Note: Descriptions are shown in the official language in which they were submitted.
~ 3 ~ 7
TITL~ O~ THE INVENTIO~
Process and apparatus for monitoring the freshness of
edible meat.
BACKGROU~D OF THE I~VENTION
S Perishable edible meat such as ra~, frozen and
canned beef, poultry and fish represent an important part
of the diet of worldwide populations as well as important
market goods for a number of nations.
Fish, for example, lose its freshness more
quickly than mea~. Furthermore, the quality of canned
salmon, tuna, crab and the like is largely dependent upon
the freshness of the fish or shellfish used for
processing. Noteworthy is the fact that in the case of
fish, freshness can rarely be visually determined because
it is often sold in frozen or processed form.
From the standpoint of consumer protection and
food hygiene, extensive research has been focused on the
development of reliable and inexpensive methods of
determination of fish freshness. The development of such
methods is urgently required in food industries since fish
freshness is an important factor in the manufacture of
high-quality products. Indicators of fish freshness such
as ammonia, amines, volatile acids, catalase activity,
trimethylamine (TMA) and nucleotides have SQ far been
proposed. Among these chemicals, nucleotides produced by
adenosine triphosphate (ATP) decomposition are considered
the most reliable and useful indicators. In recent years,
,'- '~
--2--
considerable attention has been focused on nucleotide
degradation in fish muscle as a reliable indicator of the
freshness of raw fish.
Immediately after death, ATP in fish muscles is
hydrolyzed to uric acid through the followiny autolytic
path~ray:
ATP~ ADP--~ AMP--~ IMP--~ HxR--~ Hx ~ X ~ U ~1)
10 wherein
ATP is adenosine triphosphate
ADP is adeno~ine diphosphate
AMP is adenosine monophosphate
IMP is inosine monophosphate
HxR is inosine
Hx is hypoxanthine
X is xanthine
U is uric acid.
Several researchers have recognized that
simultaneous determination of each nucleotide is necessary
for a rapid estimation of freshness. From these
observations, the concept of the K value was developed, in
which:
K ~ _ lHxR] ~ [Hxl
[ATP] + IADP] + lAMP] + [IMP] + [Hx] ~ [HxR]
~3~2~1~
--3--
In several fish species, however, ATP and ADP
concentration6 rapidly decrease and are usually inexistent
24 hours after death. Similarly, a rapid decline of AMP
is also observed and itæ concentration is somewhat less
than 1 ymol/g. In contrast to such behavior, IMP
increases in the period ranging hetween 5 and 25 hours
after death and then gradually decreases while the
concentrations of HxR and Hx increase proportionally. In
practice, the first measurements of fish freshness are
usually performed at least 24 hours after death, thereby
simplifying the determination of the K value in the
following manner:
K = _ ~HxRl + [Hxl
[IMP] + [HxR] ~ [Hx]
A low K value should be expected for fresh fish.
It is generally believed that fish having a K value of
less than 0.2 has excellent freshness qualities while fish
exhibiting a K value ranging between 0.2 and 0.4 has good
freshness qualities. The increase in the rate of tAe K
value depends on the type of fish since changes in the K
value are based on the enzymatic reac~ions within the fish
meat. The K value also varles appreciably with
temperature even amonq the same fish species.
Based on these facts, various freshness
determination methods have been developed. For example,
Uchiyama et al. tBulletin of tha Japanese Society of
~L 3~2~ri~
Scientific Fisheries, Vol. 36, 977 (1970)) made an
analysis of the various nucleotidic compounds found in
fish muscle by using liquid chromatography to show that a
deterioration in freshness can be detected from an
increase in the K value.
K = __ ~HxR] + IHxl _ x 100%
[ATP] ~ [ADP] + ~AMP3 ~ [IMP] ~ [HxR3 + ~Hx]
It was later determined by Nunata et al. in
Journal of Japanese Society of Food Science and
Technology, Vol. 28, 542 ( 1981~ and by Kitada et al. in
Journal of Japanese Society of Food Science and
Technology, Vol. 30, No. 3, 151~154 (1983), that this
method could also be used to determine the degree of
freshness of poultry such as chicken.
However, the Uchiyama method has serious
drawbacks, namely the necessity to use expensive liquid
chromatography equipment that must be operated by skilled
technicians, the time consuming separation and column
regeneration as well as the difficulty in separating
inosine from hypoxanthine.
Fujii et al. (Bulletin of the Japanese Society
of Scientific Fisheries, Vol. 39, 69-84 (1373)1 developed
a method to estimate fish freshness based on the
determination of the concentrations of IMP, ffxR and Hx
through enzymatic reactions. This method is based on the
following equations:
5 l3 ~
IMP ratio - _ [IMPl x 105%
lIMP] + [HxR] ~ ~Hx]
HxR ratio = ~HxRl _ x 103%
lIMP] ~ [HxR] + [Hx]
Hx ratio = [Hxl x 100%
[IMP] + [HxR] + ~Hx]
The IMP ratio has a high value when the degree
of ~reshness is high and decreases as the degree of
~reshness decreases. For example, canned tuna having an
IMP ratio of 40% or higher can be judged as having been
processed from raw tuna having a high degree of freshness.
Unfortunately, although it can be used for fish
and poultry, this method also presents serious drawbacks.
Hence, an expensive ultraviolet spectrophotometer must be
used to conduct certain measurements and two expensive
enzymes are necessary in order to conduc~ certain
measuremen~s and this enzymatic reaction is time
consuming. Furthermore, corrosive perchloric acid must be
used as the extractant since the ultraviolet absorbing
properties of trichloroacetic acid render the latter
unsultable for use as the ex~ractant. Finally, the
extract solution must be clarified by time-consuming
centrigufation techniques.
The determination of the X value by monitoring
oxygen consumption using a Clark oxygen electrode has been
commercially exploited by Oriental Electric Co. Ltd. The
2 ~ ~7
apparatus is known as the KV-101 freshness meter
(hereinafter referred to as the K-meter) and comprises a
Clark oxyqen electrode attached to a reaction chamber.
Although functional, there are a number of
disadvantages to such a system. For example, the current
of the Clark oxygen electrode will depend not only upon
the metabolite concentration but also on the partial
oxygen tension (Po2) of the solution, which means that a
reliable application of this probe is only possible if
bo~h the pH and P2 of the solution can be carefully
controlled. There is also a mass transfer diffusional
limitation if the enzyme xanthine oxidase is immobilized
for repeated uses since both the metabolite and oxygen
must diffuse throuqh the enzyme boùnd membrane.
Therefore, an inexpensive and rapid method
useful in monitorinq fish freshness would be highly
desirable.
SUMM~Y OF T~IE INVE~TION
In accordance with tha present invention, there
~O is provided a method for determininy the degree of
freshness of raw, frozen and proceæsed edible meat by
moni~oring the degradation of adenine triphosphate to
inosine monophosphate, inosine and hypoxanthine. The
method comprises:
(a) breaking the cell membrane of said meat to
produce an extract;
~.2~ ~
(b) contacting a first portion of said extract
with the enzymes xanthine oxidase and nucleoside
phosphorylase and electrochemically measuring through an
amperometric prGbe, comprising an anode and a cathode, a
value dl a ~HxRl ~ [Hx] from the simultaneous
determination of the amount of hydrogen peroxide and uric
acid resulting from the degradation of hypoxanthine and
inosine in said first extract portion by said enzymes,
wherein [HxR] is the concen~ration of inosine and ~Hx] is
the concentration of hypoxanthine;
(c) contacting a second por~ion of said extract
with the enzymes nucleotidase, nucleoside phosphorylase
and xanthine oxidase, and electrochemically measuring
through an amperometric probe, comprislng an anode and a
cathode, a value d2 ~ [IMP] ~ [HxR] I [Hx] from the
simultaneous determination of the amount of hydrogen
peroxide and uric acid resultiny from the degradation of
inosine monophosphate, inosine and hypoxanthine in said
second extract portion by said enzymes, wherein [IMP] is
the concentration of inosine monophosphate, ~Hx~] is the
concentration of inosine~ and lHx] is the concentration
hypoxanthine; and
(d) determining the index of freshness from the
formula K = dl/d2, wherein K represents the index of
freshness.
