A judgment on postmortem aging in Longissimus dorsi based on a peptide substrate library

Article abstract of DOI:10.1271/bbb.60208

We attempted to develop a method to determine easily and effectively the degree of postmortem aging of pork longissimus dorsi (LD) by measuring the activity of proteases in the LD using fluorogenic peptide substrates. LD was used to measure the change with time in the protease activity detected with these substrates. Determining the variations within the LD muscles, strong positive correlations were found between changes in hardness and fluorescence intensities against Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-MCA (P < 0.005), and strong negative correlations were found between changes in total amounts of free amino acids and Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-MCA (P < 0.001). Negative correlations were also observed between changes in the amounts of free Ala, Arg, Lys, Leu, Met, Phe, and Tyr and the fluorescence intensities against Ala, Arg, Lys, Leu, Met, Phe, and Tyr-MCA respectively (P < 0.001).

Full text of DOI:10.1271/bbb.60208

Biosci. Biotechnol. Biochem., 70 (12), 2836–2843, 2006
A Judgment on Postmortem Aging in Longissimus Dorsi
Based on a Peptide Substrate Library
y
Eiichiro ONITSUKA,1; Tomoyuki OKUMURA,1 Hiroshi MURAKAMI,1
1
Norikazu NISHINO,2 and Fumiki MORIMATSU
1Research and Development Center, Nippon Meat Packers, Inc., 3-3 Midorigahara,
Tsukuba, Ibaraki 300-2646, Japan
2Department of Biological Functions and Engineering, Graduate School of Life Science
and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino,
Wakamatsu-ku, Kitakyushu 808-0196, Japan
Received April 11, 2006; Accepted September 4, 2006; Online Publication, December 7, 2006
[doi:10.1271/bbb.60208]
We attempted to develop a method to determine
easily and effectively the degree of postmortem aging of
pork longissimus dorsi (LD) by measuring the activity of
proteases in the LD using fluorogenic peptide substrates.
LD was used to measure the change with time in the
protease activity detected with these substrates. Deter-
mining the variations within the LD muscles, strong
positive correlations were found between changes in
hardness and fluorescence intensities against Ac-Ala-
MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and
Ac-Ala-Phe-MCA (P < 0:005), and strong negative
correlations were found between changes in total
amounts of free amino acids and Ac-Ala-MCA, Ac-
Met-MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-
Phe-MCA (P < 0:001). Negative correlations were also
observed between changes in the amounts of free Ala,
Arg, Lys, Leu, Met, Phe, and Tyr and the fluorescence
intensities against Ala, Arg, Lys, Leu, Met, Phe, and
Tyr-MCA respectively (P < 0:001).
Bowers2) reported that storage of pork LD muscle at
2 ꢀC for 8 d increased the levels of several amino acids,
including glutamic acid. Moreover, Feidy et al.3)
reported that storage of beef longissimus dorsi, triceps
brachii caput longum, and rectus femoris at 4 ꢀC for
14 d increased levels of several free amino acids, and
Okumura et al.4–6) reported that storage of vacuum-
packed LD muscle at 4 ꢀC for at least 20 d increased the
tenderness of the meat and the amounts of free amino
acids and peptides, as well as enhancing the taste and
flavor. As mentioned above, meat with an optimum
degree of postmortem aging is the most palatable.
However, the degree of postmortem aging is not actually
appreciated at meat counters. Because the expiry date is
set on the basis of microbial safety, people tend to eat
meat when it is fresh and not at the stage when it is most
palatable. This is partly because consumers do not
realize that postmortem aging increases the taste, and
partly because a complicated procedure is required to
evaluate the postmortem aging process. Although
common conventional physical and chemical analyses,
such as the measurement of hardness using a Tensi-
presser (Taketomo Electric, Tokyo) and the amounts of
free amino acids can be used to determine the degree of
postmortem aging, these methods are not practicable
because of the long times and high costs involved in
measurement. An easier method that takes only a short
time is desirable.
Key words: 4-methyl-7-coumarylamide (MCA) sub-
strate; peptide substrate library; pork long-
issimus dorsi; postmortem aging; protease
Meat is aged by storing it at a low temperature for
a certain period of time to increase its palatability.
Various biochemical changes occur during postmortem
aging. Nishimura et al.1) reported that postmortem aging
of bovine rounds at 4 ꢀC for 12 d, pork longissimus dorsi
(LD) muscle at 4 ꢀC for 6 d, and chicken pectoralis
superficialis muscle at 4 ꢀC for 2 d significantly increas-
ed the amounts of free amino acids and free peptides.
