Food Chemistry 220 (2017) 362–370

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Target identification of volatile metabolites to allow the differentiation

of lactic acid bacteria by gas chromatography-ion mobility spectrometry
Janneth Gallegos a,b, Cristina Arce d, Rafael Jordano a, Lourdes Arce c,⇑, Luis M. Medina a
a
Food Science and Technology Department, University of Córdoba, Campus de Rabanales, 14071 Córdoba, Spain
b
Escuela Superior Politécnica de Chimborazo, Facultad de Ciencias. Riobamba, Ecuador
c
Analytical Chemistry Department, University of Córdoba, Campus de Rabanales, 14071 Córdoba, Spain
d
Animal Production Department, University of Córdoba, Campus de Rabanales, 14071 Córdoba, Spain

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

Article history: The purpose of this work was to study the potential of gas chromatography-ion mobility spectrometry
Received 26 May 2016 (GC-IMS) to differentiate lactic acid bacteria (LAB) through target identification and fingerprints of vola-
Received in revised form 3 October 2016 tile metabolites. The LAB selected were used as reference strains for their influence in the flavour of
Accepted 4 October 2016
cheese. The four strains of LAB can be distinguished by the fingerprints generated by the volatile organic
Available online 5 October 2016
compounds (VOCs) emitted. 2-butanone, 2-pentanone, 2-heptanone and 3-methyl-1-butanol were iden-
tified as relevant VOCs for Lactobacillus casei and Lactobacillus paracasei subsp. paracasei. 2-Butanone and
Keywords:
3-methyl-1-butanol were identified in Lactococcus lactis subsp. lactis and Lactococcus cremoris subsp. cre-
Lactic acid bacteria
Metabolites
moris. The IMS signals monitoring during a 24–30 h period showed the growth of the LAB in vitro. The
Volatile organic compounds results demonstrated that GC-IMS is a useful technology for bacteria recognition and also for screening
IMS fingerprints the aromatic potential of new isolates of LAB.
Lactobacillus Ó 2016 Elsevier Ltd. All rights reserved.
Lactococcus

1. Introduction improve flavour and texture in various types of cheeses (Sahingil,

Bacteria produce volatile organic compounds (VOCs) by meta-
bolism, which are of great importance in the aroma profile of many
foods. Microbial VOCs are currently used as markers for several
purposes, especially for rotten food detection (Lemfack, Nickel,
Dunkel, Preissner, & Piechulla, 2014). In fermented or matured
foods, such as cheese, it is highly important to know and under-
stand which flavour components determine the ultimate percep-
tion by the consumer. Aroma development in ripening cheese
results from the metabolic activities of the dominant microbiota,
largely composed of lactic acid bacteria (LAB). These types of bac-
teria are characterised by a wide biodiversity, offering good possi-
bilities to extend the flavour and diversity of dairy products, i.e.
cheeses. Considering their role in cheese production, LAB can be
divided into two groups: starter LAB (SLAB) and nonstarter LAB
(NSLAB) (Steele, Broadbent, & Kok, 2013). Lactococcus lactis subsp.
cremoris and Lactococcus lactis subsp. lactis are included in
starters for their acidifying and proteolytic activities. In general,
Lactococcus spp. are strong acidifiers (Hussain, Rouch, & Britz,
2009), while Lactobacillus spp. are the most widely used genus to
⇑ Corresponding author.
E-mail address: lourdes.arce@uco.es (L. Arce).
Hayaloglu, Simsek, & Ozer, 2014).
During cheese ripening, enzymes released from starter and non-
starter LAB hydrolyse caseins in peptides or amino acids (aa),
resulting in the development of cheese flavour (Settanni &
Moschetti, 2010), mainly from relevant VOCs. In this sense, for
cheese production the use of LAB (selected as started culture) as
well as NSLAB strains (to improve flavour and texture) is very
important. According to Cavanagh, Fitzgerald, and McAuliffe
(2015), Lactococcus spp. are the most important SLAB responsible
for the production of lactic acid from lactose and for converting
casein into flavour components. L. lactis subsp. lactis is able to
metabolise arginine via arginine deiminase pathway influencing
cheese flavour (Brandsma, van de Kraats, Abee, Zwietering, &
Meijer, 2012). Lactococcus spp. also contribute to the development
of improved texture (Mills, O’Sullivan, Hill, Fitzgerald, & Ross,
2010). Furthermore, Lactococcus has also been linked to the inhibi-
tion of rotten cheese and the pathogenic microorganism effect, due
to the production of certain chemical agents (Coelho, Silva, Ribeiro,
Dapkevicius, & Rosa, 2014). L. lactis subsp. cremoris has also been
incorporated as starter because of its acidifying and proteolytic
activities; the aa being involved in the production of metabolites
associated with flavour and aromatic properties (Kamalrul,
Syarul, & Normah, 2012). Moreover, Lactobacillus spp. are involved
in the improvement of flavour and texture in various types of

