THE DETERMINATi-ON OF 5‘ -MO;N.ONU.CLEOTIDES V
IN MEATS ' '

Thesis for rthe Degr’e’epf PH. VD. . 7
{MICHIGAN STATE UNIVERSITY "
RoberfD‘al’e‘ ‘rDaninert
‘ 19:66, , , .-

LIBRARY

Michigan State
University

 

This is to certifg that the

thesis entitled

THE DEPBRMINAFION OF 5'—MONONU3L£OTID£5 IN MdATS

presented by

ROBERT DAL “a D Ad‘vFT-I‘CTZT

has been accepted towards fulfillment
of the requirements for

Ph.D. Food Science

degree in

\‘4 $2 ’- J7/i . l\ ,,,f‘_(¢/’1fli‘;()“_/

Majoi“? professor

Date “’Ta'y Li" 1966

 

O~169

 

 

ABSTRACT
THE DETERMINATION OF 5'-MONONUCLEOTIDES IN MEAT

by Robert Dale Dannert

This study was designed to assess the level of 5'—mononucleotides
in the muscle of meat animals. Following hot water extraction, the
nucleotides were separated on a Dowex l K 8, 200—400 mesh (formate),
ion-exchange resin column. As the nucleotides were eluted from the
column, peaks were located with an 1500, Model UA, UV analyzer. Identi-
fication of the individual nucleotides was made with thin—layer and
paper chromatography as well as from the absorption Spectra and from
the ratio of absorbance at 250/260 and 280/260 mp. Quantitative
evaluation of the 5'—mononucleotides present in each peak was ac-
complished by using a periodic acid — 2,4-dinitr0phenylhydrazine
(2,4uDNPH) color reaction, which effectively differentiates the 2'-
and 3'-nucleotides from the 5'—isomer.

The first portion of this study dealt with the concentration of
5'-mononucleotides in three muscles from each of six pork carcasses.

The muscles examined were the longissimus dorsi, biceps femoris, and

 

semimembranosus. The concentration of inosine monophOSphate (IMP)

 

was 3.23, 3.12 and 2.98 pM./g. of tissue, reSpectively, for these
muscles. The differences were not statistically significant. The
standard deviation was 0.33, 0.42 and 0.40 pH./g. of tissue, for the

longissimus dorsi, biceps femoris and semimembranosus muscles, re.

 

 

 

spectively. Since the muscles had similar IHP concentrations and

the longissimus dorsi had the smallest standard deviation, it was

 

Robert Dale Dannert

used in all subsequent studies. It was also easily accessible and less
damage was done to the carcass on removing the sample. The level of
adenosine monOphOSphate (AMP) was considerably lower than the level of
IMP, with values of 0.33, 0.90 and 0.33 pM./g. tissue being recorded
for the longissimus dorsi, biceps femoris and semimembranosus muscles,
reSpectively. Guanosine monOphOSphate (GNP) was not detected, while
cytosine monophOSphate (0MP) was found in trace amounts only. The
level of uridine monophOSphate (UMP) was 0.13, 0.20 and 0.11 uM./g.

tissue, reSpectively, for the longissimus dorsi, biceps femoris and

 

semimembranosus muscles. These values as well as all subsequent values
were the average of duplicate determinations of the samples being
analyzed.

Part two of this experiment was designed to elucidate the change

in concentration of IMP and AMP in the lopgissimus dorsi muscle from

 

the carcasses of six Holstein heifers during a 28 day aging period at
33-35OF. Samples were frozen in liquid nitrogen after removal at 0,

l2 and 24 hours and at 4, 7, l4 and 28 days post—mortem. The level

of IMP was 4.71, 5.44, 4.86, 4.47, 3.20, 2.17 and 0.75 pE./g. tissue,
reSpectively, as the aging period was increased. The peak concentration
of IEP occurred at 12 hours. The level then declined until at 24 hours
the IMP content approximated that at 0 hours. It remained fairly
constant until the 4th day, after which a linear decrease occurred
until the 28th day of post-mortem cold storage, when less than 153 of
the IMP remained. Statistical analysis revealed that all means were
significantly different (P<;0.01) except for the values at 0 and 24

hours, and for 24 hours and 4 days. This suggests that, except for

Robert Dale Dannert

the peak IMP concentration at 12 hours, only small changes occurred
during the first four days of cold storage. Consistent decreases were
noted thereafter. The concentration of AMP was much lower than that
of IMP, with the 0 hour sample containing only, 0.94 uM./g. tissue.

The AEP content then decreased to approximately 0.37 pM./g. tissue by
4 days storage. This value remained relatively stable during the
remainder of the aging period. CMP and UHP were found in trace amounts
and GMP was not detectable.

In a third study, the nucleotide content of the longissimus dorsi

 

muscle of samples of pork removed at 0 and 48 hours, beef at 0 hours,
bull and lamb carcasses at 7 days and pork heart muscle at 0 hours was
compared. Due to the wide range in sampling and handling procedures,
comparisons between Species was difficult. The concentration of IMP
was 4.38, 3.23, 4.71, 2.8', 2.24 and 0.13 pH./g. of tissue, reSpectively,
for the above listed Species. The level of AEP was 0.70, 0.33, 0.90,
0.76, 1.21 and 2.30 uh./g. tissue, reSpectively, for the same Species.
Low levels of CHP and UKP were observed, while GNP was not detectable.
In order to assess the flavor enhancing prOperties of IMP, MSG
and IMP + GEP, these flavor potentiators were added to a standard
frankfurter formulation. This formulation utilized pork heart muscle
as the meat source, since it was demonstrated that pork heart contained
little or no TIP. A 20 member taste panel evaluated the frankfurters
using the 9 point hodonic scale. Scores of 6.40, 6.20, 5.55, 5.35,
and 4.75 were recorded for formulas containing IMP + GJP, EIP, MSG,
pork heart muscle alone (negative control) and all lean beef (positive

control), reSpectively. Statistical analysis demonstrated that the

Robert Dale Dannert

addition of IMP + GMP to frankfurters made from pork heart increased
their acceptability over the beef frankfurters (P<10.0l) and over the
frankfurters containing heart muscle with no added flavor potentiator
(P‘K0.05). When IMP was added to pork heart frankfurters, they were
preferred over the all beef frankfurters (P<:0.01). All other differ—
ences were not statistically significant, even though there was a trend
which indicated that the panel preferred the frankfurters containing
pork heart muscle with added flavor potentiators. Thus, results
suggest that the flavor enhancers improved the flavor and texture of

frankfurters made from pork hearts.

THE DETERMINATION OF 5'-MONONUCLEOTIDES IN MEATS

By

Robert Dale Dannert

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

Department of Wood Science

1966

JCKNOWLEDGMiiTS

r"he author wishes to express his gratitude for financial support
to the United States Department of Interior, Bureau of Commercial
Fisheries for the graduate fellowship and to Michigan State University
for the graduate research traineeship.

The author is eSpecialIy indebted to his major professor, Dr. A. M.
Pearson for his guidance in selecting the author's course of study and
for directing the research problem. Gratitude is also expressed to the
other members of the author's guidance committe, Dr. B. S. Schweigert,
Chairman of the Department of Food Science, Drs. J. R. Brunner and J. F.
Price, Department of food Science and Dr. C. H. Suelter, Department of
Biochemistry for their guidance in selecting the author's course of
study.

The assistance of the fierck Chemical Company, Rahway, flew Jersey for
providing samples of Hertase, monosodium glutamate and inosine mono-
phOSphate is gratefully acknowledged.

The author wishes to express his deepest appreciation to his wile
Sue for her faith, loyalty, understanding and confidence throughout the
duration of his graduate studies. Eurther, gratitude is expressed for
her assistance in the preparation and typing of this manuscript. The
long standing encouragement and inSpiration of the authors parents is

also acknowledged.

ii

TABLE OF CONTENTS

D] TROD UC TI ON 0 O O O O O O O O O O O 0 O 0 O O O O O O

EPERIE-liquMJ OBJmTIViflS o o o o o o o o o o o o o o o 0

LITERATURE REVIEN . . . . . . . . . . . . . . . . . . .
5'—mononucleotides in flavor . . . . . . . . . . .
Synergistic action between MSG and 5'-nucleotides
Effect of heat on nucleotide concentration . . . .
Effect of freezing on nucleotide concentration . .
Effect of enzymes on nucleotide concentration . .
Effect of stress on nucleotide concentration . . .
Effect of muscle on nucleotide concentration . . .
Effect of storage on nucleotide concentration . .

Fish . . . . . . . . . . . . . . . . . . . .
Meats . . . . . . . . . . . . . . . . . . . .
Nucleotide concentration in other foodstuffs . . .

Pathway of inosine monOphOSphate formation and
degradation in muscle. . . . . . . . . . . . . . .

Methods of evaluating nucleotide concentration . .

Hater soluble flavor precursors. . . . . . . . . .

EXIDEME':EJTIQL PROCLJD U33 0 o o o o o o o o o o o r o o o 0

Experimental material . . . . . . . . . . . . . .
Muscle investigation . . . . . . . . . . . .

Beef aging study. . . . . . . . . . . . . . .

iii

Page

10
12
15
17
18
20
21
21
23
27

27
31

38

38
38
38

Species investigation . . . . . . .

Methods of evaluating nucleotide

Extraction

Chromatographic separation

Elution

Quantitative evaluation

Qualitative analyses. .

Sensory evaluation . . . . .

1‘ ‘1
hflPbi

IENTAL RESULTS AND DISCUSSION

content

Evaluation of techniques and procedures.

Muscle investigation .

Beef aging study . . .

Species investigation

Sensory evaluation

SUHNARY AHD COthUSIONS

BIBLIOGRAPHY.

APPENDIX.

0

0

iv

Page

39
4O

4O
41
42
42

47

5o
50
59
65
7 3
79

83

86

95

LIST OF TABLES

Table Page

1 Stability of inosine monophOSphate and guanosine mono—
phOSphate at different pH values and temperatures. . . . 12

2 Nucleotide content for beef muscle held at 5°C. and
37000 "' u\:O/gl tissue. 0 o o o o o o o o o o o o o o o o 23

3 Quantity of adenosine phOSphate after storage in
percent of quantity present immediately after
Slaughter O O O O O O O O O O O O O O O O O O O O 0 O O O 26

4 Sample time and location for beef aging study. . . . . . 39
5 Recipe for frankfurters. . . . . . . . . . . . . . . . . 48
6 Level of added flavor potentiators . . . . . . . . . . . 49
7 Recovery of 5'-nucleotides by ion exchange chroma-

tographic separation and colorimetric analysis . . . . . 53

8 Effect of extraction procedure on inosine 5'—
mOIlOphOSphate content 0 o o o. o o o o o o o o o o o o o 511”

 

 

 

9 Spectrophotometric analysis of known and unknown

nucleotide peaks — pH 7. . . . . . . . . . . . . . . . . 56
10 Results of thin-layer and paper chromatography

analyses of nucleotides. . . . . . . . . . . . . . . . . 58
11 Inosine 5‘-monoph08phate content of 3 pork muscles —

)«fi‘.o/:o tj-SS1\J.e. . O O I . O 0 O O O O O O O O O O O O O O 60
12 Inosine 5'—monoph05phate content of lonfiissinus dorsi

muscle in beef aging study - pn./g. tissue . . . . . . . 65
13 Statistical analysis of the inosine nonophOSphate

concentration means of beef aging study. . . . . . . . . 66
14 Thin-layer chromatography of nucleosides and bases from

a sample of the longissimus dorsi muscle of beef . . . . 7O
15 Inosine 5'—mon0ph05phate content of longissimus dorsi

muscle of bull, beef, lamb and pork and of pork heart

muSCle "' p.:o/(:o tissue 0 o o o o o o o o o o o o o o o 0 71}
16 Results of taste panel evaluation of added flavor

potentiators . . . . . . . . . . . . . . . . . . . . . . 80

Figure

N

O’\

LIST OF FIGURES

Structure of 5'—mononucleotides involved as flavor
ehhmcers O O O 0 O O O O O O O O O O O O O O O O O O 0

Diagram of the gradient elution apparatus. . . . . . .
Standard curve for inosine 5'—monophosphate. . . . . .

Chromatographic separation of a synthetic mixture of
5‘-mononucleotides . . . . . . . . . . . . . . . . . .

Chromatographic separation of 5'-mononuc1eotides in an
unknown sample of Biceps femoris muscle from pork. . .

 

Thin-layer chromatogram of mononucleotides . . . . . .

Level of inosine 5'—monoph08phate in the longissimus
dorsi muscle of beef during a 28 day storage period
at 33"3503‘0 o o o o o o o o o o o o o o o o o o o o o

 

Thin-layer chromatogram of nucleosides and bases . . .

51
52

57

67
71

Table

LIST OF APPENDIX TABLES

Nucleotide content of 3 pork muscles (48 hours post-

mortem) - )fl‘Eo/go tissue. 0 o o o o o o o o o o o o o o o

Adenosine 5'~mon0ph05phate content of the longissimus

 

dorsi muscle in aging study - pM./g. tissue. . . . . . .

Nucleotide content of the longissimus dorsi muscle of

 

bulls -' my]. 0 /g 0 tissue 0 Q C O O O O O O O O O O O O O O 0

Nucleotide content of the longissimus dorsi muscle
Of 13.1an - PIE. 0 /g 0 til—S sue O O O O O O O O 0 O O O O O O O

 

Nucleotide content of the longissimus dorsi muscle of
pork (0 hour) and of pork heart - pM./g. tissue. . . . .

 

Results of individual tasters from taste panel
evaluation of flavor potentiators — (9 = highest value).

Analysis of variance of panel evaluation . . . . . . . .

vii

95

96

96

97

IETRODUCTION

Since man first found he could improve the flavor of food, scientists.
the world over have been attempting to make our foods more palatable.
Great strides have been made in this area. Pasteurization, aseptic
canning, dehydration and freezing, and more recently freeze drying and
irradiation, have all contributed in giving the food processor wide
versatility in the products he manufactures. Thus, a variety of nutri-
tious and flavorable products is available to satisfy the needs of the
consumer.

Flavor is a primary factor in acceptance of food products. As a
consequence, interest in isolation and characterization of flavor in
various foodstuffs has grown rapidly. Even though meat is one of the
basic commodities which has been extensively studied, relatively little
is known about the flavor of meat due to the complexity of the chemical
reactions occurring on heating. The elucidation of meat flavor is com—
plicated by the fact that research has been directed toward both the
volatile or non—volatile components. Further complications have occurred
since experimental studies have been carried out on isolation of the
flavor precursors of raw meat, as well as in studying the flavors
developed during the cooking process.

The flavor industry has played a fundamental role in enhancing the

n

acceptability of food products. Recent research of;orts have pointed
out that some compounds can blend or modify flavors and are known as
”flavor potentiators", which may be defined as compounds that augment or

suppress the reSponse of the sensory orqans to the stimulus of food. They

are further characterized as being effective at very low levels and by

}.J

the fact that they add no flavor of their own. The presence of inosinic
acid in meat was first reported in the classical work of Justus von
Liebig (1803-73). He isolated and named inosinic acid after the Greek
word meaning muscle. Disodimn inositate, disodium guanylate and mono-
sodium glutamate have proven useful in improving flavor scores of some
meat and vegetable mixtures (Caul and Raymond, 1963, Kurtzman and
Sjbstrbm, 1963, Terasaki 32 gl., 1965). The precise flavor effects
obtained with inosinic acid depend to a large extent on the particular
food to which it is added. In products such as soups, addition of
inosinic acid results in a fuller flavor and an impression of increased
Viscosity. Disodium inositate also can suppress undesirable sulfide
flavors in foods (Jagner gt gl., 1963) and radiation off-flavors asso-
ciated with sulfur compounds (fierrit §t_§l., 1959).

Until recently, methods of analysis made elucidation of the chemical
components contributing to meat flavor a tedious and difficult task.
flavor studies have depended primarily upon subjective measurements of
taste panels. However, recent developments in instrumentation provide
both accuracy and sensitivity, which can be used to supplement standard
organoleptic and chemical procedures. Physical methods have become in-
diSpensible to the food scientist. Volatile materials are new readily

.1

characterized ey gas chromatography, infrared and/or ultraviolet Spec-
trosc0py and mass Spectroscopy.

Separation of non—volatiles can be accomplished by dialysis through
membranes of known pore size, ion exchange or molecular sieve chromato—
graphy. Ionophoresis is available for rapid separation of nucleotides

(Bergkvist, 1959). Identity can be verified by paper or thin layer

Chromatography and by K-ray diffraction. Undoubtedl', new and more

accurate procedures will be evolved in the future to further classify

meat flavor and its precursors.

 

EXPERIMENTAL OBJECTIVES

The primary objective of this experiment was to determine the
concentration of inosine 5'—mon0ph05phate (IMP) in muscle tissue.
More Specifically, the objectives were as follows;

1. To determine the concentration of IMP in different muscles
from the sane animal.

2. To determine the concentration of IhP in samples of beef
muscle taken at varying times during aging at cooler temperatures.

