Monitoring ethanol production during wine fermentation

processes by a pervaporationenzymic derivatisation approach

F. Delgado-Reyes, I. Papaefstathiou, J. M. Fernndez Romero and M. D. Luque de
Castro*
Department of Analytical Chemistry, Faculty of Sciences, University of Crdoba, E-14004
Crdoba, Spain. E-mail: qa1lucam@uco.es
Received 25th June 1998, Accepted 3rd September 1998

A new procedure for on-line monitoring of ethanol production in the wine fermentation bioprocess is presented.
The method combines the use of a pervaporation unit inserted in a continuous flow system with the use of
enzymic derivatisation and fluorimetric detection as a way of improving both selectivity and sensitivity. The
derivatisation reaction was based on a two-step reaction involving alcohol oxidase (AOD) and horseradish
peroxidase (POD) with fluorimetric detection at lex = 310 nm and lem = 415 nm of the dimer formed. The
efficiency of the derivatisation reaction was tested using different flow injection approaches with either solution
phase or immobilised biocatalysts (in normal or stopped-flow modes in both cases). Finally, the approach based on
the stopped-flow mode and AOD/POD immobilisation was selected for application to the continuous monitoring
of ethanol production in an experimental laboratory built bioreactor prepared using bakers yeast as active cells.
The method shows different linear ranges depending on the situation of the catalyst and the mode used, with
excellent precision (RSD 3.05.5%) and a sampling frequency of 5 h21. The use of this biosensing system was
also tested by the standard addition method in the fermentation product with acceptable recoveries in all instances
(90105%). A fully automated approach to on-line bioprocess sample collection, dilution and monitoring of the
fluorimetric product is also proposed.
Pervaporation has long been employed in industry, in competition with other traditional processes (e.g., distillation, extraction, sorption) but it has rarely been used in the analytical
laboratory. Analytical pervaporation is used as an advantageous
alternative to non-chromatographic continuous separation techniques for implementing preliminary steps of the analytical
process in order to accommodate the raw samples in the
measuring intruments.1 This situation is mainly due to the
design of analytical pervaporators25 that have been used for the
development of methods based on continuous flow approaches
for a variety of analytes,28 which have shown the versatility of
this separation technique.
Automatic methods based on either segmented (SFA9) or
non-segmented flow injection (FI1014) have sometimes included enzymic derivatisation in order to endow analytical
methods with the required selectivity. Separation based on gas
diffusion has also been used when volatile analytes have been
involved. 1114
This paper reports the development of a new automated
spectrofluorimetric method for monitoring ethanol during wine
fermentation based on the coupling of a pervaporation unit
inserted in a continuous flow system. A derivatisation reaction
based on the use of two enzymes (alcohol oxidase and
horseradish peroxidase) was developed in order to endow the
method with the appropriate selectivity. Alcohol oxidase
(AOD) catalyses the oxidation of low molecular mass primary
alcohols according to the following reaction:
AOD

CH3CH2OH ? CH3CHO + H2O2

In a second step, the reaction between 4-hydroxyphenylacetic
acid (4-HPAA) and the hydrogen peroxide formed in the
presence of peroxidase (POD) to yield a dimer which exhibits
high fluorescence was developed as follows:
POD

4-HPAA + H2O2 ? (4-HPAA)2 + H2O

The product formed in the condensation reaction was monitored

fluorimetrically at lex = 310 nm, lem = 415 nm.
The efficiency of the derivatisation reaction was tested using
different flow injection approaches with solution-phase and
immobilised biocatalysts in both the normal and stopped-flow
modes.

Experimental
Instruments and apparatus
A Kontron (Zurich, Switzerland) spectrofluorimeter furnished
with an 18 ml flow cell and equipped with a Knauer (Bad
Homburg, Germany) recorder was used. A Gilson (Worthington, OH, USA) Minipuls-2 four-channel peristaltic pump
with a rate selector, two Rheodyne (Cotati, CA, USA) Model
5041 injection valves and Teflon tubing of 0.5 mm id were also
used. A PC system equipped with a DAS-8PGA interface
(Metrabyte, Taunto, MA, USA) was employed for acquisition
and processing of the relative fluorescence intensitytime
data.
The pervaporation cell, designed in this laboratory, was
similar to that described by Mattos et al.,2 which consisted of
two chambers (a donor and an acceptor chamber), fitted with
inlet and outlet orifices and a thin membrane support. The whole
module was made of methacrylate. The upper and lower
chambers and the membrane support were held together by
means of two aluminium rods and close contact was achieved
by screwing four screws between the two aluminium supports.
Reagents
Two buffer solutions were prepared for the two-step enzymic
reactions. Buffer 1 (used as both donor and acceptor solution)
Analyst, 1998, 123, 23672372