http://dx.doi.org/10.1016/j.foodchem.2016.10.022
0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.
 J. Gallegos et al. / Food Chemistry 220 (2017) 362–370
cheese. These characteristics are difficult to assess through routine
analyses, due to the complex analytical methodology and instru-
mentation necessary for this purpose.
One of the main standard techniques for microbial VOCs deter-
mination is gas chromatography-mass spectrometry (GC–MS). Sev-
eral studies have used other techniques for the same purpose, such
as: selected ion flow tube mass spectrometry, ion–molecule reac-
tion mass spectrometry, and electronic noses (Bos, Sterk, &
Schultz, 2013). Nowadays, fast and simplified analyses are needed
in the food industry and analytical laboratories. For this reason, it is
appropriate to consider innovative methods for rapid detection
and determination in real time of VOCs generated by LAB, such
as ion mobility spectrometry (IMS). At the beginning, IMS was
mainly used in the military and security field, but since then IMS
has also been applied in broader fields such as medical diagnosis,
environmental monitoring and food safety monitoring (Wang, Li,
Yang, Ruan, & Sun, 2016). Also, IMS or field asymmetric IMS has
been used in the food safety field (Zhao et al., 2015). The possibility
of enumerating Escherichia coli based on the determination of o-
nitrophenol in foodstuff by IMS was reported (Ogden & Strachan,
1993).
A previous study demonstrated the usefulness of headspace
sampling coupled with multi-capillary column-ion mobility spec-
trometry for the extraction and identification of some volatile
metabolites from different types of goat cheese samples
(Gallegos, Garrido-Delgado, Arce, & Medina, 2015). IMS is a tech-
nique that separates gaseous ions in a weak electrical field at atmo-
spheric pressure. The main components of an IMS are an injector,
an ionisation chamber, a shutter grid, a drift region, a detector
and a system that permits gas to flow inside the instrument
(Eiceman & Karpas, 2005). The use of a gas chromatography (GC)
column to pre-separate the sample before IMS improves the reso-
lution of the volatile compounds. In the instrument used in this
study, the sample is firstly ionised via charge transfer from ionised
reactant ions produced by a b-radiation source. In the drift tube
and under an electric field, the ionised molecules are separated
by collision with the drift gas flowing in the opposite direction.
The ionised molecules, depending on their ion mass, structure
and shape, are accelerated towards a Faraday plate, where the
impact of the single ions produces an electrical signal that is
detected, amplified and recorded. Moreover, GC combined with
IMS allows analysis of complex samples, such as bacterial broth
cultures. A further advantage of this system is its high sensitivity,
with detection limits from lg L–1 to ng L–1 (Perl et al., 2011). For
all these reasons, a GC-IMS device was selected.
The main goal of this study was to check the potential of GC-
IMS to differentiate LAB strains (widely used in cheese making)
by using the global information obtained in the topographic plot
Fig. 1. Summarised scheme of LAB strains preparation for analyses.
363
or using some target VOCs. The specific objectives of the present
study were: i) to analyse four LAB strains (L. casei, L. paracasei
subsp. paracasei, L. lactis subsp. lactis and L. lactis subsp. cremoris)
by GC-IMS and to verify whether it is possible to differentiate
between the four strains using the information found in the
GC-IMS profiles; and ii) to study the dynamic evolution of
2-butanone, 2-pentanone, 2-heptanone and 3-methyl-1-butanol
during incubation, since they were identified as relevant VOCs
produced by reference strains of Lactobacillus and Lactococcus.
2. Materials and methods
2.1. Reference bacterial strains and preparation of cultures for GC-IMS
analysis
Four reference strains (L. casei, isolated from commercial fer-
mented milk; L. paracasei subsp. paracasei CECT 4022; L. lactis
subsp. lactis CECT 185; and L. lactis subsp. cremoris CECT 7100)
were used. CECT strains were obtained from the Spanish type cul-
ture collection (Burjassot, Valencia, Spain). Lyophilised strains
were reactivated and subcultured in Man Rogosa Sharpe (MRS)
broth (Oxoid CMO359). The cultures were incubated for 24 h,
either at 33 or 37 °C as required for the strain. Afterwards, bacterial
cultures were grown on MRS agar (Oxoid CMO361). L. casei was
isolated from a commercial product (ActimelÒ) by spreading
0.1 mL of the dilution 1:105 on the surface of MRS agar media.
The cultures were incubated at 37 °C and single colonies from all
cultures were isolated. One colony of LAB collected from MRS agar
plate was suspended in 60 mL of MRS broth and incubated for 24 h.
The optical density at 540 nm (OD540) was recorded as an indirect
measurement of cellular concentration. A scheme of this procedure
is summarised in Fig. 1.
2.2. Chemicals and standards for VOCs identification
The different VOCs tested were chosen according to their
importance in flavour production in cheeses (Marilley & Casey,
2004). These compounds were acetaldehyde, diacetyl, hexanal,
heptanal, octanal, nonanal, 1-pentanol, 3-methyl-1-butanol, 2,3-
butanediol, 2-butanone, 2-hexanone, 2-octanone, propionic acid,
hexyl acetate, ethyl butanoate, ethyl hexanoate, benzaldehyde
and 2-propanol; all of them analytical grade (Sigma-Aldrich, St.
Louis, MO). Stock standard solutions of the analytes were prepared
in Milli-Q water (Millipore, Madrid, Spain) at a concentration of
1 g L–1 and stored away from light at 4 °C. Standard solutions of
0.1 mg L–1 and/or 1 mg L–1 were prepared by dilution of the stock
solutions in the same solvent.
364 J. Gallegos et al. / Food Chemistry 220 (2017) 362–370