It is known that endogenous protease is involved in
postmortem aging.7,8) Nishimura7) reported that calpains
and cathepsins in meat after slaughter degraded meat
proteins, such as Z lines, and thereby increased the
y
To whom correspondence should be addressed. Fax: +81-29-847-7824; E-mail: e.onitsuka@nipponham.co.jp
Abbreviations: LD, longissimus dorsi; Ac, acetyl; MCA, 4-methyl-7-coumarylamide; Boc, t-butoxycarbonyl; Fmoc, 9-fluorenylmethyloxycar-
bonyl; DCC, N,N0-dicyclohexyl carbodiimide; TFA, trifluoroacetic acid; DMF, N,N-dimethylformamide; HPLC, high performance liquid
chromatography; FAB-HRMS, fast atom bombardment high-resolution mass spectra; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-phosphate; HOBt, 1-hydroxy-1H-benzotriazole; DIEA, diisopropyl-ethylamine; pmc-,2,2,5,7,8-pentamethylchroman-6-sulfonyl-; MES,
2-morpholinoethanesulfonic acid

A Judgment on Postmortem Aging Based on Peptide Substrate Library
2837
tenderness of the meat, and that aminopeptidases caused
increases in the amounts of free amino acids, and hence
an improvement in taste. This suggests that the degree of
postmortem aging can be evaluated by quantifying
endogenous proteases over time.
carbodiimide (DCC) in dichloromethane, as in the
methods of Zimmerman16) and Kanaoka18) to give
Boc- or Fmoc-X-MCA. The Boc- group was removed
with trifluoroacetic acid (TFA) at 0 ꢀC for 30 min to give
.
X-MCA TFA, while the Fmoc- group was removed
Of the proteases involved in postmortem aging,
calpains,9) cathepsins,10,11) and aminopeptidases12,13)
have been purified, but the purification of proteases
requires considerable effort and cost. Furthermore,
because postmortem aging of meat is caused by the
combined effects of proteases, it is not practical to
determine the degree of postmortem aging using only
one isolated protease.
We attempted to develop a new and simple method
to estimate the degree of postmortem aging of pork LD
by adopting the following steps: (i) preparing a library
of fluorogenic peptide substrates consisting of a number
of candidate substrates that can react with proteases, (ii)
reacting these substrates with LD muscle extracts to
measure some protease activities simultaneously, and
(iii) investigating the correlation between the physical
and chemical properties of LD and its degree of
postmortem aging.
Nishino et al.14,15) proposed that complex character-
istics of protease mixtures can be elucidated from a
limited number of measurements performed using a
series of synthetic protease substrates arrayed on a plate.
By applying this concept, the activities of some pro-
teases contained in the extract should be measurable
immediately and simultaneously. Moreover, it is known
that the use of 4-methyl-7-coumarylamide (MCA)-
labeled peptide substrates, which fluoresce (ex. 380
nm, em. 460 nm) when cleaved, allows sensitive mea-
surements to be made of protease activities.16–18)
In this study, to establish a simple system to analyze
the degree of postmortem aging, (i) a fluorogenic
peptide substrate library (endopeptidase substrates: Ac-
X-MCA, Ac-X-Ala-MCA, Ac-X-Val-MCA, Ac-X-Phe-
MCA, Ac-X-Lys-MCA, and Ac-X-Arg-MCA; amino-
peptidase substrates: X-MCA and Leu-X-MCA; X,
standard amino acids omitted cysteine) was prepared
and used to measure protease activities in pork LD, and
(ii) the relationships between the protease activities and
physical and chemical properties, which constitute
indicators of postmortem aging, were investigated. The
results confirmed that the physical and chemical proper-
ties correlated with protease activities. Further, on the
basis of this correlation, several substrates were identi-
fied that might be useful in estimating the postmortem
aging of pork LD.
with 20% piperidine in N,N-dimethylformamide (DMF)
for 2 h to give H2N-X-MCA, which were successively
reacted with acetic anhydride and triethylamine in DMF.
The products were purified by silica gel chromatography
to give Ac-X-MCA. The protecting groups of Ac-X-
MCA, if any, were finally removed by TFA. In the case
X was Lys or Arg, Ac-X-MCAs given as TFA salt. The
19 Ac-X-MCAs thus synthesized were analyzed by
HPLC and FAB-HRMS (data not shown).
Synthesis of Ac-X-Ala-MCA. Fmoc-Ala-MCA was
deprotected with 20% piperidine in DMF, as described
above, and coupled with Fmoc-amino acids to give
Fmoc-X-Ala-MCA using 2-(1H-benzotriazole-1-yl)-1,1,
3,3-tetramethyluronium hexafluorophosphate (HBTU),
1-hydroxy-1H-benzotriazole (HOBt), and diisopropyl-
ethylamine (DIEA) as coupling agents. Furthermore, the
Fmoc- group of Fmoc-X-Ala-MCA was deprotected,
acetylated as described above, and purified by silica gel
chromatography to give Ac-X-Ala-MCA. Final side-
chain deprotection of X was done by TFA. The 19 Ac-
X-Ala-MCAs were analyzed by HPLC and FAB-HRMS
(data not shown).
