Sensors and Materials, Vol. 13, No. 3 (2001) 129-136

MYUTokyo
S &M0436

Enzyme Reactor System for the Determinationof the Quality of Chicken
Yuji Suzuki*, Ron Usami*, Koki Horikoshi and Hirokazu Okuma
Department of Life Sciences, Faculty of Life Sciences,Toyo University*Graduate School of Engineering,Toyo University
1-1-1 Izumino, ltakura, Ora, Gunma374-0193, Japan(Received October 15, 2000: accepted March 25, 2001)

Key words: reactor type enzyme sensor, freshness, K-value, spoilage, putrescine oxidase, chicken
quality

An enzyme reactor system was developed for the determination of the quality ofchicken over a wide range including both freshness (K-value) and spoilage (diaminecontent). The system was constructed by fusing a K-value sensor and a diamine sensor in
the same channel. In fresh chicken muscle, little diamine is present, and the diamine sensor
shows no response. Therefore, the diamine sensor was placed upstream to the K-value
sensor and the effects of chemical cross-talk caused by both sensors were examined.
Sufficient precision in the measurement was obtained by correcting the K-value for the
relationship between the putrescine concentration and cross-talk. One assay required 5
min. The immobilized enzymes were stable over 100 days at 4'C.

1.

Introduction

With the introduction of hazard analysis and critical control point (HACCP) to food
manufacturing, the establishment of a monitoring system for the rapid examination ofchicken quality is anticipated. As parameters of freshness, viable count, volatile basic
nitrogen,0-2l ammonia,<3Al amines,<5l volatile acids,<6l pH<7-9l and K-value<10-t5J have been
proposed for the conversion of sensory freshness into a numerical value. However, themethods for measuring these parameters have associated problems. Therefore, the fresh­ness of chicken is difficult to measure from these parameters. The development of a reactor
system for measuring fish and chicken freshness has been reported.<1 6• l With this system
it is possible to determine K-values (the most common index of freshness) rapidly andsimply but not to clarify the spoilage caused by microbial contamination. Therefore, todetermine the incipient stage of spoilage, we focused on the amount of diamines (pu17

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Sensors and Materials, Vol. 13, No. 3 (2001)

trescine and cadaverine) present, which are metabolites produced by growing microorgan­
isms. Considering that the incipient stage of spoilage can be determined by measuring
these diamines, we developed an enzyme reactor sensor that can easily.measure the amountof diamines present. By combining the proposed reactor sensor with the K-value sensor
system, we widened the range of the freshness sensor in order to be able to measure fromthe reduction of freshness to the incipient stage of spoilage of chicken.

2.

Materials and Methods

2.1 Enzyme and reagentsThe enzyme used in the immobilized enzyme reactors in the K-value sensor werealkaline phosphatase (ALP: E.C.3.1.3.1, from calf intestinal mucosa, Biozyme; UK),
nucleoside phosphorylase (NP: E.C.2.4.2.1, from calf spleen, Boehringer Mannheim;
Germany) and xanthine oxidase (XOD: E.C.1.1.3.22, from cows' milk, Biozyme; UK).
The enzyme used in the immobilized reactor in the diamine sensor was putrescine oxidase(PUO: E.C.1.4.3.10, from microorganisms, Toyobo; Japan). Glass porous beads(LCA00500B) and chitosan porous beads (Chitopeal 3001, diameter 0.1 mm) were ob­tained from Funakoshi and Fuji Spinning (Japan), respectively. Fifty millimolar phosphatebuffer, pH 7.8, and 50% glularaldehyde (GA) were obtained from Kanta Chemical (Japan).
Putrescine was obtained from E. Merck (Germany). Cadaverine, tyramine and isoamylarnine
were obtained from Tokyo Kasei Co. (Japan). Spermidine, omithine, histidine, lysine andhistamine were obtained from Wako Chemical Industries, Ltd. (Japan). Spermine wasobtained from Sigma (USA). The various amines used as model samples for diamine
sensors were dissolved in aqueous 0.8% NaCl solution. All other chemicals were analyti­
cal-reagent grade.
2.2 Preparation of enzyme reactors
A pair of enzyme reactors was used for the K-value sensor. To 2.5 ml of chitosan beads,
twenty milliliters of phosphate buffer (0.05 M, pH 7.8) and 1060 µl of 50% glutaraldehyde
solution were added and mixed, then the mixture was shaken for 2 h at 30 ° C. After
activation with glutaraldehyde, the beads were washed well with distilled water anddehydrated by vacuum filtration. To these beads, two milliliter of phosphate buffer wasadded, followed by the addition of NP (60U) and XOD (20U), then the mixture was shaken°
for 2 h at 30 C to simultaneously immobilize these two enzymes on the chitosan beads withglutaraldehyde through the formation of a Schiff' s base. The immobilized enzymes were°
preserved in 0.05 M phosphate buffer solution (pH 7.8) at 4 C. The enzyme-bonded beads
were packed into a polypropylene reactor tube (50 mm x 3 mm i.d.) and used as a reactor
for measuring inosine and hypoxanthine. To prepare the immobilized enzyme reactor formeasuring inosine monophosphate, inosine and hypoxanthine, the chitosan beads with thetwo immobilized enzymes and 850 mg of glass beads in 20 ml of phosphate buffer in which
14400U of ALP was immobilized were packed in layers at a 1:1 ratio.
The reactor for diamine measurement was prepared by the same technique. In a
mixture of 1 ml of chitosan beads and 10 ml of phosphate buffer, 500U of PUO wasimmobilized, and the beads were packed into a polypropylene reactor tube (50 mm x 2 mmi.d.).

