Food Control 32 (2013) 262e269

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Food Control
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Electronic nose investigation of Alicyclobacillus acidoterrestris inoculated apple

and orange juice treated by high hydrostatic pressure
Piroska Hartyáni a, *, István Dalmadi b, Dietrich Knorr c
a
Central Food Research Institute, Unit of Technology, H-1022 Budapest, Herman Ottó út15, Hungary
b
Department of Refrigeration & Livestock Products’ Technology, Faculty of Food Science, Corvinus University of Budapest, 1118 Budapest, Ménesi út 43-45, Hungary
c
Department of Food Biotechnology and Food Process Engineering, Berlin University of Technology, Koenigin-Luise-Str., 22, 14195 Berlin, Germany

a r t i c l e i n f o a b s t r a c t

Article history: There is an increasing consumer demand for fresh, shelf stable and healthy products with the favourable
Received 10 May 2012 sensory properties of the original material food. In the beverage industry the appearance of Alicyclo-
Received in revised form bacillus acidoterrestris spp. presents a problem which has to be solved. In this study the effectiveness of
16 October 2012
combined high hydrostatic pressure and heat treatment was investigated to examine its microbiological
Accepted 23 October 2012
reducing ability regarding the A. acidoterrestris DSMZ 2498 in commercial pasteurized apple- and orange
juice. In addition it was studied whether the electronic nose is able to show the differences during the 4
Keywords:
weeks storage time regarding the decreasing number of microorganisms which is in connection with
Alicyclobacillus acidoterrestris (DSMZ 2498)
High hydrostatic pressure
aroma changes.
Electronic nose It was established that the combined heat- and high pressure treatment caused microbial inactivation
Fruit juices of acidoterrestris at different levels dependent on the combination of applied treatment parameters.
These differences could be followed also by the electronic nose.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction
In the early 1980’s the fruit juice industry encountered
a spoilage problem which was caused by the growth of a bacterium
in shelf stable products, named originally acidoterrestris (Cerny,
Hennlich, & Poralla, 1984; Deinhard, Blanz, Poralla, & Altan, 1987).
Later it was reclassified as Alicyclobacillus, as u-alicyclic fatty acid
was the major membrane fatty acid component of its cells (Lee,
Dougherty, & Kang, 2002). The spoilage which is induced by this
microorganism can occur in apple juice and other fruit juices and
beverages, as well as in tea and other herbal drinks (Splittstoesser,
Lee, & Churey, 1998; Wisse & Parish, 1998), causing a light sediment
(Brown, 1995). Consequently, in the product off-flavour can be
observed which is due to the production of 2-methoxyphenol,
the guiacol (Yamazaki, Teduka, & Shinano, 1996) respectively 2,6-
bromophenol (Borlinghaus & Engel, 1997) and 2,6-dichlorofenol
(Jensen & Whitfield, 2003).
A. acidoterrestris is a spore-forming, non-pathogenic and ther-
moacidophilic bacterium with a central or terminal oval spore. It
* Corresponding author. Tel.: þ36 1 355 8244, þ36 1 355 8121; fax: þ36 1 355
8928.
E-mail address: piroska.hartyani@hotmail.com (P. Hartyáni).
grows between the pH values at 2.5 and 6.0 and at a temperature
range of 25e60
C (Savaş Bahçeci & Acar, 2007; Silva & Gibbs, 2001).
The conventional heat treatment ensures safety and extends the
shelf life of the juices, but it can lead to undesirable changes both in
nutritional and in sensorial properties, such as colour, odour,
texture and flavour. From the consumers point of view any unde-
sirable changes in the juice attributes are unacceptable. The
increased consumer demand for fresh-like products manifested in
the research of new mild preservation techniques. Besides their
effectiveness, by applying these new technologies minimal vitamin
and flavour losses can be reached (Mertens & Knorr, 1992).
High hydrostatic pressure (HHP) technology is one of the mild,
so called “novel” preservation technologies where the solid or
liquid foods are exposed to a given pressure, ranging from 100 MPa
to 800 MPa (Mermelstein, 1998). Earlier studies demonstrated that
by using the HHP technique the flavour, colour with high retention,
taste and nutritional properties can be maintained and a destruc-
tion of microorganisms achieved (Cheftel, 1995; Mertens & Knorr,
1992; Oey, Lille, Van Loey, & Hendrickx, 2008). Additionally, it
preserves nutritional values (Oey, Van der Plancken, Van Loey, &
Hendrickx, 2008) and the delicate sensory properties of fruits and
vegetables.
Many studies were applied concerning the effectiveness of HHP
technique in the inactivation of A. acidoterrestris, respectively in the