Synthesis of Ac-X-Val-MCA, Ac-X-Phe-MCA, Ac-X-
Lys-MCA, and Ac-X-Arg-MCA. Ac-X-Val-MCA, Ac-
X-Phe-MCA, Ac-X-Lys-MCA, and Ac-X-Arg-MCA
were synthesized using Fmoc-Val-MCA, Fmoc-Phe-
MCA, Fmoc-Lys(Boc)-MCA, and Fmoc-Arg(pmc)-
MCA (pmc-,2,2,5,7,8-pentamethylchroman-6-sulfonyl-)
respectively by the same procedure as described for the
synthesis of Ac-X-Ala-MCA. All of the dipeptide-
MCAs were analyzed by HPLC and FAB-HRMS (data
not shown).
.
Synthesis of X-MCA TFA. Boc-X-MCA was depro-
tected by TFA treatment to give X-MCA TFA. In
.
the case of X having side-chain protection, Fmoc-X-
MCA was used, and side-chain deprotection using TFA
was performed, followed by Fmoc-deprotection.
.
Synthesis of Leu-X-MCA TFA. H2N-X-MCA or X-
MCA TFA as synthesized above were coupled with
.
Boc-Leu hydrate by the HBTU/HOBt method to give
Boc-Leu-X-MCA, and were purified by silica gel
chromatography. Finally, they were treated with TFA
to remove Boc- and side-chain protection to give Leu-X-
.
MCA TFA. The 19 Leu-X-MCAs were analyzed by
HPLC and FAB-HRMS (data not shown).
Materials and Methods
Selection of substrates that react with pork LD
extract. Each of the synthetic MCA substrates (0.05
mmol) was dissolved in dimethyl sulfoxide (500 ml) to
give a 100 mM solution. Each well of a 96-well plate was
charged with 90 ml of 100 mM MES (2-morpholinoeth-
anesulfonic acid)–NaOH buffer (pH 6.0), and then the
various substrate solutions (10 ml each) were added to
Synthesis of MCA substrates.
Synthesis of Ac-X-MCA. t-Butoxycarbonyl-(Boc-) or 9-
fluorenylmethyloxycarbonyl-(Fmoc-) amino acids were
coupled with 7-amino-4-methylcoumarin by the sym-
metrical anhydride method using N,N0-dicyclohexyl

2838
E. ONITSUKA et al.
make a detection plate with a final substrate concen-
tration of 10 mM in each well. Minced LD muscle (15 g)
was homogenized with 3 volumes of 0.1 mM MES–
NaOH buffer (pH 6.0) in a juicer (National MX-X-60,
Matsushita Electric.) for 3 min. The homogenate was
centrifuged at 10;000 ꢁ g for 15 min. After an extract
was pre-incubated at 37 ꢀC for 5 min, 100 ml of the
solution was poured into each of the wells containing the
substrates. After 30 min, the fluorescence intensity (ex.
380 nm, em. 460 nm) was measured using a microplate
reader (ALVOꢀ MX 1420 multilabel counter, Perkin
Elmer, Wellesley, MA.). To check for the presence of
proteases that require metal ions for activation, EDTA
(5 mM) was added to an extract buffer solution and the
reactions were investigated.
et al.4) Five g of LD muscle was homogenized with
22 ml of 0.1% 2-mercaptoethanol for 1 min, and then
3 ml of 50% (w/v) trichloroacetic acid was added. The
mixture was homogenized again for 1 min, and then left
on ice for 3 h. The homogenate was centrifuged at
10;000 ꢁ g for 20 min and the supernatants were filtered.
After the pH of the filtrates were adjusted to pH 2.3, free
amino acids were analyzed with an amino acid analyzer
(JLC-500/V, JEOL).
Statistical analysis. The T test was used to identify
significant associations of fluorescence intensity, hard-
ness, and amount of free amino acids within the LD
muscles during 2–30 d of storage. Multiple regression
was used to determine variations between fluorescence
intensity against endopeptidase-substrates and hardness
or total amounts of free amino acids. We used
fluorescence intensity as the outcome variable, and
hardness or total amounts of amino acids of LD muscle
as predictor variables. LD muscle was treated as a
categorical factor using a dummy variable with 8
degrees of freedom. The P value from the t test for the
regression slope of hardness or total amounts of amino
acids was used to determine the probability of the
analysis. The magnitude of the correlation coefficient
between fluorescence intensity and hardness or total
amounts of free amino acids within the LD muscle was
calculated as the square root of (sum of squares for
hardness, or total amounts of free amino acids)/(sum of
squares for hardness, or total amounts of free amino
acids + residual sum of squares). The sign of the
correlation coefficient was given by that of the regres-
sion coefficient for hardness or total amounts of free
amino acids.19) This method was similarly used to
determine the variations between fluorescence intensity
against aminopeptidase-substrates and free amino acids.
