n-w- - o—‘a _o“-—
.-

‘ Vwol
. w'u-Io

" S -f '4’"-
A...

......

. ....

THE ROLE-OF MEGRGORGANISMS‘IiN‘THEEl‘
mama» A‘Nil QXIDATIQNQF mwmgmgwes. if ' f : -‘ 4"

Thais far flu Don‘t» 0H?!» D.
.mcHIGAN STATE umvmsm’. * --
Donald Lewis Robach

19.59

 

d TIE-DID

This is to certify that the

thesis entitled
Phe Adjn of licroorqanisms in the fiedUCtTOH

and Pxidation of Veg Ti~mcnts
presented by

”anald lewis Robach

has been accepted towards fulfillment
of the requirements for

_Ph°_Dsdegree inMiQLQIIinOfi'v 9- Pub, p

/P 4%)) fig: gm
ajor professor

Date MEL

 

LIBRARY

Michigan State
University

 

11:1; 1.0L; 01- L lClthlichlglfiu In ‘41:; itLglLCrl'IL/n
{Lin-b Oxxlljtr;l.'.‘lClk c: mail’i' PlLl.._.i-.lo

Douala Levis hoba ch

3 ‘ r‘ '; «:jr"“w a him
an Bipolhnbl

Submitted to the School ior Advanced braduate Studies of
hichigan State University of Agriculture and
Applied Qcience in partial fulfillment of
the requirements :or the degree of

DOClOlt Cl“ FI'ZILOQOI'IIY

Department of hicrcbiology and Public Health

1959

ass 'l'ri £1. U '1'

an iron porphyrin

heat color is cue to Lyoglotin,

pigment. In the reduced form, the iron or the heme is

in the ferrous state, and the color is gurple. Upon

exposure to oxygen the gignent LacoLes Oiycunated, tut

the iron remains in the ferrous st:te age the color is

bright red. Lnder certain corditions

oxidized to netiziyoglotin, a Lirovm gigment.

3

tortion oi this congourd is in the ferric state. the

protlem oi Ciscoloration then involves the loss of oxyken
to lorm reduced nyoglotin (gurtle) and onidation to form
netKyOLlotin (brown).

Various investigators have bthh tnrt bacteria are
instrumental in the tiscoloration of iresn beef; Losever,

the exact nethod or nethods involved are not lnown.

This investigation has an attempt to elucidate

the

actual role of microorganisns in yignent changes.

Initial studies of a hunter oi species of bacteria

and of a yeast showed that only those organisms which
metabolize aerobically (possess the trek cycle enzymes)
mould bring stout the oxidation of avoglotin to

Igetmyoglohin. Further, only those aerobic orLanisns
which were atle to metabolize at low temperatures were

shown to cause this reaction at refri¢erathitemperatures.

F’J

Plate counts and nanonetric studies with Lonogenates

\ 1' c+ Jr“ (*3 Ava; '
OJ. 5.) bbcruu. Q-A'\_"n‘v . C:

m

of tissue slices from the surfac
direct correlation letueen nu hers Ol nicrooreanisms,

oxygen uptzne rate, and color changes. Various Lacterio-

static agents a;rlied LO steal inhilited Lacterial growth
and resulted in lower WlJLCL uit he rates and yio"onted

color retention; hotever, tne sane correlation hetneen the
n ee ', ”ahles nus a'a': IC'J‘ ‘rsn ext need 'bO a! .
t r vari L“ 19 L 11 ten u‘o e s r Ce
Cell-free extracts grepared L3 sonic oscillation of
heavy cell susyensinns of rseudcncn;s aeru=inosa and

F

i . - ,: P. A” r‘ r a a. . —| ,— P . n -r ., -V- -6 .s ,1: _ ‘ n1. -'- J
F Bhuuuencs &a£lCulcta Were thLu b0 Lllnt chhL tie

U3

oxidation of n oilolin to netnyotlolin on neat suiiaces,

e_ .1

and any substance il‘ililiting oyg en uptane ‘n-v these enzynes
also inhilited gituent oxidation.

Tissue removed erh leef nuscle and glaced in re~
duced oxygen atnOSpheres usin; sterile techni ues unner-
went pignent oxidaticn ;t oxygen tensions from 6 nu to
20 mm (neasured by a nercury nanoneter) titnout the
rresence of bacteria. In an all L2 atmosphere only
reduction occurred. it higher 02 tensions,oxidation
occurred only after tacterial contamination was evident.
lnese data along with experiments involving glucose
oxidase, peroxidase, and specific enzyne inhititors led to
the conclusion that the role or lacteria in neat pigment
tie dissolved oxygen

chantes is sinply that of lowering

I

level in the surface tissue. The level nay be reduced

to the point where natural changes in the neat cause
pitnent oyidation, and further limitation results in

pigment reduction.

TEL LOLL CI nlcnocns Lions in ;1; nLubCllbn
"Ii‘."" -: “r“. t-v~‘x-~ :>\
“in; L’J‘xiurxliUh LI libnl 1' .1111”th 11L)

Ly

Donald Lewis hobach

["1

A lKLSIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

DOCIOh OF PEILOSOPEY

Department of nicrobiolOgy and Public health

1959

r ‘.'I/\‘ :'4'"'".'T rt.
kinda; “LILAJUHI “.414 la

The author is indebted to br. s. n. Costilow,

Associate Professor of Licrotiology and Iublic health,

U)

for his genuine interest, guidance, and uggesticns Cur-
inC this investigation. Dr. dostilow's valuable

criticisns during the .reparation of the nanuscrigt are

k—J

also sincerely appreciated.
Tne author uishes to exrress thanls to
Dr. L. L. Sadoff and Dr. n. l. h;errum for tneir critical

reading of this nanuscri t. the nany Lélprl suggestiOns

l—'
U)
0
on
O
:x
:7)
Fl
(D
C‘.
(1')
D;

of Dr. Sadoff are a
To his wife, Lorothy, tne author wishes to eXpress
his deepest tratitude for the innunurable sacrifices and
encouragements which have made this study possible.
The iinancial assistance of Larch and Company in the
form of a Graduate hesearch Assist ntship is gratefully

acknowledged.

TALnL Cr LChlnnTS

InllfldfijCiIOb. . . . . . . . . . . . . . . . . .
REV 12".; Cl" nI'lLI-té.’i‘UhE . . . . . . . . . . . . .
i Cne istry of nyoglobin . . . . . . . . .
Physiological hole of hyoglotin . . . . .
Reactions of hyoglobin . . . . . . . . .

The Color of Fresh heat . . . . . . . . .

Factors Influencing the Color of Fresh
PrepaCl‘tggeQ Pleat e o a o o o e o o e e 0

Oxygen leision and
Packaging material
Chemicals . . . .
Effect of lacteria

heat Enzymes . . . . .

Tenperature .

lneir anyn

e
EXPLIX Ii @131 All P110 UEULJhE . o . 0
Meat SOche o o o o o o o

it
is

[U R) R-“
(7’3 R) O

LU
H

l‘letllOd Of lint-3.11126 cultures 0 o o o e o o
Intracellular anyne Preparation . . . .
heasurenent of Lacterial Crowth . . . . .

Preparation of Solutions for Pigment

DGtemfliIlEtiOn o o o o o o o o o o e e a .

Method of Piément Determination . . . . .
Spectrophotometric Analyses . . . .

llUIlsell lJiSK COlOI’iKiEtI’y o o o a o e

W1
0')
(m.
(D

REL; ULELID ‘ O O O O C O O O O O O O O C I C O C O O O C 39
The Effects of hicroorcanisms and Cell—Free
EltrECtS on iUc t PiQMChtS o o o o o o o o o o o 39

\O

Qtech Qhrface o o o o o o o o o o o . o o

45’ LA)
[U

Leat 1:01'HCL VILELES o o o o c o o O o o o o o

C:\

l'iUSCle Pi :1 Jrit ultrEC-ts c o c o o o o o o L}

Correlaticn of of Demand ci tLe surface
Tissue of heat and Pigment LLanges . . . . . . 47

Influence of ies piratory grzy‘e InLiLitors on
'ftj I‘v

Fitmext Unar‘ es 5:Lc bestira Activity . . . 51
Steaks Held at nefri aeratec lemgeratures . 51
Steaks Held at Loom Ten 36 erature . . . . . 60

Huscle Pi ment fixtracts field at boom

Temperature . . . . . . . . . . . . . . . . . . 62

heat Homogsnates . . . . . . . . . . . . . . . 66

Leaf Tissue blices . . . . . . . . . . . . . . 70

Effects of £202, Peroxicase, and Glucose

Oxioase . . . . . . . . . . . . . . . . . . . . 72
Effects of Llucose Ogidase . . . . . . . . 7%
Effects of Fe roxidase . . . . . . . . . . 76

Effects of 02 Tensions . . . . . . . . . o . . 79
Steak Surface Pigments . . . . . . . . . . 79
huscle Pigment thracts . . . . . . . . . 93

Effects of Antioxidants . . . . . . . . . . . . 96

Effects of As corLic Acic and of a hiiture of
Ascorbic Acid and bodium Licotinate on the
COlOT Of PrepaCLE£€d steaks c o o o c o o o o o 1C0

uS cusslch . . . . . . . . . . . , , , , , , , , , , 105

o‘Ul-il-iAle .
Alei-LLC .

rusr'isnisn one

LIST OF TALLES AhD FICLhflS

Page
Figure 1. Chemical structure of myoglobin . . . . 6

Figure 2. Effects of intracellular enzymes
and cells of gs. peniculata on pre-
packaged beef held at room temperature. Ml

Table l. The effects of fig. geniculata, Es.
aeruginosa, and a cell-free prepara-
tion of the intracellular enzymes of
these organisms on tLe color of
fresh prepackaged beef . . . . . . . . 43

Table 2. Effects of fig. geniculata and aureo-
mycin on meat homogenates held at
3000. O O O O O O O O I O O O O O O O 0 1+5

 

Table 3. The effects of the intracellular
enzymes and cells of Pa. geniculata
and Es. aeruginosa on the rate of
pigment change of oxymyoelotin
GXtTaCtS o o o o o o o o o o o o o o o #8

 

Figure 3. The effects of fig. geniculata on
prepackaged beef held at 30°C. . . . . 50

 

Figure H. Effects of various levels of
aureomycin applied to the surface of
prepackaged beef held at 4°C. . . . . . 52

Figure 5. Effects of aureomycin on fresh pre-
packaged beef LBld at 40C. 0 o o o o 0 SM

Figure 6. Comparison of effects of sodium
malonate and aureomycin applied to
prepackaged beef held at °C. . . . . . 55

Figure 7. Effects of iodoacetate on inoculated
prepackaged beef steaks held at 4°C.. . 56

Table h. Effects of kCn and Pg. geniculata
inactivated with hCm and Nab3 on fresh
prepackaged beef held at h°c. . . . . . 58

Figure 8a. The effects of iodoacetate and sodium
and 8b. azide on inoculated prepackaged steaks
held at room temperature 0 o o o o o o 63

Table 50

Figure 9.

Figure 10.

Figure ll.

Talle 7.

Table 5.

Table 90

Table 10.

Table 11.

Table 12 o

Lffects of active aId inactivated

cells 01 Ps. penicu la ta on Ado lonin
pigment extracts Leld at room
t81.1p(:retuI‘e o o o o o o o o o o o o o 0

Effects of sodium fluoride and iodo-
acetate on tke respiratory activity of
cells and intracellular enzymes of

EEO firliculillé o o o o o o o o o o o o

Lffects of sodium malonate and sodium
azide on the respiratory activity of
cells and intracellular enzymes of

Q. QIIiC‘ulE‘ta o o o o o o o o o o o o

 

Effects of sodium fluoride and sodium
nalonate on the respiratory activity of
cells and intracellular enzymes of

.Eé’ LED‘LlEelSta o o o o o o o o o o o o

i‘le effects of ICL on the O uptake by
cells of is. L_niculata at various
O};:}"E_‘Url till-11810115 o o o o o o o o o o o o

 

Effects of sodium azide and potassium
cyanide on the respiratory activity
0: tCEf tissue 0 o o o o o o o o o o o

l

Lifects of hydro gen peroxide on fres:
arc aged lyoglobin extracts Leld at
room temperature . . . . . . . . . . .

The effects of Iydrogen peroxide,
glucose 0} :idase, and a contination of
Lot}; on tne surface color of unwrapped
steaks Leld at room temperature . . . .

Effects of active and heat-inactivated
glucose oxidase and peroxidase on
nyoglobin extracts Leld at room
temperEtureoo000000000000

Effects of nitrogen and oxygen
atmospheres on uninoculated prepackaged
beef Leld at H°C. . . . . . . . . . . .

Effects of Litrogen and oxygen
atmospkeres on inoculated prepackaged
beefheldEtL‘roC..ooo00.0000

(‘1)

0"?

61+

67

68

69

71

73

75

50

El

Page

Figure 12. The effects of various oxygen
tensions on the color of pre-
packaged beef held at 4°C. . . . . . . 83

Table 13. The effects of reduced oxygen
atmospheres on sterile excised muscle
tissue held at H00. 0 o o o o o o o o o 90

Table 1%. The effects of reduced oxygen
atmospheres on sterile excised muscle
tissue compared to aseptically handled
Steaks held at 4°C. 0 o o o o o o o o o 92

Table 15. Effects of various oxygen tensions on
oxymyoglobin extracts with and without
the presence of bacteria . . . o . . o 97

Table 16. Effects of ascorbic acid and ascorbic
acid plus sodium nicotinate treatments
on the rate of discoloration and
bacterial growth on steaks . . . . . . 102

Table 17. Effects of ascorbic acid and ascorbic
acid plus sodium nicotinate treatments
on the rate of discoloration and
bacterial growth on steaks . . . . . . 103

Figure 13. Comparison of effects of a miXture
of ascorbic acid and sodium nicotinate
on inoculated and uninoculated pre-
packaged beef held at 4°C. 0 o o o o 0 10h

IhThODUCTION

The discoloration of fresh prepackaged beef items is
of great economic importance. Voegeli (1952) has shown
that almost 26% of the prepackaged meat was removed from
self-service cases due to discoloration. This results in
financial losses due to the labor of reworking the product
which terminates in cheaper cuts as well as weight losses.
Ball gt gl. (1957) stated that shelf life of prepackaged
fresh meat is only slightly over 48 hours and that the
product seldom has a satisfactory appearance for as long
as 72 hours. To lengthen the period of marketability,
color retention must be prolonged.

Discoloration is called "loss of bloom" by the trade.
This means that the meat loses its bright red appearance.
Meat color is due to myoglobin which is a pigment re-
sembling hemoglobin in that it is an iron porphyrin, and
the heme prothetic group is attached to a globin protein
fraction. In the reduced form, the iron of the heme is in
the ferrous state and its color is purple. Upon exposure
to unlimited oxygen the iron remains in the ferrous state
but the pigment is oxygenated. This pigment called oxy-
myoglobin is bright red. The heme portion of myoglobin
may be oxidized by various methods to metmyoglobin, a
brown pigment. The iron portion of this compound is in
the ferric state. The problem of discoloration or "loss

of bloom" involves the loss of oxygen to form reduced

myoglobin (purple) and oxidation to metmyoglobin (brown).

There are three factors to consider when one thinks
of discoloration of meats. The factors are physical,
chemical, and biological. Physical factors include
oxygen tension, temperature, and humidity.

The maximum rate of reduction of myoglobin occurs in
the absence of oxygen, but Brooks (1933) found that max-
imum oxidation occurs at an oxygen pressure of about
H mm Hg. at 0°C. He further found that as the temperature
increased, the rate of the above reactions also increased.
Landrock and Wallace (1955) have observed that packaging
materials which were coated to prevent excessive loss of
moisture resulted in color preservation.

Chemical factors affecting pigment changes include
hydrogen ion concentration, antioxidants (ascorbic acid),
reducing agents, and oxidizing agents. At a pH of 5.5
and above the surface pigment of meat becomes darkened
while at a pH between H.S and 5.h the pigment is a
lighter red color. Various authors have observed that
ascorbic acid in dilute solutions may preserve "bloom,"
but in high concentrations it brings about oxidation and
discoloration. It is known that reducing agents such as
sodium dithionate (Na2820h) reduces all myoglobin
derivatives to reduced myoglobin, and oxidizing agents,
such as potassium ferricyanide (K3Fe(CN)6) oxidize all

myoglobin derivatives to metmyoglobin.

IA third group of factors which must be considered is
the biolOgical. This includes the active meat enzymes
and the effects of bacteria and their enzymes. From the
results obtained by various authors it appears that in
fresh meat the full complement of glycolytic enzymes are
present in active form as well as the Kreb cycle enzymes.
There is little doubt that at least some of these enzymes
are important in color changes. Several investigators have
suspected that bacteria and bacterial enzymes influence the
color of fresh meats; however, the mechanism(s) involved is
not understood. Organisms belonging to the Pseudomonas-

Achromobacter group have been associated with fresh beef

 

discoloration and Butler gt a1. (1953) indicated that these
organisms may bring about discoloration of fresh prepackaged
beef by lowering the available oxygen to a critical pressure
where the formation of metmyoglobin is optimum. The
metabolic formation of H202 by organisms without immediate .
destruction has also been postulated as causing metmyoglobin
formation. It was thought that reSpiring organisms cause
discoloration of fresh prepackaged beef by lowering the
oxygen tension; however, other systems may also be active.
The present study was undertaken to determine the

actual role of bacteria in the discoloration of fresh pre-

packaged beef.

REVIEW OF LITERATURE

A knowledge of the chemistry of the muscle pigment
myoglobin is essential to understand the effects of various
factors on the color of meat. The color of prepackaged
beef is due to the chemical state of pigment myoglobin.
Many interesting theories as to the true character of this
pigment have been set forth. The first theory was that the
red color of meat was due to blood and could be washed
away (Boerhave, 1739). Bichart (1803) agreed, but believed
that the muscle obtained its color from the blood which was
deposited in the tissue rather than from circulating blood.
In the latter part of the nineteenth century much work was
done, but it was not until the early part of the twentieth
century that Gunther (1921) suggested the term myoglobin
for muscle pigments rather than hemoglobin. He was
thoroughly convinced that muscle pigment was not identical
with the blood pigment hemoglobin.

