Enzyme-based biosensor system for monitoring the freshness of fish

The present invention relates to a method for determining the degree of freshness of raw, frozen and processed edible fish by monitoring the degradation of adenine triphosphate to inosine monophosphate, inosine and hypoxanthine. This method comprises simultaneously determining, by use of a suitable amperometric electrode such as platinum vs. silver/silver chloride polarized at 0.7 V, the amount of uric acid and hydrogen peroxide resulting from the degradation of hypoxanthine by xanthine oxidase, the degradation of inosine by the combined action of nucleoside phosphorylase and xanthine oxidase and the degradation of inosine monophosphate by the combined action of nucleotidase, nucleoside phosphorylase and xanthine oxidase. Also within the scope of this invention is a method for the immobilization of nucleotidase on the walls of a polymeric tube such as polystyrene tube and the co-immobilization of nucleoside phosphorylase and xanthine oxidase on a porous polymeric membrane such as a nylon membrane.

BACKGROUND OF THE INVENTION 
Fish and other types of marine organisms lose their freshness very rapidly 
after death. 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. Freshness of fish 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. This is urgently required in 
food industries since fish freshness is an important factor in the 
preparation of high-quality products. Indicators of fish freshness such as 
ammonia, amines, volatile acids, catalase activity, trimethylamine (TMA) 
and nucleotides have so far been proposed. Among these chemicals, 
nucleotides produced by adenosine triphosphate (ATP) decomposition are 
considered the most reliable and useful indicators. In recent years, 
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 autolytically degraded to 
hypoxanthine/xanthine through the following autolytic pathway: 
EQU ATP.fwdarw.ADP.fwdarw.AMP.fwdarw.IMP.fwdarw.HxR.fwdarw.Hx.fwdarw.X(1) 
wherein 
ATP is adenosine triphosphate 
ADP is adenosine diphosphate 
AMP is adenosine monophosphate 
IMP is inosine monophosphate 
HxR is inosine 
Hx is hypoxanthine 
X is xanthine. 
Whereas IMP is one of the major contributing factors to the pleasant flavor 
of fresh fish, the accumulation of Hx and/or X during the storage results 
in an "off-taste". 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: 
##EQU1## 
In several fish species, however, ATP and ADP concentrations rapidly 
decrease and are negligible within 24 hours after death. Similarly, a 
rapid decline of AMP is also observed and its concentration is somewhat 
less than 1 .mu.mol/g. In contrast to such behavior, IMP increases in the 
period ranging between 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: 
##EQU2## 
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 the K value depends 
on the type of fish since changes in the K value are based on the 
enzymatic reactions within the fish meat. The K value also varies 
appreciably with temperature even among the same fish species. 
Based on these facts, various freshness determination methods have been 
developed. For example, Uchiyama et al. (Bulletin of the Japanese Society 
of Scientific Fisheries, Vol. 36, 977 (1970)) made an analysis of the 
various nucleotides 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. 
##EQU3## 
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 (1973)) developed a method to estimate fish freshness based 
on the determination of the concentrations of IMP, HxR and Hx through 
enzymatic reactions. This method is based on the following equations: 
##EQU4## 
The IMP ratio has a high value when the degree of freshness is high and 
decreases as the degree of freshness 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, 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 conduct a 
blank measurement 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 unsuitable for use as the extractant. 
The determination of the K value by monitoring oxygen consumption using a 
Clark oxygen electrode has been commercially exploited by Oriental 
Electric Co. Ltd. The apparatus is known as the KV-101 freshness meter 
(hereinafter referred to as the K-meter) and comprises a Clark oxygen 
electrode attached to a reaction chamber. 
A major drawback of this technique is the low sensitivity and the 
requirement of a rigid control of pH and oxygen tension. In addition, the 
K-meter uses soluble enzymes which cannot be reused and there is a gradual 
loss of the probe's sensitivity, presumably due to the fowling of the 
electrode by the enzymes and/or compounds in fish extract. Other 
techniques of fish freshness determination have also been developed in 
recent years. 