Preparation of pork loins. Nine loins of ordinary
hybrid pork line Landrace ꢁ Large white ꢁ Duroc
were purchased from the local slaughterhouse. A portion
of pork loins was cut into six equal pieces on the day of
slaughter and the surfaces were sterilized with a burner.
The burned surfaces were later removed on a clean
bench and the LD muscle samples were vacuum packed
into sterile bags in a sterile environment and stored in a
refrigerator at 4 ꢀC.
The samples were stored for up to 30 d. On the 2nd,
5th, 10th, 20th, and 30th days of storage, the protease
activity, the amounts of free amino acids, and physical
hardness were measured for each piece of LD muscle.
Measurement of the response of substrates to changes
in pork LD properties with time. Of 152 synthetic MCA
substrates that were examined in the first analysis in
the samples stored for 2 d, 58 that showed a measured
fluorescence intensity of 1,000 or more following
reaction with the extract were selected (Table 1). These
58 selected substrates were used to measure changes in
LD properties with time. The procedures for preparing
substrate plates and measuring the reaction of substrates
were as described above. On the basis of the results of
the substrate-selection test, the times for fluorescence
intensity measurement were set at 30 min for endopep-
tidase substrates (the same as those described above) and
1 min for aminopeptidase substrates, which give faster
reactions.
Results
Selection of substrates for determination of postmor-
tem aging
First we selected substrates that react intensely with
LD muscle extracts. Using 152 synthesized MCA
substrates of eight combinations such as Ac-X-MCA,
Ac-X-Ala-MCA, Ac-X-Val-MCA, Ac-X-Phe-MCA,
Ac-X-Lys-MCA, Ac-X-Arg-MCA X-MCA, and Leu-
X-MCA (X, standard amino acids omitted cysteine),
protease activities were measured for an extract pre-
pared from the LD muscle 2 d after slaughter. Among
these substrates, fluorescence intensities against 58 of
six combinations such as Ac-X1-MCA (X1 ¼ Ala, Met,
Ser, Thr), Ac-Ala-Phe-MCA, Ac-X2-Lys-MCA (X2 ¼
Val, Leu, Ile, Met, His, Phe, Tyr, Trp), Ac-X3-Arg-
MCA (X3 ¼ Val, Leu, Ile, Met, Ser, Thr, His, Phe, Tyr,
Trp), X4-MCA (X4 ¼ Asp), and Leu-X5-MCA (X5 ¼
Asp, Thr) were as high as 1,000 or more (Table 1).
Although the fluorescence intensities against Ac-X1-
MCA, Ac-Ala-Phe-MCA, X4-MCA, and Leu-X5-MCA
Pork LD hardness. Pork LD hardness was tested by
the method of Okumura et al.4) The LD muscles were
sliced to 20 mm-thick meat and vacuum sealed. They
were heated in a water bath at 70 ꢀC for 20 min and
cooled in an ice bath for 20 min. After removal of
excessive surface water, hardness values were deter-
mined with a Tensipresserꢀ (TTP-50BXII, Taketomo
Electric, Tokyo), using a cylindric plunger with an outer
diameter of 5.5 mm.
Free amino acid analysis. A free amino acid analysis
in LD muscle was performed by the method of Okumura

A Judgment on Postmortem Aging Based on Peptide Substrate Library
Table 1. Selection of the Substrates by Fluorescence Intensity (F. I.)
Substrates were reacted with extract from LD muscle (A) or extract containing 5 mM EDTA (B). — Not detected.