0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2012.10.035
 P. Hartyáni et al. / Food Control 32 (2013) 262e269
thermal inactivation of its spores, and its combined treatment
possibilities (Lee, Chung, & Kang, 2006; Lee et al., 2002; Silva, Gibbs,
Vieira, & Silva, 1999).
Lee et al. (2002) demonstrated that the spores of A. acid-
oterrestris were successfully destroyed by a treatment combining
the HHP technique with mild to high temperatures. The microor-
ganism had been suspended in commercial apple juice. The authors
reported that 3.5 log microbial reductions were achieved by
applying 207 MPa pressure at 45
C for 10 min and 4 log reductions
with the parameter of 621 MPa applied pressure at 71
C for 1 min
and over 5.5 log reductions by applying 621 MPa pressure at 71
C
for 10 min.
Besides the importance of novel technological methods, the
monitoring of the olfactory properties of treated products plays
also a main role. The field of application for an electronic nose is
very wide spread, e.g. food and drink, perfumery, household
products, environmental monitoring, tobacco, smoke, health and
pharmaceuticals (Hodgins, 1995), as well as quality control, process
monitoring, shelf life and food industrial investigations (Reid,
O’Donnell, & Downey, 2006; Dalmadi et al., 2007; Dalmadi,
Polyák-Fehér, & Farkas, 2007).
The electronic nose consists of three almost equivalent
elements: the odour sensor array, data pre-processor, and pattern
recognition (PARC) engine. The electronic nose sensors are able to
detect only the chemical groups which are present in the headspace
of a test tube and are released by solid or liquid samples. Current
electronic noses are comparative instruments and produce classi-
fications or fingerprints of the volatile in the headspace over or
around a sample.
According to the studies of Gobbi et al. (2010), Paolesse et al.
(2006), it had been established that the key-point in the skill of
electronic noses is to identify contaminated products which derive
from the alteration of the original samples volatile profile induced
by microbial presence, as far as this alteration can be exploited as
a marker of the contamination. The alteration can be due to either
the appearance of new compounds or the variation of the relative
amount of the original volatile compounds without changes in the
qualitative composition (Concina et al., 2010).
Many studies demonstrated the use and importance of elec-
tronic nose for the measurements of A acidoterrestris (Concina et al.,
2010; Gobbi et al., 2010).
The research of Gobbi et al. (2010) was focused on the early
detection of this microorganism in different fruit juices. Concina
et al. (2010) tested the electronic nose as a diagnostic tool
towards Alicyclobacillus spp. in three commercial soft drinks. The
identification of the contaminated samples was successful with an
almost 100% of correct classification rate.
Taking into account the above mentioned facts and the results of
an earlier study of Hartyáni et al. (2011), the objective of this study
was to investigate the survival rate of A. acidoterrestris DSMZ 2498
in apple and orange juices which were treated by combined heat
(20, 50, 60
C) and high hydrostatic pressure (200, 400, 600 MPa for
10 min) and stored for 0e4 weeks. The different levels of high
pressure treatment at each temperature levels were applied and in
each case the treatment time was set to 10 min.
Beside the microbiological analysis, physical-chemical and
colour parameters were measured. Furthermore, the electronic
nose was applied to detect possible changes in the volatile
compounds. The main question was whether the electronic nose
would be able to distinguish between fruit juice samples which
were treated with different parameters concerning pressure and
