Monoamine oxidase electrode in freshness testing of meat

Monoamine oxidase electrode in freshness testing of meat Isao Karube, Ikuo Satoh, Yuichi Araki, Shuichi Suzuki Research Laboratory o f Resources Utilization, Tokyo Institute o f Technology, Nagatsuta-cho, Midori-ku, Yokohama, 227, Japan

and Hideaki Y a m a d a Department o f Agricultural Chemistry, K y o t o University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto, 606, Japam

(Received 29 January 1979; revised 9 October 1979) Monoamine oxidase (monoamine : oxygen oxidoreductase, EC 1.4.3.4 from Aspergillus niger and b e e f plasma) was immobilized in a collagen membrane. An e n z y m e electrode consisting o f a monoamine oxidase collagen membrane (10 units) and an oxygen electrode was prepared for the determination o f monoamines, Monoamines were oxidized to aldehydes by the immobilized e n z y m e and oxygen consumption was monitored amperometrically by the oxygen electrode. The response time o f the electrode was 4 rain. The optimum conditions for the e n z y m e electrode were pH 7.4 and 30°C. A linear relationship wasobserved between the amine (tyramine) concentration in the range 5 0 - 2 0 0 pM and the difference in current. No decrease in the output current was observed over an observation period o f one week. The difference in current was reproducible with an average relative error o f 8%. Monoamines in meat extracts were determined by the e n z y m e electrode.

Introduction The determination of meat freshness is important in food industries. Various compounds, such as amines, carboxylates, aldehydes, ammonia and carbon dioxide are produced in the meat putrefaction process.~ Several methods are used to measure meat freshness, 2'3 the most conventional being the measurement of volatile basic nitrogen in meats. 4 However, this method requires complicated operations, such as extraction, centrifugation, steam distillation and titration. Therefore, simple, reproducible and continuous methods are still required for use in the food industry. Recently, many methods have been developed for the electrochemical monitoring of enzymatic reactions. Since various kinds of amines are produced ,in the meat putrefaction process, they can be used as an indicator of meat freshness. This paper describes an enzyme electrode consisting of a monoamine oxidase-collagen membrane and an oxygen electrode for the determination of monoamines. Furthermore, the enzyme electrode is used for the determination of monoamines in meat pastes.

Experimental

previously, and the other, from beef plasma, was purchased from Miles Biochemicals (21.9 units/g) and used as received. Amines were obtained from Tokyo Kasei Kogyo Co. Other reagents were commercially available analytical reagents or laboratory grade materials. Collagen was obtained from steer hide and was purified by the method described previously. 6 Deionized water was used in all procedures. Preparation of enzyme-collagen

membrane

Collagen fibril suspension was prepared as described previously. 6 Enzyme solution (5 ml) containing 10 mg of monoamine oxidase was dialysed against 51 of deionized water at 4°C for the removal of salts. This procedure was repeated three times. The cofactor was not removed from the enzyme during dialysis. Monoamine oxidase solution (1.3/~g protein, 6.2 units) was added to 30 g of 1.0% collagen fibril suspension. The suspension was cast on a Teflon plate and dried at room temperature. Then, the monoamine oxidase-collagen membrane was cut into small sections (2 × 2 cm) which were treated with 1% glutaraldehyde solution, pH 7.4, for 30 s because the enzyme is denatured with glutaraldehyde. They were then washed with 0.1 M phosphate buffer, pH ;/.4, and again dried at room temperature.

Materials

Enzyme

Two kinds of monoamine oxidase (monoamine : oxygen oxidoreductase EC 1.4.3.4) were employed for the experiments. Monoamine oxidase from Aspergillus niger (5000 units/g) was purified by the method described

The activity of native monoamine oxidase and of the monoamine oxidase-collagen membrane was determined by the method of Tabor et al. 7 using benzylamine as substrate. The amount of enzyme that leaked out from the

0141-0229/80/020117-04 $02.00 © 1980 IPC BusinessPress

assay

Enzyme Microb. Technol., 1980, Vol. 2, April 117

Papers membrane during the glutaraldehyde and washing treatments was also determined. 8 Activity yield was calculated

from the amount of entrapped enzyme and the activities of the native enzyme,and the membrane.

80

Sample

Histamine solution

-

Preparation o f meat pastes Meat paste was prepared by homogenizing different

meat samples in equal volumes of 0.1 M phosphate buffer, oH 7.4, with, a homogenizer (Nihon Seiki Co SM-I ) for 20 min at 5°C. The meat fat was not removed from the sample but this did not interfere with the electrode operation. The paste was centrifuged prior to insertion of the electrode.

Determination o f volatile bases The pastes described above were centrifuged at 2000g for 10 min at 4°C. To 10 ml of supernatant, 30 ml of 20% trichloroacetic acid (TCA) solution was added and the precipitate removed by filtration. The solution obtained from these treatments was used for the experiments. Sample solution (10 ml) was placed in a Parnas-Wagner steam distillation apparatus, 10 ml of 30% NaOH solution was added and a sample solution distilled. Volatile bases produced during steam distillation were absorbed into an excess of 0.02 N H2SO 4 solution and the amount of distillates determined by titration. Methyl red-bromocresol green indicator was employed for the titration.

