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This is to certify that the

thesis entitled

SPECTROPHOTOMETRIC ESTIMATION OF

METMYOGLOBIN IN FROZEN MEAT EXTRACTS

presented by
J. Perry Lane

has been accepted towards fulfillment
of the requirements for

flat/we

Major prof?

Date December 14, 1960

 

0-169

LIBRARY

Michigan State
University

 

 

ABSTRACT

SPECTROPHOTOHETRIC ESTIMATION OF METMYOGLOBIN
IN FROZEN HEAT EXTRACTS

by J. Perry Lane

The changes in the oxidative state of myoglobin, the primary pig-
ment of lean muscle, are directly associated with changes in the color
of lean meat. Townsend (1958) reported that fluorescent lights acceler-
ated the rate of color deterioration and the formation of metmyoglobin
in beef steaks packaged in a transparent wrapper and stored at 0° F.-

The present study was initiated to determine if frozen meat extracts
would react to light in a manner similar to meat cuts. The use of water
extracts would provide a series of samples with greater homogeneity than
was obtainable by using replicate cuts of meat. Another objective of
this study was to adapt the spectrophotometric method as described by
Hangel (1951) and Townsend (1958) to the estimation of the per cent met-
myoglobin in the frozen meat extracts. Finally, investigations were con-
ducted in an attempt to inhibit or characterize the light-catalyzed in-
crease in the rate of metmyoglobin formation of the frozen solutions.

The solutions were prepared by blending portions of beef rounds with
eight parts distilled water. The solutions were then centrifuged, fil-
tered and frozen. The samples were stored at 0° F. in either a walk-in
freezer under 25 foot-candles of fluorescent illumination or in a frozen
food display case under approximately 60 foot-candles of light. The
samples were periodically removed, thawed and analyzed in a Beckman

DU Spectrophotometer. From the Optical density the per cent metmyoglobin

J. Perry Lane

was estimated. The effects on the frozen solutions of: fluorescent
lights; dialysis; addition of the concentrated dialysate; addition of
various metal ion solutions; and addition of glucose were observed.

Frozen meat-water extracts served as a satisfactory medium for
following the increase in per cent metmyoglobin. The spectrophotometric
method previously referred to proved satisfactory for estimating the
per cent metmyoglobin in the solution. It was observed that fluorescent
lights caused a significant increase in the formation of metmyoglobin
in the solution, similar to that reported for frozen steaks.

Dialyzing the extract inhibited the increase of metmyoglobin in the
frozen solution when stored under light. Addition of copper, manganese
or calcium ions in the form of the chloride salts to the dialyzed solu-
tion did not cause an appreciable increase in the rate of metmyoglobin
formation. The addition of chloride salts of magnesium and iron con-
tributed to an increase in the rate of metmyoglobin formation.

When the dialysate was concentrated and added to the dialyzed resi-
due there was a significant increase in the rate of metmyoglobin forma-
tion. The addition of one cc. of a 1% or 2% solution of glucose to an
undialyzed solution did not inhibit metmyoglobin formation in meat solu-

tions when frozen and stored under light.

 

 

i _ J. Perry Lane

REFERENCES

Mangel, Mhrgaret. 1951. The determination of methemoglobin in beef
muscle extracts. Mo. Res. Bul. 474:1-24.

Townsend, William E. 1958. Effect of temperature, storage condition
and light on the color of prepackaged frozen meat. Unpublished
Ph. D. thesis, Michigan State University, East Lansing, Michigan.

SPECTROPHOTQIETRIC ESTIMATION OF WYOGLOBIN

1N FROZEN MEAT matters
By

0Wn
J. Perry Lane

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Animal Husbandry

1960

VITA
John Perry Lane
Candidate for the Degree of
Doctor of Philosophy
Thesis: Spectrophotometric Estimation of Metmyoglcbin in Frozen'Meat
Extracts
Outline of Studies:

Major Subject: Animal Husbandry
Minor Subjects: Biochemistry, Physiology

Biographical:
Born: September 18, 1928, at Chelsea, Massachusetts.

Undergraduate Study: University of Massachusetts, Animal
Husbandry Department, 1946-50.

Graduate Studies: Oklahoma State University of Agriculture
and Applied Science, 1956-58.

Michigan State University, 1958-1960

Experience: Graduate Assistant, Department of Animal Husbandry,
Oklahoma State University

Graduate Assistant, Department of Animal Husbandry,
Michigan State University

Member: Institute of Food Technologists
American Society of Animal Production

Date of Final Examination: December 12, 1960

ii

ACKNOWLEDGEMENT

The author wishes to express his appreciation to Professor Lyman J.
Bratzler for the initiation of this study and for his understanding and
patience throughout. The author is further indebted to Professor Bratzler
for his aid in the preparation of this thesis and for his assistance in
obtaining the graduate assistantship from Michigan State University
which made this study possible.

Acknowledgement is given to Dr. A. M. Pearson, Professor of Food
Science, Dr. W. D. Collings, Professor of Physiology and Pharmacology,
and Dr. R. U. Byerrum, Professor of Chemistry, for their critical reading
of this manuscript.

To Dr. B. S. Schweigert, Head of the Department of Food Science.and
Dr. W. D. Baten, Agricultural Experiment Station Statistician, the author
wishes to express his gratitude for suggestions and advice.

Appreciation is also extended to Dr. D. E. Ullrey, Assistant
Professor of Animal Husbandry, and to Dr. J. R. Brunner, Professor of
Dairy Technology, for the use of their spectrophotometric equipment.

The author wishes to dedicate this thesis to his wife, Constance,
without whose encouragement and assistance throughout four years of
advanced study and in preparation of this manuscript, successful comple-
tion of this study would never have been possible, and to his sons,

Frederic and Patrick, who have made it all worthwhile.

iii

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1

REVIEW 0? LITERATURE . . . . . . . . . . . . . . . . . . . . . . 3

o
o
o
o
o
e
U

Characteristics of the Muscle Pigment Myoglobin
1. Historical Development . . . . . . . . . . . . . . . 3

2. Physiological Function . . . . . . . . . . . . . . . 5

3. Chemistry of Myoglobin . . . . . . . . . . . . . . ;' 6

a. Structure and Composition . . . . . . . . . . . 6

b. Oxidative Reactions and Relation to Enzyme
Systems in Muscle . . . . . . . . . . . . . . 11

c. The Relationship of Myoglobin to the Color
Of Meat 0 O 0 0 fi 0 O O O O O O O 0 O O O O O 14

Methods of Purification and Measurement of Myoglobin . . . 17

The Relationship of Various Storage Conditions to Color
of Prepackaged Meat . . . . . . . . . . . . . . . . . . . 20

1. Fresh Meat Color . . . . . . . . . . . . . . . . . . 20

2. Frozen Meat Color . . . . . . . . . . . . . . . . . . 22

3. Effect of Light on Meat Color . . . . . . . . . . . . 24

Methods of Color Measurement . . . . . . . . . . . . . . . 27
EXPERIMENTAL OBJECTIVES . . . . . . . . . . . . . . . . . . . . 29
EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . . . . . . 30

Extract Preparation and Processing Prior to Metmyoglobin
Determination O O O O O O O O O O O O O O O O O 0 0 O O O 31

Metmyoglobin Determination . . . . . . . . . . . . . . . . 32

The Munsell Spinning Disc Method of Color Notation . . . . 35

iv

Individual Trial Procedures

RESULTS AND DISCUSSION .

SUMMARY AND CONCLUSIONS

LITERATURE CITED . . . .

APPENDIX . .

O

O

Page
38
44
79
81

88

 

Table

II.
III.

IV.

VI.

VII.

VIII.

IX.

LIST OF TABLES

Molecular Weight and Iron Content of Hemoglobin . . . .
‘Molecular weight and Iron Content of Myoglobin . . . .
Analysis of Variance of Per Cent Metmyoglobin: Trial 3

Dialysate Ash Determination . . . . . . . . . . . . . .
Analysis of Variance of Per Cent Metmyoglobin: Trial 8

Analysis of Variance of Per Cent Metmyoglobin: Trial 10
Analysis of Variance of Per Cent Metmyoglobin: Trial 11
Analysis of Variance of Per Cent Metmyoglobin: Trial 12

Analysis of Variance of Per Cent Metmyoglobin: Trial 13

vi

Page
7
8
48
55
6O
66
67
70

72

 

Figure

10.

11.

12.

13.

LIST OF FIGURES

Oxidative changes of myoglobin . . . . . . . . . . . . . .

The relationship between the various states of myoglobin
‘nd meat co lor O O O O O O 0 O O 0 O O O O O O O O O O 0

Diagram to illustrate the Munsell three-dimensional
description of color . . . . . . . . . . . . . . . . . .

Trial 1: Absorption spectrum of a beef steak packaged
under petroleum ether and stored at -20° F. under ten
foot-candles of illumination . . . . . . . . . . . . . .

Trial 2: Absorption spectrum of a meat-water solution
stored unpackaged in darkness at 0° P. for 45 days . . .

Trial 3: Per cent metmyoglobin of frozen meat solutions
stored in a 0° F. display case . . . . . . . . . . . . .

Trial 4: Per cent metmyoglobin of meat solutions stored
under 35 foot-candles of fluorescent illumination in a
0° F. walk-in freezer . . . . . . . . . . . . . . . . .

Trial 5: Per cent metmyoglobin of dialyzed meat-water
solution stored under 30 foot-candles fluorescent
illumination in 0° F. walk-in freezer . . . . . . . . .

Trial 6: Per cent metmyoglobin for a dialyzed and non-
dialyzed group of samples of meat solutions stored in
a 0° F. walk-in freezer under 30 foot-candles of
fluorescent illumination . . . . . . . . . . . . . . . .

Trial 7: Per cent metmyoglobin in dialyzed control samples
to which Mn (.17 mg./gm. meat) was added--a11 samples
stored in 0° F. walk-in freezer under 30 ft.-candles
illumination . . . . . . . . . . . . . . . . . . . . . .

Correlation between index of fading of thawed solutions
and per cent metmyoglobin for all treatments in Trial 8

Trial 8: Per cent metmyoglobin for all treatments stored
in 00 F. Sherer display case under 60 foot-candles of
illuuimtion O O O O C O O O O O O O 0 O O O O O O O O 0

Trial 9: Per cent metmyoglobin for a dialyzed group vs. a
reconstituted solution stored in a 0° F. display case
under 60 foot-candles of light . . . . . . . . . . . . .

vii

Page

11

16

36

45

47

49

51

53

54

57

59

62

64

Figure

14.

15.

16.

17.

18.

19.

Trial 10: Per cent metmyoglobin for varying levels of
manganese solution stored under 25 foot-candles of
illumination in a 0° F. walk-in freezer . . . . . . .

Trial 11: Per cent metmyoglobin of meat solutions treated
with various metal ions and stored in a 0° F. display
case under 60 foot-candles of light . . . . . . . . . .

Trial 12: Per cent metmyoglobin of solutions treated with
glucose and stored in a 0° F. display case under 60
foot-candles of illumination . . . . . . . . . . .

Trial 13: Per cent metmyoglobin of ion treatments in meat
solutions stored in a 0° F. display case under 60 foot-
candles of fluorescent illumination . . . . . . .

Trial 14: Absorption spectra for dialyzed and undialyzed

meat solutions after two and eighteen days storage under

50 foot-candles illumination in a 0° F. display case .

Per cent metmyoglobin of frozen steaks and meat solutions
stored at 0° F. under fluorescent lights . . . .

viii

Page

65

68

71

73

75

77

Table

LIST OF APPENDIX TABLES

Page

Data collected from Trial 1: Spectrophotometric readings
from a steak packaged under petroleum ether and stored
at -20° F. under 10 ftc. illumination . . . . . . . . . . .

Data collected from Trial 2: Beckman DU Spectrophotometer
readings on a meat-water solution stored unpackaged in
darkness for 45 days in a -20° F. walk-in freezer . . . . .

Data collected from Trial 3 on meat extracts stored in
darkness and under 50 ftc. fluorescent illumination in a
0° F. Sherer display case . . . . . . . . . . . . . . . .

Data collected from Trial 4: Samples stored under 35 ftc.
light in 0° F. walk-in freezer . . . . . . . . . . . . . .

Data collected from Trial 5: Dialyzed solutions stored
under 35 ftc. in 0° F. walk-in freezer . . . . . . . . .

Data collected from Trial 6: Dialyzed and undialyzed samples
stored under 30 ftc. illumination in 0° F. walk-in
freezer O O O O O O O O O O O O O 0 O O O O O O O O O O O 0

Data collected from Trial 7: Manganese experimental samples
stored under 30 ftc. of light in 0° F. walk-in freezer . .

Data collected from Trial 8: The effects of metal ions on
meat solutions stored under 60 ftc. light in Sherer
display case at 0° F. . . . . . . . . . . . . . . . . .

Data collected from Trial 9 on a dialyzed solution and
a reconstituted dialyzed solution stored under 60 ftc.
at 0° F. in a Sherer display case . . . . . . . . . . . .

Data collected from Trial 10: Effect of varying levels
of manganese on dialyzed solutions stored under 25 ftc.
in a 0° F. walk-in freezer . . . . . . . . . . . . . . . .

Data collected from Trial 11: The effects of metal ions
on meat solutions stored at 0° F. in a display case
under 60 ftc. . . . . . . . . . . . . . . . . . . . . . .

Data collected from Trial 12: The effects of glucose on

meat solutions stored under 60 ftc. in a 0° F. display
use . O . O C . Q . . C O 0 O . . O . O I O O . . . O 0

ix

88

89

90

91

92

93

94

95

98

99

. 100

. 102

Table Page

M. Data collected from Trial 13: The effect of metal ions on
meat solutions stored under 50 ftc. light in a 0° F.
display case . . . . . . . . . . . . . . . . . . . . . . 103

N. Data collected from Trial 14 with a Beckman Recording
Spectrophotometer on samples stored under 50 ftc. in a
0° F. display case . . . . . . . . . . . . . . . . . . . 105

INTRODUCTION

Desirable color, while not necessarily affecting the palatability
or nutritive value of meat, is one of the primary quality attributes
demanded by the consumer. Uiner (1954) stated that consumer accept-
ability of meat as governed by color was both real and psychological.
It was psychological in that off-colored meat was associated with a
lack of freshness and created a negative response as to its desira-
bility. The meat color-acceptability relationship was real in that
discoloration might indicate improper handling, storage temperature,
sanitary conditions, etc.

The introduction of prepackaged fresh meats in the 1940's opened
up a new method of merchandising meat products. This method was
greeted with considerable consumer acceptance as evidenced by the tre-
mendous increase in the number of stores which have converted to this
method of retailing meat. Although there were several technical dif-
ficulties; such as color development, maintenance of the product,
wrapper durability, display life, etc., connected with this merchan-
dising method, they have been overcome to the extent that prepackaging
is the generally accepted manner of retailing fresh meat in this
country at the present time.

Shortly after the advent of prepackaged fresh meat, an attempt was
made by at least one large meat packing company to launch the sale of
frozen meat in a similar manner. It was thought that freezer storage
of meat would tend to equalize the meat supply throughout the year.

1

Certain other advantages were also envisaged, such as the centralized
fabrication of meat cuts and more efficient utilization-of by-products.
however, due to a combination of several factors, the retailing of
frozen meat cuts did not encounter the consumer acceptance enjoyed by
prepackaged fresh meat, and at the present time only a limited supply
of fresh-frozen meat items are to be found in most larger food stores.

One of the problems encountered by the distributors of frozen pre-
packaged meat was the accelerated rate of discoloration of the product
when placed under ordinary lighting in the display case. This discol-
oration is due to a photodynamic phenomenon which accelerates the oxi-
dation of the muscle pigment, myoglobin, to metmyoglobin (Townsend, 1958).
The oxidative states of myoglobin are directly associated with meat
color. Reduced myoglobin is the dark red-purple color of freshly cut
meat: the oxygenated form, oxymyoglobin, is the bright desirable color;
and the oxidized form, metmyoglobin, is the undesirable brownish color
(Grant, 1955).

A prior study (Townsend, 1958) has shown that the rate of metmyo-
globin formation in prepackaged frozen beef was associated with certain
portions of the visible spectrum of fluorescent light and was correlated
with a deterioration of the meat color. The present study was initiated
in an attempt to adapt the spectrophotometric method of analysis to the
study of metmyoglobin for-tion in meat-water solutions packaged and
stored under light. Any information regarding the mechanism or preven-
tion of this light-catalyzed discoloration would benefit the meat in-
dustry in reapproaching the problem of retailing prepackaged frozen

meat.