temperature levels and were stored for different periods. This
would mean that the effectiveness of HHP treatment and the
microbial inactivation of A. acidoterrestris could be measured by
electronic nose.
263
2. Materials and methods
2.1. Bacterial strain and growing enumeration medium
The strain of A. acidoterrestris DSMZ 2498 used in this research
was obtained from DSMZ (Deutsche Sammlung von Mikroorga-
nismen und Zellkulturen; Braunschweig, Germany). The medium is
composed of three solutions which were mixed after sterilization
process (121
C, 10 min). Solution A: CaCl22H2O, 0.25 g;
MgSO47H2O, 0.5 g; (NH4)2SO4, 0.2 g; yeast extract 2.0 g; glucose,
5.0 g; KH2PO4, 3.0 g; and 500 ml distilled water. The solution A had
to be adjusted to pH 4 (WTW Series Inolab, Terminal 740) with
H2SO4.
The Solution B consisted of 1.0 ml of trace element SL-6 solution
(ZnSO47H2O, 0.1 g; MnCl24H2O, 0.03 g; H3BO3, 0.3 g; CoCl26H2O,
0.2 g; CuCl22H2O, 0.01 g; NiCl26H2O, 0.02 g; Na2MoO42H2O, 0.03 g)
and 1000 ml distilled water. The Solution C consisted of 15 g agar
and 500 ml distilled water. Sol A, B, C had to be sterilized separately
and then mixed in a ration of 500 ml Solution A, 1 ml Solution B,
500 ml Solution C in case of solid medium. The inoculated plates
were incubated for 3 days at 46
C.
2.2. Preparation of fruit juices
Since a large amount and microbiologically stable fruit juice
basis was needed for the measurements, commercially available
100% content fruit juices (apple, orange) were selected. The juice
was inoculated directly with the Alicyclobacillus suspension in the
quantity of 10 ml pro 1000 ml fruit juice. The final microbial
concentration of the inoculated juices was 106 CFU/ml. The aim was
to measure the effectiveness of the applied treatments through the
determination of the number of survival microorganisms.
After the inoculation the samples were set for 1 h in room
temperature for further measurements which was determined as
a required adaptation time according to the earlier measurements.
Inoculated but not treated juices were used as control samples.
Before the HHP measurements the juices were filled in 15 ml
screw cap plastic bottles. During filling it had to be paid high
attention for closing without air. After filling the bottles were
placed in plastic packaging and full vacuum was applied (Komet
PlusVac 23, Germany) to close the package without air.
3. High hydrostatic pressure treatment of fruit juices
The HHP processing was carried out with U4000HP instrument.
After filling the screw capped plastic bottles packed in plastic
packages were subjected to the high pressure vessel (U4000, Uni-
press, Poland, 50 mm internal diameter  366 mm internal height).
The working volume of the mentioned unit was 700 ml and the
maximum recommended pressure limit was 800 MPa. As a pres-
sure transmitting medium a mixture (50:50 v/v %) of distilled water
and 1,2-propanediol was used. All treatments were carried out in
triplicate. After the treatment the samples were taken out from the
chamber and were placed immediately on ice. Finally they were
stored at 4
C degree until further analysis.
Three different temperatures (20, 50, 60
C) were selected as
fixed temperature during the HHP measurements. The samples of
each temperature level were also treated in different pressure levels
(200, 400, 600 MPa for 10 min). The U4000HP equipment was
pre-heated before the measurement. The adiabatic temperature
increase was not higher than 5
C. The initial temperature of the
control and the samples to be treated changed according to the
treatment types. The samples were pre-heated to the selected
treatment temperature (20, 50, 60
C). For the samples to be stored
always new cycles were applied, so for each treatment there was an
264 P. Hartyáni et al. / Food Control 32 (2013) 262e269