3. To determine and compare the level of IMP found in samples of
bull, heifer, lamb, pork, and pork heart muscle.

4. To assess the flavor enhancing properties of IAP.

LITERAT _T RE EH

Sjbstrbm and Cairncross (1955) divided the term flavor into four
broad groups. They designated the first as taste. It included the four
well known basic sensations of sweetness, sourness, saltiness and bitter—
ness. The second aSpect, aroma, was used to describe sensations per—
ceptible to the nose. The term body was reserved for texture, even
though it makes no contribution toward taste or aroma. Finally, mouth
satisfaction was characterized by increased salivation, pleasantness
and smoothness of blending with very little contribution to taste or
aroma.

Crocker (1937) defined flavor as that property of a food or bever-
age that excites the senses of taste and smell. He went on to state that
a food might rate high in every aSpect except one, but because of that
fault might be hastily refused by the consumer. Kramlich and Pearson
(1958) also emphasized the importance of flavor and indicated that little
is known concerning the chemical components contributing to flavor. As
a result many researchers have attempted to characterize the components

of meat flavor.

5'—menonucleotides in flavor

The nucleotides involved in flavor are termed flavor enhancers and
more recently flavor potentiators. This term, a though new in the food
industry is well known in some fields, such as pharmacy. A flavor po—
tentiator has been described as a compound which augments or suppresses
the reSponses of the sensory organs (odor, taste and mouth feel) to the

stimulus of food. They are further characterized as being effective at

0\

very low levels, and by the fact that they do not add any flavor of their
own. This definition is in agreement with Sjbstrbm and Cairncross (1955),
who have defined a seasoning agent as a material, which, on being added
to a food, alters and corrects the aroma and flavor, mainly by blending
the character notes and by augmenting the total flavor 'apression without
being necessarily detectable.

In 1913, Kodama first reported that the histidine salt of inosinic
acid (IMP) was the major flavoring substance in dried bonito, an impor—
tant seasoning material used in Japanese cookery. However, IMP was not
produced as a seasoning iniredient on a large scale until quite recently,
because both the biochemical background and the flavor enhancing preper_
ties of IMP are quite complicated (Kuninaka.§t'gl., 1964). Recently,re—
markable advances in the biochemistry and organic chemistry of nucleic
acids have made it possible to industrialize the production of the 5'—
ribonucleotides in Japan. This has resulted in the application of these
compounds to food as seasonings.

Gaul and Raymond (1964) used samples of dried beef—flavored noodle
soup to assess the flavor enhancing properties of inositate added at a
level of 0.015 to the reconstituted soup. A flavor—profile analysis and
a consumer home—use test were used for evaluation.

The flavor-profile analysis revealed the following:

a) The inositate sample created an impression of fuller flavor,
which was attributed in part to the sensation of greater viscosity and
in part to a higher degree of blending of the flavor notes. In contrast,
the control soup seemed thinner, and the individual flavor notes were

more discernable.

b) The inositate soup had a flavor which was more brothy, or more
like meat stock, and which was less identifiable by its hydrolyzed vege—
table protein character. The control soup gave the impression of a
"bouillon cube".

c) The vegetables in the inositate soup had more individual flavor
identity than in the control soup.

d) The needles seemed to taste less starchy when inositate was
added.

e) The salty tastes were different in that the control soup had a
long lasting effect, while the soup containing inositate appeared to
reach a peak and then the salty taste decreased rapidly.

Only 102 of the families failed to discern or report flavor differ-
ences between the 2 soups. In general, the panel recognized l or more
differences correSponding to the flavor-profile analyses.

Jagner gt 31. (1963) reported that to a large extent the precise
flavor effects obtained with disodimn inositate depend upon the particular
food to which it is added. Thgrsuggested that there are, however, certain
effects which seem to be characteristic. These effects included a better
blending of the individual flavor notes, a fuller flavor and an impression
of increased viscosity on adding to soups. furthermore, disodium inositate
can also suppress undesirable sulfide flavors in food, as well as to modify
sourness in many products, which results in an over-all decrease in harsh-
ness.

The flavor enhancing ability of the nucleotides is entirely dependent
upon structure, which is very Specific. In 1960 Kuninaka summarized the
tastes of various PEA derivatives. In the process, he demonstrated that

these compounds are made of 3 components, namely, a nitrogen base,

CD

a ribose sugar and inorganic phOSphate combined in a ratio of 1:1:1.
Figure 1 shows the general fonaula for the 5'—ribonucleotides, which are
effective as flavor enhancers. The base moiety is a purine containing
a hydroxyl group at its number 6 carbon atom, while the 5‘—position of
the ribose is esterified to phOSphoric acid. when inorganic phOSphate
and inosine or ribose 5‘-phosphate and hypoxanthine were combined in
equimolar mixtures no flavor enhancing ability could be detected.
Kuninaka therefore concluded that ribosidic and phOSphate ester linkages
were essential for flavoring action. fiHe was also able to demonstrate
that the 2'— and 3'—mononucleotides imparted little or no flavor. Thus,
according to his scheme, 3 compounds possessed flavor activity, namely
5'—inosinic acid, 5'—guanylic acid and 5'—x thylic acid. Guanylic and
Xanthylic acid were found to be of lesser importance in muscle, since
they occur in extremely low concentrations.

Toi gt a1. (1960) and later Jagner gt 31. (1963) reported that the

recognition threshhold for disodium inositate was 0.02Q or 200 ppm.
Fujita gt a1. (1961) reported this level to be 0.012; or 120 ppm. and
0.00353 or 3.5 ppm. for disodium inositate and disodium guanylate,
reSpectively.

Schimazono (1963) also reported that the threshhold values for
disodium inositate and disodium guanylate in aqueous solution were 120
ppm. and 3.5 ppm., reSpectively. Disodiwn guanylate has about 2.1 to
5.5 times as much flavoring capacity as inositate according to Schimazono.
He noted that this ratio varied with their concentrations and with the

constituents in the food utilized.

 

 

 

 

If

>4
ll

H = 5'-inosine monOphOSphate

><
ll

NHZ = 5'-guanosine monophOSphate

>4
ll

OH = 5'-xanthine monophOSphate

Figure 1. Structure of 5'—mononucleotides involved as flavor enhancers.

 

 

 

10

Synergistic action between M80 and 5f—nucleotides

 

In 1964, Kuninaka et a1. pointed out that there is an additive
effect when two acids or sugars are mixed. A contrasting effect occurs
on adding a small amount of acid to a sugar. Kuninaka (1960) pointed
out that there is an additive effect between guanylate and inositate,
and that the taste of the two compounds is qualitatively the sane.

He also indicated that both these compounds have a synergistic effect
with MSG.

Kuninaka gt 31. (1964) demonstrated that succinate and the basic
amino acids, such as histidine, lysine and arginine, do not significantly
enhance the flavor or taste of either inositate or guanylate. On the
other hand, MSG greatly enhanced the flavor of these nucleotides. The
reverse was also true. These authors went on to show that only small
amounts of the nucleotides have remarkable capabilities for enhancing
the flavor of M38. In order to assess the flavor enhancing ability of
the 5'-nucleotides on £33, various ratios of IAP (or GEP) and KEG were
prepared in a solution of 1.2} sodium chloride. The flavors of the re—
sulting solutions were compared by paired comparison tests with salt
samples containing 0.3$.3SG as a standard. It was clearly demonstrated
by this experiment that there is a significant synergistic effect due to
IMP or 3MP. For example, when £53 and IAP were combined in a ratio of
100:1, 505 less 183 was required to duplicate the taste of the standard
0.35 H80. This work also indicated the synergistic

solution containing

1 3

v' a
1‘ L“" «‘rr u‘

action between use and 03? was about 4 times greater than that of not
and IHP.

Fujita gt 31. (1961) claimed that other compounds have a synergistic

ll

effect on the taste threshhold of IMP. They demonstrated that the thresh—
hold recognition values for disodium inositate in solutions of 0.1% sodium
aSpartate, MSG, beta hydroxyglutamate, sodium alpha amino adipinate and
homocysteinate were reduced considerably, when compared with inosinic
acid mixed with water. The threshhold levels for recognition of sodium
aSpartate, MSG, sodium beta oxyglutamate and sodium alpha amino adipinate
in 0.01% of 5'-disodium inositate solution were reported to be 1/16, 1/15,
1/15 and 1/100 of those reported in water, reSpectively (Toi gt 31., 1960).
The interrelationships between disodium inositate, disodium gua—
nylate, M80 and beef extract were examined by Schade 32 31. (1963a).
Various levels and combinations of M33, IMP and/or GNP were subjected to
taste panels for evaluation by using ranking, triangle, multiple compari—
son preference and/or difference tests. When IMP (0.005fl) was compared
with MSG (0.15%) in commercial vegetable bouillon soup, which already
contained MSG, the taste panel found a significant difference in flavor
using a multiple comparison test. The untreated commercial product was
used as a control. Triangle tests were conducted to compare bouillons
containing high levels of hSG against bouillons containing low levels of
H80 and 5'-nuc1eotides. Results indicated that replacement of 0.155 fidd
by 0.0055 IEP (30:1) or replacement of O-ZOE [5G by 0.0015; GKP (133:1)
gave significant and highly significant differences in flavor, reSpectively.
In this study 505 of the panel detected the substitution by IMP. Of
those finding the difference, 67} preferred the product with IMP.
Utilizing a multiple comparison preference test, beef extract at a
level of 0.25$ was compared by Schade gt g1. (1963a) with 0.0255 IEP.
Omission of 0.255 beef extract from the standard bouillon resulted in a

product which was significantly less acceptable, whereas, the substitution

12

of 0.025$ IMP for the beef extract resulted in a product which was signi—
ficantly more acceptable. They concluded that IMP can be used to replace
beef extract in a ratio of 1:10 and will still improve the acceptance of
the product. However, the authors noted that the flavor of the product
containing IMP was distinctly different from that containing beef extract.
Therefore, the 5'-nucleotides may find their most advantageous use in

extending the desirable effects obtained with beef extracts.

Sffect of heat on nucleotide concentration

 

Schade gt 31. (1963b) examined the stability of IMP and GMP by using
commercial food processing conditions of time and temperature of heating,
pH and storage. Solutions of IMP and GNP in buffer solutions at pH 3.0,
3.5, 4.0, 4.5 and 5.0 were heated at 100°C. for 30 or 60 minutes and
analyzed for nucleotide content to assess stability. Results are shown
in table 1.

Table 1. Stability of inosine monophOSphate and guanosine mono—
phOSphate at different pH values and temperatures

 

5 Recovery

 

 

 

 

 

30 minutes 60 minutes
p_I:I_ GMP mp GMP IMP
3.0 95 92 90 88
3.5 95 100 91 95
4.0 97 100 89 97
4-5 97 99 89 99

5.0 100 100 94 98

 

 

 

13

The data indicate that near neutrality the nucleotides are quite
stable, but as the pH is lowered into the acid range they become less
stable. In addition, similar buffer solutions were sterilized and stored
at room temperature for 3 to 6 months. Schade gt g1. (1963b) were unable
to detect any loss of nucleotides during storage. This would indicate
that the destruction of IfiP and GMP in solutions is dependent on pH,
heating time and temperature. Furthermore, results would suggest that
IMP is quite stable in water extracts of meat tissue, since the pH would
be above the low critical pH where destruction commences.

In order to investigate the effects of commercial processing and
storage conditions on the flavor enhancing activity and chemical stability
of IMP, 5 soups were prepared by Schade gt 31. (1963b). Each soup was
prepared without adding IIP and at two levels of added IKP. The soups
were stored for either 6 months at room temperature or for 5 months at
3800. The soups were then analyzed for IMP content and evaluated
organoleptically for flavor differences and preference rankings. The
initial analysis indicated that the heat processed condensed soups
showed an IMP loss, which varied from 125 to 355, with the higher losses
occurring in the samples with low INP levels. There was no loss of IhP
in dehydrated soups, since they were not heat processed. There was no
appreciable IMP loss in any of the 5 soups following 5 months storage at
3800. 'Hith one exception, this was also true for soups stored for 6
Inonths at room temperature. The one loss observed occurred with heat
jprocessed condensed chicken soup. The author felt that reanalysis could
solve the discrepancy in the data, since the level of IiP in this soup
liad not deteriorated after 5 months storage. According to taste panel

Immiults, all IIP treated soups were significantly different at the 15

14
level from the untreated soups, initially and throughout the study. As
a general trend, the panel preferred the soups containing the higher level
of added IMP.

-Fujita gt 31. (1961) reported similar findings. These authors
heated IMP and GMP at 10000. for 1 hour at pH values ranging from 4 to
6. No decomposition occurred with either product under these heating
conditions. They also discovered that heating IfiP or QEP at 1000C. at
pH 5 for 30 minutes in the presence of oxidizing or reducing agents
resulted in very little or no destruction, and that less than 105
destruction occurred during 1 hour of heating. Both IMP and GMP were
quite stable in aqueous solutions during storage for 1 year at near
neutral pH.

Various other workers have shown that relatively mild heat does
not inactivate IHP. Hanaoaki and Higashi (1963) separated IIP from the
boiling liquor of mackerel. They demonstrated that boiling for l to 2
hours caused no marked influence on the yield of HP. Longer periods
of boiling reduced IEP to inosine and phOSphoric acid.

Take and Otsuka (1964) extracted samples of katsuobushi (dried bonito)
under varied heating conditions. Although all s mples showed the presence
of IflP, a sensory panel found the samples heated to 6000. for 5 minutes
to be most acceptable. In 1965, these same authors prepared extracts
by heating dried fish at 6000. for 5 minutes, or by heating for 10 minutes
at 10000. They demonstrated the presence of IAP, inosine, hypoxanthine
and adenine. However, they were unable to correlate organoleptic scores
‘with the levels of IMP found in the extracts.

Shimazono (1963) found that hot water extracts of mushrooms

(Cortinellus shiitake) contained significant amounts of guanylic,

 

15
adenylic, cytidylic and uridylic acids in contrast to the low levels
found when the more conventional perchloric acid extraction procedure
was utilized. Further investigations of this phenomenon revealed that
the mycelium of the mushroom yielded a typical vegetable nucleotide
pattern with no CflP or QHP and fairly low levels of AIP and UMP. During
extraction with hot water, however, the 4 nucleotides were produced by
the breakdown of intracellular RNA due to the action of the relatively
thermo—stable RNA decomposing enzymes which were contained in the

mushroom.

“1.9 D

ulleCt of freezing on nucleotide concentration

 

Saito and Arai (1957a) examined the post—mortem changes in the
content of polyphOSphates in carp muscle stored at various temperatures
(160C., 000., - OC. and liquid air). The rate of conversion of ATP
appeared to be directly related to the temperature, with the slower
rates of freezing resulting in more rapid breakdown of ATP. The ATP
content was stable if frozen in liquid air. Later reports by the sane
authors (1958b, 1959) reported on the formation of IdP in carp muscle
when frozen at -30C. (1803.). They found it was possible to classify
the changes occurring in the nucleotide concentration of carp muscle
into three stages. During stage I, which occurred during he first 3
hours after death, only small changes took place. Large amounts of
ATP were converted to IMP during stage II (after 5 hours), while in
stage III (after 28 hours) only small quantities of II? were formed.
Erom this work the authors concluded that the pathway to IMP formation

'was: ATP -—-) ADP --—> AMP ---) IHP.

16

Another paper by Saito and Arai (1957b) traced the change in
concentration of IMP in carp and squid muscle after storage for several
months at —5°C. to —8°C. During prolonged storage, the IMP was gradually
converted to inosine and finally to hypoxanthine. The authors reported
that these changes were not microbial, but were due to enzymatic degra—
dation. Squid muscle appeared to be lacking the enzyme, AMP-deaminase,
since AMP was not converted to IMP at low temperatures.

Szentkiralyi (1957) used rabbit psoas muscle strips to demonstrate
the effect of freezing on ATP concentration. The excised muscle was
frozen immediately in an ether-dry ice mixture. The samples were thawed
in Kreb's solution and the rate of conversion of ATP to AMP was determined.
Other strips of muscle were analyzed immediately, while a third set of
fibers were immersed in the oxygenated Kreb‘s solution as a control.
Results demonstrated that freezing and thawing caused a rapid deamination
of the adenosine nucleotides along with a simultaneous Splitting of ATP.
In most cases, 75% of the adenosine was deaminated after thawing for 3
to 4 minutes, while the control strips of muscle showed only a small
change. From this experiment, which demonstrates thaw rigor, the authors
concluded that inosinic acid is produced almost instantaneously, if there
is maximal contraction and the recovery processes are inadequate. The
work of Okamoto gt El. (1957) is in agreement.

Jones and Murray (1961b) studied the effect of freezing on cod
muscle stored at temperatures ranging from -l4OC. (10°F.) to —300C.
(—l4°F.). The authors found that changes varied with the temperature
and time of storage. In general, the changes were very slow at lower

temperatures, if ATP conversion to IMP was used as a criterion of

17
measurement. Although the concentration of these compounds was not
markedly different at —14°C., the relative rate of change was more rapid.
The level of inosine rose to 3.01 uM./g. after 62 weeks at -l4°C., while
at —30°C. there was little change. At —l4°C., hypoxanthine increased
from trace amounts to 0.569 uM./g., while no changes were observed at
-30°C. Results suggest that samples stored at —30°C. are relatively
stable for an indefinite period of time. Other papers, which report
the effect of freezing, have been published by Sawant and Magar (1961a,

1961b) and Sameshima (1965).
Effect of enzymes on nucleotide concentration

The enzymes phosphatase and 5'—nuc1eotidase, which are found in
animal and plant tissues, cause the phOSphate group to be Split off the
nucleotide moiety (Chargaff and Davidson, 1955). When this occurs the
flavoring activity is lost. Inactivation procedures before nucleotide
addition, such as blanching, will prevent enzymes from causing nucleotide
destruction. Shimazono (1963) also observed that 5'—ribonuc1eotides
were rapidly decomposed when added to Soybean sauce containing a high
level of the enzyme phosphomonoesterase. This enzyme can be inactivated
by heat treatment at 80°C. Consequently, soybean sauce is heated prior
to the addition of nucleotides.