Assembly o f the enzyme electrode The schemes of the electrode and the block diagram system for the enzyme electrode are shown in Figure 1. The electrode consists of double membranes where one layer is an enzyme-collagen membrane (thickness 120/~m) and the other layer consists of an oxygenpermeable Teflon membrane (thickness 27 ~m), an alkaline electrolyte, a platinum cathode and a lead anode. The double membranes are contacted directly with the platinum cathode and tightly secured to the electrode with rubber rings. Dissolved oxygen in a sample solution diffuses through the Teflon membrane of the oxygen probe and is reduced on the surface of the cathode. At the same time, the lead anode is oxidized to lead oxide and produces a current at the

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50 0

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Figure 2 Response curves of the electrode for various histamine concentrations: A, 0.5 mM; B, 1.0 mM; C, 3.0 mM. The reactions were carried out in 0.1 M phosphate buffer, pH 7.4 at 30+-1°C. Fungal MAO electrode was employed for the experiments

electrode. The current obtained depends on the oxygen concentration in the sample solution.

Assay procedure The electrode was placed in 30 ml of 0.1 M phosphate buffer solution saturated with dissolved oxygen. When the steady-state current was obtained, 0.5 ml of a sample solution containing various amounts of amine was added to the solution and stirred magnetically while measurements were taken. The current was measured by a milliammeter (Kikusui Electric, Model 114) and the signal was displayed on a, recorder (Riken Denshi, Model SP-J3C). The difference in current was calculated from the initial and final steadystate currents.

Results

Properties of a m i n e oxidase-collagen membrane Amine oxidase was immobilized in a collagen membrane to give an enzyme-collagen membrane of thickness 120+20 ~m. The activity yields of the enzyme collagen membranes were 22.4% for the beef plasma enzyme and 2.4% for the enzyme from A. niger. In the case of fungal amine oxidase, the enzyme may be denatured during the immobilization procedure, as reported previously. 9

Response o f the electrode Figure 2 shows the response curves of the enzyme elecAir--~

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Magnetic stirrer

Figure 1 Scheme of the electrode and the block diagram system for amine determination. A, Anode (Pb); B, electrolyte (KOH solution); C, cathode (Pt); D, Teflon membrane; E,,monoamine oxidase (MAO)-collagen membrane; F, rubber ring

118 Enzyme Microb. Technol., 1980, Vol. 2, April

trode at 30°C and pH 7.4. The current at zero time is that obtained in buffer solution saturated with dissolved oxygen. When a sample solution containing amine was injected into the buffer solution, oxygen consumption by amine oxidase began in the collagen membrane. The consumption of oxygen for amine oxidation caused a decrease in dissolved oxygen around the membrane, resulting in a marked decrease in the electrode current with time until steady-state was reached. The steady-state current resulted from the equilibrium between the consumption of oxygen by the enzymatic reaction and the diffusion of oxygen from the bulk solution to the membrane. In the case of histamine, the response time of the electrode was about 6 min at 30°C. The response of the electrode to various amines is summarized in Table 1. Amine oxidase from A. niger was employed for the electrode. As shown in

Monoamine oxidase electrode in freshness testing o f meat: I. Karube et aL Table 1 Response of the electrode to various amines Amines (0.1 mM)

~/(kLA)

n-Amylamine n-Butylamine n-Hexylamine Histamine Isobutylamine* Propylamine Tyramine

6.9 4.2 10.9 3.1 1.0 2.2 5.0

*0.25mM

The influence of temperature on the output of the electrode was also examined by inserting the electrode into a sample solution at various temperatures. The amount of dissolved oxygen decreased with an increase in temperature. Therefore, the results were corrected on the basis of the amount of dissolved oxygen determined at various temperatures. The optimum temperature of the electrode was 30°C, A further increase or decrease in.temperature increased the current of the electrode. The optimum temperature of the immobilized enzyme agreed with that of native monoamine oxidase (30°C).

Reusability o f the electrode 2O

A

B

The reusability of the electrode was tested on benzylamine (1.7 mM) ~.nd fungal amine oxidase electrodes, Benzylamine was determined at least three times each day. No decrease in the output current was observed over an observation period of one week (S.D. = 1.70 -+ 0.07mM).

Determination of a m i n e s in meat These experiments were carried out on pork meat. The meat pastes were prepared as described above and electrode tests made directly upon them. Figure 5 shows the time course of amine content in meat paste as determined by the enzyme electrodes, compared with that determined by the conventional method. The difference in current obtained with the electrode is converted to histamine concentration

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I I

Concentration (mM) Figure 3 Calibration curves for the electrode. Ihe reactions were carried out under the same conditions as shown in Figure 2, using a fungal M A O electrode. A, Tyramine; B, histamine; C, isobutylamine

Table 1, the difference in current obtained from the electrode depended on the type of monoamine. As reported previously, diffusion rates of low molecular weight substances were almost the same. 1o This may, therefore, be caused by differences in the oxidation rate of various amines by the immobilized enzymes.