6 6

5 5

4 4

Log10 CFU/ml
Log10 CFU/ml

3 3

2 2

1 1

0 0
0 day 14 days 28 days 0 day 14 days 28 days

A. acidoterrestris spores in apple juice A. acidoterrestris vegetative cells in apple juice

6
6

5
5

Log10 CFU/ml
4
Log10 CFU/ml

3 3

2 2

1 1

0 0
0 day 14 days 28 days 0 day 14 days 28 days

A. acidoterrestris spores in orange juice A. acidoterrestris vegetative cells in orange juice

Fig. 1. At 20
C applied treatment temperature the microbial survival rate of Alicyclobacillus acidoterrestris (DSMZ 2498) spores and vegetative cells in case of different pressure
levels and the control one as a function of the three different storage times in case of apple and orange juice. (A e 0 MPa, - e 200 MPa, : e 400 MPa, C e 600 MPa pressure
level).

extra sample of each storage time. According to the plan, the anal- 3.2. Microbiological analysis of fruit juices
yses of the 0 day samples were carried out the same day with the
treatment. The other samples for storage were put in refrigerator at After the high pressure treatment, the samples were collected
4
C for the given time (2, 4 weeks). for electronic nose analysis and also for further microbiological
analysis. Additional samples were treated by high pressure in
order to do afterwards heat treatment which was carried out at
3.1. Physical-chemical analysis and instrumental colour
80
C for 10 min. For this purpose 1.5 ml Eppendorf Safe-Lock
measurement of fruit juices
Tubes were filled and placed in a pre-heated water bath. The
aim of the microbiological analysis was to determine the ratio of
The pH was determined by Inolab pH 740 (WTW Series, USA)
spores and vegetative cells. It was supposed that after the HHP
conductometer and the Brix
with the help of a refractometer (RFM
treatment spores and in some cases vegetative cells can survive,
80, Winopal Forschung, Germany). The colour properties were
and due to the additional heat treatment all the vegetative cells
determined by using a hand-held tristimulus colour analyser
would be inactivated. The suspension of HHP treated and heat
Chromameter (CR 200, Minolta, Japan) to measure the total colour
treated fruit juice was dropped-plated in petri-dishes on the solid
differences of control, respectively the treated fruit juice samples.
medium of A acidoterrestris (DSMZ e Deutsche Sammlung von
Before analysis, the instrument was calibrated on a white standard.
Mikroorganismen und Zellkulturen- nr. 402; Braunschweig,
A special glass was filled with the samples with white background.
Germany).
The colour was recorded through the special glass using the
For the dilution phase Ringer solution was used. The petri dishes
CIELab uniform colour space at room temperature. Colour deter-
were placed afterwards in incubators at 46
C for 48 h before
mined by CIE (Commission Internationale l’Eclairage) classifies
enumeration. The difference in the number of survival microor-
colour in three dimensions; L*, brightness, a*, red to green colour
ganisms of HHP treated respectively HHP and heat treated samples,
and b*, yellow to blue colour. Total colour differences (DE ¼
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi gave the number of spores and vegetative cells.
ðL*  L*0 Þ2 þ ða*  a*0 Þ2 þ ðb*  b*0 Þ2 ) evaluate the overall colour
difference of a processed sample compared to a reference one. Each 3.3. Analysis of volatile compounds of fruit juices by electronic nose
fruit juice sample was measured three times. After this colour
measurement, according to an international scale, the following The HHP treated samples in plastic bottles were opened at the
ranges can be applied in the total colour difference (DE): not day of electronic nose measurement. For this purpose they were
noticeable (0e0.5), slightly noticeable (0.5e1.5), noticeable (1.5e filled to special glasses which were used to e-nose measurements
3.0), well visible (3.0e6.0) and great difference (6.0e12.0). and contained a septum.
 P. Hartyáni et al. / Food Control 32 (2013) 262e269 265