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Sensors and Materials, Vol. 13, No. 3 (2001)

2.3 Measurement principle and enzyme sensor system

After fish die, ATP-related compounds in the fish decompose to form uric acid asshown in Fig. 1. Saito had reported a high correlation between the percentage of inosine
and hypoxanthine in the total ATP-related compounds and the early freshness of fish.(lO)
Therefore, the ratio of inosine and hypoxanthine to the total amount of ATP-related
compounds is designated as the K-value and used as an index of fish and chicken freshness
in Japan. However, ATP, ADP and AMP are changed to inosine monophosphate in a very
short time. Actually, the Ki-value, which represents the ratio of inosine and hypoxanthine
to the total amount of inosine monophosphate, inosine and hypoxanthine, is almost equal to
the K-value. Therefore, in this study, the Ki-value was measured by the K-value sensor as
the indicator of early freshness.
The K-value sensor was assembled with an enzyme reactor system consisting of two
types of immobilized enzyme reactors with oxygen electrodes placed behind each reactor,
and the K-value was calculated from the amount of oxygen consumed in each immobilized
enzyme reactor.
In the measurement of diamines, the amount of hydrogen peroxide produced by the
enzyme reaction was detected using three electrodes. The working platinum anode waspolarized at 500 mV vs Ag/AgCL

2.4 Preparation of samples
The chicken meat used in the experiment was commercially available white chicken
meat (product of Iwate, Japan). The chicken muscle was cut into approximately 1 cm°
segments and stored under aerobic conditions at 5 and 15 C. Samples (10 g) werearbitrarily gathered at 24 h intervals and physiological saline (90 g) was added to homog­
enize. The homogenized suspensions were used as is for the measurement of the viablecount. For sample preparation in the enzyme reactor, the homogenized suspensions were
centrifuged (15000 pm for 10 min at 4°C).

ATPaseMyokinaae

AMPdeaminase

5'·Nucleotidase

Nucleoside phosphorylase

Xanthine oxidase

ATP�ADP �AMP� IM� HxR� H� UA· \ NHa\ Pi\ Pi o/ \ H2O2
\ Pi
\Pi
K value(%)= (HxR+Hx)/(ATP+ADP+AMP+IMP+HxR+Hx) x 100
K value(%) =: Ki value(%)= (HxR+Hx)/(IMP+HxR+Hx) x 100
IMP : Inosine monophosphate
Hx : Hypoxanthine

HxR : Inosine
UA: Uric acid

Fig. 1. Changes of ATP and definition of K-value.

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3.