~~ -7a- 131~7
Also within the scope of the present in~Jention
is an apparatus for determining the degree of freshness of
raw, frozen and processed edible meat, said apparatus
comprising a reaction cell; means in said cell for
S detecting uric acid and hydrogen peroxide, means for
amplifying signals produced by said detecting means and a
device for recording said signals whereby said freshness
may be determined. The instruments and reagen~s required
-8- 131 2~17
1) a measuring device for determining the amount of uric
acid and hydrogen peroxide,
2) a reaction cell provlded with a hydrogen peroxide and
uric acid sensor,
3) extractants, enzymes and buffer solutions.
Edible meat, when used herein, is intended to
include edible animal meat such as poultry, beef, veal,
pork, fish such as salmon, sole and trout as well as crab
meat, lobster and the like.
I~ TH~ DRAWI~&S
Figure 1 is a diagram of the apparatus used in
the present invention.
Figure 2 represents the effect of pH on the
actlvity of the enzymes xanthine oxidase, nucleotidase and
lS nucleoside phosphorylase.
Figure 3 illustrates ~he effect of temperature
on the activity of the enzymes xanthine oxidase,
nucleotidase and nucleoside phosphorylase.
Figure 4 represents the effect of phosphate ions
on the activity of the enzymes xanthine oxidase,
nucleotidase and nucleoside phosphorylase.
Figure 5 represen~s Lineweaver-Burk plots for
determination of the Michaelis-Henten constants for
xanthine oxidase, nucleotidase, nucleoside phosphorylase
and alkaline phosphatase.
, . .
1 3 ~
Eigure 6 represents the response of
polarographic electrode to uric acid and hydrogen
peroxide.
Figure 7 represents the response of the X-meter
to hypoxanthine concentrations in hypoxanthine containing
samples, inosine containing samples and inosine
monophosphate containiny samples.
Figure 8 represents the response of the
polarographic electrode to hypoxanthine concentrations of
hypoxanthine containing samples, inosine containing
samples and inosine monophosphate containing samples.
Figure 9 represents the difference between the
K values obtained by the polarographic elec~rode and by
the K-meter.
Figure 10 represents khe time course change of
the K value at different storage temperatures.
Figure 11 is a diagram of the apparatus used in
the context of the present invention.
Figure 12 represents the effect of
glutaraldehyde concentration on the activity of the
immobilized enzyme ~measured as ~A290/min; A290
absorbance at 290 nm) by following uric acid produced from
inosine by the action of nucleoside phosphorylase and
xanthine oxidase immobilized on the membrane.
Figure 13 illustrates the effect of the amount
of bovine serum albumin on the activity (measured
-lo- ~3~ 7
as ~A2g0/min) of nucleoside phosphorylase immobilized on
the membrane.
Figure 14 represents the relationship between
the amount of protein (specific activity of nucleoside
phosphorylase 36 IU/mg protein) and the activity (measured
as ~A290/min) of immobilized enzyme.
Figure 15 represents the activity vs. pH profile
of immobilized enzymes: (~) nucleotidase; and immobillzed
xanthine oxidase and nucleoside phosphorylase for (0)
hypoxanthine and (~) inosine as substrate.
Figure 16 illustrates the reproducibility of
analyses for fish extract (A) Hx with immobilized NP and
XO membrane (B) HxR with immobilized NP and XO membrane;
(C) IMP with immobili7ed NT tube.
Figure 17 represents the time course change of
the K value of trout at different storage temperatures.
Figure 1~ represents the time course change of
the K value of lobster at different storage temperatures.
Figure 19 represents the time course change of
the K value of shrimp at different storage temperatures.
Figure 20 represents a comparison between K
values determined with the biosensor sys~em and the
conventional enzymatic method.
D TAILED DESCRIPTIO~ OF THE INVE~TION
The present invention is concerned with a new
method useful in monltoring the freshness of various
perishable edible f ish by the determination of their
respective K value. The determination of the K value is
obtained by using a polarographic electrode which can
detect the presence of both hydrogen peroxide and uric
acid. For example, after the death of many fish species,
inosine monophosphate (IMP) contained in their muscle i5
degraded in the following manner:
NT
10 IMP ~ Hx~ (4)
~P
HxR + Pi ~ Hx + Ribose -1- Phosphate (5)
~O
Hx ~ 202 ~ ~ Uric acid + 2H202 (6)
wherein NT, NP, XO and Pi are nucleotidase, nucleoside
phosphorylase, xanthine oxidase, and inorganic phosphate,
respectively.
As demonstrated above, each mole of inosine
monophosphate consumed will ultimately requlre two moles
of oxygen and liberate two moles of hydrogen peroxide as
well as one mole of uric acid. It is therefore possible
to determine the concentration of hypoxanthine, inosine,
or inosine monophosphate by following either the rate of
oxygen consumption or the rate of hydrogen peroxide
formation. As mentioned above, the monitoring of oxygen
consumption presents serious drawbacks.
Amperometric datection of enzymatically
generated hydrogen peroxide has been widely performed by
using a Clark hydrogen peroxide electrode ~referred to
.
-12- ~2~
hereinafter as the polarographic electrode). Basically,
this electrode consis~s of a platinum anode and a
silver/silver chloride cathode where the anode is
polarized at +0.7 volts with respect to the cathode. The
polarographic probe oxidizes a constant portion oi the
hydrogen peroxide at the platinum anode at such a
polarized potential.
2 2 ~ 2H + 2 + 2e (7)
The current thus created is directly
proportional to the hydrogen peroxide level formed during
the oxldation of Hx to uric acid by the enzyme xanthine
oxidase as shown in equation 6. However it should be
noted that various reducing substances such as ascorbic
acid, glutathione, uric acid, etc., may considerably
influence the oxidation of H202. Consequently, there is
a problem for determining the level of H2O2 formed during
the oxidation of Hx si~ce the polarographic electrode will
respond to both H202 and uric acid. As experimentally
confirmed by Nanjo and Guilbault in Anal. Chem. 46, 1769
(1974), uric acid is electroactive and provides a limiting
current at the same potential (0.7 V) where hydrogen
peroxide is oxidized. The electrochemical oxidation of
uric acid can be described by the following reaction.
~ 3 ~ 7
-13-
Uric acid ~ 2 ~ 3H~O 2e
Allantoin H202 ~ HC032 (8)
Any attempt to separate the currents by pH
variations is not advisable since the current-potential
(i-E) curves of uric acid and hydrogen peroxide behave
similarly with changes in pH.
It has been discovered that the polarographic
electrode responds to a sample containing both uric acid
and hydrogan peroxide in an additiva manner. Therefore,
this electrode can be used $or monitoring the hypoxanthine
concentration in edible meat such as fish, poultry, beef
and the like. Therefore, the following equations have
been derlved,
~I = Kl [Ul (141
~I ~ K2 [HP] (15)
and ~I ~ Kl[U~ ~ K2lHP~ (16)
wherein ~I, U and HP respectively represent the electrode
output, the uric acid concentration and the hydrogen
peroxide concentration. Kl and K2 are the proportionally
constants for uric acid and hydrogen peroxide.
When it is desired to monitor the degradation of
hypoxanthine, the enzyme xanthine oxidase is added to the
sample and the following equation is derivad:
-14- 131~7
alHx~ ~ Kl[Ul] + K2[HPl] (10)
wherein ~I1, U1 and HP1 are respectively the electrode
output and the concentrations of uric acid and hydrogen
peroxide liberated during the enzymatic reaction.
When it is desired to monitor the degradation of
both inosine and hypoxanthine, the enzymes nucleoside
phosphorylase and xanthine oxidase must be sequentially
added to the sample. The following equation is derived:
~I2 a[Hx] + [HxR] ~ K1[U2] + K2[HP2] (11)
whereln ~I2, U2 and HP2 are respectively the electrode
output and the levels of uric acid and hydrogen peroxide
released as a result of the two enzymatic reactions.