Karube et al. (J. Agric. Food Chem. 32, 314-319, 1984) described an enzyme 
sensor system for the determination of the K value. The system combined a 
double membrane consisting of a 5'-nucleotidase membrane and a nucleoside 
phosphorylase-xanthine oxidase membrane with an oxygen electrode. A small 
anion-exchange resin column was also connected with the enzyme sensor for 
separation of nucleotides. This biosensor system is less desirable for 
practical application since the three compounds in the mixture (IMP, HxR 
and Hx) had to be separated by an elaborate scheme using four different 
buffers and an anion exchange column, amounting to a very complicated 
procedure. 
Ohashi et al. (U.S. Pat. No. 4,650,752) disclosed an enzymatic method for 
determining the K-value for fish and molluscs using soluble enzymes 
together with an oxygen electrode. The enzymes used for determining 
inosine and hypoxanthine concentrations are nucleoside phosphorylase and 
xanthine oxidase. To determine the concentration of the decomposition 
products of adenosine triphosphate, the enzymes alkaline phosphatase, 
adenylic acid kinase, AMP deaminase and adenosine deaminase in a crude 
extract obtained from calf intestine as well as nucleoside phosphorylase 
and xanthine oxidase are used. Again, the main drawback of this technique 
is the low sensitivity, the requirement of several enzymes and the costly 
enzymes that cannot be reused. Although Ohashi et al. state that the 
measurement can be determined electrochemically from the amount of 
hydrogen peroxide produced, no experimental data were presented to 
substantiate this statement. 
Therefore, an inexpensive and rapid method useful in monitoring fish 
freshness would be highly desirable. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, there is provided a method for 
determining the degree of freshness of raw, frozen and processed edible 
fish by monitoring the degradation of adenine triphosphate to inosine 
monophosphate, inosine and hypoxanthine. The method comprises firstly 
extracting edible fish with a solution of an acid useful to break cell 
membranes such as trichloroacetic acid, in order to produce an extract. A 
first portion of this extract is contacted with the enzymes xanthine 
oxidase and nucleoside phosphorylase and a value d.sub.1 is 
electrochemically measured through a single electrode from the 
simultaneous determination of the amount of hydrogen peroxide and uric 
acid resulting from the degradation of hypoxanthine and inosine by these 
two enzymes. 
This is followed by contacting a second portion of the fish extract with 
the enzymes xanthine oxidase, nucleoside phosphorylase and nucleotidase 
and electrochemically measuring through a single electrode a value d.sub.2 
from the simultaneous determination of the amount of hydrogen peroxide and 
uric acid resulting from the degradation of inosine monophosphate, inosine 
and hypoxanthine by these three enzymes. 
Based on values d.sub.1 and d.sub.2, the index of freshness is determined 
from the formula K=d.sub.1 /d.sub.2 where K represents the index of 
freshness, d.sub.1 represents the combined concentrations of inosine and 
hypoxanthine and d.sub.2 represents the combined concentrations of inosine 
monophosphate, inosine and hypoxanthine. 
It has been discovered that the simultaneous determination of both the 
amount of uric acid and hydrogen peroxide may be achieved through the use 
of an amperometric electrode polarized at +0.7 V. The simultaneous 
determination of both uric acid and hydrogen peroxide concentrations 
enables accurate measurements of both values d.sub.1 and d.sub.2 and thus 
accurate determination of the index of freshness. Preferably, the 
electrode to be used is a platinum vs. silver/silver chloride electrode 
polarized at +0.7 V. 
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 in the freshness determination 
of edible fish that is: xanthine oxidase, nucleoside phosphorylase, and 
nucleotidase. The method comprises immobilizing a first enzyme, such as 
nucleotidase, on a polymeric support. The immobilization is accomplished 
by contacting this support with a polyethyleneimine solution, a solution 
containing a crosslinking agent and a solution containing the enzyme to be 
immobilized. 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. 
The term "edible fish", when used herein, is intended to include a variety 
of marine organisms comprising various fish species such as salmon, sole 
and trout as well as crab meat, lobster and the like.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention is concerned with a new method useful in monitoring 
the freshness of various edible fish by the determination of their 
respective K value. The determination of the K value is obtained by using 
an amperometric 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 is degraded in the 
following manner: 
##STR1## 
wherein NT, NP, XO and P.sub.i are nucleotidase, nucleoside phosphorylase, 
xanthine oxidase, and inorganic phosphate, respectively. 