 

 

REVIEW 0! LITIRAWRE

Characteristics of the Huscle Pigment Myoglobin

1. Historical Development

Earlier workers in the field of biology believed that the color of
meat was due to residual blood hemoglobin. Several of the early studies
on this subject have been reviewed by Townsend (1958). Lawrie (1950)
stated that the first evidence of the existence of a pigment separate
from blood hemoglobin was presented by Homer (1897). Homer studied
water extracts of muscles of dogs, cattle and horses, and found that the
muscle pigments and blood hemoglobin did not have the same absorption
spectrum.

whipple (1926) perfused dogs with Ringer's solution to remove the
residual blood and then extracted the muscle hemoglobin, as myoglobin
was then called. He used a colorometric technique and concluded that
there was considerable variation among different muscles in the relation-
ship of muscle hemoglobin to total hemoglobin. Muscle hemoglobin con-
centration ums also noted to increase with age. About this same time,
Kennedy and Whipple (1926) reported that muscle hemoglobin and blood
hemoglobin were chemically identical and that muscle hemoglobin was
necessary for life of the dog.

Since hemoglobin and myoglobin behave similarly in any respects,
it is of interest to note that Conant (1923) reported that the change
from reduced hemoglobin to methemoglobin involved only one hydrogen

3

 

 

equivalent per mole. It was reported by the same author that the con-
version of hemoglobin to oxyhemoglobin was one involving oxygenation and
not oxidation.

Neill and Isstings (1925) studied the influence of the tension of
molecular oxygen on the oxidation of hemoglobin. They reported that two
reactions were involved between oxygen and hemoglobin. The first reac-
tion was the formtion of an oxidizing agent by molecular oxygen, and the
second was the oxidation of the iron in hemoglobin by the agent formed
in the first reaction. In the second reaction, the oxygen tension lim-
ited the concentration of reduced hemoglobin, which was the substance
actually oxidized to methemoglobin. They reported that the optimm oxy-
gen tension for the formtion of methemoglobin was about 20 —., which
permitted the for—tion of the oxidizing agent but allowed over half the
hemoglobin to mum in the reduced form.

Brooks (1929), in one of a series of studies on muscle hemoglobin,
stated that the color of red muscle was due to the hemoglobin present as
muscle hemoglobin, plus any blood corpuscles remaining in the capillaries.
The amount of muscle hemoglobin present was reported to be independent
of the amount of blood removed.

In 1932, Theorell succeeded in crystallizing myoglobin fron horse
heart. He thus proved that there was a protein in muscle resemuling
the hemoglobin of blood but distinct from it (Morgan, 1936). Shenk,

Ball and King (1934) contributed further evidence that muscle hemoglobin
and blood hemoglobin were two separate entities. They used a Iausch
and bomb spectrophotometer, and reported that the maximum absorption
for blood hemoglobin occurred at 577 and 542 m‘, whereas for muscle hemo-

globin the similar peaks were at 582 and 543 mu. They reported no sig-

 

 

nificant correlation between muscle hemoglobin content and hemoglobin in
the blood stream.

In one of the more recent studies, Lawrie (1950) reported on sev-
eral factors which affected the myoglobin concentration in muscle. Hide
variation among the individual muscles was cited, with the L. dorsi con-
taining .4651 myoglobin (on a moist tissue basis), diaphragm muscle
.6101, and the Psoas .7051. He also reported that species, age, and
exercise all influenced myoglobin concentration. It was concluded that
activity was the fundamental factor responsible for controlling the

amount of the pigment present in any one muscle.

2 . Physiological Mction

Hill (1936) observed that whereas hemoglobin supplied molecular oxy-
gen directly to the tissues by diffusion, myoglobin served as a means of
storing oxygen within the muscle cell during an intermittent supply or
consumption of oxygen. luewrie (1953a) found that the presence of myo-
globin in muscle was accompanied by a high concentration of cytochromes,
and this allowed a high rate of respiration. liillikan (1939), however,
reported that the highest concentration of myoglobin was found in those
muscles whose complete cycle of activity required about one second, such
as the heart muscle of ma-als, breast muscle of large flying birds, and
leg muscles of running animals. Muscles that regularly contracted sev-
eral times a second had little, if any, myoglobin, but a high concentra-
tion of cytochromes. The role of myoglobin in the body was reported by
the same author to be one of oxygen storage, with the affinity of myoglo-
bin for oxygen being greater than that of hemoglobin but less than the

oxidases. Since myoglobin acted as an oxygen reservoir, it enabled the

 

cell or organ to nke varying deamnds for oxygen under fluctuating con-
ditions.

The nnner in which myoglobin acted as an oxygen reservoir within
the cell was by the conversion of reduced myoglobin to oxymyoglobin.
Carbon monoxide was reported to interfere with this function, and acted
as a respiratory poison by combining with both hemoglobin and myoglobin
to form carboxyhemoglobin (carboxymyoglobin), thus preventing the oxy-
genation of these substances (Penrod and Baker, 1954).

Bill (1933) established the dissociation curves for muscle hemglo-
bin and blood hemoglobin and concluded that the affinity of muscle hemo-
globin for oxygen was considerably higher than was the case with blood
hemoglobin. The respiration of cells which had, in the case of red
muscle, a large amount of the oxidase-cytochrome system, could thus con-
tinue at very low oxygen pressure. The myoglobin acted as the intermedi-
ate carrier of molecular oxygen from the blood to the oxidase-cytochrome
system in the cells. Kill (1933) noted that the distinctive properties
of muscle hemoglobin were due to the globin rather than the heme portion

of the molecule.

3. Chemistry of liyoglobin

a. Structure and Composition

hemoglobin and myoglobin are conjugated proteins that contain a
heme moiety (an iron-containing porphyrin compound) attached to a globin
protein (Schweigert, 1956). Cranick and Cilder (1947) stated that the
function of the iron porphyrins associated with certain proteins was to

mke oxygen, the ultimate electron acceptor in protoplasmic processes,

 

 

available to the cell. All naturally occurring hemoglobins have the
common feature of binding oxygen reversibly to the iron of the porphyrins.
These authors reported that hemoglobin contained four heme groups per
globin molecule, whereas myoglobin had only one. watts (1954), however,
stated that each of the four hemes of hemoglobin was associated with
globin.

A considerable volume of work has been done on the physical and
chemical properties of both hemoglobin and myoglobin. A search of the
literature revealed varying figures for the molecular weights of hemo-

globin and myoglobin. Some of the values are given in Tables I and II.

IAILB I

Molecular weight and iron content of hemoglobin

 

 

 

Molecular Height Per Cent Iron Source
---- 0.339 Drabkin (1945)
68,000 ---- Svedberg (1939)
App. 67,000 0.350 Hyman (1948)
68,000 ---- watts (1954)
65,500 - 66,700 ---- Hawk, Oser and

Summerson (1954)
67,000 ---- Kendrew .e_§ 31. (1958)
67.000 ---- Adair (1939)

Ave. 67,183

 

Both myoglobin and hemoglobin are water soluble proteins (Hawk,

Deer and Summerson, 1954). These authors stated the globin portions of

   

 

IABLB 11

Molecular weight and iron content of myoglobin

 

 

Holecular Height

Per Cent Iron

Source

 

App.

17.200

Ave.

16,850
17,200
17,500
17,534
17,600
17,288
17,000

16,900

17,500
17,000
17,000

16,700

17,165

0.331 (calculated)
0.325 (calculated)
0.319 (calculated)
0.318 (calculated)
0.317 (calculated)
.345
.323

0.345

.340

Roche and Vieil (1940)*
Polson (1939)*

Polson (l939)*

Roche and Derrien (l942)*
Pedersen (1940)*
Theorell (l932)*

Bowen (1948)

Kendrew .e_t_ .a_1.. (1958)

Hawk, Oser, and
Summerson (1954)

Watts (1954)
wyman (1948)
Svedberg (1939)
Conant (1923)
Drabkin (1945)

Millikan (1939)

 

and iron content.

*Cited by Bowen, Hilliam.J. 1948. Notes on myoglobin preparation

J. Iiol. Chem. 176:747-751.

 

 

myoglobin and hemoglobin differed, although the heme moieties were the
same. Kendrew‘g£,gl, (1958) stated that myoglobin was one of the smaller
proteins having a globin portion containing about 152 amino acid residues
with no free sulphydryl groups. They also noted that myoglobin had only
one terminal amino group and was probably composed of a single polypep-
tide chain. Roughton and Kendrew (1949) reported that myoglobin had less
arginine and more lysine and tryptophan than hemoglobin. Myoglobin con-
tained isoleucine, but no cystine, whereas the reverse was true for hemo-
globin (Schweigert, 1956). Theorell (1947) observed that the globin
portion of myoglobin was of much lower weight than that of hemoglobin.

Kendrew (1959) discussed protein structure in general, and stated
that there were three levels of structure which should be considered:
the primary structure which was the sequence of amino acids along the
polypeptide chains; the secondary structure which was the type of folding,
coiling, etc. adopted by the peptide chain; and the tertiary structure
which was the manner in which the folded or coiled peptide chains were
disposed to form the protein molecule as a three-dimensional object in
space. The primary structures of myoglobin in different species were
similar but not identical.

Roughton and Kendrew (1949) postulated the crystalline structure of
horse myoglobin as being composed of two discs about 9 2 thick and not
over 57 X in diameter. The two discs in each unit cell lie parallel to
one another and are separated by a layer of crystallization 6.6 2 thick.
Kendrew 25.31, (1958) used the x-ray analysis technique to formulate a
three-dimensional model of the myoglobin molecule. They reported that
the single polypeptide chain was folded to form a flat disc of 43 X x

o o
35 A x 23 A. Inside the disc, the branching chains followed a complicated

 

 

 

10

course. The heme group was held in the structure by links to at least
four chains, but one side was exposed and readily accessible to environ-
mental oxygen. The most outstanding features of the myoglobin molecule
were reported to be its complexity and lack of symmetry. Scouloudi (1959)
also studied the myoglobin molecule by x-ray analysis. She found that the
electron density patterns of seal myoglobin and sperm whale myoglobin

were very similar. This pointed to the close relationship of myoglobin
configurations in two species known to have different amino acid compo-
sitions in their myoglobin makeup.

Rossi-Panelli and Antonini (1956) studied the homogeneity of human
myoglobin using both crystallized myoglobin and muscle extracts. They
reported that there were three components present with similar, but
different absorption curves. They designated these components as Mb 1,
Mb 11, and Mb III, and found Mb I to account for the largest proportion.
Lewis and Schwaigert (1955) noted three components in crystalline horse
myoglobin when studied electrophoretically. They also reported on the
homogeneity of crystalline beef myoglobin using electrOphoretic and
ultracentrifugal analysis. Two distinct components were observed here
and were called substances A and B. Substance A was similar to metmyo-
globin in its absorption spectrum.and present to the extent of 15-201,
while substance 8 was less than 11. Both substances were shown not to
be hemoglobin. These two substances did not separate out with the ultra-
centrifuge, and it was concluded that ultracentrifugal homogeneity alone
cannot be considered sufficient evidence for assumption of complete

homogeneity.

   

11

b. Oxidative Reactions and Relation to Enzyme Systems in.Mhscle

Both hemoglobin and myoglobin can combine reversibly with oxygen
without undergoing oxidation. There is something about the delicately
balanced electronic structure uniting heme and globin which makes possible
this remarkably labile bond with oxygen. However, it is only when the
iron is in the reduced ferrous state that this combination occurs without
any true oxidation. The oxidized ferric form cannot function as an oxygen
transport (in the case of hemoglobin) or as an oxygen reserve (myoglobin),
according to Hyman (1948).

The oxidative changes of myoglobin were depicted by brown and

Tappel (1958) in the following mnner:

 

 

 

 

n\;{+n1 02 n\ 115*" oxidation Au\’I,
C I \ / e
3/1 \H H/|\H reduction H [\N
Globin Globin Globin
Oxymyoglobin (Reduced) Myoglobin Metmyoglobin
figure 1

Oxidative changes of myoglobin

The authors further noted that the combination of oxygen with myo-
globin to form oxymyoglobin was a reversible step and was primarily a
function of oxygen availability. This change was oxygenation and not
oxidation, and was thus not subject to control by anti-oxidants. The
oxymyoglobin was quite stable at high partial pressures of oxygen.
Schweigert (1956) noted that the presence of reducing conditions kept
myoglobin in the reduced state and also converted metmyoglobin to myo-

globin.

   

12

Brooks (1929, 1931, 1935, 1948) has done considerable work on the
oxidation of hemoglobin and myoglobin. He stated that the conversion of
hemoglobin to methemoglobin involved a valence change of the iron atom
and required one hydrogen equivalent of oxidizing agent for each.atom‘of
iron in the hemoglobin molecule. The establishment of an equilibrium
between oxygen, hemoglobin and oxyhemoglobin was very rapid in comparison
with the rate of oxidation of hemoglobin to methemoglobin. Furthermore,
this oxidation to methemoglobin occurred at the maximum rate when the
oxygen pressure was low. There were three possible pathways postulated
for the reaction between hemoglobin and oxygen, depending on the partial
pressure of oxygen: 1. spontaneous decomposition of oxyhemoglobin to met-
hemoglobin; 2. the oxidation of reduced hemoglobin by oxyhemoglobin;

3. the oxidation of reduced hemoglobin by oxygen.

Neill and Hastings (1925) reported that methemoglobin was an oxide-
tion product of reduced hemoglobin. Brooks (1929) observed that oxy-
hemoglobin was not an intermediate step in the conversion of hemoglobin
to methemoglobin. Pirko and Ayres (1957) stated that before metmyoglobin
could be formed, oxymyoglobin was dissociated into oxygen and myoglobin
and the myoglobin was then oxidized to metmyoglobin. Neill and Hastings (1925)
noted that methemoglobin was formed more slowly from.carboxyhemoglobin
than from oxyhemoglobin. This was due to the fact that reduced hemoglobin
was the substance actually oxidized to methemoglobin. It was much more
difficult for the dissociation of carboxyhemoglobin to take place, since
carbon monoxide had about 250 times the affinity for hemoglobin that
oxygen had.

Neill and Hastings (1925) also reported that although certain bac-

teria can oxidize hemoglobin to methemoglobin, the spontaneous formation

   

13

of methemoglobin was only rarely due to this cause. Butler (1953) stated
that in the case of fresh meat the bacteria normally present increased
the rate of oxidation of myoglobin to metmyoglobin. Jensen (1945) ob-
served that micro-organisms, both living and dead, and their enzymes,
when present on meat surfaces, may oxidize both fresh and cured meat
pigments to metmyoglobin. Another factor which could increase the rate
of oxidation of myoglobin to metmyoglobin was the presence of salt
(Coleman, 1951).

Brooks (1934) produced methemoglobin in live rabbits by the injec-
tion of sodium nitrite. She found that l or 2 cc. of a 11 solution of
' glucose reconverted the methemoglobin to oxyhemoglobin. If the glucose
was injected before the nitrite was administered, no methemoglobin was
formed.

Grant (1955) reported that the oxidative changes of myoglobin in
meat were intimately related to the functioning enzyme systems present.
Of all the changes undergone by meat, the most important were those re-
lating to oxygen. This interaction between oxygen and meat was mediated
by the external supply of oxygen, the concentration of myoglobin, and
the activity of the surviving respiratory apparatus. This author also
stated that meat had initially the same level of enzymes as functioning
muscle, but as time passed, irreversible changes occurred. Any given
enzyme present could be: 1. inactive, making no demands on oxygen;

2. one link in a chain of enzymes making a liadted demand on oxygen;

or, 3. the sole active enzyme draining off the bulk of the oxygen stored
by myoglobin. This same author reported that succinic dehydrogenase was
quite active in beef but that phosphoglucomutase, phosphatase and enolase

(three of the glycolytic enzymes) had very limited activity. L-glutamic

   

14

acid oxidase, catalase, phosphorylase, glucose, fructose and formate
dehydrogenase were all found to be inactive. He concluded that only
succinic dehydrogenase, alpha glycerophosphate dehydrogenase and cyto-
chrome oxidase had appreciable activity in beef.

