
muscle pigments by the oxidation of the iron to the ferric state (26. 25).
Also the ferrous form of denatured hemoglobin formed by donaturation of
the protein moiety of hemoglobin under reducing conditions is much more
susceptible to oxidation than is hemoglobin itself (111.158).

In addition to brown and gray discolorations due to formation of
methemoglobin. very objectionable greenish pipsnts may appear in meats.
Loderg and Logge (159) attributed the green color to oxygen attack on
the porphyrin ring. usually at an a methane bridge. It is not essential
that the ring he ruptured at this point. in fact. there is good evidence
that at least two green coqounds. choleglobin (159) . and sulfehoms-
globin. retain their closed porphyrin rings. Further oxidation. involv-
ing opening of the ring with splitting at the a-methano carbon atom.
can then occur (Verdochroms) .

Brown and Tappel (28. 29) reported on oxidative changes in hastin
pipents of meat and fish. The non-extractable brown pig-onto of
cooked beef were identified as mixed denatured globin nicotinamide
honichrues. nernofsky gt _a_l_. (16) studied the donaturation of myo-
globin upon cooking. The pigments extractable from cooked beef con-
sist mainly of oxymyoglobin. Lane and lratzler (162) used spectro-
photometric methods to determine metmyoglobin in frozen meat extracts.
Also Luh _o_§ :1. investigated the effect of aseptic and retort heat
sterilisation processes on hematin compounds in canned strained beef
(166).

In meats which are freeze-dried. oxymyoglobin is apparently

15
deoxygenated while the meat is in the dry state (28). The resulting
myoglobin is oxidized to brown mstmyoglobin.

The development of oxidative racidity in meat has been shown to
cause and to enhance the loss of meat pigments. The hematin coaounds
are dominant catalysts of the free radical chain reaction oxidation of
unsaturated fate to form peroxides and subsequently carbonyls which are
responsible for the rancidity. Concurrently the hematin cosmoumds may
react with the free rad icalo produced resulting in degradation of the
iron protoporphyrin and loss of hematin compound color (28). Both of
these undesirable reactions. oxidativo fat racidity and destruction of
the hematin pigments. can be inhibited by fat antioxidants and reducing
capounds (238).

Stability of Heat Proteins. Insole proteins may be divided into
four groups. according to their solubility: l- Corpuscular proteins,
soluble in dilute salt (of the order of 0.15 g) at neutral pH. They
are largely enzymic in nature and include the heme-proteins. 2- Proteins
of the muscle fibre . so for established as a complex of myosin. actin
and tropomyosin. soluble in stronger salt solution (ionic strength about
0.6). 3- Proteins soluble in 0.1 3 New. and insoluble in high concen-
trations of salt solution. 4- Collagen and elastin. insoluble in 0. l I!
New.

During dehydration, sass portions of foodstuff will dry more
quickly than others (except when dielectric heating is used). The wet
areas will remain ate lower teqerature than the dry. due to evapora-
tive cooling. Proteins are known to be more thermostable when dry tha
when hydrated (13.104) and so it is probable that a portion of them at

16
least will not be damaged by dehydration.

After slaughter, actin and myooin combined to form actomyosin. which
is present in greater quantity in muscle tissue than any other protein
(8). Actomyosin. therefore. is of particular interest. It is rather
labile so its survival of any particular treatment means that many other
proteins and enzymes will also raain undamaged. Actomyosin is itself
an enzyme. for it can hydrolyse adenosine triphosphate to one of the
adenosine diphosphate and inorganic phosphate. Another property of
actomyosin lost on denaturation is its solubility in 0.5 g potassium or
sodium chloride solutions. This characteristic may be used to follow
denaturation of muscle proteins (118).

Deterioration of texture of animal tissues occurs during storage
and it seemed probable that this might be associated with changes in
proteins. For example. it has been found that casein becomes insoluble
when stored in the dry state in presence of glucose (151.153).

The possible survival of enzymes during storage is of interest
since they might contribute to more rapid deterioration in dehydrated
raw beef as opposed to that which has been cooked. Cole and Smithies
(38) have shown that the amount of actomyosin extractable from freeze-
dried beef by normal procedures is only slightly less than that extract-
able fra fresh beef. However. the actomyosin coalex extracted fru
freeze-dried beef appears to sediment faster in the ultracentrifugo than
that extracted from fresh beef (48).

they investigators (63.M.65.162,191) observed that freeze-dried
meat and fish become tougher and drier in texture during storage at

rates which depend on the toqerature of storage. and the moisture content

17

of the seals. There is an agreement among many investigators (63. 167,
162,191) that cross links between the myofibrillar protein is responsible
for the increased toughening and loss of juiciness during storage.

any research workers (36.65.162.191. 266) proved that the major
deteriorative tactics involving proteins of stored freeze-dried beef
was carbonylamine browning reaction. Browning reactions of this type
lead to insolubility of proteins. probably through the introduction of
inter-protein bonds.

zlaver Volatiles. The volatile carbonyl coqoundo believed to be
associated with food flavors have been studied by converting the carbon-
ylo to their 2-6-dinitropheny1-hydrazono derivatives. Identification of
the carbonyls has been accoqlished using paper chromatography and by
melting point determination a the isolated compounds. This technique
has been employed by several investigators (32,113,266) to study the
flavor of neat.

Pippen _o_§ g. (198) separated and identified eighteen separate
carboql compounds present in chicken. Batter _o_t_ g1. (16) reported
the presence of at least four 2-6-dinitrepheny1-hydrazonos in both the
fat and the loan of beef subjected to sterilizing doses of' gene
radiation. lo attempt was made to identify these carbonyl compounds.

Burnett .o_t_ _a_l. (32) workiu with the volatile agenda of vacui-
packed dehydrated pork, isolated and identified acetaldehydo as being
the only volatile carbonyl present in pork samples stored at both ~20?
and 96?. A-snia was detected in eagles. stored at 100’! and 160.2.

Tush and Strong (266) studied the volatile fraction fr. fresh lean
beef cooked in boiling water. Hydrogen sulfide. a-onia. acetaldehydo.

18

acetone and diacetyl were detected. In addition. the presence of formic
acetic. propionic. butyric and isobutyric acid. and dimethyl sulfide was
tentatively established. however. volatile alcohols and esters were
absent.

Bornstein and Crowe (113) isolated from cold wnter extract of lean
beef acetone. propanal. acetaldehydo. hexanal. 2-enala. and 2-4-dienals.

Fortunately. the last few years have brought the practical develop-
ment of powerful new methods of attack on the problemn particularly
in the area of the chemistry of odors.

Gas-liquid chromatography equipped with ultra-sensitive beta ray.
flame ionization. or other sensing devices is capable of detecting
quantities of 10"12 to 10'1‘ mole. rurthermore. the utility of gas-
liquid chromatography in the identification of volatile compounds is
strikingly enhanced by direct coupling with infrared analysis. and
especially by coupling with conventional or tine-flight mass spectrometry.
The latter combination is still so new that its great potentialities

are only beginning to be explored.

EXPERIMENTAL

Material

M. The Semimembranosus. Semitendinosus and Biceps-
fermoris muscles of 14-16 month old ‘ Shorthorn steers were chosen as
raw material for this investigation. The animals were slaughtered in
the Meat Laboratory. Food Science Department. nichigan State University.
The carcasses were aged at 34'! for ten days. The muscles were dissected
from the hindquarters of the animals and were closely trimmed of external
fat. wrapped in laminated freezer paper and chilled at 28‘? for 30
minutes. The meat then was cut into one half inch slices using an
electric meat slicer. The slices were chilled again for. a short period
of time to 20‘s and then were cut 1m. 1 x 1 x 1/2 inch cubes. All
cutting processes were carried out in the cold room at 34'1'. Under
these conditions of preparation of the meat. tissue fluids did not leak
out and no expressed fluid was noticed on the surface. The meat cubes
were then quick frozen to oil-0‘! and stored at this temperature until
they were freeze-dried.

Q micals and Meats. Oxygen-nitrogen gas mixtures were obtained
from )istheson Company. East Rutherford. New Jersey. Diethyleneglycol
adipate (Lac-2-R-446)and chromosorb P and H were made by applied Science
Laboratory Inc.. State College. Pa. Wood's metal was obtained from
Allied Chemical Corp.. New Jersey. The methyl esters used as standards
for gas-liquid chromatography were manufactured by California Corp. for
Biochemical Research. California. The molecular sieves were obtained
from Lida Company. New York. nexuethyl-phosphoro-enide (MA) was

made by Fisher Scientific Co. Apieson l. grease was manufactured by
19

20
intropolitan Vickers Electrical Co.. Ltd.. England. All other reagents
were analytical reagent grade. The water used throughout this investi-

gation was deionised water.

llothods

reaso- . A Stokes Treats-Dryer. liodel 2003-12 (I'd. Stokes
machine Coqany. Philadelphia) was used. This freeze-dryer has four
hollow shelves 13 x 26 inches. The lower two shelves. which are the
condensers. are cooled by a refrigeration compressor using Freon 12 as
the refrigerant. These condenser shelves can also be used for freeaing
the material prior to drying. The upper two shelves are heated by water
which is recirculated frtn a reservoir which is thermostatically
toqerature control led. The froaon material was placed on alumina
foil trays on these upper shelves during the drying process. The
absolute pressure in the drying chamber was maintained at approximately
0.13 ms of mercury by a positive displacement vacuun pump. The pressure
was neaeured with a Stokes-McLeod gauge. During the drying. the con-
denser shelvee were maintained at about o35’C and the heating shelves at
35°C. Ono layer of froaen pieces of raw beef were dry after 24 hours
under these conditions. The dried neat was transferred to a polyethylene
bag which was than air evacuated and sealed. The packages containing
dried meat were stored at 40‘! until all the prepared meat cubes were
dried.

The packages of stored meat were renoved from the cold roon and
permitted to warm to room t-perature before being opened. The dried
pieces were mixed by hand by piling. quertering and repiling several
times. This was necessary to reduce the seqling errors. for there are

21
significant variations in composition between muscles from different
animals.

Preparation for Storage

The material was then divided into different portions of the sise
needed for sample in a particular study and placed in no. 2 lacquered
cans. The cone were hermetically sealed by an automatic sealing
machine at room temperature and at atmospheric pressure.

Etmtness of the Containers. Tinplate cans in which the side
so- ie soldered and the ends rolled on have proved very satisfactory
in co-orce for a great variety of packing purposes. However. in a can
filled with a dry material. no self-sealing of small leaks by fluid con-
tents of the can is possible. The and seams of all cane used in this
investigation were therefore protected by a continuous film of Hood' s
metal. It was applied by rotating the rim of the can in the metal.
melted under a layer of lactic acid (as flux). followed by washing with
water. This procedure produced in effect. an all metal seal. which has
proved completely gas tight (146). Precautions of this nature were con-
sidered necessary to ensure that no oxygen other than that originally
present in the can could possibly gain access to dry material. otherwise
erroneous conclusions might have been drawn.

Misc-sent of Air Inside the Cans with Desired Atmosphere. To
obtain sales with different gas content. a drop of solder was placed
on the top of each can and a mall hole was punched through the solder
and the lid. The cans were placed in a vacuin chamber and this was
evacuated until the licheod gauge showed 100 p of pressure. The chmsber

was then filled with the desired gas or gas mixture. The process was

22
repeated three times for each sample. After the last filling with the
desired atmosphere. the chamber was opened and the cane were sealed by
heating the. solder puncture with a soldering iron. while still standing
in a purified nitrogen or oxygen-nitrogen mixture. These samples were
stored for 6 months in nitrogen atmospheres containing 0. 0.1. 0.5 and
2.01 oxygen at room temperature. 75-80'!.

At intervals of 4-6 weeks. samples were removed from storage and
analyzed or placed in the cold room at -40 'P with control samples until
analysis could be made.

For most of the studies reported here. freeze-dried raw beef was
ground before analysis. unless otherwise indicated. This precaution
was taken to further reduce sampling errors. Also. most of the
analytical methods used requred relatively small particle size. The
dry meat was cut into strips of about one square centimeter in cross
section and ground in a Wiley mill to a particle size of which most
would pass through No. 10 mesh wire screen. Smell batches of meat were

ground at a time to overcome overheating effect of the mill on meat

componmts.

Preparation of Nitrogen Compound Extracts
Eater Extract. One gram of freeze-dried beef was rehydrated with
five milliliters of water at room temperature for 20 minutes. The re-
hydrated meat was minced in a Virtis 54' blender with 20 ml of water.
After a few minutes. the entire slurry was poured into a 50 ml centri-
fuge tube and spun for 15 minutes in a clinical centrifuge at 1500 G.
The supernatant liquid was decanted. and the pellet re-entracted with

15 ml of water by macerating with a glass rod. The mixture was then

23
centrifuged. the supernatant removed and combined with the first extract.
The extract was diluted with water and brought to values in a 50 ml
volumetric flask. One milliliter of the total water extract was used
for nitrogen determination by a micro-[jeldehl method (127).

Soluble Nitrggen. One gram of ground dry beef and one grmn of
powdered glass were placed in a mortar. Enough 0.5 g potassium chloride
was added to thoroughly wet the material and the mixture was ground.

The mixture was then transferred to a test tube with three 25 m1
portions of 0.5 g potassium chloride for extraction. The extraction was
allowed to proceed with occasional shaking. At the end of two hours.
the solution was centrifuged at 1500 ‘G for 15 minutes. The super-
natant was removed and diluted with 0.5 g potassium chloride in a 100 ml
volmetric flask. The soluble nitrogen was determined on 1 ml of the
total extract by micro-Kjeldahl.

Soluble Protein and Soluble Non-Protein Nitrggon. The soluble non-
protein nitrogen was also determined on the potassium chloride solution
extracts. To 4.0 ml of the extract. in s 12 m1 graduated centrifuge tube.
6.0 ml of 101 TCA was added. mixed well and was let to stand at room
tonperature for 20 minutes. The solution was then centrifuged at 1500 C
for 15 minutes. The soluble non-protein nitrogen was determined on 2 m1
of the supernatant which was carefully removed by a capillary pipette.

The soluble protein nitrogen was calculated by difference from the
total soluble nitrogen.

Amino Nitrggen. The method used was a formol titration with modi-
fications suggested by Shipstead (202). One gram of freeze-dried ground
beef was placed in the cup of a Waring Slender with 250 ml of 112

24

sodium salicylate solution as a peptiaing agent and two drops of silicone
antifoall. The mixture was blended at reduced speed for 30 minutes. The
solution was then transferred to a 600 m1 beaker using 50 m1 of the
salicylate solution to complete the transfer. The solution was titrated
with 0.1 g sodium hydroxide to pH 9.0 using a pH meter. Ten m1 of
reagent grade formaldehyde were added and the solution retitrated to pH
9.0. A maximum of five minutes were allowed to casplote the titration
in both cases. A reagent blank was run to allow for correction for the

formic acid which is present in the formaldehyde solution.

attraction of lipids. The extraction procedure was essentially
that of Polch 35 3;. (68). Twenty-five grams of powdered freeze-dried
raw beef were blended with three 200 ml port ions of cold 2:1 chloroform-
methsnol (VIV) in a Waring Blendor at roan temperature. Each extraction
was carried out for 3 minutes and the slurry was filtered on a Buchner
funnel using No. 4 Whatman filter paper. The combined extracts were
then mixed with 0.2 of its volimie distilled water in a separatory funnel
and allowed to stand overnight at 35 ‘P. The separation into two phases
after storage in the cold roan was clean cut and no fluff was formed.