2839
Substrate
F. I.
F. I.
F. I.
Substrate
F. I.
F. I.
F. I.
Substrate
F. I.
(A)
170
(B)
16
(A)
—
(B)
—
(A)
(B)
Ac-Gly-MCA
Ac-Ala-MCA
Ac-Val-MCA
Ac-Leu-MCA
Ac-Ile-MCA
Ac-Met-MCA
Ac-Asp-MCA
Ac-Glu-MCA
Ac-Ser-MCA
Ac-Thr-MCA
Ac-Asn-MCA
Ac-Gln-MCA
Ac-His-MCA
Ac-Lys-MCA
Ac-Arg-MCA
Ac-Pro-MCA
Ac-Phe-MCA
Ac-Tyr-MCA
Ac-Trp-MCA
Ac-Gly-Ala-MCA
Ac-Ala-Ala-MCA
Ac-Val-Ala-MCA
Ac-Leu-Ala-MCA
Ac-Ile-Ala-MCA
Ac-Met-Ala-MCA
Ac-Asp-Ala-MCA
Ac-Glu-Ala-MCA
Ac-Ser-Ala-MCA
Ac-Thr-Ala-MCA
Ac-Asn-Ala-MCA
Ac-Gln-Ala-MCA
Ac-His-Ala-MCA
Ac-Lys-Ala-MCA
Ac-Arg-Ala-MCA
Ac-Pro-Ala-MCA
Ac-Phe-Ala-MCA
Ac-Tyr-Ala-MCA
Ac-Trp-Ala-MCA
Ac-Gly-Val-MCA
Ac-Ala-Val-MCA
Ac-Val-Val-MCA
Ac-Leu-Val-MCA
Ac-Ile-Val-MCA
Ac-Met-Val-MCA
Ac-Asp-Val-MCA
Ac-Glu-Val-MCA
Ac-Ser-Val-MCA
Ac-Thr-Val-MCA
Ac-Asn-Val-MCA
Ac-Gln-Val-MCA
Ac-His-Val-MCA
Ac-Lys-Val-MCA
Ac-Arg-Val-MCA
Ac-Pro-Val-MCA
Ac-Phe-Val-MCA
Ac-Tyr-Val-MCA
Ac-Trp-Val-MCA
—
—
—
—
—
—
—
—
—
—
10,236
391
345
—
2,531
—
924
381
345
—
144
139
219
933
163
100
266
224
256
—
—
—
2,388
—
—
705
—
20
—
—
—
—
—
—
—
—
—
—
—
2,883
3,561
154
77
569
727
—
—
55
157
29
53
—
—
—
—
—
—
—
—
—
—
247
1
88
15
8
100
119
915
367
674
407
—
—
—
—
—
—
—
108
—
—
—
—
—
850
380
595
441
945
180
143
225
103
108
—
158
159
20
219
Substrate
Substrate
Substrate
F. I.
(A)
(B)
(A)
(B)
(A)
(B)
Ac-Gly-Phe-MCA
Ac-Ala-Phe-MCA
Ac-Val-Phe-MCA
Ac-Leu-Phe-MCA
Ac-Ile-Phe-MCA
Ac-Met-Phe-MCA
Ac-Asp-Phe-MCA
Ac-Glu-Phe-MCA
Ac-Ser-Phe-MCA
Ac-Thr-Phe-MCA
Ac-Asn-Phe-MCA
Ac-Gln-Phe-MCA
Ac-His-Phe-MCA
Ac-Lys-Phe-MCA
Ac-Arg-Phe-MCA
Ac-Pro-Phe-MCA
Ac-Phe-Phe-MCA
Ac-Tyr-Phe-MCA
Ac-Trp-Phe-MCA
113
1,999
189
184
332
746
128
144
354
787
—
135
—
Ac-Gly-Lys-MCA
Ac-Ala-Lys-MCA
Ac-Val-Lys-MCA
Ac-Leu-Lys-MCA
Ac-Ile-Lys-MCA
Ac-Met-Lys-MCA
Ac-Asp-Lys-MCA
Ac-Glu-Lys-MCA
Ac-Ser-Lys-MCA
Ac-Thr-Lys-MCA
Ac-Asn-Lys-MCA
Ac-Gln-Lys-MCA
Ac-His-Lys-MCA
Ac-Lys-Lys-MCA
Ac-Arg-Lys-MCA
Ac-Pro-Lys-MCA
Ac-Phe-Lys-MCA
Ac-Tyr-Lys-MCA
Ac-Trp-Lys-MCA
—
—
Ac-Gly-Arg-MCA
Ac-Ala-Arg-MCA
Ac-Val-Arg-MCA
Ac-Leu-Arg-MCA
Ac-Ile-Arg-MCA
Ac-Met-Arg-MCA
Ac-Asp-Arg-MCA
Ac-Glu-Arg-MCA
Ac-Ser-Arg-MCA
Ac-Thr-Arg-MCA
Ac-Asn-Arg-MCA
Ac-Gln-Arg-MCA
Ac-His-Arg-MCA
Ac-Lys-Arg-MCA
Ac-Arg-Arg-MCA
Ac-Pro-Arg-MCA
Ac-Phe-Arg-MCA
Ac-Tyr-Arg-MCA
Ac-Trp-Arg-MCA
—
633
—
611
354
364
75
93
1,852
2,163
1,360
1,017
101
2,131
2,628
1,494
1,348
213
2,267
3,441
1,767
2,219
510
3,065
4,538
2,371
2,749
712
96
247
45
—
—
257
418
466
503
794
482
2,546
1,096
395
1,836
636
379
—
—
231
120
324
365
—
—
—
—
385
511
620
627
2,588
859
479
3,111
1,050
620
3,048
993
567
3,705
1,156
535
—
—
47
61
—
—
105
—
72
173
225
497
882
44
3,954
4,592
2,846
4,768
5,941
3,706
4,194
4,032
2,034
5,720
5,153
2,635
321
767
Substrate
Substrate
(A)
(B)
(A)
(B)
Gly-MCA
Ala-MCA
Val-MCA
Leu-MCA
Ile-MCA
1,855
24,106
10,462
28,307
24,611
23,909
335
754
2,723
1,641
20,474
18,622
13,368
—
Leu-Gly-MCA
Leu-Ala-MCA
Leu-Val-MCA
Leu-Leu-MCA
Leu-Ile-MCA
Leu-Met-MCA
Leu-Asp-MCA
Leu-Glu-MCA
Leu-Ser-MCA
Leu-Thr-MCA
Leu-Asn-MCA
Leu-Gln-MCA
Leu-His-MCA
Leu-Lys-MCA
Leu-Arg-MCA
Leu-Pro-MCA
Leu-Phe-MCA
Leu-Tyr-MCA
Leu-Trp-MCA
4,213
28,442
10,294
27,268
17,715
27,216
—
860
2,453
462
12,906
6,535
12,760
—
Met-MCA
Asp-MCA
Glu-MCA
Ser-MCA
Thr-MCA
Asn-MCA