6 6

5 5

4 4

Log10 CFU/ml
Log10 CFU/ml

3 3

2 2

1 1

0 0
0 day 14 days 28 days 0 da y 14 days 28 days

A. acidoterrestris spores in apple juice A. acidoterrestris vegetative cells in apple juice

6
6

5
5

Log10 CFU/ml
4
Log10 CFU/ml

3 3

2 2

1 1

0 0
0 day 14 days 28 days 0 day 14 days 28 days

A. acidoterrestris spores in orange juice A. acidoterrestris vegetative cells in orange juice

Fig. 2. At 50
C applied treatment temperature the microbial survival rate of Alicyclobacillus acidoterrestris (DSMZ 2498) spores and vegetative cells in case of different pressure
levels and the control one as a function of the three different storage times in case of apple and orange juice. (A e 0 MPa, - e 200 MPa, : e 400 MPa, C e 600 MPa pressure
level).

The headspace analysis of samples was performed by an
NST3320 type electronic nose (Applied Sensor, A.G., Sweden) with
a built-in headspace autosampler unit for 12 samples. In the elec-
tronic nose instrument the sample chamber contains 23 different
sensors, software for collecting and processing the data of the
specimen. NST3320 is provided with 10 MOS-FET (metal oxide
semiconductor field effect transistor) sensors, 12 MOS (metal oxide
semiconductor) sensors, and a sensor for relative humidity
measurements. During the measurement, ambient air was used as
a reference gas for the sensors, which was filtered through a silica gel
column and a combined moisture/hydrocarbon filter. The gas flow
rate of the dynamic sampling was set to 50 ml/min. The electronic
nose measurement sequence started with sample equilibration at
60
C for 30 min. Then reference air was pumped over the sensor
surfaces for 10 s (baseline) followed by the infusion head-space for
45 s (sampling time) while the sensor signals were recorded. After
sample analysis, the recovery phase of the sensors was set to 350 s
including the flush time of the gas lines (90 s) with filtered air prior
to the next sample injection to allow the re-establishment of the
instrument baseline. The total cycle time per sample was 430 s.
The results provided by the electronic nose were evaluated by
multiple statistical analyses.
In case of the longer period electronic nose measurements the
sensor drift phenomenon has to be taken into account. Drift is
defined as the temporal shift of sensor response under constant
physical and chemical conditions (Holmberg et al., 1997). The
periodical calibration of the sensors could help in the handling of
this phenomenon. To achieve it, the changes of the condition of the
calibration samples have to be registered during the measurement
series. Assumed that the actual condition of the calibration samples
would not change or only in smaller rate during the measurement
time, the deviations in the sign responses of sensors are due to the
sensor drift. With the help of mathematical transactions the sensor
responses of actual samples can be compensated on the basis of the
changes of calibration samples.
For this purpose Multiplicative Drift Correction (MDC) was
applied which was selected according to the work of Tomic, Eklöv,
Kvaal, and Haugen (2004). The method relies on reference
measurements in regular intervals within a sequence. The selected
MDC method is a univariated one which compensates drift for each
sensor individually. As it was stated in the study of Tomic et al.
(2004), the temporal signal variations of the reference samples
are exploited to generate unique drift compensation models for
each sensor in the array. The drift compensation models are based
on regression models as follows: fnk ¼ (a  n þ b)k where fnk
represents a correction factor. This will be multiplied with the drift
containing response of the sensors (k). The sample means the n. The
parameter a is dependent on where the measurement sequence
sample is positioned. The value of the parameter b will be always 1.
The calibration sample was the given juice (apple or orange) which
was frozen at the beginning of the experiment. They were untreated
and not contaminated with A. acidoterrestris DSMZ 2498. For further
measurement the calibration samples were stored at 20
C.
3.4. Data analysis
The analyses of measured physical-chemical properties were
repeated three times. The three sets of data were analysed by
variance analysis and student’s t-test. The significance was
expressed at p  0.05 level. Principal component analysis (PCA) was
266 P. Hartyáni et al. / Food Control 32 (2013) 262e269