2367

was an aqueous solution containing 10 mmol l21 dipotassium

hydrogendiphosphate (Merck, Darmstadt, Germany; No. 5109)
adjusted to pH 8.0 and 8.5, respectively, with 0.1 mol l21
sodium hydroxide (Merck, No. 6498). Buffer 2 was an aqueous
solution containing 100 mmol l21 ammonium chloride (Merck,
No. 1145) adjusted to pH 9.5 with 1 mmol l21 ammonia solution
(Merck, No. 5432). A solution containing 7.0 mmol l21
4-hydroxyphenylacetic acid (Sigma, St. Louis, MO, USA; No.
H-4377) was prepared in buffer 2. Alcohol oxidase (AOD,
alcohol : oxygen oxidoreductase; EC 1.1.3.13) from Pichia
pastoris (250 U, A-2404) and peroxidase (POD, donor : hydrogen peroxide oxidoreductase; EC 1.11.1.7) type VI from
horseradish (5000 U, Sigma P-8375) were used. Two separate
stock standard solutions containing one enzyme each were
prepared by dissolving the contents of each vial in 2 ml of 100
mmol l21 potassium dihydrogenphosphate (Merck, No. 4871)
buffer adjusted to pH 7.0 with 0.1 mol l21 sodium hydroxide
(Merck, No. 6498). The donor stream was an aqueous solution
into which the sample solution was inserted. All solutions were
prepared using doubly distilled water of high purity obtained
from a Millipore (Bedford, MA, USA) Milli-Q plus system.
Absolute ethanol (Merck, No. 8067) was used as a standard
after suitable dilution. PTFE membranes (47 mm diameter and
1.5 mm thickness) were purchased from Trace (Braunschweig,
Germany).

Enzyme immobilisation
Alcohol oxidase and peroxidase were immobilised separately
on controlled-pore glass, CPG-75, 200 (from Sigma) using the
glutaraldehydebovine serum albumin (GABSA) method
proposed for immobilisation of proteins such as enzymes and
antibodies.15 Teflon tubing of different lengths and 2.0 mm id
was then packed with the support-peroxidase conjugate and
stored in potassium dihydrogenphosphate buffer (100
mmol l21, pH 7) at 4 C. Under these conditions the peroxidase
activity remained constant for at least 6 months. However, the
activity of alcohol oxidase decreased by 50% in 15 d as a
consequence of the temperature of the pervaporator. The
location of the alcohol oxidase in the reactor after the outlet of
the upper chamber unit increased the lifetime of the AOD
reactor by at least 1 month (more than 85% of its activity
remained after this period).

these conditions, fermentation was complete in 10 d. Aliquots

were taken at pre-set times during the fermentation process.
Manifolds and procedure
Different continuous approaches were developed with the aim
of testing the efficiency of both the pervaporation and the
derivatisation system. Fig. 1 depicts the hydrodynamic approaches used, which consist of a flow injection manifold
divided into two subsystems where the pervaporation unit was
inserted in the sample loop of a low-pressure injection valve.
Parts A and B depict the arrangement for two ways of using
alcohol oxidase (in solution and immobilised, respectively).
Fig. 1(A) depicts the manifold in which the oxidase is
injected simultaneously with the sample into the upper
subsystem via the injection valve IV1. The lower subsystem acts
as a donor manifold, which consists of a channel provided with
an injection valve (IV2) that injects the samples into an aqueous
stream which circulates through the lower chamber of the
pervaporation cell, which is thermostated at 40 C. The ethanol
evaporates to the air gap between the donor solution and the
membrane and diffuses through the hydrophobic membrane to
the upper chamber of the pervaporation cell where it is
collected. The upper manifold includes the acceptor chamber of
the pervaporation cell, which is located in the loop of an
auxiliary valve (IV3). This valve is filled with the acceptor
solution, which contains the enzyme oxidase. The pervaporated
analyte is collected in the acceptorAOD solution, with
simultaneous development of the oxidation reaction.
Two modes, continuous and stopped-flow (designed cf and sf
modes, respectively), can be selected depending on the position