Whipple (1926) employing many extraction techniques,
estimated the myoglobin content of various muscle tissue.
More comprehensive analysis of the occurrence of myoglobin
and its chemical characteristics occurred during the next
few years. It was the contributions of Theorell (1932,
193%) which gave the researchers new grounds for continued
investigation, for he succeeded in crystallizing myoglobin.
Schenk at al. (193%) working with beef rib eye muscle found

no parallelism between the myoglobin content and hemoglobin

L.

content. In more recert gears, research has made arail-
able a more clear understahdihg of the chemical nature
of this pignent and the reactiOLs it uLdergoes in the
muscle.

“\P f“: Y7 \" - .— 1-1 q- .
LLCLlStT‘ g LgoinLin

W

 

Q)

kyoglobin, the principal pigmeht of red heats, is

chromoprotein belonging to the group of hemo-proteihs.

5..

“he arosthetic group is protOporphrin, and the protein

*

fr

(—f.

0 ion is glotin. The prOphyrin family ihcluoes two

03

other very important members, chlorophyll and henoglotin,
both essential for life. Lemberg and Legge (1929) wrote

a book dealing with this family of compounds and cited

3,182 references. The prosthetic group of myoglotin, proto—
phorphrin, consists of four pyrrole rings which are coupled
to a central iron atom through the nitrogen atoms. ”his
then is an iron porphyrin, and it is the iron atom which
makes possible its function as a respiratory pigment; i.e.,
it can form a dissociable conpound with oxygen, the iron
remaining in the ferrous state. According to hendrew (19h9),
the four valences of the iron atom are connected with the
nitrogen of each of the pyrrole rings, and the other two
seem to be bound to the protein component. however,
according to Schwiegert (1956), the fifth valence of the
iron molecule is attached to the imidazole ring of
histidine (Figure 1). This ring is bound to the other four
pyrrole rings through the iron molecule. The protein

5

Globin

 

 

l 5 I

EH2 hQO fag
[H2 fHZ
CP3 CH3

Heme Structure of Myoglobin
Figure ‘
Schweigert, B. 8., 1956. Proc. of the Eig:hth hesearch Conference,
Univ. of Chicago, Chicago, Illinois, P. 61-6H

fraction is attached to histidine and not to the iron
molecule. The sixth valence of the iron molecule is
satisfied by a water molecule, and this seems to be the
active site of the heme structures.

Hemoglobin, the blood oxygen carrier of mammals,
has a molecular weight of 66,000 - 68,000 according to
Haurowitz (1950), and consists of four "units" of the proto-
heme-globin complex. He observed myoglobin to have a
molecular weight of 17,000 and consists essentially of one
of the "units" of hemoglobin, except that the globin or
protein fractions differ.

Myoglobin was isolated, crystallized, and character-
ized by Theorell (1932), and he offered absolute proof that
hemoglobin and myoglobin are two different pigments. This
proved that Kennedy and Whipple (1926) were wrong when they
indicated the pigments were identical compounds. Theorell
(1932, 193%, and 19H7) elaborated the chemistry of myoglobin
showing among other things that the iron content of myo-
globin and hemoglobin was identical, 0.3h5%.

The spectrophotometric studies of Hill (1933) demonstrated
that the sharpest absorption band for myoglobin was 5800 2
while hemoglobin was 5700 2. Under more precise conditions
Hill (1936) found the absorption bands for hemoglobin were
sharpest at 5770 Z and 5420 3, compared to 5815 X and 5HH6 i
for myoglobin, and that myoglobin had a greater affinity for

oxygen than did hemoglobin. He also noted that myoglobin

gave a hyperbolic oxygen dissociation curve compared to
a sigmoid curve for hemoglobin. Millikan (1939) sub-
stantiated the oxygen dissociation curves of the two
pigments as found by Hill, and related the hyperbolic
shape of the curve to the physiological oxygen storing
function of myoglobin.

Shenk, Hall, and King (1934) noted that myoglobin
gave maximum absorption at 582 mp. This was substaniated
by Bowen (19h9) and others. Bowen (19H9) also observed
that myoglobin gave a second absorption peak at Shh mp.

The work of Bowen appeared to be the best of all the
literature reviewed on spectrophotometric analysis of
myoglobin, so his data for extinction coefficients are

used in this study for determinations of percent myoglobin
and percent metmyoglobin. Austin and Drabkin (1935)
published similar information for hemoglobin. Mangel (1951)
in her study of myoglobin used the procedures of Austin and
Drabkin.

The advantages of using metmyoglobin cyanide as a
reference standard for spectrophotometric analysis in
preference to gasometric techniques was demonstrated by
Drabkin and Austin (1935). They gave evidence that the
reagents used for conversion of the pigments to the cyanide
derivative had no significant effect on the absorption spectra

of the derivatives at the critical wave lengths.

Generally, there is some blood residue in meats, Lut
Shenk gt gt. (193%) found that 90% of the meat pigments is
usually myoglobin, and hemoglobin was not usually over 10%.
They also set up a method for estimating the percentage of
these two pigments in a muscle extract by use of the ratio
of the optical density measured at wave lengths of 577 my
and 582 my.

Drabkin gt gt. (1950) studying the distribution of
myoglobin concluded that the concentration of myoglobin is
not correlated with body size but is more concentrated in
the muscles of animals that run fast or work hard such as
the dog or the horse. Lawrie (1950) found that a high con-
centration of myoglobin is usually found in muscles of high
physiological activity. Using the figures of Drabkin gt g1.
(1950), Butler gt g; (1953) calculated the concentration of
myoglobin in the skeletal muscles of the heifer to be about
0.h0 to 0.H7%.

A physical structure for myoglobin was proposed by
Kendrew (19H9). He states that it consists of two disks,

9 1 thick and 57 8 diameter, parallel to each other and
perpendicular to their axis, separated by a layer of liquid
of crystallization 6.6 1 thick. Each of the disks is formed
by one polypeptide chain, folded on itself in four equal
sections. The prosthetic group is perpendicular to the

plane of each disk and extends above and below it. The thick-

ness at either point is about 15 i greater than the disk

9

itself. He obtained a sedimentation constant for myoglobin
of 2.0 x 10"13 in Svedberg's ultra centrifuge.
Myoglobin was first found to have a molecular weight
of 3200 by Theorell (1932). However, other workers have
obtained different molecular weights for myoglobin. Bowen
(1948) in his review states that workers have found the
molecular weight of myoglobin to be from 16,850 by osmotic
pressure to 17,600 by the rule of simple multiples. He
found the molecular weight of myoglobin to be 17,300 and
have an average iron content of 0.323%. Rossi-Fanelli
(1950) states that the iron content is 0.3h%. The isoelectric
point of myoglobin was found to be at pH 6.99 by both authors.
The composition of the globin fraction of myoglobin of
different species of animals has been analyzed by Rossi-
Fanelli (l9h0, iahi, 19h2, 19u7, 19MB, 195%, 1955 and 1956).
He noted that myoglobin has isoleucine but no cystine as
contrasted with hemoglobin which contains cystine but no
isoleucine. Comparing human myoglobin and hemoglobin, (1955),
he noted that myoglobin is richer in glutamic acid, lysine,
glycine, and methionine; poorer in threonine, alanine, valine,
and arginine. In general, the data showed that the proteins
are formed by a significantly different mixture of amino acids.
It was observed by Bossi-Fanelli (1950) that myoglobin
forms a hyperbolic dissociation curve which is only slightly
affected by pH. The pigment has a large affinity for oxygen
and is very easily oxidized to metmyoglobin, and it demon-
strates a large resistance to denaturation by alkali. He

10

also states that hemoglobin has an isoelectric point of
6.78, a molecular weight of 68,000, forms a sigmoid
dissociation curve which is moderately affected by pH, has
a moderate affinity for oxygen, less easily oxidized to
the "met" form than is myoglobin, and has only a small
resistance to denaturation by alkali. Thus, the physio-

chemical constants of the two pigments differ also.

Physiological Role of hyoglobin

The basic facts as to the physiological function of
myoglobin were presented by Theorell (193%) and Hill (1933,
1936). Millikan (1936, 1939) and Biorick (l9h9) have
elaborated these basic facts. Summarizing their findings,
it appears that myoglobin becomes oxygenated at the expense
of the oxyhemoglobin in the peripheral capillaries. The
myoglobin as oxymyoglobin transports the oxygen to the cells
where it is used in oxidative enzymatic activity. Thus
myoglobin acts as an intermediate between the circulatory
system and the actively metabolizing cells. The reduced

myoglobin may then be reoxygenated to complete the cycle.

Reactions of Myoglobin
Since the reactions of hemoglobin and myoglobin have
been found to be similar, the reactions of both will be

discussed.

t al. (1950) hemoglobin is

According to Haurowitz

an "aqui-compound" and forms a coordinate bond with water

11

and not oxygen. He also indicates that oxyhemoglobin does
not dissociate into hemoglobin and oxygen in the absence
of water and postulates that the following equation demon-
strates the true pathway.
Hb.(H2O) + 02 ——9 Hb02 + H20

This may be postulated for myoglobin also.

Much of the early work on the changes of pigments in
meat was performed by Brooks (1929, 1931, 1933, 1935,
1936, 1938, 19H8, and 1955). He demonstrated that myoglobin
was oxidized by oxygen, for in an all nitrogen atmosphere
(essentially free of oxygen) the oxidation of myoglobin to
metmyoglobin did not occur to any appreciable extent. He

postulated the reaction to proceed in the following manner:

Globin N N Globin N l/N Globin N\\ I/N
-0 Oxid.
Histidine-Fe+f-——a—3 Histidine-Fe++ —————a» Histidine-Fe ++

“<0th / \ WEE:- / \

N N N N
Oxymyoglobin (MbO ) Myoglobin (Mb) ietmyoglobin (Mmb)
(bright red) (purple) (brown)

The four nitrogen atoms represent the pyrrole rings which
form porphyrin. The protein fraction is connected through
histidine to the iron molecule. This reaction is essentially
as given by Brooks with formula modifications by Schweigert
(1956).

By potentiometric studies, Conant (1923) showed that
hemoglobin followed the same pathway. This pathway has met
with some disagreement, but the work of George and
Stratmann (1952 a,b) tends to validate it. The reduction

12

of oxymyoglobin to myoglobin or the reverse reaction in-
volves no electron transfer, but the oxidation of myoglobin
to metmyoglobin or the opposite reaction involves electron
transfer. The iron in myoglobin is in the ferrous state,

but the iron in metmyoglobin is in the ferric state.

The Color of Fresh heat

As stated previously some of the early workers
(Boerhave (1739), Bichart (1803)) believed that the red
color of meat was derived from the blood which remained in
the tissue. However, since the work of Gunther (1921) it
is generally accepted that the color of fresh meat is due
to myoglobin. The meat of animals which have been prOperly
bled contains myoglobin (purple), oxymyoglobin (bright red),
and metmyoglobin (brown). The color of the meat depends
upon the relative amount of each of these three compounds
present, which depends upon the storage conditions of the
meat (Rickert, 1957 a).

Brooks (1933) and Winkler (1936 b) have observed that
the discoloration of fresh meat was caused in general by
two factors; viz., desiccation and oxidation of myoglobin
to metmyoglobin. The darkening effect due to drying was
caused primarily by a concentration of pigments at the sur-
face, with the optical properties of the desiccated tissue
perhaps being altered. If the meat is prepackaged and
moisture loss is minimized, the reaction which would account
for the primary changes in the meat color would then be
myoglobin oxidation.

13

The desired color of fresh prepackaged meat is the
bright red of oxygenated myoglobin (Mb02). Freshly cut
meat when first exposed to air is a shade of purple, and
the myoglobin is primarily in the reduced state (Mb). Upon
exposure to air, oxygenation occurs, and as the myoglobin
is changed to oxymyoglobin, the meat becomes a bright red.
Mackintosh and Hall (1936), Allen (19MB) and Bratzler
(1955) pointed out that oxygenation or "blooming" of meat
occurs very rapidly within the first 30 minutes after

cutting and is accelerated by reducing the temperature.

Factors Influencing the Color of Fresh Prepackaged Meat

Oxygen tension and temperature: The papers by Brooks
(1929, 1931, 1933, 1935, 1938) go to great lengths in an
effort to discover and explain the factors which influence
the reactions of myoglobin. He noted that both the
oxygen tension and temperature are very important factors.
Temperature is of importance because as it is increased the
reaction rate is increased and also the oxygen becomes less
soluble, and consequently the partial pressure of oxygen
in the meat decreases. To cite a specific example, Brooks
(1931) noted the rate constant for Hb __) MHb to be about
four times as great at 25°C. as at 15°C. Brooks (1933)
demonstrated that at 30°F. oxyhemoglobin dissociates
rapidly to hemoglobin when the oxygen pressure drops below

80 mm of mercury. However, at this reduced oxygen tension

in

the hemoglobin is soon oxidized to methemoglobin. He also
observed that metmyoglobin formation proceeds more rapidly
at low oxygen tensions. heill and Hastings (1925 a,b)

have obtained similar results using hemoglobin solutions.
They observed maximum oxidation to take place at an oxygen
tension of about 20 mm of mercury. Brooks (1938) discovered
that the maximum rate of oxidation of Hb occurred at an
oxygen pressure of about 4 mm Hg. at 0°C. This estimate by
Brooks was made by noting the rate of color change in a
tissue section by use of a microspectrosc0pe.

George and Stratmann (1952 b) employing a more refined
gasometric technique observed that the maximum rate of
metmyoglobin formation occurred between 1.0 and 1.h mm
oxygen pressure at 30°C. The fact that the rate of oxida-
tion of myoglobin was h.25 times faster than that of hemo-
globin was also noted by these authors. Brooks (1931)
suggested that at a constant oxygen pressure the rate of
oxyhemoglobin formation is monomolecular with respect to
hemoglobin concentration. Neill (1925 c) noted that no
oxidizing agent that he used could oxidize hemoglobin to
methemoglobin in the absence of molecular oxygen. Both
Brooks (1931) and Neill (1925 d) noted that hemoglobin was
not oxidized to methemoglobin in the absence of molecular
oxygen. From the data of Brooks (1929, 1931) it is evident
that at a temperature of 30°C. and an oxygen pressure of

20 mm Hg, the oxygen concentration is low enough to permit

15

the dissociation of oxyhemoglobin into hemoglobin and yet high
enough for oxidation of hemoglobin to methemoglobin. Neill
(1925 d) demonstrated further proof of the effect of oxygen
tension on hemoglobin when he found that in a pure oxygen
atmosphere the concentration of oxyhemoglobin increased and
therefore the formation of methemoglobin was retarded. hangel
(1951) stored frozen meat samples under atmospheres of nitrogen,
oxygen, and carbon dioxide, with air as a control. She found
no significant difference. However, the metmyoglobin formation
tended to be slower when the samples were stored under oxygen.

Brooks (1938) found that muscle retains a residue of
respiratory enzymes long after slaughter of the animal, and
when exposed to air a steady state is reached where the depth
to which the oxygen penetrates is determined by the relative
rates of its diffusion and uptake.

The following formula for determining the oxygen

penetration has been given by Brooks (1938):

 

d= Facd D

\l A

where d = depth of oxygen penetration
Co = pressure of oxygen at the surface of tissue
D = coefficient of diffusion of oxygen through tissues
A = oxygen consumption

The value of A for the muscle of fresh beef as reported by
Brooks (1929, 1936) is roughly 10-h cc per gram per minute at

0°C.

Brooks (1935) found the depth of oxygen penetration
increased slowly with time, and it decreases with increasing
temperature. He found that the depth of oxygen penetration
varied from 2 to 5 mm. He also showed that discoloration
was confined to the layers where oxygen was present, and
metmyoglobin was formed rapidly in the inner surface of the
oxymyoglobin layer where oxygen pressure was lowest. The
myoglobin in the interior portion of the muscle remained in
the reduced form, unchanged. It was estimated by Brooks
(1938) that the surface color was affected by about a 2 mm
layer of tissue, for no light was reflected from layers
deeper than this.

The effect of temperature and vacuum on fresh meat was
tested by Rickert 23 al. (1957 c). They found that meat
stored under a vacuum of 20 inches or more underwent an
initial loss of redness and returned to redness more rapidly
than meat stored under a vacuum of less than 20 inches. In-
creasing the vacuum under constant storage temperature or
increasing the storage temperature while maintaining a con-
stant vacuum increased the rate of initial darkening and
shortened the time for the subsequent return to redness.
They state that vacuum appears to be necessary for the

return to redness, but not for the initial darkening. A

lower vacuum resulted in the formation of a gray color which

turned red upon increased vacuum.

17

Rickert g3 g1. (1958) working with atmospheres
containing various amounts of 02 and N2, noted that in-
creased N2 caused an increased rate of discoloration of
fresh beef. However, the return to redness was faster than
at lower N2 tensions and higher 02 tensions. Further, they
noted that low pressures of CO2 proved to be more effective
in maintaining good color, from both the standpoint of re-
tarding initial discoloration and from the standpoint of
holding color thereafter.

Packaging Material: Various wrapping materials were
tested by Lavers (19MB). He found that as oxygen
permeability of the material decreased, metmyoglobin forma-
tion increased. He noted that MSAT cellophane, which is
oxygen impermeable, caused rapid browning of the meat. LSADT
cellophane, which is oxygen permeable, caused less dis-
coloration. The rate of discoloration could be lessened
even more by moistening the MSADT cellophane which increases
oxygen permeability. He hypothesized that the development
of an oxygen permeable transparent sheet which was moisture
proof would solve the problem. Landrock and Wallace (1955)
performed a detailed study on the relationship of the oxygen
permeability of films and discoloration of fresh red meat.
They state that cellophanes used for packing fresh meat are
unique in that they are coated with a moisture-proof coating

on the side to be kept away from the meat. The inner side

18

then becomes wetted by the meat juice and consequently

increases oxygen permeability sufficiently to maintain

bloom. The coating prevents excessive loss of moisture,

thus, minimizing desiccation. The great oxygen permeability

of MSAT 80 cellophane paralleled the increased bright red

color of the meat as compared to other less permeable

wrapping material. They postulated that the minimum oxygen
permeability requirement is about 5000 ml 02/sq. met./2H hr/atm.
at 75°F.

Vinylidene copolymer (cry-o-vac) and hSAT 80 cellophane
(coated side out) were found to be superior to all other
wraps tested in preserving the flavor and organoleptic
quality of fresh meat by Clauss gt gl. (1957). However, no
color observatidns were recorded.