Gln-MCA
His-MCA
Lys-MCA
Arg-MCA
Pro-MCA
Phe-MCA
Tyr-MCA
Trp-MCA
5,525
11,671
9,849
2,516
3,543
5,967
7,828
14,956
5,985
7,152
17,249
56
3,430
10,381
503
897
2,384
343
20,424
19,008
9,716
10,408
17,608
11,380
31,373
32,522
3,375
2,127
8,974
4,745
8,127
13,582
3,216
4,876
5,500
6,275
26,973
32,884
3,545
28,915
33,660
36,968
4,003
3,910
4,578
35,080
38,961
40,199

2840
E. ONITSUKA et al.
5,000
4,000
3,000
2,000
600
500
400
300
200
100
0
4
3
2
1
0
Intensity against Ac-Ala-Phe-MCA
Hardness
Total free amino acids
0
5
10
15
20
25
30
Day
Fig. 1. Changes in Fluorescence Intensity against Ac-Ala-Phe-MCA, Hardness and Total Volume of Free Amino Acids of LD Muscle Stored at
4 ꢀC.
The bar indicates the standard error.
were as high as 1,000 or more when treated with an
EDTA-free extract, they showed reduced fluorescence
intensity when treated with an EDTA-containing extract.
The fluorescence intensities against Ac-X-Ala-MCA,
Ac-X-Val-MCA, and Ac-X-Phe-MCA (except for Ac-
Ala-Phe-MCA) were as low as 1,000 or less.
fluorescence intensity against Leu-MCA decreased
(P < 0:01). A significant decrease in fluorescence
intensity against X4-MCA substrates for X4 ¼ Ala,
Arg, Lys, Met, Phe, Tyr or Leu, and significant increases
in the amounts of free amino acids except for Gln, were
observed. As Table 3 indicates, a negative correlation
was observed between the amount of free Ala, Arg, Lys,
Leu, Met, Phe, and Tyr and fluorescence intensity
against Ala, Arg, Lys, Leu, Met, Phe, and Tyr-MCA
(P < 0:001); the corresponding correlation coefficients
were 0.780, 0.782, 0.831, 0.892, 0.891, 0.852, and 0.821
respectively (P < 0:001).
The 58 substrates were used to measure changes in
LD muscle properties.
Correlation between measurements on the substrate
library and the physical and chemical properties of pork
LD
Figure 1 shows changes with time in fluorescence
intensity against Ac-Ala-Phe-MCA, hardness, and total
amounts of free amino acids measured for LD muscle
samples stored for 2, 5, 10, 20, and 30 d. The
fluorescence intensity against Ac-Ala-Phe-MCA de-
creased during storage. This tendency was similar for
other Ac-X1-MCA substrates (data not shown), but no
significant changes were observed in fluorescence
intensity against Ac-X2-Lys-MCA or Ac-X3-Arg-
MCA. From the 2 d through the 10 d, the hardness of
LD muscle decreased by about 1:6 ꢁ 106 N/m2, al-
though this decrease was not statistically significant.
Determining variations within the LD muscles, strong
positive correlations between changes in hardness and
fluorescence activities against Ac-Ala-MCA, Ac-Met-
MCA, Ac-Ser-MCA, Ac-Thr-MCA, and Ac-Ala-Phe-
MCA were observed correlation coefficients of 0.779,
0.746, 0.778, and 0.775 respectively (P < 0:005,
Table 2). The total amounts of free amino acids,
however, increased significantly. As Table 2 indicates,
the change in total amounts of free amino acids shows a
negative correlation with changes in the intensities
against Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-
Thr-MCA, and Ac-Ala-Phe-MCA; the corresponding
correlation coefficients were 0.779, 0.756, 0.782, 0.681,
and 0.821 (P < 0:001).