Finally, the monitoring of inosine
monophosphate, inosine and hypoxanthine requires the
sequential addition of the enzymes nucleotidase,
nucleoside phosphorylase, and xanthine oxidase to the
measured sample. In this case, the electrode output (~I3)
can be expressed as follows,
~I3 [IMP~ + [HxRl + [Hx] ~ K1[U33 + K2lHP3] (12)
The K value for the freshness index can thus be
defined as the ratio between ~I2 and ~I3.
. . . .
-15- ~3~2~ ~
K ~ [HxRl -~ [Hxl = AI2
[IMPl ~ ~HxRI ~ ~Hx] ~I3 (13)
~or a reliable applicakion of this method, it i5
obvious that the proportionally constants Kl and K2 must
be constant throughout the measurements of such
metabolites for each K value determina~ion. This is a
logical expectation since the determination of the K ~alue
is completed w~thin 6 to 10 minutes.
Referring now to the drawings, Figure l shows an
example of the instrument used in the present invention.
In Figure 1, the sample measurement chamber 1, the volume
of which is preferably ranging from 0.3 to 0.~ ml,
comprises a stopper 2 provided with a capillary 3 used for
liquid injection in the center thereof, said capillary
having, for example, a diametex of about 0.125 mm. The
sample measurement chamber 1 is hermetically sealed by a
ring 4 and the samples contained in the measurement
chamber 1 are stirred by an air driven silicon diaphragm
5 which is used to provide both adequate mixing of the
solution and abundan~ supply of oxygen to support the
reaction. The reaction chamber 1 also contains a
polarographic electrode 6 which consists of a pla~inum
anode polarized at ~0.7 volts in a silver/silver chloride
cathode. ~oth the electrode 6 and a ~emperature probe 7
are mounted in the sample measurement chamber 1. The
-16-
sample measurement chamber 1 is surrounded by a block
heater 8 used to provide adequate temperature control.
It is noted that the electrode 6 used in the
context of the presen~ invention, may be any suitable
probe specific for the detection of hydrogen peroxide.
A suitable amplifier 9 is used to amplify the
signal delivered by the electrode. Also, the recorder lC
may be any commercially available mV recorder, and
preferably should have a full range of 500 mV. The system
19 of the present invention will preferably be computerized
and the computer is identified by the numeral 11. It is
noted that the instrument used in the context of the
present invention is small and light enough to be used on
site in a processing plant or other field locations.
Reaqents
a) Enzymes
The pH and temperature at which the activity of
an enzyme is optimal vary widely. The presence of some
ions in a solution may also have an influence on the
ultimate activity of the enzyme. The enzymes that are
used in the context of the present invention are xanthine
oxidase, nucleoside phosphorylase, and nucleotidase.
Therefore, it is necessary to perform assays on these
enzymes in order to determine the optimal conditions at
which the three enzymes can be used concurrently.
Thus, assays for the three above-mentioned
enzymes were performed by following the absorbance of uric
-17-
acid released at 290 nM using a Beckman DU-7
spectrophotometer. It is worth mentioning that assays for
nucleoside phosphorylase contained excess xanthine oxidase
while assays for nucleotidase contained excess xanthine
oxidase and nucleoside phosphorylase.
The pH effects on the activlty of the enzymes
was monitored between 6.5 and 3. As it can be seen in
Figure 2, at pH 7.5, all the enzymes attained a maximal
activity. Ik is important ko note that while xanthine
oxidase and nucleoside phosphorylase exhibited a broad pH
optimum, nucleotidase was very sensi~ive to acidity
variations.
Another series of experiments needs to be
performed in order to address the thermal effect on the
enzyme actlvity. The activity versus temperature profiles
were plokted between 10C and G0C, from which Arrhenius
plots could be constructed and Q1o values determined. The
temperature-activity profile shown in Figure 3
demonstrates that the maximal acti.vity of the enzymes is
achieved at 42C while it decreases very sharply beyond
45C. Furthermore, the Arrhenius plot results in straight
llne relationships and the Q1o values for xanthine
oxidase, nucleoside phosphorylase and nucleotidase were
determined to be respectively 1.65, 1.55 and 1.88.
Therefore, although temperatures ranging from 20 to 42C
can be contemplated in the context of the present
inventionr a temperature of 37C is preferred since all
13 ~ 2~3 r~
-18-
the enzymes remain stable up to 10 minutes at this
temperature.
The effect of phosphate ion on the activity of
nucleoside phosphorylase must also be investigated and
quantified since the degradation of inosine by this enzyme
requlres such an ion to produce hypoxanthine. Figure 4
shows that the effect is of the conventional substrate
inhibition kinetics which accounts for phosphate
stimulation at low concentrations and phosphate inhibition
at high concentrations. The maximal activity of
nucleoside phosphorylase is aahieved with 10 mM P0~3
while 80% of Vmax is obtained at 2 mM P043~. Above 100
mM, phosphate is inhibitory since the activity of
nuclecside phosphorylase decreases with a further increase
in the phosphate concentration. Nucleoside phosphorylase
retains only 40~ of its maximal activity at 1 M P043 .
Phosphate lons also have a pronounced effect on
nucleotidase. The experimental data shows that while 80~
of Vmax is attained at 5 mM P0~3 . In contrast to such
behavior, at a concentration up to 1.5 M, phosphate ion
exhibits no effect on the xanthine oxidase activity.
It was further observed tha~ up to a 500 mM
concentration of salts such as NaCl and ammonium sulfate
does not affect the activity of xan~hine oxidase. As far
as nucleoside phosphorylase action is concerned, the
enzyme activity is affected by both ammonium sulfate and
NaCl. Ammonium sulfate, however, exhibits a more
- 19~ 2 ~ ~ 7
pronounced inhibi~ory effect than NaCl (45~ Vmax ~t 500 mM
(NH4)2SO4 vs 80% Vmax at 500 mM NaCl). The reverse trend
i5 observed for nucleotidase since this enzyme retains 50%
and 25% of the activity at 500 mM (NH4)2SO~ and 500 mU
NaCl, respectively.
Therefore, the electrode chamber should contain
between 200 mM and 500 mM NaCl and from 20 mM to 50 mM of
phosphate ions while the pH of the solution should be
maintained at 7.5.
Xanthine oxidase is very unstable if diluted in
buffer (0.2 U mL). However, the dilu~ed enzyme can be
effectively stabilized by using 1.0 to 3.0 M (NH432S04 or
1.0 to 3.0 M NaCl. Under such conditions, xanthine
oxidase can retain up to 92~ of its activity after 1 day.
The addition of EDTA alone to the diluted enzyme is less
effective since xanthine oxidase only exhibits 70% of its
activity after 1 day. Similarly, nucleotidase is very
unstahle when diluted in buffer (2 U/mL). At this
concentration, the enzyme retains only 35% of the maximal
activity after 1 day. However, this enzyme can be
stabilized using 5 to 10 mM of MgC12 (90~ of Vmax). As
for nucleoside phosphorylase, it remains stable for at
least 6 days when diluted in buffer ~0.9 U/ml), and
requires no stabilization.
Based on the optimal activity conditions
established for these enzymes, a series of experiments may
be conducted to develop the kinetic data for xanthine
-20- ~2~7
oxidase, nucleo.side phosphorylase, and nucleotida~e. A~
determined from Figure 5 where 1/V was plotted against
l/S, the Michaelis-Menten constant (Kml for xanthine
oxidase with respect to xanthine and hypoxanthine is
respectively 2.2 ~M and 1.2 yM. When its concentration
exceeds 10 ~M, hypoxanthine was observed to inhibit
xanthine oxidase, as reflected hy the retention of only
65% of the maximum velocity at 50 ~M. The Km for
nucleoside phosphorylase with respect to inosine is 17.5
yM while that of nucleotidase with respect to IMP is
estimated to be 31.4 yM. The Km value for alkaline
phosphatase with respect to IMP is 281 yM. It should be
noted that this enzyme is also used with the K-meter for
determination of IMP.
All these enzymes are commercially available.
b) ~xtraction acids.
Extraction of a compound from tissue samples may
be accomplishecl by using perchloric acid, hut
trichloxoac0tic acid is preferred for safety reasons and
because no precipitation is formed on neutralization.