6
6

5
5

Log10 CFU/ml
4
Log10 CFU/ml

3 3

2 2

1 1

0 0
0 day 14 days 28 days 0 day 14 days 28 days

A. acidoterrestris spores in apple juice A. acidoterrestris vegetative cells in apple juice

6
6

5
5

Log10 CFU/ml
4
Log10 CFU/ml

3 3

2 2

1 1

0 0
0 day 14 days 28 days 0 day 14 days 28 days

A. acidoterrestris spores in orange juice A. acidoterrestris vegetative cells in orange juice

Fig. 3. At 60
C
applied treatment temperature the microbial survival rate of Alicyclobacillus acidoterrestris (DSMZ 2498) spores and vegetative cells in case of different pressure
levels and the control one as a function of the three different storage times in case of apple and orange juice. (A e 0 MPa, - e 200 MPa, : e 400 MPa, C e 600 MPa pressure
level).

used to detect patterns and to visualize the information present in
the data of the electronic nose measurements. Without losing
useful information it is able to reduce multidimensional data sets to
lower dimensions for analysis. PCA is an orthogonal linear trans-
formation that transforms the data to a new coordinate system.
Each principal component (PC) is a linear combination of the
original measured variables, which are uncorrelated and ordered
such that the first few retain most of the variations present in all of
the original variables (Kovács et al., 2010).
Linear discrimination analysis (LDA) was used in this work to
obtain classification rules for differentiation fruit juice samples based
on electronic nose measurement data. It provides a classification
model, characterized by a linear dependence of the classification
scores with respect to the descriptors (groups defined previously),
which maximize the ratio between-class variance and minimize the
ratio of within-class variance. LDA assumes an a priori knowledge of
the group membership of each sample in a training set. The classi-
fication power of the model derived can be evaluated using the
original grouped cases or using a ‘leaving one-out’ cross-validation
procedure. In the ‘leaving one-out’ cross-validation procedure, the
sample data minus one observation are used for the estimation of the
discriminant functions, and then the omitted variable is classified
from them; the procedure was repeated for all observations and so
each sample was classified by discriminant functions which were
estimated without its contribution (Kovács et al., 2010).
Besides eigenvalues of LDA were determined to get more
information on the relation of the factors in the electronic nose
analyses. Eigenvalue is the ratio of between-groups sums of squares
to the within-groups sums of squares. A large eigenvalue is
associated with a strong function. Multivariate statistical analyses
were performed by SPSS for Windows Release (ver.11.0.1.) statistical
software.
4. Results and discussion
4.1. Physical-chemical measurement and colour analysis
According to our expectations, no significant differences were
observed in the pH values, which stayed quite stable even with the
increasing of the pressure levels and temperature concerning the 4
weeks storage time. The colour is very important factor from the
point of consumers. For them colour is one of the first signs which
means the preference or acceptability of a fruit juice (Tosun, 2004).
Furthermore it is very important to investigate the colour changes,
because Alicyclobacillus sp. can cause light sediment as it was
demonstrated in many studies (Brown, 1995). By examining the DE
values, which is a more interpretable factor by examining the
colour attributes, only slightly noticeable differences were
observed in comparison to the control samples.
For example, in case of apple juice untreated samples at the “day
0” of storage the following L* values were measured in the order of
20
C, 50
C, 60
C temperature ranges: 49.75  0.07; 50.15  0.05
and 55.65  0.03. The same increasing tendency was observed in
cases of different pressure levels. Applying 200 MPa pressure level
the L* values of the samples at the “day 0” of storage were as
follows: 48.79  0.02; 49.69  0.05 and 55.70  0.05 in case of
increasing treatment temperature levels (20
C, 50
C, 60
C). This
result can be due to the Maillard-reaction.
 P. Hartyáni et al. / Food Control 32 (2013) 262e269 267

4.2. Results of microbiological analysis 5,0

20°C
4,0 50°C
In Figs. 1e3 the microbiological survival rate of spores and 60°C
3,0
vegetative cells of A. acidoterrestris are represented over the storage
times (0 day, 14 days, 28 days) at three different pressure levels 2,0