Yeast strains
Saccharomyces cerevisiae var. cerevisiae E1, S. cerevisiae var.
bayanus F12 and S. cerevisiae var. capensis G1 (which are
typical flor-veil-forming strains) were used. These strains were
isolated from fermented must and from the flor veils of dry,
sherry-type wine from the MontillaMoriles region of southern
Spain as described by Mauricio et al.16 and preserved on YEPD
(0.3% m/v yeast extract, 0.5% peptone, 1.0% glucose and 2.5%
agar) at pH 6.5.

Culture medium and fermentation conditions

Musts from Vitis vinifera grapes of the Pedro Ximnez variety
supplied by the Experimental Food Technology School (Cabra,
Crdoba, Spain) with a fermentable sugar content of 245 g l21
were used as culture media. The pH of the must was adjusted to
3.3 with tartaric acid (Merck, No. 0804), then filtered and
sterilised.
Fermentation was carried out in a 5 l bioreactor at 25 C,
which was inoculated with a 48 h culture grown in must. Under
2368

Analyst, 1998, 123, 23672372

Fig. 1 Manifold (A) with the AOD in solution and (B) with the AOD
immobilised in the upper part of the pervaporation cell (B.1) or in the
subsequent reactor (B.2). P = persistaltic pump; IV1, IV2 and IV3 =
injection valves; PC = pervaporation cell; T = thermostat; L1 = open
reactor; POD = peroxidase immobilised reactor; AOD = alcohol oxidase
immobilised reactor; a = merging point; D = detector; w1, w2 and w3,
wastes; R = reagent solution; AODS = alcohol oxidase solution; AS =
acceptor solution; DS = donor solution; and S = sample.

of valve IV3. When valve IV3 is in the injection position (cf

mode), the reactant plug (which includes the hydrogen peroxide
formed) is carried out from the upper chamber, and merges at
point a with the 4-hydroxyphenylacetic acid stream and passes
through the POD reactor, where the auxiliary enzymic reaction
takes place. Finally, the reactant plug is driven to the flow cell
where the fluorescent dimer is monitored. When valve IV3 is
switched to the filling position, the loop contents remain static
during a pre-set interval, Dt1, (sf mode), during which the
analyte passes through the membrane and is collected into the
AODacceptor solution, with simultaneous development of the
oxidation reaction. After a pre-set pervaporation time, Dt2, IV3
is switched again to the inject position and the reaction plug is
driven away from the upper chamber, merged at point a with the
4-hydroxyphenylacetic solution, passed through the POD
reactor and the fluorescent product is finally monitored in a
similar way as in the continuous procedure.
Fig 1(B) depicts the manifold developed using both enzymes
(alcohol oxidase and peroxidase) immobilised in the upper subsystem. This approach also includes two possibilities, depending on the location of the immobilised AOD (see B.1 and B.2,
with immobilisation of the oxidase either in the upper chamber
of the pervaporation unit or in the subsequent reactor,
respectively). The procedure in both cases is similar to that in
the stopped-flow mode with AOD in solution.

Results and discussion

Optimisation of variables
The optimisation of the variables involved in the overall
process, grouped into physical, chemical and hydrodynamic,
was performed using the univariate method. Table 1 shows the
ranges over which each variable was studied and the optimum
values found. The specific variables of the continuous and
stopped-flow modes were studied separately.
Physical variables. Temperature causes a dual effect on the
system as this variable influences both the pervaporation
efficiency and the two-step enzymatic reaction. The positive
effect of increasing the pervaporation efficiency was studied