Reflectance spectrophotometry was employed by Pirko
and Ayres (1957) to study the influence of different
packaging materials on the discoloration of fresh meat.

They observed that films with highest gas permeability (300
hSAT 80-cellophane, polyethylene 0.0015 inches and 80 FM-l
pliofilm) resulted in maximum metmyoglobin formation on

the 6th day of storage. As permeability decreased, the rate
of metmyoglobin formation increased; however, after one day's
storage, the total amount of metmyoglobin was reduced. They
consider films of high oxygen permeability to be inferior to
films of very low oxygen permeability. It appears that they

wish to sacrifice the bright red color of oxymyoglobin for

19

the purple color of myoglobin to obtain an increased sales
life.

Vinylidene copolymer (cry-o-vac) was found to be the
best of the various packaging materials used for maintain-

t 810 (1957 a).

ing good color in lean ground beef by Hickert
They determined redness by the Hunter color and color-
difference meter. The author doubts if these authors made
a distinction between the briiht redness of oxymyoglobin
and the purple-redness of myoglobin.

Rickert 23 £1. (1958) substantiated the results of
Rickert _§ g1. (1957 a). They explain that hSAT 80 cello-
phane and cellulose acetate both of which have high gas
permeability resulted in high redness of the packaged meat
for 2 days, then discoloration followed. Steaks wrapped
in cellophane-pliofilm laminate film which has low oxygen
transmission prOperties were higher in redness than steaks
wrapped in the highly permeable films except for the first
few days.

Rickert gt g1. (1957 a, 1958) and Pirko and Ayres
(1957) believe that good color of fresh beef could be
maintained by packing it in a completely impermeable film

in the absence of oxygen.

Chemicals: The chemistry of the red meat color change

 

having been fairly well characterized, many workers began
trying to influence the change. Winkler (1939 b) studied

the effects of lowering and raising the pH of meat. He

20

found that a pH above 5.5 caused darkening and a pH

between H.5 - 5.5 caused lighter colors. This agrees with

the findings of Hall, Latscher, and Mackintosh (194%) work-
ing with dark cutting beef. However, they believe that the
main reason for dark cutters is that the oxygen demand is
greater than can be supplied by normal transfer into the
tissues, resulting in oxymyoglobin being reduced to

myoglobin. The fact that muscle tissue darkens with in-
creased pH was noted by Hill (l928) and Bate-Smith (l9h8 a,b).

Some chemicals have been shown to effect the oxidation
of myoglobin to metmyoglobin. Stilles and Foster (1922),
Brooks (1930, 1931, 1936) were among the first to observe
that salt increased the rate of oxidation of oxymyoglobin
and oxyhemoglobin, and/or myoglobin and hemoglobin to the
brown metmyOglobin and methemoglobin respectively.

Ascorbic acid has been shown to bring about the rapid
oxidation of hemoglobin solutions (Chang and Watts, l9h9)
and reduce methemoglobin to hemoglobin (Gibson, 19h3).
However, Watts and Lehman (1952 a) state that ascorbic acid
protects hemoglobin solutions when it is added in low con-
centrations and at a low temperature. But at high concen-
trations and high temperatures, it brings about oxidation
and discoloration. In another paper (1952 b) they state
that ascorbic acid may be useful in preserving the red

color at the surface of refrigerated prepackaged meat.

21

These results have been confirmed by Costilow gt a1.
(1955). Studying the degradation of hemoglobin in living
tissue, Garner, Mills and Christman (1951) concluded that
ascorbic acid catalyzes the degradation of hemoglobin to
choleglobin. This has been substantiated by Lemberg and
Legge (19M9), Chang and hatts (19H9), and Watts and

Lehmann (1952). hicotinic acid has been reported by
Coleman (1951) to transform the interior color of meat from
purple to a bright red.

Studying the effects of ascorbic acid on meat color,
Clauss at £1. (1957) noted that when ascorbic acid crystals
were added to beef samples they caused an immediate darken-
ing which remained throughout the storage period. The
addition of 0.005% solution of ascorbic acid had better
color preserving properties than did a 0.01% solution.
These results were substantiated by Rickert 23 a1. (1957 b).
They also noted that hDGA (Nordehydroguaiaretic acid -
0.05%) had no beneficial effect on surface meat color.

Broumand at al. (1958) in their study of myoglobin
reported that sodium dithionite (Na2820g) reduces all
myoglobin derivatives to myoglobin, and potassium ferri-
cyanide (K3Fe(Ch)6) oxidizes all myoglobin derivatives
present in meat extracts to metmyoglobin.

Effect 9: bacteria and their enzymes: The influence
of bacteria on the color of fresh meats has been
demonstrated by several investigators, but their role in

22

discoloration has not been clearly defined. Neill (192 a)
and Neill and Hastings (1925) have observed that washed

Pneumococcus cells do not have the ability to reduce

 

methemoglobin to hemoglobin unless other substances such
as glucose or meat infusion are added to the system, but
the cells have oxidizing ability without the addition of
these substances. They also noted that cell-free extracts

of Pneumococcus had both the ability to oxidize and reduce

 

hemoglobin solutions. Another series of experiments per-
formed by heill (1925 c) demonstrated that anaerobic bacilli
brought about the oxygen dissociation of oxyhemoglobin.
Urbain and Greenwood (l9h0) compared dilute hemoglobin
solutions treated with toluene to inhibit bacterial growth
to untreated controls. Loth sets of tubes held at 10°C were
shaken daily for maximum aeration. They noted after 60
days' storage the bacteria-free tubes contained no methe-
moglobin, but contaminated (control) tubes showed reduced
oxygen capacity after two weeks, which was further reduced
to about 50% at 60 days. The loss of oxygen capacity
coupled with color change of the solution (turned brownish)
was proof of methemoglobin formation. The authors believe
microorganisms have similar effects on the sensitive heme
pigments of meat.

Jensen (19H5) was of the opinion that microorganisms
both living and dead, as well as their enzymes may oxidize

myoglobin to metmyoglobin. He further stated that desicca-

tion intensified this reaction. Allen (1948) stated that

23

there is a type of meat discoloration where a gray brown
color develops when the coloring matter is destroyed by
bacteria. Further work by Allen (19H9) showed that
bacterial growth on prepackaged boneless round steaks
held at 3H° - H0°F. increased rapidly uhtil the ninth day,
after which it remained essentially unchanged.

Further evidence of the oxidation-reduction
capabilities of bacteria was presented by Eddy gt al. (1952)
when they demonstrated that Escherichia coli had the ability
to reduce dehydro-ascorbic acid to ascorbic acid. This
substantiated the work of Hewitt (1950). Hewitt noted that
generally the cultures of bacteria caused reducing con-
ditions which were greatest during the logarithmic growth
phase. He noted the lowest O/R potentials when metabolic
activity was the greatest. He further explained that once
growth is established in a culture, oxygen donators and
hydrogen acceptors are taken up by the cells as they carry
on their normal metabolic activities. After the log growth
phase, metabolic activity decreases and as the oxygen from
the air diffuses back into the culture, the O/R potential
begins to rise.

Kraft and Ayres (1952) noted that meat samples held
at H0°F. generally discolored before any other evidence of
spoilage became apparent. They employed surface swabbing
techniques and demonstrated that when the count reached

about 2 x 106 organisms per square cm. the first detectable

2H

off odor appeared. They concluded that discoloration
could not be correlated with organoleptic or microbio-
logical evaluations as a means of determining storage
end points.

Initial contamination as well as the temperature of
the self-service case have a great influence on storage
life of prepackaged meat (Ayres, 1951 a,b). Short steaks
could be stored four days longer when the bacterial count
was very low. Initial counts on meat cuts prepared in
accordance with good sanitary practices had far lower bac-
terial counts than similar items purchased from local
retail outlets. He urged that studies which not only
determine the number of organisms present, but also their
contributions to its ultimate spoilage, be made.

Achromobacter-Pseudomonas types of organisms were
associated with spoilage of fresh beef by Ayres (1951 a).
He stated that since the revision of Bergey's Manual, some
of the strains previously reported as Achromobacter would
be classified as members of the genus Pseudomonas. The
majority of strains isolated from spoiled ground beef
belonged to this group. At spoilage time in one test, more
than 98% of the flora belonged to the Achromobacter-

Pseudomonas group. He used off-odors as the principal

 

criterion for spoilage detection, and observed it corre-

lated with slime production and increase in 002 production.

25

The Achromobacter-Pseudomonas type accounted for
about 85» of the population during the early storage of
meat (first 2 weeks) at 3H-38° and H0—50CF. (Halleck
et a 1958). During the latter part of storage
Pseudomonas fluorescens type organisms constitute approx-
imately 80% of the total count. During the entire storage
period lactobacilli accounted for about 5% of the total
population.

A slippery condition on poultry due to spore-forming
capsulated bacilli closely resembling Bacillus mesentericus
was described by Hallman (1932). Sulzbacher (1952 a) com-
pared the generation time for Pseudomonas-type organisms
in ground beef and ground pork held at 7°C. A rather heavy
inoculum was added to the meat before grinding. He noted
the generation time in ground beef to be h.5 hours and
H.81 and 5.99 hours in ground pork. hirsch gt g1. (1952)
working with commercial hamburger found initial counts
varied from 1.H x 106 to 9.5 x 106 per gram. Upon sub-
sequent storage (0-2°C.) the organisms multiplied rapidly
so that after 6 days' storage 5 x 108 or more organisms
were present, and after 8 to 12 days off odors were
apparent. Maximum counts ranged from 5 x 108 to 1 x 1010
per gram. In general the off odor was nonputrefactive but
typically stale and sour. A taxonomic study of the
isolates demonstrated that the great majority belong to

the Pseudomonas genus.

26

It has been reported by Butler at £1. (1953) that
bacteria commonly found on meat cuts caused discoloration
due to increased rate of metmyoglobin formation and caused
the production of off odors and slime formation. They
state that the changes usually happen in the order
mentioned. Shelf life of prepackaged meat was signifi-
cantly prolonged by lower initial bacterial contamination,
and by reducing the storage temperature. They indicated

that the organisms (Pseudomonas sp.) may bring about dis-

 

coloration by lowering the oxygen to a critical pressure
where metmyoglobin formation is ideal. The metabolizing
organisms lowered the oxygen tension still further which
resulted in reducing conditions and the metmyoglobin was
reduced to myoglobin.

Myoglobin solutions held at 45°F. were studied by
Broumand g3 g1 (1958). They noted that nonsterile water
extracts contained 100% metmyoglobin within 8 hours while
the sterile extract did not become 100% metmyoglobin until
about 28 hours. All solutions were in hermetically sealed
cuvettes. After 28 hours storage at 48°F. the cuvettes
were stored at 95°F. for an additional 148 hours. Within
60 hours the nonsterile cuvette contained all myoglobin,
but it took 120 hours for the sterile extract to become
entirely reduced. They used Spectrophotometric analysis
to calculate the amount of various pigments. Their con-

clusion was similar to that proposed by Butler t g1.

(1953); i.e., microorganisms increased the rate of
formation of metmyoglobin and upon subsequent holding

increase the rate of myoglobin formation.

ME AT El 21?,iES

 

Lemberg and Legge (1949) state that the metabolic
formation of H202 without immediate destruction, leads to
metmyoglobin formation. However, Brooks (1938), Lavers
(1948) and Urbain (1951) believed that the mere lowering
of oxygen tension, without peroxide formation will lead
to the same results.

Employing manometric techniques, Schneider and
Potter (1943) demonstrated the presence of cytochrome
oxidase in beef muscle. They also proved the presence of
succinic dehydrogenase by methylene blue reduction.
According to Bate-Smith (1942) beef contains about 0.005%
cytochrome-C which is about 0.4 micromoles per 100 grams.
Grant (1955 a) substantiated the results of Schneider and
Potter (1943). He found high alpha-glycerophosphate
dehydrogenase activity in steer muscle, but it was absent
in cow muscle. Using various enzyme inhibitors in con-
junction with frozen ground beef, he noted that only
malonic acid of the enzyme inhibitors used provided a
bright red interior color. This indicates that succinic
dehydrogenase is active in beef. He further found by

the use of inhibitors that the following enzymes may show

a limited activity, phosphoglucomutase, phOSphatase,
enolase, lactic dehydrogenase, malic dehydrogenase, Co-A
system and enzymes converting citrate to oxalosuccinate,
citrate to malate, alpha-ketoglutarate to succinyl Co-A,

and succinyl Co-A to succinate. He presumed L-glutamic

acid oxidase, catalase, phosphorylase, and glucose,

fructose and formate dehydrogenases to be inactive in frozen
ground beef.

In another study Grant (1955 b) inspected the stability
of succinoxidase (succinic dehydrogenase plus cytochrome
oxidase) in beef tissue. {e found that the enzyme is
better preserved if the tissue is frozen, is more stable
in ground as compared to unground muscle, and is readily
destroyed by heat.

Andrews 33 gl. (1952) demonstrated that
adenosinetriphosphate, succinic dehydrogenase and glycolytic
system enzymes were active in beef muscle, (Longissimus

gorsi and Semitendinous). The enzymes within the meat

 

 

remained active after 4 weeks' storage at 2°C. and only
aldolase was found to lose part of its activity. After
four weeks its activity was but half that of the original.
The aldolase system was shown, therefore, not to be the
limiting enzyme in the glycolytic system, since the
stabilities varied independently. They concluded that the

lack of available substrates in the intact muscle tissue

29

is the limiting factor in excised muscle tissue metabolism,
rather than the instability of specific enzyme systems.

The enzymatic activities of cytochrome oxidase and
succinic-oxidase in various excised muscle tissues from
a variety of animals were shown by Lawrie (1953). He also
found correlation between the percentage of myoglobin in
the muscle and the activity of the corresponding enzyme
preparation and both were related to the extent of muscle

exertion.

PLOCEDURE

Steaks used in this study were from the Longissimus
ggggi muscle of U. S. Good grade beef ribs. The muscle was
removed from the ribs and cut into l/2 inch thick steaks.

The wrapping material employed was DuPont cellophane
300 MSAT-80 which is for fresh meat packaging.

Cells of fig. geniculata were grown in nutrient broth

 

shake cultures or on nutrient agar slants. All cells were
washed in saline before being inoculated on steak surfaces
by the use of sterile atomizers. Washed cells were used
for intracellular enzyme preparations also. Cells were
broken in a haytheon, 50 watt, 90 kilocycle sonic oscillator
type B-22-3. The suspension was centrifuged to remove
cellular debris, and the supernatant was applied to steak
surfaces by means of a small brush.

The various enzyme and bacterial inhibitors which
were used on the steak surface were applied by sterile
atomizers.

Most myoglobin solutions were prepared from Longissimus

dors; muscle; however, some solutions were prepared from

 

beef trimmings. The myoglobin solutions were prepared
exactly as the pigment solutions used for spectro-
photometric analysis. This procedure which consists of
making a water extract of the meat tissue will be discussed

in detail later.

31

Initial studies determining bacterial growth on
steak surfaces demonstrated that a surface slicing
technique gave more reproducible results than did the
conventional swabbing technique. The surface slicing
technique consisted of cutting a 3 mm slice from the
surface of the experimental steak with a Hobart 50
slicing machine. The machine was cleaned and sterilized
before each cutting as follows: washed thoroughly with
hot detergent, rinsed with distilled H20, followed by
hypochlorite (KOCL, 500 ppm), finally rinsed with sterile
distilled water, and the rotating blade was allowed to
spin dry. Swabbing the machine at various times demonstrated
that the portion which came in contact with the meat slice
was essentially free from bacteria.

The thin slice from the surface was placed on MSAT 80
wrapping paper, weighed and placed in a sterile, chilled
Waring Blender. Refrigerated sterile distilled water was
added to give a 1:5 dilution. After blending for 30 seconds,
a sample of the homogenate was plated out in tryptone
glucose extract agar (TGE) (Difco) and observed for
bacterial growth. The rest of the homogenate was used for
determining oxygen uptake by the Warburg method and for
determining the pigments present by spectrOphotometric
analysis.

Two incubation temperatures for the TGE agar plates

were tested, and it was noted that similar results were

32

obtained from platings held at 4°C. for one week and at
20°C. for three days. For the sake of convenience the

shorter time, higher temperature of incubation was used
for all bacterial counts.

Refrigerated neat used in this study was stored in

a walk-in cooler at 4°C. (i 1°C). Shelf life was noted
to be similar to that obtained in a household type
refrigerator.

A sample of the homogenate was prepared for pigment

determinations as follows:

a. The suspension was centrifuged in a Servall
SPX centrifuge at full speed for 10 minutes.

b. The supernatant was filtered through Whatman
No. 40 filter paper.

0. The filtrate was diluted using 7 ml. of
filtrate to 3 ml of distilled water.

d. The diluted material was then subjected to spec-
trophotometric analysis. A Beckman DU with blue
filter and a slit width of 0.1 mm was employed.

For some Spectrophotometric analysis a Bausch and

Lomb spectronic 20 colorimeter was used employing optically
matched Thunburg tubes rather than cuvettes. For this
procedure the filtrate obtained in b. above was not further
diluted.

Two methods of analysis have been tried based on the

Lambert-beers Law which follows:

33

Log To = A a a b c (1)

i-
where Io = intensity of the incident light

I = intensity of the emergent light

A = absorbency or optical density from
the spectrophotometer

a = molar extinction coefficient

b = length of light path through the
solution in cm.

0 = molar concentration

c ==,_A_ when b = 1.0 cm (2)

a
The first method was essentially that of Butler gt al.

(1953). The optical density was measured in a Beckman
Model DU spectrophotometer at wave lengths of 544 and S82
millimicrons. The total pigment concentration was obtained
by conversion of the pigments to the CN derivative by
adding a drop of 4% solution of potassium ferricyanide

and a drop of 13 potassium cyanide to each cuvette after
readings were obtained. The resultant metmyoglobin cyanide
derivative was measured to 544 millimicrons and the
millimolar extinction coefficient of 11.3 given by Bowen
(1949) for metmyoglobin cyanide was used to calculate the
concentration by the following formula:

A
11.3 x 103 (3)

To estimate the percent metmyoglobin, the difference

between the extinction coefficients of oxymyoglobin and

34

metmyoglobin at 544 and 552 millimicrons was taken as

the maximum possible change at these wave lengths. These
figures were 9.6 at 544-mu.and 12.1 at 582 mH_as given by
Bowen (1949). At each wave length, the change due to
metmyoglobin was equal to the molar extinction coefficient
(a) of oxymyoglobin minus that of the sample. By dividing
the figure obtained by the total change in (A) from
oxymyoglobin and metmyoglobin and multiplying by 100, an
estimation of the percent myoglobin was obtained. The
estimations of the percent metmyoglobin at 544 mfiland 58219&
were averaged to obtain the final estimate of the percent
metmyoglobin.