As demonstrated above, each mole of inosine monophosphate consumed will 
ultimately require two moles of oxygen and liberate two moles of hydrogen 
peroxide as well as one mole of uric acid. It is therefore 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 detection of enzymatically generated hydrogen peroxide has 
been widely performed by using a Clark hydrogen peroxide electrode 
(referred to hereinafter as the amperometric electrode). Basically, this 
electrode consists of a platinum anode and a silver/silver chloride 
cathode where the anode is polarized at 0.5 V to +0.7 V with respect to 
the cathode. The amperometric probe oxidizes a constant portion of the 
hydrogen peroxide at the platinum anode at such a polarized potential. 
EQU H.sub.2 O.sub.2 .fwdarw.2H.sup.+ +O.sub.2 +2e.sup.- (11) 
The current thus created is directly proportional to the hydrogen peroxide 
level formed during the oxidation of Hx to uric acid by the enzyme 
xanthine oxidase as shown in equation 10. However it should be noted that 
various reducing substances such as ascorbic acid, glutathione, uric acid, 
etc., may considerably influence the oxidation of H.sub.2 O.sub.2. 
Consequently, there is a problem for determining the level of H.sub.2 
O.sub.2 formed during the oxidation of Hx since the amperometric electrode 
will respond to both H.sub.2 O.sub.2 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 
(0.5 V to +0.7 V) where hydrogen peroxide is oxidized. The electrochemical 
oxidation of uric acid can be described by the following reaction. 
##STR2## 
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. 
In the present invention, it has been discovered that the amperometric 
electrode responds to a sample containing both uric acid and hydrogen 
peroxide in an additive manner. 
The [Hx+HxR] concentration in tissue extract can therefore be measured by 
using nucleoside phosphorylase and xanthine oxidase which are preferably 
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+HxR+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 polyethyleneimine. Again, 
the nature of the polymeric material is not critical but polymers such as 
polystyrene and the like should be employed. The [IMP+Hx+HxR] 
concentration is then measured by the aforementioned electrode. 
Referring now to the drawings, FIG. 1 shows an example of the instrument 
used in the present invention. In FIG. 1, the sample measurement chamber 
1, the volume of which is preferably ranging from 0.3 to 0.4 ml, comprises 
a stopper 2 provided with a capillary 3 used for liquid injection in the 
center thereof, said capillary having, for example, a diameter 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 abundant supply of oxygen to support 
the reaction. The reaction chamber 1 also contains an amperometric 
electrode 6 on which is affixed a porous polymeric membrane on which the 
nucleoside phosphorylase and xanthine oxidase enzymes have previously been 
immobilized. 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. 
Description of a Preferred Embodiment Using Immobilized Enzymes for the 
Determination of Fish Freshness Materials and Methods 
a) Immobilization of Nucleotidase on the Wall of a Polystyrene Tube. 
Nucleotidase (NT) was immobilized on the wall of a 1-mL polystyrene 
centrifuge tube. The tube was filled with 1 mL of 5% polyethyleneimine 
solution and incubated at room temperature (20.degree.-24.degree. 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 temperature 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 nucleotidase dissolved in 4 mM, pH 7.8, 
phosphate buffer and incubated overnight at 4.degree. C. The solution was 
then removed and the tube was washed extensively with the buffer and 
stored filled with buffer at 4.degree. C. 
b) Co-immobilization of Nucleoside Phosphorylase and Xanthine Oxidase on a 
Membrane. 
A prewetted Immunodyne .TM. membrane (1.5.times.1.5 cm) was stretched on 
the tip of a hollow plastic cylinder (1 cm diameter) and held in place by 
an O-ring. The preactivated Immunodyne.TM. nylon 66 membrane (pore size of 
3.mu.m) was obtained from Pall BioSupport Division (Glen Cove, N.Y.). 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 .mu.l of nucleoside phosphorylase (NP, 5.1 g/l 
and 3.6 U/mg), 4 .mu.l of bovine serum albumin (BSA, 400 g/l), and 18 
.mu.l of buffer (200 mM) pH 7 phosphate), 8 .mu.of glutaraldehyde (12.5% 
w/v) was added to initiate the crosslinking. It should be noted that the 
final volume of the resulting solution is 50 .mu.l and contained 2% w/v 
glutaraldehyde, 1.6 mg BSA and 102 .mu.g NP. 35 .mu.l of the resulting 
solution was then layered on the prewetted membrane and the solution was 
allowed to crosslink at room temperature (20.degree.-24.degree. C.) 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 .mu.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 FIG. 2. The NP activity decreased drastically at 
glutaraldehyde concentrations above 2%. However, the enzyme layer obtained 
under such a condition was much stronger and slightly yellow. 
