Biosensotv 6 Bioel~mnia
7 (1992) 127-131
Lactase-based biosensor for determination of polyphenols: determination of catechols in tea A. L. Ghindilis, V. P. Gavrilova 81A. I. Yaropolov A N. Bakh Institute of Biochemistry of the Academy of Sciences of the USSR Leninsky 33, Moscow 117071, Russia (Received 17 January 1991; revised version received 15 April 1991; accepted 15 May 1991)
Aba&&r A new method of amperometric determination of phenolic compounds using an enzyme electrode is proposed. The latter represents the combination of the oxygen electrode and immobilized lactase. Analytical systems of flow injection and batch types were considered. A method of immobilization was developed that provided an increase in the stability of the enzyme. Optimal conditions for biosensor operation were found. The time needed for analysis in the flow injection mode was below 100 s. A column with immobilized enzyme could be used for up to 500 determinations of phenolic compounds without decrease of the enzyme activity. The practical validity of the method was demonstrated by tannin analysis in tea of different brands. Keywords: arn~mrnet~~ biosensor, lactase, tea catechds.
INTRODUCTION A fast and easy method of analytical determination of polyphenolic compounds is of great importance for various industries and monitoring of en~ronmental pollution. In many cases it is more impo~nt to measure the total content of pol~henolic compounds (the total phenol equivalent) than to determine each of them individually. For this reason the use of phenol-oxidizing enzymes in biosensors looks very promising. Methods for the amperometric determination of aromatic diamines and diphenols using tyrosinase (Macholan & Jilek, 1984) and pig ceruloplasmin (Machola & Schanel, 1977) have been reported in the literature. However, tyrosinase-based enzyme electrodes suffer from ~~~5~?~92~~~~.~ Q 1992 Elsevier Science Publishers Ltd.
low enzyme stability and significant inhibition of the enzyme by reaction products; both these factors deteriorate electrode characteristics in the analysis of phenols. In the case of immobilized ceruloplasmin it was possible to reduce the response time of the electrode to 2-5 min; however, such perfo~an~ could be achieved only when significant amounts of the enzyme were immobilized on the electrode because of the low catalytic activity of ceruloplasmin. In our opinion, lactase is the best candidate for use in analytical systems for the determination of polyphenolic compounds. This enzyme catalyses the oxidation of polyphenols by molecular oxygen according to the following scheme: lactase
AH2 + ;OZ -
A+ H20
127
A. L. Ghindilis et al.
where AH2 and A are reduced and oxidized states of phenol, respectively. In this reaction oxygen is reduced directly to water without intermediate formation of hydrogen peroxide. Lactase can catalyse oxidations of both para- and orthosubstituted phenols, it is not subjected, as a rule, to product inhibition, and possesses a catalytic activity (in terms of the catalytic constant) exceeding that of ceruloplasmin by two or three orders of magnitude. One of the practically important applications of polyphenol analysis is the determination of total reduced catechols (tannin) at different stages of tea processing, including quality control of the final product. Therefore, the aim of the present work was to verify the applicability of the suggested analytical method for tea-processing technology.
MATERIALS
Biostmsors & Bioelectronics injection 1
Fig. 1. Measuring cell of the batch biosensor. 1, container with immobilized lactase; 2, oxygen electrode; 3. thermocouple and heater: 4, outlet: S, mechanical stirrer.
AND METHODS Instrumentation
Lactase from Coriolus versicolor was supplied by the Armenian division of IREA. Enzymes from Coriolus hirsutus and Cerrena maxima were isolated using a modified procedure of Ghindilis et al. (1988). The enzyme was immobilized on threadlike DEAE-cellulose (Biolar, USSR) using Woodward’s reagent (Sigma). Tannin was kindly supplied by Professor G. Pruidze (Institute of Plant Biochemistry, Georgia, USSR). Enzyme immobilization To 1.6 ml of lactase solution (0.2 mg/ml) 0.5 ml of aqueous solution of Woodward’s reagent (20 mg/ml) were added and the pH of the mixture was adjusted to 5.0 with diluted sulphuric acid. After 10 min the mixture was supplemented with 200 mg of the threadlike DEAE-cellulose and 20 ml of 0.5 M sodium phosphate buffer, pH 7.8, and incubated for 1 h at 310 K. After incubation the catalyst was rinsed with distilled water. Immobilized lactase was stored at 277 K in 0.1 M sodium acetate buffer, pH 4.5.