Fujita and Hasimoto (1960) found that fresh or frozen meat from
marine vertebrates contained high concentrations of IMP, while seaweeds
and invertebrate meats were quite low in IMP. Boiled, dried or canned
fish, generally contained quite high levels of IMP. The authors assumed

that the higher levels were due to heat inactivation of the enzyme

18
systems during processing. Results further substantiated the stability
of IMP during heat processing. These authors also followed the degra-
dation of IMP in jack mackerel. Although inosinic acid disappeared
completely during autolysis, a considerable amount remained in dried fish
of the same Species. These results suggest that rapid dehydration may

also be effective in preserving IMP.

j

t

=ffect of stress on nucleotide concentration

 

According to Hedrick (1965), stress may play an important role in the
acceptability of meat. Stress may be the result of hormonal or chemical
injections, fatigue and exercise, excitement, preslaughter feeding or
dietary restrictions. A major effect of stress is a decrease in the
muscle glycogen reserve, which after death results in a lowered production
of lactic acid and a higher ultimate pH. In turn, ATP and phOSphocreatine
are rapidly broken down, with the ATP being reduced to IMP. If the animal
is stressed, 'he level of IflP at death will be higher.

Reay (1949) observed that fish differ from the muscle of meat animals
in that the level of glycogen in resting muscle (0.8%) is considerably
lower than for meat animals. fish, which are normally caught by trawling,
rapidly use up the glycogen reserve, and as a result the initial level of
ATP is low while the IMP content is quite high. In addition, the pH is
also quite high--usually in the range of 6.2-6.6. Reay suggested that
the high pH may be reSponsible for the fact that fish are more susceptible
to bacterial Spoilage than meat animals, where the pH is usually lower.

Fujimaki and Kojo (1953) divided line caught frigate mackerels

(Suxis tgpeinosoma) into two groups; one which was killed instantly and

19

a second which were allowed to struggle until they died. As expected,
ATP decreased very rapidly in stressed muscle in contrast to the slower
rate in unstressed muscle. In addition, struggling markedly increased
AMP and IEP and resulted in more ammonia production. The authors theo—
rized that the early appearance of ammonia was a result of glutamine
deamination, while during autolysis APP was deaminated.

Guardia and Dollar (1965) in a comprehensive series of experiments
attempted to elucidate the effects of exercise and feeding on the

nucleotide concentration in English sole (Porophrys vetulus). The

 

initial IMP level of the rested fish was 0.01 uw./g. and 0.08 di./g. for
fed and starved fish, reSpectively. This correSponds to values of 1.66
um. g. and 2.11 ui./g. tissue, reSpectively, for fed and starved fish
which had been exercised. The authors concluded that feeding did not
cause any significant change in the total amount of nucledtides, although
the amount of nucleotide did vary considerably with exercise. Active
fish had lower levels of initial ATP and increased levels of IEP, but
the difference was not significant after 7 days storage of ice.

fork by Zhivkov (1963) with chickenznuscle seems to indicate that

e

the season of the year may affect the concentration of nucleotides. F
noted that there was a rise in the amounts of ATP and ADP in the spring
of the year, which was accompanied by a decreased level of IIP.

Other papers concerning the biochemistry of fish and muscle,‘!ncluding
effects of stress, nucleotide concentration and level and effect of free
sugars have been published by Sharp (1935), Bate—Smith (1948),'3arr
(1950, 1954, 1958), Jones and Hurray (1957, 1961a), Jones (1958),

Sreelman and Tomlinson (1960), Fredholm (1960), Jones (1960), Jones and

20

Burt (1960), Tomlinson and Creelman (1960), Sorgstrom (1961), Burt (1961),
Tomlinson pt 31. (1961), Tomlinson and Geiger (1962b), Visioli (1964),

‘\ o /
eraser et a1. (1965) and Terasakl et 1. (1905).
Lifect of muscle on nucleotide concentration

The changes in concentration of ATP and its intermediate compounds

were measured in pork and beef muscle (fiillo, 1964). The samples were

’i

. . . . . .o .
taken from four muscles during a 15 day aging period at 3-4 a. Results

~ m--~

from the psoas major and lattissiius dorsi muscle were similar. Air

 

fell from 16.57 to 0.27 pl. of 13/23. after 30 hours, and to 0 after 4 days.
There was a simultaneous decrease of ADP and ALP, which were both still
present in small amounts as long as 7 days. The concentration of IhP
increased from 6 to 14 uh. P/g. in 7 hours, but commenced to decline
after the 4th d y post—nortem. A similar pattern of degradation was

observed with the longissimus dorsi and pectoralis profundus muscles,

 

but the rate of cxange was considerably faster. The muscles with the
slower rate of metabolism were organoleptically superior. The author
could not explain why these muscles differed, although he postulated

that perhaps it was due to the physiological activity of the muscles

Saito pt 21. (1959b) found a considerable difference in ILP level
between the dorsal and red lateral muscle of rainbow trout. The dorsal
muscle contained considerably higher (2 ph./g.) concentrations of ILP,
and correSpondingly lower levels for inosine and hyp xanthine. These

authors also noted that the rate of change varied between muscles.

21

Arai (1960a, 1960b) and Arai gt El' (1961) assessed the nucleotides
content of the foot and adductor muscles of shellfish. The level of
nucleotides was higher in the adductor muscles than in the foot muscle.
This higher level was accompanied by a more rapid rate of decomposition.
Generally, the authors found no hypoxanthine or inosine, which would
indicate the absence of IMP in shellfish.

Kazeniac (1961), Minor (1964), Zhivkov (1964) and Terasaki (1965)
have all reported that light muscles contain relatively larger amounts
of inosinic acid than do samples from dark colored muscles. Minor went
on to show that breast and leg muscles from heavy hens had the highest
concentrations of IMP, while samples from light weight hens contained
the lowest levels. He suggested that the higher inosinic acid content

the heavy hen samples probably contributed to their superior taste.

F?)

O

T Q .-~....

it is interesting to note that heart muscle contains no th. Visioli

CD
('1—

'gl. (1964) found hat there was a rapid decrease of all triphOSphates

in the myocardium of severely exercised rats. however, no ILP was detected.
Also in 1964, Imai gt fil’ explored the nucleotide content of skeletal

and cardiac muscle from anemic rabbits. The level of 0.06 pn./g. for
skeletal muscle appears very low. IEP, adenosine, inosine and hypo-

xanthine were all conSpicuously absent from cardiac muscle.
Effect of storage on nucleotide concentration

Eish

 

7“ \ .0

Jones and Murray (l95/) followed the autolysis o- nucleotides in

w 11 7

n . / O a. "o _ --‘. . n
the muscle 01 cod stored for 0 days at 2 6. inc levels 01 afr, ADP, and

AMP fell and became stable at 0.10, 0.14, 0.14 uk./g., reSpectively. In

22

exhausted muscle, the level of IMP decreased constantly from an initial
level of 4.34‘pM./g. to 0.66 pM./g. after 6 days. These authors also
noted the effect of exercise. There was over an 80% conversion of ATP
to IMP if the cod were exhausted by struggling prior to killing. Jones
and Murray in a later paper (1962) on cod muscle again demonstrated the
effects of storage and stress on nucleotide concentration during a 12
day storage period. Results presented in this paper were difficult to
analyze. values for fish caught in February differed from those caught
in July, which might indicate a seasonal effect. In both cases, the
maximum concentration of IMP occurred within one day of killing. There
was then continual IMP degradation, until none remained after 12 days
storage. In this paper Jones and Murray postulated that the major
breakdown pathway of ATP was as follows: ATP mm: ADP ----> AMP -—--)
IMP ----b Inosine ----v hypoxanthine.

Kassemsarn El.§l° (1963) followed the autolysis of nucleotides in
three Species of fish; i.e., haddock, lemon sole and plaice. The fish
‘were eviscerated, stored in ice at 200. and held for periods up to 24
days. Samples were extracted with perchloric acid and separated on Dowex—
1 X 8 columns. The authors found that the initial ATP level was low,
while IMP was present in higher amounts in all three Species. IMP
completely disappeared in plaice in 11 days and more slowly in lemon
sole. A slight increase occurred in haddock prior to degradation. The
course of IMP degradation varied widely between Species. Inosine concen—
tration reached a peak in 10 days for haddock, 7 days for plaice and was
present in traces only in lemon sole. Hypoxanthine reached peak values

at 17 days, 12-14 days and at 14 days for haddock, lemon Sole and plaice,

reSpectively. Kassemsarn postulated a breakdown pathway for ATP, which
correSponded to that suggested by Jones and Murray (1962).
Takeda and Shimeno (1964b) found that the nucleotide degradation

of Auxis tapeinosoma was Similar to that for other Species of fish.

 

They also found that inosine predominated in fish, while hypoxanthine

predominated for the other Species, which have been studied.
11 eats

Dvorak (1958) studied the degree of nucleotide decomposition in
o 0
meat from beef bulls aged at either 37 C. or 5 C. Samples were taken

from the psoas major. The muscles were irradiated and stored at 1005

 

relative humidity at either 37°C for 20 hours, or at 5°C. for 15 days.

Results are summarized in table 2.

Table 2. Nucleotide content for beef muscle held at 5°C.
and 37OC—pH./g. tissue

 

 

 

 

Temperature
3200. 50c.
Nucleotide 3 hrs. 10 hrs. 20 hrs. 1 dgy 2 days 10 days 15 days
ATP 1.5 0 0 1.5 0.1 0.2 0.2
ADP . 0.9 0.7 0.2 0.9 0.7 0.8 0.7
IiP 1.7 2.9 0.9 1.7 3.7 1.9 1.7
Hypoxanthine 1.7 2.9 4.5 1.7 .8 3.4 4.5

The authors suggested that at 500,, the remaining ADP was probably
bound to the myofibrils. They postulated that the enzyme systems were
less active at the lower temperature, and as a result the ADP remained

intact. This agrees with Perry (1951‘. He hypothesized that the acid

 

 

2b.

labile phOSphorous, arising mainly from the ADP in rabbit muscle, was
bound to the myofibril in such a way that it is not acted upon by the
enzymes present in the myofibrils. He did not Speculate as to what
factors could protect the bound nucleotides.

Tomlinson and Geiger (1962a) reported that there were no differences
in the quantity of adenine nucleotides bound in washed muscle extracts
from freshly frozen (pro—rigor) lingcod and in those from muscle severly
denatured during freezer storage. However, when similar samples of lingcod
muscle were heated, almost complete release of the bound nucleotides
occurred. Jones and Iurray (1961a) reported similar values. The stable
ADP level was higher in rested fish muscle (pH 6.3). while the levels of
ATP and AMP were lower than for the exhausted codling (pH 6.8). These
stable values were achieved in the presence of high enzymatic activities
capable of degrading free nucleotides, Since added nucleotides were
rapidly degraded in an analogous homogenate system. It was therefore
apparent to the authors that the nucleotides were present in post-rigor
muscle in some protected form. The effect did not appear to be that of
a Simple equilibrium of "free" nucleotides, under the action of the enzyme
systems to which th-y were subjected, as the level of IKP produced by the
action of AHP deaminase was falling rapidly throughout the duration of the
eXperiment due to the action 5'-nuc1eotidase. Rather, it seemed likely
that the stable fraction of adenine correSponded to the "bound" nucleotide
content of the myofibrils.

Jones and hurray (1961a) suggested that the proportion of the different
adenine nucleotides bound to the myofibrils are determined by pH dependent

enzymatic activities, Since myokinase‘is most active at p1 7.3. This in

25

turn may be part of the reason for the lower levels of ADP at the higher
pH (6.8) of exhausted muscle as compared to rested muscle (pH 6.3).
Rhodes (1965) followed the autolysis of nucleotides in beef and
lamb irradiated at 0.4 Megarads during storage for 70 days at 200.
Irradiation effectively prevented bacterial Spoilage throughout storage,
without affecting the nucleotide content or natural enzyme activity of
the meat. He found that IMP was completely reduced to hypoxanthine in
30—40 days in some beef cuts, but more Slowly in others. In addition,
the rate of IMP degradation was considerably Slower in leg of lamb,
although the total nucleotide content was Similar. Attempts to correlate
levels of IMP and flavor scores for beef and lamb were unsuccessful. The
author therefore concluded that his results do not support the theory
that IMP plays a major role in meat flavor, either as a precursor or as
a potentiator. furthermore, he suggested that measurement of IMP or

'+
u

l

U)

breakdown products appears to offer little or no promise for
measuring the freshness of red meats.

.. . / . -1_ . 1 .
Terasaki ~ 'E1. (1905) also examined he rate of nucleotide reduction

'0
('1‘

on i ... . . .
t t. in addition, the authors determined

r‘.

in chicken and pork stored t

93

the level of IhP in breast muscle of lamb and thigh muscle of horse meat.
Analysis revealed that the maximum concentration of inosinic acid occurred
about 8 hours post—mortem with chicken and between 2—3 days post—morten

with pork. However, it was noted that ‘he maximum concentration of IEP

in chicken breast muscle was approximately twice that of the longissimus

 

."11

dorsi muscle from pork. lne ILP concentration of lamb and horse was
/ “ '.- - 0"- ’1
2.30 and 3.07 pm./g. muscle reSpectively. The validity cl these last

2 values may be questioned Since the samples utilized were taken from

N
U\

frozen lamb and horse meat imported from Australia and Argentina,
reSpectively. These authors utilized a paired comparison sensory test
to demonstrate that pork samples, which contained maximum levels of IHP
were preferred over samples taken when the IMP content was less than
:iaximum.

dndo pt 31. (1965) reported that 50} of the IIP in carp muscle
was degraded in 2 days, while it required 2 weeks storage atl CC. for
beef or chicken muscle to be degraded to the sane extent.

In 1963, Fredholm studied the rate of nucleotide degradation in
beef and pork stored at 203. for 5 days. Che nucleotides were separated
by the ionophoresis method of uOr”kV1ot (1% 7) and qt lantitative measure—

ments re made :ro:n the color intensity ol the resulting Spots.
Iredholm' S result (t able 3) differ appreciably from pl revious work in
this field, Since he found the nucleotide content to be relatively

Table 3. Quantity of adenosine phOSphate a t er storarge in percent
of quantity present immediatelya Itw Slaughter

I") I“:

 

; remaining in echn iarison to 0 hours
storage

 

 

tin e C 01111.) e und
‘:.' *\ . ‘ ‘4 C \ .I [TVD I‘ h 7.! :‘I l‘
.4 ;. eel es (:1 0111 0] Al 1 AD: He'll"
. ..., A I ~ .v ‘
.see: id 100 75 100
/
72 50 O 00
120 2° 23 ho
’3 I r», f. r. r
york 2% 100 50 77
'2 35 50 31
120 l? 30 9

 

Stable. goth beef and pork ASP levels were practically the same after 24

liours post-mortem as immediately following Slaughter, and even aftel 3 days

27

these values were reported to be 50} and 35}, reSpectively. The rate of
degradation for ADP and AEP were Similar. The nucleotides appeared to

be more stable in beef than in pork.
Nucleotide concentration in other foodstuffs

Fujita et 31. (1959) evaluated the nucleotide concentration of
various foodstuffs. Samples were separated by ion exchange chromatography
and the concentration of each nucleotide was determined Spectrophoto—
metrically from their molecular extinction coefficients. Jack mackerel
and bonito contained 737 and 853 mg.§ of IHP on a dry basis, reSpeetively.
Ham contained 67 mg.) on the sane basis, while IMP levels were not
detectable in dried mushrooms or miso (bean paste). Using similar
techniques, Nakajima pt al. (1961a, 1961b) also demonstrated that dried
mushrooms and aquatic invertebrates contained no IIP. The IMP content
of dried bonito was 18.1 pfi./g., and dried sardines contained 8.73.p§./g.

' 1

The authors suggested that IMP acted as a flavor component in fis-

b

Saruno and Sasaki (1955) precipitated the nucleotides of fermented sake
with uranium acetate, converted the precipitate to the cepper salt,
decomposed the salt with sulfuric acid to liberate the bases and then
identified the bases with paper chromatography. The presence of hype-
xanthine indicated that inosinic acid was present in fermented sake.
These authors then postulated that INP together with succinic acid are

the chief components of sake flavor.

Pathway of inosine monophosnhate formation and degradation in muscle

 

Bendall and Davey (1957), Saito and Arai (1958a, 1933b, 1959),

Sawant and Hagar (1961b), Jones and Qurray (1962), Kassemsarn (1963),

2a

, huninaka et al, (1964) and

\_.l

SLi mazono (1963),1Tarr and Leroux (1%

Jones and hurray (1964) have Speculated on the most probable post—nortea
pathway of In? formation in muscle. The general consensus is t‘n at 'he
ATP present in muscle at death is degraded DJ successive steps to AD?
and AMP by the enzgrqes AEEa.se and myokinase. an? is rapidly deahinated
by the enzyme AMP—deaminase to IMP, which is then reduced to inosine.
Inosine in t urn breaks down to hypox nthine, whicl in the presence of

L

ct- erial oxidases is degraded to Xanthine.