The lower phase was drained and. without further washing. dried
over anhydrous sodium sulfate. The extract was concentrated using a
fast evaporation device. It consists of a Rinco rotary evaporator with
a trap cooled in chloroform-carbon tetrachloride (2:1) and dry ice and
reduced pressure which is provided by a positive displacement vacuum
pump. Concentration was carried out at room temperature. The concentrate
was then quantitatively transferred with about 20 ml of chloroform to a

tared 50 ml Erlenmeyer flask and dried using the evaporation unit. The

25

residue was then dried overnight in a deseicator under vacuu. The
weighed dried residue is reported as total crude lipids (Table l).

Fractionation of Crude lipid Extract. Preparation of silicic acid
column: Pifty grams of silicic acid heated overnight at 130‘C were
slurried with chloroform using a magnetic stirrer until a homogeneous
translucent mixture was obtained. The mixture was poured in a 2.5 x 60
on colunm fitted with a sintered-glass disc. Care was taken that no air
bubbles remained trapped inside the column. The silicic acid was
allowed to settle and the chloroform drained under slight nitrogen
pressure. When the level of the solvent in the colu- reached a height
of 3-5 cm from the silicic acid bed. a filter paper was placed to cover
the top of the packed colunm. Anhydrous sodium sulfate was added to
form a 2-3 cm thick layer on the top of the silicic acid. The column.
after washing with 250 m1 of chloroform. was ready to be used.

Etral and Phospholipid Fractions. The entire crude lipid extract

was applied to the tap of the colunm with care. The column was then
washed with 300 ml chloroform. 200 ml acetone. and 300 ml methanol
successively. A flow rate of approximately 5 ml/min. was obtained by
applying about three pounds of nitrogen pressure to the top of the column.
Chloroform and methanol fractions were concentrated and dried by the same
technique described above for preparation of the total lipid extract.

The residue obtained from the chloroform eluate was weighed and reported
as neutral lipids (Table l). The methanol fraction was reported as

phospholipids (Table l). The acetone eluate was discarded.

Fatty Acids Preparatig. Each of the dried residues of neutral and

26
phospholipids was transferred to a 25 ml volumetric flask with the least
required anwnt of chloroform. The solution was diluted with chloroform
and brought to volt-e. The sample containing 300-400 mg dried material
was taken to dryness. as described above for preparation of the total
lipid extract. in a 50 ml round bottom flask and 25 ml of 0.5 g
alcoholic petassiue hydroxide were added. The mixture was refluxed
gently for six hours. The contents of the flask were then transferred
to a 125 ml separatory funnel using approximately 50 ml of water. The
solution was made acid to phenclphthelein by dropwise addition of con-
centrated hydrochloric acid and then one milliliter excess was added.
The solution then was extracted with three successive 25 ml portions of
petroleum ether. and the combined ether extracts were washed three
times with 25 ml of cold water. The ether extract was dried by filtra-
tion through a bed of anhydrous sodium sulfate on No. b Whatmen filter

”1":-

Hethyl lister Preparation

[aeration of Resin. To 10 grams of Amberlite m 600 weighed in
a 125 ml Brlemeyer flask. 25 ml of 1.0 g sodium hydroxide were added
and stirred with a magnetic stirrer. After five minutes. the resin was
allowed to settle and the supernatant liquid was discarded. The resin
was washed. on a Buchner funnel. with water to remove free alkali.
Additional washings consisted of three portions of 25 ml of anhydrous
ethyl alcohol and finally three 25 ml portions of petroleum ether.

3starification. The petrolem ether solution of fatty acids was
decanted onto the prepared resin in a 250 ml eremyer flask. Two 5

ml portions of petroleum ether were used to wash the residual solution

27

into the flask. After five minutes stirring with a magnetic stirrer.
the resin was allowed to settle and the supernatant was discarded. The
residue then was washed by stirring with three successive 25 ml portions
of petroleum ether and the washings were discarded.

The adsorbed fatty acids were converted to their methyl esters
directly on the resin by the following procedure: Twenty-five ml of
6.0% dry as). in anhydrous methanol were added to the residue and the
mixture was stirred for 25 minutes. The methanol-hydrochloric acid
solution was removed by filtration through No. lo Uhatman filter paper
into a 250 ml separatory funnel. The resin was then washed by stirring
for 5 minutes with two successive 15 ml portions of anhydrous methanol-
hydrochloric acid and each was iecanted onto the filter paper in a
Buchner funnel. To the combined methanol hydrochloric acid. 10 ml of
water was added. The diluted solution was extracted with 50 ml
petroleum ether. The aquems phase was drained in another separatory
funnel. and extracted twice with 20 ml portions of petroleum ether. The
three petroleum ether extracts were combined and the aqueous phase was
discarded. The combined petroleum ether extract was washed with water
until free from acid and then dried over anhydrous sodium sulfate. The
methyl ester extract was concentrated by evaporation over a water bath
and bubbling nitrogen through the solution. The concentrated aliquot

was used for gas-liquid chromatographic separation and identification.

fis-Liguid Chromatograpm. An F and M Model 500 temperature
progressed gas chromatograph (r and 14 Scientific Corporation. Avondale.

Pennsylvania) was used for the analysis of methyl esters. The
chromatograph is equipped with both thermal conductivity and flame

28

ionisation (Model 1609) detectors. The detectors. in conjunction with a
Wheatstane bridge. generates an electrical signal proportional to the
amount of any component in the carrier gas. This signal is fed to a
potentiometric recorder which produces the chromatogrsm. Also. the
recorder is equipped by "The Series 200 Disc Integrator" which auto-
matically computes the area under a strip chart curve and presents this
inferential an the same chart.

The chrometagraph was provided with a five feet coiled copper
column of 114" dimeter. The column was packed with 201 by weight Lac-2-
3-446 on chromosorb w. 60-80 mesh as a support phase. The calm was
conditioned at 215 'c for at least 24 hours before being used for
fractionation of methyl esters of fatty acids. The thermal conductivity
detector was used for this purpose. The operating conditions used for
this study were: Column tmnperature. 210's; injection port tempera-
ture. 225; detector block temperature. 225'0; carrier gas. helium;
carrier gas flow rate. 100 ml/min.; reference gas flow rate. 180 ml/min.;
attenuator setting. 1. An aliquot containing 600-800 microgram of

total esters was used for gas chromatography analysis.

gelatin Co_n_xpo_\_1nd . Absorption spectra were used for identification
and for following the changes of the hematin compounds in freeze-dried
beef during storage. A one gram sample was extracted twice with 20 ml
portion of diethyl ether and then dried in a dessicatar under vacuum.
One half gram of the dried sample was transferred to a test tube and
mixed with powdered glass. Ten milliliter of distilled water were
added and the meat tissue was mascarated with a glass rod. The mixture

was centrifuged at 1500 G for 15 minutes. The supernatant was removed

29

and the spectra in the region of 380-480 '11 were determined inediately
in a Bechnan DK-Z recording spectrophotometer. The extract was diluted
15 times for measurement of the spectra in the region 380-450 mu against

water as the reference.

W. The reducing compounds produced in the freeze-
dried raw beef during storage were determined colorimetricelly using a

ferricyanide reduction method which was essentially the same as that of
Chapman and McFarlane (37). Twenty-five ml of water. 25 ml of 0.1 g
phthalate buffer at pH 5 and 25 ml 11 potassium ferricyanide were added
to 50 mg ground freeze-dried meat. The mixture was allowed to react at
70‘s for 20 minutes with occasional shaking. The flask was then cooled
in an ice bath to 25": and 25 ml 10% trichloraacetic acid were added.
After standing for 5 minutes. the mixture was filtered through No. 2
Whatmsn filter paper. Five ml of the filtrate and 5 m1 of water were
placed in a calorimeter tube. the ml of fresh 0.11 ferric chloride
solution added and the color was measured in a Coleman Universal
Spectrophotometer Model 14 (Coleman lnstrunents. lnc.. mywood. Illinois).
after one minute, against a reagent blank adjusted to 1002 transmission.

A standard curve was established using glutathiane.

ggterminatian of Fluorescence. Since fluorescence is characteristic
of many browning reactions and the soluble intermediates are probably
responsible for this property. the fluorescence developed in the various
samples was determined as follows: One gram of ground freeze-dried
meat was mixed with 30 ml of 0.5 5 KCl - 0.3 )3 $3003 and macerated in a

Virtis Blender for a few minutes. The slurry was transferred. with two

30
35 ml portions of the solvent. into a centrifuge tube. Five milliliters
of 601 trichloraacetic acid were added to the extract and stirred
thoroughly. After 5 minutes. the mixture was centrifuged at 1500 C for
10 minutes to get rid of the precipitated proteins. The fluorescence
of the supernatant was measured in an L-Turnor Electronic Fluorometer
(0.x. Turner Association. Falo Alto. California) using 47-8 and 7-60
filters and 0.2 mg] liter solution of quinine sulfate as a standard of
100.

Manon. Exactly weighed pieces of freeze-dried beef
approximately 1 x l x 2 inches were placed in 25 ml beakers. A quantity
of water at 72'7. equal to three times the weight of the meat was added
to each piece. A few large glass beads were placed on top of the pieces
of neat to prevent their floating during rehydration. The beakers were
placed in a thermostatically controlled water bath adjusted to 72°F.
After 15 minutes. the pieces of most were drained and the excess water
blotted off. The rehydrated meet was weighed and the rehydration
ratios calculated (Table l) .

at of Dehydration. The use of the heat of rehydration as a
possible criterion for the deterioration of freeze-dried meat was
suggested by the studies of Urbin and Harland (249). The adiabatic
calorimeter used (Figure 1) consisted of a Dewar flask surrounded by
thick layers of insulating pads. and had styrofaam cover lined by
aluminum foil. The cover had two openings: A center opening which could
be sealed with a No. 10 rubber stopper. and a sech smaller opening
imediately adjacent to it. Two holes were bored in the No. 10 rubber

stopper so that a Bechnan differential thermometer and polyethylene

 

  

g ——lrlJ-ll"'ll“s I Il'lll’lliil

é flW/IIIIIH

 

 

WWW/[Jill] A

Figure I- Design of the adiabatic calorimeter used for
determining heat of rehydration of freeze-dried row
beef. o-ponetherne tube, b- wire (sample), c- Beckman thermometer,
d-polyethylene stirrer, e- rubber stopper, f_aluminum cup,

9. insulated cover, h- Dewar flask, l- insulation‘

31

32

stirring rod could be admitted into the calorimeter. A maell polyethylene
stopper‘was used to seal the second opening and a piece of wire was in-
serted through this stopper ta hold.the sample. 3y attaching the

sample to one end of the wire. it could be placed in the calorimeter for
temperature equilibration without wetting and. at a later time. lowered
into the water in the calorimeterwwithout opening the system» The
stirring rad was attached to a motor. and the speed of stirring regulated
‘with a powerstat.

A small aluminum cup was attached to the cover and 200 m1 of
deionized water were placed in it. When the calorimeter cover was set
in place. the Beckman thermometer. stirring rod. and‘wire for introducing
the sample fit into the aluminum cup.

The heat capacity of the calorimeter was determined by introducing
a known amount of heat in the form of electrical energy. The heater
consisted of a coil of nichrae wire with a resistance of 3.5 obs. It
was inersed in 200 m1 of deionized water in the aluimas cup of the
calorimeter to approximately the same level as that of the freeze-dried
samples. The leads fro-xthe heater were brought out through the smaller
opening in the calorimeter cover. Two dry cell batteries. ”each l-ll2
volts. wired in series. were used as a power source and a direct current
voltmeter and direct meter were used to measure volts and snares.
respectively. The circuit was closed and opened with a knife switch.
and the two minute heating period accurately timed with a stop watch.
The heat capacity of the calorimeter was calculated using the following
formula:

Specific heat (calories/degree tenerature rise) - TVt - (Tenanture

33

rise X weight of water 1: specific heat of water)! temperature rise where
I - current in snares. V - volts. t .- time in seconds.

All calorimetric determinations were run in a relatively constant
temperature room (at 20°C). The sample of freeze-dried meat. exactly
weighed was fastened to the wire inserted through the stopper in the
calorimeter cover and the cover set in place. Timing with a stop watch
was begun immediately. After 5 minutes. stirring was begun using a
relatively low Speed and the temperature measured by a Bockman thermo-
meter at 5 minute intervals for 25 minutes. After 30 minutes. the
sample was lowered into the water and allowed to rehydrate. The temper-
ature was measured every five minutes until a new equilibrium had been
achieved.

The heat of rehydration in calories per gram of meat was calculated
using the following formula:

Heat of rehydration (calories/gram) - (temperature rise/gram of
sample) (heat capacity of calorimeter X weight of 1120 X specific heat of

water) .

gead Secs Gas Cmsition. Determination of oxygen and nitrogen
in the head space of cans used for storing free: e-dried beef was carried

out by gas- liquid chromatography. A parallel calm system (23).
(Figure 2a) was built to permit the passage of a portion of an injected
gas sample into each of two flow paths 3 and 13. The systen consisted
of two separate calms: A one meter column. llli" in diameter. packed
with 301 MA on 60-80 mesh coltmmpack. and a 2 meter column. 1/4" in
daimeter. packed by 131! molecular sieve. The shorter column had a

35

metering valve to regulate the quantity of gaseous nterial entering
the column Sensing at head space gases was accoqlished by means at
the thermal conductivity detectors- The operating conditions for the
instrument were: Colt-m temperature. 21. 2‘s (ran teqerature);
injection port tqerature. 225‘s; detector block teqerature. 225");
detector temperature. 21. 2'0 (room temperature); gas carrier. helim;
carrier gas flow rate. 100 ml/min.. reference gas flow rate. 180 mllmin..
attemator setting. 1.

need space angles were removed from the sealed cans using the
sampling device developed by Bradley and Stine (21). The can was first
held loosely in the apparatus as shown in Figure 2b all! the screw
tightened until a seal was accomplished between the rubber stopper and
the container. The contaminating atmospheric gases in the device were
rammed with a positive displacement vacuu pump. The screw was
tightened further. «greasing the rubber stopper until the can was
punctured by the piercing tip. A gas tight syring: with a fouroinch
hypodermic needle (23-27 gs) was inserted through the rubber system. An
internally contained hypodermic needle (16 ga) provides a passage way
to guide the inserted needle through the can piercing tip into the can.
At least two rinsings of the syringe with the head space gas are
necessary to obtain reproducible results when the sale is injected into
the gas chromatograph. The quantitative analysis was based upon the use
of air as a standard. A standard curve was established to relate
concentration of oxygen per unit height at the chrmaetogram chart. The
curve was then used directly for inadiate calculation of cuponent
concentration in unknom mixtures removed from the head space of stored

 

 

 

 

 

 

 

 

 

 

 

J

 

      

 

Figure Za— Design of the parallel column system
used for head space gas analysis.