1. The apparatus for batch analysis was designed at the A. N. Bakh Institute of Biochemistry in Moscow. The reaction chamber of the apparatus was equipped with a mechanical stirrer, microthermostating device, and container with immobilized enzyme and oxygen electrode (Fig. 1). Samples were injected directly into the reaction chamber through a channel in the chamber cap. 2. Flow injection analysis was performed using an ‘Arfa’ glucose analyser (product of Biosensor, Erevan) with an oxygen electrode as detector. Polyphenols were analysed using a column reactor containing lactase immobilized on DEAE-cellulose. 3. Lactase activity was determined spectrophotometrically at 298 K by means of a Hitachi557 spectrophotometer or polarographically using a Radelkis OH-105 polarograph (Hungary) equipped with an oxygen electrode. As a substrate 10 mM pyrocatechol in 0.1 M acetate buffer pH 4.5 was used.
Preparation of tea extracts RESULTS AND DISCUSSION Dry tea leaves (400 mg) were boiled on a water bath in 40 ml of distilled water for 30 min. After that the volume of the extract was brought to 50 ml with water and tea leaves were separated by filtration. The filtrate was used for analysis. 128
The pH profile of lactase activity is shown in Fig. 2. It is seen that the optimal pH lies between 4.5 and 5.0, which is quite convenient for the determination of polyphenols because in this pH
Biosensors dt Bioelectronics
1
ACTIVITY
-3
(relatlw
4
Biosensor for determination of polyphenols
units)
6
5
7
PH
Fig. 2. The pH profile of lactase activity. Conditions: 001 M citrate phosphate buffet substrate pyrocatechol.
range the latter are practically spontaneous oxidation. Properties
not subjected
to
of immobilized lactase
The method of lactase immobilization should provide a heterogeneous catalyst with high operational and storage stability. It is to be noted that certain polyphenolic compounds possess very high degrees of polymerization (e.g. lignin). For this reason enzyme immobilization by the entrapment into a polymer gel cannot be used for the preparation of a versatile catalyst. A method of lignin determination based on the use of the enzyme immobilized on the surface of glass bearing amino groups has been reported in the literature (Malovik et al., 1984). However, in this case there is practically no improvement in the stability of the immobilized enzyme as compared to the native lactase. In the present work we did not investigate in detail the mechanism of lactase binding by the threadlike DEAE-cellulose in the presence of Woodward’s reagent. However, there is no doubt that this binding is covalent and cannot be explained by electrostatic interactions, because lactase remains bound to the support even after treatment with solutions with high ionic strength (0.5 M phosphate buffer). An important advantage of the catalyst obtained is that it possesses good hydrodynamic characteristics, which is especially signiticant when the flow injection method of analysis is employed. The storage stability of the native and immobilized lactase from the three sources was
Fig. 3. Dependence of catalytic activity on incubation time for native lactase from Coriolus hirsutus (l), Cerrena maxima (2). Coriolus versicolor (3) andfor laccasefrom Coriolus hirsutus (I’), Cerrena maxima (2% Coriolus versicolor (3’) immobilized on threadlike DEAE-cellulose at room temperature.