Table 1

between 30 and 90 C. A temperature of 70 C was appropriate

for transfer of the analyte through the membrane. However,
temperatures over 45 C caused dramatic destabilisation of the
biocatalyst owing to denaturation of the proteins. Obviously,
this effect was more acute when the enzyme was immobilised
because of the continuous action of the variable. Hence, a
temperature of 35 C was selected as a compromise.
Chemical variables. The influence of the pH of both the
donor and acceptor solutions was studied. An aqueous solution
adjusted to different pH values was used as the donor stream. A
higher pervaporation efficiency was achieved using phosphate
buffer of pH 8.5 as the donor stream. As commented upon in a
previous paper,17 the pervaporation efficiency is higher when
the acceptor is a basic solution; however, owing to the
derivatising biochemical system, the pH of the acceptor solution
must be compatible with the alcohol oxidase reaction. An
aqueous solution containing 10 mmol l21 dipotassium hydrogendiphosphate adjusted to pH 8.0 with 0.1 mol l21 sodium
hydroxide (buffer 1) provided the best conditions for both
pervaporation efficiency and development of the biocatalysed
reaction.
A solution containing 6 U ml21 of alcohol oxidase was
prepared in buffer 1 in order to provide sufficient concentration
of the biocatalyst for development of the oxidation reaction in
both the continuous and stopped-flow modes.
The second enzymic reaction catalysed by peroxidase
occurred over a wider range of pH. However, the final
condensation product is fluorescent at pH values higher than
that provided by buffer 1. An aqueous solution containing 100
mmol l21 ammonium chloride adjusted to pH 9.5 with 1
mmol l21 ammonia solution (buffer 2) provided an appropriate
medium for maximum fluorescence of the derivatisation
product. A 4.0 mmol l21 concentration of 4-hydroxyphenylacetic acid was selected as optimum. A higher concentration of
the substrate promoted the uncatalysed reaction. On the other
hand, a lower concentration resulted in poor development of the
catalysed reaction.
Hydrodynamic variables. The flow rate had a dramatic
influence on the performance of the system. As the flow rate of
the donor solution determined the time during which the analyte

Optimisation of variables influencing the method

With AOD/POD immobilised
With AOD solutiona

Type

Variable

Range studied

cf mode

sf mode

Physical

Temperature/C

3090

35
0.3
0.6
500
2000

0.5

35
0.3
0.6
500
2000
70
420

0.5

Donor flow rate/ml min21

0.21.6
0.21.6
Acceptor flow rate/ml min21
AODa volume injected/ml
501000
Sample volume injected/m1
505000
Switching time/s
3080
Pervaporation time/s
30600
IMERb length (AOD)/cm
2
IMER length (POD)/cm
0.52
IMEPCc area, cm2
0.255
Chemical
Buffer 1: [Na2HPO4/NaOH]/mmol l21
5500
10
10
pH1 (acceptor/AOD solution)
59
8
8
0.510
6
6
[AOD]/U ml21
Buffer 2: [NH3/NH4Cl]/mmol l21
50500
100
100
612
9.5
9.5
pH2
[4-HPAA]d/mmol l21
0.510
4
4
a cf and sf denote continuous and stopped-flow, respectively. b Immobilised enzyme reactor (for AOD and POD).
upper chamber of the pervaporation cell. d 4-Hydroxyphenylacetic acid.
Hydrodynamic and pervaporation

In pervaporation
cell

In external
reactor

35
0.3
0.6

35
0.3
0.6

2000
70
420
1.0
0.5
1.0

2000
70
420
1.0
0.5

10
8

10
8

100
100
9.5
9.5
4
4
Immobilised area of AOD in the

Analyst, 1998, 123, 23672372

2369

is in the pervaporation module, a flow rate of 0.3 ml min21 for

the donor stream was selected as optimum (lower flow rates
yielded non-reproducible results).
The flow rate of the upper chamber also influenced decisively
the pervaporation efficiency in the continuous mode owing to
the continuous removal of the analyte, which always finds fresh
solution on the acceptor side of the membrane. A flow rate of
0.6 ml min21 provided the best results owing to a higher
collection in the AODacceptor solution. An injection volume
of 500 ml provided sufficient AOD concentration for development of the oxidation reaction.
As expected, the flow rate had no decisive influence on the
upper FI subsystem in both stopped-flow modes (with and
without immobilisation of AOD), as the collection step
occurred in static conditions. When the flow was re-started the
reaction plug was driven to the detector. A flow rate of 0.6
ml min21 was also appropriate to flush the flow manifold.
In order to obtain the best analytical signal with an acceptable
sampling frequency, the stopped-flow variables were also
studied. The switching time (defined as the interval elapsed
between switching valves IV2 and IV3) and stop-flow time were
studied in the ranges 3080 and 60600 s, respectively. A 70 s
switching time and a pervaporation/stopped-flow time of 420 s
were chosen as a compromise between the best signal and an
acceptable sampling frequency. Sample injection volumes over
2 ml did not increase the analytical signal significantly.