Andrews 35 _a_1. (1952) observed no reduction in ATPase, succinic de-
hydrogenase or glycolytic enzyme activity in either the longissimus dorsi
or semitendinosus muscles of beef after four weeks storage. Brown and
Tappel (1958) reported that in the interior of fresh meat, respiratory
enzymes, particularly succinoxidase, consumed oxygen and tended to deoxy-
genate oxymyoglobin to the reduced form. Lawrie (1953b) reported that

‘the succinic dehydrogenase-cytochrome system catalyzed intracellular oxy-
gen uptake. He also found a correlation between the per cent myoglobin
and the activity of cytochrome oxidase. In a study of the intact red
blood cells, Watts (1954) noted that the reduction of methemoglobin was
dependent on the glycolytic system. Reduced DPN was reported to indirectly
reduce methemoglobin. Gutmann gs_gl. (1947) observed that the reduction
of methemoglobin within the intact red blood cells or in solution appeared

to be due to the interaction between methemoglobin and reduced DPN.

c. The Relationship of Myoglobin to the Color of Meat

The oxidative changes of myoglobin are one of the main causes of
variation in the color of meat. Reduced myoglobin is the dark purple-red
color characteristic of freshly cut meat; the oxygenated form, oxymyoglo~
bin, is bright red, while the oxidized form, metmyoglobin, is brownish
(Brooks, 1947; Brown and Tappel, 1958; Glidden 35 21,, 1960; Grant, 1955;
Levers, 1948; Penrod and Baker, 1954).

Reduced myoglobin is found in very adnute amounts on meat surfaces

   

15

since it is readily oxygenated to oxymyoglobin or oxidized to metmyoglo-
bin (Clidden.££.al,, 1960). The "blooming" of meat is the brightening
to a red color due to this conversion of the reduced myoglobin to oxy-
myoglobin. Brooks (1933) stated that the depth of the color of meat
depended on the concentration of myoglobin and on the tissue through
which light was reflected to the eye. The thicker the surface layer
and the greater the concentration of myoglobin, the deeper was the color.
Brooks (1955) reported that lean meat after rigor could still con-
sume oxygen at a slow rate. The rate of oxygen diffusion from the atmos-
phere through the meat was not fast enough to supply more than a thin
surface layer. The result was that most of the meat was completely
devoid of oxygen except for the external few millimeters. This meant
that the oxymyoglobin was confined to the surface and was bright in
color, while the interior retained the dark purple color of reduced myo-
globin. Also, the deeper the oxygen penetration, the redder the meat
appeared. This layer was deeper at 0° C. than at room temperature, since
the lower temperature permitted deeper oxygen penetration by decreasing
the rate of oxygen consumption. Brooks (1937) observed that the color
of lean meat appeared to be brownish when about 601 of the myoglobin
present in the surface layer had been oxidized to metmyoglobin. The rate
of oxidation to metmyoglobin increased with decreasing oxygen pressure.
Another factor which caused discoloration of fresh meat was dessica-
tion (Brooks, 1955). Penrod and Baker (1954) found that in fresh meat
during the first few hours after storage, discoloration occurred more
rapidly than dehydration, but after a few hours the rate of discoloration
paralleled dehydration.

Bernofsky‘g£_gl, (1959) stated that color changes in cooked beef

 

16

were related to an alteration in the state of myoglobin as it was af-
fected by heat. The insoluble brown pigments of cooked meat were dena-
tured ferric hemichromogen. These authors reported that soluble cooked
meat pigments were 951 oxymyoglobin and 51 metmyoglobin, whereas the
uncooked pigments were 801 oxymyoglobin and 201 metmyoglobin. This
indicated that there were conditions that developed during cooking which
reduced metmyoglobin to myoglobin with a purple-grey color, or that met-
myoglobin was more rapidly denatured than the reduced pigment.

Cured meat color is due largely to the reversible combination of
myoglobin with nitric oxide to form nitric oxide myoglobin which is
pink in color (watts, 1954).

Myoglobin 02 (oxygenation) X Oxymyog lobin
(purplish red) \' (bright red)

 

 

Nitrous acid

 

 

 

 

 

(reducing
agents)
/
Nitric oxide myoglobin oxidation lietmyoglobin
(pinkish red) (-N0) " (brown)
heat oxidation
(irradiation)
V v
Nitric oxide denatured oxidation \_Green compounds

 

myoglobin (pinkish red) (02, H202, light) (on
6339’t

Colorless compounds

Figure 2
The relationship between the various states of myoglobin and meat color*

*Schweigert, B. S. 1956. Chemistry of meat pigments. Proc. 8th
338. 0011f. Fe 61‘ 66s

 

 

 

17

Ginger 3; g . (1955) studied changes in myoglobin associated with
irradiating meat and meat extracts with gs- rays. They found that
irradiation of myoglobin extracts caused a greenish discoloration which
was associated with the alterations of the porphyrin structures of the
compounds. when fresh meat was packaged in oxygen-impermeable material,
little discoloration occurred on irradiation, but when packaged in oxy-
gen-permeable material, irradiation produced i-sdiate discoloration.

Hall g_t_ 9.1:- (1944) noted that another factor, not related to myo-
globin, that affected meat color was the pH of the tissue. They stated
that light colored beef had a pH of about 5.4, whereas dark beef was
around 6.4 in pH. They concluded that the oxygen dennd in dark colored
beef was greater than could be supplied by nor-i diffusion through the
tissues. This resulted in depriving oxymyoglobin of its oxygen and re-
ducing it to the darker myoglobin. Bate-Smith (1948) reaffirmed the
observation that beef with a higher pH was darker in color. He stated
that at the pH of living muscle, myosin had the consistancy of a weak
jelly. As the pH fell this jelly shrank, until at a point between pH
6.5 and 6.0, the muscle fibrils began to shrink and scatter light. This
scattering effect caused the muscle to appear lighter in color although
the actual pigment content was unchanged. Watts (1954) gave the range

in pH of normal meat as 5.2 to 6.6.
Methods of Purification and Measurement of Myoglobin

Various methods or adaptations of methods to isolate myoglobin have
been reported since 1932, when Theorell first performed the successful
isolation of crystalline muscle hemoglobin (myoglobin) from horse heart.

Methods for preparing human crystalline myoglobin have been described

   

 

 

18

by Drabkin (1945), Roughton and Kendrew (1949) and Theorell (1947).

These authors, as well as Morgan (1936), utilized the difference in solu-
bility of hemoglobin and myoglobin in ammonium.sulphate as a means of
separating these two closely related pigments. Myoglobin from beef was
isolated using a modification of the method of Theorell by Ginger g£_gl.
(1954), Ginger and Schweigert (1954) and Schweigert (1954). The latter
author reported values for beef ranging from 2.26 to 5.41 milligrams of
myoglobin per gram of fresh tissue.

The use of spectrophotometry has been quite widely accepted as a
means of measuring the concentration of various forms of myoglobin pre-
sent at a given time. The underlying principle of this method is that
under standard conditions the molar extinction coefficient (designated
as K or<E) is a function of the concentration and the optical density
of the solution. Mellon (1945) showed this relationship, an adaptation
of the Lambert-Beer law, as K a blob, when D a the optical density
measured on a spectrophotometer, c - the concentration in moles per
liter, and b a the thickness of the solution in centimeters. This prin-
ciple was first used to study hemoglobin concentration by Drabkin and
Austin (1932). They found that by the addition of potassium ferricyanide
and potassium cyanide, all the pigment could be converted to methemoglobin
and then to a cyano derivative for which the extinction coefficient
could be determined. In further studies, Drabkin and Austin (1935-1936),
and Austin and Drabkin (1935-1936) stated that their method could be used
to determine the concentrations of the various forms of hemoglobin (oxy-
hemoglobin, hemoglobin, methemoglobin) present at a given time. They
also reported that nitrosohemoglobin could be converted to methemoglobin,

but not to the cyano derivatives as could hemoglobin, oxyhemoglobin,

   

19

carboxyhemoglobin and methemoglobin. They reported a millimolar extinc-
tion coefficient of 11.5 for cyanmethemoglobin when read at 540 mu.

Shenk e_t. 2.1;- (1934) used this method to distinguish between hemoglobin
and myoglobin in a study to determine how important residual blood might
be in effecting the color of lean meat. They reported the maximum light
absorption for blood hemoglobin was at 577 and 542 mi, whereas for muscle
hemoglobin (myoglobin) the points were at 582 and 543 mu. These same
authors prepared solutions of myoglobin free from blood hemoglobin and
observed that these gave absorption curves similar to the curves obtained
from blood hemoglobin, but were displaced towards the red portion of

the spectrum. Bowen (1949) reported the molar extinction coefficient to
be 11.3 x 103 for cyanmetmyoglobin rather than the 11.5 x 103 reported
earlier for cyanmethemoglobin. Mangel (1951) compared the spectrophoto-
metric method .used by Austin and Drabkin with Bowen's method, and reported
considerably higher estintes of the metmyoglobin content with the former
method. Ginger .e_t L1.- (1954) reported an average of 3.7 mg. myoglobin
per gram of beef tissue using Drabkin and Austin's method for the con-
version of metmyoglobin to cyanmetmyoglobin.

Glidden gt 9.1. (1960) noted that systematic variations in the ex-
tinction coefficients obtained by the spectrOphotometric method may have
been due to the presence of a sulphur-containing myoglobin pigment.

Several authors have used the ratios of the optical density at var-
ious wavelengths to calculate the per cent metmyoglobin. This method
was known as the absorbency ratio method and was used by Brooks (1934),
Shenk 5_t_ L1. (1934), Brou-nd 3.}. _a_l. (1958), and Dean and Ball (1960).
The latter authors also reported on a method utilizing reflectance rather

than absorption to measure the proportions of met-, oxy- aml reduced

   

 

20

myoglobin. They observed that the absorbancy ratio method gave higher
values for oxymyoglobin than did the reflectance methods, which indicated
that some of the metmyoglobin was converted to oxymyoglobin in the extrac-
tion process used in the absorbency method.

Rossi-Panelli (1949) used a spectrophotometric method to make simul-
taneous determinations of myoglobin and hemoglobin concentrations. This
method involved using K20r202 and CuSO4 at a controlled pH, and reading
spectrophotometrically at 579 and 577 an. Huaaini 35,31, (1950) reported
a simplified method of determining total hemoglobin (blood hemoglobin
plus myoglobin) in muscles. This procedure utilized the addition of
sodium carbonate to the pigment solution and then the determination of
the optical density at 542 mu, Sane and Hashimoto (1958) reported on a
spectrophotometric method for measuring metmyoglobin in tune fish meat.
This method entailed conversion of the myoglobin by treatment with carbon
monoxide, and then conversion to metmyoglobin with ferricyanide. The
optical density at 568 mu_was determined, and the relative amount of met-
myoglobin to total myoglobin was calculated.

The Relationship of Various Storage Conditions
to Color of Prepackaged Meat

1. Fresh Meat Color

Such factors as storage temperature, bacteria, oxygen pressure, and
type of wrapping material have an effect on the color of prepackaged
fresh meat. Brooks (1939, 1937) and Levers (1948) stated that when fresh
meat was subjected to low oxygen pressure the change of myoglobin to met-
myoglobin (with the resulting discoloration) was increased. For this

reason the latter author recommended an oxygen-permeable wrapper for

   

21

non-frozen meat. Pirko and Ayres (1957) reported a direct relationship
between discoloration due to metmyoglobin and the gas permeability of

the package. There was a sharp increase in metmyoglobin forntion in
beef steaks packaged in films having a low gas permeability as compared
with films having a higher permeability. The greatest difference in rate
of metmyoglobin forntion was observed during the first 24 hours of stor-
age at 6° C. They also noted, however, that fresh meat packaged and
stored in gas-impermeable film would reoxygenate when the package was
opened to give the desired bright color of oxymyoglobin. Rikert _e_§_ §_1_.
(1957) noted that a decrease in oxygen penetration permitted more rapid
metmyoglobin formation near the surface of the meat. Ramsbottom 35 .a_1_.
(1951) showed that if fresh meat was packaged in an oxygen-impermeable
wrap the oxymyoglobin would revert to myoglobin with the resulting dark
color. Dean and Ball (1960), however, stated that an excess of oxygen

in prepackaged beef after 24 hours of storage increased the rate of met-
myoglobin formation, while vacuum packaging (reduced quantity of oxygen)
enhanced oxymyoglobin forumtion. Bratzler (1955) noted that treatment
of fresh meat with oxygen enhanced fresh meat color but did not improve
its stability.

Rikert _e_g _a_1. (1957) reported less color loss at low temperatures
than at higher temperatures. They found that increasing the temperature
caused oxymyoglobin to be more easily dissociated to myoglobin at a con-
stant oxygen pressure. This resulted in an increase in the rate of met-
myoglobin formation. Kraft and Henderstock (1950) observed that the
best storage temperature for fresh meat was 33° F.

The effect of antioxidants in retarding color degradation of fresh

meat was reported by Kraft and Handerstock (1950). They found that

   

 

22

reducing agents such as sodiumnbisulphate retarded the discoloration due
to the formation of metmyoglobin. Antioxidants were most effective in
preventing color changes during the first one or two days of storage.
Butler g§_gl, (1953) studied the effect of bacteria on the color
of prepackaged beef. They found that bacterial growth shortened the
shelf life of the meat. It was concluded that bacterial growth hastened

discoloration by increasing the rate of metmyoglobin formation.

2. Frozen Meat Color

In one of the earlier reports, Brooks (1929) noted that repeated
freezing and thawing increased the rate of metmyoglobin formation. In
another early report on the effect of freezing on meat color, Brooks (1930)
stated that when red muscle was thawed after being rapidly frozen in a
cold solution with sodium chloride, there was a superficial layer that
was dark brown due to methemoglobin formation. It was also reported that
if muscle tissue was frozen in air the rate of methemoglobin formation
was retarded. when the muscle was frozen in brine the penetration of
sodiwm chloride decreased the oxygen uptake of the tissue and increased
the rate of metmyoglobin formation by: 1. increasing the oxidation rate;
and 2. increasing the superficial layer in which oxidation is possible.

Luzet (1959) discussed the freezing of tissue at the molecular level.
He observed that it required about 27 billion molecules to form.a crystal
of ice large enough to be well observed under the microscope, and that a
crystal of such size could be formed in about one second. Ice formation
in an aqueous solution resulted in a great variety of crystalline forms,
i.e. hexagonal, irregular, dendrites, lobed formations, spherulites and

recrystallization clouds. When ice was formed in a centrifuged muscle

   

 

23

extract, the structureless muscle components behaved like solutions con-
taining solutes of high molecular weight. In discussing the formation

of ice in intact muscles, it was reported that the number and diameter

of ice columns formed in a muscle fiber of a given water content increased
at lower freezing temperatures. Rapid freezing did not distort the fibers
as greatly as slow freezing at lower temperatures, since the crystals thus
formed were smaller in size. Ramsbottom and Koonz (1941) observed that
slowly frozen meat appeared darker due to the presence of these large ice
crystals. 1n steaks frozen at ~30° P., most of the ice crystals were
intrafiber. The authors also noted that there was greater discoloration
due to metmyoglobin formation when steaks were frozen and held at 100 P.
than when freezing and storage took place at -30° F.

Robertson (1950) observed that there were two problems in regard to
frozen meat color. The first was to develOp the desired color initially,
and the second was to maintain this color during the freezing process
and subsequent storage of the meat. He suggested that frozen meat should
be wrapped in an oxygen-impermeable material to prevent discoloration
due to metmyoglobin formation. The packaging material should also pro-
vide a good moisture-vapor barrier to prevent discoloration due to dehy-
dration. Brooks (1937) stated that discoloration of frozen meat due to
metmyoglobin formation was not a problem unless excessively long storage
periods were involved. His study dealt with meat frozen and stored in
the dark, however. An article on retailing frozen meat (Anonymous, 1958)
stated that appearance was of prime importance to the consumer. This
would preclude the use of "blind" packages.

Ramabottom (1947) studied the effect on meat quality of various

freezer storage conditions. It was found that when different cuts of

   

 

24

seat were frozen at -20° F. and then held at temperatures ranging from
-20° to 26° P., the lower temperatures resulted in better color of meat
for longer periods of time than for similar cuts of meat stored at higher
temperatures. The meat was not exposed to light during storage in this
study.