Apparently, a low glutaraldehyde concentration resulted in an insufficient 
protein crosslinking and led to washing away 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 FIG. 3. 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 albumin 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 FIG. 4. Below 20 .mu.g 
NP, there was a linear relationship between the activity of the 
immobilized enzyme and the amount of enzyme used. Beyond 70 .mu.g 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 .mu.g NP was used for enzyme layer preparation. 
After NP was immobilized, the membrane was washed extensively with 50 mM 
phosphate buffer and it 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 
Geratebau, Hamburg, FRG) for 4 h. at 4.degree. C. The membrane was then 
washed several times with cold phosphate buffer (50 mM, pH 7.8, 4.degree. 
C.) to remove unbound xanthine oxidase. A circular 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.degree. 
C. in the same buffer containing 1.0 mM Mg.sup.2+. 
Effect of pH on the Activity of Immobilized Enzymes 
The effect of pH on the activity of the resulting enzymic membrane is 
illustrated in FIG. 5. The enzyme xanthine oxidase exhibited a maximum 
activity at pH 7.8 when hypoxanthine was used as substrate. Similarly, for 
the inosine substrate, the pH optimum for both xanthine oxidase and 
nucleoside phosphorylase was also about 7.8. The immobilized enzyme 
nucleotidase exhibited a broad optimum pH (7.5 to 9). Therefore, pH 7.8 
was recommended for analysis using the newly developed enzyme sensor 
system in this invention. 
Response of the Biosensor System to Samples Containing HxR or Hx 
An excellent linear relation existed between the electrode output and HxR 
concentration up to 143 .mu.M. The slope was determined to be 11.3 mV 
.mu.M.sup.-1 with a correlation coefficient of 1 (standard deviation of 
.+-.4.8). The minimum detectable concentration of HxR was determined to be 
3.6 .mu.M. The reproducibility was .+-.4% for repeated analyses of 7.14 
.mu.M of HxR as illustrated in FIG. 6B. The standard deviation for 40 
repeated assays was .+-.0.14 .mu.M. Similarly, a good reproducibility 
(.+-.3%) (FIG. 6A) and a low standard deviation (.+-.0.13 .mu.M) were 
observed when 7.14 .mu.M Hx was assayed repeatedly. The membranes were 
stable at least up to two months with respect to NP activity when stored 
at 4.degree. C. in 50 mM, pH 7.8, phosphate buffer containing 1 mM 
magnesium. Under similar conditions, there was a 20% decrease in XO 
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 monitored the products of degradation, hydrogen peroxide and 
uric acid, and exhibited a 125-fold higher sensitivity than the 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. 2 g) were homogenized with about 10% 
trichloroacetic acid (4 ml) using a homogenizer. It has been found that a 
trichloroacetic acid concentration 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 obtained after centrifugation at 27,000 g 
force was neutralized with 2M sodium hydroxide solution. The sample was 
then diluted up to 5 fold using 50 mM glycine+5 mM MgSO.sub.4 buffer (pH 
7.5). It should be noted 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 mM glycine+5 mM MgSO.sub.4, pH 7.5 buffer was 
used for fish sample analyses. 
The numerator in Eq. (3) or [Hx+HxR] was determined by injecting 25 .mu.l 
diluted extract in a reaction chamber equipped with the xanthine 
oxidase-nucleoside phosphorylase enzyme electrode described above. The 
output of the electrode increased and approached a plateau in 90-120 
seconds (d.sub.1). For [IMP+Hx+HxR] measurements, 500 .mu.l of diluted 
extract was reacted with the immobilized nucleotidase for 5-10 min. under 
constant shaking on a vortex mixer and 25 .mu.l of the resulting product 
was injected to the reaction chamber. The result recorded after 2 minutes 
(d.sub.2) was used together with d.sub.1 to calculate the K value d.sub.1 
/d.sub.2. 
The process referred to above is also described in the publications 
entitled "Development and application of a biosensor for hypoxanthine in 
fish extract", Analytica Chimica Acta, 221 (1989), 215-222 and 
"Development of a biosensor for assaying postmortem nucleotide degradation 
in fish tissues", Biotechnology and Bioengineering, Vol. 35, pp. 739-734 
(1990), which are hereby incorporated by reference. 