Function 2
(200 MPa, 400 MPa, 600 MPa) and the control sample (0 MPa) in 1,0
case of apple- and orange juice. In each diagram the three pressure
0,0
levels and control samples are highlighted with different symbols.
As a result, the survival rate of spores and vegetative cells in all -1,0

treated samples were not strongly reduced by high pressure at -2,0

room temperature (Fig. 1), however the survival rate changed with
-3,0
the increase of storage time.
In the case of A. acidoterrestris inoculated apple juice the -4,0
-6,0 -4,0 -2,0 0,0 2,0 4,0 6,0
application of 50
C treatment temperature (Fig. 2) and 200 MPa
Function 1
pressure level resulted 2 log reduction in the number of spores. By
applying 400 MPa and 600 MPa pressure level 3 log reduction was Fig. 5. LDA score plot of electronic nose signals of samples grouped on the basis of
achieved. Concerning the vegetative cell number, the survival rate treatment temperatures.

decreased to detection limit with the application of increasing

pressure levels.
In case of inoculated orange juice the treatment at 60
C
temperature (Fig. 3) using 200, 400 and 600 MPa for 10 min caused
1.1 log; 2.5 log respectively 3 log reduction in the spore number.
At this temperature level the survival rate of vegetative cells
decreased to detection limit in case of all applied pressure levels.
4.3. Electronic nose measurements
According to the earlier studies of volatile compounds (Hartyáni
et al., 2011) it was established that chemosensor arrays, namely the
electronic nose and tongue, seemed to be promising equipment to
investigate the effects on the quality of the juice due to the application
of minimal processing. Additionally these instruments were able to
differentiate each applied treatment types from the control samples.
In Fig. 4 the results of principal component analysis can be seen
which is illustrated space bounded by the first two principal
components. During the analysis which had been made for all data
it was recognized that the signal responses of the electronic nose
offered the possibility to distinguish successfully the fruit juices
from each other (orange, apple). However, this mathematical-
statistic method was not able to differentiate the samples
regarding to varying treatment parameters (temperatures, pressure
levels) or storage periods. For this purpose the supervised LDA
method was selected.
Concerning the treatment temperature (Fig. 5) it was observed
that within all samples each particular treatment temperature level
(20, 50, 60
C) was distinguishable independently from other factors
such as: fruit juice types, applied pressure levels, storage times.
Setting the storage time into the focus (Fig.6) the electronic nose
was able to differentiate clearly the three periods (0, 14, 28 days), no
matter what factors had been used during the treatment (fruit juice
types, applied pressure levels, treatment temperature).
In contrast, different pressure levels seemed to be less relevant
as far as in this study no significant differences were observed in
the separation (Fig. 7).
The trends plotted on Figs. 4e7 are confirmed by analyses of
leave-one-out cross validation and eigenvalue of the first discrimi-
nant function represented in Table 1. According to these two statis-
tical output values the following order of the parameters can be set
on concerning their ability to separate: the most dominant factor is
the type of the juice, followed by the treatment temperature, the
storage time and at last the applied pressure during the treatment.
For example, in the case of fruit juice type as separation
parameter, the eigenvalue of the first discriminant function is 19.62.
This means that the difference between the groups is 19.62 times
bigger than within the groups. In case of pressure this value is only
0.16. As a result, whilst the fruit juice types (Fig. 4) can be distin-
guished from each other nearly without any proof of overlapping,
the samples treated with variable pressure levels cluster undefin-
able (Fig. 7).
The same sequence of parameters concerning their separation
ability is determined through the cross validation (Table 1).
The electronic nose was able to distinguish both fruit juice types

5,0 4,0
0day
apple
14day
4,0 orange
3,0 28day

3,0
2,0
PC2 (16.5%)

Function 2

2,0
1,0
1,0
0,0
0,0
-1,0
-1,0

-2,0
-2,0

-3,0 -3,0
-3,0 -2,0 -1,0 0,0 1,0 2,0 3,0 -6,0 -4,0 -2,0 0,0 2,0 4,0 6,0
PC1 (49.9%) Function 1