Sensors and Materials, Vol. 13, No. 3 (2001)

Results and Discussion

3 .1 Substrate specificity of the immobilized putrescine oxidase

The PUO used in the diamine sensor shows substrate specificity only to putrescine.
Therefore, to simultaneously detect putrescine, cadaverine and spermidine, which was theobjective of this study, the glutaraldehyde concentration used in the immobilization of
enzymes and the amount of putrescine oxidase were changed, and the optimal immobiliza­tion conditions for obtaining equal sensor responses to these three substances were
investigated. As the substrate, ten amines adjusted to 0.25 mM were injected into thesensor, and the sensor response to each amine was measured.
The results are shown in Table 1. As the enzyme amount increased, the relative
responses to cadaverine and spermidine compared to the response to putrescine increased,
and this increase became marked upon treatment with glutaraldehyde. This sensor
responded only to these three amines.. Based on this observation, the enzyme immobiliza­tion conditions used in this sensor system were set to 2.5% glutaraldehyde concentrationand 500U putrescine oxidase.
The calibration curves for the determination of putrescine, cadaverine and spermidine
were determined. Linear relationships were observed between the sensor response and all
three compounds up to 1 mM. The lower limit of detection for the compounds was 0.01mM.

3.2 Application of the diamine sensor to the detection of the incipient stage of
spoilage of chicken
Changes in the diamine concentration in chicken extracts during storage at 5 and 15° C
are shown in Fig. 2. The response to diamine rapidly increased after 4 days and 1 day at 5
and 15 ° C, respectively, and the viable counts at these points were 106- 107 cells/g. ThereTable 1
Effects of putrescine oxidase concentration on relative sensor response (relative activity).
Subustrate

Immobilized enzyme (glutaraldehyde: 2.5%)
lOU
lO0U
300U 500U (GAO%)
5U (GAO%)

Putrescine
Cadaverine
Spermidine
LysineHistamine
Tyramine
Isoamylamine
Omithine
Spermine
Histidine

100 (100)
14.5 (7.1)
30.6 (11.4)
0 (0)
0 (0)
2.41 (1.05)
0 (0)
0 (0)
0 (0)
0 (0)

Substrate: 0.25 mM

100
22.7
46
0
0
2.16
0
0
0
0

100
78.4
92.8
0
0
1.03
0
0
0
0

100
94.8
104.7
0
0
2.37
0
0

1.12
0

100 (100)
96.9 (33.8)
102.4 (48.7)
0 (0)
0 (0)
1.09 (1.61)
0(0)
0 (0)
1.09 (0)0 (0)Native

100
1.9
14
0
0
0
0
0
0.38
0

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Sensors and Materials, Vol. 13, No. 3 (2001)
200'bl) 1800-0-5"C-+-15"C

0

160
140
__§
...,A 120
100
A
80
'-'
Q)
60
'§ 40
20
0

0

5

10

15

Days of storage

Fig. 2. Changes in diamine content of chicken muscle during storage at 5 and 15 C. 0: Chicken°
extract during storage at 5 C. e: Chicken extract during storage at 15 °C.
°

fore, the incipient stage of spoilage was detected. The levels of diamines further increased°°
very rapidly when the samples were stored at 15 C in comparison to 5 C. These results
depended on the optimum growth temperature of the microorganisms. Microorganisms°
generally have optimum growth temperatures of around 30 C. Therefore, the growth°velocity of the microorganisms was greater in chicken muscle stored at 15 C than in those°stored at 5 C. Accordingly, the microbial metabolic velocity increased, resulting in an
increase in the concentration of diamines, which are metabolic products.
The sensor response was observed in chicken muscle stored for more than 4 days at°
5 C. Therefore, it is difficult to monitor the early stage of the reduction of freshness by thissensor alone.

3.3 Determination of the freshness of chicken meat by the K-value sensor°

The results of K-value determination in chicken muscle stored at 5 .and 15 C are shownin Fig. 3. The K-value increased more rapidly in the chicken muscle stored at 15'C than in°that stored at 5 C because the velocity of the reactions of the enzymes that decompose°
ATP-related compounds is greater in the chicken muscle stored at 15 C. Accordingly, thevelocity of the decomposition of the ATP-related compounds was increased in the meat
stored at 15'C, resulting in a rapid increase in the K-value. However, regardless of thestorage temperature, after the K-value rose by almost 70%, it did not increase further. Thisfinding showed that the early reduction of freshness can be measured by determining theK-value, but spoilage thereafter is difficult to determine from the K-value.