investigated. Figure 3 shows the dependence of enzyme activity on the incubation time. It is clearly seen that in all cases the enzyme becomes more stable upon immobilization on the threadlike DEAE-cellulose. The stabilization effect is most pronounced for lactase from Coriolus versicolor and Coriolus hirsutus. Both immobilized preparations retain their activity completely for one month, whereas the activity of the native enzymes decreases after the same period by more than 70%. Based on the above results one can conclude that lactase from Coriolus hirsutus possesses the best stability properties. For this reason, this enzyme was used in further experiments. Flow injection analysis of polyphenols Determination of certain polyphenols (such as lignins or tannins) representing the mixture of numerous individual compounds cannot be performed by measuring initial reaction rates. Therefore, quantitative determination should be based on the overall response of the enzyme 129
A. L. Ghindilis et al. RESPONSE
(arbitrary
Biosensors & Bioelectronics units)
RESPONSE
400
TIME (s) - 200
the same for both diphenols and are linear in the concentration range up to at least 10 mM. The electrode response time does not exceed 100 s, and the column can be used 500 times without decrease in activity. Batch sensor for polyphenol determination
50
loo-
0 4
2
5
SO
FLOW RATE (ml/min)
Fig. 4. Dependences of response amplitude (I) and sensor response time (2) onflow rate in theflow injection sensor.
electrode. This value reflects, in fact, the amount of oxygen required for the complete enzymatic oxidation of polyphenols in the sample. In the case of flow injection analysis all polyphenols present in the sample must be oxidized while the sample is flowing through the column. Dependences of the sensor response time and the response amplitude on the flow rate are shown in Fig. 4. It is seen that response time rapidly decreases with increasing flow rate, whereas the response amplitude remains unchanged up to a flow rate of 6 ml/min. If the flow rate is increased still further, the amplitude decreases, probably because of the incomplete oxidation of hydroquinone during its stay in the column. Thus, for the present experimental setup the flow rate of 6 ml/min represents the critical upper limit. Calibration graphs of sensor response versus concentration of hydroquinone and pyrocatechol are shown in Fig. 5. It is seen that the graphs are RESPONSE
(arbitrary
The batch sensor for the determination of polyphenols is characterized by a high response time (up to 5 min). However, it possesses several advantages: it is very compact and simple in construction, and less sensitive to the stability of the catalyst. The dependence of the sensor response on the concentration of diphenols is shown in Fig. 6. Determination of tea catechols The determination of reduced polyphenolic compounds (tannin) present in tea leaves is an important task in most steps of tea production. The lowest amount of reduced catechols is found in tea leaves after the completion of all technological operations. Therefore, the method of determination of tannin can be used not only for the analysis of the final product but also at different intermediate steps of tea production. As a standard, we used the extract of tannin (reduced tea catechols). The dependence of responses from flow injection and batch sensors on tannin concentration is shown in Fig. 7. The data obtained allow one to determine the phenolic equivalent of tea catechols. The sensor response to 1 mg/ml tannin corresponds to that observed for 3 mM diphenol (e.g. pyrocatechol). Extracts from several tea samples were RESPONSE
units)
(arbitrary
unlts)
100
200,
* SO
0
[DIPHENOLI (mmolll)
Fig. 5. Dependence of jlow injection sensor response on concentration of hydroquinone (0) andpyrocatechol(*). 130
4
1
IDIPLENOLl
5
(~molll)
Fig. 6. Dependence of batch sensor response on concentration of hydroquinone (0) and pyrocatechol (*).
Biosensors & Bioelectronics RESPONSE
(arbitrary
Biosensor for determination of polyphenols
un~tsl
I
0’
halogenide ions on lactase. For example, chloride ions at concentrations higher than 1 mM can significantly decrease the rate of enzymatic reaction. However, if the determination is based on the overall response, the presence of small amounts of chloride ions results only in increase of response time and does not affect the accuracy of the analysis.
I
0
2
CONCLUSION
ICATEC&LSI(mg/ml) Fig. 7. Dependence of the response of batch (*) and flow injection (0) sensors on the concentrations of catechols.
TABLE 1 Tannin
contents Tannin
Brand of tea
in different brands
of tea
content (mg/g of dry substance)
From flow injection analysis
From batch analysis
157 f 6
159 + 5
169 f 8
173 f 5
‘Cha Kvi Georgian black tea
102 f 6
look4
Ridgeways Ceylon orange pekoe tea
189 f 7
199+66
Milford tea/ Ceylon tea
130 f 6
Georgian tea (highest grade) Georgian tea
green
126 + 5
Interfering factors When the above-described sensors are used for analysis of food products (not only tea), it should be kept in mind that lactase can vigorously oxidation
and juices as well as iignins
in inbustrial
wastes.