35

 

.aa«>am wawaaamm mam aomam can; any «a awfimao .nm aunwfim

X

‘1“!
V

 

 

‘1“
V

 

fizz/z;

A /////// /
\ amend $9.9m m
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&@ l / madd.2..~>x..m\.
" / mezqe 9. 3852 cezmmcodir .mme

35.55%

 

 

 

 

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36

cans. Also. the initial gas mixtures used in this investigation were
analysed. The sise of the eagle injected was l-S ml.

glavor Volatig s. ligure 3 shows the set up used for isolation and
trapping the flavorous volatile We of freeze-dried beef.
Three hundred grams of ground dry uterial and 1500 ml of distilled
water were placed in a three liter. two neck Pyrex round bottom flask
jacketed with a heating mantle. One neck of the flask was fitted with
an adapter that mounted an inlet glass tubing extended to about one inch
from the bottom of the flask. whereby highly purified nitrogen could be
bubbled through the slurry. The other neck was fitted with a reflux
condenser. Beading from the condenser were a series either of cooled
traps or gas washing tubes containing the following reagents: lead
acetate. 42 mercuric chloride solution. 41 mercuric chloride. acidic
hydroxylemine or basic hydromylemine. The traps and the washing bottle
were arranged in such a way to obtain the total volatiles or the volatiles
remaisina (tar certain cmapounds were r-oved with selective qualitative
reagents (206). Hydrogen sulfide vms absorbed by lead acetate.
liercaptsns react with mercuric cyanide and mono- or disulfides react
with mercuric chloride. Acidic hydroucylamine was used to remove carbonyl
colqaunds. Esters and carbonyls were absorbed by basic hydromylemine.

The traps were instead in Dewar flasks containing chloroform-
carbon tetrachloride (2:1) mixture and dry ice or liquid nitrogen.
Nitrogen gas was passed through the system before cooliu with liquid
nitrogen to avoid condensation of oxygen which may result in blocking
the system.

The meat slurry was heated to boiling for four hours. At the end

no mace: on E8

53256 -e 6:2 223; .. £8:
8:8 -u 52380-0 18: no... oééozoaecae A. 6:85 8:8,. -o coon 32 metal enact

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38

of the process. nitrogen gas flow was stopped. and the washing bottles
were removed for further chemical analysis. The cold traps were dis-
connected. the stapcacks closed. and placed aside in the cooling reagent
for subsequent fractionation of the volatile components by gas-liquid
chromatography.

The flame ionization detector was used for separation and detection
of the volatile Wdrivea from the freeze-dried raw beef. A ll "
x 13' coiled copper column packed with 152 by weight Apiezon l. grease
absorbed on 60-80 mesh Chromosorb W was used. The following operating
conditions have been used during these analyses: Column temperature .
lOO'c; injection port temperature. 223'6; detector block temperature.
225.0; detector temperature. 200%; carrier gas. helium; carrier gas
flow rate. 100 ml/min.; hydrogen flow rate. 35 mllmin.; scavenger air
flow rate. 250 mllmin.; attenuator setting. 16; range setting. 1;
sensitivity. one part per million.

Each cold trap was removed from the cooling reagent and warmed up
to room temperature. A few crystals of anhydrous sodium sulfate were
introduced into the trap to salt out the volatiles. A sample of l-2 ml
trapped volatiles were withdrawn using a Hamilton gas-tight syringe and
injected into the gas chromatograph. At least three runs were carried
out for each trap. Five rinsings of the syringe with trapped volatiles
are necessary to obtain reproducible results.

The peaks resolved by gas chromatography were tentatively identi-
fied by retention times for each unknown peak compared with the
retention time resulting from the gas chromatographic analysis of known

volatiles. Also. identification of the peaks as groups was attempted

 

 

 

39
using the selective qualitative reagents.

Portions of the precipitate collected in the washing tube con-
taining the mercuric cyanide were treated with I. l 801 to detect the
presence of hydrogen sulfide and] or mercaptans. Portions of the
precipitates from the mercuric chloride absorption trap were treated
with either 4 g 361 or 102 m to detect the presence of disulfide and
monosulfide respectively. Chemical trappim of sulfur compounds was
necessary because hydrogen sulfide is not detected by the flame
ionization detector. Moreover. it is necessary to confirm identifica-
tion of the other sulfur caspounds which are detected by gas
chromatography as to whether they are disulfide. dimethyl sulfide.

or mercaptsn.

 

 

 

 

Rams

grease-Dried Raw Beef. The Seminambranasus. Semitendinosus and
Biceps-femoris muscles were chosen as a raw material for this investi-
gation because their physical and chemical properties are about average
for beef muscles and have been studied extensively. The freeze-dried
raw beef obtained for this investigation was light pink in color when it
came out of the drier.

Table 1 shows some of the chemical and physico-chemical character-
istics of freeze-dried raw beef stored at 40’? and under an atmosphere
of nitrogen. All results reported in this investigation were on dry
basis and were an average of triplicate runs.

Water attract. The water extract of freeze-dried raw beef contains
the sarcoplasmic group of protein which consists. in addition to myo-
globin and hemoglobin. all the enzymes of the glycolysis and citric acid
cycles. Results were reported as changes in water extractability of
freeze-dried raw beef during storage under different ratios of oxygen
to nitrogen at room temperature. Figure 4 shows that the total nitrogen
of the water extract of the dried meat decreased as a result of two
functions. namely. time of storage and the initial concentrations of
oxygen in cans atmosphere. It was found that a total of 45. 55. 67 and
701 of water extractable compounds were obtained from dried meat stored
under 2.0. 0.5. 0.1 and 0.02 oxygen in nitrogen. respectively. and

after six months of storage at room temperature.

fialuble nitrogen. There was a decrease in the total nitrogen

40

 

 

 

Table I

Some Chemical and Physico-Chemical Characteristics of

Freeze-Dried Raw Beef Stored at -40°C and Under an
Atmosphere of Nitrogen.

 

(7.)
Moisture

2.85
Total Soluble Nitrogen

l2.53
Soluble Protein Nitrogen 8.65
Soluble Non-Protein Nitrogen 3.88
Total Crude Lipids l9.55
Neutral Lipids l6.23
Phospho- Lipids 3. l5
Reducing Capacity (uMole/GmMeat) l85
Rehydration Ratio 0.8l
Heat of Rehydration (Calories /Gm.Meat) l5.4

61

 

 

 

PERCENT

a)
0,
'I

/

 

100

ZN \0 Initial 02 (96)
. o 0.0
B V 0.:
g e 0.5
'__ 80 _ x 0 X 2.0
X
LIJ
m \\o
|.—
g x \V\

 

4O 1 1

 

 

0 8 I6 24
TIME (Weeks)

Figure 4- Changes in water extract nltrogen during
storage of freeze-dried raw best at room temper-

ature and under different Oxygen-nitrogen atmo-
spheres.

 

 

 

NITROGEN

PERCENT TOTAL SOLUBLE

I00 _

80

60

 

 

initial 02 (’l.)

0.0
O. I

0.5
2.0

 

8 l6
TIME (Weeks)

Figure 5- Changes in total soluble nitrogen (0.511 KCI)
beef at room
temperature and under different oxygen-nltrogen

during storage of freeze—dried raw

atmospheres,

‘3

24

 

 

 

 

 

44

soluble in 0.5 g potassium chloride solution during storage of freeze-
dried raw beef at room temperature and under different percentages of
oxygen in nitrogen. The total soluble nitrogen content of the control
sample. stored at 40’! and under purified nitrogen. was 46.8 mg
nitrogen for one gram of dry beef. This is equivalent to 37.432 of the
total nitrogen. The solubility of soluble nitrogen compounds in 0.5 g
potassium chloride decreased rapidly as the ratio of oxygen to nitrogen
increased. Figure 5 shows the changes in the extractability of soluble
nitrogenous material of raw dried beef in 0.5 5 potassium chloride
during storage.

§oluble Protein and Nam-Protein llitrggen. Figures 6 and 7 show a
decrease in potassium chloride soluble protein and soluble non-protein
nitrogen. The changes are dependent both on time of storage and the
initial oxygen content of the storage atmosphere. The initial soluble
protein of freeze-dried beef in 0.5 g potassium chloride is equivalent
to 18.132 of the dried weight. However. the initial value for soluble
non-protein nitrogen was 9.64% of the freeze-dried raw beef muscles.

[roe Amino Nitrogen. The free amino nitrogen of the freeze-dried
raw beef stored at ~40"? and under an atmosphere of purified nitrogen.
as determined by the formal titration method. is 48 mg for each gram of
total nitrogen. A decrease was found to occur. during storage at room
temperature. in the free amino groups of the samples stored either
under purified nitrogen or a mixture of oxygen and nitrogen. The
decrease was dependent both on the concentration of oxygen and the
storage atmosphere and on the time.

Figure 8 represents the curves which show the changes of the free

 

 

 

 

PERCENT

SOLUBLE PROTEIN NITROGEN

 

I00

80.

 

60.

 

Initial 02 ('lo)

—o— 0.0
—v— f 0. I
—s - 0.5
—x— 2.0

 

 

.\0_
4O '- x \
X\x~
20 J i 4
O 8 IS I 24

TIME (Weeks)

Figure 6- Changes In soluble protein nitrogen (0.511

KCI ) during

temperature
atmosphere 3 .

storage of freeze-dried raw beef at room
and .under different oxygen-nitrogen

45

 

 

 

 

PERCENT SOLUBLE NON-PROTEIN NITROGEN

 

  

 

 

 

IOO
Initial 02 (’l.)
—o— 0.0
—v— 0.!
-e'-- 0.5
—x— 2.0
X\ .\ N~
90 - x\
80 _
0 8 IS 24

TIME (Weeks)

Figure 7- Changes in soluble non-protein nitrogen
(0.5 11 KCI) during storage of freeze-dried raw

beef at room temperature and under different
oxygen - nitrogen atmospheres.

’06

 

 

 

 

PERCENT TITE R

IOO

 

 

 

\ o
.§g\
X .\0\
\'\\ 0"“ ~0-
..\‘\ \V\'-
9 O — x\::‘
Initial 0\2 (7.)
-o— 0.0
.47.... 0|
—e— 05
—x—- 2.0
80 L l A
O 8 I6 24

TIME (Weeks)

Figure 8- Changes inthe free amino groups of

freeze-driedjaw beef,determined by formal
titration, during storage at room temperature

and under different oxygen-nitrogen atmospheres.

£7

 

 

 

 

 

48

amino nitrogen available during storage of freeze-dried raw beef.

2' ipidsI Extraction. The total crude lipids extracted with cold 2:1
chloroform-methanol (v/v) represent 19.551 of the control sample of
freeze-dried raw beef. The crude lipid extract was fractionated on
silicic acid into neutral lipid and phospholipid fractions. The neutral
fraction was eluted from the silicic acid column with chloroform. The
column was then washed with acetone to remove non- lipid compounds.
oxidized substances and pigments. Finally. the column was washed with
methanol to obtain the phospholipid fraction. The yields of the neutral
lipids and phospholipids recovered from silicic acid column were 16.23
and 3.151 of the freeze-dried raw beef respectively. The acetone

fraction was discarded.

fighzl Esters. Methyl esters of free fatty acids were prepared
by direct esterification on Anberlite IRA 400 resin using anhydrous
methanol-hydrochloric acid. The methyl esters were resolved by gas
liquid chromatography using a five foot column packed with 202 by
weight of LAC-Z-R 446 an chromosorb W. The thermal conductivity
detector was used at 210'0. The methyl ester peaks recorded were
identified by the retention time for each unknown peak compared with
the retention time resulting from gas chromatographic analysis of known
methyl esters under the same operating conditions.

Figure 9 shows a typical chromatogram of methyl esters of neutral
fatty acids and phospholipid fractions of the control sample of freeze-
dried raw beef stored at -40’P and under an atmosphere of purified

nitrogen.

 

 

 

 

 

49

“any investigators (23.56) demonstrated. by using thermal con-
ductivity detectors. that the peak area percent can be used as a close
approximation to concentration by weight of the individual fatty acid
esters.

Table 2 shows the fatty acid composition of neutral and phospho-
lipids of the control sample stored at «40’! and under an atmosphere of
purified nitrogen. The results show that the unsaturated acids con-
taining two or more double bonds make up 6.12 and 33.811 of the neutral
and phospholipid fractions. respectively. However. the actual weights
of polyunsaturated acids contributed by the two fractions are similar
since the ratio of triglycerides to phospholipids is 5:1 in freeze-dried
beef. The major quantitative difference is the large amount of
arachidonic and docosa-trienoic acids contributed by the phospholipid
fraction that has no counterpart in the neutral lipid fraction.

Oxidation and hydrolysis are probably the main mechanisms of
deterioration in lipids of stored tissues. Since. oxidative degradation
of unsaturated fatty acids was expected to exert most damaging effects
during storage. lipid fractionation and fatty acids analysis was used
to follow the changes in coqosition of tissue lipids. Therefore. the
changes in composition of the unsaturated acids.conteining two or more
double bonds in neutral and phospholipids were followed during storage
at room temperature and different oxygen in nitrogen atmospheres.

Results plotted in figures 10 and ll show that oxidation of
tissue lipids seems to be a two-step process; the phospholipids are
oxidised first and the neutral fat autoxidises somewhat later. It was

noticed that there was a very marked loss of the unsaturated fatty acids

.couoEc coated cove:

«teams 3:2: no £525 8.3238825

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to .328: 2.: 22.23.23 .23 is 32. .23

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50

Table 2
Analysis of Methyl Esters of Fatty Acids of Neutral and

 

 

 

 

 

Phospholipids Fraction s of Pro e ze - Dried Raw Beef
Stored at —40°F and Under an Atmosphere of Nitrogen.
. 2 I
, Area Percent f
Fatty Acids

Neutral Phos ho-

Llpids Lip ds

l220 0.2l 0.32

I420 3.72 2.l3

I4Zl 2.29 I.63

I 620 22.24 l7.27

l61| 6.!3 4.96

I810 I 6.20 l2.4l

I8Il 42.98 27.44

l822 3. l5 l5.29

I813 0.97 2.45

2034 — 9.47

2223 2J0 3.65

Total sotUrated acids 42.37 32.l3
Total mono-unsaturated acids 5|.40 34.03
Total dienoic acids 3-l5 I5.29
Total trienoic acids, 3.07 9.05
Total tetraenoic acids — 9.47
Total polyunsaturate acids 622 33.8I

' :X indicates carbon chain length and degree of unsaturatlon.

2Area calculated by Disc integrator.

51

 

 

 

 

52
in the phospholipid fraction after a short time of storage. whereas these
enchants disappeared more gradually in the neutral lipids of dried
beef. All sales stored in purified nitrogen or a mixture of oxygen
and nitrogen followed the same pattern of change but at different rates.

The amount of arachidonic acid decreased from 28.12! of the poly-
unsaturated fatty acids in phospholipids in the eagle at the beginning
of storage to 16. 252 of polyunsaturated fatty acids in phospholipids in
samples stored for six months at room teqerature under an atmosphere of
purified nitrogen. This clearly demonstrated the oxidative deterioration
due to oxygen adsorbed during the short initial exposure to air iamadiately
following freeze-drying. The trienoic fatty acids obtained free the con-
trol sample and the sample stored at rem temperature for six months de-
creased from 26.76 to 16.56%. respectively.

Generally. all sanples stored under different oxygen in nitrogen
atmospheres showed a greater decrease in the unsaturated fatty acid
composition by increasing the oxygen concentration in the initial
atmosphere of storage and by time as shown in Figures 10 and ll.