Caldwell 35 21. (1960) reported on the addition of antioxidants to
frozen ground beef. They found that ascorbate alone, or in combination
with other antioxidants (NDGA, BDTA, sodium citrate, and dihydroxy
quescetin) aided in the maintenance of desirable color during six weeks
of storage at -23° C. The antioxidants other than ascorbate did not,
when used individually, protect the color of frozen ground beef during

the storage period.
3. Effect of Light on Meat Color

According to Taylor and Pracejus (1950), "Light is an indispensable
factor in the display and sale of merchandise, but long exposure to high
intensities of illumination may result in damage to the articles dis-
played". These workers noted that in order for a photochemical change
to take place energy must have been absorbed, but the effect on the ab-
sorbing material may not have been proportional to the amount of energy
absorbed. Blum (1941) observed that in photochemical reactions the
"energy of activation" was supplied directly to the atom.or molecule
involved when it was raised to an excited state by the absorption of radi-
ant energy. He also stated that many photochemical reactions were vir-
tually unaffected by temperature. Furthermore, photochemical reactions
were irreversible and independent of light intensity. An outstanding

characteristic of photodynamic processes was that they required oxygen;

   

 

25

however, the oxidations fundamental to photodynsmic action ware entirely
distinct from those of nornl cellular metabolism. Watts (1954) noted
that light accelerated all oxidative changes in meat provided that oxygen
was available.

Display case lighting had little effect upon the color of fresh meat
(Bratzler, 1955). Watts (1954) and Ramsbottom g_t_ 2.1.- (1951) reported no
discoloration of fresh meat due to the effects of light when the storage
period did not exceed three days. Kraft and Ayres (1954) stated that the
intensity of soft white fluorescent light was not important in influenc-
ing the course of discoloration of prepackaged fresh meat. They noted
that ultraviolet lights, while retarding bacterial growth, did cause con-
siderab 1e discoloration by increasing the rate of oxidation of myoglobin
to metmyoglobin. Brown and Tappel (1958) confirmed this observation in
regard to ultraviolet lights. Voegeli (1952) and Ramsbottom _e_t _a_l_. (1951)
observed that the main cause of color degradation of fresh meat stored
over three days was microbial in nature. Rikert £5 21. (1957) noted that
light had a beneficial effect on the color of fresh pork.

Cured meets, on the other hand, were noticeably "faded" in color
when exposed to light (Rikert 5; $1., 1957; Allen, 1949; Kraft and
Henderstock, 1950). Ramsbottom at, $1. (1951) stated that light acted
as a catalyst, and the amount of discoloration or "fading" of cured meat
was proportional to the light intensity. Filtering out the ultraviolet
rays emitted by fluorescent lights did not lessen the discoloration of
cured meats. Draudt ( 1956) observed that the desirable pipent of un-
heated cured meats was nitric oxide myoglobin, and that nitric oxide was
lost rapidly from the pigment in the presence of light and oxygen.

Kampschmidt (1955) noted that light hastened the dissociation of nitric

   

26

oxide in cured meats and thus rendered the myoglobin more susceptible to
oxidation. It was further reported that wavelengths of light between
400 and 550 mu provided most of the energy for this dissociation.

Allen (1949) found that all portions of the spectrum of visible
light were about equal in producing discoloration of cured most if the
same light intensity was used. Light in the ultraviolet range caused
more rapid "fading" of cured meat than did visible light.

There was little information available in the literature in regard
to the effect of light on frozen neat color. Raumann 35.3}, (1957) pack-
aged beef round steaks in flexible transparent film and then stored them
in the dark at 0° P. for O, l, 2, and 4 weeks prior to illuminated dis-
play. The. samples were then placed under 25-50 foot-candles of illumina-
tion for up to 7200 foot-candle hours. They found that the stability of
the color of frozen beef was related to the length of dark storage, foot-
cendle hours of illumination and temperature of the storage cabinet.

Townsend and Bratzler (1958) and Townsend (1958) observed that the
type of light used had a aarked effect on the stability of color in pack-
aged frozen beef. Steaks frozen and stored in the dark had a lower per
cent aetmyoglobin and a brighter color than steaks stored under fluores-
cent light. Have lengths of about 5800 K were absorbed by the myoglobin
and as a result a photochemical reaction was produced resulting in the
conversion of oxymyoglobin and myoglobin to metmyoglobin. The yellow
portion of the spectrum in white fluorescent lamps seemed to be the chief

cause of the color deterioration.

 

 

 

27

Methods of Color Measurement

There are many methods in use at the present time to describe, nasa-
ure, evaluate and detect differences between various colors. One of the
subjective methods of evaluating color is the use of a panel of judges
who rate color according to some arbitrary scale. Ramsbottom (1947)
used a panel of three judges and a scale ranging from 1 (extremely poor)
to 10 (excellent) to rate the color of beef.

Nickerson (1948) discussed several methods of describing color.

One method considered was the use of color chips to which.the sample being
studied could be compared. There were other methods eentioned which at-
tempted to be more objective in their approach. Hunter (1948) described

a photoelectric color difference meter (now known as the Hunter Color and
Color Difference Meter) which was a photoelectric tristimulus calorimeter
used to measure color on three scales. winkler (1939) discussed the use
of a photoelectric color comparator that measured the amount of light
scattered in red, green and blue portions of the spectrum.

Hiner (1954) presented a paper on the use of the Hunsell spinning
disc method of color notation. This method entailed adjusting the pro-
portions of colored discs to give a blended color that matched the sample
being studied. Methods for the conversion of the proportions of the indi-
vidual discs to a color notation based on the concepts of hue (the name
of a color), value (the degree of lightness or darkness of a color) and
chron (the degree of departure of a color from a gray of the same value)
were given by Nickerson (1946). Townsend (1958) obtained correlations
ranging from..777 to .930 between the index of fading calculated from

the Hunsell discs and the per cent metmyoglobin in frozen beef samples.

   

28

Butler (1953) obtained correlations ranging from .830 to .882 in a 01-19
lar study using fresh beef. Voegeli (1952) presented detailed instruc-

tions for the application of the Munsell system to measurement of meat

color.

 

EXPERIMENTAL OBJECTIVES

The objectives of this study were as follows:

I.

II.

III.

To determine if frozen.neet-water extracts stored under lights
would undergo color deterioration due to an increase in the
formation of metmyoglobin.

To adept the spectrophotometric method as used and described by
Butler (1953) and Townsend (1958) to the determination of met-
myoglobin in frozen meat extracts.

To investigate possible methods for the inhibition of the

light-catalyzed formation of metmyoglobin in these extracts.

29

   

EXPERIMENTAL PROCEDURE

It had been observed in one of the preliminary trials, as well as
by other workers at Michigan State University, that when whole blood was
treated with potassium oxalate as an anti-coagulant, oxygenated and fro-
zen, there was en almost immediate extreme darkening of color. The whole
blood apparently underwent a type of color change different from that of
fresh meat. This indicated that whole blood would not be a suitable me-
dium.for the study of light-catalyzed color deterioration of frozen meat.
At the same time, if it were possible to develop some type of solution
that could be used for this work.a more homogeneous series of samples
could be obtained. In any study that makes use of seat cuts, the deter-
mination of the per cent metmyoglobin ends the usefulness of a particular
sample. This allows the random variation between individual cuts to have
an effect on the final results of any study which utilizes these cuts.

If, however, a single solution prepared from one large cut of meat
could be analyzed for changes in metmyoglobin, a whole series of orig-
inally uniform samples could be prepared. Once the replicate samples
were prepared, various treatments could be applied with a rather strong
assurance that any changes in the final results would be due to the
treatment and not to individual variation among the samples themselves.

The main problem involved in this study was one of methodology,
involving the development of meat extracts that could be adapted satis-
factorily to spectrophotometric metmyoglobin determination. In this sec-

tion, the procedure finally adopted will be described, as well as the

30

   

 

31

procedure for spectrOphotometric metmyoglobin determination and the use
of the Munsell spinning discs for color characterization. In the inter-
ests of clarity, the individual variations in procedure and treatments
will be discussed for each trial in a chronological order, insofar as

this lends itself to a logical presentation.

Extract Preparation and Processing Prior to

Metmyoglobin Determination

Portions of beef rounds obtained either from the Michigan State
University Meat Laboratory or the University Food Stores were used
throughout this study. The meat was generally frozen prior to the pre-
paration of the extract. Slices were sawed from the larger pieces of
round at the time of preparation. All external fat and connective tis-
sue, as well as the outside layer of lean, were trimmed off. The samples
were then cut into small cubes, weighed to the nearest tenth of a gram,
and placed in a chilled Haring Blender. Distilled water was added at
the rate of eight parts water to one part meat, and the mixture was
blended for two minutes at maximum.speed.

The slurry was centrifuged at 2500 r.p.m. at 0° C. for 30 minutes.
The supernatant was decanted through absorbent cotton to remove any fat
that had collected. The clear solution was filtered through Number #2
Hhatman filter paper in a chilled Bushner funnel. This filtration,
which took place in a suction flask, was the only step in the preparation
that was not performed in a cooler at 34° F. The filtered solution was
then in a proper condition for freezing or for the application of the
various treatments utilized. If the treatment involved dialysis, the

solution was placed in cellulose No-Jax casings and dialyzed against

   

32

distilled and de-ionized water for three days with two changes of water
per day.

When the solution was ready to be frozen, it was poured into either
a metal ice cube tray or into individual 50 ml. beekers. The containers
were placed in a walk-in freezer at -20° F. and allowed to remain there
from four to fourteen hours in the dark. At the end of this time the
frozen cubes or portions were removed from the containers, placed in
clear Dow Saran freezer bags, and sealed under vacuum. The samples
were stored at 0° F. either in a walk-in type freezer under approximately
25 foot-candles of fluorescent light, or in a cycling 0° F. Sherer dis-
play case under 50-60 foot-candles of fluorescent illumination.

The samples were removed after varying storage periods and melted
in a 37° C. incubator. This process required 30-45 minutes and raised
the temperature of the solution to 8-10° C. The solution was next cen-
trifuged for 30 minutes at 2500 r.p.m. in a refrigerated centrifuge at
0° C. The supernatant was filtered through Number 42 Hhatman filter paper
through a Buchner funnel into a chilled suction flask. The extract was
then ready for spectrophotometric analysis. The time involved between
removal of the frozen samples from the freezer to spectrOphotometric
analysis was from one and one half to two and one half hours. In any
given experiment the various treatments were rotated on successive deter-

minations to equalize the holding time among all treatments.
Metmyoglobin Determination

The estimation of the per cent metmyoglobin was essentially the
method described by Mangel (1951) and adapted by Butler (1953) and

Townsend (1958) for use with fresh meat samples. This method involved

   

 

33

differential spectrophotometry and was used to estimate the per cent
metmyoglobin in a solution which was assumed to contain only the two pig-
ments, oxymyoglobin and metmyoglobin. The procedure was based on the
fact that each of these two pigments, if taken individually under stand-
ard conditions, gave a characteristic absorption curve. Then at any
given wavelength (544 and 582 mu, the absorption peaks for oxymyoglobin,
were the wavelengths used) the difference between these curves could be
measured. This difference could be compared to the maximum possible
change which would occur if all the pigment in the solution were converted
to the metmyoglobin form and thus the per cent metmyoglobin actually pre-
sent could be derived. This was done by converting all the pigment to
cyanmetmyoglobin by the addition of a drOp of a 21 solution of potassium
ferricyanide and a drop of .51 potassium cyanide to each cuvette before
reading on the Beckman DU Spectr0photometer at 540 mu. All optical den-
sity readings on the Beckman DU Spectrophotometer were taken with slit
openings between .01 and .02. Drabkin and Austin (1932), in a spectro-
photometric study of hemoglobin, noted that the potassium ferricyanide
oxidized hemoglobin to the methemoglobin form and the potassium.cyenide
then formed a cyanmethemoglobin derivative. Bowen (1949) gave the milli-
molar extinction coefficient for the cyanmetmyoglobin derivative as ll.3.

The calculations to estimate the per cent metmyoglobin in the orig-
inal mixture whose optical density was measured were based on the

Lambert-Beer law as given by Bowen (1949).

10 - incident light

I I emergent light

   

34

él- molar extinction coefficient

0.
I

optical density (from the spectrophotometer for the cyanmetmyoglobin
derivative)

c a molar concentration of the solution

pa
I

length of the light path through the solution in cm. (which equals
one for the cell used)

Therefore, c - d/e , and using Bowen's coefficient, c a d/11.3 x 103.
The molar extinction coefficient (ea) was computed for the solution con-
taining the mixed pigment using the value for c obtained in the above
formula, so 6 :- d/c.

The per cent metmyoglobin was calculated by obtaining the difference
between the molar extinction coefficients of oxymyoglobin and metmyoglobin
at 544 and 582 mu for the sample, and the molar extinction coefficient
when all the pigment was in the oxymyoglobin form. These values for
oxymyoglobin as given by Bowen (1949) were: 6 a 14.6 at 544 mu,and
é: a 15.1 at 582 mu. Thus, at either wavelength the change due to met-
myoglobin was equal to the molar extinction coefficient of the oxymyo-
globin minus that of the sample. This difference was then divided by
the total possible change between the molar extinction coefficients for
oxymyoglobin and metmyoglobin (9.6 and 12.1 respectively) and multiplied
by 100 to give the per cent metmyoglobin in the original solution. The
final value was determined by averaging the values obtained at the two
wavelengths. The concentration was then multiplied by the molecular
weight of myoglobin and the dilution factor. The resulting value divided
by ten gave an estimation of the concentration of the pigment in the

original sample expressed as per cent myoglobin.

   

35

The Munsell Spinning Disc Method of Color Notation

The Munsell color system was developed over the years 1900-1912 by
the Boston artist and art teacher, Albert 8. Munsell. The system was
based on three attributes of color: hue, which is the name of a color;
value, the degree of lightness or darkness of a color; and chrome, the
degree of departure of a color from a gray of the same value (Nickerson,
1948).

These three color attributes may be best visualized in the form of
a sphere (see Figure 3). The vertical axis of this sphere has an eleven
step scale of value ranging from 0 at the bottom.(black) to 10 at the
top (white). The circumference of the sphere is composed of various hues
which radiate out like the spokes of a wheel from the vertical value
scale. The hues are red, yellow-red, yellow, green-yellow, green, blue-
green, blue, purple-blue, purple and purple-red. The chroma scale is a
horizontal scale which extends from 0, or neutral gray, at the vertical
value scale, outward to 10 or even 20 for very vivid colors. The color
notation is written as hue, value/chroma,‘i.g. 5R 4/10 (Biner, 1954;
Nickerson, 1948).

Detailed instructions were given by Voegli (1952) for the applica-
tion of this method for the description of fresh meat color. This method
was used in the present study with one or two adaptations. when color
readings were to be obtained on a frozen solution, the cube was removed
from its package and affixed with lard to the matching platform in such
a manner that the top surface of the cube was as nearly level with the
spinning discs as possible. The readings on the frozen material were

obtained in a 00 P. walk-in freezer. When the thawed solutions were

   

 

36

 

 

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37

used, a measured amount was placed in a circular metal container which
had been painted gray.

The solutions or frozen cubes were placed on a platform in a match-
ing booth. This booth was constructed in the Michigan State University
Meat Laboratory from plywood and was painted black to minimize light re—
flectance. The light source was a Macheth BOO-watt daylight lamp. The
colored discs were spun rapidly with an electric motor so that a single
"color" was visible. The discs and the sample were viewed through a
Bausch and Lomb optical eyepiece, and the proportions of the discs ad-
justed so that the discs and the sample appeared as nearly matched as
possible. The pr0portions of each colored disc were recorded after the
match had been completed.

The individual discs have known tristimulus values x, Y, Z, estab-
lished by the International Commission on Illumination. The x, y, z val-
ues were derived by computation of the fractional X, Y, 2 values. The
value portion of the notation was obtained by referring to the table of
I. C. l. equivalents of the Munsell value scale. The hue and chroma
were obtained by plotting the x and y values on Munsell charts.

The formula presented by Nickerson (1946) was used to determine the
difference of the samples from a standard. This color differential is
expressed as an Index of Fading (I):

I - c/smaa) + 6AV + 3/_\.c

C I chroma of the sample

13H I difference in hue between the standard and the sample
45V I difference in value between the standard and the sample
(LC I difference in chroma between the standard and the sample

The standard used for the solutions was an oxygenated meat-water

   

 

38

extract obtained by bubbling oxygen through the solution for ten minutes.
The color readings were determined by placing the standard solution in
containers similar to those used for the sample readings. The renotations
in the two containers used were 4.7YR.3.2/4.8 and 7.9YR.4.4/4.2. The
standard used for the frozen cubes was the Munsell notation with the

lowest value. This was 4.0YR.2.7/3.2.
Individual Trial Procedures
Trial 1:

Five bottom round steaks were cut and treated as follows: Steak 1
was trimmed and allowed to bloom.thirty minutes, then packaged under vac-
uum in a Cry-O-Vac bag; Steak 2 and Steak 3 were trimmed and packaged
under petroleum ether to prevent any contact with oxygen, then vacuum
packaged; Steak 4 was packaged under petroleum ether, then nitrogen gas
was bubbled into the bag before it was sealed; Steak 5 was handled the
same as Steak 4 except that a vacuum‘was drawn on the package after the
steak had been exposed to nitrogen.