Determination of the freshness of various perishable
edible meats
If it i5 desired to u~e the method of the
present invention to determine the degree of freshness of
perishable edible meat such as fish, poultry, beef, pork
and the like, a tissue sample having a weight ranging from
1.0 to 3.5 g may be homogenized with 3.0 ml to 10 ml of
1 31 1 ~J ~
-21-
10% trichloroacetic acid. After centrifugation, the
supernatant solution may be neutralized with 4 to 5%
volume of a suitable base ~uch as 0.1 mM NaOH.
10 to 50 ~l of the resulting solution may then
be incubated in a solution containing from 0~45 to 0.9 ml
of 2 mM P043 , 2 mM MgCl2 at pH 7.5 for 10 to 20 minutes
at a temperature ranging from 25 to 37C in the presence
of 0.03 U to 0.10 U of nucleotidase and 0.009 U to 0.030
U nucleoside phosphorylase. Another similar sample may
then be incubated in a solution containing from 0.45 to
0.9 ml of lO mM P043 at a pH of 7.5 for S to 1~ minutes
at a temperature ranging from 25 to 37C in the presence
of 0.009 U to 0.030 U nucleotide phosphorylase. A
solution containing 50 to 100 ~l of 5 M NaCl and 500 mH
P04 at pH 7.5 is then added to result in a final
concentration of 500 mM NaCl and 50 mM P04 . The
solution may then be delivered to the electrode chambe
where .0025 U to .0075 U of xanthine oxidase is added to
initiate the reaction. A steady state output of the
electrode may be obtained within two minutes.
As mentioned above, uric acid itself is
electroactive, and provides a limiting current of the same
potential where hydrogen peroxide is oxidized. Therefore~
any hydrogen peroxide probe can be used for the detection
of uric acid. The response of the H202 electrode to both
uric acid and H202 is demonstrated in Figure 6. It should
be noted that the results obtained are quite unexpected
-22- ~3~ 7
since the pro~e is found to be more sensitive to uric acid
than hydrogen peroxide. The fact that the electrode
response to uric acid and hydrogen peroxide is additive
leads to a method of very high senæitivity.
It iæ also noted that the electrode response to
uric acid and hydrogen peroxide is affected by the ion
strength of the measured sample. Hence, the response to
uric acid can be 3 to 5 times higher if 10 to 500 mM of
phosphate or NaCl is added to the sample.
When used for many measurements, the electrode
appears to lose its sensitivity to both uric acid and
H O . In fact, the sensitivity loss is more rapid for
2 2
H202 than it is for uric acid. However, the elecrode
response can be easily restored by washing the probe with
a 8 m urea/1 M NaOH solution for 10 to 20 minutes.
Thorough washing with distilled water must then follow in
order to remove NaOH since this base interfers with the
electrode performance.
It is noted however that the electrode needs to
be washed only after several measurements have been
conducted.
Comparison between the ~olaroqraPhic electrode and the X-
meter
a) Estimation of the K value
A series of experiment was conducted to
establish the calibration curves for the polarographic
electrode and the K-meter by total digestion of
-23- ~2~ ~
hypoxanthine to uric acid. In accordance with the assay
procedures described above, samples containing different
Hx concentrations and xanthine oxidase were applied to
both detecting devices. As expected, deyradation of Hx to
urlc acid consumed oxygen and liberated hydrogen peroxide.
Thix may be observed in Figures 7 and 8 where the
responses of the detectiny devices were plotted against
the total concentration of hypoxanthine digested. For the
polarographic electrode, a linear relationship ~as
obtained between the probe and [Hx] in the range of 0.5
~M. For the K-meter, a linear relationship was observed
in the range of 0-100 yM, which means that the K-meter is
much less sensitive than the polarographic electrode for
detecting hypoxanthine. Such results obtained were not
completely unexpected since the uric acid produced is
electroactive and produces a limiting current at the same
potential where hydrogen peroxide is oxidized. As a
result, the polarographic electrode will respond to both
uric acid and hydrogen peroxide while the K-meter only
detects the rate of oxygen consumption in the reaction.
The calibration curves could also be established
by using inosine or IMP as the substrate. Of course, such
a metabolite was converted to hypoxanthine and then to
uric acid by the appropriate enzymesO As shown in Figures
7 and 8, the calibration curves established by using three
different metabolites resulted in only one line, as
indication of total digestion of hypoxanthine, inosine, or
~ `t
' ' ,' ' '
~ 3 ~ 7
-2~-
IMP to uric acid and therefore of the applicability of the
detecting systems for monitoring the presence of such
metabolites. Samples containing various known
concentrations of IMP, HxR, Hx and the appropriate enzymes
were then applied to the detecting devices for estimation
of the K value. Good comparative results were observed
between the K values determined by the polarographic
electrode and the K-meter. By plotting the K value
obtained by one method versus that of another, a s~raight
line relationship with a slope of 0.98 resulted with a
correlation coefficient of 0.99 as shown in Figure 9.
There was also excellent agreement between the expected
and experimental K values as demonstrated in Tables 1 and
2. The marginal error of the polarographic electrode and
the K meter was determined to be 6.0~ and 5.6%,
respectively.
h) Economical considerations
In terms of cost effectiveness, the apparatus of
the present invention demonstrates considerable advantages
over the K-meter. ~irst, the method of the present
invention is much more sensitive, thereby requiring about
times less sample than necessary for effective
freshness determination by the K-meter. Consequently,
since the sensitivity toward hypoxanthine is much higher
when using the method of the present invention, smaller
amounts of enzymes are required. In fact, 40 times as
-25- ~ 7
much xanthine oxidase, the most costly enzyme, is required
to perform successful analysis using the K-meter.
Furthermore, in addition to the enzyme cost
savings, apparati associated with sample handling and
preparation as well as the reaction chamber are compact
and can be easily integrated with the polarographic
electrode to form a portable sensing device suitable for
field work.
The following examples are intended to
illustrate rather than limit the scope of the present
invention.
Example 1
A 3.5 g tissue sample taken from the muscle of
freshly caught rainbow trout was homogenlzed wlth 10 ml of
10% trichloroacetlc acid. After centrifugation, the
supernatant was neutralized with 20 ml of 0.1 M NaOH. A
10 ~1 aliquot of the neutralized solution was first
lncubated ln a volume of 0.9 ml 2 MM P043 , 2 mM MgCl2
buffer pH 7.5 for 10 minutes at 37~C in the presence of
0.03 U nucleotidase and 0.009 U nucleoside phosphorylase.
Another 10 ~1 aliquot of the dlluted solution was
incubated in a volume of 0.9 ml 10 mM P043 buffer pH 7.5
for 5 minutes at 37C in the presence of 0.009 U
nuclsoside phosphorylase. A solu~ion containing 100 ~1 of
5 M NaCl and 500 mM P043 buffer pH 7.5 was then added to
result in a ~inal concentra~ion of 500 mM NaCl and So mM
-26- ~ 7
P04 . The resulting solution was then peristaltically
delivered to the electrode chamber where 0.0025 U xanthine
oxidase was added to ini-tiate the reaction. The steady
state response of the electrode was obtained within two
minutes. The K value was determined to be approximately
O . 1 .
Example 2
The procedure described in Example 1 was
repeated on a tissue sample taken from a rainbow trout 24
hours after death. The fish had been maintained at room
temperature. The recorded K value was estimated to be
approximately 1.
Example 3
The procedure described in Example 1 was
repeated using a tissue sample taken from a rainbow trout
24 hours after death. The fish had been maintained at a
temperature ranging hetween 0 and 5C. The K value was
estimated to be 0.61.
Exa~plè 4
The procedure described in Example 1 was
repeated using a tissue sample taken from a rainbow trout
72 hours after death. The fish had been maintained at a
-27- ~3~2 ~ ~ 7
temperature ranging between 0 and 5C. The estimated K
value was determined to be 1.
Exampl0 5
The procedure described in Example 1 was
repeated using a tissue sample taken from a rainbow trout
2 weeks after death. The fi6h had been maintained at a
temperature of -20C. The estimated K value was
determined to be 0.15.