0n the other hand. the changes in the unsaturated fatty acids of
the neutral fractions changed considerably less than those of the phos-
pholipid fractions. The decrease in the trienoic fatty acids accounted
only for 401 of the changes which occurred in the phospholipid fraction
for the same samples stored under purified nitrogen for six months.

mtin Casuals. The changes in the hematin coupounds of freeze-
dried beef stored at room tmerature and under different mixtures of
oxygen and nitrogen were characterised by the absorption spectra of

a water extract of meat using a Beckman DK-2 recording spectrophotometer.

 

 

 

 

 

 

PERCENT POLYUNSATURATED FATTY ACIDS

IOO

90‘-
Initial 02 (ole)
—o— 0. 0
_v_ 0. I
—e— 0.5
—-x-— 2. 0

go . L .

O 8 IS 24

 

    

V\

v\
x\
'\

 

 

 

TIME ( Weeks)

Figure l0- Changes in unsaturated fatty acids
(two or more double bonds) in the neutral

fipld fractions of freeze-dried raw beef during
storage at room temperature and under different
oxygen .. nitrogen atmospheres .

53

 

 

 

 

FAT TY ACIDS

PERCENT

POLYUNSATURATED

 

 

 

 

 

Initial 02 (7o)
IOO - -—o— 8'?
-7... .
x \V -e— 0.5
--x-- 2.0
80 .
60 ..
4O -
3 O a . .
O 8 IS 24

TIME (Weeks)
Figure ll- Changes in polyunsaturated fatty acids in'
the phospholipid fractions of freeze-dried raw
beef during storage at room temperature andgunder
different oxygen-nitrogen atmospheres.

54

 

 

 

 

 

55
The control sample of dried meat. stored under an atmosphere of nitrogen
at -40"l‘. appeared virtually to retain most of its initial pink-red
color. The absorption spectra of its aqueous extracts are shown in
Figure 12. The maxima of the oxygenated myoglobin absorption bands
appear at 540 m and 580 mp. however. matmyoglobin and mathemoglobin
are characterized respectively at 500 Ian and 630 In.

Generally. all samples stored under nitrogen or nitrogen-oxygen
mixtures showed increasing losses of the characteristics of moglobin.
i.e. a decrease in the maxima at 540 mu and 580 mu. and relatively
high concentration of matmyoglobin. It seems that the decrease in
spectral absorption characteristics are a function of increasing the
storage time and increasing the initial concentration of oxygen in
the atmosphere of the cans.

Lemberg and Legge (158) indicated that the absorption maxim of
hematin compounds give quantitative information on the amount of
oxidation during storage. The results of this study were reported as
changes in abserbance at 540 mp and 580 mn for oxynyoglobin as shown
in Figure 12. The curves show typical absorption spectra of aquems
extracts of freeze-dried beef. stored at 4°“! (Curve 1). and at room
temperature for two months (Curve 2). four months (Curve 3) and six

months (Curve 4). under an atmosphere of purified nitrogen.

Figure 13 shows that a greater rate of oxidation occurred by in-
creasing the initial oxygen concentration in the storage atmosphere
and increasing storage time. Moreover. there is a linear relationship
between the change in abserbance and time.

 

 

 

 

 

ABSORBANCE

 

 

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0. 0L b . n . I 0.0 s . . - o
«.3 u soo sea do 3o woo woo moo 3o
xsfirmzoqx rt £2993... gt.

39.3 .m- .25 33332. «323 on 3:35 8:233 on 133$.on at .31. moan?

5 3.2.2. 3 unoooLoBEaaAyu one 3 «33a 3 32... 33.3333 has 393....
one 2x 33:6 3132?; can $82. 3433: 338253.

56

 

 

 

 

ABSORBANCE

IOO

75

0|
0

25

 

 

 

 

 

Initial 02W.)
—0- 000
—v- 00 I
—o— 0.5
O 8 I6 24

TIME (Weeks)

Figure l3— Change in the absorbance maxima at

540 and 580 MB of water extracts of freeze-
drled raw beef during storage at room temperature
and under different a xygen - nitrogen atmospheres.

57

 

 

 

 

58

Reducigg Compounds. A substantial increase in reducing compounds
in the water extracts of freeze-dried raw beef was observed during storage
at room temperature and under different oxygen in nitrogen atmospheres.
The reducing compounds were determined by the reduction of ferricyanide.
Under the experimental conditions used in this study. glucose does not
reduce ferricyanide. Thus. the changes occurring during storage must be
due to the formation or umnasking of groups or compounds in the meat
tissues which are stronger reducing agents.

Porter (199) reported that thermal denaturation of proteins causes
umasking of sulfhydryl groups. presumably by unfolding of the molecule.
Also. the active carbonyl-amine browning compounds are quite strong re-
ducing agents (255). The sulfhydryl groups that could be released by
denaturation of the protein. however. could account for only a small
portion of the observed increase. This is similar to the observation
made by Lee 046) in dried milk.

Figure 14 shows the increase in the reducing compounds. during
storage at room temperature and under different concentrations of oxygen
in nitrogen. expressed in 3: mole reductants when glutathione solution
was used as a standard. Results show that the increase of reducing
compounds is dependent upon the initial oxygen concentration in storage
atmospheres.

gluorescigg Cmnds. The fluorescence of 0.5 g K01 - 0.03 )1

NaHCO3 extracts of freeze-dried raw beef samples was canpared with 0.2
mg/liter of quinine sulfate solution as a standard of 100. Fluorescence
is a characteristic of browning reaction and in this system. of the

soluble intermediates or products.

 

 

 

 

59

Figure 15 shows that fluorescence of freeze-dried raw beef
increased during storage at room temperature and the changes are
dependent on the length of storage time and the initial.concentration
of oxygen in the atmosphere of the can. Although it has been found
in this study that increasing the amount of oxygen in the storage atmos-
phere caused more fluorescence in the stored meet. it has been reported
(133. 241) that oxygen is not necessary for browning reactions. More-
over. for the data recorded in this study. it is noticed that the
patterns of increase in fluorescence are similar to those of the forma-
tion of the reducing compound of dried meat during storage at room tenor-
ature. This may correlate both characteristics to meat deterioration.

Manon. The freese-dried beef used in this investigation
contained 2.87% water on a dry basis. The rehydration ratio of the
dried.matarial decreased quite significantly during storage at room
teqerature and different oxygen-nitrogen atmospheres. The rehydration
ratio is defined as the ratio of the weight of water taken up by the dry
tissues to that which would be necessary to make rehydratod meat 75%
water. The rehydration ratio for the control sample. stored at o‘O’!
and under an atmosphere of purified nitrogen was 0.81.

figure 16 shows the changes in.rebydration ratios of fracas-dried
raw beef during storage at room teqerature and with different initial
concentration of oxygen in nitrogen in the storage atmosphere.

lost of Rahzgratigg. The hosts of rehydration of freese-dried raw
beef during storage were determined in an adiabatic calorimeter. The

average heat capacity of the calorimeter was 26.98 calories per degree

 

 

 

 

REDUCTION! GRAM DRY MEAT

.u MOLES

 

800-

600

400‘

 

 

 

 

lnitiol 02 (7.)
--o- 0.0
-e — 0.5
- —x- 2.0
'00 l I l e ' a
O 8 IS 24

TIME (Weeks)

Figure I4- Production of reducing compounds during
storage of freeze-dried row beef at room temp-

rotu re

pheres.

and under different oxygen-nitrogen atmos-

 

FLUORESCENCE

60

 

 

 

 

N x
40. //. 7/,
O/ /O
x
v
/o
x/ ‘ Initial 02W.)
20- / —o— oo
' 0 -v- O.l
-e— 0.5
-x- 2.0
IO 4 L .
O 8 IS 24

TIME (Weeks)
Figure l5- Change in fluorescence during storage
of freeze-dried raw beef at room temperature

and under different oxygen-nitrogen atmOSpheres.

61

 

 

Centigrade. A satisfactory agreement between duplicate runs was
obtained. the heat of rehydration of the control sqle was 15.“
calories per gr- dried meat.

figure 17 shows the results obtained for the heats of rehydration
of the freon-dried raw beef during storage at room temperature and
under different percentages of oaygen in nitrogen. lomlts reported in

this study were an average of triplicate runs.

lead Sgce Gas Anal; sis. mrygon and nitrogen cqosition in the
head space of the can in which freese-driod beef was. was determined by

gas-liquid chromatography using the thernal conductivity detector. Air
was used as a standard containing 20.8% of oaygen under the sons condi-
tions used for this study.

It was observed that all sanplos stored at room toqsrature and
under either purified nitrogen or oxygen-nitrogen mixtures. had signi-
ficantly higher anounts of oxygen than was present in the initial
storage gas. ‘i'ho increase was alnoat constant within experimental
error. the average excess oxygen in the can atmosphere was 1.5! after
four weeks of storage at roa temperature. Since no analysis was
carried out before four weeks of storage. all of the results in this
investigation were related to the nount of oxygen found in the head
space of the cans after this period of time.

rheandseusofallcaneused inthis studyworeprotectedbya
continuous film of the Hood's metal and were cqletaly gas tight.
thus. it may be assumed the only source of oxygen is the dried most. In
the preparation of the freeso-driad beef. the vacui- in the froste-
drier was released by atmospheric air. after the meat was dry. the air

 

 

 

REHYDRATION

PERCENT

m
0

'60

 

 

 

 

.- r
—o- 0.0
-v- 0. I
—e- 0.5
-x- 2.0
40 . . _ ' .
O 8 l6 24

TIME (Weeks)

Figure l6- Changes in rehydration capacity of
freeze-dried raw beef during storage at room

temperature and under different oxygen-nitrogen
atmospheres .

63

 

 

 

 

 

 

HEAT OF REHYDRATION

PERCENT

60

50

 

 

 

Initial 029/.)

 

 

—o— 0.0 K
—v— O.I
-o- 0.5
-x- 2.0
1 l l
O 8 l6 24
TIME (Weeks)
Figure l7- Changes in heat of rehydration of

freeze - dried

row beef during storage at room

temperature and under different

atmosphe re s.

615

oxygen-nitrogen

 

 

 

 

65
probably replaced the water in dried tissues and after storage. a
state of equilibrit- of adsorbed air and the head space gas or gases
was attained. '

Any .o'unts of oxygen less than the .ount detected in the head
space of the control a-ples after four weeks of storage were con-
sidered used up by chcical reactions in tho dry meat.

Results are reported in Figure 18 as changes of head space oxygen
contnt in percent by assigning that oxygen found after four weeks of
storage in each condition value of 100. It was observed that the rate
of chemo was dependent upon the initial concentration of oxygen in the
head space.

glsvor Volatiles. The flavor volatile cqonents of freeze-dried
raw beef were generated by bubbling purified nitrogen in a boiling
slurry of rehydrated meat and water for four hours. The volatiles were
collected in traps cooled with liquid nitrogen. They were then
fractionated on a calm packed with 151 Apieson adsorbed on 60-80 mesh
chraosorb it at loo‘c using a hydrogen flame imitation detector on the
gas- liquid chruatograph.

Results shown in Figures 19 and 20 indicate that the volatile flavor
compounds increased in concentration as they were measured by the heights
of their peaks. Also. the amber of volatile camounds in all the samples
stored under purified nitrogen or oxygen-nitrogen atmospheres at room
toqoratura. moreover. the volatile compmnds were increased in amber
and concentration as the initial atygen concentration in the storage
atmosphere was increased.

figure 19 shows gas-liquid chromatograms obtained by direct in-
jection of 1 m1 of cooled trapped volatiles of fresco-dried raw beef;

 

 

 

 

 

 

 

 

PERCENT OXYGE N

I00

60.

20

 

   
   
 

Initial 0207.)

—o- 0.0
—v— O.I
—s— 0.5
'4" 2.0

 

 

 

TIME (Weeks)

Figure I8- Changes in head space oxygen

during storage of freeze dried row beef
at room temperature and under different
oxygen-nitrogen atmospheres.

66

 

 

 

 

RECORDER RESPONSE

 

Air
Mo-mercoptan

 

 

 

file-disulfide

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0

39.3

.ml

9 too. m
.5393 .

jam Azsimmv

329. 3.02.3 oaqoaonoaaém on 283-38
:5 can 303 ingooqoncam A3 3... my. Erato

32 amen £0me
:32 0:122.

g m
.m i 3 m
m
a m
m
.M.
a
j m
T M
>
fiz/L )4, )
_O NO we 8

67

 

 

 

68

(a) stored at ~40" and under an atmosphere of purified nitrogen; (b)
stored at room tqerature for six maths. under the some gas.

l‘igure 20 shows gas liquid chm-“grams obtained by direct injec-
‘ tion of 1 ml of cooled trapped volatiles of dried beef stored at room
tonerature and under an atmosphere containing 21 oxygen in nitrogen
after four (a) and six months of storage (b).

At least fifteen components were resolved by gas liquid chromato-
graphy when the stean volatiles of the control a-ple of freeze-dried
raw beef was injected directly to the fractionation column. Attempts to
classify those cowomds into different groups were carried out.

A purely tentative identification of the peaks resolved by gas-
liquid chromatography was made. the retentim tines for each unknown
peak were counted with retentia time resulting from the gas
chromatogrqhy analysis of known volatiles. Acetaldohyde. propanel.
hexansl. pentanal. acetone and methyl disulfide were identified as
shown in Figure 20 .

An attempt to identify the peaks using the technique suggested by
bassette. Ozeris and Whitnah (206) was carried out. selective qualita-
tive reagents were used to eliminate certain class of compounds and
thus. the peaks could be classified into groups. Peaks corresponding to
carbonyls were eliminated when the steam volatile components were
bubbled through a tube containing acidic hydroxylamine prior to collecting
then in the cooled trap. Esters as well as carbonyl cowounds were
removed by basic hydroxylamine. Using both acidic and basic hydroxyl-
anime reagents it was necessary to establish that esters were not among

the components of dried beef volatiles as detected by gas-liqixid

 

 

 

 

 

 

 

RESPONSE

RECORDER.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. m, m

z. j

m 3 ,1. 4 a, m
N A m a

i
r]. | Z,
3 e ,
mug _¢ we we .IIIIIIW so

4.3m «2:333

39:6 No: 233 33:5» 9333833». on 333.93... 3: of:" «3qu on
32: 33.5333 can can: on 338268 on mg. 92%: 3.. 353:2.

ax Amy :533 on £28?

69

 

70
chraaatography. All the peaks resolved by gas chromatography. except
peaks 2 and 3 either were thawed or decreased in sine when volatiles
were bubbled through acidic or basic hydroxylamine.

Hydrogen sulfide is not detected by gas-liquid chrontography when
the hydrogen flame detector is used. Ch-ical analysis was carried out
for hydrogen sulfide as well as other sulfur coqouuis which might be
present in freeze-dried raw beef steam volatiles. ‘l'he volatiles
were bubbled throufl: washing tubes containing lead acetate. mercuric
chloride and mercuric cyanide. A black precipitate was obtained in the
lead acetate tube indicating the presence of hydrogen sulfide. 0n the
other hand. a white precipitate was formed when mercuric chloride was
used in the washing tube. The white precipitate shows the presence of
mono- or disulfides among the volatile compounds obtained by steam
distillation. When aqueous mercuric cyanide was used for washing the
volatiles. a yellowish green color was produced.