The second method of analysis used was that of
Broumand (1956) which is as follows. According to Bowen
(1949) the millimolar extinction coefficient for
oxymyoglobin and myoglobin are the same (5.6) at a wave
length of 507 mewhile that of metmyoglobin is 9.9 at this
wamelength. At a wave length of 573 QR.b0th oxymyoglobin
and myoglobin have a millimolar extinction coefficient of
10.6 while that for metmyoglobin at this wave length is 3.0.
Theoretically, at these two wave lengths, if the percent
metmyoglobin remains constant, so does the millimolar
extinction coefficient of the solution; that is, at these
wave lengths, the millimolar extinction coefficients of

the extract are independent of the relative percent of

35

myoglobin and oxymyoglobin as long as their combined
percentage is constant. Therefore, at 507 my, if the
millimolar extinction coefficient (E mm) is 9.9, then the
extract is 100» metmyoglobin and 0; oxymyoilobin and
myoglobin or if at 507 my the E mm is 5.6, then the
extract is Oh metmyoglobin and 100p oxymyoglobin and
myoglobin. If E mm at 573 mp is 3.0, then the extract
contains 100» metmyoglobin and 0% oxymyoglobin and
myoglobin or if the E mm is 10.6, then the extract is 0%
metmyoglobin and 100$ oxymyoglobin and myoglobin. If E mm
is greater than 9.9 or less than 5.6, another pigment must
be present, but if E mm is equal or greater than 5.6 and
equal to or less than 9.9, then the extract contains X%
metmyoglobin and lOO-Xfi oxymyoglobin and myoglobin.

A graph for the determination of k metmyoglobin and
the p of the combined pigments, oxymyoglobin and myoglobin,

can be constructed. Value of a)_at 502 mu_of a
a) at 573 mp

solution containing 100% myoglobin and oxymyoglobin and

0% metmyoglobin is (100 x 5,6) + (0 x 9391-5,6 = 528
(100 X 10.6) + (O x 3) 10.6 °

A solution containing 75% (oxymyoglobin and myoglobin) and
25% metmyoglobin would equal:

Lea) at 502mu=fl5x 5.6) +(25x 9.91_ 665: w
(a) at 573nm (75x10.6)+(25x3) -gf-7g .767

36

50% (oxymyoglobin and myoglobin) and 50p metmyoglobin

£50 x 5.6) + (50 x 9.9) _ . _
30 x 10.67 + (50 x 3) - gtgg - 1°1“O

 

25% (oxymyoglobin and myoglobin) 75% metmyoglobin

(25 X 5.6) + (75 x 9.9)_ 8.82 _
(25 x 10.6) + (75 x 3.0)‘ 5T9'5 - 1.801

 

0% (oxymyoglobin and myoglobin) 100% metmyoglobin

(0 x 5.6) + (100 x 9.9)

T 9.9 _ 3 3
(O x 10.6) + (100 X 3.0) °

3.0 _

Results of the above calculations for all percentages of
metmyoglobin from 0 to 100 can be put into graph form in

which (a) at_507mu is plotted versus relative concen-

(a) at 5731wx
trations of metmyoglobin and oxymyoglobin plus myoglobin.
Thus, by finding the ratio of absorbancy (O.D.) at

507 mu and 573 mH|(0.D, at 507 mu) and referring to the
(0.3. at 573 mp)

graph, the relative percent of metmyoglobin can be
obtained.

Bowen (19H9) found that the E mm for metmyoglobin
and oxymyoglobin are both 7.6 at a wave length of h73xmr
and the E mm of myoglobin is H.H; also, the E mm of
metmyoglobin and oxymyoglobin are both 3.0 at a wave
length of 597rmranmitiet of myoglobin is 5.1. By follow-

ing the procedure as outlined just previously, a second

 

a) at M73 mg:is plotted
a) at 5971m&

graph can be constructed in which (
(

versus relative concentrations of myoglobin and metmyoglobin

plus oxymyoglobin.

37

From these two graphs the relative percentages of
metmyoglobin and myOglobin can be determined. Presuming
not more than 3 pigments are present, the percent of
oxymyoglobin can be determined by adding the percent
‘myoglobin and percent metmyoglobin and substracting
from 100.

Voegeli (1952) has given detailed instructions for
the measurement of the surface color of meats by the use
of Munsell spinning disks. The same procedure and similar
equipment was used in this study.

The standard color employed for calculations of the
index of fading (I) by the formula of Nickerson (l9h6),
as applied by Voegeli (1952) was 7.0 red in hue, %.0 in
value, and 8.0 in chroma. The standard selected for the
plotting of color readings of the fresh prepackaged meat
was the color of steaks which had been oxygenated under
an oxygen pressure of 30 pounds per square inch for one
hour. This color was believed by Eutler (1953) to approx-
imate closely the color of fully oxygenated myoglobin. It
was then possible to find the position of subsequent
sample readings in relation to the standard by using the
Nickerson (l9h6) formula. Thus, as the steaks discolor,
less pigment is in the oxygenated form, and the index of
fading (I) increases in numerical value. Lower values
for the index of fading indicates that the steaks have a
color more close to the standard steak which was presumed
to have its surface pigment fully oxygenated.

38

RESULTS

The Effects 9f Microorganisms and Cell-Free Extracts on
heat Pigments

 

 

Steak Surface
Pure cultures of Pseudomonas fluorescens, Pseudomonas
aeruginosa, Archromobacter liquefacians, Flavobacterium

rhenanus, Lactobacillus plantarum, Saccharomyces cerevisiae

 

and two strains of Pseudomonas geniculata were tested for

 

their effect on the surface color of wrapped beef steaks at
room temperature. All the aerobic strains of bacteria
tested, including three untyped strains isolated from dis—
colored meat, caused rapid discoloration as did the one
strain of yeast. These bacteria are active in meat dis-
coloration at refrigerated temperatures also. L. plantarum,
a strain which is microaerophilic and does not contain the
Kreb cycle enzyme system, caused no discoloration.

In all subsequent experiments Es. geniculata was used
since it was shown to cause discoloration of fresh pre-
packaged meat by Butler gt g1. (1953). Various levels of

inoculum of fig. geniculata were tested for effects on pre-

 

packaged meat at H9O. Data demonstrated that as the
bacteria level increased, so did the rate of discoloration.
However, the steaks which discolored most rapidly became
the purple color of myoglobin with only little evidence of
metmyoglobin formation. As the initial level of organisms

decreased, so did the rate of discoloration, but these

39

steaks became brown before turning purple. In all cases
the initially bright red steaks became dark red before
turning purple or brown.

The effects of intra- and extracellular enzymes

 

of a strain of fig. geniculata on the pigmeht of beef steaks
were studied. At refrigeration temperature up to nine days
there was little or no difference between control steaks
and steaks treated with an extracellular enzyme prepara-
tion, but those treated with an intracellular enzyme
preparation darkened slightly. Initially, the steaks
treated with the intracellular enzymes and cells were
slightly darker, but at the end of two days only the
inoculated steak had discolored completely. A preliminary
experiment using the above four trea ments conducted at
room temperature (23-2H°C.) demonstrated that after 15
minutes the inoculated steaks were visually darker, and within
one hour those treated with intracellular enzymes were also
darker. Both the inoculated steaks and the steaks treated
with the intracellular enzyme preparation turned a definite
purple after 2 hours. At 2% hours the control steaks and
steaks treated with extracellular enzymes began to turn
brown. The inoculated steaks remained purple, but the
steaks treated with intracellular enzymes were purple-brown
at the 2H-hour interval. A repeat of this gave similar

results. (Fig. 2) Visual observations of steaks held at

H0

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EFFECTS OF INTRACELLULAR ENZYMES AND CELLS 0F
PLGENICULATA 0N PREPACKAGED BEEF HELD AT ROOM
TEMPERATURE

nun: 2
A1

30°C. indicated that the changes were similar to those
obtained at room temperature.

Cell-free extracts and cells of fig. aeruginosa

 

also have the ability to cause the discoloration of fresh
prepackaged steaks held at room temperature. From the

data in Table 1 it can be seen that cells of fig. aeruginosa

 

cause discoloration at about the same rate as do cells of
Ps. geniculata. Cell-free extracts of both organisms

bring about discoloration at equal rates but slower than the
intact cells.

Attempts were made to determine if fig. geniculata

 

or a crude preparation of intracellular enzymes of this
organism could cause discoloration of unwrapped steaks at
room temperature. No color differences were noted after

1 hour. Even after 2 hours, no clear differentiation could
be made. However, at 3 hours the inoculated steak was
slightly darker than the control or enzyme treated steaks,
but darkening due to desiccation was quite advanced and
only a slight differentiation could be made. Wrapped steaks
which had been inoculated with cells or treated with the

same enzyme preparation discolored within 15 to 30 minutes.

Meat Homogenates
Four hundred ml of sterile distilled water was
added to 80 grams of meat in a sterile Waring blendor and

blended for 1 minute. One hundred ml. of this homogenate

H2

.mmzpm ammsm p :ooa #0 omaaomamg max paviaao *

 

 

 

 

 

 

 

magnum M\H mamasm :\H qsoan m\w
among M\w queen :\m zxoapnwamasm manage 6mm m\H :m
mmmnw T can mmmm agony
won Aamw 0mm memo qzoaplmamasm magnum pom ma
6mm Ammo won name pmmlmflmadg qaoauloagasg ewe NH
eon Ahab ema mate awn ammo emu same awn m
sea hump can Ammo mm
unsaaa panama awn wasp awn paaaa; eta phaaan a
eta shaman awn entaah eta pnqaan can ensawn awn phaaha o
mu chaiid 0 .WM mpufiJOflan .MM maouflwsmom .MM mpnflgoflnow .ml Hoapgoo masom
mo mmsmmqm mo mm :2 sz SH maHB

 

 

 

‘Il 1-1'Ill E |‘III I... ‘II. .A

*wmop ovumgommmam among Ho aoaoo mm“ :o manaqmwao omwgp go magma so amasaamomapqfl
map mo Soapmammmag mmmwuaamo m ham namesawflamm .WM ampmHJOfinww . mm mo mpoogmm mga

 

 

H magma

was placed in each of h - 500 ml Erlenmeyer flasks and
plugged with cotton. Three flasks--inoculated, untreated,
and treated with aureomycin (final concentration of

100 ppm)--were placed on a rotary shaker, and the fourth
was held as the untreated, unstirred control. Ten ml
samples were taken at different intervals, spun down,
filtered, and the percent metmyoglobin and percent myoglobin
determined spectrophotometrically. At zero time, all
samples contained some metmyoglobin, and at 6 hours,
samples from the 3 shaken flasks had increased in percent
metmyoglobin. The untreated sample which was not shaken
was essentially unchanged. Eo myoglobin could be deter-
mined. The percent metmyoglobin in the three flasks which
had been shaken increased from about 35% at 6 hours to
about 50» at 9 hours. After 2 days'shaking, the control
had 15% metmyoglobin and la myoglobin, and the inoculated
had 2km metmyoglobin anc 18m myoglobin, but the sample
with aureomycin added contained 905 metmyoglobin and no
myoglobin. The standing untreated sample contained 27%
metmyoglobin and no myoglobin. Upon further shaking the
inoculated sample and control developed a green color
typical of choleglobin while the aureomycin-treated sample
turned straw color and had developed mold growth. The un-
stirred untreated meat homogenate developed high
populations of bacteria and after H days contained 17%

metmyoglobin and 3km myoglobin but no choleglobin.

w+

 

memos w: pm

 

 

 

 

pom pmaoaoo Bmapm :moam Smoam mQOHpm>ameo
HmSmH>
0 am 0 om ma mm H ma w:
o a; 0 mm o Hm o o: m
0 ea 0 o: 0 mm 0 mm o
m 0H 3 aa H ma H jH 0 MW
.0»: Q .pma 1 .omg Q .pmq & .oma & .me & .ozn a .pma a
HOHPQOQ Gaongomnsm wopmHSoouH Hompgoo masom
wnfiwgm n mommnn SH maHH

 

 

.ooom pm cam; mmpmgmmomOA anon no aflohnomasm paw mpmHsoHnmw .wm mo mpoomwm

m mHQmB

From this data (Table 2) it would appear that
bacteria did not play an important role in the change of
pigments of shaken meat homogenates until after 2 days at
which time they began to increase the myoglobin content.
Aureomycin seemed to make conditions ideal for the
formation of metmyoglobin.

Attempts to measure the oxidation-reduction potentials
of inoculated and uninoculated meat homogenates were made.
The extracts were placed in cotton plugged flasks into
which a platinum electrode and salt bridge were inserted.
The salt bridge extended lLtO a beaker containing saturated
hCl and the reference electrode. Variance in potential
was measured by a galvanometer to which both electrodes
were connected. ho significant difference between the
inoculated and uninoculated extracts was observed because
the differences between replications were quite large.
However, the Eh of all extracts decrease rather rapidly.
huscle Pigment Extract

The addition of fig. geniculata to a solution of oxy-
myoglobin held at room temperature caused an initial
formation of metmyoglobin. However, within five minutes
the percent myOglobin was twice that of metmyoglobin, and
this ratio existed until all the pigment was a mixture of
these two forms. At the end of 3 hours the ratio remained

unchanged.

M6

Cell-free enzyme preparations of is. geniculata and

 

Es. aer‘ginosa were tested for their effects on oxymyo—
globin solutions at normal atmosphere and at room
temperature (Table 3). It appeared that both enzyme
preparations caused an initial increase in metmyoglobin
formation; however, it is quite probable that the enzyme
preparation absorbed in the range used for measuring
metmyoglobin as indicated by the O-time reading giving
false results. The cells probably affected both the range
for measuring metmyoglobin and the range for measuring
myoglobin for at O-time an increase in both metmyoglobin
and myoglobin was observed. Assuming that after the
initial absorption the enzymes and cell no longer interfere,
the following observations were made: first, the enzymes

of fig. geniculata showed some action up to H hours while

 

the enzymes of fig. aeruginosa caused a steady increase in

 

both metmyOglobin and myoglobin, and second, the cells

of fig. geniculata reduced oxymyoglobin to myoglobin at a

 

faster rate than did is. aeruainosa but demonstrated less

 

ability to oxidize oxymyOglobin to metmyoglobin in a H-hour
interval.

.Q

0)

Correlation of 92 gema_; of the Surface Tissue f Meat

 

 

 

Pigment Changes
These studies were undertahen to see if there was a
correlation between the activity of the reapiratory

enzymes present and discoloration. Manometric studies

w

magpmmmdamp Soon pm wamn who: mSOHpSHON Sfinoamomamxo*

 

 

 

 

 

 

0H H: OH mN Hm N: mo mm o NH omH

NH m a mH Nm :1 no mm o m oNH

NH mN : mH mm :: mo mm o m 00

:H mN 0 MH a: H: mm mm o m m:

N NN 0 HH om mm ow mm o O on

NH :N H :H a: Hm :0 NN o o mH

NH NN 0 NH ON uN wN HN o O OH

HH NN o mH NN :N wN ON 0 o m

0 0H 0 @H :N :N mN NN o o o

o H o o o o c o o N mwoumH
.oaa a .pwa a .0»; a .pma a .0»; a wuma a, .dw: a .Hm; a .omala .pma a! mmpsuHm
dmoqfluunoml¢mm NHNHJufiumweqwu

mmimmEAqu mo moahmqm mwoqfldsaom qmw NQNHJoHSow‘.mm donuqoo CH mafia

 

*.mpown

pxo choanomemxo wo obsmno pumnwfld wo mama mnp so
dwdmwdjwmm .wm cum Npmamw

fidww .MM mo maamo paw moammcm Amazaaoomhpqfl onp mo mpomgmm one

 

m mfinme

1+8

were employed in addition to visual observations, index

of fading, and bacterial counts. Various lots of steaks
inoculated and uninoculated were tested over a period of
ll days. After visual observations and Munsell disk
notations were made, a 3 mm. slice from the surface of each
steak was placed in a sterile Waring blendor, diluted 1 to
5 with sterile distilled water and ground for 30 seconds.
Two and eight tenths ml. of this homogenate was used to
determine’pl of 02 uptake by the conventional Warburg
manometric method. Results (Fig. 3) demonstrated that the
number of bacteria of the inoculated steaks did not in-
crease greatly, but as the steaks discolored (fading index

increased) the pl of O uptake increased. There was a lag

2
in the oxygen uptake by the bacterial cells since even
though the counts were high initially, the pl of 02 uptake
was no more than that of the uninoculated control. However,
as the steaks discolored, the oxygen uptake rate increased.
A good correlation between the number of bacteria
present, rate of discoloration, and oxygen consumption was
obtained from the uninoculated group of steaks, i.e. as the
bacteria increased in numbers and the oxygen uptake in-
creased, steaks discolored. From these data, it appears
that there is good correlation between respiratory activity

measured by oxygen uptake and steak discoloration.

F9

IIIEX IF Flllll

ll! 0F llllEIS/EI.

#2 u, mm m at m:

 

 

 

 

 

 

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7‘1

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301 I

20"

 

 

 

 

 

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I r. v r t t r I ""fi
0 1 2 3 3 3 6 7 E 9 10
TIIE ll .11:
EFFECTS OF P:.GENICULATA ON PREPACKAGED
BEEF HELD AT u°C
"all! 3

Influence of Respiratory Enzyme Inhibitors on Pigment
ghanges and hespiratory Activity

Steaks Held at Refrigerated Temperatures

 

To study the fundamental principles of discoloration
of fresh meat by bacteria, various inhibitors were applied
to the surface of beef steaks. Various concentrations of
aureomycin ranging from 5 to 100 ppm were sprayed on the
surfaces of the steaks. From Figure 4 it can be seen that
as the concentrations were increased, the bacterial growth
was controlled more completely, and the bright red color
of oxymyoglobin was retained longer. Further experimenta-
tion using aureomycin at 100 ppm demonstrated good control
of bacteria as compared to the control and resulted in
preservation of a desirable color 3 or a days longer than
the control (Fig. 5).