All steaks except Steak 3 were stored at -20° F. under approximately
ten foot-candles of fluorescent light. Steak 3 was stored at -20° F. in
the dark. Steak 2 was used after a three-month storage period for met-
myoglobin determination utilizing the procedure outlined by Townsend
(1958). All steaks were checked visually over a two-month period for

color change.
Trial 2:

A solution using lean cow round was made up in the standard manner.

The procedure was as previously described except that before being frozen,

   

 

39

the solution was centrifuged eight minutes at 2000 r.p.m., and then fil-
tered through Number 40 Nhatman filter paper. The solution was frozen,
but not packaged, and then stored in the dark at 0° F. The sample was
held unpackaged for 45 days, then thawed and handled in the standard man-
ner except that it was centrifuged at 7500 r.p.m. for fifteen minutes

at 2° C.
Trial 3:

A standard solution was divided into two groups. Group 1 was stored
under approximately 50 foot-candles of fluorescent light and Group 2 was
stored in darkness. Both groups were packaged and held in a 0° F. Sherer

display freezer over a 19-day period.
Trial 4:

One hundred grams of hanging tenderloin were prepared in the
standard manner, except that the solution was centrifuged fifteen minutes
at 2000 r.p.m. before being filtered and then the supernatant was recen-
trifuged at 7500 r.p m. at 1° C. for fifteen minutes. The frozen cubes
were stored in a walk-in freezer at 0° F. under 35 foot-candles of illum-
ination. Munsell color readings were taken on the frozen samples in the
0° freezer and also on the thawed solutions after the spectrophotometric

metmyoglobin determinations. Readings were taken over a 40-day period.

Trial 5:

The solution was made up in the standard manner, except that only
six parts distilled water were used, and then dialyzed for three days in

No-Jax casings. At the end of this time the solution was centrifuged for

   

40

thirty minutes at 7500 r.p.m. at 1° C. The solution was then recentri-
fuged at 9000 r.p.m. for an additional thirty minutes. An aliquot of the
unfrozen solution was read on the Beckman Recording Spectrophotometer.
The remaining solution was placed in an ice cube tray and mixed with a
mechanical agitator in a -20° F. blast freezer until the solution began
to freeze. Munsell color readings were taken on the frozen samples and
on the thawed solutions. Storage was in a walk-in freezer (0° F.) under

approximately 30 foot-candles of light, and extended over a 60-day period.

Trial 6:

Two 100 gm. portions of beef round were each combined with 800 ml.
distilled water and the solution made up in the standard manner. One
portion was dialyzed three days and then packaged, while the other portion
was packaged immediately without dialysis. Both sets of cubes were stored
in a 0° F. walk-in freezer under 30 foot-candles of light. One sample
from each group was removed periodically over a 66-day period, the per
cent metmyoglobin determined in the usual manner, and the pH of the
thawed solution determined. The dialysate was concentrated down and per
cent ash determined. A partial analysis was conducted on the ash using

8 spectroscope .

Trial 7:

A dialyzed solution was made up and divided into two portions. One
portion, the control, was packaged with no further treatment. The other
portion had MnClz added at the rate of .170 mg. per m1. of meat solution,
or .5 mg. manganese per gram of meat. Both groups were stored in a 0° F.

walk-in freezer under 30 foot-candles of illumination, and were period-

   

 

4l
ically examined spectrophotometrically over a 44-day period.
Trial 8:

A standard meat solution was prepared and divided into seven groups.
Group 1 was an undialyzed control; Group 2 was a dialyzed control. The
remaining five groups had metal ions added in the form of the chloride
salt. The ion concentration was made up according to the concentrations
reported for beef by Mitteldorf and Landon (1952) as follows: Group 3,
manganese, .00002 mg. per gram of meat; Group 4, copper, .00045 mg. per
gram of meat; Group 5, magnesium, .21 mg. per gram of meat; Group 6, iron,
.031 mg. per gram of meat; Group 7, calcium, .026 mg. per gram of meat.

The ion solutions were so constituted as to require .71 ml. per
50 ml. aliquot of meat solution. An equal volume of de-ionized distilled
water was added to the two control groups. All but one of the samples
(seven samples per group) were stored in the 0° F. display freezer under
approximately 60 foot-candles of fluorescent light. One sample per group
was stored in the 0° walk-in freezer under 25 foot-candles of illumina-
tion. Thirty-five milliliter portions of the thawed solution were used
for Hunsell color determinations after the spectrophotometric analysis

was performed.
Trial 9:

A standard dialyzed solution was divided into two portions of seven
samples each. Group 1 was the control. To the samples in Group 2 were
added 2.5 ml. of the concentrated dialysate per 50 ml. aliquot of meat
solution. The control group had 2.5 ml. of de-ionized distilled water

added to each aliquot. All samples were stored in the 0° P. display case

   

42

under 60 foot-candles of illumination for a total period of 48 days, with

routine spectrophotometric analysis.
Trial 10:

A dialyzed solution was divided into four groups. Group 1 was the
control. The other three groups had a solution of MnClz°9HZO added to
give: .0002 mg. manganese per gram of meat (Group 2); .002 mg. manganese
per gram of meat (Group 3); and .02 mg. manganese per gram.of meat
(Group 4). The manganese solutions were added at the rate of .81 ml. per
50 ml. aliquot of meat solution, and a like amount of de-ionized distilled
water was added to the control samples. All samples were stored in the
0° P. walk-in freezer under approximately 25 foot-candles of light, and

periodically examined spectrophotometrically over a 56-day period.
Trial 11:

This was a replication of Trial 8, with the exception that the ion
solutions were added in exactly double the amounts used in Trial 8. This
required .86 m1. of the ion solutions per 30 ml. aliquot of meat solution.

The period covered in this trial was fifteen days.

Trial 12:

A standard undialyzed solution was prepared and divided into three
groups. Group 1 was the control. Group 2 had one ml. of a 12 glucose
solution added per 35 ml. aliquot. Group 3 had two ml. of a 11 glucose
solution added per 35 ml. aliquot. All samples were placed in the 0° P.
display case under approximately 60 foot-candles of light, and metmyoglo-

bin determinations made over a lS-day period.

   

 

 

43

Trial 13:

A standard solution was prepared and divided into six portions. All
portions but those in Group 1 were dialyzed. Group 1 was the undialyzed
control; Group 2, the dialyzed control; Group 3 had manganese added in
the same proportions as used in Trial 8; Group 4 had magnesium added in
the same proportions as used in Trial 8; Group 5 had iron in the ferrous
form (PeClz'4H20) added to the extent of .031 mg. per gram.of meat;

Group 6 had iron in the ferric form (rec13-6nzo) added at the rate of
.031 mg. per gram of meat (Mittledorf and Landon, 1952). All samples
were stored in the 0° P. display case under approximately 50 foot-candles

of fluorescent light over a period of 14 days.

Trial 14:

A standard solution was prepared and divided into two portions.
Group 1 was the control and Group 2 was dialyzed. Both groups were
packaged and stored in the 0° !. display freezer under approximately
50 foot-candles of fluorescent light. All spectrophotometric determina-
tions for both groups were performed on a Beckman Recording Spectropho-

tometer over a period of 18 days.

Statistical analyses for all trials were performed according to

Snedecor (1956).

   

RESULTS AND DISCUSSION

The results will be presented in the same sequence in which the re-
spective trials were considered in the previous section. The complete

data obtained for the various trials are listed in the appendices.

Trial 1:

The elimination of oxygen from beef round steaks by packaging under
petroleum ether and vacuum had no effect on the color of lean as rated
by visual appraisal. The addition of nitrogen to the package also failed
to prevent discoloration when the steaks were stored under light in the
freezer. At the same time, the steak which had been stored in darkness
retained a desirable bright color after three months in storage at 0° P.

A sample from a steak packaged in petroleum ether and stored under
light was analyzed spectrophotometrically for metmyoglobin after a stor-
age period of two months. Individual optical density readings were taken
from 500 mu to 590 mu, The results of these observations appear in
Figure 4. It may be noted that these estimations of the per cent met-
myoglobin were within 71 at the two primary wavelengths 544 and 582 mu,
These were not as close as most of the later trials, but were in closer
agreement than the per cent metmyoglobin estimations in several prelim-
inary trials. The average per cent metmyoglobin, 53.5%, was high enough
to cause a noticeable discoloration of the steak. This confirms the ob-
servation made by Townsend (1958) that fluorescent lights have an adverse

effect on the color of frozen meat.

44

   

45

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Trial 2:

The higher centrifugation speeds used in this trial, 7500 r.p.m.,
did not improve the clarity of the final solutions. The filter paper
(N. 40, Whatman) was too porous to do a satisfactory job of removing pre-
cipitous material from.the thawed solution.

The solution was analyzed over a range of 500 to 650 mm on the
Beckman DU Spectrophotometer. The results of these readings appear in
Figure 5. The per cent metmyogldain was estimated as 28.11 at 544 I4
and 33.11 at 582 mu. The concentration of myoglobin in the original tis-
sue was .541 or 3.5 x 10’5 moles.

This sample had been stored in the dark and exposed to atmospheric
oxygen for 45 days. This condition resulted in an average of 31.21 met-
myoglobin, which was not as high as would be expected if the sample had
been exposed to light. The agreement between the pri-ry wavelengths as
to the per cent metmyoglobin was quite close. The absorption curve agreed
‘well with Hangel (1951), Bowen (1949), and Townsend (1958), with peaks at

544 and 582 mu.
Trial 3:

In this trial all frozen samples were stored in a 0° P. Sherer dis-
play freezer. Group 1 was exposed to approximately 50 foot-candles of
light, while Group 2 was stored in darkness. The temperature in the dis-
play case fluctuated considerably more than did the temperature in the
walk-in freezer, which was also kept at 0° F. The display case tempera-
ture went as high as 55° F. every six hours when the case was defrosting.

The temperature remained above freezing long enough to partially melt the

   

 

Millimolar extinction coefficients

47

 

Average per cent metmyoglobin 31.15

Estimated per cent
metmyoglobin 28.1

12 —

ll

10 _

 

o l L I J... __ 1

500 520 540 560 580 600

wavelengths in millimicrons

Figure 5

Trial 2: Absorption spectrum of a meat-water
solution stored unpackaged in darkness at 0° P. for 45 days

n—u—a—-—-—'~ ~_..-—-—-———-.--~-T- m- -_ . I- ~-- an—r—_ ~m.——- a ——-*I-~ - u.“ 1

Estimated per cent_
metmyoglobin 33.1

620

 

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48

solutions. Between the defrosting cycles the temperature varied between
-4° F. and +l° F. The walk-in freezer, on the other hand, operated at
0° F. with a mximm fluctuation of 2'31".

Figure 6 presents the variation in per cent metmyoglobin between the
two groups. It my be observed that after the first day in storage the
group exposed to light was consistently higher in per cent metmyoglobin.
Table III presents the analysis of variance for these data. The differ-

ences in per cent metmyoglobin were significant at the one per cent level.

TABLE III

Analysis of variance of per cent metmyoglobin: Trial 3

 

Source D/ F SS HS F

 

 

Total 19 1,413.25
Between days 9 1,242. 15 138.02 24.3
Between treatments 1 120. 10 120. 10 21.2”
Error 9 51.07 5.67

**F < .01

As would be expected, and as was the case in all trials, the per cent
metmyoglobin increased as the storage period increased. This trial indi-
cated that light caused an increase in the rate of metmyoglobin formation
in frozen meat solutions. Thus the solutions were satisfactory media for

following the color changes in frozen meats.

Trial 4:

In this trial, a series of 14 samples was prepared and stored under

light in the 0° F. walk-in freezer. Some of the cubes were shattered in

   

49

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50

the packaging process so no Hunsell readings were obtained from.them.
Also, some of the spectrophotometric readings had to be duplicated and
this did not leave sufficient solution for a thsell color reading.

The standard used for the frozen cubes was the reading with the low-
est value obtained in Trial 5. This was 4.0YR 2.7/3.2. The standard
for the thawed solutions was an oxygenated solution with the notation
7.9YR 4.4/4.2. The correlation between the index of fading of the frozen
cubes and the per cent metmyoglobin was .37. The correlation between the
index of fading for the thawed solutions and the per cent metmyoglobin
was .28. Neither of these correlations was statistically significant.

The properties of frozen cubes were not well adapted to Hunsell color
notations. The reflective properties of the cubes themselves and the
irregular surfaces had an adverse effect on the color notations. The
thawed solutions were placed in a glass insert in a metal container.

This utilized only 22 m1. of solution (about all that was available after
the spectrophotometric analysis). Apparently this was not sufficient to
mask the color of the container itself.

The spectrophotometric analysis showed an excellent increase in per
cent metmyoglobin as the storage period increased. This increase is
shown in Figure 7. The high speed centrifugation utilized before freezing

had no appreciable effect on the clarity of the thawed solution.
Trial 5:

This trial involved dialysis of the solution before freezing. It was
thought that possibly the removal of metal ions might have some effect on
the light-catalyzed increased rate of metmyoglobin formation. In addition

to dialysis, the solution was mechanically agitated in a -20° F. blast

 

 

51

 

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52

freezer until freezing began, in an attempt to increase the homogeneity
of the individual samples.

The procedure for the Munsell readings was the same as in Trial 4.
Correlation coefficients obtained were .29 for the index of fading of
the frozen cubes versus per cent metmyoglobin, and .31 for the index of I
fading of the thawed solutions versus per cent metmyoglobin. Again,
both of these correlations were not statistically significant. In view
of this fact, it was decided to discontinue the‘nunsell readings on the
frozen cubes. The readings for the thawed solutions would also require
a different approach if they were to be of any value.

The dialysis process did prove effective in retarding the rate of
metmyoglobin formation as is shown in Figure 8. After a 62-day storage
period, there was only 5.91 metmyoglobin as compared to over 381 for an

undialyzed solution used in Trial 4.
Trial 6:

One half of the solution was divided into 16 portions and frozen
immediately. This was Group 1, and served as an undialyzed control. The
samples in this group were packaged and stored at 0° F. for three days
in darkness. The other half of the solution (Group 2) was dialyzed for
three days and then packaged and frozen. Both groups were stored at
00 F. in the walk-in freezer under 30 foot-candles of fluorescent illum-
ination. Samples from.both groups were removed periodically from the
freezer and analyzed spectrophotometrically for metmyoglobin, Figure 9
shows that the undialyzed control was considerably higher in per cent
metmyoglobin throughout the test period. It was also noted that the ph

of the dialyzed group tended to be slightly higher than for the non-

   

 

 

 

 

 

 

 

 

53

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55

dialyzed group. This was not a consistent effect, however, and in some
subsequent trials dialysis had no effect on pH. A £.test was performed
on the data from the two groups and a value of 6.2 with 29 degrees of
freedom obtained. This showed that there was a significant difference
(P<:.001) between the per cent metmyoglobin for the two groups.

The dialysate was retained and concentrated from an original volume
of 14,470 ml. to a final volume of 595 ml. Four 25 ml. aliquots were
dried at 100° C , then ashed at 1000° F. in a muffle furnace. The results,

in terms of grams of ash per aliquot, appear in Table IV.

TABLE IV

Dialysate ash determination

 

 

Aliquot Number
1 2 3 4 Ave.

 

Ash (gma.) .0347 .0345 .0342 0339 .0343

 

From this it was estimated that there were .8163 gas. ash in the
total dialysate from 100 gms. of meat, or .821 of the original sample was
removed as ash.

One sample of ash was given a partial analysis on a spectroscope.
While this analysis was not complete, the following metals were definitely
identified: iron, bromine, phosphorus, magnesium, manganese, calcium and
copper. The results of this analysis were used as a basis for treatments

in future trials.

   

56
Trial 7:

This was the first trial in which solutions were prepared entirely
according to the standard procedure previously described. The combination
of centrifugation and filtering through Number 42 Whatman filter paper
before freezing and after thawing resulted in a clear solution for spec-
trophotometric analysis.