Practical Considerations 
In terms of cost effectiveness, the method of the present invention 
demonstrates several advantages. First, the method of the present 
invention offers a rapid, simple and accurate method for K value 
determination, the freshness indicator of edible fish 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 addition to the low cost of analysis, apparati 
associated with sample handling and preparation as well as the reaction 
chamber equipped with an amperometric electrode are compact and suitable 
for field work. 
The following examples are intended to illustrate rather than limit the 
scope of the present invention. 
EXAMPLE 1 
The procedure described under the heading "Determination of the freshness 
of various edible fish" was repeated on a tissue taken from a freshly 
caught rainbow trout. The K value was determined to be approximately 0.1. 
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 on a tissue sample taken 
from a rainbow trout 24 hours after death. The fish had been maintained at 
a temperature ranging between 0.degree. and 5.degree. C. The K value was 
estimated to be 0.61. 
EXAMPLE 4 
The procedure described in Example 1 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.degree. and 5.degree. C. The K value was 
determined to be 1. 
EXAMPLE 5 
The procedure described in Example 1 was repeated on a tissue sample taken 
from a rainbow trout 2 weeks after death. The fish had been maintained at 
a temperature of -20.degree. C. The estimated K value was determined to be 
0.15. 
EXAMPLE 6 
The procedure described in Example 1 was repeated using six samples taken 
from the muscle of frozen sole. The average K value was determined to be 
approximately 0.65. 
EXAMPLE 7 
The procedure described in Example 6 was repeated using a tissue sample 
taken from sole which had been maintained at -20.degree. C. for 2 months. 
The estimated K value was determined to be 0.65. 
EXAMPLE 8 
The procedure described in Example 6 was repeated using a tissue sample 
taken from sole which had been maintained at 5.degree. C. for 24 hours. 
The estimated K value was determined to be 1. 
EXAMPLE 9 
The procedure described in Example 6 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. 
EXAMPLE 10 
The procedure described in Example 9 was repeated on a tissue 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 in Example 9 was repeated on a tissue sample taken 
from the frozen salmon and maintained at 0.degree.-5.degree. C. for 24 
hours. The recorded K value was estimated to be approximately 0.76. 
EXAMPLE 12 
The procedure described in Example 9 was repeated on a tissue sample taken 
from the frozen salmon and maintained at 0.degree.-5.degree. C. for 48 
hours. The recorded K value was estimated to be approximately 1. 
EXAMPLE 13 
The procedure described in Example 9 was repeated on a tissue sample taken 
from the frozen salmon and maintained at -20.degree. C. for a further 2 
weeks. The recorded K value was estimated to be approximately 0.75. 
EXAMPLE 14 
The procedure described in Example 1 was repeated on a tissue sample taken 
from the muscle of freshly caught carp. The K value was determined to be 
0.31. 
EXAMPLE 15 
The procedure in Example 14 was repeated on a tissue sample taken from a 
carp 24 hours after death. The fish had been maintained at a temperature 
ranging between 0.degree. and 5.degree. C. The K value was estimated to be 
0.78. 
EXAMPLE 16 
The procedure in Example 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.degree. and 5.degree. C. The K value was estimated to be 
1. 
EXAMPLE 17 
The procedure in Example 14 was repeated on a tissue sample taken from a 
carp 1 week after death. The fish had been maintained at a temperature of 
-20.degree. C. The K value was estimated to be 0.29. 
EXAMPLE 18 
The procedure in Example 1 was repeated on a tissue sample taken from a 
live lobster. The estimated K value was very close to zero (0.03). 
EXAMPLE 19 
The procedure in Example 1 was repeated on a tissue sample taken from 
lobster 12 hours after death. The lobster had been maintained at a 
temperature of 20.degree. C. The K value was estimated to be 0.24. 
EXAMPLE 20 
The procedure in Example 1 was repeated on a tissue sample taken from 
lobster 24 hours after death. The lobster had been maintained at a 
temperature of 20.degree. C. The K value was estimated to be 0.94. 
EXAMPLE 21 
The procedure in Example 1 was repeated on a tissue sample taken from 
lobster 24 hours after death. The lobster had been maintained at a 
temperature of 4.degree. C. The K value was estimated to be 0.24. 