2.3. GC-IMS analysis

197.58 ± 0.99
100.98 ± 1.05
For GC-IMS analysis, a FlavourSpecÒ (G.A.S. Gesellschaft für
analytische Sensorsysteme mbH Dortmund, Germany) was used.

tr

–
One millilitre of bacterial cultures (24 h, 33 °C) was poured into a

11.32 ± 0.03
9.42 ± 0.05
20-mL vial closed with a magnetic screw-cap and septum. Vials

Dimer
were subjected to heating at 35 °C for 5 min with speed agitation
Lactococcus lactis subsp. cremoris

td

–
of 500 rpm. The injector temperature was 80 °C. These conditions

197.58 ± 1.04
correspond to the automatic sample unit.
A 500-lL aliquot of the headspace was injected in the heated
injector and then transferred to a non-polar GC column (SE-54-
tr

–
–

– CB from CS-Chromatographie Service GmbH (Düren, Germany)),

Monomer

9.29 ± 0.0

with a stationary phase formed by 94% methyl-5% phenyl-1% vinyl-

silicone, with 0.25 lm film thickness, 30 m length  0.25 mm
td

–
–

–
199.97 ± 1.26

internal diameter. The GC column operated at a constant tempera-

100.7 ± 0.9

ture (40 °C) and N2 purity grade 5.0 was used as sample gas at a
flow rate of 5 mL min–1. N2 was also used as drift gas at a constant
tr

flow of 250 mL min–1. The drift gas enters the device in the oppo-
11.32 ± 0.02
9.51 ± 0.05

site direction to the ions, in order to prevent non-ionised molecules

Dimer

or analytes from entering the ionisation chamber. N2 was supplied

td

by Abelló Linde S.A. (Barcelona, Spain). Once ions are formed in the
Lactococcus lactis subsp. lactis

197.58 ± 2.72

ionisation chamber (tritium source) and are focused to a shutter

grid (BN gate, grid pulse width 100 ls), they start to move along
the drift tube of 5-cm length maintained at 65 °C, under a constant
tr

–
–

electrical field of 400 V cm–1. The separated ions reach the detector
9.29 ± 0.06
Monomer

(Faraday plate). Data were acquired and recorded during 30 min of

analysis. The spectrometer was driven in the positive drift voltage
td

–
–

mode.
148.51 ± 0.65

610.15 ± 1.61
195.89 ± 1.00
98.32 ± 0.36

Table 1 contains the GC-IMS characteristics of VOCs emitted by

LAB reference strains. Reference compounds were used for con-
firming unequivocally the presence of particular volatile com-
tr

11.36 ± 0.08
12.42 ± 0.02
10.39 ± 0.05

pounds. For that, the identification of the volatile metabolites

9.42 ± 0.03
Lactobacillus paracasei subsp. paracasei

present in the samples was confirmed by spiking an established

Dimer

amount of each pure analyte onto the culture medium followed

td

by GC-IMS analysis. In addition, GC  IMS Library version 1.01

146.16 ± 1.58

611.35 ± 1.26
197.88 ± 0.98

(G.A.S. Gesellschaft für analytische Sensorsysteme mbH) was also

used to confirm the compounds identified by GC-IMS, comparing
td: drift time in milliseconds; tr: retention time in seconds; Ko: reduced ion mobility in cm2
V–1
s–1
tr

the coordinate positions (drift time and retention time) of a partic-

8.45 ± 0.04
9.34 ± 0.05
9.50 ± 0.05

ular signal used to identify the associated compound (metabolite).