The treatment in this trial was the addition of manganese ions in
the form of the chloride salt to the dialyzed solution. An arbitrary
level of .5 mg. of manganese per gram of beef was selected. This was
added to the dialyzed solution on the basis of the weight of meat origi-
nally used and the final volume of the residue after dialysis.

The per cent metmyoglobin in the manganese-treated group went to a
high level after only five days of storage. This level remained high and
fairly constant through the 45-day storage period (Figure 10). It was
later ascertained that the HnClz in the solution was reacting with either
potassium cyanide or potassium.ferricyanide to form a complex that inter-
fered with the light absorption through the cuvette. It was concluded
that this increase in the per cent metmyoglobin was an artifact and not
a real change in the amount of myoglobin in the metmyoglobin form. Fur-
thermore, Mittledorf and Landon (1952) stated that the concentration of
manganese in beef was approximately .00002 mg. per gram of tissue. This
meant that the level used in this trial was in great excess. Due to these
factors it cannot be concluded that manganese had an actual effect on in-
creasing the rate of metmyoglobin in frozen meat solutions stored under

light.

   

57

 

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58

Trial 8:

The treatments for this experiment were the chloride salt solutions
of manganese (Group 3), copper (Group 4), magnesium.(Group 5), iron
(Group 6) and calcium.(Group 7). The concentrations used were given in
the section on experimental procedure. Group 1 was an undialyzed control
and Group 2 was a dialyzed control. All solutions were prepared and ana-
lyzed spectrophotometrically in the standard manner.

Seven samples were prepared for each group. All but one of these
were stored in a 0° F. display case under 60 foot-candles of fluorescent
light. The seventh sample in each group was retained in a 0° F. walk-in
freezer under 30 foot-candles of light. The latter samples were analyzed
after a 77-day storage period.

Hunsell color readings were made on all the thawed solutions.
Following spectrophotometric analysis, a 35 ml. portion was placed in a
circular metal container painted a darker gray than previously used. The
same oxygenated standard used in the earlier trials for thawed solutions
was utilized, but consisted of a 35 m1. aliquot read in the new, darker
container. The renotations for this standard were 4.7YR 3.2/4.8. The
value, 3.2, for the standard was higher than most of the values for the
experimental samples. This resulted in an index of fading that, in gen-
eral, decreased as the per cent metmyoglobin increased instead of the
reverse as is generally the case. When the results from all seven groups
were combined the correlation was -.71 and was significant at the one
per cent level (Figure 11). This was the only instance in which a sig-
nificant 5 value was obtained between either the frozen or thawed solu-

tions and an index of fading. It may be observed that even here there

   

 

 

 

59

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60

is considerable individual variation among the samples.

In order for Munsell color notations to have any meaning, when work-
ing with solutions, two conditions must be standardized. First, the
same container must be used for all readings, and secondly, the volume
of solutions used should be held constant and be large enough to ade-
quately mask the color of the container. In connection with this last
requirement it was felt that a 35 ml. aliquot was still not large enough,
but several practical experimental considerations limited the size of
each individual portion.

The individual treatments did present some variation in the per cent
metmyoglobin formation. Table V shows the analysis of variance and the
significant differences between means for the per cent metmyoglobin of

a l 1 treatments .

TABLE V

Analysis of variance of per cent metmyoglobin: Trial 8

r - i
*7 rm ‘4

 

 

-_ ‘— ‘-
‘.

 

 

Source D/F SS HS
Total 48 11,430.94
Between days 6 4,996.35 832.73 10.20**
Between treatments 6 3,495.24 582.54 7.13**
Error 36 2,939.35 81.65
**P‘(.01

Significant differences (5% level) between treatment means

 

 

 

Mean per cent 10.47 10.67 117.10 11.81 16.07 21.69 35.23
metmyoglobin

Treatment 2(Dia.) 7(Ca) 3(Mn) 4(Cu) 6(Fe) 5(Hg) 1(Und.)

 

 

   

61

The undialyzed control had the highest average per cent metmyoglobin
(35.231) and was significantly higher than all other treatments. The dia-
lyzed control had the lowest average per cent metmyoglobin and was not
significantly different from any other treatment except the undialyzed
control and magnesium. The solution with the magnesium ions added had
the second highest average per cent metmyoglobin, 21.691. This may mean
that magnesium is involved in the light-catalyzed increase in metmyoglo-
bin formation. However, it may be noted that there was an exceptionally
high reading on the 34th day (Figure 12), which may largely be responsible
for the significant difference in per cent metmyoglobin of magnesium from
the dialyzed control. Iron had the third highest average per cent met-
myoglobin, with 16.07, but was not significantly different from the dia-
lyzed control. None of the other treatments were significantly greater

than the dialyzed control group.

Trial 9:

A dialyzed solution was prepared in the usual manner and the dialy-
sate concentrated to a volume of 25 ml. The concentrated dialysate was
added back to one half the residue solution at the rate of 2.5 ml. per
50 ml. sample. The other half of the solution was divided into 50 ml.
aliquots and constituted the control group. All samples were stored in
the 0° F. display case under fluorescent lights. A pair of samples was
removed periodically over a 48-day period and analyzed spectrophotometri-
cally for metmyoglobin.

The purpose of this experiment was to determine if there was a sub-
stance, or substances, being removed in the dialysis process that was

responsible for the increased rate of metmyoglobin formation in the

   

 

57

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33

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Per cent metmyoglobin

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Days in storage

Figure 12

Trial 8: Per cent metmyoglobin for all treatments stored
in 0° F. Sherer display case under 60 foot-candles of illumination

   

63

undialyzed solution, or if the improved keeping qualities of the dialyzed
solutions were due to some mechanical change caused by the process of
dialysis itself.

The results of this trial appear in Figure 13. It can be readily
ascertained that the reconstituted group had a much higher rate of met-
myoglobin formation. A 5 test was performed on the data and a value of
3.72 with ten degrees of freedom obtained. This was highly significant
(P<<.005) and indicates that there is something actually removed during
dialysis which acts as an activator for the light-catalyzed increase in
rate of metmyoglobin formation in frozen meat solutions. The fact that
the levels of metmyoglobin formation are even higher for the reconstituted
group than for the undialyzed groups in other trials may be due to the
fact that the materials in the dialysate were added back at greater than

the original concentrations.

Trial 10:

In this trial a dialyzed solution prepared in the standard manner
was divided into four groups: Group 1, control; Group 2, .0002 mg. man-
ganese per gram of meat; Group 3, .002 mg. manganese per gram of meat;
Group 4, .02 mg. manganese per gram of meat. All samples were stored in
the 0° F. walk-in freezer under 25 foot-candles of fluorescent illumina-
tion and periodically examined spectrOphotometrically over 56 days.

Figure 14 presents the results of this trial. It may be noted that
the rate of metmyoglobin formation was very low for all groups. This
again emphasizes the fact that the fluctuating temperatures of the display
case greatly increase the rate of metmyoglobin formation in frozen meat

solutions as compared to the constant 0° F. temperature of the walk-in

   

64

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Days in storage

Figure 13

Trial 9: Per cent metmyoglobin for a dialyzed group vs.a reconstituted
solution stored in a 0° F. display case under 60 foot-candles of light

 

 

 

 

Per cent metmyoglobin

65

: “——' Gr.1 Control

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g ‘“°" Gr.3 .002 mg. Mn
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Days in storage

Figure 14

Trial 10: Per cent metmyoglobin for
varying levels of manganese solution stored under
25 foot-candles of illumination in a 0° F. walk-in freezer

_...___. .— ~_ ”Q'I-d

60

   

 

66

freezer. This is in agreement with several reports in the literature for
frozen meat. Table VI presents the analysis of variance performed on the

data from this trial.

TABLE VI

Analysis of variance of per cent metmyoglobin: Trial 10

 

 

 

Source D/F SS HS P
Total 23 38.62
Between days 5 16.55 3.31 6.16**
Between treatments 3 14.02 4.67 8.70**
Error 15 8.05 .54
**P<.Ol

Significant differences (5% level) between treatment means

 

 

‘Mean per cent .83 .98 1.19 2.74
metmyoglobin
Treatment 3(.002 ngin) 1(Control) 2(.002 ngin) 4(.02 ngln)

 

Although the 3 test was highly significant, the range test shows
that the only treatment that was significantly different from the others
was Group 4, with the highest level of manganese (.02 mg. per gram of
meat). This treatment never had over 51 metmyoglobin even after 56 days
of storage. The higher level of manganese used may have caused a coloro-
metric reaction with the cyanide solutions and thus gave relatively higher

readings than were obtained in Trial 8.

 

   

67

Trial 11:

This experiment duplicated Trial 8, but used exactly twice the con-
centration of the manganese, copper, magnesium, iron and calcium ions.
All samples were stored under 60 foot-candles of illumination in a 0° F.
display case and analyzed spectrophotometrically for metmyoglobin over a
lS-day period. No Munsell color readings were taken in this trial.

Figure 15 presents the results of the metmyoglobin determinations.
An analysis of variance of the per cent metmyoglobin for the various

treatments appears in Table VII.

TABLE VII

Analysis of variance of per cent metmyoglobin: Trial 11

 

 

 

 

Source D]? 88 as P
Total 48 5,103.92
Between days 6 1,009.49 168.25 5.31**
Between treatments 6 2,954.31 492.39 15.55**
Error 36 1,140.12 31.67
**P‘<.01

Significant differences (5% level) between treatment means

 

Mean per cent 1.66 2.06 2.06 2.52 3.62 12.80 23.72
metmyoglobin

Treatment 301:.) 7(Ca) 4(Cu) 2031:.) 5043) 6(Pe) 1(Und.)

 

As in Trial 8, the undialyzed group had the highest per cent metmyo-
globin, with an average of 23.72%. This was significantly greater than

any other treatment. The next highest per cent metmyoglobin was in the

   

 

Per cent metmyoglobin

68

 

 

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H—HH‘ Gr.2 Dialyzed control
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Days in storage

Figure 15

Trial 11: Per cent metmyoglobin of meat
solutions treated with various metal ions and
stored in a 0° P. display case under 60 foot-candles of light

   

 

69

group to which the iron had been added (12.801 metmyoglobin). This was
significantly different from all other treatments. In Trial 8, the group
with the magnesium ion was the only one which was significantly higher

in per cent metmyoglobin than the dialyzed control. In this trial, while
not significantly greater than the dialyzed control, it had the third
highest average per cent metmyoglobin (3.621). As in Trial 8, neither
cOpper, manganese nor calcium had a significant effect on the rate of
metmyoglobin formation.

It appears that both magnesium and iron have some effect on the in-
crease of metmyoglobin in frozen meat extracts stored under light. The
role of the iron may be simply that of an oxidizing agent. The iron was
added in the ferrous form (Fe012°4H20), but this is capable of changing
to the ferric form, which should be more effective as an oxidizing agent.
It would not be expected that magnesium functions in a similar manner,
but may exert some separate influence on the conversion of myoglobin to

metmyoglobin in the presence of light.

Trial 12:

Matilda Brooks (1934) reported that a 12 solution of glucose was
effective in preventing induced methemoglobin formation in live rabbits.
The experiment performed in Trial 12 was carried out to determine_if
glucose would have a similar effect on the rate of metmyoglobin formation
in frozen meat solutions. A 11 and a 22 solution of glucose were pre-
pared and added to 35 ml. samples of a previously prepared undialyzed
meat solution. A series of untreated samples constituted the control
Group 1; one ml. of the 11 glucose solution was added to the samples in

Group 2, and one ml. of the 2% solution to the samples in Group 3. All

   

70

samples were stored in the 0° P. display case under 60 foot-candles of
illumination, and periodically examined spectrophotometrically for met-
myoglobin.

The results of this trial appear in Figure 16. All three groups
reached a high level of metmyoglobin in the course of the 15-day storage
period. The analysis of variance of the data from this trial appears in

Table VIII.

TABLE VIII

Analysis of variance for per cent metmyoglobin: Trial 12

 

 

 

 

Source D/F 55 us 1?
Total 14 2,011.77
Between days 4 1,938.01 484.50 80.90**
Between treatments 2 25.81 12.91 2.16
Error 8 47.95 5.99
**P~<.01

The differences between the treatments were not significant. It may
be concluded that glucose, in the concentrations used,was not effective
in inhibiting the formation of metmyoglobin in frozen meat solutions

stored under light.

Trial 13:

In this trial the effects of ion solutions of manganese, magnesium
and iron were studied. The concentrations used were the same as in
Trial 7, only in this case, two iron solutions were prepared. One was

the ferrous form using PeClz°4H20, and the other was the ferric form

   

42
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14 ' H6 ‘T‘O' Gr.3 2% glucose

12 _.. -

10 :_._-1 -.l_.__-1... l l 1 .1 l l .1... ..l I 1 I .

123456 78910111213141
Days in storage

Figure 16

Trial 12: Per cent metmyoglobin of solutions treated with glucose
and stored in a 0° F. display case under 60 foot-candles of illumanation

   

72

using FeCl3-6H20. A meat solution was prepared in the usual manner, with
Group 1 as an undialyzed control. The remaining solution was dialyzed
and the treatments allocated as follows: Group 2, dialyzed control;
Group 3, manganese; Group 4, magnesium; Group 5, iron (Fe‘I); Group 6,
iron (Fe+++). All samples were stored in the 0° F. display case under
60 foot-candles of fluorescent illumination and examined for metmyoglobin
at intervals over a 14-day period.

The results of this trial appear in Figure 17. The analysis of

variance on the data appears in Table XI.

TABLE IX

Analysis of variance of per cent metmyoglobin: Trial 13

 

 

 

Source o/r ss as r
Total 41 2,599.61
Between days 6 1,412.97 235.95 20.0**
Between treatments 5 833.05 166.61 l4.l**
Error 30 353.59 11.79
**P‘<;Ol

Significant differences (51 level) between treatment means

 

 

 

 

 

Mean per cent 3.37 4.87 8.63 11.24 12.83 16.22
metmyoglobin
Treatment 2(Dis.) 3(Mn) 4(Mg) 6(Fe+++) 5(Fe+*) 1(Und.)

 

The differences between treatments were highly significant. As in
Trials 7 and 8, the undialyzed control had the highest average per cent

metmyoglobin, 16.22%. This was significantly higher than all the other

   

 

73

 

 

 

3—’ —1 I I T r— I" I I I a —'T‘ ”I I l
32 _ 1r‘H‘J-H'Grd Undialyzed control
- -Gr.2 Dialyzed control '91
O-‘O— Gr.4 143 4
—~- "Gr.5 Fe++ 4
28 "’ ..... 6:06 re d
4
- 4
26 -_ <3
4
24 -. 4
d.
C 4
.,.q A
A 22 .- 4
.9. .. .\
I /
g 20 I... ' / \ \ -'
u ‘ 0
<1 /
3 18 .- q / .' . - . . . _
<3 / '
‘3 < - - 2 .'
0 i q .- , ’ I -
o 16 - j/ I ' o /.
H I //. o ' . D
0 ' s
a. 14 _ fifl .,. o/ -
v 0’ /.’
' <1 .' /0 ”D
12 {- 4c/ .0 fl / . ‘
; ' 1 / J/U
f / 0 /.O
10 3— 7°; ‘- o -
I 4- ' [I
I /( o o ' I.
8 .. ,24 ' o -
= / a . I .O
1 s 4 - O «9
6 l ’4 . 1’, __..".'“"~
L .T. J ' WWW" '-
s / V.
, hag ,0 .34.. I
4 ._ ”.63 0 x 4 -
I .f'
/.‘3 o/ / JI‘ (
.Q . .’ I
2 <1 / we“
'- /< o ‘15.”... , “’1 3
s/ "W‘- - ~- 4
”00 .5‘7w "Jr.
/ ~-_::'~____ -..L... I 3 I I . i ' I l

 

0 l 2 3 4 5 6 7 8 9 10 ll 12 l3 14 15
Days in storage

Figure 17

Trial 13: Per cent metmyoglobin
of ion treatments in meat solutions stored in
a 0° F. display case under 60 foot-candles of fluorescent illumdnation

 

   

74

treatments except the ferrous ion. The FeII and Fe+++ had the next
highest percent metmyoglobin, and were not significantly different from
one another. Magnesium.was next and was not significantly different

from Fe+++, but was significantly higher than the dialyzed control. Man-
ganese and the dialyzed control had the lowest levels of metmyoglobin
and were not significantly different from one another but were signifi-
cantly lower than all the other treatments.