1024 and 0.1% v/v were injected in triplicate into each manifold
(according to Fig. 1) with the AOD in solution in both cf and sf
modes [Fig. 1(A)], and with AOD immobilised either in the
pervaporation unit or in the subsequent reactor [Fig. 1(B)].
Table 2 summarises the figures of merit, which include
equation, regression coefficient, linear range, detection limit
and RSD. As can be seen, the method shows different linear
ranges depending on the situation of the catalyst and the mode
used. In all instances the regression coefficients were excellent
( > 0.9912). The use of AOD in solution provided wider linear
ranges than the immobilised enzyme. The sensitivity of the
method (expressed as the slope of the calibration graph) was
higher when the oxidase was immobilised either in the

Features of the method

Calibration graphs were constructed using the optimum values
of the variables established in the previous section. Standard
solutions containing ethanol at concentrations between 1 3

Table 2

Features of the method

AOD in solution

cf mode

sf mode

Equationa
r2
Linear rangeb
Detection limitc
RSD (%)d

y = 6.48 + 288.5x
0.9930
0.00250.007
0.006
5.5

y = 2.31 + 1822.3x
0.9997
0.00080.002
0.002
3.5

AOD immobilised

In pervaporation cell

In reactor

Equation
r2
Linear rangeb
Detection limitc
RSD (%)d

y = 8.34 + 10 837x
y = 1.09 + 3287.1x
0.947
0.9912
0.0010.002
0.001 0.002
0.0004
0.0003
3.9
3.0
a y denotes fluorescence intensity and x ethanol concentration (% v/v).
b Expressed in % v/v. c Calculated as 3s blank signal deviation. d For
0.002% v/v of ethanol.

Table 3

Fig. 2 Application of the method. (A) Refractometric method (-, solid in

suspension; and 5, saccharose remaining) and (B) proposed method.

Application of the method

Recovery (%)d

Sample No.

Fermentation
time/h

Dilution
factor

Sugar
concentrationa

Calculated ethanol
concentrationb

Ethanol
foundc

Error (%)

Addition 1

Addition 2

1
2
3
4
5
6

0
30
45
53
75
115

10
150
500
1500
2000
2500

239
192.5
132.4
86.1
72.8
52.9

2.19
3.51
4.21
5.08
8.51

1.61
3.68
4.08
4.76
8.52

27.7
+4.8
23.1
26.3
20.1

97.1
98.9
104.4
90.6
103.6
106.0

105.2
103.7
108.9
102.6
98
93

In g l21 (refractive index method). b In % v/v, by conversion from the sugar concentration values. c In % v/v. d From 0.002 and 0.01% v/v for addition
1 and 2, respectively.

2370

Analyst, 1998, 123, 23672372

pervaporation unit [10 837 arbitrary units (% v/v)21] or in the

reactor [3287.1 arbitrary units (% v/v)21] in comparison with
the use of the biocatalyst in solution, as a consequence of
a higher effective concentration of the biocatalyst in the
system.
The precision, expressed as RSD and calculated using 11
ethanol solutions injected in triplicate, was acceptable for the sf
mode and when the enzyme was immobilised. However, a
lower precision was obtained in the continuous mode with the
enzyme in solution.
The study of potential interferents was aimed at those
commonly present in wine and fermentation musts which are
susceptible to evaporating through the air gap between the
donor and the membrane and diffusing through the hydrophobic
membrane during the pervaporation process. Sulfur dioxide,
carbon dioxide, acetaldehyde and ammonia were studied. The
selectivity of alcohol oxidase was also tested using different
primary aliphatic alcohols. All potential interferents were added
to the sample at concentrations higher than those usually found
in wines and fermentation musts.
The results showed a 500 : 1 tolerance ratio of interferent to
analyte for ammonia and carbon dioxide, 25 : 1 for acetaldehyde
and 5 : 1 for sulfur dioxide. These ratios are much superior to
those which exist in fermentation musts. The catalytic action of
alcohol oxidase on aliphatic alcohol substrates increases as the
chain length decreases. Thus, taking the analytical signal
provided by a given concentration of ethanol as 1, the same
concentration of methanol and propanol yielded signals of 3 and
0.8, respectively.
The estimated sampling frequency under the optimum
working conditions was 5 h21.

