International Journal of Food Microbiology 160 (2012) 65–75
Contents lists available at SciVerse ScienceDirect
International Journal of Food Microbiology
journal homepage: www.elsevier.com/locate/ijfoodmicro
Effect of O2\CO2 enriched atmospheres on microbiological growth and volatile
metabolite production in packaged cooked peeled gray shrimp (Crangon crangon)
Bert Noseda a, b, Joke Goethals a, b, Lies De Smedt a, b, Jo Dewulf c, d, Simbarashe Samapundo a, b,
Herman Van Langenhove c, d, Frank Devlieghere a, b,⁎
a
Laboratory of Food Microbiology and Food Preservation (LFMFP), Ghent University, Coupure Links 653, 9000 Ghent, Belgium
Department of Food Safety and Food Quality, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
Laboratory of Environmental Organic Chemistry (ENVOC), Ghent University, Coupure Links 653, 9000 Ghent, Belgium
d
Department of Organic Chemistry, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
b
c
a r t i c l e
i n f o
Article history:
Received 4 March 2012
Received in revised form 14 September 2012
Accepted 22 September 2012
Available online 28 September 2012
Keywords:
Crangon crangon
MAP
Volatile spoilage metabolites
SIFT-MS
Shelf life
Microbial activity
a b s t r a c t
This study evaluated the effect of modified atmospheres (MAs) with different O2 concentrations on microbial
growth and volatile metabolite production in gray shrimp (Crangon crangon) during storage at 4 °C. Eight
MAs were evaluated in total. Four of the MAs evaluated were without CO2: 0/0/100, 0/10/90, 0/30/70, 0/
50/50 (% CO2/O2/N2) whilst the other four MAs all contained 50% CO2: 50/0/50, 50/10/40, 50/30/20, 50/50/
0 (% CO2/O2/N2). Volatile spoilage metabolites were identified by thermal desorption GC-MS and quantified
during storage by selective ion flow tube mass spectrometry (SIFT-MS). In comparison to microbial growth
observed with an atmosphere of 100% N2, microbial growth was stimulated by the addition of O2 in the
MAP in the absence of CO2. Under these conditions the total psychrotrophic counts exceeded 7 log cfu g−1
after just 3 days of storage. However, in the presence of 50% CO2 the total psychrotrophic count exceeded
7 log cfu g −1 after 5 days of storage. The combination of 50% CO2 and 50% O2 significantly inhibited microbial
growth. For this MA condition, a diminishing effect on the production of metabolites was also observed, especially for amines and sulfur compounds, which constituted the major fraction of components causing the
offensive odor.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Gray shrimp, also referred to as brown shrimp, (Crangon crangon)
are for the North–West of Europe a very important commercial fishery
product with an annual market value of € 70–90 million (Catchpole et
al., 2008). Although German and Dutch vessels catch approximately
85% of the total European landings (Catchpole et al., 2008), substantial
landings are also made in Denmark, Belgium, France, the UK, and in
the central Mediterranean (Dore and Frimodt, 1987). This crustacean
is typically cooked on board of the fishing vessel and sold either
whole cooked or as cooked and peeled tails. Gray shrimp are generally
treated with preservatives in order to prolong the shelf life. Mostly
benzoic acid (0.6%) and/or sorbic acid (0.6%) or their derived salts are
added. Often organic acids such as citric acid, acetic acid, lactic acid,
glucono delta-lactone or their derived salts are added too since the
above mentioned preservatives function more effective at lower pH
⁎ Corresponding author at: Laboratory of Food Microbiology and Food Preservation,
Ghent University, Coupure Links 653, 9000 Ghent, Belgium. Tel.: + 32 9 264 61 64;
fax: + 32 9 264 55 10.
E-mail addresses: [email protected] (B. Noseda), [email protected]
(J. Dewulf), [email protected] (S. Samapundo),
[email protected] (H. Van Langenhove), [email protected]
(F. Devlieghere).
0168-1605/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijfoodmicro.2012.09.018
values. But a recent tendency towards the use of less preservatives
has expressed itself in the commercialization of gray shrimp without
chemical preservation, only relying on modified atmosphere packaging
(MAP) to extend the shelf life.
The microbiological spoilage process of air stored gray shrimp results
in the rapid formation of unpleasant and unacceptable off-odors and
off-flavors, resulting in a very limited shelf life during refrigerated storage. Most shrimps have high initial microbial counts (10 5–107 cfu/g) at
the time of receipt in the processing plant (ICMSF, 2005). The
cooking step constitutes a strong barrier, efficiently reducing the initial microbial content. Several studies investigating the cooked
shrimp microbiota, demonstrated that the microbiota was the result
of direct recontamination from the processing equipment and environment (Jaffres et al., 2009; Valdimarsson et al., 1998; Zapatka
and Bartolom, 1973). Broekaert et al. (2012) revealed that the specific predominating microbiota on C. crangon during aerobic storage at
7.5 °C merely consists of members of the genera Psychrobacter and
Pseudoalteromonas, despite differences in area and season of catch,
in early handling and processing procedures or storage conditions.