Monomer

The repeatability values for the determination of VOCs by GC-IMS

td

are summarised in Table 2, showing the good precision of the

198.77 ± 1.26
607.76 ± 1.26
100.72 ± 0.96
147.33 ± 0.05

method.
Blanks (empty sterile flasks) were measured to check if there
were some retained compounds in the GC column and in the IMS
tr

drift tube. If so, the next sample could not be analysed and a clean
11.29 ± 0.03
12.42 ± 0.05
10.39 ± 0.03
9.44 ± 0.03

method was run before sample analysis. Under the experimental

Dimer

conditions used in this project, all blanks tested were free of inter-
td

ferences. The MRS broth (culture medium) was also analysed to

144.96 ± 1.79
196.39 ± 1.76
607.76 ± 2.45

identify the signals corresponding to this solution. Any of the sig-

Identification of VOCs in LAB reference strains using GC-IMS.

nals identified in this sample interfere with the signals of the target
Lactobacillus casei

compounds. The signals identified in the culture medium appeared

tr

at 10.6 ms and 127.7 s and their intensities decreased due to the

9.29 ± 0.04
9.42 ± 0.03
8.40 ± 0.03
Monomer

effect of bacterial growth.

td

–
1.72 ± 0.01
1.56 ± 0
1.31 ± 0
1.43 ± 0
Average values of Ko

Dimer

Table 2
1.93 ± 0.01
1.72 ± 0.01
1.75 ± 0.01

Precision values for the determination of VOCs by GC-IMS.

Monomer

Compound Between-day precision (% RSD, n = 9)

–

Retention time Drift time Peak height

3-Methyl-1-butanol

2 butanone (m, d) (5.9, 6.3) (0.04, 0.04) (0.13, 0.31)

2-Heptanone
2-Pentanone

2 pentanone (m, d) (7.1, 8.6) (0.03, 0.04) (0.1, 0.24)

Compounds

2-Butanone

2 heptanone (m, d) (9.9, 8.8) (0.2, 0.3) (0.1, 0.33)

Table 1

3-methyl-1-butanol (m, d) (5.2, 5.9) (0.03, 0.04) (0.2, 0.4)

m: monomer; d: dimer; RSD: relative standard deviation.

 J. Gallegos et al. / Food Chemistry 220 (2017) 362–370 365

Fig. 2. (A) Topographical plots and (B) three-dimensional topographical plot, corresponding to GC-IMS signals detected in LC: L. casei, LPP: L. paracasei subsp. paracasei, LLL: L.
lactis subsp. lactis and LLC: L. lactis subsp. cremoris. X-axis: drift time in ms; Y-axis: retention time in s, Z-axis: intensity in V. The identified VOCs were: (a2) 2-butanone dimer,
(b1) 2-pentanone monomer, (b2) 2-pentanone dimer, (c1) 3-methyl-1-butanol monomer, (c2) 3-methyl-1-butanol dimer, (d1) 2-heptanone monomer, (d2) 2-heptanone
dimer. RIP: reactant ion peak.

