Development of a system with double enzyme reactors for the determination of fish freshness

93

Analytica Chimica Acta, 260 (1992) 93-98

Elsevier Science Publishers B.V., Amsterdam

Development of a system with double enzyme reactors for the determination of fish freshness Hirokazu Okuma * , Hitoshi Takahashi, Seiichi Yazawa and Shuichi Sekimukai Research and Development Laboratory, New Japan Radio Co. Ltd., 2-l-l

Fukuoka, fimifukuoka,

Saitama 356 (Japan)

Etsuo Watanabe Department of Food Science and Technology, Tokyo University of Fisheries, 4 Konan, Minato-ku, Tokyo 108 (Japan)

(Received 2nd September 1991; revised manuscript received 23rd December 1991)

Abstract A continuous system for the determination of fish freshness with double enzyme reactors was developed and applied to the determination of the freshness indicator (Ki = [(HxR + Hx)/(IMP + HxR + Hx)] X 100) in many types of fish, where IMP, HxR and Hx are inosine monophosphate, inosine and hypoxanthine, respectively. The system was prepared from two combinations of oxygen electrodes and reactors. One reactor for the determination of the total amount of HxR and Hx was packed with nucleoside phosphorylase (NP) and xanthine oxidase (XOD) immobilized simultaneously on chitosan porous beads. Similarly, another reactor for IMP, HxR and Hx was packed with 5nucleotidase (NT), NP and XOD immobilized simultaneously on chitosan beads. The system was prepared from two combinations of oxygen electrodes and reactors. One assay could be completed within 5 min. The system for the determination of fish freshness was reproducible within 2.1% (n = 30). The immobilized enzymes were sufficiently stable for at least 7 months at 4°C. More than 200 samples could be analysed in about 1 month by using these enzyme reactors. The results for fish meat (13 types) correlated satisfactorily with those obtained by liquid chromatography (r = 0.989, n = 253) and ion-exchange column chromatography (r = 0.973, n = 50). These results suggest that the proposed sensor system provides a simple, rapid and economical method for the determination of fish freshness (Ki). Keywords: Biosensors; Enzyme reactor; Fish freshness

The establishment of a simple, rapid and accurate method for the determination of fish freshness is required in the food industry. Various indicators of spoilage such as volatile basic nitrogen [l], ammonia [2], amines [3], volatile acids [41 and pH [5] have been reported. However, fish freshness is difficult to measure satisfactorily from these indicators and, further, they require pretreatment and complicated procedures. Immediately after the death of a fish, adenosineJ’-triphosphate (ATP) begins to degrade to uric acid through the following pathway: ATP-,ADT+AMP+IMP+I-IxR+ I-Ix+UA

where ADP is adenosined’-diphosphate, AMP is adenosine-5’-monophosphate, IMP is inosine monophosphate, HxR is inosine, Hx is hypoxanthine and UA is uric acid. The relative concentrations of these compounds change drastically after death. To indicate fish freshness, a K value is defined: K=

.HxR+Hx ATP+ADP+AMP+IMP+HxR+Hx x 100

The K value is based on the degradation of these compounds in fish meat [6]. It has been shown that fish meats with K values below 20 (very

0003~2670/92/$05.00 0 1992 - Elsevier Science Publishers B.V. All rights reserved

H. Okuma et al. /AnaL Chim. Acta 260 (1992) 93-98

94

fresh) are suitable for “sashimi” (edible raw fish meats). On the other hand, those with K between 20 and 40 (fresh) have to be cooked and those with K above 40 (not fresh) are not suitable for human consumption. As there is a good correlation between the K value and sensory evaluation, the K value is widely used as an indicator of fish freshness in Japan. ATP, ADP and AMP generally disappear around 24 h after death, and usually fish are obtained from the market at least 24 h after the death. Therefore, another freshness indicator, Ki, is defined as [7]: Ki =

HxR+Hx IMP+HxR+Hx

x100

Enzyme sensors for the determination of Hx, HxR and IMP have been developed for the purpose of Ki determinations 18-101. These enzyme sensor systems, however, have the disadvantages of the limited long-term stability of the sensors and the intractability of immobilized enzyme membranes. In this paper, an attempt to produce a simple and long-lived enzyme sensor for the determination of fish freshness and its use for the determination of freshness in many types of fish are described. The measurement system consists of two immobilized enzyme reactors with two oxygen electrodes positioned close to the reactors. IMP, HxR and Hx determinations are based on the following enzyme reactions: IMP=HxR~I-Ix

