CA1312117C

CA1312117C - Process and apparatus for monitoring the freshness of edible meat

Info

Publication number: CA1312117C
Application number: CA000591169A
Authority: CA
Inventors: John H.T. Luong; An Lac Nguyen; Keith Bradford Male
Original assignee: National Research Council of Canada
Current assignee: National Research Council of Canada
Priority date: 1988-02-17
Filing date: 1989-02-15
Publication date: 1992-12-29
Anticipated expiration: 2009-12-29
Also published as: JPH0210155A

Abstract

ABSTRACT

A method for determining the degree of freshness of raw, frozen and processed edible meat from the values obtained by a composition analysis of the decomposition products of adenosine triphosphate. This method comprises determining by use of a suitable electrode 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 the present invention is an apparatus for determining the degree of freshness of raw, frozen and processed edible meat. This apparatus comprises a reaction cell provided with a suitable uric acid and hydrogen peroxide sensor, an amplifier for sensing signals produced by said sensor and a recording device.

Description

~ 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, ,'- '~

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~

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 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 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 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~

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 ~

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 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 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 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 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 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 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 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 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 ~

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 ~

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 . , . ~

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 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 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 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 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 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 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 ~

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 equations (8-10). The K value was of course calculated according to Fquation ~3).

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

Claims

1. A method for determining the degree of freshness of raw, frozen and processed edible meat by monitoring the autolytic degradation of adenosine triphosphate to inosine monophosphate, inosine and hypoxanthine, said method comprising:
(a) breaking the cell membrane of said meat to produce an extract;
(b) contacting a first portion of said extract with the enzymes xanthine oxidase and nucleoside phosphorylase and electrochemically measuring through an amperometric probe, comprising an anode and a cathode, a value d1 = [HxR] + [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 concentration of inosine and [Hx] is the concentration of hypoxanthine;
(c) contacting a second portion of said extract with the enzymes nucleotidase, nucleoside phosphorylase and xanthine oxidase, and electrochemically measuring through an amperometric probe, comprising an anode and a cathode, a value d2 = [IMP] + [HxR] + [Hx] from the simultaneous determination of the amount of hydrogen peroxide and uric acid resulting 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, [HxR] is the concentration of inosine, and [Hx] is the concentration hypoxanthine; and (d) determining the index of freshness from the formula K = d1/d2, wherein K represents the index of freshness.

2. A method according to claim 1, wherein said extract is produced by extracting said meat with a solution comprising an acid in sufficient concentration to break the cell membrane of said meat and to produce an extract.

3. A method according to claim 2, wherein said solution is a 10% trichloroacetic acid solution.

4. A method according to claim 1, wherein said amperometric probe consists of a platinum anode and a silver/silver chloride cathode, wherein said anode is polarized at +0.5 V to +0.7 V with respect to said cathode.

5. 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 detecting uric acid and hydrogen peroxide resulting from the degradation of inosine monophosphate contained in said meat; means for amplifying signals produced by said detecting means; and a device for recording said signals whereby said freshness may be determined.

6. The apparatus of claim 5, wherein said detecting means is a polarographic electrode.

CLAIMS SUPPORTED BY THE SUPPLEMENTARY DISCLOSURE

7. A method according to claim 1, wherein said enzymes xanthine oxidase and nucleoside phosphorylase are co-immobilized on a polymeric membrane, said polymeric membrane being on said probe to form an enzyme electrode.

8. A method according to claim 7, wherein said enzymes are co-immobilized through glutaraldehyde cross-linking with bovine serum albumin and deposited on a nylon 66 membrane having a pore size of about 3 µm.

9. A method according to claim 1, wherein said enzyme nucleotidase is immobilized on a polymeric support.

10. A method according to claim 9, wherein said nucleotidase is immobilized through a glutaraldehyde activation on the wall of a polymeric tube precoated with a thin layer of polyethyleneimine.

11. An apparatus according to claim 5, wherein said means for detecting uric acid and hydrogen peroxide resulting from the degradation of inosine, monophosphate comprise in combination an amperometric electrode and a porous membrane having xanthine oxidase and nucleoside phosphorylase immobilized thereon.

12. An apparatus according to claim 11, wherein said porous membrane is a nylon membrane.

13. An apparatus according to claim 11, further comprising nucleotidase immobilized on the wall of a polystyrene tube precoated with a thin layer of polystyrene coated with polyethyleneimine.