Challenger (36) showed that when both hydrogen sulfide and mer-
capten are present. the color of the precipitate with aqueous mercuric
cyanide could be yellow. yellowish green. or black depending on the
relative quantities of the two col-pounds. when a portion of the pre-
cipitate fron the nercuric cyanide absorption trap was warned with I. g
ml. a faint sulfurous odor was detected. this may indicate the presence
of mercaptan in dried beef volatiles but in an anount which is not
sufficient to be detected by this technique. However. the volatiles
W after being washed in aqueous mercuric chloride and mercuric
cyanides showed that peaks were greatly decreased in size whereas peaks
were coqletely disappeared. Ithese two peaks may be identified as

 

 

 

 

7 l
methylmsrcaptan and methyldisulfide respectively. n- Quantitative
studios have been carried out on sulfur compounds which were found in
beef volatiles obtained by steam distillation.

DIM!!!“ m COMMSIONS

In assessing the results of this investigation. it is clear that
many of the important characteristics of the freeze-dried raw beef.
stored at room temperature and under different ratios of oxygen to
nitrogen. deteriorated appreciably after a few months under the condi-
tions of storage. The control samples. stored under an.atmosphere of
purified nitrogen at -40'F. were comparable to fresh freeze-dried meat
so far as color. odor. and texture were concerned.

All samples including those packed in nitrogen. showed a constant
increase in the amount of oxygen when head space was analyzed after four
‘weeks of storage at room temperature. Since all the containers were
completely air-tight. it may be assumed this represented oxygen desorbcd
from the meat. This conclusion is in agreement with.Thommon. Fox. and
Landmmn.(246). who studied the effect of water and temperature on the
deterioration of freeze-dried beef during storage. However. their
results showed that the samples at 0. 0.1 and 1.0% oxygen. stored at O'F
had 13-141 oxygen after storage. .Also. it has been reported that the
amount of oxygen desorhed from dried meat was dependent on the initial
‘moisture content of the dried beef and the storage temperature. The
actual amount of oxygen absorbed by freeze-dried beef after releasing
the vacuum in the freeze-dryer was not determined in this study.

The low moisture content of foods prevents the growth of micro-
organisms. However. enzymes survive in freeze-dried fresh meat and fish
products. Adenosine triphosphate (ATP-see) and lipoxidase have been
identifiedtmmong others (38. 43. 44. 45). Cole and Smithies (38) have
recovered practically all the original ATP-ass of beef muscle in the

freeze-dried products; however. this facet has not yet been thoroughly
72

 

 

 

 

73
investigated. Precooked foods would not be expected to have residual
enzymes. but Olley and Lovern (188) report that the phospholipase in cod
flesh is still active after 30'minutes at 100'.

Storage of freeze-dried beef progressively reduces its ATP-ass
activity (38.43.120) and the measurement of this has actually been pro-
posed as a criterion of quality. It has been shown recently (52) that
the blocking of a very small number of the amino groups of myosin
suffice to eliminate its enzymatic activity. therefore. one cannot rule
out the possibility that the loss in activity observed on storing freeze-
dried beef is the result of browning reactions involving these groups.
Also. there may be some thermal decomposition of compounds such as
amides or keto acids. This would not be expected to be very extensive
under the pH. moisture. and temperature conditions existing during the
storage of the freeze-dried beef.

All samples showed some brown discoloration when compared with
control samples stored at «40'? and developed an odor like that of
slightly rancid fat.

An increase in reducing groups and decrease in solubility are
characteristic of the thermal denaturation of many proteins (43.44.45).
In the native state. some proteins have a number of cysteine sulfide
groups hidden within their folded structure. When the protein is un-
folded in denaturation. these groups become exposed and reactive. Also.
the unfolding of protein molecules in denaturation exposes polar groups
which can interact with those of other molecules to cause aggregation
and result in decreased solubility. An increase in reducing groups and

decrease in solubility is also characteristic of the amine-active

 

 

74
carbonyl browning of proteins (202). In this case the increase in re-
ducing groups is due to the femation of reductones and similar coupounds.
As browning progresses. the polymerization of these unsaturated materials
causes a decrease in the protein solubility.

Although from previous studies (133. 241. 242) it has been reported
that in some cases browning deterioration is greater in nitrogen or in
vacuum than in air. it is known that such a reaction may use up oxygen.
Results in this study show that after longer storage. a noticeable de-
crease in head space oxygen was observed. A portion of this oxygen
could possibly be accounted for by oxidation of browning reaction re-
ductants.

There is little discussion in the literature of the heat denaturation
of essentially dry proteins with which one can compare results. If
thermal denaturation did occur during storage. there probably would be
an increase in the digestability of the proteins by proteolytic enzymes
(238). However. Regier and Tappel (203) found that digestibility of
freeze-dried beef proteins decreased during storage. These results
pointed to emine- carbonyl browning as an important deteriorative reaction.

This study has shown. regardless of the initial concentration of
oxygen in storage atmosphere. that nearly all the changes associated
with active-amine browning occurred during the storage period. The
protein solubility and free amino groups decreased while the reducing
compounds increased. It has been shown that there is also an increase
in fluorescing materials. The reactants for the browning reaction are
certainly available in meat since proteins. free amino acids. glucose.

hexose phosphates. organic acids and other metabolic intermediates are

 

 

 

 

 

75

present. Regier and Tappel (203) found that added glucose increased
the deterioration. the carbonyl trapping compounds. bisulfite and semi-
carbazide decreased the deterioration.as measured by the decrease in
soluble nitrogen. (Also. it has been suggested by Regier and Tappel
that the carbonyl compounds are the limiting reactants in the browning
reaction in freeze-dried beef. However. in this study it has been found
that there is a substantial increase in the carbonyl compounds.

0n the other hand. Regier and Tappel (203) concluded that the inter-
‘mediates of the browning reaction in freeze-dried beef are not free
carbonyl compounds for the lack of free water prevents diffusion which
is necessary for the formation of the carbonyl additional compounds.

In.dehydrated meet such as used in.these studies. the moisture is
so low that diffusion through an aqueous phase should be quite limited.
In such a system. one can visualize that some of the initial reactants
of the intermediate are relatively volatile. In this case the diffusion
barrier would be small and the reaction could proceed very rapidly. This
study showed that deterioration.of freeze-dried raw beef produces
volatile carbonyl compounds which may play a role in the initiation of
browning reaction and produce basic compounds which are strongly reducing.

Among the tentatively identified volatile compounds which increased
greatly during storage is n hexanal. The autoxidation products of
linoleate and linolenate have been reported (73.129) and a fairly
strongly supported mechanism has been established (55) for the formation
of hexanal fron linoleate. It has been reported recently (33) that the
rates of increase in 1»me emcentration in the volatiles of food

product can be used to cupare susceptibility to

 

76

oxidation of dehydrated products. However. the author does not imply
that hexanal is the main carbonyl compound which may be used as a
measure for progressive deterioration in dried meat or is the product
mainly responsible for the characteristic odor of rancidity.

Unlike the stored triglycerides of adipose tissues. muscle lipids
are not arranged in large globules within the cell. Instead. they are
integral parts of various cellular structures. including the cell wall
(135). the myofibrils (189). the mitochondria and microsomes (169).
These intercellular fatty substances include most of the phospholipids
of the tissue and are at least partially associated with proteins.
Recent work by Folch and others (68. 112) has shown that there are in
animal tissues a number of different types of lipOproteins varying con-
siderably in their solubility characteristics and ease of decomposition
from those of blood plasma. Lipoproteins usually denature more readily
than ordinary proteins. This results in lesser solubility.

The results of this study agree with Bernstein (115) that the
large: proportion of unsaturated fatty acids are in the phospholipid
fraction of raw beef.

It was observed that phospholipid fractions are more easily oxidized
than the triglycerides. It is not known why the bound lipids are more
susceptible to oxidation than neutral fats. but a possible explanation
may be that fatty acids of the complex lipids are more unsaturated than
those of triglycerides. Also. the former are more closely associated
with iron-containing heme compounds of tissues which can act as pro-
oxidants. The oxidation is home catalyzed in beef (263). It does not

occur in invertebrate muscle such as shrimp and crab which do not have

 

 

 

 

 

77
heme pigments. and is strongly inhibited in muscle meats by converting
the heme compounds to the cured meat pigments.

Luyet and Melanie (165) studied the mechanism of rehydration of
freeze-dried muscle. In the-rehydration of freeze-dried mscle tissue.
water penetrates either through large channels formed by ice crystals
during slow freezing or through the minute cavities formed within the
fibers during rapid freezing. This is followed by an inhibition of water
by the tissue solids. Recognition was given to the lack of infometion on
the binding of water by tissue solids. Several obstacles to the penetra-
tion of water was noted. such as water repellent surfaces which inter-
fere with wetsbility. impermeable membranes which resist transmission of
water. and trapped air which may prevent entry of water into dry tissue
cavities.

In this study. the rehydration characteristics of the stored freeze-
dried beef were affected by a decrease with time of storage in the final
snout of water taken up by the dry tissue. Also. it has been observed
that the experimental variation was greater in the more badly deteriorated
samples. a reflection of the way in which these samples rehydrated. The
effective rehydration is a process whereby penetration of water into the
tissue is fast enough to wet all the fibers before leaching of tissue
components can form gel layers to block the progress. rat may play an
important part in the rehydration of freeze-dried meat was suggested by
Luyet and McKenzie (165) . who proposed that the water-repellent material
they observed in channel of freeze-dried meat was fat.

Both heat of rehydratien and amount of water taken up are measures

of the state of protein in the dried tissue. The former is probably

 

 

 

 

 

78

more directly related to protein. since the latter includes water not
bound to protein. but taken up by capillary action intercellularly and
by cell rehydration.

The Q10 of the browning reaction in dehydrated meat was found to
be 3.2 and 4.3 over the range of 15 to 50's (220). The observed
activation energy for the deterioration of freeze-dried raw beef was
found to correspond to Q10 of about 4 (203). In general terms this
might be expressed as follows: An increase in the storage temperature
of 10.0 would cause the storage life to decrease to one fourth of the
time at the lower temperature. This emphasizes the fact that no
matter what other techniques may be used to increase the storage life
of dehydrated foods. the control of the temperature of storage should
always be one of the prime considerations.

The essential complete removal of the water from freeze-dried
beef does not completely eliminate the storage deterioration. However,
the moisture level is quite important in determining the rate of
deterioration. lbreover. to reach very low levels of water in the final
dried product is very expensive process.

It is known that under normal conditions of handling meat. the
concentration of reducing sugar increases with time post-marten and
with the object of reducing the potential browning in dehydrated meat
during subsequent storage. the aim should be to process meat in which
the reducing sugars are present in low concentrations. This can be
achieved in certain animals by lowering the glycogen reserves in
the muscle by starvation and] or fatigue before slaughter. Callow

(34) has shown this can be done quite effectively for pigs. but

 

 

 

 

79
according to Reward and Lewrie (117) it has proved to be iqractical for
beef animals in which the stored glycogen are maintained at relatively
high levels. Insulin shock might be used to decrease the concentration
of glucose in the tissues and thus lower the. level of at least one of
the reactants in the storage deterioration reactions. These types of
treatment. however. will introduce a number of other problems. both
technical and esthetic. which may be unsurmountable.

The possible use of glucose oxidsso in freeze-dried beef should not
be overlooked. A large portion of the glucose in beef exists as the
phosphate esters which are not oxidized by the enzyme (202). If hexose
phosphate kinsse were added to the enzyme preparation. much of the
glucose- l-phoiphsto would be converted to free glucose. The glucose
would be then oxidized to gluconic acid which doeo'not enter into the
browning reaction. however. there are a number of other factors which
may be important in the treatment of the beef with glucose exidsse
which were not explored. The hydrogen peroxide added to the material
or produced in the onidstion of glucose may be sufficient to cause some
oxidation of the hone commands or the protein. These side reactions
are ; undesirable and could ruin the product if they were very extensive.

In the second place. the moat could be prepared and dehydrated
soon after the death of the animal and before any appreciable
eccmlation of reducing sugars had taken place. This procedure.
although Quito practicable in small scale production. is not very
suitable for large scale production. and. in any case. may lead to
the production of a rather tough product.

If the meat is to be held for one to three days before being

 

 

 

 

 

80
processed. the rate of production of free sugars in the meat can be
reduced by lowering the temperature as quickly as possible post-mortem
and holding the meat in the chilled. state. If the meat is to be held
for longer periods. the acculmllation of free sugars can be almost
completely inhibited over a long period by freezing the meat innediately
post-mortem and holding it at temperatures of ~10'c or below. This
last procedure would appear to be particularly appropriate to the
latest development in freeze-drying as described by Brynko and
Smithies (31).

The amount of oxygen detected after four weeks of storage. in the
head space gas of freeze-dried raw beef canned under an atmosphere of
nitrogen. exceeded the minimum level oxygen used in this investigation.
Thus. the desirable minimum level of oxygen for the storage of freeze-
dried raw beef could not be determined. However. most of the changes
which took place during storage of freeze-dried raw beef in this study
could be related to oxidative changes.

Further studies are needed to follow the non-oxidativo changes
during storage after taking all the measurements possible to eliminate
the free and the adsorbed oxygen from the storage atmosphere. Releasing
the vacuum in the freeze-drier with purified nitrogen and flooding the
samples with inert gas during packaging and storage are reconmended to
increase the storage life of freeze-dried raw beef. 4

Also. further work is needed to identify the volatile compounds
produced during storage. The identification of such compounds quali-
tatively and quantitatively may lead to the understanding of the

mechanism of their formation.

 

 

 

l.

3.
4.

5.
6.

7.

9.

10.

ll.

12.

13.

14.

BIN-1mm

Adachi. R. keg ShC‘f’rp Log I“ Specter. H. ("58)o m
in vitro digestibility and nutritional quality of dehydrated
beef. fish and beans. Peed Res. 23. 401.

Altmsnn. R. (1890). Die Elnentarorgani-en und ihre Desieh
ungen an den Zellen. Viet Leipzig.

Anonomous. (1958). Dehydrated meat. Pond [London] a. 218.

Anonomous. (1959). Economic study of food preservation methods.
11.8.0. Equipment Corporation. Newton highland 61. lines. D.S.A.

Anemone”. (1962). Presseodried foods. Conan. Rpt. g. 338.

Arnold. 1.. 1.. and am. P. B. (1958). Drying fish and beef
prior to solvent extraction. J. Agr. Peed Chem. _6_. 231.

Auerbsch. 3.. Wang. 3.. Maynard. 11.. Doty. D. it. and
Kraybill. H. R. (1954). A histological and histochomical
study of beef dehydration. V. Some factors influencing the
rehydration level of frozen dried muscle tissue. Peed
”Co _l_gp 557o

Bailey. K. (1954). Structure of proteins. II. Muscles.
Chapter 24. in The Proteins. Rd. 11. Neurath and K. Bailey.
11 3. Academic Press. Inc. low York. 11.!” p. 951.

man, a. D... Lowry. J. a. and Theiosen. 3.. Jr. (1951).
Some effects of processing on the nutritive properties of
prot31n.o Food R..o 12. 1070

Dallsntyne. R. 11.. Drynho. 0.. Ducher. A. J.. and Smithies. w. R.
(1958). Dehydrated cooked meat products. Pood Technol. .13. 398.

Ballantyne. R. lb. Blahley. T. S.. Ducher. A. J.. and
Smithies. W. R. (1961). Factors influencing the quality of
freeze-dried foods. Peed Trade Rev. 3;. 56. '

“full... No Go, “$13.. On 6.. M AIWGIp lee H. (1936).
The influence of retarded growth in lambs on flavor and
other characteristics of the moat. Proc. Am. Soc. Animal
Prod.. 29th Ann. Meeting. 289.