Fig. 4. PCA score plot of electronic nose signals of samples coloured on the basis of Fig. 6. LDA score plot of electronic nose signals of samples grouped on the basis of
fruit juice types. storage times.
268 P. Hartyáni et al. / Food Control 32 (2013) 262e269

8,0 control groups. Consequently a complete separation of the groups was

200MPa conducted in all possible ranking levels. The results are illustrated
6,0 400MPa in Fig. 8. The indicated percentage values show the goodness of
600MPa
group ranking during the cross validation. It verifies that no matter
4,0 how deeply the model can be disintegrated, no acceptable sepa-
Function 2

ration level can be reached via the pressure treatment.

2,0
5. Concluding remarks
0,0
Because of the increasing consumer demands, food technology
-2,0
innovations meet continuous changing challenges. A. acidoterrestris
spp. causes such problems for the beverage industry which have to
be solved. Obviously innovative techniques should also ensure that
-4,0
-4,0 -2,0 0,0 2,0 4,0 6,0 8,0 the sensory properties of foods should not deteriorate during
processing and storage.
Function 1 In this study commercially pasteurized 100% apple- and orange
Fig. 7. LDA score plot of electronic nose signals of samples grouped on the basis of juices were inoculated with A. acidoterrestris spp. (DSMZ 2498). After
pressure levels. inoculation, high hydrostatic pressure treatment (200, 400, 600 MPa
for 10 min) was applied with selected fixed temperatures (20, 50,
Table 1 60
C) and afterwards the juices were stored up to four weeks. Beside
Output values of linear discriminant analysis based on different grouping of e-nose the investigation of physical-chemical properties and microbiolog-
data. ical measurements, in order to check the olfactory changes after the
Basis of separation Eigenvalue Percentage of correct classification treatment and storage, an electronic nose analysis was applied.
of cross validation With the increase of the treatment temperature changes in the
Fruit juice type 19.62 100.00 volatile components were observed. The differences were most
Temperature 3.26 84.70 obvious in case of 60
C treatment temperature. Although the elec-
Storage time 3.08 82.20
tronic nose could show differences in this range, it would be inter-
Pressure 0.16 27.30
esting to check and compare the results with human olfactory
experiments. Furthermore it was established that the combined heat-
with 100% efficiency, in case of the treatment temperature and and high pressure treatment caused microbial inactivation of A.
storage time, the correct group ranking appeared in more than acidoterrestris in different ratios depending on the combination of the
80% of the cases. On basis of pressure level little more than one applied parameters. These differences remained stable also during
fourth of the samples of four groups were successfully ranked the storage at 4
C. The number of the microorganism could be only
in the appropriate group. On this account the question arises reduced under the detection limit with the combination of heat and
whether the samples with different pressure levels would be pressure. Consequently the combined heat- and high hydrostatic
distinguishable if the samples of both fruit juices or with different pressure treatment followed by storage of the samples at 4
C proved
treatment temperature would have been analysed in separate to be a successful strategy.

Fig. 8. Percentage of correctly classified samples treated at different pressure levels.

 P. Hartyáni et al. / Food Control 32 (2013) 262e269
It was established that the electronic nose is useful tool for
tracing sensor changes after preservation treatments and storage,
in connection with the aroma changes as an effect, of inactivation of
problematic microorganism in fruit juices.
Acknowledgement
This study had been carried out in the frame of the Integrated
Project NovelQ FP6-CT-2006-015710, Framework 6, Priority 5 ‘Food
Quality and Safety’, supported by the Commission of the European
Communities and National Development Agency of Hungary
(TÁMOP 4.2.1/B-09/1/KMR/-2010-0005) and with support of the
DAAD (Deutscher Akademischer Austausch Dienst) in the form of
research scholarship at the Department of Food Biotechnology and
Food Process Engineering at the Berlin University of Technology,
Berlin, Germany.
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