Exa~ple 6
Six samples of 3.5 g each were taken from the
muscle of frozen sole and were each homogenized with 10 ml
of 10~ trichloroacetic acid. After centrifugation, the
supernatant was neutralized with 20 ml of 0.1 M NaOH. A
20 ~1 aliquot of the neutralized solution was first
incubated in a volume of 0.9 ml 2 mM PO43 , 2 mM MgCl2
buffer pH 7.5 for 10 minutes at 37~C in the presence of
0.030 U nucleotidase and 0.009 U nucleoside phosphorylase.
Another 20 ~l aliquot of the diluted solution was
incubated in a volume of 0.9 ml 10 mM PO~3 buffer pH 7.5
for 5 minutes at 37C in the presence of 0.009 U
nucleoside phosphorylase. A solution containing 100 ~l of
5 M ~aCl and 500 mM PO43 buffer pH 7.5 was then added to
rasult in a final concentration of 500 mM NaCl and 50 mM
PO43 . The resulting solution was then peristaltically
delivered to the electrode chamber where 0.00~5 U xanthine
-28-
oxidase was added to initiate the reaction. The steady
sta~e response of the electrode was obtained within two
minutes. The K value was determined to approximately
0.65. Results are summarized in Table 3.
Exa~ple 7
The procedure described in Example 6 was
repeat~d using a tissue sample taken from sole which had
been maintained at -20C for 2 months. The estimated K
value was determined to be 0.65.
Ex~mple 8
The procedure described in Example 6 was
repeated using a tissue sample taken from sole which had
been maintained at 5C for 24 hours. The estimated K
value was determined to be 1.
~xample 9
A 3.5 g tissue sample from the muscle of salmon
frozen for 3 weeks after being caught was homogenized with
10 ml of 10~ trichloroacetic acid. After centrifugation,
the supernatant was neutralized with 20 ml of 0.1M NaOH.
A 20 yl aliquot of the neutralized solution was first
incubated in a volume of 0.9 ml 2 mM P04 , 2 mM MgC12
buffer pH 7.5 for 10 minutes a~ 37~C in the presence of
0.03 U nucleotidase and 0.009 U nucleoside phosphorylase.
Another 20 yl aliquot of the neutralized solution was
-29-
incubated in a volume of 0.9 ml 10 mM P043 buffer p-H 7.5
for 5 minutes at 37C in the presence of 0.009 U
nucleoside phosphorylase. A solution containiny 100 ~l of
5 M NaClo and 500 mM P043 buffer pH 7.5 was then added to
reæult in a final concentration of 500 mM NaCl and 50 mM
P04 . The resulting solution was then peristaltically
delivered to the electrode chamber where 0.0025 U xanthine
oxidase was added to initiate the reaction. The steady
state response of khe electrode was obtained within two
minutes. The K value was determined to be 0.37.
Example 10
The procedure descri~ed in Example 9 was
repeated on a tlssue sample taken from the frozen salmon
and maintained at room temperature for 24 hours. The
recorded K value was estimated to be approximately 1.
Example 11
The procedure described ln Example 9 was
repeated on a tlssue sample taken from the frozen salmon
and maintained at 0-5~C for 24 hours. The K value was
estimated to be 0.76.
Example 12
The procedure described in Æxample 9 was
repeated on a tissue sample taken from the frozen salmon
-30-
and maintained at 0-5C for 48 hours. The K value ~Jas
estimated to be 1.
Example 13
The procedure described in Example 9 was
repeated on a tissue sample taken from the frozen salmon
and maintained at -20C for a further 2 weeks. The
estimated K value was determined to be 0.75.
Exa~ple 14
A 3.5 g tissue sample from the muscle of freshly
caught carp was homoyenized with 10 ml of 10%
trichloroacetic acid. After centrifugation, the
supernatant was neutralized with 20 ml of 0.1 M NaOH. A
50 ~1 aliquot of the neutralized was first incubated in a
volume of 0.9 ml 2 mM PO~ , 2 mM MgC12 buffer pH 7.5 for
10 minutes at 37C in the presence of 0.03 U nucleotidase,
and 0.009 U nucleoside phosphorylase. Another 50 yl
aliquot of the neutralized solution was incubated in a
volume of 0.9 ml 10 mM P043 buffer pH 7.5 for 5 minutes
at 37C in the presence of 0.009 U nucleoside
phosphorylase. A solution containing 100 yl of 5 M NaCl
and 500 mM P043 buffer pH 7.5 was then added to result in
a final concentration of 500 mM NaCl and 50 mM P04 . The
resulting solution was then peristaltically delivered to
the electrode chamber where 0.0025 U xanthine oxidase was
added to initiate the reaction. The steady state response
131 ~ ~7
-31-
of the electrode was obtained within two minutes. The K
value was determined to be 0.31.
Example 1~
The procedure in Example 1~ was repeated on a
tissue sample from a carp 24 hours after death. The fish
had been maintained at a temperature ranging between 0 and
5C. The K value was estimated to be 0.78.
Example 16
The procedure in ~xample 14 was repeated on a
tissue sample taken from a carp 48 hours after death. The
fish had been maintained at a temperature ranging between
0 and 5C. The K value was estimated to be 1.
Example 17
The procedure in Example 14 was repeated on a
tissue sample taken from carp 1 week after death. The
fish had been maintained at a temperature of -20DC. The
estimated K value was determined to be 0.29.
rl
- 32 -
Table 1 - Esti~ation of the K v~lue by the polarographic ~lectrode
~ . _ . ~ . ~ _ __
Sample composition Polarogr~phic K value Di~ference
~oncentration (u~) el~ctrode response
_ . _ _ . __ . _ _ . _
Hx HxR I~P ~I~ ~I3 experimental theoreti~l
_ _ . _ . .. _
0 0 2 <5 165 0.03 0 3.0
0 2 0 165 160 1.03 1.0 3.0
2 0 ~ 155 170 0.91 1.~ ~.8
1 1 1 155 235 0.66 - 0.67 1.0
I 2 1 245 305 0.80 0.75 7.1
2 1 1 225 315 0.71 0.75 4.8
1 ~ 0 150 155 0.97 1.0 3.2
1 0 1 80 155 0.52 0.5 3.2
0 1 1 80 160 0.50 0.5 0
2 1 1 230 275 0.84 0~75 12
2 1 0 220 230 0.96 1.0 ~l.3
2 0 1 160 235 0.68 ~.67 2.1
0 1 2 80 225 0.36 0.33 6.9
1 0 2 80 230 0.~5 0.33 4.5
1 0 3 80 305 0.2~ 0.25 4.8
0 3 1 235 285 0.83 0.75 10
1 2 2 ~25 335 0.67 0.60 12
~. _ .
-33~ 7
TAble 2 - ~stimAtion of the X value by thQ X machine.
._ . _
Sa~ple co~position K-machlne K v~lue Difference
concentra~ion (u~) response
. . . _ _, _ . _
Hx ~xR IKP ~I, ~I, exper~mental th~oretical
.__ ._ . . ._ ._ . _ ,. I
0 ~ 180 1~9 0.95 1.~ 4.8
~ 20 0 16.7 17.8 0.94 1.0 6.2
0 0 20 <1.0 16.0 0 0 0
36.1 49.5 0.73 O.S7 9.3
2~ 0 32.g 32.9 1.~ 1.0 0
0 20 18.8 34.7 0.54 0~5 8.4
0 20 2~ 18.0 33.4 0.54 0.5 7.8
Z0 0 49.5 48.4 1.02 1.0 2.3
0 20 35.9 49.4 0.73 0.67 9.0
0 40 2~ 33.7 ~8.5 0.70 0.67 4.2
0 50.0 49.3 1.01 1.0 1.4
0 20 40 17.0 47.3 U.36 0.33 7.8
0 40 17.5 49.5 0.~5 0.33 6.3
_ .- _ ._ _ . _ . _ . ,_ ,
~3~ 7
-34-
able 3 - EstLmation of the X value of fro~en ssle fillet by the
polarographic elec~rode
~ _
Sample ~ Dilution Polarographic K value
_ fDctor electrod~ reKponse
1 60X 113 170 0.66
30X 235 355 0.66
2 60X 1~ 175 0.74
30X Z28 345 0.66
3 60X 125 198 0.63
30X 233 355 0.66
4 60X 105 150 0.70
30X 200 28D 0.71
60X 113 1~5 0.61
30X 223 360 0.62
6 30X 258 353 0.73
~35_ ~ 3~
SUPPLE~E~T~XY DISCLOSURE
While the disclosure o~ the present application
contemplates any means of using enzymes to monitor ATP
degradation as a determination of the freshness o~ edible
meat, lt has been determined that one preferred embodiment
consisted in using at least one enzyme immobilized on a
porous substrate.