.0.)

dendall and Davey (1957) examined the changes occurring in rabbit

- ’1 ‘ - \ O "1 '. .“\ 1 "\ a y
inuscle during the course or rigor mortis at 0 e., lZOe. anc )7Ot. fhe

 

authors concluded that the appearance of anM01ia, which was liberated

by deamination of the adenine nucleotides, was directly related to their
disappearance and to the formation of '1osine nucleotides. 110 disap—
pearance of the adenine nucleotides and the appearance of the inosine
nucleotides occurred in nearly equimolar proportions. Two atoms 0;
labile—P disappeared for each mole of ATP degraded or for each mole of
In? that appeared. it 3700., the authors indicated that ATP reduction

.1.

occurred in three stages as shown below:

 

Egaption ihgyme involved
a) if? -—-9’ADP + I? ATPase
1)) 2 5L? 3 ATP + ILL? ljyokinas e
c) ALP ---+IIJP + J£3 Deaninase

‘ O-‘u . .0 —~«"- |-.--- 1 _ _- - 9 - _
at 17 9. small amounts or id? and lTr probaolj originate erfl two re-

 

actions as sug sted by Bendall and Dave? (195?) and as outlined below:
Reaction Lizgtle involved
a) ADP -—--> ID? + LL13 Sound deaninase

13/ ID? + ifl':==3:3fl3 +.aue ‘ ransaios:hor'l.

29

Bendall and Davey (1957) postulated that at higher pH values ADP is
tightly bound to the myofibrils and is therefore resistant to attack. As
the pH falls during development of rigor, it becomes progressively more
vulnerable to deamination by some bound deaminase, which is more active
at lower pH's.

Tarr and Leroux (1963) attempted to elucidate the major pathway of
glycolytic intermediates in salmonoid fish. Intravenous introduction of
Ola-labeled glucose or glucose é—phOSphate resulted in rapid labeling of
such intermediates as fructose 6—ph05phate, fructose di—phOSphate, and
lactate in both muscle and liver. On using Ola-labeled muscle glycogen,
results suggest that the glycogen is degraded to maltose and glucose post—
mortem, both by an amylase and the Embdem—Ieyerhof system, and that very
little glucose arises by hydrolysis of glucose 6—ph08phate. when Clu-
labeled ATP was introduced into fish muscle post—mortem, it yielded
radioactive IMP and ribose, but glucose 6—ph08phate and glucose were
unlabeled. Radioactive ribose-E—phOSphate (or ribose) was never found
in fish muscle injected with 014—labeled glucose or Clu-labeled glucose
6-ph05phate, indicating that these compounds are not formed via the
hexosemonOphOSphate shunt system. Ribose—B-phOSphate was not found in
the muscle of living fish, and only in comparatively small amounts post—
mortem. From the evidence these authors presented, it is quite obvious
that ATP is degraded to IMP post—mortem, and that IEP is reduced further
to inosine and then to ribose and hypoxanthine.

Saito and Arai (1958a) found that the reduction of ATP was related

quantitatively to the formation of IMP. The authors demonstrated this

by measuring the levels of ATP and AJP, both before and after homogenizing

 

30

fresh carp muscle for 10 minutes in’a 0.13 M. NaCl solution with ice.
During the homogenizing procedure, 2.90 pM./g. of AMP was decomposed
while 2.09 ,ul-l./g. of 11-2? was formed. The deamination of the AMP did
not appear to be affected by the low temperature. Keir and Davidson
(1958) and Davey (1961) have also published papers dealing with the
metabolism of IHP.

Jones (1961) reported a high inosine 5'—mon0ph05phate content in
fresh fish and showed that it was one of the major flavoring components.
He desribed IMP as possessing a strong saltyeacid flavor with overtones
described as "meat extract," "yeasty" or "almonds". He went on to
describe the taste of hypoxanthine as strong_y bitter at the level at
which it occurs in fish after 10 days cold storage. he also stated
that the removal of glucose, the major hexose phOSphates and inosine
5'-monoph05phate account for part of the loss of the initial sweet,
meaty flavor in fish. Realizing that the concentration of hypoxanthine
increased with cold storage, Jones and Murray (1964) and Jones EE.§;'
(1964) postulated that procedures for following the course of nucleotide
degradation would seem to be an excellent Opportunity for obtaining
valid useful indices of quality. host of the currently available
chemical indices are measuring bacterial Spoilage as pointed out by these
workers. By assaying for hypoxanthine, certain initial autolytic phases
of deterioration can be observed. As a result, this assay could be
quite useful in assessing the quality of fish or meat stored at cooler
temperatures. This measure would probably be of more value in the fish
industry, since nucleotide decomposition occurs at a faster rate than

microbial Spoilage. Several researchers have used this hypothesis and

31

developed rapid and accurate methods for determining the freshness of
stored fish muscle. Among these, Saito gt 2l° (1959a), Spinelli gt fil’
(1964), Jones and Murray (1964) and Jones 23.§l- (l96h) have published

papers in this field.

Methods of evaluating nucleotide congentration

Nucleotides are generally separated by ion-exchange chromatography
and then quantitatively evaluated from their molecular extinction
coefficient. In general, only the ion—exchange resins or the elution
system being utilized are changed to meet a particular researchers need.
Most methods for separation of nucleotides by ion-exchange are patterned
after that of Bergkvist and Deutch (1954) and Hurlbert 32 El- (1954).
Other variations have been employed by Saito and Arai (1958b), Jones and
Murray (1961a), Nakajima gt 9;}: (1961a) and Takeda and Shimeno (1964a).

Cohn (1950) discussed conditions which are necessary for successful
ion-exchange chromatography. He noted that the net charge per ion was
the most important factor in determining the ion affinity for an ion—
exchange resin, when a closely related family of compounds was being
separated. Since both acid (phosphate) and basic (amino) groups are
present on a nucleotide, the pH of the medium determines the net charge
by affecting the degree of dissociation of these groups. Cohn further
noted that a sample of nucleotides could be adsorbed onto an ion-exchange
resin at pH values greater than 6 and could then be removed by lowering
the pH in a stepwise fashion. He suggested increasing the anion
concentration by adding salt near the end of elution to prevent

hydrolysis of the nucleotides.

 

32

In 1957, Bergkvist noted that ion—exchange chromatography has
several advantages, since it is capable of handling both large and small
quantities of material. In addition, it is suitable for preparative
work, great resolving power is attained and contaminants are of little
importance. Bergkvist cited two disadvantages, first, it is a very slow
procedure, and second, large volumes of liquids are required during
elution.

Nmnerous workers have reported other more conventional procedures
for the separation of nucleotides. Bergkvist (1957) developed a method
based on chromatography together with ionOphoresis on filter paper.
Smillie (1959) described a procedure for separating nucleotides on ion—
exchange paper. More recently, Strickland (1965), Randerath (1965),
Randerath and Randerath (1965), and Grippo 33 El. (1965) have described
procedures for the separation, identification and quantitative measurement
of nucleotides using thin-layer chromatography. fieinstein (196d) described
an automated method of analysis for individual samples as they are eluted
from ion—exchange columns. lacy and Bailey (1966) developed a rapid
procedure for separation of nucleotides on a Dowex ion—exchcnge resin
column. They successfully shortened the time for complete resolution

of a sample from 12 to 5 hours.

fiater soluble flavor precursors

 

In a review of the literature on meat flavor, Doty et a1. (1961)
observed that the purely subjective approaches which were used in earlier
flavor research have been replaced by modern objective procedures. They

stated that two approaches have been used in meat flavor research. One

 

33

technique involves the isolation and identification of flavor components,
both volatile and nonvolatile, from cooked meat. The second method
depends upon the isolation and characterization of flavor precursors

from raw meat. There are obvious advantages and disadvantages inherent
in either approach. Thgrsuggested that many compounds may be isolated
from cooked meat, yet there is no way of determining the relative
importance of these compounds to overall meat flavor. Thus, if isolation
of flavor components from cooked meat is utilized, the various components
must be estimated quantitatively and then recombined in the correct
concentration to again achieve the original flavor. Doty further pointed
out that if one isolates and identifies the precursors of cooked meat
flavor from raw meat, it is still necessary to elaborate the chemical
changes which are responsible for the development of typical cooked

meat flavor.

With reference to precursors, first Crocker (1948) and then Kramlich
and Pearson (1958) and Hornstein.g£‘§l. (1960) demonstrated that flavor
precursors can be leached frmn raw beef, although none of these workers
attempted to establish their identity. In 1948, Crocker stated that the
typical meaty flavors apparently were contained in the fibers of cooked
meat rather than in the expressible juices, and were released by chewing.
Barylko—Pikielna (1957) by taste panel evaluation demonstrated that the
typical flavor of roast beef was present in the water insoluble residue,
whereas, the water soluble fraction had a very intense but atypical
beef flavor. Kramlich and Pearson (1958) worked with press fluids from
raw and cooked beef. Their results indicated that press fluids of raw

1

meat had a highly concentrated flavor on cooking. Cooking prior to

 

34

extraction increased the flavor, indicating that the full flavor develop-
ment may be due to heating the juice and the fiber together.

Using paper chromatography, wood (1956) demonstrated the presence
of inosine and hypoxanthine in commercial meat extracts. This would
suggest the presence of inosinic acid, even though it was not identified.
It was interesting to note that no sugars or carbohydrates were detected
in the extract. Wood surmised that they may have combined with some
nitrogenous constituent in the extract during browning.

In 1957, Wood and Bender undertook the formidable task of isolating
and identifying the constituents of commercial ox—muscle extract. In
the process, they detected over 100 compounds. This list included,
inosinic acid, inosine and hypoxanthine. In a later report, Bender
g: 31. (1958) compared the constituents of an extract of fresh ox—muscle
with commercial beef extract. Despite different methods of extraction
and subsequent treatment, the composition of the two extracts was
remarkably similar. Inosine and hypoxanthine were present in both
extracts. The most notable difference was the loss of approximately
80% of the amino acids in the commercial product as well as 100% of the
reducing sugars. The authors postulated that these losses were related
to the higher yield of colored pigments isolated from the commercial
extract, such as would be the case with the Maillard—type browning
reaction.

The American Meat Institute Foundation has studied the flavor
precursors from raw beef and their studies have been summarized by Doty
g: 31. (1961). The authors selected this approach after observing that

the fat fraction from the third acetone extract of raw ground beef gave a

35

typical broiled steak odor on heating. Subsequent research on this
fraction revealed that the flavor was not due to the fatty material
pg; g2, but was caused by substances that difqued through a semi—
permeable membrane upon dialysis with water. On separation of this
fraction, they isolated a white granular material, which was unstable
and became a brown, tarry mass if stored under vacuum. Ammonia and/or
amines were released as browning progressed, and the material assumed
a characteristic stale meat odor. wood (1961) reported that a similar
acetone—soluble material from ox—muscle extract decomposed in the same
manner. He went on to state that nucleic acid decomposition was an
important factor in browning and development of meat flavor. He
isolated inosinic acid and ribose—5‘—ph05phate as the active ingredients.
Wood described the taste of inosinic acid as meaty.

Batzer gt 31. (1960) undertook the task of trying to characterize
and identify the components in the water—soluble fraction that yielded
the typical meaty odor on heating. Several fractions were obtained by
the extraction procedure utilized. Fraction A, which contained the
flavor components, was obtained by filling cellulose dialysis tubing
with distilled water and inserting the tubes into the water-soluble
extract of beef. Upon further fractionation of the diffusate by dialysis
through a sausage casing, two fractions were obtained. The material
remaining in the sausage casing (Aa) was primarily protein in nature,
while the material that diffused through the sausage casing (Ab) was
composed prhnarily of small molecular weight compounds, such as sugars,
amino acids and small peptides. Two more fractions resulted when

fraction Aa was separated on a Sephadex G-25 column. One was a protein

36

fraction, and the other was characterized as the basic meat flavor
precursor in beef. Chicken or pork loin tissue extracts treated in

an identical manner resulted in similar fractions. rhese fractions

had almost the same basic odor as that isolated from beef muscle. In

a classical series of ex;eriments, the same investigators showed that
the basic meat flavor precursor was a glycoprotein, which gave a strongly
positive carbohydrate reaction prior to hydrolysis with perchloric acid
and a strongly positive phOSphate test after hydrolysis. Ultra—centri—
fugation at 60,000 rpm. resulted in no distinct peak, suggesting that a
relatively low molecular weight compound was involved. hinhydrin tests
were positive after 3 Spots had been separated by paper chromatography

f the acid hydrolysate. Two of the ninhydrin positive spots could not

0

1

be positively identified, but the others revealed the presence of leucine,
proline, isoleucine, alpha alanine, valine, serine and beta alanine,

with trace amounts of glycine and glutamic acid. Uhen the diffusate
fraction of the secondary dialysis (Ab) was separated on Dowex—5O ion—
exchange resin and eluted with acid, a spectra of nucleotide peaks
matching published results for hypoxanthine, inosine and inosinic acid
were obtained. further studies by Batzer gt fil‘ (1962) resulted in the
identification of inosinic acid, inosine and a glycoprotein having a
glucose moiety. These authors concluded that these simple water Soluble
compounds in combination with certain amino acids are important components
of basic meat flavor.

fasserman and Gray (1965) reported data that varied significantly

from results obtained by Latzer et a1. (1960, 1962) on utilizing the

same fractionation procedure for isolatinr precursors of beef flavor.

a)

 

 

. ..¢....’...1...
vi. . I.

 

37

In contrast to Batzer, these authors were unable to effect a second
separation of Fraction A, when it was subjected to dialysis in Visking
sausage casings. Instead, there was essentially an equal distribution

of the casing contents across the membrane. Three fractions rather than
the two reported by Batzer gt El' Were obtained when Aa was separated on
Sephadex G-25. Only one fraction (Aaz) had the characteristic aroma of
commercial meat extract. Fraction Ab was separated on Dowex-5O to yield
three fractions. After freeze drying, the meaty aroma was found only in
the heat decomposed solutions of AbZ. They demonstrated that this fraction
contained only amino acids, hypoxanthine and a trace of inosine. Inosinic
acid was conSpicuously absent. These authors also went on to show that
fraction Ab2 contained no glucose or ribose sugars as clahned by Batzer
pt 31. (1960, 1962), even though a meat-like aroma existed.

Macy gt El' (1964a, 1964b) demonstrated the presence of inosine
and hypoxanthine in water—soluble extracts of beef, lamb and pork.

They went on to suggest that inosine may play a major role in the
browning of meat, and that it could be important as a flavor and/or
odor precursor in meat products.

In 1961, Kazeniac discussed chicken flavor and some of the components
reSponsible for flavor. He noted that hypoxanthine and inosine have a
bitter taste, whereas, inosinic acid makes a major contribution to mouth
satisfaction by intensifying the flavor effects of other compounds. He
attributed the fact that light meat broths from chicken have a stronger

taste and mouth satisfaction to their higher inosinic acid content.

 

maimuxrm Pacer? [BE

This investigation was necessarily broken down into several phaseS.
The first study was designed to find the concentration of nucleotides in
each of three muscles from pork carcasses. The second phase dealt with
the nucleotide levels in samples taken from beef carcasses during post-
mortem aging. The third phase investigated the concentration of nucleo—

tides from various Species of meat animals. The final investigation

3

was desi

,

)

‘ned to assess the flavor enhancing properties of inosine mono—

phOSphate.

dxperimental material

 

Iuscle investigation

 

Six Hampshire barrows with an average weight of 210 lbs. were used
for this portion of the study. Three muscles were sampled from the
right side of each carcass. They were the 10th to 13th rib section of
the lpngissinus dgpsi muscle of the loin, and the seniuembpépgsus and
piggps fepgpis:nuscle from the ham. The samples were removed from the
carcasses approximately M8 hours DOStAWOTtCH, in an attempt to approxinate
commercially existing conditions. All fat and connective tissue were

r *n

removed, and the samples were ground twice through a 2 mm. plate. The
. . , ,on .
sanples ware placed in sample bottles, frozen and Stored at —20 :. until

further analysis.

7 c an, A J-
fee]; a, Li; _s_ tudy

Six Lolstein heifers, approximately 2M months of age were used in

this investigation. Baubles of the loniissinus dorsi huscle were removed

33

 

 

39

according to the schedule shown in table 4.

Table 4. Sample time and location for beef aging study

 

Sampling time

 

 

post-mortem Sample location
0 hr. 12th rib section
12 hr. llth rib section
24 hr. lOth rib section
4 days 9th rib section
7 days 8th rib section
14 days 7th rib section
28 days 6th rib section

 

The wholesale rib from the right side of each carcass was removed
and stored at 33—350F. The sample at 0 hour was removed as rapidly as
possible following stunning. In all cases, no more than 15 minutes
elapsed before the initial sample was removed. All 0 hour and subsequent
samples were immediately frozen in liquid nitrogen and stored at —200F.
until removed for analysis. Before freezing, all fat, connective tissue
and any exposed lean areas were trimmed away in order to avoid contami-
nation or excessive moisture loss. Considerable trimming was necessary on
all samples aged for either 1% or 28 days, because they were contaminated

by molds and other microorganisms.