Manganese, when used in physiological amounts, apparently has no
effect on the rate of metmyoglobin formation in frozen meat solution.
There is evidence to indicate that both iron and magnesium may have an
effect on the increase in metmyoglobin formation in frozen meat extracts
stored under light. The two forms of iron, ferric and ferrous, seem to
be equally effective in increasing the metmyoglobin content of the dia-
lyzed solution. Further investigation is needed to determine the mech-

anism of the reactions involved between iron and magnesium and light.
Trial 14:

A series of readings were taken on a Beckman Recording Spectrophoto-
meter in order to observe the entire spectra for dialyzed and undialyzed
solutions. The data from this trial appear in Appendix N. Again, the
undialyzed group had a much greater increase in metmyoglobin formation
than did the dialyzed group.

Figure 18 presents the absorption spectra for both groups after two
and 18 days storage in the 0° F. display case under approximately 50 foot-
candles of light. It may be observed that the absorption maxima are dis-
placed slightly towards the shorter wavelengths from the expected peaks

at 544 and 582 mu“ All data obtained from the recording spectrophotometer

 

   

 

75

16 f————-—-——- . - - I --- -- ~--—-~~ ,--» »-
15L. _”\
147 ,1 \1 " I

13;.

‘Hillimolar extinction coefficient

 

53 Undialyzed control: 0
—' ‘2 days in storage
‘0”0'18 days in storage

a Dialyzed group: O
3 '*-'"'2 days in storage
L mmwmnlB days in storage 09

L 07:
2 --_._..--_...._- _.. I _____ _ - I - '3

500 525 550 575 600
wavelengths in millimicrons

O
—— —-m ._ -‘v -'—- *M --.e -

n———.

l
O-—-—-——. “4....

Figure 18

Trial 14: Absorption spectra for dialyzed
and undialyzed meat solutions after two and eighteen days
storage under 50 foot-candles illumination in a 0° F. display case

 

   

 

76

were displaced two to four millimicrons to the left. When duplicate
readings were taken on the Beckman DU Spectrophotometer the peaks were
at the expected wavelengths. In estimating the per cent metmyoglobin,
the absorption maxima as determined on the recording spectrophotometer
were used but calculated as if they had occurred at 544 and 582 mu.

There were no other peaks besides the two primary ones, from 400
to 900 mm. This indicates that the pigments involved are restricted to
oxymyoglobin and metmyoglobin, and serves to justify one of the assump-
tions on which this spectrophotometric analysis is based.

In order to present the similarities in rate of metmyoglobin for-
mation of the frozen meat solutions used in this study and frozen steaks,
Figure 19 was compiled. This utilizes data on frozen steaks stored in
the 0° F. display case under 56 foot-candles of fluorescent illumination
as presented by Townsend (1958). Data from the present study include
untreated meat-water solutions from Trial 4 (stored in the 0° F. walk-in
freezer under 35 foot-candles), Trial 6 (stored in the 0° F. walk-in
freezer under 30 foot-candles) and Trial 14 (stored in a 0° F. display
case under 50 foot-candles).

The same general pattern of increased metmyoglobin formation with
increased length of storage may be observed for all four groups. The
samples in Trials 4 and 6, which were stored in the walk-in freezer,
present a lower rate of increase in metmyoglobin formation than do the
steaks or the solutions (Trial 14) stored in the display freezer. This
is in agreement with Townsend's findings (1958) that fluctuating storage
temperatures increased the rate of metmyoglobin formation in frozen

steaks.

 

 

   

77

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78

It may be also noted that the per cent metmyoglobin estimates vary
more for the steaks than for the solutions. This tends to support the
observation made earlier in this study that meat solutions should pro-
vide a more homogeneous series of samples for following the myoglobin
oxidation than would a series of steaks. Figure 19 also presents evi-
dence that the rate of metmyoglobin formation is similar for frozen

steaks and frozen meat extract solutions.

 

   

 

SUMMARY AND CONCLUSIONS

A method for the preparation of meat-water extracts, and the
spectrophotometric estimation of the per cent metmyoglobin in these ex-
tracts has been presented. Using frozen solutions, the effects of fluo-
rescent lights, dialysis, addition of the concentrated dialysate, addi-
tion of various metal ions, and the addition of glucose were investigated.
From these experiments the following conclusions were drawn:

1. Frozen meat-water extracts showed an increase in per cent met-
myoglobin during storage at 0° F. The increase resembled that of frozen
beef steaks stored under similar conditions.

2. The spectrophotometric method of metmyoglobin estimation as
adapted for use in this study gave satisfactory results for detecting
color deterioration of meat-water extracts as related to the increase in
metmyoglobin.

3. Storage of meat-water extracts under fluorescent lighting in-
creased the rate of metmyoglobin formation as compared to similar extracts
stored in darkness.

4. Dialyzing the extracts greatly retarded the rate of metmyoglobin
formation as compared to undialyzed solutions.

5. The addition to the dialyzed solution of copper, manganese or
calcium ions as the respective chloride salts did not have an appreciable
effect on the rate of metmyoglobin formation. The addition of the chlo-
ride salts of magnesium and iron (both ferric and ferrous form; con-

tributed to an increase in the rate of metmyoglobin formation of the

79

   

 

80

dialyzed solutions.

6. Addition of the concentrated dialysate to the dialyzed residue
greatly increased the rate of metmyoglobin formation of frozen extracts
stored under fluorescent lights.

7. The addition of one cc. of a 1% or 21 solution of glucose had
no effect on reducing the rate of metmyoglobin formation of the frozen

meat-water solutions.

 

 

   

 

 

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88

Appendix A

Data collected from Trial 1: Spectrophotometric readings from a steak
packaged under petroleum ether and stored at
-20° F. under 10 ftc. illumination

 

 

 

 

Mixedggigment Cyanide derivative
an 02:, MEG J55 OD
500 .278 8.7 500 .254
510 .277 8.7 510 .252
520 .259 8.1 520 .286
530 .270 8.4 530 .340
540 .306 9.6 539 .365
542 .309 9.7 540 .367*
543 .311 9.7 541 .367
544 .312 9.8* 547 .365
545 .308 9.6 550 .361
550 .287 9.0 560 .325
560 .203 6.3 570 .284
570 .193 6.0 580 .239
580 .254 7.9 590 .182
581 .259 8.1 600 .132
582 .261 8.2* 610 .088
583 .261 8.2 620 .063
584 .258 8.1 630 .048
590 .183 5.7 640 .040
600 .102 3.2 650 .060
610 .087 2.7
620 .088 2.8
630 .094 2.9
640 .094 2.9
650 .083 2.6

 

*Absorption maxima; OD I Optical density, MEC II Millimolar
extinction coefficient

Molar myoglobin concentration - .367 - .000032 moles
11,300

Estimated per cent metmyoglobin: 544 mm 50.0
582 In, 57.0
Ave. 53.5

Per cent myoglobin (moist tissue basis) .491

   

 

89

Appendix B

Data collected from Trial 2: Beckman DU Spectrophotometer readings
on a meat-water solution stored unpackaged in darkness
for 45 days in a -20° F. walk-in freezer

 

 

 

 

 

Mixed Mt Cyanide derivative
nu; on e: x10'3 m on
500 .259 7.4 500 .268
510 .258 7.4 510 .261
520 .256 7.3 520 .300
530 .308 8.8 530 .366
540 .397 11.3 538 .393
542 .408 11.7 539 .394
543 .411 11.7 540 .396*
544 .415 11.9* 541 .396
545 .414 11.8 542 .396
550 .384 11.0 550 .382
560 .269 7.7 560 .350
570 .278 7.9 570 .305
580 .372 10.6 580 .251
581 .378 10.8 590 .189
582 .389 11.1* 600 .129
583 .388 11.1 610 .078
584 .378 10.8 620 .044
590 .246 7.0 630 .031
600 .092 2.6 640 .020
610 .064 1.8 650 .018
620 .061 1.7
630 .063 1.8
640 .061 1.8
650 .051 1.5

 

*Absorption maxima; OD - Optical density

Molar myoglobin concentration I .396 - .000035
11,300

Estimated per cent metmyoglobin: 544 mu 2
582 mu 3
Ave.

to
.43....
O 0 O
HHH
U'U

Per cent myoglobin (moist tissue basis) .541

 

 

 

 

90

Appendix C

Data collected from.Trial 3 on meat extracts stored in darkness and under
50 ftc. fluorescent illumination in a 0° F. Sherer display case

5 1110‘5
D M_MC 544 582 2 Rib .1. Mb pH

 

Group 1: Stored under 50 ftc. illumination

0 3.71 15.39 15.63 0 .57 6.11
l 3.64 14.89 15.16 0 .56 6.24
4 2.58 13.80 13.64 10.21 .39 6.17
6 2.64 13.83 13.67 9.92 .40 6.23
8 2.84 13.49 13.17 13.76 .43 6.22
11 2.67 13.18 12.66 17.48 .41 6.15
13 2.39 13.01 12.47 19.14 .37 6.14
15 3.06 12.61 11.96 23.34 .47 6.27
17 2.46 12.28 11.54 26.80 .37 6.32
19 2.45 12.53 11.84 24.25 .37 6.25
Group 2: Stored in darkness
0 3.71 15.39 15.63 0 .57 6.11
l 2.81 14.91 15.10 0 .43 6.20
4 2.88 14.58 14.51 2.55 .44 6.23
6 2.41 14.23 14.19 5.69 .37 6.18
8 3.56 13.74 13.51 11.05 .54 6.10
11 4.23 13.17 12.81 11.58 .62 6.15
13 2.64 13.67 13.41 11.83 .40 6.08
15 2.54 13.43 13.03 14.65 .39 6.10
17 2.96 13.11 12.70 17.68 .45 6.19
19 2.99 12.84 12.27 20.86 .46 6.08

 

D I Days in storage; MMC -rMo1ar myoglobin concentration x 105;
Z.MMb - Average per cent metmyoglobin; Z.Mb - per cent myoglobin

   

 

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a xqwconn<

 

“a?

 

 

 

93

Appendix F

0
Data collected frou.Tria1 6: Dialyzed and undialyzed samples stored
under 30 ftc. illumination in 00 P. walk-in freezer

6 x105

.__1_

 

 

 

D 1910 544 mL 582 m1 2, Hub 2 Mb p11
Group 1: Undialyzed control
0 2.17 15.30 15.50 0 .33 5.50
2 3.82 13.95 13.87 8.47 .58 5.50
4 3.35 13.43 13.16 14.10 .51 5.42
7 3.97 12.57 12.09 20.49 .61 5.50
9 2.27 12.82 12.29 20.88 .35 5.40
11 2.05 12.05 11.32 28.90 .31 5.48
14 1.72 12.27 11.40 27.43 .26 5.50
16 3.24 11.36 10.28 36.79 .49 5.49
18 2.08 11.54 10.43 34.83 .32 5.34
21 2.23 11.08 9.96 39.58 .34 5.50
23 2.19 10.87 9.54 42.40 .34 5.42
25 2.32 10.65 9.31 44.50 .35 5.62
33
36 2.30 9.87 8.17 50.77 .35 5.60
66 1.82 8.13 5.93 71.60 .29 5.75
68* 1.88 8.94 8.02 58.70 .29 5.80
Group 2: Dialyzed

O 2.07 15.21 15.30 0 .32 5.70
2 3.92 14.49 14.59 2.68 .60 5.70
4 2.93 14.60 14.74 1.49 .45 5.90
7 2.62 14.58 14.96 0.67 .40 5.70
9 1.39 15.00 15.18 0 .21 5.90
11 2.63 13.95 14.07 7.64 .40 5.91
14 3.07 14.40 14.46 3.69 .47 6.00
16 2.62 14.27 14.35 4.82 .40 5.70
18 2.87 14.70 14.91 0.79 .49 5.70
21 2.65 14.45 14.57 2.97 .41 5.81
23 2.30 14.48 14.39 3.56 .35 5.85
25 3.02 14.27 14.24 5.28 .46 5.87
33 2.63 14.26 14.22 5.41 .40 5.98
36 2.67 14.60 14.57 2.19 .41 5.84
66 2.77 14.19 14.15 6.06 .42 6.40
68* 2.77 14.44 14.26 4.31 .42 6.08

 

*Stored in darkness; D - Days in storage; HMO I Molar myoglobin
concentration x 105; 1 Hub . Average per cent metmyoglobin; 1 11b II

per cent myoglobin

 

 

 

 

94

Appendix G

Data collected from Trial 7: Manganese experimental samples stored
under 30 ftc. of light in 0° F. walk-in freezer

6 x10“:

 

 

D me 544 m 582 mu 7. mb 7. Mb p11
Group 1: Dialyzed control

0 2.62 14.89 15.04 0.25 .40 6.13
3 2.45 15.35 15.51 0 .37 6.40
5 2.32 14.91 15.17 0 .35 6.50

7 2.93 14.78 14.98 0.50 .45 6.67
10 2.97 14.92 15.05 0.21 .45 6.59
12 2.77 15.05 14.91 0.79 .42 6.62
14 2.88 14.97 15.21 0 .44 6.40
19 2.70 14.89 15.19 0 .41 6.75
21 2.64 15.23 15.34 0 .40 6.86
24 2.69 14.68 14.91 0.79 .41 6.62
31 2.67 15.10 15.17 0 .41 6.90
33 2.80 14.82 15.03 0.29 .43 6.65
35 2.68 15.15 15.26 0 .41 6.70
38 2.69 14.98 15.10 0 .41 6.52
45 2.86 14.90 14.97 0.54 .44 6.72

Group 2: Dialyzed + .5 mg. Mn/gm. meat

0 2.78 15.04 15.32 0 .43 6.20
3 2.23 15.07 15.29 0 .34 6.38
5 2.89 11.14 11.21 34.10 .44 6.00

7 3.50 11.54 11.51 30.79 .54 6.68
10 3.12 11.79 11.76 28.44 .48 6.35
12 3.48 10.83 10.78 37.49 .53 6.52
14 3.42 10.26 10.26 42.60 .52 6.30
19 3.43 10.96 10.85 36.52 .52 6.89
21 3.64 10.85 10.55 38.33 .56 6.32
24 3.99 10.15 9.85 44.87 .61 6.69
31 3.10 12.32 11.94 24.94 .47 6.42
33
35 3.15 11.46 10.98 33.38 .48 6.40
38 3.05 11.51 11.38 31.47 .47 6.19
45 2.98 11.54 11.34 31.43 .46 6.22

 

D 8 Days in storage; MMC c Molar myoglobin concentration x 105;
1 MMb - Average per cent metmyoglobin; Z‘Mb - Per cent myoglobin

9S

 

 

 

 

 

 

 

 

4.43 4.4 4.4 444.4 45.4 45. 44.3 44.43 44.43 44.4 4455
43.4 54. 44.44 45.33 44.43 44.4 44
4.4 4.4 3.4 444.3 44.4 54. 45.43 43.43 44.43 44.4 44
4. 4.4 4.4 444.4 44.4 54. 43.44 44.33 44.43 44.4 44
4. 3.4 4.4 444.3 44.4 44. 44.4 44.43 44.43 44.4 43
4. 4.4 4.4 44.4 44.4 45. 45.4 54.43 44.43 45.4 4
4. 4.4 4.4 44.4 44.4 45. 4 44.43 44.43 54.4 4
44444444: 34 nacho
4.43 4.4 4.4 444.4 44.4 45. 43.4 44.43 44.43 54.4 4455
44.4 44. 44.43 44.43 33.43 44.4 44
4. 4.4 3.4 444.3 44.4 44. 44.43 44.43 34.43 44.4 44
4. 4.4 4.4 444.4 44.4 44. 44.34 44.43 45.43 53.4 44
4. 4.4 4.4 445.3 54.4 44. 44.33 54.43 44.43 34.4 43
4. 4.4 4.4 44.4 44.4 45. 54.4 44.43 44.43 44.4 4
5. 4.4 3.4 444.4 45.4 44. 4 44.43 44.43 34.4 4
3044400 44443434 34 45044
4. 4.4 4.4 444.4 45.4 44. 44.45 44.4 44.4 34.4 4455
35.4 44. 44.44 44.4 44.4 44.4 44
4.4 4.4 4.4 444.4 44.4 44. 44.54 44.4 43.43 44.4 44
4.4 4.4 4.4 444.4 45.4 44. 44.44 43.43 44.33 44.4 44
4.4 3.4 4.4 444.4 44.4 44. 54.44 44.33 34.43 44.4 43
4.4 3.4 4.4 444.4 44.4 44. 44.43 45.43 45.43 44.4 4
4.43 4.4 4.4 444.4 44.4 45. 44.4 44.43 45.43 44.4 4
3644400 444434344: 33 4:044
11:33 o > 4F 33.3 e3 .5 A? s :3 4.44 a. 444 low. 113.3
3334303304 -4333 w

