Etsuo Watanabe

Flow system for fish freshness determination based on double multi-enzyme reactor electrodes

A double reactor system for the determination of fish and shellfish freshness using the freshness indicator, K-value (K=[(HxR+Hx)/(ATP+ADP+AMP+IMP+HxR+Hx)]x100), was developed, where ATP, ADP, AMP, IMP, HxR and Hx are adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, inosine and hypoxanthine, respectively. The system consisted of a pair of enzyme reactors with an oxygen electrode positioned close to the respective reactor. The enzyme reactor (I) was packed with nucleoside phosphorylase and xanthine oxidase immobilized simultaneously on chitosan beads (immobilized enzyme A). Similarly, the enzyme reactor (II) was packed with immobilized enzyme A and immobilized enzyme B (co-immobilized alkaline phosphatase and adenosine deaminase). Moreover, this reactor consisted of two layers, the enzyme A and enzyme B (1:1). A good correlation was obtained between K values, which were determination by the proposed system and by the HPLC method. One assay could be completed within 5 min. The signal for the determination of K value of fish and shellfish was reproducible within 2.3%. The long-term stability of the enzyme reactors was evaluated at 30 degrees C for 28 days.

Determination of phosphate ions with an enzyme sensor system

An enzyme sensor system for the determination of phosphate ions was constructed using immobilized enzymes and an oxygen electrode. The principle of this method is based upon the nucleoside phosphorylase catalyzed reaction for which the presence of inorganic phosphorus is indispensable. One assay could be completed within 3 min. This enzyme sensor was able to withstand at least 70 assays. This system was applicable to simple, rapid and continuous determination of phosphate ions in food.

Induction of TNF-α production from human peripheral blood monocytes with β-1,3-glucan oligomer prepared from laminarin with β-1,3-glucanase from Bacillus clausii NM-1

Abstract We prepared a β-1,3-glucan oligomer (DP/>-4) from laminarin (DP: 25–30) derived from Laminaria digitata with β-1,3-glucanase, and examined its effect on human peripheral blood monocytes. Conditioned medium prepared by incubating monocytes (MC-CM) with the β-1,3-glucan oligomer showed strong inhibitory activity against the proliferation of human leukemic U937 cells. Since the β-1,3-glucan oligomer had no direct cytotoxic effect on U937 cells up to 1000 μg/ml, the cytotoxicity of the MC-CM may be due to cytotoxic cytokines produced from monocytes stimulated by the β-1,3-glucan oligomer. On the other hand, the MC-CM prepared with original laminarin had little effect on the growth of U937 cells. The cytotoxicity of the MC-CM prepared with the β-1,3-glucan oligomer was significantly reduced by an anti-TNF-α antibody, but the anti-TNF-β antibody had no effect. Our results suggest that the enzymatically depolymerized β-1,3-glucan oligomer induces TNF-α production from human monocytes.

Development of a d-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of d-amino acid oxidase and pyruvate oxidase

A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA). The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala. The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction. The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala. It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1). A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM. The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala. The D-Ala content in some fish sauces was also determined using the proposed sensor system. The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method. From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx).

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

Abstract A continous system for the determination of fish freshness with double enzyme reactors was developed and applied to the determination of the freshness indicator ( K i =[(HxR + Hx)/(IMP + Hx)/IMP + Hxr + Hx)]× 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 5-nucleotidase (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 ( K i ).

Enzyme sensor for hypoxanthine and inosine determination in edible fish

SummaryAn enzyme sensor for hypoxanthine (Hx) and inosine (HxR), consisting of an enzyme membrane and an oxygen electrode, was constructed, Xanthine oxidase (XO) and nucleoside phosphorylase (NP) were both immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane and glutaraldehyde. The enzyme sensor responded to Hx and HxR in the presence of phosphate, while it responded only to Hx in the absence of phosphate. A linear correlation was observed between current decrease and the concentrations of Hx and HxR in the range 0.5–2.0 mM respectively. Correlation coefficients between the present enzyme sensor and a conventional enzymatic method were 0.98 and 0.94 for Hx and HxR respectively. The standard deviation was +-1.5 μM and 0.75 μM for Hx and HxR respectively in 100 experiments. A simple and rapid determination of Hx and HxR in fish meat was possible within 3 min with the enzyme sensor.

Simultaneous determination of hypoxanthine and inosine with an enzyme sensor

Abstract A sensor for the simultaneous determination of hypoxanthine and inosine was prepared by a combination of the enzyme system (shown below) and an oxygen electrode. Xanthine oxidase and nucleoside phosphorylase, respectively, were covalently immobilized on triacetyl cellulose membranes containing 1,8-diamino-4-aminomethyloctane. Xanthine oxidase membrane, three sheets of the triacetyl cellulose membrane described above, and nucleoside phosphorylase membrane were placed in that order on the tip of the oxygen electrode. The optimum conditions for simultaneous determination of hypoxanthine and inosine were pH 7.8, 303 K, and a flow rate of 1.4ml min −1 . Calibration curves for hypoxanthine and inosine were linear up to 0.4 mM and 4 mM, respectively. The relative errors were 6% and 1.5% for hypoxanthine and inosine, respectively, in 24 assays.

Effect of culture conditions on lactic acid production of Tetragenococcus species

Aims:  To investigate the effects of the salt concentration, incubation temperature and initial pH of the medium on the fermentative ability of the halophilic lactic acid bacteria, Tetragenococcus muriaticus and T. halophilus.

Use of a channel biosensor for the assay of paralytic shellfish toxins.

Gonyautoxin (GTX), saxitoxin (STX) and tetrodotoxin (TTX), also known as paralytic shellfish poisons (PSP), block Na+ channels, including those in the frog bladder membrane. A tissue biosensor has been developed, consisting of a Na+ electrode covered with a frog bladder membrane integrated within a flow cell. The direction of Na+ transfer, investigated in the absence of Na+ channel blockers, established that active transport of Na+ occurs across the frogs bladder membrane from the internal to the external face. Transfer was shown to be TTX sensitive. The tissue sensor response to each of the different PSP was recorded and the results compared with toxicities determined by the standard mouse bio-assay. Using high concentrations of TTX from the puffer fish Takifugu niphobles, a linear correlation was found between the results from the two assay systems. However, the tissue biosensor system was also able to detect very low concentrations of TTX in samples from two species of puffer fish (Takifugu niphobles and Takifugu pardalis) at concentrations below the detection limit of the mouse bio-assay.

Simultaneous determination of hypoxanthine, inosine, inosine-5′-phosphate and adenosine-5′-phosphate with a multielectrode enzyme sensor

Abstract A multielectrode enzyme sensor for the simultaneous determination of adenosine-5′-phosphate (AMP), inosine-5′-phosphate (IMP), inosine (HXR) and hypoxanthine (HX)in fish meat was developed by assembling four enzyme sensors for AMP, IMP, HXR and HX in a flow cell. These compounds were determined from oxygen consumption according to the following reactions: AMP AD IMP NT HXR NP, PO 3− 4 HX XO, O 2 Uric acid where AD is AMP deaminase, NT is 5′-nucleotidase, NP is nucleoside phosphorylase and XO is xanthine oxidase. Enzymes were covalently bound to a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane and glutaraldehyde. Sensors for HX, HXR, IMP and AMP were prepared by attaching membranes of XO, XONP, XO NPNT, and of XONPNT and AD, respectively, to four oxygen electrodes. Samples extracted from sea bass, bream, flounder, abalone and arkshell were analyzed within 5 min, from the simultaneous response curves of the four electrodes. Results obtained by the multisensor system were in good agreement with those determined by each single electrode.