A preferred embodiment of the process of the
present invention consists in co-immobilizing the enzymes
xanthine oxidase and nucleoside phosphorylase on a porous
polymeric membrane, more preferably a nylon membrane.
The immobilization of the enzymes on porous
membranes is advantageous since it enables the enzymes to
be used several times, thereby substantially simplifying
the method and reducing its costs.
Also within the scope of the present invention
is a method for the preparation of the immobilized enzymes
used to monitor the degradation of ATP. Enzymes such as
xanthine oxidase, nucleoside phosphorylase, and
nucleotidase can be immobiliæed if it is desired to
monito~ the degradation of ATP to inosine monophosphate,
inosine and hypoxanthine for example. The method thus
comprises immobilizing a ~irst enzyme, su~h as
nucleotidase, on a polymeric support. The immo~ilization
is accomplished by contacting this support with a
polyethyleneimine solution, a solution containing a
crosslinking agent and a solution containing the enzyme to
-36- ~ 3 ~
be immo~ilized. A second and a third enzyme, such as
xanthine oxidase and nucleoside phosphorylase, are also
immobilized on a porous polymeric membrane by contacting
this membrane with a solution comprising the enzymes and
a crosslinking agent. Preferably, the enzymes xanthine
oxidase and nucleoside phosphorylase are co-immobilized on
a porous nylon membrane or the like and nucleotidase is
immobilized via glutaraldehyde activation on the wall of
a polymeric tube such as a polystyrene tube precoated with
a thin layer of polyethyleneimine.
Finally, also contemplated is an enzyme
biosensor system for use to monitor ~he degradation of ATP
comprising in combination an amperometric electrode and a
porous membrane having xanthine oxidase and nucleoside
phosphorylase immobilized thereon, an enzyme biosensor
wherein the porous membrane is a nylon membrane, an enzyme
biosensor system further comprising nucleotidase
immobilized on the wall of a polystyrene ~ube precoated
with a thin layer of pol~styrene coated with
polyethyleimine and a method for monitoring the
degradation of adenine triphosphate in an extract to
inosine monophosphate, inosine and hypoxanthine, the
method comprlsing:
a) contacting a first portion of the extract ~ith the
enzymes xanthine oxidase and nucleoside phosphorylase and
electrochemi~ally measuring through a single electrode a
value ~I2 from the simultaneous determination of the
-37- ~ 7
amount of hydrogen peroxide and uric acid resulting from
the de~radation of hypoxanthine and inosine by the
enzymes; and
b) contacting a second portion of the extract with the
enzymes xanthine oxidase, nucleoside phosphorylase and
nucleotidase and electrochemically measuring through a
single electrode a value AI2 from the determination of the
amount of hydrogen peroxide and uric acid resulting from
the simultaneous degradation of inosine monophosphate,
inosine and hypoxanthine by the enzymes.
Hence, the [Hx + HxR] concentration in tissue
extract can be measured by using nucleoside phosphorylase
and xanthlne oxidase which are co-immobilized on a porous
polymeric membrane. Various types of porous polymeric
materials such as cellulose, nylon and the like may be
used in the context of the present invention, although
nylon appears to be the most preferred one. The shape,
size and thickness of this membrane do not seem to be
critical to the viability of the process. In fact, what
is needed is a porous polymer suitable to immobilize one
or more enzymes. The electrode amperometrically detects
the products of the enzymatic degradation of Hx and HxR,
hydrogen peroxide and uric acid.
For the determination of [IMP] ~ lHxR] + [Hx],
IMP is first converted to HxR by nucleotidase.
Preferably, the enzyme is to be immobilized on the walls
of a polymeric tube, precoated with a thin layer of
-38-
polyethylenei~ine. Again, the nature of the polymeric
material is not critical but polymers such as polystyrene
and the like should be employed. The ~IMP ~ Hx ~ HxRI
concentration is then measured by the aforementioned
electrode.
Referring to the drawings, Figure 11 shows an
example of the instrument uæed in the present invention.
In Figure 11, the sample measurement chamber 1, the volume
of which is preferably ranging ~rom 0.3 to 0.4 ml,
comprises a stopper 2 provided with a capillary 3 used for
liquid injec~ion in the center ~hereof, said capillary
having, for example, a diameter of about 0.125 mm. The
sample measurement chamber 1 is hermetlcally sealed by a
ring 4 and the samples contained in the measurement
chamber 1 are stirred by an air driven stlicon diaphragm
5 which is used to provide both adequate mixing of the
solution and abundant supply of oxygen to support the
reaction. The reaction chamber 1 also contains an
amperometric electrode 6 on which is a~fixed a porous
polymeric membrane on which the nucleoside phosphorylase
and xanthine oxidase enzymes have previously been
immobllized. The amperometric electrode consists of a
platinum anode polarized at +0.7 V versus a silver/silver
chloride cathode. Both the electrode 6 and a temperature
probe 7 are mounted in the sample measurement chamber 1.
The sample measurement chamber 1 is surrounded by a block
heater 8 used to provide adequate temperature control.
~ 3 ~
-39-
D ~ ription of a Preferred embodiment usin~ imnobilized
enzymes ~or the determina$ion of fi~h freshne~s
Materials and methods
a) Immobilization of nucleotidase on the wall of a
polystyrene tube.
Nucleotidase (NT) was immobilized on the wall of
a l-mL polystyrene centrifuge tube. The tube was filled
with 1 mL of 5% polyethyleneimine solution and incubated
at room temperature ~20-2~C) for 2 h. The tube was then
emptied and filled with 2.5% of a crosslinking agent
solution such as a glutaraldehyde solution in 150 mM, pH
7.8, phosphate buffer. Incubation was carried out at room
te~perature for 3 h. Glutaraldehyde solution was then
removed and the tube was washed thoroughly with 150 mM, pH
7.8, phosphate buffer. The tube was filled with 1 mL
solution containing 5-6 IU of nualeotidase dissolved in 4
mM, pH 7.8, phosphate buffer and incubated overnight at
4C. The solution was then removed and the tube was
washed extensively with the buffer and stored filled with
buffer at 4~C.
b) Co-immobilization of nucleoside phosphorylase and
xanthine oxidase on a membrane.
A prewetted Immunodyne~M membrane (1.5 x 1.5 cm)
was stretched on the tip of a hollow plastic cylinder (1
cm diameter) and held in place by an 0-ring. The
preactivated ImmunodyneTM nylon 66 membrane (pore slze ~f
3 ~m) was obtained from Pall BioSupport Division (Glen
~ 3 ~ 7
-~o
Cove, NY). The membrane is intrinsically hydrophilic and
contains function groups which form covalent linkages with
a variety of nucleophilic groups of enzymes/proteins.
To a mixture containing 20 yl of nucleoside
phosphorylase (NP, 5.1 g/l and 3.6 U/mg), 4 ~l of bovine
serum albumin (BSA, 4()0 g/l), and 18 ~1 of bui~er (200 mM)
pH 7 phospha~e), 8 ~1 of ylutaraldehyde (12.5~ w/v) was
added to initia~e the crosslinking. It should be noted
that the final volume of the resulting solution is 50 ~1
and contained 2% w/v glutaraldehyde, 1.6 mg BSA and 102 yg
NP. 35 ~1 of the resulting solution was th~n layered on
the prewetked membrane and the solution was allowed to
crosslink at room temperature (20-24C) until a yellowish
hard gel layer was obtained (20-30 min). The membrane was
then removed and washed extensively with phosphate buffer
(50 mM, pH 7.8) to remove unreacted glutaraldehyde. The
final concentration of NP and BSA was thus estimated to be
71 ~g and 1.12 mg, respectively. It should be noted that
the resulting NP activity increased with glutaraldehyde
concentration used and reached maximum at 1% (w/v).