Barker. 11. A. (1933). The effect of water content upon the rate
of heat denaturation of cryotsllizable egg albumin. J. Gen
Physiol. _l_7_. 21.

33:80:. On Fog Scribe}. “on DC”. Do 11.. m smuCr‘p 3. So
(1957). Production of carbonyl cqounds during irradiation
of most and meat fats. J. Agr, good chem. 2. 700.

81

 

 

 

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.
26.

27.

28.

82

“mtg as P.. m‘u0p Ac Jog t“. l. Cop W. "o Asp CM
Schweigert. B. S. (1960). Precursor of beef flavor. J. Agr.
Food Chem. _8_. 498. _

um£.k’p Co. ’0‘. J. ’0. m smmrtg B. S. (1959).
Biochnistry of myoglobin. VII. The effect of cooking in
myohomoglobin in beef muscle. Pond Res. _25. 339.

Boggs. ll. 1‘. and Povold. ll. 1.. (1946). Dehydrated egg powders.
Pastors in palatibility of stored eggs. Ind. Eng. Chem. g.
1075. .

’M.g I. no. “4““, H. Jog “firth. Bo Gog M ["01d, H. Lo
(1946). Dehydrated egg powders. Relation of lipids and salt
water fluorescence values to palatibility. Ind. Eng. Chem.
.33. 1°82.

Bomichsen. B. K.. Chance. 3.. and Theorell. B. (1947).
Catalsse activity. Acts Chem. Scand. _l. 685.

Borgstrom. B. (1952). Investigation of lipid separation methods.
Separation of phospholipids from neutral fat and fatty acids.
Acta Physiol. Scand. 33. 101.

Bradley. B. 1... Jr. and Stine. 0. ll. (1962). Simple device for
obtaining samples of head space gas directly fr. sealed
containers for analysis by gas chromatography. J. Dairy Sci.
5;. l 259.

Brenner. 11.. and Cieplinski. B. (1959). Gas chromatographic
analysis of mixtures containing oxygen. nitrogen. and carbon
dioxide. Anals of the New York Acad. of Sci. 12. 705.

Brenner. 11.. Scholly. P.. and O'Brien. 1.. (1959). The analysis
of fatty acid esters by gas chromatography.
MI Spokesman .22. 137.

Brooks. J. (1929). Post-mortem formation of metl-oglobin in
red muscle. Biochem. J. _23. 1391. .

Brooks. J. (1938). Color of meats. Pood Res. 3. 75.

Brooks. J. (1958). The structure of the animal tissues and
daydtCC1mo '0“ ”to 22, 204.

Browse. w. J.. and Kerwin. T. D. (1954). The kinetics of
myoinsse. 1. Studies of the effects of salts and pH and of
the state of equilibrium. Arch. Biochem. Biophys. 29. 149.

Brown. 17. D.. and Tappel. A. 1.. (1958). Pigment-antioxidant
relationships to meat color stability. Proc. 10th Res.
Conference. Am. Moat Inst. Pound. Univ. of Chicago. 81.

 

 

 

29.

30.

31.

32.

33.

35.

36.

37.

38.

39.

40.

41.

42.

43.

83

3t“, We Des Tappel. Ao Leo and Olcott. 11. S. (1958). The
pigments of off-color cooked tuna meat. Food Res. 33. 86.

Barylko Pikielna. N. (1957). Gas-liquid chromatography.
Pizemysl. Spozywczy. 11.26.

Brynko. C. and Smithies. U. R. (1958). Rapid Freeze-Drying of
“ate Jo 3C1. POOd “to 2., 576.

mmttg 1!. Cop 69hr“. Co "op and Brady. Do Ea (1955)o
Volatile components of vacuum-packed dehydrated pork. J.
Agr. Food Chem. _3. 524.

Buttery. R. C. and Teranishi. R. (1963). Measurement of fat
autoxidation and browning aldehydes in food vapors by direct
vapor injection gas-liquid chromatography. Agr. Food Chem.
_l_l. 504.

Callow. 3. 11. (1938). Aftereffects of fasting. Ann. Rpt. Food
Invest. Bd.. London. p. 54.

Cavalier. 1.. F. and Wolfram. ll. 1.. (1946). Chemical interaction
of amino compounds and sugars. I. J. Am. Chem. Soc. _6_8_. 2022.

Challenger. F. (1959). "Aspects of Organic Chemistry of Sulphur"
Butterworths Scientific Publications. London.

Chapman. R. A. and Marlene. W. D. (1945). A colorimetric
method for the estimation of reducing groups in milk powders.
Can. Jo Res. 2.2!. 91o

c013, Le Jo No. and smithieSp We P. (1960)o mthOds Of
evaluating freeze-dried beef. Food Res. 2;. 363.

Cole. 1.. J. N. (1961). Cooperative effects of 2.4-dinitrophenol
on the actomyosin adenosine triphosphatase from fresh. frozen
and freeze-dried beef. Nature. _l_9_2.. 1288.

Cole. 1.. J. N. (1962). The effect of storage at elevated
temperature on some proteins of freeze-dried beef. J. Food
Sci. _21. 139.

Cole. L. J. N. (1962). A comparison of the effect of freezing
and drying in the rehydratability of freeze-dried beef. _l_g
Freeze-drying of foods; Proceedings. Natl. Acad. Sci. . Natl.
Res. Council. 217.

Connell. J. J. (1957). Some aspects of the texture of dehydrated
figho Jo Sci. 300d “to £3 526o

Connell. J. J. (1958). The effect of drying and storage in dried

state on some properties of the proteins of food. Soc. Chem.
Ind. Fundamental aspects of the dehydration of foodstuffs. 167.

 

 

 

 

 

 

65.

46.

47.

49.

50.

51.

52.

$3.

54.

55.

56.

57.

84

Connell. J. J. (1959). Aggregation of cod myosin during frozen
storage. Nature. _1_8_§. 666.

Connell. J. J. (1960). Changes in the actin of cod flesh
during storage at -14'C. J. Sci. Food Agr. , 11. 515.

Connell. J. J. (1962). the effects of freeze-drying and subse-
quent storage on the proteins of flesh foods. In Freeze-
drying of foods; Proceedings of Natl. Acad. Sci.. Natl. Res.
Council. 50.

Conroy. A. (1955). the freeze-drying and subsequent storage of
animal products. Thesis (11.8.) University of California.
Davis. California.

Cracker. B. C. (1968). Flavor of meat.
Food Res. .13. 179.

Danahy. J. P. and Pig-an. W. W. (1951). Reactions between
sugars and nitrogenous compounds and their relationship to
certain food problems. "Adv. in Food Research" (edited by
E. H. link and C. F. Stewart). Vol. III. Academic Press.
New York. -

Deatherage. F. 8.. and Ham. R. (1960). Influence of freezing
and thawing on hydration and changes of the muscle proteins.
rad u.e £2. 623e

Dacareau. R. V. (1961). Now microwaves speed freeze-drying.
Food Eng. 33(8). 34.

Doty. D. M. (1962). Increasing product storage life--Basic
deteriorative reactions. _Ig Freeze-drying of foods; Proceedings
Natl. Acad. Sci.. Natl. Res. Council. 289.

Dulkin. s. .I.. Friedemann. 'r. E. (1956). The role of dehydro-
ascorbic and dehydro-reductic acids in the browning reaction.
’0“ ”Ce 2;; 519e

Dutton. A. J. and Edwards. 3. G. (1946). Changes in color of
dehydrated eggs during storage. Ind. Eng. Chem. _3_§. 347.

Evans. C. D. (1961). "Proceedings Flavor Symposium
Campbell Soup Co. Rd. and Publisher. p. 123.

Farquhar. J. 14.. Insull. 11.. Rosen. P.. Stoffel. 14.. and
Ahrens. B. 11.. Jr. (1959). Dietary effects a: huen milk
fatty acids. Nutrition Rev. .11. 1203.

I‘VOIdg 11. Leg ma.g ‘e Ge. Dka. A. Le. “ m. “a H.
(1966). Dehydrated egg powders. Sources of off-flavors
developed during storage. Ind. Eng. Chem. _3_§. 1079.

 

 

 

 

 

58.

59.

60.

61.
62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

85

Fisher. F. R. (1962). Freeze-drying of foods. Proc. of
00010131100. Chi-“0. Ille. Mtle Ame 301e, mtle ”Be
Council. 237.

Flosdorf. R. H. and mdd. S. (1935). Procedure and apparatus
for preservation in "Lyophile" form of serum and other
biological substances. J.. I-unol. _2_9_. 389.

Flosdorf. E. W. (1945). Drying by sublimation. Food Ind.
.11. 92.

Flosdorf. E. w. (1945). Drying meat by sublimation. Heat. 22. 27.

Flosdorf. E. W. (1945). Advances in drying by sublimation:
blood plasma. penicillin. foods. J. Chem. Educ. _2_2_. 470.

Flosdorf. E. w. (1947). Freeze-drying as applied to penicillin.
blood plasma and orange juice. Chem. mg. Frog. Q. 343.

Flosdorf. R. w. (1949). "Freeze-Drying". Reinhold Publishing
Corp.. New York. ' . A

Flosdorf.. E. W. (1954). Preservation by freeze-drying. Refrig.
m. 6‘2, 49.

Flosdorf. E. W. (1957). Freeze-dried foods: what they are -
how they are prepared. Frosted field 24 15 .

Folch. J.. and Lees. H. (1955). Proteolipids. a new type of
tissue lipoproteins. J. Biol. Chem. 3.3;. 807.

POI-Chg Jen 113... “e m sm1.ys Ge He Se (1957)e A .MI.
method for the isolation and purification of total lipides
from animal tissues. J. Biol. Chem. 339. 497.

Food Machinery and Ch-icals. (1961). Freeze-drying equipment.
Food Trade Rev. 31. 61.

Forrest. J. C. (1959). Large scale freeze-drying equipment for
im.tu££.e ”rite Chue me l. 3900

Friedman. 1.. and Kline. 0. L. (1950). The relation of the amino
acid- sugar reaction to the nutritive value of protein
mr°1yuc..e J e “tut!“ £9. 295s

Friedman. 1.. and Kline. 0. 1.. (1950). Amino acid - sugar
reaction. J. Biol. Chem. .184. 599.

mat.g Ae 16.. 3111.. use “"10. Ge Te (1961). 6‘:le in
oxidising fat. V. The caposition of neutral volatile mono-
carbonyl compounds from autoaidised oleate. linoleate.
linolen'ate esters. and fats. J. Am. Oil‘Chem. Soc. .12.
371. '

 

 

 

 

 

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

85.

86.

87.

86

Cane. R. (1943). Dried meat. III. The water relations of air-
dried. precooked beef and pork. J. Soc. Chem. Ind. _63. 139.

Gene. R. (1954). Freeze-drying of foodstuffs. In "Biological
Applicationsof Freezing and Drying". Edited by R. J. C.
Harris. Academic Press. New York.

Gnu. 1.. (1962). Die Bedeutung der Cefriertrocknug fur die
Lebensmittelindustrie [The importance of freeze-drying for
the food. industry.) 1.. 0nd E )3. 11.

61133813 1e Deg “113°“, Ge De. 3‘“ Schvelgert. Be Se (1954)e
Biochemistry of myoglobin. Chemical studies with purified
metmyoglobin. J. Agr. Food Chem. 2;. 1037.

Gooding. E. G. R.. and Rolfe. E. J. (1957). Some recent work
on dehydration in the United Kingdom. Food Technol. _6_, 302.

Gooding. E. G. B. (1962). The storage behaviour of dehydrated
food. In "Recent advances in food science" Ed. Hawthorn.
J. and Leitch. J. M. Butterworths. London. 22.

Gorling. P. (1961). Der Wissenschaftliche und technische
Stand der Gefriertrocknung von Lebensmitteln. [Scientific
and technical status of freeze-drying of foods.) '2. f.
Lebensmtl.-Uter-such. U.-Forsch. 11.3. 128.

Gornhardt. L. (1955). Die nichtenzymatische Eraunung von
Lebensmitteln. Fette U. Seifen _Sl, 270.

Guss, C. 0. (1952). in Contribution of Browning Research to
Ration Item Stability, pp. 26-29. Research and Development
Associates. Food and Container Institute. Inc.. Chicago. 111.

Handy. M. R. (1958). Ion-protein interrelations affecting the
quality of dehydrated meat. U.S. Off. Tech. Ser. P3145764.

Handy. M. K. and Deatherage. F. E. (1958). Observations on the
changes produced in the freeze dehydration of meat. Food
13011301» 1.3, 49s

Randy. H. R.. Cahill. V. R. and Deatherage. F. E. (1959).
Some observations on the modifications of freeze dehydrated
melt. F0“ R38. 2.9., 79o

Hm. R.. and Deatherage. F. E. (1960). Changes in hydration
and changes of muscle proteins. during freeze-dehydration of
mate Food ”8e .32, 537e

Hanan. R. (1960). Freeze dehydration. In "Advances in Food
Research". Ed. Chichester. C. 0.. Mark. E. 11.. and Stewart. C. F.
3.2. 411.

 

 

 

 

 

 

 

 

89.

90.

91.

92.

93.

94.

95.

96.

97.

99.

100.

101.

87

Rama. R. (1962). Chemische und physikalische Veranderungen
bei der Cefriertrocknung von Fleisch. [Chemical and physical
changes with) freeze-drying of meat.) Ralte H: 202.

Ba-aer. w. B. (1911). A note on the vacuum desiccation of
“ccCrue Je N. u.e 294 5270

Rankins. O. C. and Eetzer. E. B. (1947). Rehydration of
dehydrated meat. Food Res. _13. 111.

Hansen. 8. W. F. (1958). Some observations on the problem of
space feeding. Food Technol. .13. 430.

Hansen. 8. N. F. (1959). The accelerated freeze-drying of
fade PM ‘23. 2‘5.

Hanson. S. v. F. (1961). The maintenance of life in space
ships. III. The food problem. In " Institute of Biology.
The Biology of Space Travel" 33. London.

Harper. Je Ce .nd 1W1. Ae Le (1957)e "Cele.dry1u8 0‘ EM
products. "Advances in Food Research" Academic Press. Inc..
N.Y.. Ed. E. 11. llrak and C. F. Stewart 1. 172.

Harper. J. C. and Chichester. C. 0. (1960). "Freeze-drying
application of dielectric heating" in "Freeze-drying of Foods"
The Carrad Press. Champaign. Ill.

Harper. J. 0.. and Chichester. C. O. (1961). Freeze-drying--
application of dielectric heating. In "Researeh and Development
Association. Food and Container Institute. Freeze-dehydration
0f f0d8e lle

HarpCrg Je Ce. Chichester. Ce 0e. m 3%.:th '1'. Be (1962).
Freeze-drying of foods. Agr. Eng. 43. 78.

Eaugaard. 6.. Tuermenn. I... and Silvestri. 11. (1951). A
study of the reactions of aldoses and amino acids. J.A.C. S.
1;. 4594. .

We Re R.. ‘d mrlifip re Po (196°)e m “11“” 100k. ‘t
freeze dehydration economics and engineering. Food Technol.
y... 27.

Renick. A. S. (1961). Oxygen problem in dehydrated foods.
Res. and Devlpmt. Assoc. Food and Container Inst. Activ. Rpt.
13. 200.