 

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444.4 34.4 54. 44.4 34.43 54.43 44.4 4455

44.4 44. 44.34 44.33 34.33 44.4 44

444.3 44.4 44. 45.44 44.33 44.43 44.4 44
445.3 44.4 35. 44.44 54.33 44.43 44.4 44
445.3 44.4 54. 44.43 43.43 34.43 44.4 43
44.43 54.4 44. 43.4 43.43 43.43 54.4 4
44.43 54.4 45. 4 44.43 44.43 44.4 4

4643 ”4 43644

444.4 54.4 45. 44.43 44.43 44.43 44.4 4455

44.4 54. 44.54 44.33 43.43 44.4 44

444.4 45.4 54. 44.54 34.33 33.43 44.4 44
444.4 44.4 44. 44.34 45.4 55.43 54.4 44
444.3 44.4 44. 44.44 43.43 44.43 44.4 43
44.4 44.4 45. 44.43 44.43 44.43 44.4 4
44.43 44.4 45. 4 44.43 44.43 44.4 4

59344444: 34 44644

444.4 44.4 45. 44.4 44.43 34.43 34.4 4455

44.4 44. 44.44 44.43 54.43 44.4 44

444.3 44.4 44. 44.44 44.33 34.43 44.4 44

444.3 44.4 44. 44.43 44.43 44.43 54.4 44
444.3 44.4 44. 44.5 44.43 44.43 34.4 43
44.43 44.4 45. 55.4 34.43 43.43 44.4 4
444.4 44.4 45. 4 44.43 44.43 44.4 4

444404 “4 msouu
33 44 e. a as s 433444 3.4344 433: a
ao3u4uoau4 4-43 x w

 

 

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=«-xdgs .mdmo :4 soauacqasgflfi .ouu 44 Have: vapoum44 mm.4\~.n 445.4 unavguu. souu «nowuuuoaou no 444

 

 

 

 

mu 4.44 4.4 4.4 444.4 44.4 45. no.4 45.44 54.43 34.4 4455
. 45.4 44. 54.43 om.~4 44.43 54.4 44
4.5 4.4 4.4 445.3 44.4 44. 54.44 44.43 44.43 43.4 44
4.43 4.4 4.4 444.3 44.4 44. 45.34 44.43 44.43 54.4 44
4.4 3.4 4.4 444.4 44.4 44. 43.3 54.2 45.43 54.4 2
4.43 5.4 4.4 4°.o4 54.4 45. 44.4 44.44 44.43 44.4 4
4.44 4.4 «.4 444.4 44.4 45. c 44.43 44.43 54.4 4
3540314 “5 cache
4.: o n > 4 4m 4: 4 4:: .5 31444 15444 oz: 4
:owunuoau4 .4143 x.w

 

 

Avocadocoov a xgvcoaac

 

 

98

Appendix I

Data collected from Trial 9 on a dialyzed solution and reconstituted
dialyzed solution stored under 60 ftc.
at 0° F. in a Sherer display case

 

 

6. x10“:

 

D 11146 544 am 582 m Z 1424‘: '11 Mb 911
Group 1: Dialyzed control
0 4.75 14.90 15.37 0 .73 6.22
6 4.93 14.36 14.40 4.15 .75 5.92
25 4.59 13.88 13.90 8.71 .70 6.20
32 4.68 13.03 12.88 17.35 .72 6.06
43 4.45 13.57 13.53 11.86 .68 6.01
48 4.43 13.16 12.87 16.72 .68 5.89
Group 2: Dialysate added back
0 4.87 14.48 14.58 2.78 .75 5.98
6 5.73 10.33 9.95 63.52 .88 5.99
25 3.73 8.39 6.25 68.92 .57 5.97
32 4.95 6.63 4.95 83.45 .76 5.89
43 5.27 6.51 5.24 82.88 .81 5.62
48 5.40 8.67 7.56 62.04 .83 5.60

 

D I Days in storage;‘MHC I«Molar myoglobin concentration x 105;
Z.MHb - Average per cent metmyoglobin; 1 Mb - Per cent myoglobin

99

Appendix J

Data collected from Trial 10: Effect of varying levels of manganese on
dialyzed solutions stored under 25 ftc. in a 00 P. walk-in freezer

 

 

 

 

e x 10'3
D 11110 544 In; 582 mg 7. Hub 1 Mb )1!
Group 1: Control
0 3.18 15.50 15.70 0 .49 6.88
21 3.27 14.80 15.02 0.33 .50 6.68
28 3.40 14.50 14.65 2.33 .52 6.62
39 3.29 14.83 15.02 0.33 .50 6.39
44 3.38 14.56 14.79 1.49 .52 6.61
56 3.27 14.70 14.77 1.37 .50 6.31
Group : .0002 mg. Hn/gm. meat
0 3.18 15.50 16.00 0 .49 6.10
21 3.29 14.86 14.98 0.50 .50 6.70
28 3.27 14.53 14.68 2.10 .50 6.60
39 3.37 14.30 14.33 2.32 .51 6.00
44 3.32 14.61 14.85 1.04 .51 6.53
56 3.30 14.79 14.82 1.16 .50 6.40
Group 3: .002 mg. Hn/gn. meat
0 3.17 15.40 15.80 0 .49 6.10
21 3.24 14.94 15.12 0 .50 6.29
28 3.28 14.66 14.82 1.16 '.50 6.52
39 3.24 14.60 14.72 1.57 .50 6.00
44 3.29 14.74 14.89 0.87 .50 6.50
56 3.33 14.62 14.77 1.37 .51 6.31
Group 4: .02 mg. Mn/gn. neat
0 3.19 15.61 16.00 0 .49 6.25
21 3.24 14.57 14.75 1.60 .50 6.40
28 3.29 14.38 14.53 3.50 .50 6.53
39 3.25 14.46 14.52 3.13 .50 6.20
44 3.33 14.20 14.44 4.81 .51 6.h7
56 3.28 14.42 14.51 3.38 .50 6.30

 

D I Days in storage; MHC I Molar myoglobin concentration x 105?'
1 Huh I Average per cent metmyoglobin; 1 Mb I Per cent myoglobin

 

 

 

100

 

Appendix K

Data collected fron.Tria1 11: The effects of metal ions on meat
solutions stored at 00 F. in a display case under 60 ftc.

 

 

 

 

éfx 10'37
mo 544 m 582 m 7. Mb 1 Mb A pl!
Group 1: Undialyzed control
0 3.61 15.60 16.00 0 .55 5.36
3 2.88 14.10 14.03 7.03 .44 5.38
6 3.15 12.95 12.73 18.39 .48 5.42
8 3.35 11.97 11.52 28.50 .51 5.41
10 3.24 11.91 11.51 28.85 .50 5.54
13 3.04 10.99 10.43 38.10 .47 5.63
15 3.67 10.25 9.65 45.18 .56 5.50
Group 2: Dialyzed control
0 3.57 15.10 15.30 0 .55 6.80
3 3.57 14.90 15.07 0.12 .55 6.65
6 3.56 14.55 14.83 1.38 .55 6.50
8 3.64 14.26 14.51 4.21 .56 6.34
10 3.57 14.40 14.57 3.23 .55 6.46
13 3.42 14.27 14.44 4.45 .52 6.56
15 3.34 14.34 14.40 4.25 .51 6.24
Group 3: Manganese
0 3.65 15.80 15.90 0 .56 6.80
3 3.69 14.88 15.18 0 .56 6.54
6 3.65 14.60 14.88 0.91 .56 6.43
8 3.41 14.55 14.72 1.83 .52 6.33
10 3.47 14.50 14.76 1.93 .53 6.51
13 3.32 14.79 14.85 1.04 .51 6.58
15 3.46 14.16 14.22 5.93 .53 6.30
Group 4: Copper
0 3.65 15.50 15.60 0 .56 6.82
3 3.57 14.87 15.21 0 .55 6.29
6 3.44 14.82 14.94 0.66 .53 6.36
8 2.77 14.66 14.87 0.95 .42 6.79
10 3.41 14.43 14.66 2.70 .52 6.70
13 3.56 14.38 14.55 3.42 .54 6.70
15 3.18 14.15 14.18 6.67 .49 6.38
Group 5: Magnesium

0 3.65 15.10 15.40 0 .56 6.83
3 3.44 14.88 15.06 0.17 .53 6.42
6 3.56 14.33 14.58 3.06 .55 6.59
8 2.92 14.59 14.66 1.87 .45 6.29
10 3.37 13.95 13.92 8.26 .52 6.83
13 2.84 14.47 14.40 3.57 .43 6.58
15 2.60 13.96 13.88 8.38 .40 6.30

 

 

 

 

 

 

101

Appendix K (Concluded)
6 x 10"3

 

 

D me 544 m 582 mu 1 gab 2 Mb pH
Group 6: Iron
0 3.61 15.50 15.50 0 .55 6.75
3 3.64 14.20 14.20 5.81 .56 6.38
6 3.51 13.70 13.68 10.06 .54 6.49
8 3.44 13.34 13.08 14.91 .53 6.40
10 3.39 13.21 13.01 15.88 .52 6.48
13 3.43 12.92 12.48 19.58 .52 6.81
15 3.07 12.48 12.12 23.36 .47 6.33
Group 7: Calcium
0 3.65 15.20 15.70 0 .56 6.80
3 3.51 15.10 15.36 0 .54 6.45
6 3.62 14.50 14.78 1.84 .55 6.50
8 3.48 14.57 14.71 1.77 .53 6.41
10 3.69 14.36 14.50 3.73 .56 6.68
13 3.49 14.38 14.56 3.38 .53 6.75
15 3.51 14.39 14.47 3.70 .54 6.51

 

D I Days in storage; HMC I‘Molar myoglobin concentration x 105;
2 Hub I.Average per cent metmyoglobin; 1 Mb I Per cent myoglobin

   

 

102

Appendix L

Data collected from Trial 12: The effects of glucose on meat solutions
stored under 60 ftc. in a 0° F. display case

 

 

 

 

 

é’x 10:31
D me 544 L 582 m 7. ml) 1 Mb pH
Group 1: Control
0 3.37 13.68 13.59 11.03 .52 5.52
3 2.94 12.79 12.35 20.79 .45 5.53
7 3.35 10.93 10.33 38.83 .51 5.78
9 3.28 10.61 10.09 41.48 .50 5.56
15 2.60 10.69 9.88 41.94 .40 5.51
Group 2: One m1. 1% glucose
0 3.39 13.72 13.57 10.91 .52 5.51
3 3.11 12.51 12.09 23.33 .48 5.47
7 2.86 11.33 10.70 35.23 .44 5.64
9 2.96 11.01 10.37 38.25 .45 5.49
15 3.19 10.91 10.22 39.39 .49 5.50
Group 3: One ml. 21 glucose
0 3.29 13.77 13.62 10.44 .50 5.51
3 2.89 12.73 12.32 21.23 .44 5.46
7 2.87 11.97 11.46 28.74 .44 5.63
9 2.77 11.26 10.54 36.24 .42 5.53
15 3.22 10.65 10.06 41.40 .49 5.49

 

D I Days in storage; “NC I Molar myoglobin concentration x 105;
1 Hub I Average per cent metmyoglobin; Z‘Hb I Per cent myoglobin

 

   

103

Appendix M

Data collected from Trial 13: The effect of metal ions on meat solutions
stored under 50 ftc. light in 0° F. display case

 

 

 

 

 

éIx 10:3
D M40 544% 58233; 7. Mb 2 Mb pH
Group 1: Undialyzed control
0 3.90 15.44 15.85 0 .60 5.59
3 3.29 14.22 14.22 5.67 .50 5.71
5 3.24 13.70 13.52 11.22 .50 5.58
7 2.94 13.37 12.99 15.12 .45 5.58
10 2.89 12.87 12.35 20.38 .35 5.62
12 3.31 11.72 11.24 30.95 .51 5.56
14 3.24 11.82 11.30 30.18 .50 5.54
Group 2: Dialyzed control
0 3.78 15.45 15.63 0 .58 5.62
3 3.97 14.63 14.79 1.28 .61 5.89
5 3.98 14.47 14.65 2.54 .61 5.71
7 3.80 14.66 14.68 1.74 .58 5.78
10 3.82 14.29 14.24 5.17 .58 5.84
12 3.82 14.14 14.24 5.95 .58 5.65
14 3.70 14.08 14.08 6.93 .57 5.65
Group 3: Manganese
0 3.74 15.16 15.32 0 .57 5.42
3 3.91 14.60 14.76 1.41 .60 5.77
5 3.76 14.68 14.81 1.20 .58 5.73
7 3.93 14.20 14.20 5.81 .60 5.78
10 3.79 14.06 14.09 7.36 .58 5.83
12 3.88 13.84 13.87 9.05 .59 5.68
14 3.81 13.89 13.78 9.26 .58 5.69
Group 4: Magnesium
0 3.73 15.04 15.31 0 .57 5.41
3 3.74 14.33 14.41 4.26 .57 5.73
5 3.68 14.29 14.29 4.96 .56 5.68
7 3.80 13.79 13.63 10.30 .58 6.02
10 3.65 13.64 13.40 12.03 .56 5.74
12 3.39 13.48 13.30 13.28 .52 5.71
14 3.61 13.66 12.52 15.56 .55 5.66
Group 5: Ferrous (Fe++)
0 3.73 15.07 15.34 0 .57 5.50
3 3.85 14.08 14.08 6.93 .59 5.72
5 3.78 13.76 13.65 10.37 .58 5.51
7 3.79 13.30 12.98 15.53 .58 5.70
10 3.70 13.19 12.78 16.93 .57 5.61
12 3.71 12.72 12.37 21.07 .57 5.63
14 3.53 13.00 12.52 19.00 .54 5.53

 

   

Appendex M.(Conc1uded)

 

 

 

 

e: x 1073
D 11130 544 In; 582% 7. MM!) 7. Mb LE
Group 6: Ferric (Pe+++)
0 3.76 15.60 15.24 0 .58 5.21
3 3.85 14.18 14.18 5.99 .59 5.58
5 3.72 14.01 13.87 8.16 .57 5.43
7 3.77 13.55 13.21 13.28 .58 5.55
10 3.66 13.36 12.98 15.22 .56 5.53
12 3.66 13.09 12.62 18.12 .56 5.49
14 3.58 13.10 12.65 17.94 .55 5.40

 

D I Days in storage; MMC I‘Mo1ar myoglobin concentration x 105}
Z MMb I Average per cent metmyoglobin; 2.Mb I Per cent myoglobin

 

 

 

 

 

105

Appendix M

Data collected from Trial 14 with a Beckman Recording Spectrophotometer
on samples stored under 50 ftc. in a 00 P. display case

6’x 10'5

 

D MC 544m 582m 7. Mb 1 Mb pH
Group 1: Undialyzed control

0 4.62 14.44 14.29 4.08 .71

2 3.57 13.61 13.14 13.25 .55 5.69
4 3.10 12.94 12.35 20.01 .47 5.58
6 3.44 12.59 11.92 23.61 .53 5.41
9 3.27 11.96 11.38 29.12 .50 5.56
11 2.89 11.73 11.07 31.61 .44 4.98
13 3.78 11.30 10.48 36.28 .58 5.63
18 4.31 9.65 8.79 51.86 .66 5.50

Group 2: Dialyzed

0 3.65 15.15 16.38 0 .56

2 3.88 15.15 14.95 0.62 .59 5.67
4 4.24 14.20 14.01 6.59 .65 6.18
6 4.07 14.00 13.59 9.37 .62 6.00
9 4.13 13.68 13.22 12.56 .63 5.65
11 4.04 13.69 13.19 12.64 .62 5.62
13 4.19 13.51 12.89 14.81 .64 5.68
18 4.25 13.44 12.42 17.11 .65 6.03

 

D I Days in storage; MMC I Molar myoglobin concentration x 105;
Z MMb I Average per cent metmyoglobin; 1 Mb I Per cent myoglobin

 

 

 

 

 

 

-v‘.--_..‘.,.._ _

 

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ROOM "<5 ONLY ‘

 

 

 

 

 

 

 

 

   

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