The membranes prepared with glutaraldehyde
concentrations below 1% exhibited a very soft layer which
was easily damaged/detached. Increase in glutaraldehyde
concentration beyond 1% (w/v) resulted in decreased NP
activity as shown in Figure 12. The NP activity decreased
drastically at glutaraldehyde concentrations above 2%.
~ 3 ~
-41-
However, the enzyme layer obtained under such a condition
was much stronger and slightly yellow.
Apparently, a low glutaraldehyde concentration
rasulted in an insufficient protein crosslinking and led
to washing a~ay of the enzyme. On the other hand, at a
high glutaraldehyde level, the enzyme and BSA were
extensively crosslinked to form a thick gel which causes
severe diffusional limitation and destruction of the
enzyme active sites. A concentration of 2% glutaraldehyde
was considered optimal since it represents a good
compromise between the enzyme activity and the mechanical
strength of the enzyme layer.
To a lesser extent, the activity of NP in the
enzyme layer was also affected by BSA concentration as
shown in Figure 13. The enzyme activity increased
slightly with albumin concentration, reached a maximum,
and then decreased. The decrease in the activity can be
attributed to an increased diffusional resistance of the
complex matrix. Once again, at a low albumin
concentration, the enzyme layer formed was not firm and
easily damaged~ detached. At a higher albumin
concentration, the layer was hard and yellow. As a result
of this finding, 1.12 mg of albu~in was considered
optimum.
As expected, the activity of the immobilized NP
was dependent on the amount of enzyme used for membrane
preparation as shown in Figure 14. Below 20 yg NP, there
. , . ~
-42-
was a linear relationship between the activity of the
immobilized enzyme and the amount of enz~me used. Beyond
~y NP, the activity of the enzyme membrane was
independent of any further increase in the enzyme
concentration used during immobilization. Consequently,
2.6 IU or 71 ~g NP was used for enzyme layer preparation.
After NP was immobilized, the membrane was
washed extensively with 50 mM phosphate buffer and i~ was
then immersed in a centrifuge tube containing 2 mL of 25
mM, pH 7.5, tris buffer and 0.27 IU xanthine oxidase (XO).
The tube was continuously agitated on a vortex mixer
(model 5432, Eppendorf ~,eratebau, Hamburg, FRG) for 4 h.
at 4C. The membrane was then washed several times with
cold phosphate buffer (50 mM, pH 7.8, 4~C) to remove
unbound xanthine oxidase. A clrcular disk of the size
matching with the electrode was cut out of the membrane
loaded with enzymes (henceforth referred as enzymic
membrane) and stored at 4~C in the same buffer containing
1.0 mM Mg2 .
Effect of PH on the activitv of immobillzed enzYmes
The effect of pH on the activity of the
resulting enzymic membrane is illustrated in Figure 15.
The enzyme xanthine oxidase exhibited a maximum activity
at pH 7.8 when hypoxanthine was used as substrate.
Similarly, for ~he inosine substrate, the pH optimum for
both xanthine oxidase and nucleoside phosphorylase was
also about 7.8. The immobilized enzyme nucleotidase
.... , ,..,
,
-43~
exhibited a hroad optimum pH (7.5 to 9). Therefore, pH
7.8 was re~ommended for analysis using the newly developed
enzyme sensor system in this invention.
Response_of the biosensor sYstem to samPles containinq HxR
or Hx
An excellent linear relation existed between the
electrode output and HxR concentration up to 143 yM. The
slope was determined to be 11.3 mV ~M 1 with a correlation
coefficient of 1 (standard deviation of 4.8~. The
minimum detectable concentration of HxR was determined to
be 3.6 ~M. The reproducibility was l4% for repeated
analyses of 7.14 ~ of HxR as illustrated in Figure 16B.
The standard deviation for 40 repeated assays was +0.1
~ M. Similarly, a good reproducibility (~3%) (Fig. l~A)
and a low standard deviation (-~0.13 ~M) were observed when
7.14 yM Hx was assayed repeatedly. The membranes were
stable at least up to two months with respect to NP
activity when stored at 4~C in 50 mM, pH 7.8, phosphate
buffer containing 1 mM magnesium. Under similar
conditions, there was a 20% decrease in X0 activity.
However, this activity loss did not affect the membrane
performance when used in the analyzer. The response to
HxR was approximately 81 ~2~ of an equimolar Hx sample and
the membrane was useful for at least 40 repeated analyses.
The enzyme electrode developed in this study moni~ored the
products of degradation, hydrogen peroxide and uric acid,
and exhibited a 125-fold higher sensitivi~y than Lhe
-44-
enzyme electrode based on oxygen detection. The higher
sensitivity can be attributed to the detection of three
moles of products released per mole of inosine consumed
compared to the detection of two moles of oxygen consumed
for each mole of inosine degraded and lower diffusional
resistance of the nylon membrane.
Determination of the freshness of various edible fish
Tissue samples from fish fillet (ca. 2g) were
homogenized with about 10% trichloroacetic acid (4 ml)
using a homogenizer. It has been found that a
trichloroacetic acid concentra~ion of about 10% was
suitable for the purposes of the present invention
although other acids and possible different concentrations
could be contemplated. In fact, one needs an acid in
sufficient concentration to break the cell membrane of the
fish sample to be analyzed. The supernatant ob~ained
after centrifugation at 27,000 g force was neutralized
with 2 M sodium hydroxide solution. The sample was then
dlluted up ~o 5 fold using 50 mM glycine ~ 5 mM MgS04
buffer (pH 7.5). It should be no~ed that due to the
highly acidic nature of the fish extract, it is somewhat
difficult to adjust pH 7 to the desired value. Therefore,
it was necessary to use a high ionic-strength buffer for
assay of fish samples. However, it should be borne in
mind that phosphate ions of high concentration resulted in
a high background reading in the biosensor. Therefore, 50
-45-
mM glycine + 5mM MgS04, pH 7.5 buffer was used for fish
sample analyses.
The numerator in Eq. (13) or [Hx] ~ [HxR] was
determined by injecting 25 yl diluted extract in a
reactlon chamber equlpped with the xanthine oxidase-
nucleoside phosphorylase enzyme electrode described above.
The output of the electrode increased and approached a
plateau in 9Q-120 seconds (~I2). For [IMP] ~ ~Hx] ~ lHxR]
measurements, 500 ~l of diluted extract was reacted with
the immobilized nucleotidase for 5-10 min. under constant
shaking on a vortex mixer and 25 ~1 of the resulting
product was injected to the reaction chamber. The result
recorded after 2 minute~ (~I2~ was used toyether with
~I2 to calculate the K value ~I2/~I3.
The process referred to above is also described
in the publications entitled "Development and application
of a biosensor for hypoxan~hine in fish ex~ract",
Analytica Chimica Acta, 221 (1989), 215-222 and
"Development of a biosensor for assaying postmortem
nucleotide degradation in fish tissues", Bio~echnology and
Bioengineering, Vol. 35, pp. 739-734 (1990).
Practical considerations
In terms of cost effectiveness, this method
demonstrates several advantages. First, this method
offers a rapid, simple and accurate method for K value
determination, the freshness indicator of edible fish
-46- ~ 3 ~ 7
meat. Secondly, the enzyme membrane consisting of
nucleoside phosphorylase and xanthine oxidase provides
excellent reproducible results for at least 40 repeated
assays and immobilized nucleotidase is good for at least
40 assays as well. Furthermore, in addltion to the low
cost of analysis, apparati assoeiated with sample handling
and preparation as well as the reaction chamber equipped
with an amperometric elec~rode are compact and suitable
for field work.
The following examples are intended to
illustrate rather than limit the scope of the present
invention.
Example 18
The procedure described under the heading
"Determination of the freshness of various edible fish"
was repeated on a tissue taken from a ~reshly caught
rainbow trout. The K value was determined to be
approximately 0.1.