XOD,UA

where NT is 5’-nucleotidase, NP is nucleoside phosphorylase and XOD is xanthine oxidase. The amounts of IMP, HxR and I-Ix are determined as the current decrease corresponding to oxygen consumption in the final step. Therefore, the total amount of HxR and Hx was determined by the NP and XOD immobilized simultaneously on chitosan porous beads. Similarly, the total amount of IMP, HxR and Hx was determined by the NT, NP and XOD immobilized simultaneously on chitosan beads.

EXPERIMENTAL

Materials

Nucleoside phosphorylase (E.C. 2.4.2.1 from calf spleen) and xanthine oxidase (E.C. 1.2.3.2 from cow milk) were obtained from Boehringer. 5’-Nucleotidase (E.C. 3.1.3.5 from CrotaZus adamanteous venom) was purchased from Sigma. Hypoxanthine and inosine were obtained from Tokyo Kasei. Inosine-5’-monophosphate was obtained from Sigma and 50% glutaraldehyde from Kanto Chemical. Chitosan porous beads (Chitopearl 3001, diameter 0.1 mm) were obtained from Fuji Spinning. All other chemicals were of analytical-reagent grade.

Saurel (Trachurus japonicus), sea bream (Pagrus major>, bluefin tuna (Thunnus thynnus orientalk>, plaice (Paralichthys olivaceus), skipjack (Euthynnus pelamis), flatfish (Limanda yokohamae), striped pig fish (Parapristipoma trilineaturn>, pacific mackerel @comber japonicus), sardine (Sardinops meianostictus), saury pike (Coloabis saira), marlin (Makaira mitsukurii), rainbow trout (Oncorhynchus mykiss) and yellowtail (Seriola quinqueradiuta) were purchased from a fish market and stored at 4°C or -40°C. Preparation of enzyme reactor

NP and XOD were immobilized simultaneously on chitosan porous beads with glutaraldehyde through Schiff base formation. Chitosan beads (2.5 ml> were immersed in a 2.5% (w/v) glutaraldehyde solution IO.1 M phosphate buffer (pH 7.811 for 2 h at 30°C. After the reaction, the beads were washed with distilled water, followed by washing with 0.1 M phosphate buffer (pH 7.8). To 4.6 ml of the mixed enzyme solution [O.l M phosphate buffer (pH 7.811 containing 60 I.U. of NP and 20 I.U. of XOD, 2.5 ml of glutaraldehyde-treated beads were added. The mixture was stirred for 2 h at 30°C and then for 24 h at 4°C. According to the same method, NT (500 I.U.), NP (60 1.U.) and XOD (20 I.U.) were immobilized simultaneously on chitosan porous beads. The immobilized enzymes were preserved in 0.1 M phosphate buffer solution (pH 7.8) at 4°C. The

95

H. Okumaet al./Anal. Chim.Acta 260 (1992) 93-98 enzyme (NP and XOD)-bonded beads were packed into a polypropylene reactor tube A (50 mm x 3 mm i.d., volume 157 ,ul) and nylon nets (420 mesh) were placed at both ends of the reactor to keep the immobilized enzymes within the tube. Similarly, the enzyme (NT, NP and XPD)-bonded beads were packed into reactor tube B. Apparatus and procedures The continuous system for the determination

of fish freshness consisted of two immobilized enzyme reactors, two galvani-type oxygen electrodes (Japan Storage Battery, KDF-251, an analogue-to-digital (A/D) converter (Analog Devices, AD574; 12 bits), a microcomputer (Hitachi, HD64180; 8 bits) and a peristaltic pump. The measurement system for the total amount of HxR and I-Ix was prepared by combining enzyme reactor A and an oxygen electrode A. The system for total amount of IMP, HxR and Hx was prepared by combining enzyme reactor B and an oxygen electrode B. A schematic diagram of the reactor system is shown in Fig. 1. Phosphate buffer (0.05 M) (pH 7.8) was transferred continuously into the system by a peristaltic pump at a flow-rate of 1.5 ml min-l. When the output current of the electrodes had become steady, a 20-~1 aliquot of the sample was injected directly into the flow line and the current decrease was recorded. The reactor responded only to HxR and I-Ix. The total amount of HxR and Hx was determined directly as the response of this electrode. Similarly, the sum of IMP, HxR and I-Ix was measured from the response of the reac-