Discussion
Nishimura,7) Koohmaraie,8) and other researchers
have shown that endogenous proteases in muscle
contribute markedly to the mechanism of meat tender-
ness and an improvement in meat flavor. In this study,
we focused on the change with time in the activity of
proteases in meat as an indicator that permits measure-
ments of the degree of postmortem aging of meat to be
made more easily. The use of only a few substrates to
monitor changes in the activities of the proteases that
are involved in postmortem aging may not cover the
entire range of changes that can occur during the
postmortem aging process. In this study, a large number
of fluorescently labeled peptide substrates were synthe-
sized to produce a substrate library, and this was used
to examine whether postmortem aging of LD can be
evaluated in one experiment by simultaneously measur-
ing the activities of a number of proteases in LD muscle
during postmortem aging.
Among the 152 synthesized substrates, fluorescence
intensities against 58 substrates were stronger for crude
LD muscle extract. These substrates were considered to
be easily decomposed by some of the proteases present
in LD. The remaining substrates were considered to
have a low specificity, although some fluorescence
intensities against them were detected (Table 1). The
addition of EDTA decreased the fluorescence intensity
Figure 2 shows changes with time in fluorescence
intensity against Leu-MCA and the amount of free
leucine. As the amount of free leucine increased,

A Judgment on Postmortem Aging Based on Peptide Substrate Library
2841
Table 2. Correlation Coefficients between Hardness or Total Volume of Free Amino Acids and Activity against Endpeptidase Substrates
Tenderness
Sign
Total free amino acids
Sign
Correlation
coefficient
Difference
Correlation
coefficient
Difference
Ac-Ala-MCA
Ac-Met-MCA
0.779
0.701
0.746
0.778
0.775
0.244
0.249
0.268
0.304
0.258
0.216
0.200
0.261
0.282
0.237
0.226
0.263
0.634
0.446
0.236
0.240
0.236
0.297
+
+
+
+
+
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢃꢃ
ꢃ
0.779
0.756
0.782
0.681
0.821
0.344
0.406
0.396
0.362
0.455
0.431
0.428
0.413
0.446
0.448
0.438
0.429
0.001
0.349
0.501
0.437
0.498
0.475
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
+
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢃ ꢃ ꢃ
ꢃ ꢃ ꢃ
ꢃ ꢃ ꢃ
ꢃ ꢃ ꢃ
ꢃ ꢃ ꢃ
NS
NS
NS
NS
ꢃꢃ
Ac-Ser-MCA
Ac-Thr-MCA
ꢃꢃ
ꢃꢃ
Ac-Ala-Phe-MCA
Ac-Val-Lys-MCA
Ac-Leu-Lys-MCA
Ac-Ile-Lys-MCA
Ac-Met-Lys-MCA
Ac-His-Lys-MCA
Ac-Phe-Lys-MCA
Ac-Tyr-Lys-MCA
Ac-Trp-Lys-MCA
Ac-Val-Arg-MCA
Ac-Leu-Arg-MCA
Ac-Ile-Arg-MCA
Ac-Met-Arg-MCA
Ac-Ser-Arg-MCA
Ac-Thr-Arg-MCA
Ac-His-Arg-MCA
Ac-Phe-Arg-MCA
Ac-Tyr-Arg-MCA
Ac-Trp-Arg-MCA
ꢃꢃ
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
ꢃ
ꢃ
NS
ꢃ
ꢃ
ꢃ
ꢃ
NS
NS
ꢃꢃ
ꢃ
ꢃꢃ
ꢃꢃ
Significant differences are indicated by asterisks ꢃꢃꢃP < 0:001, ꢃꢃP < 0:005, ꢃP < 0:01. NS, not significant.
Table 3. Correlation Coefficients between Total Volume of Free
Amino Acid and Activity against Aminopeptidase Substrates
30,000
25,000
20,000
15,000
10,000
50
40
30
20
10
0
Intensity against Leu-MCA
Free Leu
Correlation
coefficient
Sign
Difference
Ala
Arg
Asn
Asp
Gln
Glu
Gly
His
Ile
0.780
0.782
0.735
0.218
0.357
0.752
0.078
0.590
0.749
0.892
0.831
0.891
0.852
0.065
0.572
0.867
0.498
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
+
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
+
ꢂ
ꢂ
ꢂ
ꢃꢃ
ꢃꢃ
ꢃꢃ
NS
NS
ꢃꢃ
NS
ꢃꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
ꢃꢃ
NS
ꢃꢃ
ꢃꢃ
NS
0
5
10
15
20
25
30
Day
Fig. 2. Changes in Fluorescence Intensity against Leu-MCA and in
the Amounts of Free Leu of LD Muscle Stored at 4 ꢀC.
The bar indicates the standard error.