Species investigation

 

Samples were taken from pork, lamb and bull carcasses for this
portion of the study.
Six pork carcasses weighing approximately 220 lbs. were utilized

in this experiment. Samples were removed from the lopgissimus dorsi

 

muscle at the 10th rib within 15 minutes of stunning. Samples were

excised after removing the backfat from the loin in the 10th rib area

 

 

40

and then by using a cork borer to remove the sample. After removal of
the connective tissue the samples were frozen in liquid nitrogen and
stored at —200F. until analysis. Heart samples from the same carcasses
were removed, frozen and stored in the same manner. In addition, heart
muscle was sampled at 12 and 24 hours post—mortem. This series of
samples was alSO utilized to study different extraction procedures.

The entire longissimus dpggi muscle from the wholesale loin section
of 6 lamb carcasses was sampled. The lambs had been used for class eval—
uation, and as a result were not kept under constant storage conditions.
Samples were taken approximately 7 days post—mortem, ground through a 2
mm. plate, frozen and stored at —20°F.

Samples from 6 bull carcasses, which had been used for another study,
were analyzed. These samples had been taken from the longissimus QQEEE
muscle of the 10th rib section of the wholesale rib. They were ground
through a 2 mm. plate, frozen and stored at —200F. These samples had
been previously thawed for proximate analysis, refrozen and stored

before being analyzed for nucleotides.
Methods for evalgation of nugleotide content
Extraction

Samples were not thawed prior to analysis. Approximately 10 grams
of tissue was weighed into a VirTis homogenizer flask. A total of 25
ml. of distilled water was added, and the sample was homogenized for 2
minutes at mathmn speed. The sample was then quantitatively transferred
to a 100 ml. volumetric flask and boiled for 5 minutes. It was allowed to
cool before bringing the volume of the extract up to 100 ml. with distilled

water. Following centrifugation for 20 minutes, the extract was filtered

VI.

1+1

through a Whatman GF/B glass fiber filter pad. The resulting extract
was measured and adjusted to pH 8.0 by adding 1 N. sodium hydroxide. A
10 ml. aliquot of the extract was then separated with an ion—exchange

. , 0.
column. The remainder of the extract was frozen and stored at —20 r.
Chromatographic separation

The method of Lento gt a1. (1963) was used to separate the nucleo—
tides. Dowex—l X 8 anion exchange resin (200—400 mesh) was washed with
water to remove the fines and suSpended solids. It was then packed as a
wet slurry in a 12 mm. (inside diameter) glass column to a height of ap—
proximately 160 mm. The column was fitted with a stopcock and a plug of
glass wool in order to retain the resin. The resin was then converted
to the hydroxyl form by adding three bed volumes of l M. sodium hydroxide.
After washing the resin free of excess sodium hydroxide, it was converted
back to the formate fonn by the addition of 3 bed volumes of 6 N. formic
acid. The excess reagent was again removed by washing with distilled
water. At this point, an aliquot of the test solution was added to the
the top of the column and passed through at the rate of 1 ml. per minute.
The resin was washed free of UV absorbing materials with water until the
effluent gave a reading of approximately 100 percent transmission. The
nucleosides, bases and free sugars, which were removed from the column in
the wash water, were saved for further analyses. The resin columns were
reuseable on recharging them as described above. Cohn (1950) reported

that some columns were used as many as 30 times without any loss of

capacity or exchanging power.

 

42
Elution

The nucleotides were eluted by a 3 phase gradient system consisting
of water, 0.5 N. formic acid and 0.2 N. sodium formate. Three aSpirator
bottles were aligned above one another as shown in figure 2. The lower
bottle contained 250 ml. distilled water, the middle bottle contained
250 ml. 0.5 N. formic acid and the upper bottle contained 250 ml. 0.2M.
sodium formate. Magnetic stirrers mixed the solutions in the lower 2
bottles during elution.

To begin the elution, the screw clamps were removed from the Tygon
tubing connections between the aSpirator bottles. It was necessary for
this tubing to be airtight so repeatable gradients could be obtained
during the elution process. The solution was allowed to percolate
through the column at a flow rate of 1 ml. per minute. As the effluent
flowed from the column it passed through an Isco, Model UA, UV analyzer,
which automatically recorded the absorbance at 25” mp. The fraction
collector was wired to the monitor so that each time it moved it was
recorded on the chart paper. This System made it possible to locate

any single fraction containing the 5'-nucleotides for further analysis.
Quantitative evaluation

Since only the 5‘ isomers of the nucleotides are important as flavor
enhancers, the method of Lento gt El. (1963), which is Specific for the
5‘-ribonucleotides, was employed. This is in contrast to earlier pro-
cedures described by Beck 33 3;. (1956), in which the quantitative deter—
mination of the nucleotides is based upon their molar extinction coefficients.

One ml. of 0.01 N. periodic acid (H104) was added to a 5 ml. aliquot

 

 

 

 

L34

of the sample. The mixture was heated for exactly 3 minutes at 70°C.

This step oxidizes the ribose moiety of the nucleotide between the

number 2 and 3 carbon atoms and is specific for the 5' isomers. Oxidation
was stopped by the addition of 1 ml. of 0.05 N. sodium arsenite (NaZHAsOB).
This was followed immediately by the addition of 1 ml. of concentrated
hydrochloric acid and 1 ml. of 2,H-dinitrophenylhydrazine (2,4—DNPH) in

2 N. hydrochloric acid. After mixing, the tubes were placed in a boiling
water bath for 10 minutes to hydrolyze the oxidation products, which
facilitated precipitation of the bis—hydrazone. It was necessary to add
the reagents in the order described to prevent interaction of periodic
acid and 2,4—DNPH, which results in high reagent blanks. The precipitate
was then collected on a medium porosity fritted glass filter and washed
Wibh distilled water to remove any unreacted 2,4—DNPH. The 2,M—DNPH
derivative was then dissolved in acetone, diluted to volmne, and the
absorbance of the yellow solution was read against a reagent blanx in a
Beckman DU Spectrophotometer at #35 mp in a 10 mm. pyrex absorbance cell.
According to Lento gt 3;. (1963) the nucleotide content of each fraction
can be calculated from a single standard, since the extinction coefficient
of each of the 5'—nuclootidcs determined by this colorimetric test is
essentially the same. flowever, a standard curve using inosine monophos—

phate was developed and used for the actual determinations (Sigure 3).
Qualitative analyses

Thin—layer chromatography was employed to aid in the identification
of the eluted nucleotides by comparing the fir values of unknown compounds

.L

to those of known standards, which were run concurrently. Chromatography

was carried out using Desaca e uipment purchased from the Desaca Con any
0 u i _ a. J,

 

ABSORBANCE (435 mu.)

 

“aw—.7.“— ..

 

45

 

 

Figure 3.

CONCENTRATION (pg . /m1. acetone)

Standard curve for inosine 5'.- monOphOSphate.

 

46

Heidelberg, Germany. It consisted of a plastic aligning tray, which held
5 plates (20 X 20 cm.), a spotting template, an applicator and a rectangular
chromatography developing tank. MN Cellulose Powder 300 with no binder
was purchased from the Brinkman Instrument Company, Westbury, New York.
A total of 15 grams of absorbant was slurried with 90 ml. distilled water
and applied to the clean glass plates in a uniform layer about 250 microns
in thickness.

The plates were allowed to dry at room temperature for 15 minutes
and then activated in an oven at 100°C. for 30 minutes. The activated
plates were then stored in a desiccator until used.

For identification of nucleotides, the chromatoplates were Spotted
with 10'”3 to 10"2 pH. of the known standards and the unknown compounds.

Care had to be exercised Since tailing occurred if over 10'"2

a. of the
nucleotides were added to the plates. They were then developed for 80
minutes in an equilibrated chromatographic tank. The solvent system
consisted of n—butanol/acetone/acetic acid/5% ammonium hydroxide/water
in a #.5:l.5:l:l:2 ratio. After development, the plates were allowed
to dry at room temperature. Spots were observed by visualization under
an ultraviolet lamp.

Nucleosides and baSes were identified in a similar manner. The same
cellulose plates were utilized. The plates were spotted with 10'"2 pH.
of the substances involved and developed for 45 minutes in a solvent
system of distilled water. The plates were then air dried and observed
under ultraviolet light. After observing under UV lights, tracings of
all plates were recorded on acetate paper.

Paper chromatography on Whatman No. 1 filter paper was used as a

second method of analysis. Solvent systems utilized were adapted from

47

those described in the Pabst Circular OR—lO (1956). Sheets, 21 X 16
inches, with a base line drawn 1% inches from the bottom were Spotted
with approximately 150 pg. of the nucleotides. They were then immersed
in covered jars and developed for 16 hours at 27°C. The chromatograms
were then air dried and read under ultraviolet light. The Solvent
system consisted of isobutyric acid/concentrated ammonium hydroxide/
water (66:1:33) at pH 3.7.

Finally, the nucleotides were characterized from their ultraviolet
absorption Spectra and the ratio of absorption at 250/260 and 280/260 mu.
Speetras were determined at pH 2, 7 and 11 and compared with known
spectras given in the Pabst Circular 03—10 (1956) or Pabst Circular 0R-17
(1961). After several determinations with known samples, the elution
sequence of the nucleotides was established. The same was true of
unknown samples. Thus, it was not always necessary to make qualitative

determinations on all samples added to the column.

Sensogy evaluation

Preliminary work in this study indicated that the concentration of
IMP in heart muscle is negligible. This agrees with the findings of
Imai pt 31. (1964) and Visioli st 31. (1964), who reported finding no IMP
in heart muscle of rabbit and rat, respectively. The lack of IMP occurs
since the enzyme AMP-deaminase seems to be lacking in cardiac and Smooth
muscle of the body. For the above reason it was decided to use heart
muscle for sensory evaluations in this experiment. It was felt that due

to the lack of IMP better control could be exercised, and the flavor

enhancing ability of the nucleotide could be more clearly demonstrated.

48

Pork heart muscle was incorporated into a standard formulation of
cooked frankfurters in order to assess the effect of added flavor

enhancers. The recipe used for these frankfurters is shown in table 5.

Table 5. Recipe for frankfurters

 

 

Ingredients .Mnount
Pork heart muscle 6.5 lb.
Pork fat 3.5 lb.
Salt 136.0 g.
Sugar 23.0 g.
dhite pepper 17.5 3.
Ginger 3.5 g.
Coriander 5.0 g.
AllSpice 1.0 g.
fiarlic powder ‘ 0.8 g.
Sodium ascorbate 12.5 g.
Sodium nitrate 4.0 g.
Sodium nitrite 1.0 g.
Dried skimmilk 226.0 g.
Shaved ice 3.0 1‘.

 

Eive formulations of frankfurters were prepared, one utilizing beef
muscle as in commercial practice and four other formulas utilizing
heart muscle and varying levels of flavor potentiators as Shown in

table 6.

11,3
.r‘ 3,

Table 6. Level of added flavor potentiators

 

Formulation

 

Ingredients 1 2 3 4 5

 

Heat source heart Heart Heart Heart Beef muscle
se 0 0.2133 0 0 0
INF 0 0 0.053 0 0

1n? + 33 0 0 o 0.025,; 0

 

1 e trimmed heart muscle and fat were ground separately through a
.te. The meat was then placed in a Silent flutter and chOpped
lor about 5 minutes to a smooth consistency. The Spices and most of the
ice were added and mixed. At this point, the dried skimmilk was added.
The fat was added aad chopping was continued for approximately 3 more
minutes until the consistency was smooth. The tenpcratur was naintained
below 55—6003. by the addition of shaved ice.

inely chopped mixture was stuffed into artifical casings and
linked on the linking rack. They were then smoked for 35 minutes at
17503. The smok house temperature was lowered to 1650?. and smoking

was contihued until the internal temperature of the frankfurters

reached 1520 . The raniiurters were reioved from the smokehouse and
showered for 5 minutes in not water and l0 uinutes in cold water. After

cooling, the frankfurtcrs were evaluated organoleptically with a consuner

type taste panel utilizing the 9—point hedonic scale.

T‘rh'i

livid hdeAL RSSULTS AhD DISCUSSIOR
Evaluation of techniques and procedures

Known and unknown samples were qualitatively and quantitatively

evaluated before beginning work with regular samples, in order to assess
he effectiveness of 'he orocedures involved The chrowatoeraphic

--_. __ - .L -n. J, _ _._J. n. L..- z -4. ,.- . b L-
separation of a synthetic mixture of 5‘—nononucleotides and the order

in which these compounds are eluted by a three phase gradient elution

(‘0

system is shown in iigure 4. The elution order of these compounds was

:1 -. r:

5 ~eir, 5 —A;P, 5'—ULP, 5'-lnf and 5'_eir. The order of elution was

\

similar to that of Lento et a1. (1963). In addition, Cohn (1950)

reported that the order of elution at pl values of 2. to 3 should be

U1

all, luP, UL? and SLP according to his theory that the compounds with
the lowest net negative charge would have the least affinity for an ion—
exchange resin and as a result would be eluted first. he did not use
IE? in this theoretical discussion or in the series of experiments he
used to demonstrate nis postulation. The shape of the eluted nu;1ootide
peaks was interesting. The iirst peak was always very sharp and well
defined, while each succeeding peak decreased in height and increased

in width. JveL so, resolution was definite and it was easy to identify
each compound.

Eigure 5 illustrates the chromatographic resolution of a sample of

unknown nucleotides from the biceps femoris muscle of pork. 7t is ouite

 

evident that very good separation was obtained when this system of
gradient elution was employed, as no overlapping of the various compounds
occurred. 'he separation pattern and peak size shown in figure 5 were

pl

50

 

51

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ML. <67“.

0

L
p

.

3 age we

MM . -

"‘ ‘:"'M‘I‘ -“‘1§

 

CM‘FWMw-mr . - ‘1'! m. m M

f
r .

_ \\
flail-

o'c- mmeWmI-UF m m d’W‘W-VW" 0mm“: m "M

w-Q"

hw‘
u

if“

aPV

drawn“

(
I_\

I
F:
\.

 

 

.xhom scum oHOmSE mauosmm mmeon mo meaeswm Egoqxs: cw ca mecflpooaoscocosu.m mo soapmpdaem owneahmopmsopnm .m magmas
mHQZDz 20Heo<mm
DOOH I P b bowl“ b F til lb, VOOH h D I Pom h n L
1‘1 1 4 1 J 1‘ 1 1 l 4 O
- .YH.
4m.
am
me0
:2, is m w
Ixnu
we
g'd.
mam was mea mes
pouaawcm >5 .ap Home: oomH n umppooom ifim.

mpSCHF\.HE H u even 30am

chested esaecm .2 N.o .Hs 0mm
sacs custom .: m.o .as 0mm
have: emaaapmae .Hs 0mm . mzoapgaom

canawhmomeOhgo

.sa oma x NH .gmos ooeuoom
evMELom .p x HuxeSOQ I QESHoo

 

53

typical for other pork muscles, as well as for samples from bull, lanb
'and beef from the beef aging study. dven though IMP was the predominant
nucleotide, it was always well separated from UMP.

The recovery of known amounts of 5'—nucleotides following separation
and colorimetric analysis is shown in table 7. Recovery rates ranged from

Table 7. Recovery of 5'—nucleotides by ion exchange chromatographic
separation and colorimetric analysis.

 

 

 

 

ggdgg; Recovered . éQQEQ Recovered .
Nucleotide _pg. ng. p .Péb )g;, _#b
5'-C:P 800 701 92.6 1300 1329 102.2
5'-AMP 700 720 102.8 1300 1296 99.6
5'_U:r 700 733 100.7 1500 1470 98.0
5'-1w2 700 7&2 106.3 1400 1360 97.1
5'-GIP 300 311 103.6 1300 1311 100.3

 

92.6; to 106.3} and from 97.15 to 102.2; for low and high levels of added
nucleotides, reSpectively. These are excellent recovery rates, eSpecially
when talking in terms of 3. added and recovered. A very small weighing
error could drastically effect the results since 100 pg. is equal to 0.0001 g.
and many analytical balances are accurate only to 0.0001 5. Some difficulty
was encountered when analysis were first started. however, after a few

trial runs excellent recovery and repeatability was obtained. Known samples
were added to the column and analyzed throughout the duration of the
experiment in order to assess the accuracy of the techniques and the
resolving power of the ion exchange resins, which were being recharged

and reused. In no case was a coluin used over 20 times, even if the

 

54

resolving power of the Dowex appeared to be adequate. Cohn (1950)
reported using columns up to 30 times with no loss of capacity or
resolving power.