ACKNOWLEDGEMENTS The authors thank Professor G. Pruidze (Institute of Plant Biochemistry of the Georgian Academy of Sciences, Georgia, USSR) and Dr L. L. Simonyan (Physical Institute, Erevan, Armenia, USSR) for fruitful discussions. The authors are also grateful to A. Kosov and G. Erygin for their invaluable help in the development of the batch biosensor.
REFERENCES
analysed. The sensitivity of both sensors was quite sufficient for the determination of tannin in all cases. Tannin contents of several brands of tea determined in this way are given in Table 1.
catalyse
Biosensors based on immobilized lactase can be used for the analysis of tea at different stages of its production. Moreover, such sensors can probably be used for the analysis of polyphenols in vines
of ascorbic
also take into account
acid. One should
the inhibitory
effect of
Ghindilis, A. L., Zhazhaina, E. 0.. Baranov. Yu. A., Karyakin, A. A., Gavrilova, V. P. & Yaropolov. A. 1. (1988). Isolation and properties of lactase from the basidial fungus Coriolus hirsutus (FR) Quel Biokhimiya, 53, 735-9. Macholan, L. & Jilek M. (1984). An enzyme electrode for determination of aromatic diamines and aminophenols based on cross-linked ceruloplasmin. Collection Czechoslovac Chem. Commun.. 49, 752-6. Macholan, L. & Schanel, L. (1977). Enzyme electrode with immobilized polyphenol oxidase for determination ofphenolic substrates. Collection Czechoslovac Chem. Commun., 42, 3667-75. Malovik, V., Yaropolov, A. I. & Varfolomeev, S. D. (1984). Oxidation of lignins and their components by oxygen in the presence of lactase from Polyporous versicolor: lignin detection by enzyme electrode. Collection Czechoslovac Chem. Commun.. 49, 1390-4.
131
7 (1992) 127-131
Lactase-based biosensor for determination of polyphenols: determination of catechols in tea A. L. Ghindilis, V. P. Gavrilova 81A. I. Yaropolov A N. Bakh Institute of Biochemistry of the Academy of Sciences of the USSR Leninsky 33, Moscow 117071, Russia (Received 17 January 1991; revised version received 15 April 1991; accepted 15 May 1991)
Aba&&r A new method of amperometric determination of phenolic compounds using an enzyme electrode is proposed. The latter represents the combination of the oxygen electrode and immobilized lactase. Analytical systems of flow injection and batch types were considered. A method of immobilization was developed that provided an increase in the stability of the enzyme. Optimal conditions for biosensor operation were found. The time needed for analysis in the flow injection mode was below 100 s. A column with immobilized enzyme could be used for up to 500 determinations of phenolic compounds without decrease of the enzyme activity. The practical validity of the method was demonstrated by tannin analysis in tea of different brands. Keywords: arn~mrnet~~ biosensor, lactase, tea catechds.
INTRODUCTION A fast and easy method of analytical determination of polyphenolic compounds is of great importance for various industries and monitoring of en~ronmental pollution. In many cases it is more impo~nt to measure the total content of pol~henolic compounds (the total phenol equivalent) than to determine each of them individually. For this reason the use of phenol-oxidizing enzymes in biosensors looks very promising. Methods for the amperometric determination of aromatic diamines and diphenols using tyrosinase (Macholan & Jilek, 1984) and pig ceruloplasmin (Machola & Schanel, 1977) have been reported in the literature. However, tyrosinase-based enzyme electrodes suffer from ~~~5~?~92~~~~.~ Q 1992 Elsevier Science Publishers Ltd.