2.4. VOCs determination during LAB growth
GC-IMS measurements were performed to evaluate LAB spec-
tral fingerprints and their volatile metabolites during growth in
MRS broth (for 24–30 h). The cultures were prepared by diluting
an aliquot of a fresh culture in MRS broth until an OD540 around
0.20 was reached (approximately 15  106 CFU mL–1). Then, ali-
quots of 7 mL of this culture were placed in different tubes and
they were incubated in a water bath at 33 °C for the entire period
of study. During the incubation time, the tubes were closed. One
tube was taken for each measurement. GC-IMS measurements
were taken firstly every 30 min (during the first 2 h) and later
366 J. Gallegos et al. / Food Chemistry 220 (2017) 362–370
every 90 min, up to a total of 24–30 h. At the end of each time of
incubation, 1 mL of culture was transferred to one glass vial
(20 mL) to be analysed by GC-IMS. Each measurement was per-
formed twice for each strain. At the same time, OD540, pH, cell via-
bility and absence of contaminants were determined by streaking
10 lL of culture in MRS agar.
Before each LAB reference strain analysis, a mixture of VOC
standards was analysed by using the same GC-IMS method and
the retention and drift times were identified for each compo-
nent. In all cases, the signal of monomers and dimers of each
component was clearly distinguished in the topographic plot,
except for 2-butanone. In this case, only the dimer signal was
used to distinguish this analyte. The identification of each VOC
and their corresponding monomer and dimer were also carried
out using the standard addition method to the LAB reference
strains samples.
2.5. Data analysis
Data analysis of GC-IMS was carried out using LAV software
version 2.000 (G.A.S. Gesellschaft für analytische Sensorsysteme
mbH. Dortmund, Germany). An initial exploratory data analysis
by principal component analysis (PCA) was included using the
area of different peaks selected from the topographic plot shown
in Fig. 2A. Moreover, the differentiation of LAB by PCA was also
carried out using the whole spectral information and using
Matlab (The Mathworks Ins, Natick, MA, 2007). In a previous
work, this data treatment was fully described (Garrido-Delgado
et al., 2011).
Fig. 3. Evolution of VOCs emitted by Lactobacillus casei, Lactobacillus paracasei subsp. paracasei, Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris in MRS
broth. m: monomer, d: dimer. Intensity in V corresponded to the IMS signal intensity of each component identified in each LAB (see Fig. 1).
3. Results and discussion
3.1. Determination of VOCs from LAB reference strains
The 4 different LAB samples (L. casei, L. paracasei subsp. paraca-
sei, L. lactis subsp. lactis, and L. lactis subsp. cremoris) were analysed
by GC-IMS in duplicate. The results are shown in Fig. 2A. In
Fig. 2B, a three-dimensional topographic plot of each LAB is shown
as an example of the valuable information (signal/retention time/-
drift time) obtained after each individual analysis.
Similar spectral fingerprints were obtained for the two Lacto-
bacillus strains (Lactobacillus casei and Lactobacillus paracasei
subsp. paracasei) analysed. The resulting compounds which
permit differentiating Lactobacillus reference strains were 2-
butanone, 2-pentanone, 2-heptanone and 3-methyl-1-butanol,
according to the intensity of their signals. These four analytes
were identified calculating the reduced ion mobility of each
one, and the data were included in Table 1. Lactococcus were
also analysed under the same conditions and only 2-butanone
and 3-methyl-1-butanol were identified. The VOCs detected
from Lactococcus strains also showed different intensity in each
strain. Moreover, we observed that the metabolites are appeared
as proton-bound monomers and dimers in GC-IMS plots
(see Fig. 2A). Notice that in some cases, the monomer of
2-butanone or 2-heptanone appeared in a region along with
other signals, so it was very difficult to identify these signals
clearly, and for this reason these data were not included in
Table 1. The formation of dimers beside monomers is well
described by Borsdorf and Eiceman (2006).
 J. Gallegos et al. / Food Chemistry 220 (2017) 362–370 367

All detected VOCs are considered as products of LAB metabo-
lism and could be useful for the differentiation between the strains
examined. Ketones (2-butanone, 2-pentanone and 2-heptanone)
are the most frequent compounds detected by GC-IMS in this
study. Ketones are common constituents of a wide range of dairy
products and have a major influence on cheese aroma and low per-
ception threshold (Curioni & Bosset, 2002). 2-Heptanone has been
detected in some types of cheeses, like Emmental or Gorgonzola
(Moio, Piombino, & Addeo, 2000). The general pathway of ketone
production is explained by the fact that free fatty acids released
by lipolysis are oxidised to b-ketoacids and decarboxylated to
ketones with one less carbon atom (McSweeney & Sousa, 2000).
In addition to ketones, an alcoholic compound was also found in
this study: 3-methyl-1-butanol (produced by leucine degradation)
is related to the pleasant aroma of fresh cheese (Randazzo et al.,
2007; Smit, Smit, & Engels, 2005). This compound has also been
recognised as a key flavour in different handcrafted cheeses and
the compound is commonly produced by wild strains of LAB
(Morales, Fernández-Garcia, Gaya, & Núñez, 2003). Although the
most frequent metabolic pathway for the production of alcohols
by Lactobacillus casei is the catabolism of aa, (which includes the
degradation of linoleic and linolenic acids), the biosynthesis of 3-
methyl-1-butanol also includes lactose or lactate metabolism and
aldehyde reduction (Sgarbi et al., 2013).
such as 3-methyl-1-butanol, while lipolysis generates compounds
such as 2-butanone, 2-pentanone and 2-heptanone. In this study
the substrate was the culture medium.
Fig. 4A shows the trends of bacterial growth of Lactobacillus
casei and Lactobacillus paracasei subsp. paracasei. The biomass pro-
duction and biochemical activity over time can be observed by
changes in the optical density (OD540) of the cultures. The measure-
ment of optical density helps to obtain qualitative evidence about
kinetic behaviour of bacteria in a fermentative process. In this case,
it allows us to know the stage of maximum activity with respect to
the emission of volatile metabolites. As widely described, in all
cases, LAB growth follows an exponential curve, as seen in Lacto-
bacillus casei. Lactobacillus paracasei subsp. paracasei, however, only
shows exponential growth and stationary phase (the lag phase,
corresponding to a low growth previously to logarithmic phase,