Leu
Lys
Met
Phe
Pro
Ser
against Ac-X1-MCA and Ac-Ala-Phe-MCA. On the
basis on Davey’s report,20) which suggests that EDTA
inhibits the degradation of Z lines by chelating calcium,
it was hypothesized that the reaction of these substrates
might involve calpains, a calcium-dependent protease.
It is known that cathepsin B reacts specifically with
the substrate Z-Arg-Arg-MCA or Z-Phe-Arg-MCA,21)
and that cathepsin L reacts specifically with the substrate
Z-Phe-Arg-MCA.21) Based on this information, it is
hypothesized that a reaction with a substrate that has a
basic amino acid in the P1 part, such as Ac-X2-Lys-
MCA and Ac-X3-Arg-MCA, might involve cathepsin B
or L.
Tyr
Val
Significant differences are indicated by asterisks ꢃꢃP < 0:001. NS, not
significant.
investigated. Okumura et al.4) reported that hardness
decreased gradually until 20 d and that the total amounts
of free amino acids increased up to 20 d, when the LD
was found to have the best sensory properties. In this
study, the hardness decreased until 10 d, and there was
almost no change subsequently. Therefore, it may be
best to eat pork LD on day 10 as for hardness. The total
amounts of free amino acids in this study increased up to
The correlation between changes in the traditional
indicators of degree of postmortem aging and changes in
fluorescence intensity against the substrates were then

2842
E. ONITSUKA et al.
20 d, which was consistent with their report.4) In the
evaluation of the relationship between changes in
hardness of LD and fluorescence intensity against the
substrate, a strong positive correlation was observed
for Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-MCA, Ac-
Thr-MCA, and Ac-Ala-Phe-MCA (P < 0:005, Table 2).
Koohmaraie et al.22) reported that after a 2 weeks of
postmortem aging of beef, the remaining activities of m-
and ꢀ-calpain were about 90 and 20% respectively,
which might be sufficient to contribute to postmortem
aging. The fact that the above five substrates are cleaved
by calpains indicates that these fluorescence intensities
can be attributed to calpains. In the evaluation of the
relationship between changes in the total amounts of
free amino acids and changes in fluorescence inten-
sity against the substrate, a strong negative correlation
was observed for Ac-Ala-MCA, Ac-Met-MCA, Ac-Ser-
MCA, Ac-Thr-MCA, Ac-Ala-Phe-MCA, and other
endopeptidase substrates (P < 0:001, Table 2). It is
believed that protein is degraded initially by endopepti-
dase and subsequently by aminopeptidase to produce
free amino acids.7) This study confirmed that the two
types of protease act together to increase the total
amounts of free amino acids. Nishimura et al.7) reported
that calpain has an important role in postmortem aging
of meat. Correlations were observed between substrates
suggesting the activity of calpain and hardness or total
amounts of free amino acid, and hence the substrates can
serve as indicators of postmortem aging.
The change in free amino acids during postmortem
aging was similar to that observed by Okumura et al.4)
They reported that the taste of pork LD became better in
sensory properties on day 20. In this study, pork LD
stored for 20 d was best to eat. A negative correlation
was observed between the amount of free Ala, Arg, Lys,
Leu, Met, Phe, and Tyr and fluorescence intensity
against Ala, Arg, Lys, Leu, Met, Phe, and Tyr-MCA
(P < 0:001, Table 3) respectively. Thus, Ala, Arg, Lys,
Leu, Met, Phe, and Tyr-MCA can serve as indicators of
postmortem aging. Nishimura et al.12,13) purified amino-
peptidase C and H and investigated their abilities to
hydrolyze amino acids. They found that aminopeptidase
C tends to hydrolyze Lys, Ala, Leu, and Met, whereas
aminopeptidase H tends to hydrolyze Met, Leu, and Lys.
(Their study did not cover Arg, Phe, or Tyr.) The
decrease in measured fluorescence intensities against
Ala, Arg, Lys, Leu, Met, Phe, and Tyr-MCA in this
study can be attributed to the actions of aminopeptidases
C and H, but the involvement of other aminopeptidases,
such as leucine aminopeptidase23) and cathepsin H,21)
cannot be eliminated.
substrates will help to establish a system for analyzing
postmortem aging that can be used easily and quickly to
determine the optimum timing for eating meat. Fur-
thermore, we can determine when meat is of the best
palatability and safety to eat by combining this method
with a bacterial count test. We also expect that beef, as
well as pork, can easily be analyzed by applying our
concept. Because beef requires longer postmortem
aging, it is more important to set the timing for the
consumption of beef.
Acknowledgments
This study was conducted using part of a contract-
research fund allocated by the Consortium R & D
projects for regional revitalization, of the Kyushu
Bureau of Economy, of Trade and Industry, the Ministry
of Economy, Trade, and Industry of Japan.
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