low enzyme stability and significant inhibition of the enzyme by reaction products; both these factors deteriorate electrode characteristics in the analysis of phenols. In the case of immobilized ceruloplasmin it was possible to reduce the response time of the electrode to 2-5 min; however, such perfo~an~ could be achieved only when significant amounts of the enzyme were immobilized on the electrode because of the low catalytic activity of ceruloplasmin. In our opinion, lactase is the best candidate for use in analytical systems for the determination of polyphenolic compounds. This enzyme catalyses the oxidation of polyphenols by molecular oxygen according to the following scheme: lactase
AH2 + ;OZ -
A+ H20
127
A. L. Ghindilis et al.
where AH2 and A are reduced and oxidized states of phenol, respectively. In this reaction oxygen is reduced directly to water without intermediate formation of hydrogen peroxide. Lactase can catalyse oxidations of both para- and orthosubstituted phenols, it is not subjected, as a rule, to product inhibition, and possesses a catalytic activity (in terms of the catalytic constant) exceeding that of ceruloplasmin by two or three orders of magnitude. One of the practically important applications of polyphenol analysis is the determination of total reduced catechols (tannin) at different stages of tea processing, including quality control of the final product. Therefore, the aim of the present work was to verify the applicability of the suggested analytical method for tea-processing technology.
MATERIALS
Biostmsors & Bioelectronics injection 1
Fig. 1. Measuring cell of the batch biosensor. 1, container with immobilized lactase; 2, oxygen electrode; 3. thermocouple and heater: 4, outlet: S, mechanical stirrer.
AND METHODS Instrumentation
Lactase from Coriolus versicolor was supplied by the Armenian division of IREA. Enzymes from Coriolus hirsutus and Cerrena maxima were isolated using a modified procedure of Ghindilis et al. (1988). The enzyme was immobilized on threadlike DEAE-cellulose (Biolar, USSR) using Woodward’s reagent (Sigma). Tannin was kindly supplied by Professor G. Pruidze (Institute of Plant Biochemistry, Georgia, USSR). Enzyme immobilization To 1.6 ml of lactase solution (0.2 mg/ml) 0.5 ml of aqueous solution of Woodward’s reagent (20 mg/ml) were added and the pH of the mixture was adjusted to 5.0 with diluted sulphuric acid. After 10 min the mixture was supplemented with 200 mg of the threadlike DEAE-cellulose and 20 ml of 0.5 M sodium phosphate buffer, pH 7.8, and incubated for 1 h at 310 K. After incubation the catalyst was rinsed with distilled water. Immobilized lactase was stored at 277 K in 0.1 M sodium acetate buffer, pH 4.5.
1. The apparatus for batch analysis was designed at the A. N. Bakh Institute of Biochemistry in Moscow. The reaction chamber of the apparatus was equipped with a mechanical stirrer, microthermostating device, and container with immobilized enzyme and oxygen electrode (Fig. 1). Samples were injected directly into the reaction chamber through a channel in the chamber cap. 2. Flow injection analysis was performed using an ‘Arfa’ glucose analyser (product of Biosensor, Erevan) with an oxygen electrode as detector. Polyphenols were analysed using a column reactor containing lactase immobilized on DEAE-cellulose. 3. Lactase activity was determined spectrophotometrically at 298 K by means of a Hitachi557 spectrophotometer or polarographically using a Radelkis OH-105 polarograph (Hungary) equipped with an oxygen electrode. As a substrate 10 mM pyrocatechol in 0.1 M acetate buffer pH 4.5 was used.
Preparation of tea extracts RESULTS AND DISCUSSION Dry tea leaves (400 mg) were boiled on a water bath in 40 ml of distilled water for 30 min. After that the volume of the extract was brought to 50 ml with water and tea leaves were separated by filtration. The filtrate was used for analysis. 128
The pH profile of lactase activity is shown in Fig. 2. It is seen that the optimal pH lies between 4.5 and 5.0, which is quite convenient for the determination of polyphenols because in this pH
Biosensors dt Bioelectronics
1
ACTIVITY
-3
(relatlw
4
Biosensor for determination of polyphenols
units)
6
5
7
PH
Fig. 2. The pH profile of lactase activity. Conditions: 001 M citrate phosphate buffet substrate pyrocatechol.