3.2. Monitoring of VOCs produced by LAB reference strains during their

growth

In order to demonstrate that 2-butanone, 2-pentanone, 2-

heptanone and 3-methyl-1-butanol are produced by LAB metabo-
lism, the monitoring of these VOCs during bacterial growth was
carried out. It can be observed that the intensity of their signals
varied through the culture time (24–30 h) for each bacteria stud-
ied. The evolution of VOCs produced by the LAB studied during dif-
ferent phases of growth culture is shown in Fig. 3. The GC-IMS
signals generated by LAB during culture time revealed volatile
metabolites released by bacterial cells and they are associated with
changes of concentration in biomass production.
In the case of Lactobacillus casei (see Fig. 3A), 2-butanone and 3-
methyl-1-butanol showed the same upward trend, with a moder-
ate increase during the period studied. 2-Butanone showed higher
values than 3-methy-1-butanol. 2-Pentanone and 2-heptanone
also showed a similar trend, with a high increase in their intensity
values after 20 h of incubation. The higher value of intensity (2 V)
was reached by 2-pentanone and 2-heptanone after incubation for
30 h.
The evolution of Lactobacillus paracasei subsp. paracasei (see
Fig. 3B) showed a very similar trend for 3-methyl-1-butanol, 2-
pentanone and 2-heptanone, the two last compounds reaching
practically the same values. For this bacterium, 2-butanone also
reached the highest values.
In the case of Lactococus lactis subsp. Lactis (see Fig. 3C), only
two signals were identified: 2-butanone and 3-methyl-1-butanol.
The kinetics of the culture indicated that both compounds reached
practically 1 V of intensity before the first hour. Then, both
achieved their maximum intensity (1.2 V) at 7 h and remained
with this value practically until the end of the experiment.
For Lactococcus lactis subsp. cremoris (see Fig. 3D), the same
Fig. 4. (A) Cell growth evolution of Lactobacillus casei (LC) and Lactobacillus
VOCs as those found in Lactococcus lactis subsp. lactis were identi-
paracasei subsp. paracasei (LPP). Cell growth was monitored determining the
fied. However, the highest value was reached later (at 15.5 h) and culture broth optical density (540 nm). The data points represent the average of two
both compounds reached exactly the same values for practically independent experiments. (B) Cell growth evolution of Lactococcus lactis subsp.
the entire experiment. In summary, it can be said that, the beha- lactis (LLL) and Lactococcus lactis subsp. cremoris (LLC). Cell growth was monitored
viour of Lactococcus and Lactobacillus strains in milk fermentations determining the culture broth optical density (540 nm). The data points represent
the average of two independent experiments. (C) Evolution of pH of growing LAB
result in acidification and proteolytic activities related to flavour cultures measured at 25 °C. LC (Lactobacillus casei), LPP (Lactobacillus paracasei
production. The proteolysis is the first biochemical step involved subsp. paracasei), LLL (Lactococcus lactis subsp. lactis), LLC (Lactococcus lactis subsp.
in the degradation of substrates and can give rise to compounds cremoris).
368 J. Gallegos et al. / Food Chemistry 220 (2017) 362–370