Henrickson. R. 1... Brady. D. E.. Cehrke. C. N. and Brooks. R. F.
(1955). Dehydrated pork studies. Removal of glucose by
yeast fermentation. Food Technol. 2. 290.

 

 

 

102.

103.

104.

105.

106.

107 .

108.

109.

110.

111.

112.

113.

114.

115.

116.

88

Henrickson. R. 1... Brady. D. E.. Cehrke. C. N. and Brooks. R. F.
(1956). Dehydrated pork studies. Removal of glucose by
glucose-oxide“ azyme. Food Technol. 19. 1.

1...... r. 11.. Ron. 8. R.. Lea. c. 11.. such. J. n. a. (1949).
Protein degradation in stored milk powder. Proc. 12th Intern.
Dairy Cong. (Stockholm) _2_. 166.

Herrlinger. P.. and Riermeier. F. (1948). Inaktivierung und
Regeneration der Peronydase in winebehendelten Pflanzeneweben.
’10Chae 2g 31’.

Herrnnn. R. (1961). Die Cefriertrocknung des Fleisches und
anderer tierische Produkte. [The freeze-drying of meat and
other animal products.l Fleischwirtschaft 12. 730.

Hirschmann. D. J.. and Lightbody. H. D. (1947). Effect of
bacteria on quality of stored lyophilised egg powders.
Food R..e E. 381.

KNEW. Re 83d snidCrg Ge Ge (1%6)e Mtdtive V81“ Of
protein in dehydrated meat. Food Res. .11.. 494.

Hodge. J. E. and Rist. C. E. (1952). N-Clycosyl derivatives of
secondary amines. J. Am. Chem. Soc. 14. 1494.

Hodge. J. E. (1953). Chemistry of Browning Reactions in Model
Systems. J. Agri. Food Chem. 1. 928.

Hofstrand. J.. and Jacobson. N. (1960). The role of fat in
the flavor of lamb and mutton as tested with broths and with
d.”t f.t.e 30“ Res. £2. 706.

Holden. H. F. (1936). Nathemoglobin. a spectrophotometric
study. Australian J. Exptl. Biol. Med. 'Sci. .1_4_. 291.

Holman. R. J.. and Widmar. C. (1959). Polyunsaturated fatty
acids in beef heart mitochondria and derived enzymatically
active lypoprotein fractions. J. Biol. Chem. 2%, 2269.

HOWCCtnp Iep ”d or”. Pe F. (1960).. rl‘vor .md1CC a "Bf
w ”the Je me ’9“ Che 9's ‘Me

HomCCtup 1e. or”. Pe Re. C“ sallb‘ChCIg We lee (196°).
Constituents of meat flavors; beef. J. Agr. Food Chn. g. 65.

Hothhp 1e, Alford, Je Aeg Elliott. lee Be a Ct”, Pe Fe
(1960). Determination of free fatty acids in fat. Anal.
cm. L2’ 540.

Howe. r- 3.. and Barbells. s. o. (1937)

The flavor of meat and meat products.
Food a... _2_. 197.

 

 

 

 

 

117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

128.

129.

89

Howard. A.. and Lawrie. R. A. (1956). Spec. Rep. Food Invest.
Bd.. London. No. 63; also C.S.I.R.O. Australia. Tech. Paper
No. 2e

Hunt. S. N. V.. and Natheson. N. A. (1958). Adenosine triphos-
phatase and contraction in dehydrated muscle fibres.
Nature [London] 1.81. 472. .

Hunt. S. H. V.. and llatheson. N. A. (1958). The effect of
dehydration on actoqyosin in fish and beef muscle. Food Technol.
_13. 410.

Mt. Se )1. Veg m nth..ong Ne Ae (1959)e The relatimlhip
between the quality of dehydrated raw beef and the adenesine
triphosphatase activity after storage at various moisture
contents. Food Res. _29. 262.

Ingles. D. 1.. and Reynolds. T. N. (1958). Chemistry of non-
enzymatic browning. IV. Determination of amino acid-deoxyfructoses
in browned freeze-dried apricots. Austral. J. Chem. _l_l. 575.

Ingram. H. (1962). Freeze-drying of foodstuffs. Food Technol.
lg, 35. ‘

Jason. A. C. (1958). Fundamental aspects of dehydration of
foodstuffs; report on Aberdeen Conference. Chem. and Indus.
26. 821.

Jones. N. R. (1954). Browning reaction in freeze-dried extractives
from the skeletal mscle of codling (Cadus Callaries). Nature
[London] 3.14. 605.

Jones. N. R. (1956). Discoloration of muscle preparations from
codling (Cadus Callaries) by degradation products of i-
methylhistidine. Nature [London] .1_7_7_. 748.

Jones. N. R. (1962). Browning reactions in dried fish products.
In "Hawthorne. J. and Leithch. J. 14.. ed. Recent Advances in
Food Science" 3.. 74. * '

Johnson. J. Marvin. (1941). Isolation and progerties of a pure
7‘“: polypeptidase. J e 8101» Chen. .1-3-2.’ 5 e

Jab-um, De Aep ‘11“29 Ge We. ‘fi KOChp Be Be (1960). A study Of
physical and chemical changes in beef proteins caused by
lyophilization. Food Technol. 14. 25.

JOhflBOfl. 0. Ce. Me se Ses ‘mrws to As (1953). VOlfitllO
decaposition products of oxidative polymers of ethyl-linolenate.
J. Am. Oil Chem. Soc. .32. 317.

 

 

 

 

130.

131.

132.

133.

134.

135.

136.

137 .

138.

139.

140.

141.

142.

143.

144.

90

Rain. J. T.. Saga. H. (1963). Fatty acid composition of lecithin
from beef brain and egg yolk. J. Biochem. (Tokyo) 24;. 403.

Ratchalsky. A. and Sharon. N. (1953). Kinetics of aldose-amino
acid interaction. Biochin. at. Biophys. Acta .12. 290.

Kline. R. w. and Stewart. G. F. (1948). Glucose-protein reaction
in dried egg albumin. Ind. Eng. Chem. 42. 919.

K11”. Le. Ge“, Je Beg ‘fi Swede. T. I. (1951)e R013 0f
glucose in the storage deterioration of whole egg powder. II. A
browning reaction involving glucose and cephalin in dried .whole
e388. Food reabmle a. 1810

Koch. R. B. (1961). Dehydrated foods and model systems. Symp.
Foods [Proc.] _2_. 230.

None. T.. and Colowick. S. P. (1961). Isolation of skeletal
muscle cell membrane and some of its properties. Arch. Biochem.
am Blophys. 2. 5200

Kraft. R. A. and Morgan. A. F. (1951). The effect of heat treat-
ment on the nutritive value of milk proteins. IV. The biological
value of unheated and autoclaved dried skim milk. J. Nutrition
45. 567.

Kremlich. W. E.. and Pearson. A. M. (1958). Some preliminary
studies on meat flavor. Food Res. 32. 567.

Rramlich. w. E.. and Pearson. A. N. (1960). Separation and
identification of cooked beef flavor components. Food Res..
25. 712.

Kraybill. H. R. (1943). The dehydration of meat. Proc. Inst.
Food Technol. 90.

Rretovich. V. 1.. and Tohareva. R. (1948). Interaction of amino
acids and sugars at high temperature. Biokhimiya g. 508.

Robots. T. (1941). Reactions of sugars with amino acids under
mild conditions. J. Biochem. (Japan) 34. 119.

Lane. J. P.. and Bratzler. 1.. J. (1962). Spectrophotometric
estimation of metmyoglobin in frozen meat extracts. J. Food
Sci. a. 343.

Long. 0. (1961). Die Cefriertroclmung von Lebensmitteln. [The
freeze-drying of foods.] Ralte 41. 653.

Lang. 0. (1962). Die Cefriertrocknung von Fleisch. [The freeze-
drym 0f ”.tel “It. lg. 195e

 

 

 

 

91

145. Lang. 0. (1962). Die Cefriertrockung von Fleisch. [The freeze-
drying of meat-. Schlact .-u.Viehhof-Ztg. .6_2_. 167.

146. Les. C. H.. Moran. T.. and Smith. J. A. (1943). The gas packing
and storage of milk powder. J. Dairy Sci. _13. 162.

147. Lee. C. H. (1943). Dried meat. V. The storage of dried meat.
Je SOCe Chan. we 2. 200.

148. Lee. C. H. (1947). A note on the Chapman and NcFarlane method
for the estimation of reducing groups in milk powder.
Analyst ]_2_. 336.

149e 1.08. Ce 1!. m White. Je Ce De (1%7)e n3 amino-aldehyde ‘
reaction in the deterioration of stored foods. with particular
reference to milk powder. Proc. 11th Intern. Cong. Pure and
Applied Chem. (London) 2. 519.

150. Lea. C. H. and Hannan. R. S. (1949). Reaction between proteins
and reducing sugars in the dry state. I. Effect of water. pH and
temperature on the primary reaction between casein and glucose.
Biochin. et Biophys. Acta _3. 312.

151. Lea. C. H. and Hannan. R. S. (1950). Biochemical and nutritional
significance of the reaction between proteins and reducing
sugars. Nature _1_6_5. 438.

152. Lea. C. H.. Hannen. R. S. and Creaves. R. I. N. (1950). Reaction
between proteins and reducing sugars in the dry state. Dried
human blood plasma. Biochem. J. 51. 626.

153. Les. C. H. and Hannan. R. S. (1950). Reaction between proteins
and reducing sugars in the dry state. 11. Nature of the protein
groups reacting. Biochim at. Biophys. Acta 5. 433.

154. Lee. C. H.. Hannan. R. S. and Rhodes. D. N. (1951). Reaction
between proteins and reducing sugars in the dry state. IV.
Decomposition of nine-sugar complex and the reaction of
acetylated casein with glucose. Biochim et Biophys. Acta 1. 366.

155. Lee. C. H. (1957). Deteriorative reactions involving phospho-
lipids and lipoproteins. J. Sci. Food Agr. _8_. l.

156. Lee. C. H. (1958). Chemical changes in the preparation and
storage of dehydrated foods. In "Society of Chemical Industry.
Fundamental aspects of the dehydration of foodstuffs" p. 178
London.

157. Lee. C. H.. and Parr. 1.. J. (1961). Some observations on the
oxidative deterieration of the lipids of crude leaf protein.
Je Sci.. ’0“ Agr. . 1'22 785e

 

 

 

 

 

158.

159.

160.

161.

162.

163.

164.

165.

166.

167 Q

168.

169.

170.

171.

172.

92

Lemberg. R. and Legge. S. H.
piglents.

Lamberg. R. and Legge. J. N. (1950). Pyrrole pigments. Ann. Rev.
BiOMe .12.. ‘31.

(1949). Hematin canpounds and bile
Interscience Publishers. Inc.. New York.

Lepper. H. A. Official Nethods of Analysis of the Association of
Official Agricultural Chemists. 7th Bd.. 1950. Washington. D.C.

Leslie. J. (1811). Nathede Nouvelle de produice et d'entretenir
la congeletion. Am. de Chin. at de Physique ]_§. 177.

Love. R. N. (1956). Influence of freezing rate on denaturation of
cold stored fish. Nature. 118. 988.

Lowry. J. R. and Thiessen. R.. Jr. (1950). Nutritive impairment
of proteins heated with carbohydrates. II. In vitro digestion
studies. Arch. Biochem. _2_._5_. 148.

m, Be 8.. GmICPAC‘m. Ce Ge. umrdg Se m 31m, “e
(1964). Aseptic canning of foods. VI. Hematin and volatile
sulfur compounds in strained beef. Food Technol. 18. 216.

Luyet. B. J.. and Mackenzie. A. P. (1960). Rehydration of
freeze-dried meat. Q” and CI Container No. DA 19-129-QN-1413.
File No. A-334. Progr. Rpt. No. 4.

Hans. J. (1957). Contribution a l'etude de la viande de boeuf
cryodessechee. [Contribution to the study of freeze-dried
beef.l Alfort. Ecole Natl. Vet. These 56.

Macara. T. J. R. (1943). Dried meat. 11. The growth of moulds
on dried meat. J. Sci. Chem. Ind. 93. 104.

NacDonnell. 1.. R.. Silva. R. B.. and Feeney. R. E.
sulfhydryl groups of ov-albumin.
93. 288.

Arch. Biochem. And Biophys.

Nacfarlane. u. c.. Cray. o. 14.. and Nheeldon. 1.. w. (1960).
Fatty acids of phospholipids fraa mitochondria and microscmes
of rat liver. Biochem. J. 23. 430.

Marteml Yanova. R. B. (1958). Opytnoe thanenie ryby uysushenoi
metodcm sublmtsii [Experimental storage of fish dried by
the freeze-drying method. In Nakarova. T. 1.. ed.
Tekhnologiya Rybnykh Produktov (Selected articles from
technology of fish processinsn 161. Noscow. Pishchepromizdat.

Natheson. N. A. (1959). Absorption of atmospheric moisture by
freeze-dried pork and fish. Nature [London] _193. 1949.

( 1961).
PM PfOCe P‘ckge _32. 87.

Natheson. N. A. and Penny. J. F.
cod.

Storage of dehydrated

 

 

 

 

 

 

173.

174.

175.

176.

177.

178.

179.

180.

181.

182.

183.

184.

185.

93

Natheson. N. A. (1962). Enzymatic activity at low moisture
levels and its relation to deterioration in freeze-dried foods.
J. Sci. Food Agr. - 1.3. 248.

Natheson. N. A. (1962). Enzymes in dehydrated meat. In "
Hawthorn. J.. and Leitch. J. 11. Recent advances in food
science. 2. 57. '

Natsumoto. J. (1961). Denaturation of fish protein by freeze-
drying. (Jap.) Reito (Refrigeration) 26. 1110.

Neryman. H. T. (1960). Principle of freeze-drying. N.Y. Acad.
Sci. Am. .82. 630.

Meyer. B.. Nysinger. N. and Buckley. R. (1963). Storage effects
on beef. effect of 3 years of freezer storage on thiamine.
riboflavine and niacin content of ripened and unripened beef.
Jo Agr. PM 0116!. ll». 5250

Milne. C. R. and Patric. D. S. (1961). User experience with a
large- scale freeze-drying plant for drying hmn plasma.
Symposium on user experience of large scale industrial vacuma
plant. Institution of Mechanical Engineers. London.

Ministry of Agriculture Fisheries and Foods (1961). The
Accelerated Freeze-drying (A.F.D.) Method of Food Processing
11.11. 8.0.

Mitchell. J. H. (1954). Control of changes occurring in beef
during dehydration and subsequent storage. Res. and
Devlpmt. Assoc. Food and Container Inst. Activ. Rpt. 2. 296.

Mohamed. A.. Fraenkel-Conrat. H. and Olcott. H. S. (1949). The
browning reaction of proteins with glucose. (Archiv. of
Biochem.) Arch. Biochem. _23. 157.

Norse. R. E. (1962). Preparation and storage stability character-
istics of dehydrated sausage products. 0. 8. Off. Tech. Serv.
0.8. Govt. Res. Rpt. 37.. 146.

Nestor. J. B. and Chapun. R. A. (1949). Browning reaction in
dried “111‘ WCre cme Jo Ra’s E’ 429.

Nemitz. C. (1962). Vargleichende Untersuchungen uber die Nasser-
wiederaufnahmc gefriergetrockneter und warmluft getrockneter
Census [Comparative research on the water rec-uptake in freeze-
dried vegetables.) Indus. Obst.-u. Cemuseverwert. __4_7_. 409.