range the latter are practically spontaneous oxidation. Properties
not subjected
to
of immobilized lactase
The method of lactase immobilization should provide a heterogeneous catalyst with high operational and storage stability. It is to be noted that certain polyphenolic compounds possess very high degrees of polymerization (e.g. lignin). For this reason enzyme immobilization by the entrapment into a polymer gel cannot be used for the preparation of a versatile catalyst. A method of lignin determination based on the use of the enzyme immobilized on the surface of glass bearing amino groups has been reported in the literature (Malovik et al., 1984). However, in this case there is practically no improvement in the stability of the immobilized enzyme as compared to the native lactase. In the present work we did not investigate in detail the mechanism of lactase binding by the threadlike DEAE-cellulose in the presence of Woodward’s reagent. However, there is no doubt that this binding is covalent and cannot be explained by electrostatic interactions, because lactase remains bound to the support even after treatment with solutions with high ionic strength (0.5 M phosphate buffer). An important advantage of the catalyst obtained is that it possesses good hydrodynamic characteristics, which is especially signiticant when the flow injection method of analysis is employed. The storage stability of the native and immobilized lactase from the three sources was
Fig. 3. Dependence of catalytic activity on incubation time for native lactase from Coriolus hirsutus (l), Cerrena maxima (2). Coriolus versicolor (3) andfor laccasefrom Coriolus hirsutus (I’), Cerrena maxima (2% Coriolus versicolor (3’) immobilized on threadlike DEAE-cellulose at room temperature.
investigated. Figure 3 shows the dependence of enzyme activity on the incubation time. It is clearly seen that in all cases the enzyme becomes more stable upon immobilization on the threadlike DEAE-cellulose. The stabilization effect is most pronounced for lactase from Coriolus versicolor and Coriolus hirsutus. Both immobilized preparations retain their activity completely for one month, whereas the activity of the native enzymes decreases after the same period by more than 70%. Based on the above results one can conclude that lactase from Coriolus hirsutus possesses the best stability properties. For this reason, this enzyme was used in further experiments. Flow injection analysis of polyphenols Determination of certain polyphenols (such as lignins or tannins) representing the mixture of numerous individual compounds cannot be performed by measuring initial reaction rates. Therefore, quantitative determination should be based on the overall response of the enzyme 129
A. L. Ghindilis et al. RESPONSE
(arbitrary
Biosensors & Bioelectronics units)
RESPONSE
400
TIME (s) - 200
the same for both diphenols and are linear in the concentration range up to at least 10 mM. The electrode response time does not exceed 100 s, and the column can be used 500 times without decrease in activity. Batch sensor for polyphenol determination
50
loo-
0 4
2
5
SO
FLOW RATE (ml/min)
Fig. 4. Dependences of response amplitude (I) and sensor response time (2) onflow rate in theflow injection sensor.
electrode. This value reflects, in fact, the amount of oxygen required for the complete enzymatic oxidation of polyphenols in the sample. In the case of flow injection analysis all polyphenols present in the sample must be oxidized while the sample is flowing through the column. Dependences of the sensor response time and the response amplitude on the flow rate are shown in Fig. 4. It is seen that response time rapidly decreases with increasing flow rate, whereas the response amplitude remains unchanged up to a flow rate of 6 ml/min. If the flow rate is increased still further, the amplitude decreases, probably because of the incomplete oxidation of hydroquinone during its stay in the column. Thus, for the present experimental setup the flow rate of 6 ml/min represents the critical upper limit. Calibration graphs of sensor response versus concentration of hydroquinone and pyrocatechol are shown in Fig. 5. It is seen that the graphs are RESPONSE
(arbitrary
The batch sensor for the determination of polyphenols is characterized by a high response time (up to 5 min). However, it possesses several advantages: it is very compact and simple in construction, and less sensitive to the stability of the catalyst. The dependence of the sensor response on the concentration of diphenols is shown in Fig. 6. Determination of tea catechols The determination of reduced polyphenolic compounds (tannin) present in tea leaves is an important task in most steps of tea production. The lowest amount of reduced catechols is found in tea leaves after the completion of all technological operations. Therefore, the method of determination of tannin can be used not only for the analysis of the final product but also at different intermediate steps of tea production. As a standard, we used the extract of tannin (reduced tea catechols). The dependence of responses from flow injection and batch sensors on tannin concentration is shown in Fig. 7. The data obtained allow one to determine the phenolic equivalent of tea catechols. The sensor response to 1 mg/ml tannin corresponds to that observed for 3 mM diphenol (e.g. pyrocatechol). Extracts from several tea samples were RESPONSE
units)
(arbitrary
unlts)
100
200,
* SO
0
[DIPHENOLI (mmolll)
Fig. 5. Dependence of jlow injection sensor response on concentration of hydroquinone (0) andpyrocatechol(*). 130
4
1
IDIPLENOLl
5
(~molll)
Fig. 6. Dependence of batch sensor response on concentration of hydroquinone (0) and pyrocatechol (*).