One percent solutions of both sorbic acid and sodium
sorbate were sprayed on the surface of the beef steaks.
heither form had any effect on color, nor did they control
the bacteria. However, preliminary studies using
cellophane impregnated with sorbic acid as a wrapping
material showed that color retention could be extended
somewhat.

Sodium malonate is a competitive inhibitor of
succinic dehydrogenase. To test the effect of this
respiratory enzyme inhibitor on color retention, a group of

steaks was treated with sodium malonate and compared to an

51

IIIEX 0F Fill"

L06 0F IUIIEIS/GI.

 

 

O
./ I -t / .
o -’ s¢h~ O
’0 ’I/ fi.‘ ~‘ .0

    
  

 
 

/.-'
/_~

(\c-‘Do—o—O‘.’ '0‘

 

 

 

 

 

 

 

 

T I r T ,
2 n c a 10‘
TIIE II I":
EFFECTS OF VARIOUS LEVELS OF AUREOMYCIN APPLIED
TO THE SURFACE OF PREPACKAGED BEEF HELD AT 11°C
Control
“""'" 100 pp- Aureoucln
"""""' 50 pp- Macro-yam
— — — 25 pp- Anton-yam
00.00.. 5 ”AMONG“!
, noun n

52

IIIEX IF FIDIIG

-L06 IF IUIIEIS/GI.

PfllPlEfi

no" 1

IRIBHT

 

 

 

 

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V‘-

 

104

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7a

8n

 

 

 

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Control

  
 

 

 

I
I”
I l 3 '0 10
TIIE ll DIV:
Erasers or Auaaonvcun ON FRESH PREPACKAGED
BEEF HELD AT u°c
FIQURE 5

l‘3

aureomycin (100 ppm) treated group and a control group.
The steaks treated with aureomycin had low bacteria counts,
low metnyoglobin formation, low 02 uptake, and low index
of fading. Sodium malonate demonstrated some bactericidal
or bacteriostatic activity, and the index of fading and
oxygen uptake were less than the control. However, the
percent metmyoglobin formed was practically the same.
(Fig. 6). .The results illustrated that sodium malonate
had some effect on the microflora and color retention of
prepackaged beef, but its action was inferior to
aureomycin.

Iodoacetate (0.1M) sprayed on steak surfaces proved
much more effective than the 20p malonate. The iodoacetate
was applied to inoculated steaks and compared to inoculated
untreated and to uninoculated steaks. The inoculated
steaks having high bacterial counts and high respiratory
activity discolored within 2 days. (Fig. 7). The inoculated
steaks sprayed with iodoacetate had a low bacterial popula-
tion, low respiratory activity, and prolonged color
retention. In fact, the action was so pronounced that the
inoculated steaks treated with iodoacetate gave counts,
respiratory activity, and color notations very comparable
to the uninoculated control.

Warburg data demonstrated that cells treated with

haN3 and hCN had far lower respiratory activity on meat

5A

L06 OF IlllElS/fll. IIDEX 0F HIIIG

5 IETINEUIII

A1 or I, "HIE/30 ms.

     
 

 

 

‘{\
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IO-I

 

     

 

-- ---"’-

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v"

0—0—0—0 “3 Inlonsto (M)

 

10d

 

 

7 0' . 9’ so 12
”IE II I":
COMPARISON OF EFFECTS OF SODIUM MALONATE AND
AUREOMYCIN APPLIED TO PREPACKAGED BEEF HELD
AT lI’C noun: a

‘55

I.“ IIF IUIBEIS/EI. IIDEX 0F FIN"

s Istivoslosll

,ul or o, mus/an mg.

 

$14
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I’d F’ "

   
  

28-

‘ . .. . _ . .. Inoculutod ”hm”l
2’ - Iodocootuto (lo I)
21 -

1’

 

 

 

 

 

 

 

 

 

 

 

 

704 I

    

 

 

 

 

—
I
2

, III! II 911:
Erracrs or IODOACETATE ON INOCULATED PREPACKAGED
BEEF HELD AT u°c

FIGURE 7

‘56

substrate than did untreated cells of fig. geniculata.
Cells treated with Nah3 (0.01 M and th (C.l M) spun out
washed, and resuspended in sterile saline, showed about
73% inhibition. haN3 and hCN added to the meat substrate
before the addition of bacteria resulted in complete in-
hibition of the respiratory enzymes. Final concentration
of Nak3 was 1 x 10-3 molar and hCh was 1 x 10'2 molar.
(3.3 x lO'3M th gave similar results.)

Cells treated as above were sprayed on the surface
of steaks and these compared to steaks treated with the
inhibitors and inoculated, treated but uninoculated, and
to a control lot of untreated steaks. All steaks were
then packaged and held at 4°C. for visual observations.
Results presented in Table 4 indicate that cells pre-
treated with hCh or Nak3 as well as cells added in con-
junction with these inhibitors caused no discoloration.
This demonstrates that respiratory enzymes play an im-
portant role in the discoloration of fresh prepackaged

beef. It was observed, however, that Nah per ge caused

3
discoloration of the meat. This might be expected for
Nah3 reacts chemically with porphyrins and the prosthetic
group of myoglobin is a porphyrin. The exact reaction
involved was not studied in this investigation. When

hCh was added to steaks without inoculation, a bright red

color was still present after 10 days.

57

mama naHoa Ho.o**

 

 

 

58

 

 

 

 

304 nwao: H.o*
mamasm m\m madame m\H czopp mmem Q3099 szopp ewe
CSOQQ M\H csoen m\H magnum ewe manage pnmflmn QSOHQ OH
macaw N\H qsonn ewe £3099 ewe ewe
qzomp ewe meme m\H magnum pnmfinp banana pguflnp game e
ewe ewe madam emm mzomp emn emn
xnme Meme madman pnwfimfl damage pzmflan same J
emu ewe ewe ewe ewe ewe
pannn pmmflnp magnsm pmmflnp manage pgmflnn emm m
ewe ewe ewe ewe ewe emu emu
pannp pannp mama pgmHnn meme pgdan pannp H
ewe ewe ewe ewe ewe ewe ewe
panwp pannp pannn pannn pnmann pnNan pngnp o
empw>fluomqfl empN>Hpomqfl mama 304 m mama
am; gem msam mafia aw; avg Honpqoo :fi
NpNHSonmw .WM NpmHsoflmmw .mm NpmHSOHQMm .WM NpmHSoflqmw .mm mafia

 

.00: um eHmw Hemp ewmwmomampm Ame 9
so mzmz ecm aux mpfla empm>fipowqfi NpmHSofimuw .mm emmw ** zmz .*434 Mo mpommem

 

: mHnma

Further studies were completed comparing the
activity of treated and untreated cells of fresh prepackaged
meat at various oxygen tensions held at H° C. he various
oxygen tensions were obtained by drawing a vacuum in
dessicator jars and readmitting N2 and 02. before ad-
mitting the final gas mixture the jars were flushed twice
with nitrogen gas. Steaks of the various treatments
described above plus inoculated untreated steaks were placed
in the dessicators at the various oxygen tensions. In an
atmosphere of nitrogen all the steaks became purple within
an hour. At oxygen tensions between 20 mm and 40 mm 02 all
steaks turned brown between 3 and a days. The surface pig-
ment of steaks inoculated with dilute suspensions of
actively metabolizing cells was oxidized at a slightly
faster rate than that of uninoculated steaks; however, the
metmyoglobin of the inoculated steaks was reduced to
myoglobin within 1 day after formation. The surface
metmyoglobin of the uninoculated steaks was more stable,
remaining at least 3 days before being reduced. Steaks
treated with aureomycin,iodoacetate,and cells inactivated
with.hCh gave results similar to the untreated, uninoculated
controls. In a normal atmosphere, no metmyoglobin formation
could be observed during the first 7 days except for the
steak inoculated with active cells. This steak began to
discolor after 2 days and within 5 days had become purple
with only a slight evidence of browning. The results

SQ

indicate that under reduced 02 tensions surface pigment
oxidation is accelerated, and the reaction is not in-
hibited by either aureomycin or iodoacetate. Also dilute
suspensions of is. geniculata increase the rate of pigment
change only if their reSpiratory enzymes are active.
Steaks Held a_ Room Temperature

Early work demonstrated that at room temperature
crude intracellular enzyme preparations of fig. geniculata
caused surface discoloration of wrapped beef steaks while
extracellular preparations did not. The action of the
intracellular enzymes, however, was less pronounced than
that demonstrated by intact cells. Preliminary work using
crude enzyme extract in conjunction with sodium fluoride
(0.01 molar) applied to steak surfaces gave no significant
information. That is, at room temperature this enzyme
inhibitor had little or no effect on the enzymes causing
discoloration. The data indicate one of two things:

(1) the concentration of the fluoride was too low to be
effective, or (2) the enzymes which fluoride inhibits are
not involved in meat discoloration.

In order to obtain further krowledge as to the
mechanisms of discoloration of beef at room temperatures,
four steaks were treated as follows: (1) untreated con4
trol; (2) inoculated, but no further treatment; (3) in-
oculated and sprayed with 0.1 molar iodoacetate; and

(H) inoculated and sprayed with 0.01 molar sodium azide.

60

All steaks were wrapped with M.S.A.T. 80 cellophane.
Results (Fig. B-A) indicated that both enzyme inhibitors
slowed down discoloration up to 45 minutes. At this time,
the steak treated with sodium azide turned a dark purple
and remained such for the rest of the experiment. At

2 hours, based on final index of fading values, iodoacetate
resulted in about a 50; color preservation. That is, the
index of fading value lay midway between the index of
fading for the untreated control and the index of fading
for the inoculated control. Upon refrigeration, all the
steaks turned bright red except for the steak treated with
sodium azide. This would indicate that sodium azide forms
a stable complex with the myoglobin which seems very
probable since sodium azide reacts with metalloproteins

of which myoglobin is one.

Steaks treated as above, but not wrapped until one
hour after treatment, discolored only slightly for the
first hour. Only the inoculated control had begun to
darken sufficiently to be observed by index of fading
values. The index of fading of the uninoculated control
was only slightly less than those of the steaks treated
with sodium azide and iodoacetate at one hour. After
wrapping, all steaks discolored (became purple) rapidly as
indicated by visual observations and index of fading
values. The inoculated control steaks discolored most
rapidly. Sodium azide slowed down the discoloration only
very slightly before forming the stable dark purple pigment.

61

Iodoacetate seemed to be very effective in that it reduced
the rate of discoloration to that of the uninoculated
controls. (Fig. 8—1).

huscle Pigment nxtracts held §_ £993 Temperature

Cells of fig. geniculata have shown the ability of both
reduce and oxidize myoglobin solutions. These results were
obtained by spectrOplotometric analysis of the solutions
containin~ the organisms using a water suSpension of the
organisms as a blank. Studies comparing treated cells
(kCn, hah3 and heated) with untreated cells by the above
method gave false results, for treating the cells gave them
different optical properties which could not be compensated
for by a cell suspension blank. The results indicated
that cells which were inactivated, as demonstrated by
Uarburg studies, had the ability to oxidize and reduce
the pigment.

A subsequent study was undertaken to note the effects
of cells inactivated with hCh. In this study a sample was
taken and centrifuged before spectrophotometric analysis.
Visual observations of the solutions were also taken. After
30 minutes the myoLlobin solution containing the active
cells was a definite purple while the solutions contain-
ing cells inactivated by heat (35 minutes at 75°C.) and
hCh (10"1 M) remained red; however, spectroplotometric

analysis demonstrated essentially no difference (Table 5).

62

IIIEX 0F Fill"

IIlEX 0F Fill"

 

2L,

 

 

mma ,1
I

 

 

 

so.»

2’.
20-
11.1
26-
28..
2!...
2)..
.12.
21-1

 

 

"IMP?“ IIIPPED

I

d
’ .fl .‘ 0’.

 

 

 

U
18 ,0 u ‘0 78 90 108 120

THE II ITIUTES

EFFECTS OF IODOACETATE AND NI AZIDE ON
INOCULATED STEAK HELD AT ROOM TEMPERATURE

 

Cbntrol

“oculated

Inoculated * Iodonoototo (10' 1I)
Inoonlstod + u. Aside (10" III)

snout: o
63

.nflm mafia powpqoo
:fiw mnowpsaoa nhxxa»am ohm mow «awn moumo QM Mafifismmcwu who; noan.MofiHHfihpgmo x

.

 

(A) LA (\J

(J

g, ( J a Q\
,j' [\ U\ C\
H H r- I H
(Y) ,3‘

\J \J

L \ (\J

k J J

C) Q

C\ U\

r‘-{ Cd

'X: *

x.) C)

,j‘ (3

< \J (*3

so a o we do an ca
o ma o on o an m
o d o ma

0 Q
(q ‘
H
O

0 ma o ma

 

 

 

 

 

0 (..Jri “ 1% O Pr» M \j 0 C.\.r1_.H . a QM.” fl 0 CJHWH «Q 6 PCWH J O 44H”
an: sf . _ - a
pttt an) mHHoo Q>Hpo¢ Hospaoo ma
pigmeanowu VHHD, mafia
. onSpmncmuppIMoon pm damn meoUHQMm anonmflm
son: no antagoHuMJ .mg mo mHHoo oppm>flpomnfl was m>fipom no mpoommm

m magma

At one hour a centrifuged sample of the inoculated myo-
globin solution showed a slight increase in metmyoglobin
and myoglobin content, but not nearly as much as visual
observations would indicate. At H hours samples of the
various solutions were placed in centrifuge tubes and
rubber stoppers were inserted. The tubes were filled so
as to overflow when the stoppers were inserted so that no
free air was present in the tube. After centrifugation
the solutions were placed in the colorimeter tubes
carefully, and spectrophotometric determinations were made.
Tie data demonstrated that active cells had increased the
percent metmyoglobin slightly, but primarily the action
was reducing the pigment to myoglobin. Treated cells were
essentially inactive.

From these results it is evident that one must be
careful in his work with myoglobin solutions, especially
if spectrOphotometric analyses are to be made. The data
demonstrate that cells of fig. geniculata which have
cyanide inactivated cytochrome systems are unable to reduce
a solution of oxymyoglobin to myoglobin. This is in
agreement with the results observed in previous work with
myoglobin solutions in which the cells were left in the

solution used for Spectrophotometric analysis.

65

Meat homogenates
heat homogenates were used as a substrate in Warburg
experiments testing the effects of various enzyme in-
hibitors on cells and cell-free preparations of fig.

geniculata. Sodium fluoride at a concentration of 0.01 M

 

had no inhibitory effect on the enzyme preparation and

only a slight inhibitory effect (18p) on the cells (Fig. 9).
iowever, from the data in Fig. 10, it is evident that 0.1
molar sodium fluoride inhibited the respiratory activity of
both whole cells and enzyme preparations. This inhibitor
brought about a 50% inhibition of the enzymes and 38A in-
hibition of the cells. A 0.01 molar solution of sodium
malonate was also found to be inactive (Fig. 10), but 0.1 M
sodium malonate caused a 50% inhibition of the respiratory
activity of the enzyme preparation and the intact cells
(Fig. 11). Iodoacetate (0.01 M) reduced the respiratory
activity of the cells and cell-free intracellular enzyme
preparations by about 50fi,and sodium azide (0.01 h) com-
pletely inhibited the oxygen uptake by the enzyme
preparations and caused virtually complete inhibition of

the cells of fig, geniculata (Fig. 11). One-tenth molar

 

hCh gave results similar to those obtained with sodium
azide.

In order to find out if cells of fig. geniculata
utilized an alternate aerobic metabolic pathway at re-

duced oxygen tensions, the following Warburg eXperiment

66

 

 

 

 

 

 

 

 

 

 

'._ -'--"" Cbntrol
g 5" "T" ' n. fluoride ’55: °
N “'4 a: :’ ...—L
- __ __ .. lune plus 4.9 o ____, I
.L gr Iodoacetate ,;. d,
I" ‘hTr. a—" ‘—'-
0“ ‘ al’
N 2 ‘fi. ..., a,
’
m .
' II
206-1 Cell- plus "E" “I.“ I
"'""" u. fluoride 107’"
”y con 1 ’
.__.._—.—— I D “I .
“0" loch-octet. 10 2| 4
1'0‘ " 0”.
I /
160- 1' 1’.
I o
and I /
‘“ l
v: 1”" I
p. o
:5 1,0- l /
I O
¢:~110F /' .T” ",p
... / /
, /
‘10.! I ’0 /
r .l o
R ”‘ 1’ .I /
cl
.0 I ‘1’ ‘r'
70¢ / / /
. I 0 /
‘°' I ,/ /
00-» ,’./ /
u. ’/./ /
,0. I I, "”
’l. /
3.- "/
sou /
v j —' ' fl :‘V' —' V
I 10 1| 0. .0 II ‘0

EFFECTS or N. FLUORIDE AND

I 30 E; to
"IE II II'TES

IODOACETATE ON THE RESPIRATORY

ACTIVITY OF CELLS AND I‘NTRACELLULAR ENZYMES OF P8. GENICULATA
FIGURE 9

 

\0
L1“
\

‘\
\.
\\

\O
O

o—o— Na Inlonnto TOLD /.V

'— —" Na Azide 1'50 //.