Results of a pre—trial study on the effectiveness of two extraction

procedures is shown in table 8. Analysis of the data indicated that the

 

 

 

 

Table 8. Effect of extraction procedure on inosine
5'-mon0ph03phate content
Extraction Procedure
éamplg Perchloric acid Boilinngater
1 £1.80 5. 50
2 13.44 19.33
3 ' 13,, 51 11.57
4 3.11 3.01
Mean 4.21 4.35

 

difference between treatments was not statistically significant on using

.v

Duncan's lew.fiultip1e Range Test for evaluating means (Steel and Torrie,
1960, Snedecor, 1956). This indicates that either the perchloric acid

or the hot water extraction procedure could be utilized without seriously
affecting experimental results. These results are logical since work by
Schade et.al. (1963b), Fujita et a1. (1961), Hanaoki and higashi (1963)

and Take and 0ts*ka (1964) indicated that nucleotides are stable at 10003.,
as long as the pH is maintained near neutral. hot water extraction was
used to inactivate any enzmne systems present which might dephOSphorylate

5'-inosine monOphOSphate and thereby render it ineffective as a flavor

- ,1, a c 1 .4 -.1 n ./ l a o J. \
potentiator (onargoif and DaVioson, 1955, animazono, 1903 and JHJlba and

55

Hasimoto, 1960). The hot water extraction procedure was used not only

to extract the nucleotides and to inactivate the enzymes but also because
it was simpler and less time consuming.

Qualitative determinations of the nucleotides were made Spectro—
photometrically and with thin-layer and paper chromatogra_hy. This was
necessary in order to identify and establish the order of nucleotide
elution. Since samples were diluted during elution, he individual peaks
were collected and freeze dried to concentrate them. Freeze dried peaks
from several successive runs were then composited until enough material
was available for characterization. Generally, 1h? from one run was
adequate for qualitative analysis, while several runs were required to
accumulate ample AQP, CL? and Uh? for characterization. Once the order
of elution was established as well as the approximate position or time of
elution, it was not necessary to carry out qualitative characterization
on every sample. However, qualitative and quantitative analyses were
made whenever a new column was being used or when a different series of
samples was being analyzed.

Results of the spectrophotometric analysis of known and unknown
nucleotide peaks are shown in table 9. chellent results were obtained
in identifying the individual nucleotide peaks with this method. rhe
ratio of absorbance at 250/260 and 230/260 mp.was faster and sfmpler
to obtain than was the complete Spectra of a compound. 1L addition,
'hese results were equally as reliable. Absorption Spectras were tedious
and time consuming to measure with a jeckman DU spectrophotometer, since
the blank reading required adjustment each time the wave length was

changed during the determination.

56

Table 9. Spectrophotometric analysis of known and unknown
nucleotide peaks - pH 7

 

Maximum absorbance Ratio of absorbance

 

 

52501260 280/260

Peak Unknown £3933 Unknown Known Unknown Known Identity

1 271 271 .88 .84 .93 .98 CMP
2 259 259 .82 .79 .29 .16 AMP
3 262 262 .76 .73 .38 .39 UMP
4 249 248 1.65 1.67 .27 .25 IMP
5 251 252 1.18 1.16 .63 .66 GMP

 

Table 10 shows the results of thin-layer and paper chromatography
for identification of the nucleotides, while figure 6 illustrates the
migration pattern of the nucleotides during thin-layer chromatography.
Positive identification of the nucleotides was made when the Rf values of
the unknowns were compared to the standards, which were analyzed concurrently.
As can be seen in table 10, the relative rate of separation of the nucleo-
tides was greater if thin-layer Chromatography was utilized. As shown in
table 10, the rate of migration of UMP, IMP and GMP was similar. Sepa-
ration of these three nucleotides would not have been possible if all
three compounds had been applied to the same Spot at the origin. In the
present study, separation was achieved by ion exchange chromatography,
so that paper chromatography was utilized only for identification. Thin-
layer chromatography was superior to paper chromatography, since it was

a faster procedure (80 minutes vs. 16 hours), less tailing of the Spots

occurred, less material was required for Spotting the chromatograms and

\J‘k

Chromatoplate — MN cellulose powder 300

Solvent system - n butanol/acetone/acetic acid/5% ammonium
hydroxide/water 4.5:l.5:1:l:2

Development time - 80 minutes

Detection - Ultraviolet light

STANDARDS UNKNOWNS

 

 

Eégg‘é‘ E E2?

 

Figure 6. Thin-layer chromatogram of nucleotides.

UMP

h

58

Table 10. Results of thin—layer and paper chromatography
analyses of nucleotides

 

 

 

Thin-layer Paper
Chromatography» 3hr omatography
Peak Rf Rf Rf Rf
11.2.2. 31111219111 1212.121 12......m01-m 2112121 3211.22.11

1 .33. .33 .211 .24 C113
2 . 38 . 37 . [4’7 . [49 1h .P
3 .36 .35 .15 .15 HEP
h .30 .30 .lM .135 TWP
5 .26 .25 .13 .126 0122

repeatability was better. It can be seen from figure 6 that resolution
of the nucleotides was quite good. LOIger development time further
increased the resolution.

From the results of these three qualitative evaluation procedures
positive identi1 ication of the eluted nucleotides from samples of known
nucleotide mixtures, as well as from unknown meat samples was accomplished.
In addition, the elution order and approximate elution position were
established.

The periodic acid — 2,4—DMP3 color reaction employed for quantitative
determination of the nucleotides can be used to distinguish he 5’—nucleo—
tides frozn their 2'— a1d 3'—isome1 s. This can oe accomplish -ed by selective
oxidation of adjacent hrdroxyl groups attached to the number 2 and 3 carbon
atoms of the ribose moiety. The product of tlfi is oxidation are then reacted
with 2,4—DIPE and estimated colorimetrically. This scheme is very effective

for assessing the level of the flavor: ul 5 —nucleotides in foods. Any

U1
\0

compound, which reacts with periodic acid and/or 2,4—DNPH, could conceivably
interfere in the colorimetric determination of the 5'—nucleotides. However,
the periodate — 2,4-DNPE color reaction incorporates the use of anion ex—
change chromatography as a prerequisite to colorimetric analysis, and all
anionic compounds are retained by the resin. Interference from carbonyl
compounds, carbohydrates, nucleosides or other nonionic substances is
eliminated, since these compounds are washed from the resin prior to gradient
elution.

Other compounds of biological origin with anionic groupings, such as
1h05phorylated sugars and the 5'—di— and tri—phOSphonucleotides, interfere
with this color reaction due to the presence of the vicinal hydr xyl group.
In the latter case, however, the di- and tri—nucleotides are not eluted
from the column by the gradient systen employed. The phoephorylated
sugars are eluted in the vicinity of the 5'—nucleotides but are located
in separate fractions. Further, these compounds do not absorb in the

ultraviolet region, and would therefore not be observable by the ow

scanner on elution from the column.

luscle investigation

 

This portion of the experiment was designed to assess the nucleotide

concentration in each of three muscles from six pork carcasses. The

muscles involved were the biceps femoris, longissiuus dorsi and semi—

 

:uenbranosus. samples were separated by ion-exdhange ehnonato

‘
‘rauhy and

 

’3
'/
0.;

'wn _I o J_ _l__o _ _._ -‘ l ' 7 o_l_1 ‘ ~‘u '0 o I «3 <3“ _“.-‘”2 4. ‘0
thanblbaulwelv evaluated with a oe1iooic a01o — M—un1n COlOllmellC
c/ ,, ’
J. _r_\ -" -:-, J- ~ 0 ‘ _ o ‘ :- ‘1“ J_ 4 _ J. 0 ‘ _n 7-1‘ 0 ‘ v7 -‘ _-‘
ales 01 the 11: deuCflfllflablOfl 1202 this DorblOfl 01 the investi—

gation are given in table ll while the complete nononucleotide content

of theihree muscles is shown in Appendix table I.

Table 11. Inon

'2 1,‘
Ian ._

Animal
dumber

l

\R

O\

Kean

'./~

q

sine 5 —nonoeeosoi 'e content of 3 pork muscles -

biSSU

1
_

‘__7°

diceps Longissinus Semi
feneris dorsi membranosus

 

 

3.40 3.03 2.59
'3 to. ’3 ’3 .~.
3.59 2.31 5.11

'I " . - 3‘.
3.40 2.91 3.59’
Q /[-I I) ’(\ I")!
V.’\)) ).k)d 2.).“

N
\O
\O

{0
u)

(\D

N

V-N

{\D

U ‘\
\J1

Id
\J

Ks.)
\2.)

b.)

CO

H
N
N
\O

b.)
L.)
E
\J.)

 

 

The average inosinic acid

+h

content of one 1o issimus dorsi, biceps

 

.1 - . -
. erenosns

K—s

.—

muscles was 3.23, 3.12 and 2.98 pI./g.

 

 

tissue, re ctivel dtati tical analysis of the muscle means indicated
tiat Si'nific ant (if? rences did dot exist at eitner the l} or 53 level,
wheL1 ‘uncan‘ we: jultiole Jan 0 Test was utilizec (Rteel and Torrie, 1361,
JOGOCOI 1956). Jven though the data were not statistically significant,
a slight trend ap'eared to be evideit. inc so1h1embranos1:1uscle
contained the loweSt level of InP in four of the six pigs exaained, whicl:

1 . ‘3' 1‘ .‘ 4_'\\
0035.1. ”11° 113.6. p-10

1 1.4.x)

cle

1-1
on
v

still had highes

variation between

0y the fact that the

V‘U

p.
LJ

average value. The biceps nor: containeo the

 

J-

 

three 01 the fig gs ested Even thorgh IZ.1e longissinus
highest concentration of IIP from only two pigs it
t average IL? content. This indicates con81derable
scles from pig to p13. The variation is s Wbs an tiated
standard deviation for Lhe lon issi :us dorsi, biceps

 

feneris and senimembranosus muscles was 0.33, 0.42 and 0.40 ph./g. tissue,

 

reSpectively. rhe wide range between high and low values, of course,
agreed with the relatively high standard deviations. However, standard
deviation is a rather poor neas ure in this case, as there are many factors
which could cause wide variation from pig to pig and as a result increase
the stanC2ard deviation. Hedrick (1965) in a review article indicated that
stress could have an e11* ect on the ’ T? level. As a result of stress, the

ATP level could be altered, nhich in turn would aziect ne IIP level.

f—I—i

Iedri ck suggested the at stress nay oe a result of hormonal or chemical
injections, fatigue, exercise, excitement, preslaughter feeding and die-
tary restrictions. flhen evaluating the data in this study, it should be
remembered that they were taken 48 hours post-morten. Consequently,
inosinic acid degradation may have alrea.dy be 3un. It should be pointed
out that there may be two possible mechanisms for the degradation of
inosinic acid. First, a constant amount of II? may be decomposed during
a given time period. In this case, the range between values for two
muscles would remain constant as IhP was decomposed. Secondly, the

rate of decomposition may occur on a percentage basis. In this case,
the r anfse between values ior two muscles would decrease as degradation

w.

oroceeded. If we had two muscles, for example, with an II? content of

5 and 4,n1./3. tissue, re pectively , and a 53 reduction in In? content
occurred, the new IIP values would be ~.' .75 and 3.e0 pI ./f3 . tissue. The

.1.

difference between the two muscles would now be 0.95 nI./g. tissue rather

than the 1.0 pI./g. tissue, as was the case in the original samples.
The II? content of these three muscles a3rees well with results 01

..

Terasalci et al. (1965), who reported the In? content of the longissinus

 

O\
N

dorsi muscle frdn 154 pound Yorkshire barrows to be approximatexy 3.0

ph./g. tissue at 48 hours post—morten. Lillo (1965) found that the IMF

content was similar for the psoas major, lattissinus dorsi, longissinus

 

dorsi and Lectoralis pr fundus muscles from beef and pork, although the

 

rate of decomposition varied between muscles during a 15 day storage
period at 3—403.

Differences between muscles have been observed by Saito gt al.
(1959b), who indicated that the IIP content of the dorsal muscle of the
rainbow trout is considerably higher than the red lateral muscle. Arai
(1960a, 1960b) and Arai gt al. (1961) reported differences between the
foot and adductor muscles of shellfish. Minor (1964), Zhivkov (1965),
Kazeniac (1961) and Terasaki_gt.§l. (l965) have all reported that samples
of chicken breast muscle contained relatively higher concentrations of
ZIP than did the darker colored muscle.

One objective of this portion of the study was to determine if the
inosinic acid concentration varied from muscle to muscle. Even though
statistical analysis revealed that there was no difference between the
means of the three muscles examined, it must be remembered that relatively
small numbers were involved in +his investigation. If large numbers of
determinations had been carried out, it is possible tha‘ some of the
trends observed might have been statistically significant. Since the
muscles examined contained essentially the same ILP concentration, it was

decided that the longissimus dorsi muscle would be utilized for further

 

investigations. The lonfiissimus dorsi muscle was more accessible, less

 

damage was done on removal of the sample and the standard deviation of

'he longissinus dorsi muscle from the six pork carcasses studied was

 

smaller, thus indicatilg that less animal to animal variation existed.

(V \
L1)

she was not detec able,1hile SIP was present in only trace amounts
in all three muscles tested (Appendix table 1). The level of UhP was

only slight1/1.er. The longissimus dorsi, biceps femoris and

 

 

sen1i11emo1anosus muscles contained 0.12, 0.20 an( 0.11 uI./g. tissue,

 

reSpectively. it such low concentrations, accurate determinations of

the nucleotides are diffic1 t to obtain since it is not easy to ascertain
from the recorc1n" contained on the chart paper the exact point at which
the peak was eluted.

fhe ASP concentration in pork muscle was 0.33, 0.90 and 0.33 hi %/

tissue ior the longissimus dorsi, biceps femoris and sesinenbranosus

 

 

 

rm

nuscles, :eSpcctiv fwl ‘1050 values are consideraoly higher than other
alt 1es reported1 in the literature. Jakaji ma et.al. (1961) reported

1:1D values of 0.19, 0.03.033 and. 3.1-5.9 urn/5.1;. tiss 1e for fish, meats

and inverteorate aniials, reSpectively. Terasaki gt 31. (1965; listed

the levels of ii? for mutton, horse, broilers and pork at 0.19, 0.0,,

0.15 and 0.15 n ./3. tissue, reSpectively. Jones and Lurr.y (1961a)

found a level of 0.01 u;./3. of codling muscle.

The high average value 0.90 un./”. tissue) obtained 10“ the bicens

4.1

fenoris muscle was due primarily to the ezcceptionally high level in one
sample (Appendix table 1). Due to the one high value, the standard

0 u o o o A fl.
deviation for the biceps femoris muscle was extremely high, being 0.09

 

m1 ’3 .L

u£./g. tissue. _ne reason ior the high level in the one case cannot be

explained. ill values in this investigation are the result of avera win”

ygv 1..)

J
.1.

the concentration fr 011 duolica 1te determinations. ill duplicate measure-
ments included separate extraction, ion exchange chromatographic sepa-

ration and quantitative evaluation, which was also carried out on

 

6L1

duplicate samples of each peak. utandard deviations for the lon3iSS1Lnus

dorsi and senimembranosus muscles were 0.03 and 0.17 uh./g. of tissue,

 

reSpectivel1, indicating considerably less variability between animals
for these muscles than for the biceps femoris

Several workers (Perry 1951, Dvorak, 1958, Jones and hurray 1961a,
Tomlinson and Geiger 1962a) have indicated that some nucleotides are
bound to the myofibrils of muscle. These adenine nucleotides appear
to be protected in some manner from enzymatic breakdown at pH values
which normally occur in post-rigor muscle. Tomlinson and Geiger (1962a)
demonstrated with lingcod muscle that the bound nucleotides were released
from the nv01' ibrils bv heatin3, but they were not released if the muscle
was simply frozen and analyzed. Dvorak (l9 3) suf'ested that temperature
night cause the release of bound nucleotides, since the adenine nucleo—
tides were stable at 500., while at 3700. they were not stable. If this
is the case, extlaction with boiling water 11a y release the bound adenine
nucleotides and at the same time inactivate the LEI —deaninase enz3 he
system, so that the nucleotides are not converted to ZIP. This would
partially explain the reason for finding higher levels of ALB in these
three muscles in the present study than have been reported by other

workers, when they used the n re conventional perchloric acid extraction

procedure. Jones and hurray (1931a: and . ML all and Davey (1953) both
suggested that nucleotide bindin3 is dependent upon p1 and concluded
that less nucleotides are bound at hi 3her pi values.

lredholu (193 3) su33ested that the level of the adenine nucleotides

was more staele than p1 eviously believed. Le indicated that e-

of AL? remaining at ., '72 and 120 l1ours was 1003, 60~ and 403 for beef

6L1,

duplicate samples of each peak. Standard deviations for the longissimus

 

dorsi and senimembranosus muscles were 0.03 and 0.17 uh./"

(3.

of tissue,

 

reSpectively, indicating considerably less variability between animals

for these muscles than for the biceps femoris.

 

Several irorkers (Perry 1951, Dvorak, 1958, Jones and Lurray 1961a,
Tomlinson and Geiger 1962a) have indicated that some nucleotides are
bound to the myofibrils of muscle. These adenine nucleotides appear
to be protected in some manner iron enz3t1atic M3 eal {down at pH values
which normally occur in post—rigor muscle. Tomlinson and Geiger (1962a)
demonstrated with lingcod muscle that the bound nucleotides were released
from the nyo1ibrils by heatin3, but they were not released if the muscle
was simply frozen and analyzed. Dvorak (1953) suggested that temperature
ni3ht cause th Qe release 01 bound nucleotides, since the adenine nucleo—
tides were stable at 500., while at 3700. they were not stable. If this
is the case, extraction with boiling water ma; release the bound adenine
nucleotides and at the saw e time inactivate the :jP-oea31nase enz3 no
system, so that the nucleotides are not converted to 11?. ihis would
partially explain the reason for finding higher levels of AL? in these
three muscles in the present study than have been reported by other

1

workers, when they used the n re conventional perchloric acid extraction

’ 1

procedure. Jones and urray, (19 and Bendall and Davey (1953} be h
sug3ested that nucleotide bindin3 is dep e11dent upon p: and concluded
‘hat less nucleotides are bound at hi3her pf values.