tor. The temperature was maintained at 30°C during the enzyme reaction. As conventional methods, a liquid chromatographic (LC) method and an ion-exchange resin colwnn chromatographic method were used to determine the fish freshness (Ki value). Preparation of samples for fish freshness determination Samples for enzyme reactor method and LC method. After heat treatment using a microwave

oven (500 W, 15 s), the exudates of fish muscle (2 g) obtained by press treatment were filtered through a membrane filter (pore size 0.45 km). IMP, HxR and Hx in fish muscle were not decomposed by the microwave heat treatment. Samples for ion-exchange resin column chromatography. IMP, HxR and I-Ix were extracted

from fish muscle (2 g) with 10% perchloric acid (PCA) according to Ehira and Uchiyama’s method [ 111. After the PCA extract had been neutralized with 10 M KOH, 0.65 ml of 0.5 M phosphate buffer solution (pH 7.8) was added and the volume was adjusted to 10 ml with 10% neutralized PCA solution (pH 7.7).

RESULTS AND DISCUSSION

Efficiency of the enzyme reactors Three different types of reactor A were prepared: type I, the reactor was packed with beads on which NP and XOD were simultaneously immobilized; type II, the column was prepared with a mixture of NP- and XOD-bonded beads; type 11

Fig. 1. Schematic diagram of the reactor system. 1, Air; 2, buffer tank; 3, peristaltic pump; 4, injection port; 5, precolumn; 6, enzyme column A (co-immobilized nucleoside phosphorylase and xanthine oxidase); 7, enzyme column B (co-immobilized 5’-nucleotidase, nucleoside phosphorylase and xanthine oxidase): 8, oxygen probe; 9, A/D converter; 10, computer; 11, recorder; 12, valve; 13, waste.

H. Okuma et al. /Anal. Chitn. Acta 260 (1992) 93-98

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III, the reactor consisted of two layers of NP- and XOD-bonded beads. Similarly, three types of reactor B were prepared. The efficiencies of the different types of reactors A and B were determined by using the standard Hx, HxR and IMP solutions. These results showed that the efficiencies of reactors with enzymes immobilized simultaneously on chitosan beads were best, although the sixes of the reactor tubes and the amounts of enzymes were identical for each type. Therefore, in the determination of fish freshness, all of the enzymes were simultaneously immobilized on chitosan beads.

PH profile The optimum pH of the co-immobilized NT, NP and XOD was examined using IMP as a substrate. The optimum pH was found to be in the range 7.4-7.8 and subsequent experiments were performed using pH 7.8 phosphate buffer. Calibration graphs for IMP, HxR and Hx

IMP, HxR and Hx as standard solutions were injected into the flow system at 3O”C, pH 7.8 and a flow-rate of 1.0 ml min-i. One assay could be completed within 5 min. Linear relationships were obtained for IMP below 1.9 mM, for HxR below 1.7 mM and for Hx below 1.8 mM. The detection limits were 2 X lo-’ M for IMP, 1 X lo-’ M for HxR and 1 X 10e5 M for Hx when a 20-~1 was injected. The correlation coefficients for IMP, HxR and Hx were 0.999, 0.997 and 0.997, respectively. Precision and reproducibility

Two samples of exudates of bluefin tuna muscle with different values of Ki were repeatedly analysed under the optimum conditions. As shown in Fig. 2, the present system gave precise and reproducible results. The relative errors were 1.6% (n = 15) and 2.1% (n = 15) for samples with Ki values of 11% and 21%, respectively. Stability of co-immobilized enzyme The long-term and operational stabilities

of NT, NP and XOD immobilized simultaneously on chitosan beads were examined under the opti-

Ki-11%

X=11% CV=l.GX

nrl6

Ki=BlX

x=20%

n-15

CV=2.1%

-

,I-

5 Number of

10 analysis

I5

Fig. 2. Reproducibility of repetitive injections of the exudates of bluefin tuna muscle. A 2-4 aliquot of exudate was injected. The pH, temperature and flow-rate were 7.8, 30°C and 1.0 ml min-‘, respectively.