.0 0" N
O C O
I n 1

,4! or 0, arm:
i

‘\

I

I

 

 

§
‘\
\

N
3
l
‘\
\
O

210- I o

 

Control
m. I,/ - — - - - Calla
up. 70 o— 0— Cells plus Nu lnonste
m- I — — - cons plus n. Aside

1‘0. I
no- [I /
no. I /

if] 0F 0, mm
§
\
\

 

 

 

 

I 10 II 20 2'3 "0 3" :0 II IO SI ‘0
TIIE II IIIUTES
EFFECT OF NO MALONATE AND NI AZIDE ON THE RESPIRATORY ACTIVITY

CELLS AND INTRACELLULAR ENZYMES DF P8. GENICULATA "w“ u

68

 

 

 

 

 

 

 

 

 

25* -..---me ””
MI ”_ ’ ’ I
= 0—'--"’ III Fluoride ’ , I a
o— “ a
g A .... -— - No IQIOIIICO ’ ’ f ’9’; ’b
u 104 v ‘T .... v‘fia- ""
H I ’13:"; "'
3 a ’ ‘13-
OdL‘lfl: , .
3: ,0 o to In to Is. 39
uo‘ ‘ ’l
”0" I
I
210‘ ’
zoo- """"'" "’ Cello I,
no“ "-0-0- Cello plus NI Fluoride ’
4 1'6 ,
A” """' "" "' Cells plus Io Iolouoto ’
“Hr ,
"0" ‘ ’
«o- ’I
no" I /
r: . ’ /°
‘ 1‘0 I
z I /
= 130‘ I /.
a“ no" ” /o /
g noq ’ o /
I / /
\‘o S”. I, /0 /
I 904 I /0 /
00" [I o /
u" ,’ ./ / /
I
60" I ./ /
u" I’ .// /
IO" ’ /
I o /
30" I / /
-I I .
20 I I o/ /
II" I./
m:
o o 1 ' Io in yo

 

0 ll
TIIE II IIIIITES
EFFECTS OF NI FLUORIDE AND NI MALONATE ON THE RESPIRATORY

ACTIVITY OF CELLS AND INTRACELLULAR ENZYMES 0F P3. GENICULATA
FIBIIIE 11

60

was undertaken: cells treated with hCh were compared
to untreated cells at various reduced oxygen tensions

which were obtained by allowing mixture of 02 and N to

2
flow through the system prior to starting the Narburg
analysis. The atmospheres used were: (a) all N2,
(b) 1% 02 (99» N2), (C) 5» 02 (95% N2), and (d) 10% 02
(90% N2). These were compared to normal atmospheres.
The “pl 02 uptake was determined for a 30-minute interval
(Table 6). The respiratory enzymes of cells treated with
hCN were essentially inactive at all atmospheres. The
activity of respiratory enzymes of the untreated cells
increased as the oxygen tensions increased from 0 to 10%.
The enzymatic activity at 5 and 10% oxygen was similar to
that at normal atmospheres.
Beef Tissue Slices

Beef tissue homogenates had very low oxygen uptake
activity; therefore, tissue slices were tested for
reSpiratory activity. Both myoglobin solution and Ringer
solution were used as suSpending media in the Warburg
flasks. Both gave similar results, so Ringer solution
Jas used for its constituents are defined and controlled.

The tissue slices showed active metabolism as
measured by oxygen uptake, and gave essentially the same
activity at reduced oxygen tensions as at a normal

tension. It was noted that hCN and haN3 at final

70

ooonooon mam owned:

N

0 90 H1.**

anoE mnoaxo.o mm3,zom mo COHpmnpzoodoo Hmnfim *

 

 

 

0.0 mw.00 No.3 m0.0m 0.0 0m.0 0.s 0.:H m.s mu.m: 0m
0.0 mm.0m m0.: 5.5: 0.0 0m.0 0.m 0.mH m.s mm.sm mm
0.0 @0.53 m0.m mm.0: 0.0 sm.: 0H.0 :.w m.s ms.0m 0m
0.0 nw.fim wo.m mm.mm 0.0 0m.a No.3 0.m :m.m m0.am ma
0.0 nm.sm w0.m sm.mm 0.0 mm.a 00.m m.: :w.m ma.0a 0H
0.0 fi.0 0.0 m:.a 0.0 0.0 :m.H :.H 0:.H ..ms.m m
30a eon eon eon 20a
+ + + + + .CHE
maamo adamo mammo maamo maamo maamo maamo maamo maamo anamu ea
maa00 + moaoa mymfio + Noam maamm + mesa ma mafia

 

 

mpmfidoflsmn .WM no mHHoo an oxmpms

mQOHmnop Cowmxo mJOHnm> pm

m

o manna

0 map so *aoz mo pomuum one

71

concentration of 10"2 h greatly inhibited the respiration
action of the slices both at normal atmospheres and at an
atmOSphere.of reduced oxygen content. The inhibitory
action was essentially the same under both conditions

(Table 7).

 

held at room temperature or at refrigerated temperature,
wrapped or unwrapped, resulted in no metmyoglobin formation.
bubbling occurred indicating catalase activity,but no
evidence of pigment oxidation was visible. however, a

steak which had been held at a reduced oxygen tension

(80 mm 02) became brown when peroxidase was added to its
surface at room temperature under normal atmospheric con-
ditions. In fact, the oxymyoglobin on the surface of most
aged steaks was oxidized to metmyOglobin on addition of

H20 .

2
Both fresh and aged solutions of myoglobin pigments

2
from the results in Table 8, it can be seen that the aged

were partially oxidized by the addition of H 02. However,

solutions of myOglobin were oxidized much more readily
than fresh preparations.

heat extracts gave a positive test for peroxidase
using orthophenylene diamine (0.P.D.A.) indicator. To
further verify the evidence of peroxidase, hCN, which is
known to inhibit this enzyme, was added to the extract

72

 

m:.: mm.m 0.:u m, .Eu H.O\.HQ\NO H1.

404 mama Honpsoo so; MAm; HOHpuou

Qmwoapflg {mm cum :mwhxo 1H maozmmospa Hmaaoz

mSmme mode mo

mpfl>flpom AhopmHHAuoa one so ooflzmmo Ssflmmmpog wan mofimm Ezflwom mo muommnm

m oHQmH

{/3

and compared to an untreated control. It was noted that
as the concentration of the hCL increased, the peroxidase
activity decreased. When compared to a heat-inactivated
control, hCh completely inhibited the heat sensitive
activity. Potassium iodide plus starch, benzidine, and
gum guaiac all failed to give a positive reaction for the
presence of H202 in or on steaks whose pigments were
undergoing oxidation. Thus the presence of peroxidase has
been shown, but no free H202 could be detected. However,
any H202 produced would probably be very fleeting due to
the presence of catalase in high concentration. Therefore,
it is not likely that the tests used are sensitive enough to
detect the level of H202 which might be present.

Effect of Glucose Oxidase

Glucose oxidase when applied to the surface of steaks
wrapped or unwrapped, caused the formation of metmyoglobin
within five minutes at room temperature. However, the
discoloration reaction did not stop at this point but pro-
ceeded to the choleglobin stage. Refrigeration did not
stop this reaction though it was somewhat slower than at
room temperature.

A second trial using dilute solutions of glucose
oxidase proved that this enzyme preparation could oxidize
the surface pigment of steaks to metmyoglobin without the
formation of choleglobin. The results in Table 9 also
demonstrate that the heat-treated glucose oxidase was
inactive. This would indicate that oxidation caused by

71+

 

 

 

 

0 OJ 0 NH 0 0H 0 O 00
o m: o n o m o m om
0 mm o O 0 0H 0 OH mH
O Om O aw O NH O O O
o m o o o m o m goapaaoa
maowon
.OAn i .pon 1 .ome x .ppn & .oms A .pos Q .oafi & .pmfi R
mowu+soapmammmnm goapmnamoal momg+qoapmnmganl soapsamgmam .qHa
coda pomm gmomm gmoam SH
mafia

 

magpmmmmsop Eooa pm pawn
mpomapmm Sflnoagomn wonm mum gmoaw so mmflxoamm Genome»; mo mpommwm

r

w mHQmH

glucose oxidase was enzymatic. £202 had no effect on the
surface pigment of steaks when added alone or in con-
junction with glUCOSG oxidase inactivated by heat.
Conparing the effects of hgog, glucose oxidase and
peroxidase on oxymyoglobin solutions (Tat e 10), it was
observed that E202 caused some oxidation but was far

less active than dilute glucose oxidase. The fact that
heated glucose oxidase was active would suggest that some
of the activity of the unheated sample was nonenzymatic,
but the difference in activity was great enough to show
the enzyme system did cause oxidation of oxymyoglobin.
Glucose oxidase when nore concentrated caused the forma-

tion of choleglobin.

1

f'fect 9i Peroxidase

t

Peroxidase was obtained by filtering a comhercial
unpasteurized horseradish preparation. Peroxidase added
to the surface of fresh steaks caused no discoloration,

nor did 0.3» solution of 3202 at room temperature. how-

q
‘

ever, when added together the steak surface turned brown
within 10 minutes; and after holding at H°C. overnight,
the brown pigment became a green-brown color. E202 added
to steals previously treated with peroxidase turned brown
almost immediately.

It was noted that some honenzymatic ozidation takes
place on steaks. Peroxidase solutions inactivated by heat
still caused some surface oxidation when used in con-

junction with thg, but the reaction was slower than that of

76

 

 

 

czoan zsoan
own emu dame mama won omfi
Hhaoau HEPOHQ
pom pom mane hymn won 00
qsomn gsomn
won own dame same pom om
azsoag
wma Una . Esonfl 0mm Uma ma
pom pom
pom Una ammo yawn won 0
6mm pom pom own 0mm goapflvum
oaoamg
mmmmfiso mmoosao mwmpaAO mmoosaa mpfiaoaom Gonomwhm mmmoflao mnemoamm .qfl;
Umpm>flpomQHupmmm wmpm>flpomnHupmom + mmmefiso omOUSHo mmoozao Cowoapmu
EH m: . m.

U
+motu

 

magpmamdzmu Eoo

Qwog mo Soapmaflpsoo m

a pm pawn mxmmpm Ummmmazgs mo aoaoo momMASw mnp Go

and .mmmbfimo mmoozan .mUonamg :mtopwmn mo mpommwm mga

0 magma

77

 

 

Ili‘l“: III. II .‘ri I- 3'.

(Y)

(o
d)
N

 

 

 

 

 

a mm a am 0 0a Ha an 0 ma 0 m om
o o a N O o o o o m o o 0 ha
.oa: a .am; a .ops a .sss a .0»; 2 .e94 a .0»: a .po: a .0»; a .pm: 2 .cam 2 .pma a .mqaz
mmmCHmoamm mmmwfihOHmm mmmefls mmmoflao Noun Hoapnoo CH
mmoozwo
U®um®M Umpm A mmOOSHU mfiwfl

 

mmmuasoamm ohm m mafiao mmoosam empm>apomnflapmmn use m>apom Ho mpoowmm

OH mHJmB

the active preparation. The nonenzymatic reaction was
shown to interfere far more greatly in myoglobin
solutions, causing rapid oxidation (Table 10).

Ei____feCtS 9i 9.2 ELL-(2.12%
Steak surface Pigments

A series of experiments was designed to show the
influence of different oxygen levels on the color changes
of both inoculated and uninoculated steaks. In the first
experiment atmospheres used were oxygen, nitrogen, and
air. The wrapped steaks were placed in desiccating jars,
a vacuum drawn with a water aspirator, and the jars
flushed with the gas to be used. This process was repeated
three tines.

The results of this study are presented in Tables ll
and 12. The steaks held under an oxygen atmOSphere de—
velOped a brighter color (lower index of fading values)
and maintained it longer than the steaks held in air;
however, inoculation with Es. geniculata greatly reduced
the time necessary for a color change to occur in both
air and oxygen. The steaks in the nitrogen atmosphere
never returned to a bright red, but remained dark red
until discoloring further. The uninoculated group held
in the N2 atmosphere developed a brown color and high
metmyoglobin percentage. This may have been the result
of a low residual oxygen level in this atmosphere. The
failure to observe a brown color or a high metmyoglobin

percentage in the inoculated group under nitrOgen was

79

 

 

 

 

m.w m.m m.: o.m In: mflampomn .0; woq
w.ea m.om m.wm w.om --- annoawoaanmz a
:.mw w.om o.mm ©.mm an: mnflpmm mo waqH
GBOHQImHQHSQ qaoan magasgnzsoag madman emu name :oflpm>nmmno HmSmH>
amoogaHz
:qoa N.© ma; o.m nun wflaopomp .oz moq
m.mm 5.:m 1.9H m.ow --- manoasoaapma a
a.om m.mH m.ma m.ea --- sqaemu no ameqH
pea Q\H
mamasm m\H pea emu emu
Czoan M\H own pnnflnp pgmfipp mnm> pnnfipn mam> pgwfipn Anm> SOHpm>ammno Hmsmfl>
nu
zuswso no
m.m o.m w.: o.m m.m mfiampomn .o; woq
e.mm m.wm m.mm :.m m.aw canoanoaspma a
.H.mw m.om o.wH m.ma n.0H wgfiwmw mo NmenH
magnum M\H
Gzoan m\m won pea pnmfimn mom pnmflan pea pnnflan soaum>nmmpo HmSmH>
qeszz
HH 0 J m o mQOflum>ameo

 

00; pm easy “map was“.

momgmhq umpmfigooqflss so mmnmuamospm :mmkfio use mmwoapwz Mo mpomem

mmmc Ga mafia

AH magma

mam monogam08¢<

iii-{If

 

mpmHuoHQmu mmuouowSmmm Qua; UmpmHSUocH*

 

 

 

 

 

 

m.m o.w m.u m.m In: mHampomn .oa n04
9.5 m.aa w.ow 0.1m --- canoawosapo: a
m.mm ¢.am w.mw m.:m --- maaoou no woqu
mHstm ngnsm deasm mHQnsu own memo mQOHpm>Hmmno Hmzmfi>
amuoan:
m.OH 0.0 m.m 3.5 In: mflampomn .0: woq
:.a m.om 0.5H m.oa --- gaooamoaaooz a
m.am m.wm w.wa m.:H --- wsaoon do woqu
ooh ooh
mHaasm oHdnSQIQEOHn own unnflan muo> unofiap mno> m30apm>ammno Hmdmfi>
am; fine
H.0H 0.0 m.m 1.5 m.o wfiaopomp .oz woq
o.m o.mw o.Hm o.oa m.mm :Hooaoosapoz a
H.0m o.Hm m.mw 1.5H m.aa moaoon no woosH
eon aaao o\a
mHQMSd mHmazdnzsopp 230nm ©\m won pgoflan boa panHp mnoapm>ammno HwSmH>
Adamo;
HH 0 J m o
WNAMU SH marl—”PH. WHHOHpmwkawmfloo

Una monogamospd

 

.00: pm Ung moon omnmnommmhm *UopmHSOO:H no monogamoapm Commxo 03m ammompfis mo mpommmm

NH mHnt

El

probably due to the rapid utilization of residual oxygen
by the bacteria.

To get further information on the influence of
various oxygen levels on fresh meat color, a more com-
prehensive experiment was designed. This involved
storing prepackaged meat at various oxygen tensions.
Control steaks inoculated steaks, and steaks treated

with cell-free extracts of fig. geniculata were stored

 

under each atmosphere. The atmospheres were obtained

in vacuum desiccator jars by pulling a vacuum with a
"hi-vac” pump, measuring in the desired oxygen pressure
by the use of a mercury manometer, and bringing the
internal pressure near atmospheric pressure with nitrogen.
Visual observations of color were made during the lZedays'
storage under refrigeration. Observations of the control
and inoculated groups of steaks are shown in Figure 12.

All steaks held in the absence of O2 turned purple
within l-l/2 hours and remained purple throughout the
experiment.

The steaks held at 10 mm of 02 demonstrated some
differences as to treatments. The uninoculated steaks
began to darken within 12 hours, and at 36 hours became a
definite brown and remained brown. The steaks which
were inoculated turned dark red immediately, and after
2% hours became purple without the formation of a brown
pigment. The steaks treated with the intracellular
enzymes gave results similar to the inoculated steaks,

82

 

..--------— "IIIOOIIUTEO

 

 

 

 

 

 

 

 

‘\
IINI' __ _ _ \\
IE. : \ \\
P: O C . \ \\
o
k. . , . \ \
\ 0 \ ~~
‘ ‘.o . \\\’ "-._
\ . ° 0 , ~ ~ “’~.
"0" o o o o o o 0 o O o I o‘ofio—o
o
o
PURPLE-H
” Ilflclllnfll
K ‘ .... .— 10 no,
" 000.00 75 .02
.\ —- —- Nor-cl
\
nmJ \‘~‘~
J. \b ..‘E\
‘5 \ \
'.' \ \
'3 ‘\~ ‘\
\
PIIIPlE-d i
I r I 1 F I T I.
O 2 I C O ‘0 12 1‘ 3‘
TIIE - B":

THE EFFECTS OF VARIOUS, OXYGEN TENSIONS ON
THE COLOR 0F PREPACKAGED BEEF HELD AT u°C

FIGURE 12

83

 

but before turning purple, browning was observed.

The inoculated steaks and those treated with the
enzyme preparation held at 75 mm 02 gave results similar
to those obtained at 10 mm 02; however, discoloration
caused by the enzymes was less pronounced. The
uninoculated steak was a briiht red up to 2 hours and
then became a dark ad turning brown within 6 days and
finally began to turn purple after 16 days. It should
be noted that up to this point only the uninoculated
steak held at 75 mm 02 was bright red initially.

Inoculated steaks held at normal atmosphere began
to darken within one hour and within 2% hours turned
purple. The steak treated with enzymes demonstrated a
slight darkening for the first 2 hours, but then returned
to a bright red. Subsequent discoloration of the steaks
treated with enzymes was probably due to bacteria and
not to the enzyme preparation pg; ge. Results of the
enzyme-treated steak were similar to the results of the
uninoculated steak except for the initial darkening.

All the steaks in the oxygen atmosphere were very
bribht red for the first few hours. The inoculated
steak was first to discolor, having a brown periphery
and a dark red center by 2% hours. At h days the steak
was partially purple, and after 6 days it was all purple.
The steak treated with enzymes and the uninoculated steak
began to discolor after 6 days, probably due to bacterial
action.

8%

Two experiments were completed in desiccator jars
testing the effects of iodoacetate, aureomycin, and ka.
In the first test, steaks wrapped in hSAT 80 cellophane
were used. In the nitrogen atmosphere, all steaks turned
purple after 2 days and remained as such until the ex-
periment terminated. All hie steaks which were exposed
to an atmosphere containing 10 mm 02, except that treated
with ka, began to turn brown at 3 days and remained
brown to the tenth day at which time the steaks became a
mixed brown-purple color. The steak treated with th
began to turn purple after 3 days. The surface pigment
of the steak treated with kCn was completely reduced
after 5 days, tut at no time was the brown color of
metmyoLlobin observed. Steaks exposed to 75 mm 02 became
red upon refrigeration, but not the bright red that was
observed on steaks held at normal or complete oxygen
atmospheres. The control steak was first to discolor.
Iodoacetate afforded about 1 day of color preservation
and the aureomycin about H days as compared to the control.
The ka-treated steak retained its red color for about a
day longer than the aureomycin-treated steak and then be-
came a dark red and finally purple. Again, all the
steaks except that treated with kCh became brown before
turning purple.