Application of the proposed method

In order to apply the proposed method, the manifold based on
the stopped-flow mode with the alcohol oxidase immobilised in
the upper chamber of the pervaporation cell was selected as the
best. The method was applied to the determination of ethanol in
fermentation musts in two ways: (a) determination of the
ethanol concentration in different musts from the fermentation
tank and (b) study of the recovery afforded by the proposed
method after addition of ethanol (0.002 and 0.01% v/v).

The samples were collected by direct aspiration from the

fermentation tank at pre-set times according to the evolution of
the fermentation. The efficiency of the fermentation process
was tested both by measuring the total content of in-suspension
solids and by monitoring the sugar depletion in the fermentation
media. Both parameters were checked by measurement of the
refractive index. Fig. 2(A) depicts the monitoring of both solid
content and sugar depletion by refractometry during the
fermentation process.
All the samples were previously diluted to fit the analyte
concentration within the linear range of the calibration curve.
Table 3 summarises the concentration found and the recoveries
obtained. Fig. 2(B) also shows the evolution of the ethanol
formation and its correlation with the data achieved by
refractometry. As can be seen, the results provided by the
proposed method show excellent agreement with those
achieved by the refractometric method and also good recoveries
(between 93 and 106%).
Fully automated methods for on-line fermentation
monitoring
Based on the principles described above and using the manifold
based on the stopped-flow mode with immobilised enzymes, a
fully automated approach for on-line monitoring of the
fermentation process was developed (see Fig. 3). Full automation of the system was achieved both using a dilution unit
connected on-line with the fermentation tank (which provided
enough sample dilution to fit the evolving concentration of
ethanol within the linear calibration range) and synchronising
all the steps in an automatic fashion with the aid of an
appropriate computer program and both passive and active
interfaces.
The sample dilution unit consists of: (1) a merging point
where the sample stream (at constant flow rate) and the dilution
stream (at variable flow rate) meet and (2) a 10 cm 3 50 mm id
reactor packed with 2030 mm glass beads (a macro single bead
string reactor, SBSR18) for total homogenisation of the stream
emerging from the merging point. The effective synchronisation
between the information received from the detector and the
pump speed which propels the dilution stream permits the
control of the ethanol content in the sample which reaches the
separation unit, which remains within the linear range of the

Fig. 3 Fully automated approached for on-line fermentation monitoring. FMT = denote fermentation tank; DU = dilution unit; CS = carrier solution; SV1
= selecting valve; S1, S2 and S3 = standard solution for calibration; ai = active interface; pi = passive interface; and MC = microcomputer (for other
abbreviations, see Fig.1).

Analyst, 1998, 123, 23672372

2371

calibration curve independent of the degree of evolution of the

fermentation process.

Microbiology (University of Crdoba) for preparing both the

yeast strains and culture media.

Conclusions

References

The method proposed for monitoring ethanol production during

must fermentation processes is based on the following principles: (a) a pervaporation unit inserted in a flow injection
manifold and (b) a two-step enzymic reaction involving alcohol
oxidase and peroxidase followed by fluorimetric monitoring.
The versatility of the pervaporationFU assembly was demonstrated by presenting alternative ways of implementing the
method.
In comparison with previous work by Prinzing et al.,19,20 the
method proposed here exhibits the following advantages: higher
versatility due to the use of the enzyme in the system either
immobilised or in solutionthis is the first time in which the
enzyme has been immobilised in the upper chamber of the
pervaporation module, thus integrating pervaporation and
enzymic reaction with an immobilised biocatalyst; increased
sensitivity, expressed as detection limit (5.8 3 1022% v/v
versus 1.1024% v/v in our method); higher selectivity, probably
due to the higher dilution factor; longer use of the immobilised
biocatalysts (at least twice as stable); detailed precision study
with acceptable RSDs (not documented by Prinzing et al.);
application to natural samples (must fermentation) and recovery
study with excellent results; comparison with the refractometric
method; easier implementation of fully automatic on-line
monitoring of fermentation process.

1
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Acknowledgements

18

The Comisin Interministerial de Ciencia y Tecnologa

(CICyT) is thanked for financial support (Project No. PB97/
0505). We gratefully acknowledge P. Prez Juan of the Escuela
de Capacitacin Agraria de Cabra (Spain) for supplying the
must and J. C. Mauricio and J. M. Ortega of the Department of

19

2372

Analyst, 1998, 123, 23672372

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