3 .4 Effect of chemical cross-talk caused by fusion of the sensors

Oxidizing enzymes were used in the diamine and K-value sensor; when these flow
sensors were fused and placed on the same line, chemical cross-talk occurred in which
oxygen consumed in the oxidation reaction in the upstream sensor caused a response at the134

Sensors and Materials, Vol. 13, No. 3 (2001)
80
70

60

50
40

�

30

-0-5'C-+-15°C

20

10
0

0

5

10

Days of storage

15

Fig. 3. Changes in K-value of chicken muscle during storage at 5 and 15 ° C. 0: Chicken extract°
Chicken extract during storage at 15 ° C.
during storage at 5 C.

e:downstream sensor.. In chicken and fish, almost no diamine is produced in the very fresh
range with a K-value of 30% or lower, and the diamine sensor does not respond. Therefore,
to reduce chemical cross-talk, the diamine sensor was placed upstream in the constructionof the fused system. The fused sensor system is shown in Fig. 4.
Since the optimal pH was 7.5-8.0 for the diamine sensor and 8.0-8.5 for the K-value
sensor, tris-HCl buffer, pH 8.0, was used in the fused sensors and the flow rate was 1.2 ml/min.
To the standard samples at K-values of 15% and 40%, putrescine at a specified
concentration was added. Forty microliters of the sample was injected into the sensor
system, and the effect of chemical cross-talk on the K-value sensor was investigated. The
results are shown in Fig. 5. Deviation of the K-value occurred due to cross-talk at aputrescine concentration of 0.71 mg/100 ml. We attempted to correct the effect of cross­talk. Since the cross-talk increased linearly with putrescine concentration, the output of theK-value sensor was corrected using the slope as the correction coefficient, and the K-value
was re-calculated. The results are shown in Fig. 6. By correcting the K-value, sufficientmeasurement precision with a correlation coefficient of 0.955 was obtained even in a rangeof high putrescine concentration. One assay could be completed within 5 min. The
precision of the analysis was 2% of the coefficient of variation. The immobilized enzymes
were stable over 100 days at 4 °C.

4.

Conclusion

The K-value sensor was used to evaluate early stage freshness, and the diamine sensor
could estimate the incipient stage of spoilage by measuring diamines. An enzyme flow
reactor system could be constructed by fusing the K-value sensor and a diamine sensor for135

Sensors and Materials, Vol. 13, No. 3 (2001)

Dill
I

11

12

[] 10

waste

32 °C

Fig. 4. Construction of the fused biosensor system for determination of freshness. 1. Buffer, 2.
peristaltic pump, 3. injection port, 4. column for determination of diamine, 5. hydrogen peroxide
electrode, 6. column (A) for determination of K-value, 7. column (B) for determination of K-value,
8. oxygen electrode (ELI), 9. oxygen electrode (EL2), 10. AID converter, 11. computer, 12. recorder.
70.. •40%
O 15%
50 ..
• ••
60

40

••

..
20 • - r.o
0 10 ..

� 30

0

0

•

•

0
0

.

.

2

4

6

8

Putrescine concentration(mg/lOOml)

10

Fig. 5. Effect of chemical cross-talk on K-value. Measurement was carried out in 0.05M tris-HCI°
buffer at l.2ml/min, 32 C and pH 8.0. Forty microliter samples containing putrescine were injected.
0: Standard sample at K-value of 15%, putrescine at a specified concentration.
Standard sample
at K-value of 40%, putrescine at a specified concentration.

e:serially detecting freshness up to the incipient stage of spoilage. Sufficient precision in themeasurement could be achieved by correcting the effect of chemical cross-talk by fusing
these reactors. Therefore, an enzyme reactor system can be developed for the determina­tion of chicken quality over a wide range including both freshness and spoilage.

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Sensors and Materials, Vol. 13, No. 3 (2001)
70

015%
•40%

60
,...._

50
40

� 30

20
10
0

••••• •
V

µ VQV

•
0

2

0

.

.

4

6

8

10

Putrescine concentration(mg/lO0ml)

Fig. 6. Correlation between the corrected K-values and putrescine concentration. Measurement°
was carried out in 0.05 M tris-HCl buffer at l.2ml/min, 32 C and pH 8.0. Forty microliter samples
containing putrescine were injected. 0: Standard sample at K-value of 15%, putrescine at a
specified concentration.
Standard sample at K-value of 40%, putrescine at a specified concentration.

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