The color changes observed with steaks held at
normal atmosphere were similar to those held at 75 mm 02

except that the initial color was bright red. Steaks

85

held in an atmosphere of oxygen retained the bright red
color 3 days longer than at normal atmosphere. It was
also noted that these sterks turned a purple color with
only little evidence of metmyoglobin formation.

The various bacterial inhibitors did show some
color retention, but only at atmospheres containing high
amounts of oxygen. In an atmOSphere of all h2 and 10 mm
02, they were inactive. It should be noted that at no
time was the formation of metmyoglobin clearly defined
on those steaks treated with kCh except in an atmosphere
of all oxygen.

The above experiment was repeated using steaks
which were not wrapped. The hCh treatment was omitted.

The results were similar to those of the previous experiment.
From this information,it appears that the oxygen
present at the steak surface plays an important role in the
rate of discoloration. At lower oxygen tensions (10 mm 02),
metmyoglobin is formed within 3 days while at higher oxygen

tensions (normal) metmyoglobin is not produced until the
fifth day. It would appear that at the lower oxygen
tensions bacteria play only a small part, if any, for the
addition of bacterial inhititors (excluding hCh) does not
slow down the metmyoglobin formation. At higher oxygen
tensions, however, the opposite is true, that is, bacteria
do play an important role in metmyoglobin formation for

the addition of bacterial inhibitors, especially

86

aureomycin, slows down metmyoglobin formation
considerably.

To check the effect of temperature on these color
changes, steaks wrapped and unwrapped, with and without
the addition of hxkacetate were placed in atmospheres
of all nitrogen, nitrogen plus 10 mm 02, nitrogen plus
75 mm 02, normal, and all oxygen. Storage was at room
temperature.

Steaks in the nitrogen atmosphere became purple
immediately and remained so through the experiment. In
the other atmOSpheres browning occurred within 2 days.
The steaks held in the normal and all-oxygen atmospheres
developed a large amount of metmyoglobin as did the un-
wrapped steaks and the steaks treated with iodoacetate

held at 10 mm 0 The other steaks turned purple with-

2.
out becoming a definite brown. Thus, at room temperature
the formation of metmyoglobin was accelerated, but the
reduction to myoglobin was also accelerated. In the all
oxygen atmosphere, steCks were all brown at 2 days, while
at refrigerated temperature no definite browning could be
observed. Iodoacetate did not decrease metmyoglobin
formation, but in all cases tended to increase its
formation.

The second experiment in this series was the same

as the first except that various oxygen levels were

obtained by drawing partial vacuums in desiccating jars.

87

Considering that air contains approximately 20p oxygen,
and knowing the barometric pressure, a vacuum can be
pulled to the point where the gas left in the desiccator
jar would contain the amount of oxygen desired. (For
example, if the barometric pressure is 740 mm of mercury
and a vacuum of 6MO nm of mercury is drawn, 100 mm of
gas would be left in the jar of which 20% or 20 mm is
oxygen.) Results indicate that at room temperature
metmyoglobin formation proceeds at a maximum rate near
an oxygen tension of 20 mm 02. Again, steaks treated
with iodoacetate tended to form metmyoglobin more com-
pletely except in an atmosphere containing 20 mm 02
where all steaks became brown about the same time.

The third experiment in this series was undertaken

to test the effect of fig. geniculata on steaks held in

 

partial vacuum with various oxygen levels. Again
various oxygen levels were obtained by pulling partial
vacuums in desiccator jars. All the inoculated steaks
at the various oxygen levels turned purple within one-
half hour and remained purple. The uninoculated steaks
exposed to oxygen levels of 20 mm and #0 mm demonstrated
some browning on the second and third day, but the rest
of the steaks turned purple without browning. Es.

geniculata hastened the formation of the purple color

 

at 20 mm 02, 40 mm 02, and 60 mm 02 and kept the pigment
of steaks in the reduced form. The pigment of the

88

uninoculated steéks was partially oxidized to metmyo-
plobin before being entirely reduced to myotlolin.

It has been shown that at reduced atmospheres
especially at atmospheres containing 10 nm 02 the forma-
tion of metmyoglobin from oxymyoLlobin on steaks is
accelerated. This occurs even when the bacterial in-
hibitors are added, but it was not known if all the
bacteria had been inhibited. The following experiment
was conducted to see if metmyoglobin could be produced
on pieces of meat without bacterial growth by reducing
the amount of available oxygen. To accomplish this,
pieces of meat were extracted by sterile technique,
placed in sterile petri dishes, and in turn placed in a
sterile desiccator jar at which time petri dish covers
were removed. Each jar contained three meat samples.
The various oxygen tensions were obtained by adding
mixtures of h2 and O2 to the jars after they had been
evacuated. All gases were run through a sterile glass
wool filter to keep conditions within the desiccator
jars free of microbial growth. All jars were refrig-
erated. Results (Table 13) show that at reduced oxygen
tensions the pigment of muscle tissue can be oxidized
to metmyoglobin within 36 hours without the presence of
bacteria. Spectrophotometric analysis as well as
visual observations bears out this fact. The rate of

metmyoglobin formation was essentially the same at oxygen

89

 

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tensions of 8 mm, 10 mm, 15 mm, and 20 nm. Bacterial
counts made by plating with tryptone glucose extract
agar (Difco) demonstrated no significant growth.

A second investigation (Table 1%) was performed
using a wider range of 02 tensions comparing excised
tissue to aseptically handled beef steaks. hesults
showed that steaks did not react to the various
atmospheres as readily as the excised tissue. At 10 mm
02 and 25 mm 02 the excised tissue turned brown within
#8 hours, and the steaks did not become entirely brown
until 96 hours at 10 mm 02 and lZO hours at 25 mm 02.

The important thing is, however, that the steaks did become
brown without the presence of a significant number of
bacteria. At 50 mm 02 the steak turned brown after lhh
hours while the excised tissue began to turn brown at #8
hours. Counts indicated that the tissue was without
bacterial growth, and the steak had insignificant numbers
of bacteria present at the time of browning. However, the
steak held under a normal atmosphere and at an atmosphere
containing 75 mm 02 became brown after 12 days' storage,
and counts revealed high numbers of bacteria (1 x 108).
This would indicate that the discoloration was caused by
the bacteria. The excised tissue became brown, but after

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Muscle Pigment Extracts

The importance of oxygen levels and bacteria on
discoloration of fresh prepackaged and unpackaged beef
steaks having been demonstrated, it was thought that
further information could be obtained by subjecting
oxymyoglobin extracts to various oxygen levels with the
addition of bacterial suSpensions and various bacterial
inhibitors. The oxymyoglobin solutions were placed in
Thunburg tubes, and the bacterial suspension or bacterial
inhibitor was placed in the side arm of the top of the
tubes. The top having been put in place, a vacuum was
pulled with a vacuum pump. Various oxygen levels were
obtained by pulling partial vacuums or by pulling a vacuum
and readmitting various oxygen and nitrogen mixtures. Be-
fore removing the Thunburg tube from the vacuum system,
the top was turned so as to close the internal gas system
from the external air. Change in pigment was determined
spectrOphotometrically. All of the Thunburg tubes used
were of the same optical nature, i.e., at a given wave
length the tules filled with water gave similar optical
density values. Also, before this method of pigment
measurement was used, its accuracy was tested using myo-
globin solutions treated with sodium hydrosulfite and
potassium ferricyanide. Sodium hydrosulfite reduces the
pigment to the myoglobin form, and potassium ferricyanide

oxidizes the pigment to the metmyoglobin form. Excellent

93

results were obtained in both ases.

Oxygen tension was found to have only a slight
effect on inoculated pigment solutions; that is, oxygen
tensions from near zero to l50 mm gave comparable results.

baker's yeast under atmospheric conditions caused
little or no change of the pigment oxynyoglobin, but at
a partial pressure of 10 mm 02 the yeast apparently had
changed about 80; of the oxynyoglobin pigment after 2
hours. The pigment :.ixture was found to contain the
following relative percentages: 27p metnyoglobin, Shfi
myoLlotin, and 19p ox*myoglobin. Glucose (C.lp) was
added to the pigment solution to enhance the metalolism

f the yeast cells.
Oxygen tension had no effect on pigment solutions

inoculated with Lg. geniculata. Therefore, an attempt

 

was made to see if reduced oxygen atmospheres had any
effect on "sterile" pigment solutions. Aureomycin was
added to the oxymyoglobin solutions in an attanpt to
render them bacteria free. The solutions held at various
oxygen tensions from lC mm 02 to normal atmosphere
demonstrated no essential difference after 3 hours. Each
solution was considered bacteria free for no bacterial
growth was observed when 1 ml of each solution was
plated out in ”GE agar and incubated for 1 week.
Thessadata would tend to minimize the importance of

oxygen tension in the reaction of oxymyoLlobin being

9n

either reduced to Lyoalotin or oxidized to met yoblobin
with or without the aid of bacteria. however, an
equilibrium between the gas phase and liquid phase with—
in the ThunLurL tubes may not have been reached. This
difficulty was partially alleviated by placing the tubes
wrapped in cheesecloth on a shaker between the readings.

An oxymyoglotin solution containing cells of fig. aeniculata

 

was compared to oxymyoilotin solution containing
aureomycin (10 ppm) to inhibit microbial growth. The
solutions were placed at oxygen levels of C, 5, 10, and
20 mm 02. hesults in Table 15 indicate that as the oxygen
levels are lowered, metmyoglotin and myoglobin formation
is increased in both lot 1 (aureomycin treated) and lot 2
(inoculated). In an atmosphere containing no oxygen at
40 minutes, Loth lots COLtained 80m myoglotin and metmyo-
globin at a ratio of 2:1. In the reduced oxygen
atmospheres (5, 10, and 20 mm 02) there is essentially no
difference in metmyoLlobin formation between lot 1 and 2;
however, the organisms in lot 2 increased the myobloLin
content sutstantially. This would indicate that the
bacteria caused an increase in the reduction of oxymyo-
globin but had no significant oxidative ability under
these COLdltiOLS. Further data at 02 tensions between
20 mm 02 and normal demonstrated results similar to
those obtained at normal 02 tensions.

Pigment chances of xymyoglotin solutions caused

by reduced oxygen tension and bacteria are slowed down

by refrigeration (M°C.), tut are not inhibited 1y it.

At M°C. is. geniculata demonstrated greater pigment

 

reducing and oxidizing ability in a partial vacuum than

at normal atmospheric COLClthLS compared to uninoculated
pigment controls which were suijected to the same con-
ditions. A day was required for the cells of fig. Leniculata
to change 80p or more of the picment from oxymyoglobin to a
mixture of myoglobin and nettyoilotin. Once more the

ratio was about 1 part netmyoplobin to 2 parts KYOglOtih.
The two pigment solutions treated with aureomycin
demonstrated no myoglotin formation after 12 days, yet

the sample 161d in a vacuum contained 813 metmyoglobin,

and that held at normal atmosphere contained 66%
metmyoglobin. At 3 weeks the measurements indicated

90p and 71% metmyotlobin respectively. to myoglobin was
observed. Plating in To; agar demonstrated no growth.

This would indicate that metmyoglolin can be produced

in extracts without bacterial activity. However, the

role of aureomycin in the reaction (s) is unknown.

Effe_t of Antioxidants

 

 

Three samples of antioxidants from the Griffith
Laboratories were used: G-h containint vegetable oil,
lecithin, propylgallate, and citric acid; G-lS con-
taining vegetable oil, lecithin, butylated hydroxytoluene,
propylgallate, citric acid, and butylated hydroxyanisole;

and G-16 containing vegetable oil, nonoglycerides,

96

 

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butylated hydroxytoluene, propylgallate, citric acid,
and lutylated hydroxyanisole. all were in liquid form.
Three samples from the Lastnan Chemical Products,
Inc. were also used. Tenox II, a liruid, contained
butylated hydroxyanisole, propylgallate, citric acid,

1

and propylene glycol; Tenox REA, crystals of butylated
hydroxyanisole; and Tenox PG which was propylgallate
crystals.

The three samples from the Griffith aboratories
were darn solutions, and initially slightly Lashed the
red color of the me t. All 3 gave similar results in that

they caused uninoculated fresh prepac‘ beef steaks to

h, .
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(1‘:
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discolor about 3 days before control steaks. The treated
steaks became Lrown (metmyoglobin) and remained brown for
about 2 weeks before turnin; purple (myoglobin). The control
steak turned brown (metmyoplotin) 3 days later than the
treated steaks, yet it remained Lrown only a day before be-
coming partially purple; within 2 days the surface pigment

of control steaas was completely reduced.

Tenox II gave results similar to the uriffith
products, but when the solution was first added to the
meat, a milk-like precipitate formed.

Tenox PG and Tenon LEA did not fully dissolve, and,
therefore, a portion regained as white crystals on the meat

surface. Tenox Pt gave szimilar results to the Griffith

98

products, i. e., browning after 3 days and preservation
of the brown color ior about 2 weeks. Tenox LLA gave
slightly different results. The steak treated vith
Tenox LLA did not turn brown until after the control
stean, but it retained the bronn color for a longer
period of time than the control steak. However, the
pignent of tie steak turned purple about a week before
the other antioxidant-treated steaks.

All of the antioxidants used slowed down the initial
rate of discoloration of steaks inoculated with Es.

geniculata; however, the steaks treated hith the anti-

 

oxidants retained the bronn color of netnyo¢lobin ior a

'1 .f‘
-;

perion 0 time than did the inoculated control

In general, tLe antioxidants used did rot delay the
formation of metmyoglobin, but after its formation did,
tend to keep it from being reduced to nyo:lobin.

A In solution of sodium isoascorbate uas corpared
to a 1» solution of ascorbic acid as applied to the sur-
faces of inoculated and uninoculated steaks. Data
observed indicated that neither solution had any apparent
effect on the bacteria present nor on the n netnyoglobin
formed. The ascorbic acid solution did slow down dis-
coloration of both the inoculated and uninoculated steaks
soneuhat, but sodium isoascorbate was ineffective.

Co paring the results obtained on uninoculated
steaks treated with lg solution of isoascorlic acid and
a IN solution of ascorbic acid, it was observed that

CEO

1']

there was no significant difference in regard to bacteria
counts and p metmyoglobin formed. Each demonstrated some
color preservation. Steaks which were inoculated and
treated with the two antioxidants gave similar results, but
the color preservation was less pronounced.

The application of 10a sodium ascorbate, ION sodium
isoascorbate, or a mixture containing 5w sodium ascorbic
and 5% ascorbic acid applied to steak surfaces resulted
in no final color preservation and initially caused a
slight discoloration which remained for about 2 days. Ten
percent ascorbic acid when applied to a steak surface also
caused this initial discoloration (slight dark red) which
remained about 2 days but resulted in color preservation
of 4 days beyond that of the control.

Effect of Ascorbic Acid and of a Mixture of Ascorbic

Acid and Sodium kicotinate on the Color of
Prepackaged Steaks

A series of experiments were run comparing the effect
of la ascorbic acid (A.A.) solution and a solution contain-
ing 8% sodium nicotinate (h.k.) and 2m A.A. on the surface
pigment of meat. Visual observation, index of fading, and
bacterial counts were performed on the treated steaks and
on untreated steaks after various storage intervals.

The results of these studies are presented in
Tables 16 and 17. It was obvious that the N.h. plus A.A.
mixture resulted in a brighter red color initially and

maintained this color for a much longer period than that

100

p

of either the control group or the A.s. treated group of
steaks. This was true even when the steaks were inoculated
with a susyersion of is. geniculrta (Fig. 13). Lo effect

f the treatments on tie bacterial growth was evident.

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III! II INS

COMPARISON OF THE EFFECTS OFAMIXTURE OF

ASCORBIC ACID AND SODIUM NICOTINATE ON

INOCULATED AND UNINOCULATED PREPACKAGED
BEEF HELD AT l-I’C

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active in causip; pi rent clan;e is substantiated by
tvo fixtiiég3. First, t;¢.<: tracellnlar‘exn flies of L“.

Q
—

geniculata and Ps. aeruLinosa were round to Le inactive,

 

 

but tie intracellulfir erz mes oi alese organisms brwught

about tie discoloratior 01 fresh feat at rorm terzerature.

0)
.1
(Tx
1"
N
,4
rat
I x
(”I
O
I— '3

[he intracelltl

tie cLange oi' 3Qinw3-lolin solutions. In toth cases the

07
O
(+-
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O
5
O
I- b
(.5-
H4
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(n
H
[xx
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r
O
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a~
O
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;._
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O
p-
H
V:

slicht 0}ieative action was noted.

The second findir; mLich 2 lps to trove tLis

at

assuuptirn is tLat .ood correlation between steak cis-

coloration FLO respiratory activity of the organisms present

165

on the stash surface pas obeerved. is t1e number of
organians ant tgeir aero.ic res iration iLCSeEbed, the
steals lost tlo brit1t red color of o33myoglolin in favor
of a Car; rec color. tron itrt1er ilcrease in 1es; 111t1on

tne Exx“t ”13=a1¢t 1e c ::'irovn. sit Lnis gcxh1t the 1;:13 lo L1

pihnent was in 11c oxidi go As tbe respiratory

N
(u
}.l

1
H!

\D

(,1.
,Cf
rt
”‘3

activity contiruao to iocrease, tie oiicized flgtent,
metng,o ogloiin, ias Loon reduced to ryo‘lolin. Fi3nent

obsnges in tlis order 1ave also been 1e10“teo ly butler

...

et al. (lEE3 and Kallech 33 gl. (léit . Lotler aLo 115

(h
C”
H
1%
0)
O
(T1

COWOTLCITSJJLFtLLT obseflwmu1 correlctior11natueer

discoloration of freon yrepacl gee beef an: t1e number of

v tic LCt ELtCLfC to noasure

m

bacteria tresent, 11t t;

respiratorv activity.