Jredholu (1963) su33ested that the level of the adenine nucleotides
was more stable than previously believed. 10 indicated that

n ~--~r-
H

s . ’1 '. . , I" . ,2 _.._ . f \ - ‘ I ’\ J."- 1 -T‘
01 511 remaininc at h, (2 and 120 hours was 1003, 00‘ ane +0 _or oee1

Q

55

and 77d, 315 and 193 for pork, reSpectively, when compared to the value
at 0 hours. Fredholm used inophoresis to separate the nucleotides in
this experiment. However, his quantitative evaluation was simply the
measurement of the color intensity of the resulting Spots. Since the
adenine nucleotides occur at such low levels in meat, it appears likely
that this method of quantitative measurement would be inadequate and as

a result these values might be questioned.
Beef aging study

The aging study was designed to assess the nucleotide content of

the longissimus dorsi muscle of beef during a 28 day storage period

 

o. ,w .
at 33—35 4. oamples were analyzed at O, 12 and 2% hours and at 4, 7,
19 an‘ 28 days post—morten. The concentration of inosinic acid through—

wan—r-

out aging is shown in table 12, while Appendix table 11 lists the

Table 12. Inosine 5'—monOphOSphate content of the longissimus
dorsi muscle in beef aginfi Study — u§./g. tissue

 

 

 

 

line
Animal O 12 24 4 7 1% 28
ilusfo or hours 5121,13 hours 93273 day‘s d are d axis
1 4.73 5.50 4.39 c.34 3.'6 2.28 0.84
’ / u A , , r’ \ .
2 5.32 5.03 @.37 b.12 3.@5 2.37 1.00
3 4.30 H.35 ”.03 3.ol 2.9a 2.2? 0.79
b 4.11 5.11L 5.03 “.0? 2.31 1.96 O.M6
5 11.90 5.40 50:17 5.09 3.22 2.1.0 005'
.1 1, My 3 l 5 37 ). “A 3 r1 ‘1 ”W p ‘7
x». .J .. ._ N, . fl !.\)/ Q)... _o/ 1’ \J.
jean M.71 5.@# $.86 @.47 3.20 2.17 0.75

 

concentration of AID durin: this sane period. Figure 7 schematically
shows the formation and degwracation of 11?.

Inosinic acid reached its highest concentration 12 hours after
slaughter (table 12). The level then began to decline so that by 24

0 V

hours post—norteu it was approxinately the same as the value at 0 hours.
The concentrationo of 12P th ien remained relatively staele ntL 1 the 4th
dagr, after which a gradual but steady decline was observed. At 14 days
post—mortem, the I}? level was less than 503 of the value of 5. WI ;; 3.
found at 12 hours. At 28 days the average 1h? concentration was 0.75
ph./5., or less tian lEfl of th maxiamn concentration occurring at 12

hours. The decrease in ZAP content from w days to 23 days appeared to
be almost linear in nature (figure 7).

Table 13 shows the results 0
means in this study when Duncan's Qew nultiple lan 0 lost was utilizec
(Steel and Torrie, 1961, Snedecor, 1956). The differences between means

Table 13. Statistical a alzs sis of th LO inosin :1 nophOSphate
concentration nee us of beef aging study.

 

 

 

 

1 _ I 1 - ~ 1 ," -- ,_ r 1 __ ,I w - m —. .
12 ars. 2w. hrs. 0 hrs. v days / ca¢s 14 days 2o days
urns-M— 34“ ‘
TI’ ,’ y!” 1, :j/ I, ,~ ".1" f‘ '7 r\ r r’
{Lean DOT-4‘5 L.’.'..)O “’0 {/1 1’ 1’ J 0 iv golf U o 7;)

 

 

 

Any two neans not und erscorec or the same line are significantly
different at the lo level.

Any two means underscored bv the sane line are not significantly
di f3_erent at the 1} level.

 

.m ommnmm as voice omepopm hem. wN a maze moon. mo oHomsa
IIIIIIHMQII
amuon wdfiwmm. 0H o5 5 ovufimofiocoslfi efimofi mo Hezeq .m. made

man

 

 

mmw mH b a

 

 

 

('2/°wd) NOILVHLNEONOO

 

68

were statistically significant in all cases except for values at 24

hours and 0 hour and at 0 hour and 4 days. This suggests, that except
for the peak IMP concentration at 12 hours post—mortem, there is very
little change in the level of IMF during the first 4 days of cold storage.
The differences between means after 4 days storage were statistically
significant at the 1% level, which would indicate that consistent changes
in the level of IMP were taking place.

The rate of IMP degradation found in the present investigation
agrees with that reported by Terasaki st 31. (1965). These authors found
that the peak concentration of IMP in pork was not observed until the 3rd
day postamortem and that the level was 3 uM./g. tissue. The level gradup
ally decreased until the lOth and last day of the study, when approximately
1/3 of the inosinic acid had been decomposed. Terasaki gt al. demonstrated
that chicken breast muscle had a peak inosinic acid content at 8 hours
post—mortem with a gradual degradation through the 8th day, when the test
was concluded. Although nucleotide decomposition rates were generally
the same, the actual nucleotide content could not be validly compared
since different Species were involved in the studies. Endo 32 al. (1966)
demonstrated that 50$ of the IMP concentration of beef and chicken was
decomposed in two weeks at O—lOC. Dvorak (1958) found that the IMP con—
centration in samples of bull muscle was the greatest at two days post—
mortem and then fell until 15 days when it was approximately the same as
after one day of cold storage.

Rhodes (1965) observed that inosinic acid has completely degraded
in some beef cuts after 30 - 40 days, while the rate of decomposition
occurred more slowly in other cuts of beef and in lamb. The data are in
general agreement with the relatively low levels of IMP found following

28 days of cold storage in the present study.

 

O\
\O

The initial level of inosinic acid in beef in the present study
appeared to be high. This could be the result of two factors. Sirst,
the samples were not taken at exactly 0 hours. Samples were, however,
taken within 15 minutes of stunning and frozen immediately in liquid
nitrogen. Sven in this brief period of time, there could have been a
rapid dephOSphorylation and deamination of the adenine nucleotides.
Secondly, if the animal were excited to any degree before slaughter,
the ATP supply could have been partly depleted and as a result a
relatively high concentration of inosinic acid would be present. This
latter phenomena has been noted by numerous scientists,name1y, Terasaki
g: El° (1965), Jones and Qurray (1957, 1961a, 1961b), Eraser 23.31'
(1965), Eujhiaki and fiojo (1953) and duardia and Dollar (1965). It is
probable that the high initial level of inosinic acid could be partially
overcome by s npling immediately after bleeding and by using care not
to excite the animals prior to slaughterinj.

Results from this experiment also indicate that it may be of value
to take more samples earlier in the storage period so that the exact

r11

time of peak In? concentration could be pinointed. This could be

{ '1

accomplished by taking samples at O, 4, e, 12, 16, 20 and 24 hours, as

I/ , r3 . . . ‘ a. .
well as at Be an: 4a hours. This sampling plan would comprehenSively

cover any critical chazfies in the level of inosinic acid during early

storage when the greatest chanfes are apparently occurring.
‘Iie level of ALP was highest at 0 hour and decreased constantly
until the 5th day (Appendix table 11}. At this tine, the concentration
a

. 1 _- _- - o ‘ 1 ‘ o ‘1 1 ' If! Ar I ‘ .‘ --‘ -
stabilized in tn- HellnbOPJOOC or o.)? al./3. tissue. as with pg» per:

inuscle study, these values again are higher than other values reported

70

in the literature. From knowledge of adenine nucleotide degradation,
it appears logical that the level of Ah? would be highest at 0 hour
and then decrease thereafter as was found in the present investigation.
The Ah? level would likely be lower due to the activity of ALP—deaminase,
unless the adenine nucleotides were protected in some manner as was
previously discussed. The pattern of Ah? degradation noted in this study
is similar to that demonstrated by Terasaki §§_al. (1965), who showed
that the level of AI? reached a relatively stable level of about 0.15
pI./g. after 48 hours cold storage. Jones and nurray (1957, 1961a),
Dvorak (1958) and Kassemsarn‘pt.a1. (1963) have also demonstrated a
similar pattern of An? degradation.

Sigure 8 shows a typical separation pattern for nucleosides and

bases. Table 14 gives the R values and identity of the nucleosides

f

and bases from a sample of the longissimus dorsi muscle of beef. hypo—

 

Table 14. Thin—layer chromatography of nucleosides and bases
from a sample of the longissimus dorsi muscle of beef

 

 

Unknown Known

____lf?.-° ____ "ti... Id entigl
0.74 0.73 Inosine
0.57 0.59 hypoxanthine
0.35 0.34 Adenine
0.85 0.84 Uridine

 

xanthine, inosine, adenine and uridine were present in the effluent,
which resulted from washing the ion-exchange column with distilled water

prior to elution with the three phase gradient system. Their presence

ADENOSINE

 

Figure 8 0

‘Q
l—’

Chromatoplate - MN cellulose powder 300
Solvent system — Distilled water
Development time - 45 minutes

Detection - Ultraviolet light

CYTIDI”E
GUANOSINE
INOSINE
URIDlNE
UN NJ OWN
ADERINE
CYTOSINE

o
O

Thin-layer chromatogran of nucleosides and bases.

HYPOXANTHINE

0 C3 UNKNOWN

0

 

72

was demonstrated when thin-layer chromatography was used to separate the
components in the effluent (figure 8). Inosine and hypoxanthine were
present due to the degradation of inosinic acid, while adenine and uridine
undoubtedly resulted from the decomposition of AMP and UMP which had
previously been shown to be present in muscle (Appendix table I). Cytosine
may not have been observed since CMP is present in trace amounts only.
From figure 8, it can be seen that good separation of nucleosides and
bases was effected by using thin-layer chromatography. The ease and
simplicity of the test is evident since the solvent system consisted of
distilled water and only 45 minutes were required for the development

of the chromatogrmns, which are then air dried and read under ultra-
violet light.

Although the level of hypoxanthine and inosine were not quantitatively
measured in this experiment, it was readily evident that their concen-
tration was increasing steadily as IIP was degraded during the storage
period. The increase was evident as shown by the gradually increasing
peak size obtained when the nucleosides and bases were scanned by the
ultraviolet scanner as they left the column. There was only a very
minute hump on the base line from samples taken at 0 hours. In many
cases the peak was absent entirely. An optical density reading of over
0.5 was noted after 28 days storage, which corresponded to the maximum
reading on the chart paper. It is evident from these observations
that the accumulation of hypoxanthine from IEP degradation might be
used to assess the freshness of muscle tissue. Jones and Murray (1964),
Jones gt al. (1964), Spinelli 33 al. (1964) and Saito st 31. (1959) have

all demonstrated that the freshness of stored fish can be effectively

—.—‘—.

measured by the level of hypoxanthine. These procedures are based on
the assumption that the degradation of ILP to hypoxanthine is a linear
function. This being true, one need only to measure the hypoxanthine
content of fish muscle, compare this value to a known curve or insert
it into a standard formula and evaluate the freshness of the product.
However, Rhodes (1965) concluded that 'he measurement of IMP or its
breakdown products appeared to offer little or no promise for measuring
the freshness of red meats. There may be a greater number of problems
in developing a reliable method for assessing the freshness of red
meats, since the rate of ZAP degradation appears to proceed at a con—
siderably slower rate in meat than in fish muscle. Secondly, Spoilage
does not occur so rapidly and is more readily discernible in meat than
in fish, where the method was evolved out of necessity of evaluating

freshness.

species investigation

 

The nucleotide content of the loneissimus dorsi muscle of bull,

Q.
beef, lamb, pork and of pork heart muscle were determined and compared.

The inosinic acid concentration of these muscles is shown in table 15.

Appendix tables III, IV and V list

.L

the total nucleotide content for
bull, lamb, and pork muscle at 0 hours and for pork heart muscle. Beef
;p

n -
-,. .
.L __|-

muscle at 0 hours contained the highest level (4.71 u ./g.)

0

9

while pork heart muscle contained the least (0.13 u&./;.). The concen-
tration of IQ? in other Species fell between these values. Pork muscle
at 0 hours, pork muscle at 43 hours, bull muscle and lamb muscle contained

4.33, 3.23, 2.84 and 2.24 pH./g. of tissue, reSpectively.

74

Table 15. Inosine 5'-monophosphate content of the longgssimus
dorsi muscle of bull, beef, lamb and pork and of
pork heart muscle — nM./g. tissue.

 

 

§E§S§E§
. Pork
Animal Beef Pork Pork Heart
Number Bull 50 hour) Lamb (0 hour) (48 hours) (0 hour)
1 3.04 4.78 2.82 4.10 3.03 0.27
2 2.89 5.32 2.12 4.26 3.31 0.10
3 2.95 4.80 1.68 4.35 2.91 0.03
4 2.65 4.11 2.66 3.84 3.60 0.13
5 3.05 4.90 1.86 5.35 2.92 -—
6 2.45 4.34 2.31 -- 3.61 --
Mean 2.84 4.71 2.24 4.38 3.23 0.13

 

It is rather difficult to make comparisons between the Species
inVOlved in this portion of the study as wide variations existed in the
time of sample procurement. In addition, samples were not all treated in
a similar manner. ‘However, the samples of beef and pork taken at 0 hours
Should be comparable. Although beef contained 0.33 pM./g. more inosinic
acid than pork, this difference was not significant, when Species means
were analyzed using Duncan's New Multiple Range Test (Steel and Torrie,
1961, Snedecor, 1956). Although the mean difference between samples of
beef and pork at 0 hours was not statistically significant, a trend for
beef carcasses to have higher levels of inosinic acid appeared to be

evident (table 15).

 

-------

.......

75

The samples of pork taken at 0 hours contained 1.15 pM. more IMP per
gram of tissue than samples removed after 48 hours of cold storage. This
is over a 25% reduction in IMP content. Although this value may seem
somewhat high, different pigs were being compared at the two periods and
the handling procedures may have varied slightly. The concentration of
inosinic acid in the pork at 0 hours differs from the results obtained by
Terasaki gt 31. (1965), who reported the IMP level to be approximately
1.0 pM./g. tissue. The difference between values in the two studies
would be explainable if the samples utilized by these workers had been
removed from the animals immediately after stunning and bleeding. However,
lit does not seem likely that Terasaki gt al. could have obtained the pork
samples immediately upon death, since the pigs were stunned, bled and
scalded prior to sampling. It Should also be noted that the number of
animals utilized was not mentioned in this paper. The 0 hour samples in
the present experiment were taken within 15 minutes of stunning, and this
slight delay could explain the relatively high concentrations at 0 hours.
Terasaki gt 31. reported a level of 3.0 pM./g. tissue at 48 hours post-
mortem, which correSponds quite closely to the value of 3.23 uM./g.
tissue in the present experiment.

The concentration of IMP in the longissimus dgpgi muscle from bull
carcasses was 2.84 uM./g. of tissue (table 15). The values from the Six
bulls, all fell into a very narrow range, which extended from 2.45 to
3.05 pM./g. tissue. The standard deviation of these samples was 0.24
pM./g. tissue. No control could be exercised over the treatment of
samples in this study Since the samples were part of another investigation.

The samples were taken on the 7th day post—mortem, ground and frozen. In

76

addition, these samples had been thawed for proximate analysis and then
refrozen before being analyzed for inosinic acid concentration. The
beef aging study revealed that the IMP level after 7 days was 3.20 pM./g.
tissue which is comparable to the value found for the bulls. It was also
found in the beef aging study, that the range between the high and low
values tended to decrease as the length of the cold storage was increased.
This would partially explain the narrow range of values found for the
bulls. If the thawing and refreezing had any effect on the concentration,
it would be likely to cause a further degradation as well as to further
decrease the range of values. This would agree with Guardia and Dollar
(1965), who stated that differences in IMP content were evident between
exercised and unexercised fish, but that this difference became less as
the length of cold storage increased, until no significant differences
were observed after 7 days of post—mortem storage. The levels of IMP
found in the bull samples were comparable to values for beef bulls
reported by Dvorak (1958). He reported that the concentration of
inosinic acid after two days storage was 3.7 pM./g. tissue and that it
had decreased to 1.9 yM./g. tissue after 10 days, when the samples were
aged at 50C.

Nakajima gt al. (1961b) reported the IMP content of beef to be
3.07 pM./g. and the value for pork to be 3.51 pM./g. He also reported
the combined level of inosine-hypoxanthine to be 2.90 )1M./g. tissue,
which would indicate considerable IMP degradation. This suggests that
the meat samples were fairly old before analysis, although he did not

report the length of aging or sample source.