is almost imperceptible). VOCs described during Lactobacillus casei
growth (Fig. 3A) show that the high increase of 2-pentanone and 2-
heptanone occurs during the stationary phase. In contrast, VOCs
produced by Lactobacillus paracasei subsp. paracasei (Fig. 3B) seem
to be independent of the cell growth time course.
Fig. 4B shows Lactococcus lactis subsp. lactis and Lactococcus lac-
tis subsp. cremoris growth during the time course studied. Lactoco-
cus lactis subsp. lactis shows a stationary phase after 15 h. This is
coherent with the production of 2-butanone and 3-methyl-1-
butanol (see Fig. 3D) which increased steadily during this phase.
In the case of Lactococus lactis subsp. cremoris, the first large
increase (to 1 V) was during the logarithmic phase, while the other
large increase (3 V) was observed during the stationary phase. Lac-
tococcus strains and L. paracasei, subsp paracasei start growth with-
out a lag phase but Lactobacillus casei shows a lag phase, although
all the cultures were standardised to the same cell density. This
may be due to the physiological conditions of L. casei, resulting in
a lower metabolic activity compared with other strains.
Fig. 4C shows the pH evolution during LAB culture growing at
25 °C. A decrease can be appreciated from pH initial values for
24 h, from 6.00 to 3.57 (Lactobacillus casei), 3.86 (Lactobacillus para-
casei subsp. paracasei and Lactococus lactis subsp. lactis) and 4.70
(Lactococus lactis subsp. cremoris). The acidification of the medium
indicates the rapid utilisation of carbohydrates with accumulation

Fig. 5. (A) PCA of the LAB reference strains using the information of spectral region of drift time from 8 to 14 ms and retention time from 90 to 650 s. This PCA was built with
Matlab software. Ellipses have been drawn around (A) Lactobacillus casei, (B) Lactobacillus paracasei subsp. paracasei, (C) Lactococcus lactis subsp. lactis, (D) Lactococcus lactis
subsp. cremoris. (B) PCA using the data of the areas from 20 peaks found in the topographic plot from LAB reference strains. This PCA was built with LAV software. Ellipses
have been drawn around (A) Lactobacillus casei, (B) Lactobacillus paracasei subsp. paracasei, (C) Lactococcus lactis subsp. lactis, (D) Lactococcus lactis subsp. cremoris. PCA
explains 88% of the total variation distributed in PCA1 50% and PCA2 38%.
 J. Gallegos et al. / Food Chemistry 220 (2017) 362–370 369

of lactic and acetic acids. At low pH (i.e. 4.6), the metabolism from Table 3
hexose fermentation changes to utilisation of aa, which is favour- Results of PCA of selected GC-IMS signals.

able for aroma production in ripened cheeses (Gerrit, Smit, & PC1 PC2
Engels, 2005; Gänzel, 2015). Eigenvalues 3.244 2.888
In summary, GC-IMS signals detected in the cultures of LAB dur- % of variance 50 38
ing their culture time clearly reveal changes in the production of Cumulative % 50 88
volatile metabolites released by bacterial cells. Similar conclusions Selected GC-IMS signals Factor loadings
were obtained by Maddula, Blank, Schmid, and Baumbach (2009). a2: 2-butanone dimer 0.199 0.875
They confirmed that the volatile metabolites produced by Escheri- b1: 2-pentanone monomer 0.013 0.904
b2: 2-pentanone dimer 0.944 0.256
chia coli were products associated with biodegradation of sub- c1: 3-methyl-1-butanol monomer 0.146 0.921
strates supplied by the culture medium, and their concentration c2: 3-methyl-1-butanol dimer 0.599 0.614
could increase in parallel to cell growth. A similar trend can be d1: 2-heptanone monomer 0.985 0.096
observed in Lactobacillus and Lactococcus. They also reported that d2: 2-heptanone dimer 0.982 0.084
the concentration changes of volatiles compounds during bacterial The bold variables are the highest weight of the factor loadings.
growth can be due to the variation of activity in the metabolic
pathways, changes in solubility and volatility of the analytes in
the culture medium, but most likely correlate with the increase
in biomass. Lactococcus lactis subsp. cremoris (Nomura, Kobayashi, Narita,
In this experiment, it has been found that the production of 2- Kimoto-Nira, & Okamoto, 2006). The volatile metabolites are
butanone and 3-methyl-1-butanol by Lactobacillus and Lactococcus also considered as phenotypical characteristics of Lactococcus
follows the trend of bacterial growth during adaptation stage, lactis. Certainly, the GC-IMS volatile profiles of these strains
exponential and stationary phases of bacterial growth curve share the same volatile compounds, but differ in intensity of
(Fig. 4A and B). Thus, in general, the metabolites from LAB increase the signals. They also differ in their evolution during the growth
at the beginning of exponential growth, without changing when of cultures. The possibility of distinguishing the fingerprints
reaching the stationary phase. In contrast, 2-pentanone and 2- between Lactococcus lactis subspecies enhances the significance
heptanone from Lactobacillus casei keep a constant intensity of sig- of these results.
nal increasing at 21 h (see Fig. 3A), showing that the pathways Regarding Lactobacillus strains, it should be taken into account
involved did not entirely depend on cell growth. The different pro- that this genus is taxonomically complex; their species (over
files of VOCs emitted by Lactobacillus and Lactococcus strains sug- 170) cannot be easily differentiated according to phenotypical
gest that bacterial growth does not explain all the process that properties (Goldstein, Tyrrell, & Citron, 2015). However, this study
could be involved in the production of these metabolites by LAB reports the possibility of discriminating Lactobacillus casei from
in the culture medium, to which other authors have referred Lactobacillus paracasei subsp. paracasei, according to their relevant
(Maddula et al., 2009). volatile fingerprints.