Chinivasagam et al. reported in 1998 the spoilage bacteria and their
metabolites produced on 4 different shellfish species. Here, mainly
amines, sulfides, ketones and esters were described as spoilage metabolites. Amines were the major spoilage compounds during the
66
B. Noseda et al. / International Journal of Food Microbiology 160 (2012) 65–75
early phases of aerobic storage under ice, while sulfides and esters
became dominant later (Chinivasagam et al., 1998).
To prolong this short shelf life, modified atmosphere packaging
(MAP) has already been proven to be a successful preservation technique for fish and fishery products. The inhibitory effect of MAP on microbiological growth is generally attributed to the CO2 dissolving in the
water phase of the product, causing an extension of the lag phase and
reducing the growth rate during the logarithmic phase (Devlieghere
and Debevere, 2000). Jaffres et al. (2009) highlighted the main bacterial
genera and species related to the spoilage of cooked and peeled tropical
shrimps, packaged in 50/0/50 and 100/0/0 (% CO2/O2/N2). Lacic acid
bacteria, mainly represented by the genera Carnobacterium, Vagococcus
and Enterococcus, were found to dominate the microbiota of the studied
shrimp, followed by Brochothrix thermosphacta and Serratia liquefaciens.
Jaffres et al. (2011) inoculated cooked and peeled shrimp with six isolated bacterial species and stored the shrimp packaged under and atmosphere of 50/0/50 at 8 °C (% CO2/O2/N2). Metabolite production was
monitored with solid phase micro-extraction (SPME) GC-MS.
The addition of O2 to CO2 enriched atmospheres has also been described as a preservation method for crustaceans (Chen and Xiong,
2008; Lopez-Caballero et al., 2002; Lu, 2009; Mastromatteo et al.,
2010; Mejlholm et al., 2005). Next to preserving the red color, elevated
O2 concentrations in combination with CO2 may have an additional antimicrobial activity in fishery products (Lopez-Caballero et al., 2002).
Debevere and Boskou (1996) indicated that a modified atmosphere
containing O2 retarded the onset of trimethylamine-oxide reduction
in fishery products. But besides this aspect, not much is known on the
effect of O2 in CO2 enriched packaged cooked and peeled shrimp towards microbial growth in relation to metabolite production.
This study therefore evaluated the impact of different levels of O2 in
modified atmospheres (with and without CO2) on microbiological
growth and volatile metabolite production on packaged and stored
(4 °C) cooked and peeled C. crangon. Metabolite production was monitored by means selective ion flow tube mass spectrometry (SIFT-MS).
The gas combination 50/0/50 (% CO2/O2/N2) with a gas/product ratio
of 2/1 is currently often used in the industry to package chemically preserved as well as unpreserved cooked and peeled gray shrimp. We have
therefore opted to maintain 50 % CO2 as a fixed parameter as a reference, and vary the O2 concentration between 0 and 50%.
2. Materials and methods
2.1. Preliminary identification of spoilage related VOCs by TD GC-MS
A preliminary experiment was performed to identify the volatile
organic compounds which may attribute to the off-odors present in
the headspace of MAP shrimp at the end of the shelf life. Gray shrimp
samples packaged by the following modified atmosphere conditions
were evaluated during this preliminary experiment: 50/0/50, 50/10/
40, 50/30/20, 50/50/0 (%CO2/ %O2/ %N2), with storage at 4 °C. On a
regular basis, samples were taken for sensorial evaluation and VOC's
present in the headspace of the packages were identified by means
of thermal desorption GC-MS (TD GC-MS).
2.2. Experimental set-up
In this part of the study, eight MAP conditions to package gray
shrimp were evaluated during storage at 4 °C. Four conditions were
without CO2: 0/0/100, 0/10/90, 0/30/70, 0/50/50 (% CO2/O2/N2) and
four conditions were with 50% CO2: 50/0/50, 50/10/40, 50/30/20,
50/50/0 (% CO2/O2/N2). Samples were collected on regular basis
(days 0, 3, 5, 7, 10 and 12) for the determination of the pH, headspace
(%O2, %CO2) composition, microbiological analysis (including the
enumeration of the total psychrotrophic counts, lactic acid bacteria,
H2S producing bacteria and Pseudomonas spp.) and headspace analysis of volatile organic compounds (VOC's) with SIFT-MS. For each
investigated MAP condition three independent replicates were used.