EXAMPLE 22 
The procedure in Example 1 was repeated on a tissue sample taken from 
lobster 5 days after death. The lobster had been maintained at a 
temperature of 4.degree. C. The K value was estimated to be 0.80. 
EXAMPLE 23 
The procedure in Example 1 was repeated on a tissue sample taken from 
lobster 24 hours after death. The lobster had been maintained at a 
temperature of -10.degree. C. The K value was estimated to be 0.06. 
EXAMPLE 24 
The procedure in Example 1 was repeated on a tissue sample taken from 
lobster 2 days after death. The lobster had been maintained at a 
temperature of -10.degree. C. The K value was estimated to be 0.06. 
EXAMPLE 25 
The procedure in Example 1 was repeated on a tissue sample taken from 
lobster 20 days after death. The lobster had been maintained at a 
temperature of -10.degree. C. The K value was estimated to be 0.08. 
EXAMPLE 26 
The procedure in Example 1 was repeated on a tissue sample taken from a 
live shrimp. The K value was estimated to be close to zero. 
EXAMPLE 27 
The procedure in Example 1 was repeated on a tissue sample taken from 
shrimp 12 hours after death. The shrimp had been maintained at a 
temperature of 20.degree. C. The K value was estimated to be 0.4. 
EXAMPLE 28 
The procedure in Example 1 was repeated on a tissue sample taken from 
shrimp 24 hours after death. The shrimp had been maintained at a 
temperature of 20.degree. C. The K value was estimated to be 0.73. 
EXAMPLE 29 
The procedure in Example 1 was repeated on a tissue sample taken from 
shrimp 2 days after death. The shrimp had been maintained at a temperature 
of 4.degree. C. The K value was estimated to be 0.15. 
EXAMPLE 30 
The procedure in Example 1 was repeated on a tissue sample taken from 
shrimp 3 days after death. The shrimp had been maintained at a temperature 
of 4.degree. C. The K value was estimated to be 0.19. 
EXAMPLE 31 
The procedure in Example 1 was repeated on a tissue sample taken from 
shrimp 4 days after death. The shrimp had been maintained at a temperature 
of 4.degree. C. The K value was estimated to be 0.2. 
EXAMPLE 32 
The procedure in Example 1 was repeated on a tissue sample taken from 
shrimp 9 days after death. The shrimp had been maintained at a temperature 
of 4.degree. C. The K value was estimated to be 0.38. 
EXAMPLE 33 
The procedure in Example 1 was repeated on a tissue sample taken from 
shrimp 1 day after death. The shrimp had been maintained at a temperature 
of -10.degree. C. The K value was estimated to be 0.04. 
EXAMPLE 34 
The procedure in Example 1 was repeated on a tissue sample taken from 
shrimp 2 days after death. The shrimp had been maintained at a temperature 
of -10.degree. C. The K value was estimated to be 0.04. 
EXAMPLE 35 
The procedure in Example 1 was repeated on a tissue sample taken from 
shrimp 10 days after death. The shrimp had been maintained at a 
temperature of -10.degree. 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 the 
biosensor system developed in this invention and those determined by the 
conventional enzymatic assay as shown in FIG. 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 extract prepared as described 
above was diluted up to 40-fold. To 1 mL of diluted extract in 10 mM, pH 
7.8 phosphate buffer, 0.18 IU XO, 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 
equations (8-10). The K value was of course calculated according to 
Equation (3). 
TABLE 1 
______________________________________ 
Estimation of the K value of frozen sole fillet 
by the amperometric electrode 
Amperometric 
Dilution electrode response 
Sample # Factor d.sub.1 d.sub.2 
K value 
______________________________________ 
1 60.times. 
113 170 0.66 
30.times. 
235 355 0.66 
2 60.times. 
130 175 0.74 
30.times. 
228 345 0.66 
3 60.times. 
125 198 0.63 
30.times. 
233 355 0.66 
4 60.times. 
105 150 0.70 
30.times. 
200 280 0.71 
5 60.times. 
113 185 0.61 
30.times. 
223 360 0.62 
6 30.times. 
258 353 0.73 
______________________________________ 
This is a continuation-in-part of U.S. application Ser. No. 157,390 filed 
Feb. 17, 1988, which is hereby incorporated by reference.