3.3. Discrimination of strains by principal components analysis

The potential of the GC-IMS method to identify different LAB
can be highlighted using a global analysis (including all data in
the region of drift time from 8 to 14 ms and retention time from
90 to 650 s) from the topographic plot of each sample (see
Fig. 2A). The data set used to make the PCA was obtained incu-
bating the LAB at 35 °C. The data used to build the PCA were
normalised with respect to the reactant ion peak area. According
to Fig. 5A, LAB reference samples can be categorised into four
different groups. The first two components explained 82.06% of
variance; component PC1 contributing 54.75% and component
PC2, 27.31%. The fingerprints of Lactococcus tested were clearly
different as shown in groups C and D. The difference between
groups A and B (Lactobacillus) was not as clear as for Lactococ-
cus. Better differentiation was achieved among Lactobacillus
when a target analysis was followed (using the data of the areas
from 20 signals found in the topographic plot from LAB refer-
ence strains) to build the PCA (see Fig. 5B). The PCA showed
that 88% of the variance in the data was explained by the first
two PCs with Eigenvalues P1 (see Table 3). The highly weighted
variables under PC1 and PC2 with the highest weight of the fac-
tor loadings were b1 (2-pentanone monomer), b2 (2-pentanone
dimer), c1 (3-methyl-1-butanol monomer), d1 (2-heptanone
monomer), and d2 (2-heptanone dimer). PC1 can explain some
differences between strains of Lactobacillus and Lactococcus,
when these LAB produce volatile metabolites under the condi-
tions of this study. In contrast, PC2 shows Lactobacillus, clustered
in (A), clearly separated from clusters (B), (C) and (D). The dis-
similarities between clusters (C) and (D) are in accordance with
other taxonomical characteristics that classify Lactococcus lactis
in subspecies, among them Lactococcus lactis subsp. lactis and
4. Conclusions
GC-IMS is a technology that can be applied to detect VOCs of
LAB in an inexpensive way in comparison to GC–MS, with the
advantage of immediate detection, in real-time, and without need-
ing sample pretreatment or complex accessories for sampling. It is
a suitable methodology to make screenings of volatiles from LAB or
to control bioprocess in-line, which could be used for the selection
and development of starters. The VOCs 2-butanone, 2-pentanone,
2-heptanone and 3-methyl-1-butanol have been identified as rele-
vant VOCs from LAB analysed. Lactobacillus casei, Lactobacillus para-
casei subsp. paracasei, Lactococcus lactis subsp. lactis and
Lactococcus lactis subsp. cremoris have been characterised by GC-
IMS and the VOCs emitted lead to discriminate between them,
using a multivariate analysis technique (PCA), in which four clus-
ters were identified.
The change in intensity of IMS signals during the growth of the
strains demonstrated that these VOCs were derived from the bio-
chemical activity of the cells (or metabolic pathway). These com-
pounds would contribute to the sensory perception of the cheese.
These results indicate that their volatile profiles (fingerprints)
can be used as a pattern to search for strains with similar finger-
prints and aromatic capacity. Moreover, this methodology could
be used for identification or authentication of specific strains in
the laboratory from a specific and previously tested food matrix.
In addition, GC-IMS can be used to evaluate strains of technological
interest.
GC-IMS can assess the physiological state of the bacterial cells
during bacterial growth (i.e. active growth). Also, the technique
makes it possible to obtain data for process control in industrial
fermentations or maturations.
370 J. Gallegos et al. / Food Chemistry 220 (2017) 362–370
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
J.G. wishes to thank the Secretaria Nacional de Educación Superior
Ciencia Tecnología e Innovación (Senescyt) from Ecuador for financial
support through a predoctoral grant. The authors are also grateful
to Spain’s DGICyT (Project CTQ2014-52939R) for funding this
work.
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