mum conditions by using IMP as a substrate. The enzymes immobilized on the beads were stored in 0.1 m phosphate buffer (pH 7.8) at 4°C. The results are shown in Figs. 3 and 4. Even after 222 days, the residual activity of the co-immobilized enzyme was 70% and it was still useful for the determination of fish freshness. The reactors were stored in a refrigerator overnight; more than 200 samples could be determined in about 1 month. Application to the determination system of j%h freshness

Samples for the determination of fish freshness (Ki values) were prepared from the species listed under Experimental. The freshness was determined with the proposed reactor system and the results were compared with results obtained by the conventional LC and ion-exchange resin column chromatographic methods. Figure 5 illustrates the correlation between Ki values obtained by the present method and by the LC method. Good agreement was observed. The correlation coefficient was 0.989 for 253 assays by the mean least-squares method, and the regression equation was y = 0.98x + 2.17. As there is a good correlation between K values and sensory evaluation, the K value is widely used as an indicator of fish freshness in Japan. The ion-exchange column chromato-

97

H. Okuma et aL /Anal. Chim. Acta 260 (1992) 93-98 150

6or-----l

I

;;

n=253

.; 100 > .r( +I s

Y=O.98X + 2.17

y=o.9a9

.: 50 +r : d OO

50

100

I

I

150

200

i!5(1

Fig. 3. Long-term stability of NT, NP and XOD immobilized simultaneously on chitosan beads. The enzyme was stored at 4°C. A 20+1 aliquot of IMP (1.0 mM) was injected. Other conditions as in Fig. 2.

01 0

I

I

I

I

t

I

10

20

30

40

50

60

Ki value (X) HPLC method

graphic procedure is the accepted standard method for the determination of K values. Therefore, the Ki values obtained by the present method were compared with the K values obtained by the column method. The results are summarized in Fig. 6. A linear correlation was obtained. The correlation coefficient was 0.973 for 50 assays and the regression equation was y = 0.88x + 1.89. The present system has the advantage that fish freshness can be continuously determined. The enzyme reactors show good operational stability

Fig. 5. Correlation between K, values of fish muscle determined by the enzyme reactor system and by the conventional LC method.

during 1 month for at least 200 assays. This system gives reliable and reproducible results at low cost with simple operation.

I

I

I

I

I

n=50

y=o.913

Y=O.88X t 1.89 o 8 @em

150

8

;; k $,lOO .rl .U S >” : 50 : c? 0

0

I

t

10

20

I 30

0

I

I

I

I

I

10

20

30

40

50

60

40

Days

Fig. 4. Operational stability of NT, NP and XOD immobilized simultaneously on chitosan beads in the column. Other conditions as in Fig. 3.

K va+e (X) Column chromatography

Fig. 6. Comparison of Ki values obtained by the enzyme reactor system and K values obtained by column chromatography.

H. Okuma et aL/AnaL Chim. Acta 260 (1992) 93-98 REFERENCES 1 T. Kawabata and H. Term, Bull. Jpn. Sot. Sci. Fish., 19 (1953) 741. 2 F. Ota and T. Nakamura, Bull. Jpn. Sot. Sci. Fish., 18 (1952) 15. 3 K. Yamada, Bull. Jpn. Sot. Sci. Fish., 34 (1968) 541. 4 T. Suzuki, Bull. Jpn. Sot. Sci. Fish., 19 (1953) 102. 5 M. Yamamoto and M. Sonehara, Bull. Jpn. Sot. Sci. Fish., 19 (1953) 761. 6 T. Saito, K Arai and M. Matsuyoshi, Bull. Jpn. Sot. Sci. Fish., 24 (1959) 749.

7 I. Karube, H. Matsuoka, S. Suzuki, E. Watanabe and K. Toyama, 3. Agric. Food Chem., 32 (1984) 314. 8 E. Watanabe, K. Toyama, I. Karube, H. Matsuoka and S. Suzuki, Appl. Microbial. Biotechnol., 19 (1984) 18. 9 E. Watanabe, K. Toyama, I. Karube, H. Matsuoka and S. Suzuki, J. Food Sci., 49 (1984) 114. 10 H. Okuma, H. Takahashi, S. Sekimukai and E. Watanabe, Nippon Shokuhin Kogyo Gakkaishi, 38 (1991) 1019. 11 S. Ehira and H. Uchiyama, Bull. Jpn. Sot. Sci. Fish., 35 (1969) 1080.