<3

Calibration The relationship between the difference in current and the amine concentration is shown in Figure 3. A linear relationship was obtained between the difference in current and concentrations of histamine, tyramine and isobutylamine below 1 mrs. The current was reproducible within 8% of the relative error when a solution containing 0.5 mM of histamine was used.

Influence o f pH and temperature As the activity of the enzyme is markedly dependent on pH, the enzyme electrode current may be affected by the pH of the solution. Figure 4 shows the current differencepH curve for 0.05 mM histamine'solution at various pH values; the optimum pH was found to be ~7.4. The current difference-pH profile of the electrode almost agreed with the activity-pH profile of native amine oxidase (optimum pH 7.4).

0

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I

I 8.0

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Figure 4 Influence of pH on the difference m current using a fungal M A O electrode. The reactions were carried out at 30+-0.1°C in 0.1 M p h o s p h a t e buffer solution (pH 7.0, 7.5, 7.8, 8.1 ). The concentration o f histamine was O . 5 m M

Enzyme Microb. Technol., 1980, Vol. 2, April 119

Papers 5.0

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Incubation time ( h ) Figure 5 Determination of amines in meat. A, Fungal MAO electrode method; B, plasma MAO electrode method. The reactions were carried out at 30-+0.1°C and pH 7.4 (0.1 M phosphate buffer. C, Titration method

from a curve of histamine concentration against the current difference of the electrode in Figure 3. As shown in Figure 5, the concentration of amines in meat paste (solid line) increased with increasing incubation time at 20°C. However, the electrode using fungal amine oxidase was more sensitive than that using beef plasma amine oxidase. No activity of other oxidases or catalase was found in an extract of the paste even though it was saturated with oxygen by bubbling air during experiments. However, a slight increase in volatile basic nitrogen (determined by the conventional method) was observed during the same period.

Discussion

It is well known that various kinds of amines are produced from amino acid residues by decarboxylation. It is possible to estimate meat freshness from the amine content, but the determination of amines in meat is difficult because complicated procedures are needed. The oxidation of monoamine with dissolved oxygen in the presence of monoamine oxidase is shown in equation (1). Monoamine R.CH2NH 2 + 02 + H 2 0

120 Enzyme Microb. Technol., 1980, V o l . 2, A p r i l

References 1

2 3 4 5 6 7 8 9 10

),

oxidase R,CHO + H202 + NH3

The principle of the assay is based on monitoring the decrease in dissolved oxygen resulting from the enzymatic reaction in the presence of monoamines, and the oxygen electrode can be used to determine dissolved oxygen. It is known that several kinds of amines are produced in meat putrefaction, but tyramine and histamine are the main products. They are oxidized to the corresponding aldehydes by monoamine oxidase immobilized in collagen membrane. The potentiometric determination of monoamine has also been reported by Hsiung et al. 11 We found that the concentration of monoamines was directly proportional to the difference in current between the initial steady-state current and the final steady-state current. The amines produced during the putrefaction of meat were determined continuously by the enzyme electrode. However, an increase in volatile bases was not observed during this period, therefore the sensitivity of the conventional method was not high enought to determine the volatile bases produced during this period. The conventional titration method cannot, therefore, be used to estimate meat freshness. However, the difference in current obtained from the electrode using fungal monoamine oxidase was larger than that obtained from the electrode using beef plasma oxidase, which may be caused by the wider substrate specificity of fungal monoanaine oxidase, s In conclusion, the proposed enzyme electrode seems promising and could provide an economical and reliable method for use in the routine analysis of monoamines to estimate meat freshness.

(1)

11

Handbook o f Microbiology, 1II. Microbial Products.

(Laskin, A. I. and Lechevalier, H. A., eds, CRC Press, Cleveland, Ohio, 1973 Ferber, L. Food Technol. 1975, 11,621 Walkiewicz, W. Z. Fleisch. Milchhyg. 1936, 46, 171 Ritskes, T. M. J. Food Technol. 1975, 10, 221 Yamada, H., Adachi, O., Kumagai, H. and Ogata, K. Agric. Biol. Chem. 1965, 29, 864 Karube, I., Suzuki, S., Kinoshita, S. and Mizuguchi, J. Ind. Eng. Chem., Prod. Res. Dev. 1971, 10, 160 Tabor, C. W., Tabor, H. and Rosenthal, S. M. J. Biol. Chem. 1954, 208, 645 Nakamoto, Y., Nishida, M., Karube, I. and Suzuki. S. Biotechnol. Bioeng. 1977, 19, 1115 Karube, I., Hara, K., Satoh, I. and Suzuki, S. Anal. Chim. Acta 1979, 106, 243 Karube, I., Suzuki, S., Kusano, T., and Sato, T. J. SolidPhase Biochem. 1978, 2, 273 Hsiung, A. and Guilbault, G. G. Anal. Chim. Acta 1976, 84, 15