Biosensors & Bioelectronics RESPONSE
(arbitrary
Biosensor for determination of polyphenols
un~tsl
I
0’
halogenide ions on lactase. For example, chloride ions at concentrations higher than 1 mM can significantly decrease the rate of enzymatic reaction. However, if the determination is based on the overall response, the presence of small amounts of chloride ions results only in increase of response time and does not affect the accuracy of the analysis.
I
0
2
CONCLUSION
ICATEC&LSI(mg/ml) Fig. 7. Dependence of the response of batch (*) and flow injection (0) sensors on the concentrations of catechols.
TABLE 1 Tannin
contents Tannin
Brand of tea
in different brands
of tea
content (mg/g of dry substance)
From flow injection analysis
From batch analysis
157 f 6
159 + 5
169 f 8
173 f 5
‘Cha Kvi Georgian black tea
102 f 6
look4
Ridgeways Ceylon orange pekoe tea
189 f 7
199+66
Milford tea/ Ceylon tea
130 f 6
Georgian tea (highest grade) Georgian tea
green
126 + 5
Interfering factors When the above-described sensors are used for analysis of food products (not only tea), it should be kept in mind that lactase can vigorously oxidation
and juices as well as iignins
in inbustrial
wastes.
ACKNOWLEDGEMENTS The authors thank Professor G. Pruidze (Institute of Plant Biochemistry of the Georgian Academy of Sciences, Georgia, USSR) and Dr L. L. Simonyan (Physical Institute, Erevan, Armenia, USSR) for fruitful discussions. The authors are also grateful to A. Kosov and G. Erygin for their invaluable help in the development of the batch biosensor.
REFERENCES
analysed. The sensitivity of both sensors was quite sufficient for the determination of tannin in all cases. Tannin contents of several brands of tea determined in this way are given in Table 1.
catalyse
Biosensors based on immobilized lactase can be used for the analysis of tea at different stages of its production. Moreover, such sensors can probably be used for the analysis of polyphenols in vines
of ascorbic
also take into account
acid. One should
the inhibitory
effect of
Ghindilis, A. L., Zhazhaina, E. 0.. Baranov. Yu. A., Karyakin, A. A., Gavrilova, V. P. & Yaropolov. A. 1. (1988). Isolation and properties of lactase from the basidial fungus Coriolus hirsutus (FR) Quel Biokhimiya, 53, 735-9. Macholan, L. & Jilek M. (1984). An enzyme electrode for determination of aromatic diamines and aminophenols based on cross-linked ceruloplasmin. Collection Czechoslovac Chem. Commun.. 49, 752-6. Macholan, L. & Schanel, L. (1977). Enzyme electrode with immobilized polyphenol oxidase for determination ofphenolic substrates. Collection Czechoslovac Chem. Commun., 42, 3667-75. Malovik, V., Yaropolov, A. I. & Varfolomeev, S. D. (1984). Oxidation of lignins and their components by oxygen in the presence of lactase from Polyporous versicolor: lignin detection by enzyme electrode. Collection Czechoslovac Chem. Commun.. 49, 1390-4.
131