77

Terasaki 3t 3;. (1965) reported the inosinic acid content of mutton
(9 month old ewes) to be 2.36 pM./g. tissue. This correSponds closely to
the value of 2.24 pM./g. tissue found in this experiment (table 15). The
values reported by Terasaki gt al. for mutton are difficult to interpret,
as the meat had been imported from Australia in the frozen state. As a
consequence, nothing was known about the post—mortem treatment received
by the samples. In addition, the length of the aging period and the
freezing temperature at which the carcasses were held during shipment
from Australia were not known. If the freezing temperature was maintained
at —3OOC. or lower IMP degradation was probably negligible, while at higher
temperatures some IMP decomposition probably would have occurred. Jones
and Murray (1961b) reported that IMP was stable in cod fish muscle when
stored at -3OOC. The authors measured the level of inosine accumulation
as a criterion of IMP degradation during cold storage. At —30°C. no
inosine was detectable, while at —l4°C. the inosine content of cod fish
muscle was 3.01 pM./g. tissue following 62 weeks of storage. This corre-
sponds to an inosine content of 3.23 pJ./g. tissue for lamb muscle as
reported by Terasaki gt El. (1965). This value would indicate that
considerable IMP had been degraded in the lamb samples prior to being
utilized for their investigation.

The lambs in the present experiment had been used for classroom
carcass evaluation. As a result, the lambs were not held at a constant
temperature during the seven day holding period prior to the time that
the samples were removed. Some of the variation in IMP content can
probably be explained by the fact that part of the lambs were held
continually at low temperatures and part were held at varying periods

of time at cold and warm temperatures.

 

78

Dvorak (1958) reported that the rate of IMP decomposition varied
with storage temperature. He demonstrated that the rate of nucleotide
degradation was more rapid in bull carcasses held at 38°C. than when
aged at 5°C. Although, the temperature extremes in the present study
were not so severe, variation in the holding temperature may suggest
reasons for such differences.

The concentration of inosine 5'—monophOSphate in heart muscle was
0.13 pM./g. tissue (table 15). This value is the average determination
of four heart muscles and would have been approximately 25% lower except
for the 0.27 pM./g. tissue found in one heart. The comparatively low
level of IMP found in this study is in agreement with the results of
Visioli gt at. (1964) and Imai EE.§$° (1964). Visioli gt gt. demonstrated
that little IMP was produced in the myocardium of seVerely exercised rats,
while Imai gt fil' noted that IMP was absent in heart tissue from anemic
rabbits. It is also interesting that many invertebrate sea animals do
not possess IMP. Saito and Arai (1957a) reported that Squid contain no
IMP, while Fujita and Hasimoto (1960), Arai (1960a, 1960b) and Arai gt
al. (1961) and Nakajima (1961a, 1962b) verified the fact that invertebrate
sea animals contain little or no inosinic acid.

The AMP content in the pork heart muscle samples was 2.29 pM./g.
tissue (table 15). The high concentration of AMP may indicate that the
enzyme, AMP—deaminase, is absent in heart muscle. Suelter (Unpublished
data) has found that the enzyme AMP—deaminase is present in heart muscle.
However, the enzyme is not normally active since ATP is required to
activate the system. This would explain the reason for the high AMP

content and the low concentration of IMP in heart muscle. Davey (1961)

 

79

was able to demonstrate that dialyzed extracts of skeletal muscle were
capable of synthesizing adenylosuccinic acid by the condensation of
inosine monophosphate and aSpartic acid, from which it is converted to
adenine nucleotides. This enzyme was shown to be absent in heart, lung
and kidney tissue. It is interesting to Speculate that the absence of
this amination enzyme may be the reason why deaminase is not present in
heart muscle. For example, if AMP was deaminated to IMP during periods
of severe exercise, there would be no mechanism present for the reami-
nation of IMP. It has not been demonstrated whether the deamination—
reamination reaction is involved in the cycle of chemical events
associated with muscular activity, although skeletal muscle contains 1
both enzymes.

Guanosine monophOSphate was not detectable in the present series
of samples, while CMP was present in only trace amounts as shown in
Appendix tables III, IV and V. Since these compounds are present in
such small quantities, more accurate measurement of their concentrations
could be made by adding larger samples to the ion-exchange columns. The
level of UMP was trace amounts, 0.06 and 0.02 pM./g. of tissue, re—
spectively, for pork sampled at 0 hours and for bull and lamb carcasses
sampled at 7 days post-mortem (Appendix tables III, IV and V). The
concentration of adenylic acid for these samples was 0.70, 0.76 and

1.21 pM./g. of tissue, reSpectively.

§§nsory evaluation

The final portion of this experiment was designed to assess the flavor
enhancing properties of the flavor potentiators. It was shown earlier in

this study that the level of inosinic acid in pork heart muscle was very

80

low. Thus, a frankfurter formulation using pork heart in place of lean
beef or pork was utilized. Five different recipes were prepared. The
first was a control and contained heart muscle only, the second contained
heart muscle plus 0.2#% MSG, while the third recipe consisted of heart
muscle plus 0.05% inosinic acid. The fourth formulation consisted of
theart muscle and 0.025% Mertase (IMP + GMP, 1:1 ratio). A fifth formula
was made with beef muscle in order to give a positive control. Evaluation
of the frankfurters was made by using a 9 point hedonic scale, where 9
was the highest score and 1 the lowest. The panel members were instructed
to rate each sample for flavor only.

Table 16 shows the average values for the 20 member taste panel,
while Appendix table VI summarizes the results for each panel member.

The average values from the hedonic scale preference ratings indicate

Table 16. Results1 of taste panel evaluation of added flavor

 

potentiators.
Formulation
Pork Hegtt Egg:
1 2 3 4 5
Control M§§_ £M§_ IMP + GMP Control
Mean 5.35b’° 5.55""’b'c 6.2oa’b 6.40a 4.75"

 

l Any means with the same superscript are not significantly different
from each other at the 5% level.

that the panel preferred the sample that contained IMP + GMP. The value

of 6.40 is midway between "like moderately" and "like slightly" on the

hedonic scale. The other values were 6.20 for the sample with added IMP,

5.55 when MSG was added to heart muscle, 5.35 when pork heart was used

 

 

81

as the meat source with no added potentiator and 4.75 for beef muscle
(table 16). The value for beef falls below the median on the hedonic
scale and is between "dislike slightly" and "neither like nor dislike."
Analysis of variance (Appendix table VII) indicated that the average
values obtained from taste panel evaluation of frankfurters were sig—
nificantly different at the 1% level of probability.

Since analysis of variance indicated that significant differences
existed, Duncan's New Multiple Range Test (Steel and Torrie, 1961,
Snedecor, 1956) was utilized to determine the significance between
means. The analysis revealed that the addition of Mertase (IMP + GMP)
to pork heart frankfurters significantly increased the acceptability
of this product over the beef frankfurters at the 1% level and over
the all pork heart frankfurters with no added flavor enhancer at the
5% level of significance. When IMP was added to pork heart frankfurters,
they were significantly preferred over beef frankfurters at the 1% level.
The differences between the other means in this study were not significant
at either the 1% or 5% level of probability. Although the data are
rather limited, results suggest that the addition of IMP + GMP or IMP
to pork heart frankfurters significantly increased the acceptability of
these products. In addition, the frankfurters containing IMP or IMP +
GMP generally tended to be more acceptable to the panel members than
the formulation containing heart muscle alone or the one with added
MSG (table 16) .

Several problems were encountered in this portion of the experiment.
First of all the stability of the heart muscle emulsion was not very good.

These samples required stuffing and processing in a relatively short time

 

82

so that the emulsion did not break. Even so, some fat caps were observed
in this study. Secondly, the peelability of the frankfurters made with
pork heart was poor and as a result removal of the casings was difficult.
Third, the texture of the pork heart frankfurters was poor, being soggy
and rather coarse. It was noted, however, that the addition of any of
the three flavor potentiators, and eSpecially MSG, tended to produce a
firmer product. The beef, which was incorporated into the frankfurters,
had been frozen for approxhnately one year. This may partially explain
the poor acceptability of the beef frankfurters. A second factor, which
may have detracted from the beef frankfurters, was the relatively high
fat content. A preliminary study had demonstrated that due to the high
moisture content of heart muscle some additional fat in the recipe was
needed to make a firmer product. The increased fat content was not so
noticeable in the frankfurters containing heart, and as a result the
panel may have been influenced.

It should be pointed out that this was only a preliminary test and
that only one level of the flavor enhancer was used. In additional
testing, various levels of the flavor potentiators should be added and
their effect evaluated in order to define optimum levels. The flavor
enhancing compounds could also be added to frankfurter formulations
where red meats are being utilized. In actuality, the latter test
may have more commercial application since the amount of available

heart muscle is limited.

 

SUMMARY AND CONCLUSIONS

Known and unknown nucleotide samples were separated on Dowex l X 8,
200—400 mesh, (formate) ion-exchange resin columns and quantitatively
measured utilizing a periodic acid — 2,4hDNPH color reaction. Recovery
rates and repeatability were excellent. Qualitative evaluation of the
eluted nucleotide peaks was accomplished by using thin—layer and paper
chromatography and by measuring absorbance spectras and the ratio of
absorbance at 250/260 and 280/260 mu. Of the three methods utilized,
thin—layer chromatography was the simplest and fastest to use, and in
addition gave excellent results.

The longissimus dgggt, ttggpg tgmggtg and semimembranosus muscles
from six pigs were analyzed for their nucleotide content. The concen—
tration of inosine monophosphate in the three muscles was 3.23, 3.12 and
2.98 pM./g. of tissue, reSpectively, which was not statistically signif—
icant. The standard deviation was 0.33, 0.42 and 0.40 pM./g. of tissue,
reSpectively, for the longissimus dgtgt, RESEEE femoris and semimembranosus
muscles. Since the three muscles were similar in their nucleotide content
and the lgpgtggimtg Q2321 had the smallest standard deviation, it was
used in all subsequent studies. It was also easily accessible and less
damage was done to the carcass on removing the sample.

The level of AMP in pork was 0.33, 0.90 and 0.33 pH. g. of tissue

for the longissimus dorsi, biceps femoris and semimembranosus muscles,

 

reSpectively. The level of UMP was 0.13, 0.11 and 0.20 pM./g. of tissue
for the same three muscles, respectively, while CMP was present in traces
only and GMP was not detectable. The fact that GMP and CMP were not

present in measurable levels suggests that larger samples should be added

83

 

84

to the ion—exchange column for separation.

The second part of this study was designed to determine the level
of IMP and AMP from six beef carcasses stored at 33 - 35°F. for 28 days.
Samples were removed and analyzed at 0, 12 and 24 hours and at 4, 7,

14 and 28 days post—mortem. The peak concentration of IMP occurred at

12 hours. The level then declined until at 24 hours the IMP content
approximated that at 0 hours. It remained fairly constant until the

4th day, after which a linear decrease occurred until the 28th day of
post—mortem storage, when less than 15% of the IMP remained. The concen—
tration of AMP was highest at 0 hours and was followed by a steady decline
until the 4th day, after which it remained relatively constant.

Phase three of the present experiment was conducted to evaluate
the nucleotide content of various Species. The longissimtg QQEEE
muscle removed from bull and lamb carcasses after 7 days post—mortem
storage was compared with pork sampled at O and 48 hours, beef at 0
hours and pork heart muscle at 0 hours. The concentration of IMP for
these samples was 2.84, 2.24, 4.38, 3.23, 4.71 and 0.13 pN./g. of tissue,
respectively. Results were difficult to compare since a wide range of
sampling times and handling procedures were involved. The level of AMP
in the above samples was 0.76, 1.21, 0.70, 0.33, 0.90 and 2.30 pM./g.
of tissue, respectively. Low levels of GNP and UMP were observed while
GMP was not detectable.

In order to assess the flavor enhancing prOperties of IMP, IMP +
GTP and MSG, these potentiators were added to a standard frankfurter
formulation which contained pork heart muscle as a meat source. A

20 member taste panel evaluated the frankfurters on the 9 point hedonic

 

85

scale. Scores of 6.40, 6.20, 5.55. 5.35 and 4.75 were obtained for
recipes containing 0.02533. IMP + GMP, 0.05% IMP, 0.24% MSG, heart muscle
alone and all lean beef (control), reSpectively. Statistical analysis
revealed that the addition of IMP + GMP to pork heart frankfurters
increased their acceptability over the beef frankfurters (P210.0l) and
over the frankfurters containing heart muscle with no added potentiator
(P<L0.05). On addition of IMP to pork heart frankfurters, they were
preferred over the beef frankfurters CP< 0.01). All other differences
were not statistically significant. Thus, results suggested that the
flavor potentiators improved the acceptability of the frankfurters

containing pork hearts.

 

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APPENDIX

 

 

Table I. Nucleotide content of 3 pork muscles (48 hours post—mortem) -

fiM./g. tissue

95

 

Ec@sfmmfis

 

 

Animal
Number CMP All? mm IMP
1 Trace 0.30 0.06 3.40
2 Trace 0.68 0.27 3.59
3 Trace 0.96 0.13 3.45
4 Trace 2.09 0.23 2.65
5 Trace 0.41 0.28 2.99
6 Trace 0.96 0.21 2.65
Mean 0.90 0.20 3.12
Longissimus dorsi
1 Trace 0.34 0.09 3.03
2 Trace 0.37 0.12 3.31
3 Trace 0.45 0.18 2.91
4 Trace 0.23 0.13 3.60
5 Trace 0.35 0.13 2.92
6 Trace 0.26 0.15 3.61
Mean 0.33 0.13 3.23
Semimembranosus
1 Trace 0.43 0.12 2.59
2 Trace 0.22 0.12 3.18
3 Trace 0.55 0.12 3.58
4 Trace 0.47 0.08 2.54
5 Trace 0.13 0.13 2.81
6 Trace' 0.19 0.10 3.17
Mean 0.33 0.11 2.98

 

 

96

Table II. Adenosine 5'—monoph05phate content of the longissimus dorsi
muscle in aging study - pM./g. tissue

 

 

Anhnal 0 12 24 4 7 14 28
Numbgp hours hours hours dgys days dgys days
1 0.85 0.42 0.32 0.22 0.65 0.31 0.23

2 1.00 0.63 0.30 0.39 0.35 0.45 0.26

 

3 0.62 0.59 0.73 0.61 0.61 0.42 0.32
4 1.04 1.15 0.71 0.32 0.22 0.28 0.31
5 1.14 0.63 0.52 0.36 0.24 0.47 0.23
6 1.02 0.82 0.36 0.28 0.50 0.41 0.45
Mean 0.94 0.71 0.49 0.36 0.43 0.39 0.30

 

Table III. Nucleotide content of the longissimus dorsi muscle of
bulls — pH./g. tissue

 

 

 

Animal

N umb er GEE AMP UMP IMP
1 Trace 1.46 0.06 3.04
2 Trace 0.51 0.08 2.89
3 Trace 0.86 0.05 2.95
4 Trace 0.39 0.08 2.65
5 Trace 0.47 0.06 3.05
6 Trace 0.89 Trace 2.45

Kean 0.76 0.06 2.84

 

 

 

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97

 

 

 

 

Table IV. Nucleotide content of the longissimus dorsi muscles of
lambs - pM./g. tissue
Animal
Nutter CM? 3MP UMP IMP
1 Trace 1.41 0.03 2.82
2 Trace 0.35 0.03 2.12
3 Trace 0.98 0.05 1.68
4 Trace 2.01 Trace 2.66
5 Trace 0.61 Trace 1.86
6 Trace 1.90 0.02 2.31
Mean 1.21 2.24
Table V. Nucleotide content of the longissimus £2351 muscle of pork

__Lij___.

(0 hour) and of pork heart — pH. g. tissue

 

 

 

Animal Longissrnus dorsi Heart

Numb er CI‘TP Al 33 U1? 113? All? DP
1 Trace 0.70 Trace 4.10 2.52 0.27
2 Trace 0.86 Trace 4.26 2.60 0.10
3 Trace 0.70 Trace 4.35 1.75 0.03
4 Trace 0.44 Trace 3.84 2.34 0.13
5 Trace 0.82 Trace 5.35 -- -—

Mean 0.70 4.38 2.30 0.13

 

98

Table VI. Results of individual tasters from taste panel evaluation
' of flavor potentiators — (9 = highest value)

 

 

 

 

 

Formulattgp
Taster Heart MSG IMP IMP + GMP Beef
Number 2 3 4 j
l 3 7 7 6 4
2 3 5 4 4 5
3 4 4 4 7 4
4 6 5 7 6 4
5 6 7 7 8 6
6 8 7 7 8 6
7 5 7 7 6 5
8 7 3 4 7 6
9 6 7 5 6 2
10 5 6 7 8 4
ll 4 4 7 6 2
12 4 4 6 6 8
l3 7 6 6 7 5
14 5 4 4 5 4
15 6 6 6 6 5
16 5 7 7 6 4
l7 5 6 6 7 5
18 6 6 8 7 5
l9 7 6 S 6 6
20 5 4 7 o 5
Mean 5.35 5.55 6.20 6.40 4.75
S.D. 1.35 1.31 1.32 0.98 1.37
Table VII. Analysis of variance of panel evaluation
Source of Degrees of Sum of Mean
Variation Freedom Squares REHEEE .21.
Treatment 4 36.0 9.0 5.52**
Error 95 154.75 1.63

Total 99 190.75

 

** Significant at 13 level of probability

 

   

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