}-.J

Various 1n0111ena1tma iLbilitors terw.e131cn1mi in orcer

<19}

4“

to obtain better 1novledbc o1

C)
be
P

srec1iic en23ne :3stexs

A

of fig. LéfilChlftf are active in LLe recuction and ozioaticn

 

01 ox3nj, o lo iL. 11a inakilitj oi C.Cl h sodiur iluorioe

ES KGESMTC‘

(1

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f

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Pb
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1.1.
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H
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I01

to inhibit th"
by the harbtrg Eds expected since at low corcentratiors
sodium fluoride inhibits eLolase only. This e1L3me is not
critical in respiration L3 1118 organisn since it follows
the genzmase-simnit 3211: av. 'ILe jihab.ilijnv to iifl;ilit

respiration ras correlated with ito 112 1111 1ty to in111it

the action of ‘s. geLicolata on Test color. Sodium fluoride

...—

 

at a concentration of C.l L oehorstrated LON res3iratory

of (iscoloration

H
C
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11-1.1111 T1101": 71"}.

1

n . . ' 3 .. 1, ”-3-. ”,1 J.,, ..9 ,1. 1. 1.- ‘, -._- ., .,
OI l‘c‘uf St‘i‘ErtS ES CELiirb‘C. L; L‘cCLfldIlEo 1.4111Cc ‘31'1f3f3- 1.C!z_ll.':f::::;

.1 ~ 1
are inactivated 1: sodiur fluoride at Ligber concentration,

this was as predicteo. sodium melonsto, iodoacetate,

fanic" caused inhitition of

 

t e res iratory L1L3 s of ~s. ;-1ic;lata. The degree of
of irLilition 1v e:c1 aéert {as vell correlateo yith t1e

abilitv of t7; inLiLitor to greserva the bright red color

-0
>/-\r‘ ‘11.
~,\-lu1.-.

O
H)
(I
‘Q
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C 1.
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(D
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’17
I\‘I
H.
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LL;
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1 11

/ inii.itec tLe o yien uptgke 01 both cells

01
[\‘I
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p
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' 4

v—r .1
H
(I
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t
H

are cell—free extracts of 1;. :arictl'ta, i aiforded LO

yrotection of Oivc'o lo in. Tovever, tLe ti Leflt cnznge
tricn occurs in tie Ireserce o1 sociuy inoe is 1ot (me to

bacteria, tat is Lee to a cleiical reaction betueeh the

globin IlLLLLt 21c tLlS ageLt. T-is state eLt is urie in

n:
.4
1
5+-
IF"
I
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H
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:5
m
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n
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light of tLe fact that bacteria treats;
anc otbsehuent13 1as1ec vere oralle to eit1er retuce or

--.° -, 1 r. _ 1 3 ° ....
04‘.l\r11_LZU 04'31‘li (3 iOllll .

.L
V v"\

inhibit t1e resyirator3 snz3 es of Is. 3enice :ta vas well

 

 

correlated 11th its ability t preserve tn: bright redness
of yrepaclaged L“at. Grant (19

various iLbiEitors 1e tested, orlr ralonic acid provided

(z‘

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a bri.bt e3 interior color of frozen {TOLIC beef. Sirce

'1 ,1 1,. . 1,. T 4. 1 ‘1. -1 7 ' 1. ’3- .L. .' - 1....
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Elie) 111 .nglib 0.1- L119; 5 LOVE: OL:_‘Cl'V; tic/1111;, lb 153‘ i.OSJClL.‘.Lt 1.:

as lifiit ermg. es.

From ire rtbulth o
erogfige iiAleitoIm;:it is
is leg ae"rfi.ic rtr iret
volvec in lee: discolor
respiratior 2n-'L fege;
f: g i\/'Lli C: l'LLHnC lug LC“; 1;:
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H~

iygen pressure Lezr lC
t'smnie elgd :u.sll. 5*51£ s
ozygtn tersicrs oi lC LL
bacteria. ope to Lle Cl
teeterie Cjz rot inste;

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hesults of various

occurred

PM

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fl
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lLVOl\bu

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ll.

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tests

p

r
hr

=t

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V. ’ q > ‘.
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(D

F“

I“

P\‘

vgich OCCered;

believed that they were inactive in this reaction. As
the temperature increased, the maximum rate of oxidation
was found to occur at higher oxygen tensions. These
results agree with those of Brooks (1931, 1933, and 1938).
Iaving shown that oxidation occurred at lower oxygen
tensions, two possible mechanisms were postulated. Both
were based on the theory that bacteria cause pigment changes
by lowering oxygen tensions. It is possible that at a
critical point oxidation occurs, but on further aerobic
metabolism the available 02 is lowered still further and
results in a strictly physiochemical pigment reduction.
In short, this simply involves either a physical or

biological limitation of O which results in nonenzymatic

2
pigment oxidation or reduction depending upon the degree

of limitation. The second possibility is that the pigment
is enzymatically oxidized at the reduced 02 tension. At
reduced oxygen atmospheres the activity of the cytochrome
system of both the bacterial cells and the meat tissue

would be greatly reduced while the action of any flavin
system present would not be so greatly reduced. Thus, one
might eXpect an accumulation of I202 which in the presence
of peroxidase would oxidize the pigment.

Sodium azide and potassium cyanide poison the cyto-
chrome system but have no inhibitory effects on flavin
systems. Thus, if there was appreciable flavin enzyme
activity in either cells or meat tissue slices at low oxygen

tension, either KaN3 or hCh would not inhibit respiration

109

~ 4.—
'u

- i ,. tr ~ ,— 4., ..
LO u;.b S;h-(;‘ Urbcll

m

s at the hither oxygen levels. hesults

.L

of such studies oemohstrated that this was not the case

with either cells of La. Lghict : or with meat tissue

\—

w.)

slices. This, glus the fact that hUL treated cells would
not cause either oridation or reduction of grepachaged
steahs held in normal or recuce; o ygen atmospheres, casts
cohsiCeralle Coutt Cl; tie validity oi” the enzymatic theory
proposed.

Lirce catalase also acts as a 1ero1idese, it is
‘ossille that at recuceo oxygen tensiors catalase loses its
C to molecular oLyLen and water,
yet the peroxidase activity remairs acthe. The geroiioase
could tten decoupose f O to tater anc atonic oxygen

7
‘C

f2
4

r1

utilizing Lyoglotin as an electron conor. inis action would
result in the formation of LEtKyOLlOLiUQ honever, as the
oxygen is further limited, ho H202 mould be formed, and no
further oxiuatioh toulu tlen occur. Also, the oxidized
pignent vould he reduced in the aLsehce of oxygen. heat
extracts were shown to have Leroxifiase activity, but all
efforts to demonstrate the presehce of g in meat under-
going oxidation were unsuccessful. however, £202 may be
procuced and subsequently Lroken Conn without being
detected.

Glucose oxidase in dilute solutions brought about the
oxidation of the surface ritment of steaks. However, in

more concentrated solutions the pigment was Chfnéed to

d.

110

choleglotih. iris enz*ne system produces 1:02 from the
oxidation of glucose ahc has an active catalase present
to breah it Coun. hydrogen peroxide (Co3p) was inactive
on fresh steaks, tut the iimgtnxt at the surface of aged
steahs tas readily oxicized to netm

rcglolin. Dilute

1
V

solutiors of IleliC

(1')

Se c user t;e oxidaticn of meat piL-
tents. Tris reaction could he lastehed by the ardition of
l;02; however, inactiviteu 1ar0iitase soluticrs were also
active. in: rate (iiferehce Las larLe enouph to show that

-

the enzrne LES active in the oxidation reaction. hore

d

concentrated solutiore oi 1eroxidase plus ILOC caused
L

r.

choletlobin iorhation. These r

r?-
a
r?-
he
3
(D

:ults cencnstra

(D
{A

possilility of a yeroxiCase system causirg pigment oxidation,

but the inalility to show the yresence of free haO in

I”:
C. L

tissues where tie gi:neht was undergoiht oxidation or of

k.

enhanced ilavin ehayme activity at low c}? en tensions

made it ityossille to conclusively prove that this rechanism

7‘7
'V

5 active. The stuty of this reaction vas hot yursued

0‘.

further since the niis investigation yes to study'the
role of bacteria in yiiment oxidation and reduction, and this
reaction has not directly caused ly the bacteria.

There can he little Ccuht that the prinary role of the
bacteria in the color changes of fresh peat tissue is in the
reduction of the ozygen level in the surface tissue. This is

supported Ly a number of “acts; vi;., (a) fiihent oricstion

and recucticn can he controlled Ly Lhysical acjustnent

avel in the store e atmosphere in the atssnce

O
F
C
F
C)
O
L_.J
a;
(' ’ \
U
H
F
H
(’w

of a significant number of tacteria,(h) the oiy;en level in
the storage atmosphere greatly affects the rate of pigment

1

changes on lotn inoculatec anc uninoculated steah

(.0

7

(I\
i—Jo
(fl
c—l
F5
(T‘

(c) oxygen uptale rate of tie surfac‘ t site 0 r=at is
directly correlated hltL color change; tius, the hither the
number: of aerolic orgtnisms on a given piece of meat, the

greater the oxygen oehanc and the more rapid the color

change, (d) at ihtermediate leV”lS of oxyien demand of

(1

surface tissue, oxidation to metnyoLlohin occurs while with
higher resgiration rates, recuction to myoglobin occurs;
and this is correlated with similar changes under controlled

niration

_.
L
L

U]

oxygen atmosphere , and (e) any atent inhititiha res

will result in color preservation providing it does Lot

‘-

chenically react with the pigment itself.

A , ‘3
UL L‘A-l LISLLJ.

All tLe aerolic orgaLisrs tested HJiCh iLcluoeo

*otacter,

\D

l}—
t“:
m
<
O
v—‘J
.13
0
C4.
(1
H
L
C
”:i

\D
.I
O

straiLs oi IfiooLridLES

 

 

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to reLoce oxyivoglotin at room te Lerature; hovever, OLly

tLose orgaLisLs mlich grow resfily at low tar rI tures
were iouLL to c use Lis ol or tion of refrigerated pre-

packaged Leef. At room terysratore, tLe iLtracellula

A r V ' f' . I ~'\‘ '. .". ‘1 r“ 4‘4- - ‘ -. ~ (~ 1* .~ . . " . - ~ rs .
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discoloratiOL 01 fretL rest, but the e>tr slitlai enzymes
were iLactive.

Loco correlatioL LEtto‘U steaL oiscoloration, Lumber
of bacteria preseLt on tLe stezn surface, anc respiratory
activity of tiese organisms was observed. The agility of
various eLzyme iLLiLitors to iLLiLit the respiratory

activity of Eé' ng as Leastred Lt COLveLtioLal

 

uarturg techLicues Les correlated titl tL alilit; of these
agents to preserve tLe ”bloom” of Loth iLoCLlated and con-
trol fresh prepackaged Leef steaks. Lodiun azide Cid Lot
inLiLit changes of ozyn; o lOLin even though it coxpletely
inhibited the respiratory eLzymes of ii. ggriculata. This
pigment change was COLsioered to be due to a chemical
reaction tetveen ozymyOglotin aLo sodium azide and not due

to tacteria.

The results show conclusively th't bacteria lower
the Oiygen tension at the surface of steaks, and the
lowering oi oxygen teLsions results in the discoloration
of the steaks. The type of pig ent charge which can be
observed depends upon the rate of free oxygen reroval from
the surface area; i.e., slow reduction of oxygen tensions
resulted in pigment oxidation, and fast reduction resulted
in pigment reduction. The pigment of neat tissue was
found to be oxidized at reduced oxygen tension (10 mm to
50 Lm Hg.) without the presence of bacteria. Therefore,
it appears that the Lain function OI tne bacteria is to
lower the oxygen level, and the pigment is then oxidized
or reduced either by non—enzymatic or enzymatic reactions
which may occur in the absence of microorganisms.

A study of the possible mechanisms involved in pigment
oxidation demonstrated that there was not a high activity

system at low ox*gen tensions resulting

(D

of flavin enzyn
in the formation of £202 either with cells 01 fig. geniculata,
beef tissue slices or homogenates. Catalase and perOLidase
activity was shown to enist in neat and neat homogenates.
hydrogen peroxide (0.3”) was essentially unatle to oxidize
the pigment at the surface 01 fresh beef steaks, or
oxymyoglobin solutions ottained from fresh meat; however,
when added to aced steaks or oxynyoglotin solutions from aged

steaks, oxidation was apparent. Dilute glucose oxidase had

114

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159

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BALL, C. 0., CLLUJS, W. L. and STILh, E. F. 1957 Factors
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(E) $92 - 35-500

BhOOhS, J. 1933 The effect of carbon dioxide on the color
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The effects of bacteria on tne color of prepackaged
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An improved method of determining methemoglobin. J. Biol.
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hEnLLbT, h. P. and MEIPELE, C. I. 1926 The identification
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16%

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hnILL, J. n. 1925c Stufies on tie oxidation- reduction

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color product. Food Tech., 11 567-573.

hICthT, J. A., LhEbsLLB, L., LALL, C. 0., ALL STth, 3. F.
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LICLBhT, J. A., TALL, C. 0., and STILL. L. 1. 1958
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LCJCI-FALLLLI, A. and VLCICA, A. 19H2 Lpecificata e
co posizic1m:cnxagica di giaglpdjima e emoglotira:<ti specie
diverse. Lata II Lall. d. soc. Ital. spcrun., I], 29 .

nCoJI-LALLLLI, A., VLLICA, A. and PLLLI, C. C. 1957
LioLloLina u uona cristallizzate. I Forma cristallina
solubilita, contenite in Fe in L. Lall. d. soc. Ital.
Liol. sperun., 23, 1-2.

LCCJI-FALLLLI, A. 1958 Crystalline Lunan myoglobin some
pL3siocLemical properties and cLeAical composition.

'1 r \ r)

r- .- . , . ‘ " (
QCiCIJCC, $122., lS’-.LU.

LOoJI-FALLLLI, A. 1950 M s piéient nuscularies. PLysio
.ainualogie (11nn.ch t1’1.slat1o11\.1tn C1A11AI/ ir1fu' lisLJ,
21-26.

hCoCI—FALLLLI, A. 1954 AmiIO acid composition of
o

crystallized Lunan r; Torin anc Laeno:lotin. prerientia,
AT, 72-

LOJJI- FA1LLLI, A. 1955 Le nio looine, Ciorrate liocleniche.
a o- ElCO- e e n - Shn a 1 Ln 1
It 1 1r lr tic e, M 95 ( 1 r 11 1isL).

LOLCI-FALLLLI, A. 1956 Lateropeneity of human myoaloLin.
- ‘ 7 0 fl ' - ' (I-r ‘
Arcn. Llocheu. anu Lioch3s., 65, 567-590.

‘1?"

1r ""

LLILLL, J. C. and PCTILL, V. L. 1953 The assay of
animal tissue for respirator3 enzymes. II Cuccinic
den3orocenas and c3 tocLIome oxidase. J. Liol. Chem.,
1112. 2:17-

JCLHLICLLI, L. J. 956 C1e1istry of Le t Iiyrcnt Froc.
of tle Cth. research corference C, Univer. oi C11icato,
Chicago, Ill. pp 61-64.

62L1L, J. 3., LALL, J. L. 111C, E. L. 193% spectro-
pLotometric characteristics of lemoeloLin I beef Llood
and muscle nemoglobina. J. Liol. C1e1., l_5, 741- 752.
STILLS, M. and FCCTLL, L. L. 1922 The preservation of
food L3 freezing witL special reference to fish and meat.
Report of the food investigation Loard, Ct. Eritain,
Special report no. 7, 161.

CLLLLAC~
on tLe Crowth rate of bacteria in ground neat. Food

CLL, Wm. L. 1952a Effect of freezing and thawiLg
C
Tech., g, 3A1—3A3.

167

ILLCLLLL, I. 1932 LristalliLiscLes Lyoglolin. I
Lrista llisteren aLc TeifilLULL des ryoulolin, s wie verlaufige
" tteiILIL Lber seiL LoleLLlar gewith. LiocLem - zeitsch.,

TiLCLLLL, I. 33% LristalliLiscLes ryoglolin III.
Lie-aLsolLte lithabsorLtiOL VOL ory-, co-, Leta—LLG reduz
myOElOUiL. LiocLem. seitch., égg, 95-63.

TZLCLLLL, In ElC lAL.L,<3. lC H7 Cryst lliLe ILJEflHIIyO lOCIL
frO);l ii 6C1rt-IJUSC16/ Cl.‘(.A [AI 1148. (LTCI.. L1001lbl:.. lg, 113-12%.

ULLAIL, b. L. 1952 Facts aLOLt LLEt color. oxygen is key
to tLe color iL heat. The LatioLal Irovisioner, lHG-IHH.

CLLLIL, g. L. aLd LLLLL1CCL, L. L. l9¥0 She Lame pigment
of cured feats. II :L applicctiOL of Van bl; he Leill
Lcloretilc La.s 5: er: th to tLe deterLiLatiOL of oxyten
capacity oi CilLte LCHOblOLlL solLtiOLs. Food research,
fi DC? (1/

VCZLLLI, L. 1952 TLe measureLeLts of fresL Leef muscle
color CLELLe Ly dish colorinetry. UngullisLed PL.L.
thesis, L.L.U.

VOLLLLI, L., lLLlLLLL, L. Q. aLd LALLLLL, h. L. 1952 Flow
sLeets on grepackzgec freshx ILC.§ ts . JOLr. article 10. 139'
LicL. agr. exp. sta.

‘l'U-Al):LC.-)l, Z... 1:. FIAd L;::;$C:‘.itl‘, l... L 0 19,528 Tile ei|f€3CtS Of
ascorLic acid on tLe oxicatiOL of LeLogloLiL aid the
formation of Litric oiice LGLOLlOLIh. rood L s earcL, l2,
“ATTQ, L. h. clu LELLnLI, L. To
meet color. looo iech., é, lQh-l

m
5
Q.

52b Ascortic acid
0

NHIIILL, G. I. l926 TLe LeLoLloLin of striated Luscle
I variatiOLs CLe to age eLd exercise. II va riatiors due
to aLemia and Ea alysis. ALer. J. my ioloty, Z6, 6 93— 707

and. ILC, 7O 7- 7].

NILLLLL, C. A. 1939a COIOLr of Le t. I aygaratus for
its measurement and relation Letween pH aLC colour.
CaL. J. LesearcL, I], l-7.

NILLLLL, C. A. 1939b ColoLr of neat II effect for

desiccatiOL on tLe colOLr oi CLred pork. CaL. J. Lesearch,

$29, 29‘3“.

168

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