~xample 19
The procedure described in Example 18 was
repeated on a tissue sample taXen from a rainbow trout 24
hours after death. The fish had been maintained at room
temperature. The recorded K value was estimated to be
approximately 1.
`- ~3~21 ~ ~1
-47-
Example 20
The procedure described in Example 18 was
repeated on a tissue sample taken from a rainbow trout 24
hours after death. The fish had been maintained at a
temperature ranging between 0 and 5C. The K value wa3
estlmated to be 0.61.
Example 21
The procedure described in Example 18 was
repeated on a tissue sample taken from a rainbow trout 72
hours after death. The fish had been maintained at a
temperature ranging between 0 and 5C. The K value was
determined to be 1.
Example 22
The procedure described in Example 18 was
repeated on a tissue sample taken from a rainbow trout 2
weeks after death. The fish had been maintained at a
temperature of -20C. The estimated K value was
determined to be 0.15.
Example 23
The procedure described in Example 18 was
repeated using six samples taken from the muscle of frozen
sole. The average K value was determined to be
approxlmately 0.65.
~ 3~ 2 `~ ~ 7
-48-
Example 24
The procedure described in Example 18 was
repeated using a tissue sample taken from sole which had
been maintained at ~20C for 2 months. The estimated K
value was determlned to be 0.65.
Example 25
The procedure described in Example 24 was
repeated uslng a tissue sample taken from sole which had
been maintained at 5C for 24 hours. The estimated K
value was determined to be 1.
Example 26
The procedure described in Example 24 was
repeated using a tissue sample from the muscle of salmon
frozen for 3 weeks after being caught. The K value was
determined to be 0.37.
Exa~ple 27
The procedure described in Example 26 was
repeated on a tissue sample taXen from the frozen salmon
and maintained at room temperature for 24 hours. The
recorded K value was estimated to be approximately 1.
Example 28
The procedure described in Example 26 was
repeated on a tissue sample taken from the frozen salmon
_49_ ~3~
and maintained at Q-5C for 24 hours. The recorded K
value was estimated to be approximately 0.76.
Exa~ple 29
The procedure described in Example 26 was
repeated on a tissue sample taken from the frozen salmon
and maintained at 0-5~C for 48 hours. The recorded K
value was estimated to be approximately 1.
Exa~ple 30
The procedure described in Example 26 was
repeated on a tissue sample taken from the frozen salmon
and maintained at -20C for a ~urther 2 weeks. The
recorded K value was estimated to be approximately 0.75.
~xample 31
The procedure described in Example 18 was
repeated on a tissue sample taken from the muscle of
freshly caught carp. The K value was determined to be
0.31.
Exa~ple 32
The procedure in Example 31 was repeated on a
tissue sample taken from a carp 24 hours after death. The
fish had been main~ained at a temperature ranging between
0 and 5C. The R value was estimated to be 0.78.
1~ 2~7
-50-
Example 33
The procedure in Example 31 was repeated on a
tissue sample taken from a carp 48 hours after death. The
fish had been maintained a~ a temperature ranging between
0 and 5C. The K value was estima$ed to be 1.
Example 34
The procedure in Example 31 was repeated on a
tissue sample taken from a carp 1 week after death. The
fish had been maintained at a temperature of -20C. The
K value was estimated to be 0.29.
Example 3S
The procedure in Example 18 was repeated on a
tissue sample taken from a live lobster. The estima~ed K
value was very close to zero (0.03).
Example 36
The procedure in Example 18 was repeated on a
tissue sample taken from lobster 12 hours after death.
The lobster had been maintained at a temperature of 20C.
The K value was estimated to be 0.24.
Example 37
The procedure in Example 18 was repeated on a
tissue sample taken from lobster 24 hours af~er deatn.
-51- ~ 7
The lobster had been maintained at a temperature of 20~C.
The K value was es~imated to be 0.94.
Example ~8
The procedure in Example 18 was repeated on a
tissue sample taken from lobster 24 hours after death.
The lobster had been maintained at a temperature of 4C.
The K value was estimated to be 0.24.
Example 39
The procedure in Example 18 was repeated on a
tissue sample taken ~rom lobster 5 days after death. The
lobster had been maintained at a temperature of ~C. The
K value was estimated to be 0.80.
Example 40
The procedure in Example 18 was repeated on a
tissue sample taken from lobster 24 hours after death.
The lobster had been maintained at a temperature of -10C.
The K value was estimated to be 0.06.
Example 41
The procedure in Example 18 was repeated on a
tissue sample taken from lobster 2 days af~er dea$h. The
2S lobster had been maintained at a temperature of -lO~C.
The K value was estimated to be 0.06.
-52- ~3~
Example ~2
The procedure in Example 18 was repeated on a
tissue sample taken from lobster 20 days after death. The
lobster had been maintained at a temperature of -10C.
The K value was estimated to be 0.08.
$xample 43
The procedure in Example 18 was repeated on a
tissue sample taken from a live shrimp. The K value was
estlmated to be close to zero.
Bxample 44
The procedure in Example 18 was repeated on a
tissue sample taken from shrimp 12 hours after death. The
shrimp had been maintained at a temperature of 20C. The
K value was estimated to be 0.4.
Example 45
The procedure in Example 18 was repeated on a
tissue sample taken from shrimp 24 hours after death. The
shrimp had been maintained at a temperature of 20C. The
K value was estimated to be 0.73.
Example 46
The procedure in Example 18 was repeated on a
tissue sample taken from shrimp 2 days after death. The
~ 3 ~ 7
-53-
shrimp had been maintained at a temperature of 4C. The
K value was estimated to be 0.15.
~xa~ple 47
The procedure in Example 18 was repeated on a
tlssue sample taken ~rom shrimp 3 days after death. The
shrimp had been maintained at a temperature o~ 4C. The
K value was estimated ko be 0.19.
~xample 48
The procedure in Example 18 was repeated on a
tissue sample taken from shrimp 4 days after death. The
shrimp had been maintained at a temperature of 4C. The
K value ~as estimated to be 0.2.
Example 49
The procedure in Example 18 was repeated on a
tissue sample taken fxom shrimp 9 days after death. The
shrimp had been maintained at a temperature of 4C. The
K value was estimated to be 0.38.
Example 50
The procedure in Example 18 was repeated on a
tissue sample taken from shrimp 1 day after death. The
shrimp had been maintained at a temperature of -10C. The
K value was estimated to be 0.04.
~ 3 ~
-54-
Example 51
The procedure in Example 18 was repeated on a
tissue sample taken from shrimp 2 days after death. The
shrimp had been maintained at a temperature of -10C. The
K value was estimated to be 0.04.
Exa~ple 52
The procedure in ~xample 18 was repeated on a
tissue sample taken from shrimp 10 days after death. The
shrimp had been maintained at a temperature of -lO~C. The
K value was estimated to be 0.04.
c) Validity of the results obtained.
There was excellent agreement between the K
value determined by ~he biosensor system developed in this
invention and those determined by the conventional
enzymatic assay as shown in Figure 10. The slope was
determined to be 0.967 with a correlation coefficient of
0.998 and a standard deviation of +0.021.
For the conventional enzymatic assay, the
extrack prepared as described above was diluted up to 40-
fold. To 1 mL of diluted extract in 10 mMr pH 7.8
phosphate buffer, 0.18 IU %0, 0.036 IU NP and 1.5 IU
nucleotidase were added sequentially. The concentrations
of Hx, HxR and IMP were determined, respectively from the
three plateaus of uric acid produced according to
-55-
equations (8-10). The K value was of course calculated
according to Fquation ~3).
TABLE 4
Estimation o~ the K value of frozen 501e iillet
by the amperometric electrode
Dilution AmPerometric
Sample # Factor electrode response K value
AI2 ~I3
1 60X 113 170 0.66
30X 235 355 0.66
2 60X 130 175 0.74
30X 228 345 0.66
3 60X 125 198 0.63
30X 233 355 0.66
4 60X 105 150 0.70
30X 200 280 0.71
60X 113 185 0.61
30X 223 36~ 0.62
6 30X 258 353 0.73