Neumann. R. (1960). Les Problemss de Nssure et de Teglage en
Lyophilisation. Traits de Lyophilisation. Hermann. Paris 180.

 

 

 

 

 

186.

187 .

188.

189.

190.

191.

192.

193.

194.

195.

196.

197 .

198.

199.

200.

94

Oetjen. C. w. (1961). Continuous freeze-drying with automatic
temperature control. In Research and Development Associates
Food and Container Institute. Freeze-dehydration of foods. 78.

Olcott. H. S. (1962). Deteriorative reactions in stored freeze-
dried meat and fish. In Fisher. F. R.. ed. Freeze-drying of
foods; Proceedings of a conference. Chicago. Ill. Natl. Acad.
Sci.. Natl. Res. Council. Washington. p. 74.

Olley. J. and Lovern. J. A. (1960). Phospholipidhydrolysis in

cod flesh at various temperature.
J. Sci.. Food Agr. ‘ 1.1... We

Olley. J. N. gt; _a_1_.,. (1961). Unpublished work from Torry Research
Station. Aberdeen.

Grant-Rules. E.. Hewston. E. N. and Butler. 1.. (1946). Effects
of different methods of dehydration on vitamin and mineral
value of meats. Food Res. _1_1_. 486.

Osman. H. O. A.. and Norse. R. E. (1960). Preparation and
storage characteristics of freeze-dried sausage. II.
Mlsion stabilization and rate of freeze-drying in
cominuted sausages (frankfurters). Food Technol. 11;. 37.

Overby. 1.. R. and Frost. D. V. (1952). The effects of heat on
the nutritive value of protein hydrolyzatas with glucose.
J. Nutrition _4_6. 539.

Parkes. A. S. and “th. A. U. (1960). Recent research in
freezing and drying. Blackwell Scientific Publications. Oxford.

Patton. S. (1955). Browning and association changes in milk and
its products. A review. J. Dairy Sci. _3_8_. 457.

Pearce. J. A. (1950). Fluoresce changes in solutions of glucose
and glycine and of acetaldehyde and almonia. Food Tech. 4. 416.

Penny. I. F. (1960). The effect of accelerated freeze-drying
on the colour of beef. Food Proc. and Packg. 32. 363.

Penny. I. F. (1963). Cmparison of freeze-dried beef muscle of
high or low ultimate pH. J. Sci. Food Agr. .15" 535.

Pippen. E. l... N. Nonaka. F. T. Jones and F. Stilt. (1958).
Volatiles carbonyl caepounds of cooked chicken. I. Cmapounds
obtained by air entraiment. Food Res. 33. 103.

Porter. R. R. (1948). The unreactive amino groups of proteins.
Biochin. et Biophys. Acta 2. 105-112.

Record. B. R. and Taylor. R. (1958). Freeze-drying equipment for
large-scale laboratory use. Biochem. J. 9;. 420.

 

 

 

 

201.

202.

203.

204.

205 .

206.

207 .

208.

209.

210.

211.

212.

213.

95

Regier. 1.. H.. hereon. N. R.. Tappel. A. 1... Conory. A.. and

Stewart. 6. F. (1954). The preparation and storage
stability of freeze-dried beef. Food Techol. g. .42.

Regier. 1.. w. Physical and chemical changes in freeze-dried beef
during storage. Ph.D. Thesis. Univ. of California. (1956).

Regier. 1.. H.. and Tappel. A. 1.. (1956). Freeze-dried meat. III.
Non-oxidativo deterioration of freeze-dried beef. Food Res.
21. 630.

Regier. 1.. H.. and Tappel. A. 1.. (1956). Freeze-dried meat. IV.
Factors affecting the rate of deterioration. Food Res. _2_1.._. 640.

Rey. 1.. R. (1960). "Systemes Automatique et Nachsnismes de
Regulation" Deuxieme cours Internationauy de Lyophilisation.
Lyme

Richard Bassette. Shheyla Maria. and C. H. Hhitnah. (1962).
Gas chromatographic analysis of head space gas of dilute
aqueous 80111110118. Anal-e Chue .32. 1540.

Richards. E. 1.. (1956). Non-enzymatic browning: the reaction
between D-glucose and glycine in the "dry" state. Biochem.
J. _§_4_. 639.

Robert. 1.. and Penaranda. F. (1953) Sur 1'interaction des
aldehydes. reductones et des acides amines et proteines. II.
Cinetique de l'interaction des acides amines et de l'acetaldehyde.
suivie par la chrmatographie et l'ionophorese sur papier.
Bulletin de la Societe de Chimie biologique _3_§. 791.

Robert. 1.. and Nolla. N. (1953). Sur l'interaction des aldehydes.
reductones et des acides amines et proteines. III. Etude de
l'hydrolyseenzymatique des proteines' traitees par 1'acetaldehyde.
Bulletin dela Societe de Chimie biologique _3_§_. 807.

Rolfe. E. J. (1954). Development of Neat Dehydration in the
United Kingdom" Fod Trade Rev.. 24. 112,

Rolfe. E. J. (1956). An improved method for dehydrating meat.
Food [London] 35. 199.

Rolfe. E. J. (1958). Fundamental aspects of the dehydration of
foodstuffs. The influence of the conditions of dehydration on
the quality of vacuum dried meat. Food Nanufacture 33. 251.

Salwin. H. (1961). Moisture levels required for stability in
dehydrated foods. Res. and Devlpmt. Assoc. Food and Container
mte A¢t1Ve ‘Cpte 13. 19]»

 

 

 

214.

215.

216.

217.

218.

219.

220.

221.

222.

223.

224.

225.

226.

96

Salwin. H. (1961). The role of moisture in deteriorative reactions
of dehydrated foods. In Fisher. F. R.. ed. Freeze-drying of
foods; proceedings of a conference. Chicago. 111.. Natl. Acad.
Sci.. Natl. Res. Council. p. 58.

Schackell. 1.. F. (1909). An improved method of desiccation with
some applications to biological problems. Am. J. Physiol.
24. 325.

Schwartz. H. N. and Lea. C. H. (1952). The reaction between
proteins and reducing sugars in the "drv" state. Relative
reactivity of the and amino groups of insulin. Biochem. J.
50. 713.

Schwinmer. S. and Olcott. A. S. (1954). Reactions between glycine
and hexose phosphate. J. Am. Chem. Soc. 22.

Sharp. J. G. (1953). Dehydrated meat. Dept. Sci. Ind. Res.
Food Invest. Board Spec. Rept. no. 2. [London].

Sharp. J. G. (1957). Deterioration of dehydrated meat during
storage. I. Non-enzymatic deterioration in absence of oxygen
at tropical temperatures. J.‘Sci. Food Agr. .8. 14.

Sharp. J. G. (1957). Deterioratiai of dehydrated meat during
storage. 11. Effect of pH and temperature on browning changes
in dehydrated aqueous extracts. J. Sci. Food Agr. .8. 21.

Sharp. J. G.. and Rolfe. E. J. (1958). Deterioration of dehydrated
meat during storage. In Society of Chemical Industry (London)
Fundamental aspects of the dehydration of foodstuffs. p. 197.

Sharp. J. G. (1958). Fundamental aspects of the dehydration of
deterioration of foodstuffs. Food Nanufacture. 33. 250.

Sharp. J. G. (1958). Fundamental aspects of the dehydration of
foodstuffs. 2. Abstracts from the Aberdeen Conference ..
deterioration of dehydrated meat during storage. Food
Manufacture 23. 250.

Sharp. J. G. (1962). Non-enzymatic browning deterioration in
dehydrated meat. In Hawthorn. J. and Leitch. J. M. ed.
Recent advances in food science. Butterworths. London. _2_. 65.

Sidwell. C. G.. Salwin. H.. and Koch. R. B. (1962). The molecular
oxygen content of dehydrated foods. J. Food Sci. 27. 255.

Simonson. H. D. and Tarassuk. N. P. (1952). Fluorescence and
associated changes produced upon storage of evaporated milk.
J. Dairy Sci. .33. 166.

 

 

 

 

227.

228.

229.

230.

231.

232.

233.

234.

235.

236.

237 .

238.

239.

97

Sinitsyn. A. (1960). Snake metodom sublimatsii gotouykh K
upotrebleniyu masoproduktov (Drying by the sublimation method
of meat products ready for use.) Nyashaya Indus. SSSR 31. 25.

Smithies. W. R. (1957). New developments in the freeze-drying of
meat. Chem. and Ind. . _22. 1008.

mum... w. a. (1959). Design of freeze-drying equipsent for
the dehydration of foodstuffs. Food Tech. 1.3. 610.

anithies. W. R. (1962). The influence of processing conditions on
the rehydration of freeze-dried foods. In Fisher. F. R. ed.
Freeze-drying of foods; proceedings of a conference. Chicago.
Ill. Natl. Acad. Sci.. Natl. Res. Council. Washington. p. 191.

Speck. J. 0.. Jr. (1952). "Contributions of Browning Research
to Ration Item Stability". Research and Development Assoc.
Food and Container Institute. Chicago. Ill. 29.

Stadtman. E. R. (1948). Non-enzymatic browning in fruit products.
In "Adv. in Food Res." (edited by E. N. link and G. F. Stewart).
Vol. I. Academic Press. New York.

Stephenson. J. 1.. (1960). Fundamental problems in freezing and
drying of biological materials. Recent research in freezing
and drying. Blackwell Scientific Publications. Oxford. 121.

Stewart. C. F. and Kline. R. W. (1948). Factors influencim
the rate of deterioration in dried egg albumen. Ind. Eng.
Chem. 42. 916.

Stukis. J. N. (1954). Effect of dehydration and rehydration on
the histochemical and histological characteristics of beef.
Res. and Devlpmt. Assoc. Food and Cont. Inst. Activ. Rpt. 5. 289.

Tappel. A. l... Conroy. A.. Ernerson. N. R.. Regier. 1.. W.. and
Stewart. C. F. (1955). Freeze-dried meat. 1. Preparation and
properties. Food Technol. 2. 401.

Tappel. A. 1.. (1956). Freeze-dried meat. 11. The mechanism of
oxidative deterioration of freeze-dried beef. Food Res. 21. 1.

Tappel. A. and Harper. J. (1957). Freeze-drying of food products.
In "Adv. in Food Research" (edited by E. N. Nrak and C. F.
Stewart). Vol. VII. Academic Press. New York.

Tappel. Ae Lee ”temp Rep ‘fi P1°&.rg Be (1957). h‘.z.'
dried meat. V. Preparation. properties and storage stability of
precooked freeze-dried meat. poultry. and seafoods. Food
IOChnOIe 2p 599e

 

 

 

 

 

240.

241.

242.

243.

245.

246.

247 .

248.

249.

250.

251.

252.

253.

254.

98

Tappel. A. 1.. (1961). Spectral measurements of hematin pigments
of cooked and cured meats. J. Food Sci . 32. 269.

Terr. H. 1.. A. (1950). The Naillard reaction in fish products.
J. Fisheries Res. Board. _8. 74.

Tarr. H.1..A. (1953). Ribose and the Naillard reaction in fish
muscle. Nature 111. 344.

Terr. H. 1.. A. and Bissett. H. N. (1954). Cause and control of
the browning of heat-processed fish products. Prog. Rept.
Pacific Coast Stas.. Fisheries Res. Board Can.. No. 2_8_. 3.

Thompson. 11. (1962). Freeze-drying of foodstuffs. Food Trade
RBVo _3_2_s 37o

Mon. Jo Se m FOX. Jo Bo (1960)e EffGCt Of water ‘nd
oxygen upon deteriorative changes in freeze-dried beef.
(Sum) Amer. Neat Inst. Found. C. 22. 27.

Thomson. J. 8.. Fox. J. B.. and Landmann. W. A. (1962). The
effect of water and temperature on the deterioration of freeze-
dried beef during storage. Food Technol. 13. 131.

Turner. E. W. (1956). The future of dehydrated meat as a
cmenience food itale In Reae Conf. PrOCe £9 37.

Turner. E. W. (1956). Reservation of meet by freeze-drying
Natl. Provisioner 133. 142.

Urbin. N. 0.. and Worland. N. C. (1961). Behavior of water
phases in freeze-drying. 014 F and C 1 contract No. A 19-129-
QN-1347, file No. A-332. Rept. No. 9. Final.

Von Hippel. A. R. (1954). "Dielectric Materials and Applications"
The Technology Press of N.I.T. and John Wiley and Sons. Inc..
New Yorke .

Wang. H. (1954). Dehydration of meat (Sum.) Amer. Neat Inst.
Found. Ce & 59o

Wang. H.. Auerback. E.. Bates. V.. Andrews. F.. Doty. D. N. and
Kraybill. H. R. (1954). A histological and histochemical study
of beef dehydration. 11. Influence of carcass grade. aging.
muscle and electrolysis pre-treatment. Food Res. _12. 154.

Wang. Hep AmruCR, Eeg 38:08. Veg ”Qty. Do Me and KrCYbillg He Re
(1954). A histological and histochemical study of beef dehydra-
tion. IV. Characteristics of muscle tissues dehydrated by
freeze-drying techniques. Food Res. .12. 543.

Wang. H.. and Auerbach. E. (1954). Structure and reconstitutability
of beef muscle dehydrated by freeze-drying. Anal. Rec. .118. 366.

 

 

 

255.

256.

257.

258.

259.

260.

261.

262.

263.

264.

99

Wendland. C. (1952). Strongly reducing substances (reductones)
formed in the processing of natural products. 1. Formation of
strongly reducing substances from sugars and natural products.
Archiv. Pharm. £82. 71.

Whimreg Re Aep Palhrdg Ae Bop nQYbillg Ho Re m Flyehlem. Ce Ae
(1946). Vitamin content of dehydrated meats. Food Res. 11.419.

Whitmore. Re Asp 591183011, Deg Kraybill. Re Re and “ebb, 3. Ho
(1948). Packaging dehydrated meat. Food Res. _l_3_. l9.

Wierbicki. E.. Kunkle. 1.. E.. Cahill. V. R.. and Deatherage. F. E.
(1956). Post-marten changes in meat and their possible relation
to tenderness together with some comparisons of meat from
heifers. bulls. steers and diethyl-stilbesterol treated bulls
and steers. Food Techol. Ev .80.

Wolfram. N. L. (1952). in "Contributions of Browning Research
to Ration Item Stability" pp. 36-40. Res. and Devlpmt.
Associates. Food and Container Institute. Chicago. Ill.

Wolfram. N. 1... Kolb. D. R.. and Longer. A. W.. Jr. (1953).
Chemical interactions of amino compounds and sugars. V11. pH
dependency. J. Am. Chem. Soc. L5. 3471.

”00d, To, and Bender. Ao Ee (1957)e Aulyfltfl 0f t1..u. con-

stituents. Cc-ercial out-muscle extract.
Biochem. J. _61. 366.

Younathan, M. T.. and B. 11. Watts. (1960). Oxidation of tissue
lipids in cooked pork. Food Res. 32, 538.

Younathan. M. T.. and Watts. B. N. (1959). Relationship of
meat pigments to lipid oxidation. Food Res. _2_4_.728.

Yueh. M. H. and Strong. F. M. (1960). Some volatile con-
stituents of cooked beef. J. Agr.. _ sod Chem. _6_. 491.

 

 

 

 

 

 

 

 

 

 

    

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