On day 0, microbiological analysis was performed in triplicate on
the incoming gray shrimp before the onset of packaging. The samples
for SIFT-MS analysis were frozen in sealed high barrier PA/PE/EVOH/
PE bags (Euralpack, Schoten, Belgium) with an oxygen transmission
rate of 2 cm 3/m 2*d*bar and on day 14 subsequently thawed and analyzed at 4 °C.
2.3. Raw material
For each experiment, gray shrimp (C. crangon) were caught in the
North Sea the night before the start of the experiment (September
2011). The shrimp were cooked on-board for 3 min in salted water
(±90 °C), stored during the night at 1.0 ± 1.0 °C and brought onshore
at the Port of Ostend (Belgium), where they were mechanically
sorted according to size (6.0–8.0 cm) and peeled under standard
GMP in a local shrimp processing company. During processing, no
salts or preservatives e.g. sorbic acid or benzoic acid were added.
The shrimp were subsequently transported under ice in sterile stomacher bags to the Laboratory of Food Microbiology and Food Preservation, for the onset of each experiment.
2.4. MAP packaging
Portions of 100.0 ±5.0 g gray shrimp were packaged under a modified atmosphere with a gas/product ratio of 2/1. The trays were packaged using a Tray sealer MECA 900 (DecaTechnic, Herentals, Belgium).
Multilayer high barrier packaging materials were used: PP/EVOH/PP
trays (DECA Pac, Herentals, Belgium) and OPA/EVOH/PE/PP toppings
(BEMIS Packaging Benelux, Monceau-sur-Sambre, Belgium) with an oxygen transmission rate of respectively 0.5–13 cm3/m2 *d*bar at 23 °C,
0% R.H. and 5 cm3/m 2*d*bar at 23 °C, 50% R.H.. The packaged shrimp
samples were immediately stored in a refrigerator set at 4.0± 0.7 °C.
2.5. Sensorial analysis
Sensorial evaluation was performed by a trained panel (n = 10)
which had to evaluate the odor and appearance and state whether
the samples were still acceptable for consumption. The panel received
a reference sample (samples from day 0, which were kept frozen at −
21 °C and thawed before use) to compare with the stored sample.
When > 50% of the panel rejected the sample, the sample was considered to have exceeded the shelf life. This limit is in shelf life studies
generally considered as limit of product acceptability (Gimenez et
al., 2008; Guerra et al., 2008; Hough et al., 2003).
2.6. Identification of VOCs with TD GC-MS
For the identification of volatile compounds by headspace analysis, the sampling methodology was modified from Ragaert et al.
(2006). Twenty grams of gray shrimp were placed in a 250 mL glass
flask, connected with a sorbent sampling tube (4 mm ID), equipped
with an aluminium diffusion cap at one side and filled with 200 mg
Tenax TA (35/60 mesh) (Markes, Llantrisant, UK). Prior to use, the
tubes were conditioned during 1 h at 250 °C while flushing with
helium (50 mL min −1). After an initial equilibration time of 20 min
at 30 °C, the flask with shrimps was flushed with 50 mL helium (Air
Liquide, Aalter, Belgium) at a flow rate of 100 ml min −1. Desorption
of the analytes pre-concentrated on the Tenax TA sorbent, GC (Trace
2000, Thermo Finnigan, Milan, Italy) analysis and mass spectrometric
detection with the Trace DSQ Quadrupole MS (Thermo Finnigan,
Austin,TX USA) occurred in analogy with Walgraeve et al. (2011).
Chromatograms and mass spectra were processed using Xcalibur
software package (Thermo Finnigan, version 1.4). and identification
was carried out based on retention time and comparison with the
NIST database.
B. Noseda et al. / International Journal of Food Microbiology 160 (2012) 65–75
2.7. Microbiological analysis
From each sample, 30 g was aseptically placed into a sterile stomacher bag and diluted 10 times in physiological saline peptone solution
(PPS, 0.85% NaCl, 0.1% peptone). After homogenization for 1 min in a
Stomacher 400 Lab Blender (LED Techno, Heusden-Zolder, Belgium),
the appropriate decimal dilutions of the homogenate were made. The
total viable psychrotrophic count was determined on Marine agar MA
(Difco, Le Pont de Claix, France) by spread plating. Lactic acid bacteria
were enumerated by pour plating of appropriate serial dilutions on de
Man Rogosa Sharpe agar MRS (Oxoid, Hampshire, U.K.). MA and MRS
plates were incubated during respectively 5 and 3 days at 22 °C. H2S
producing bacteria were enumerated on Iron Agar Lyngby (Oxoid,
Hampshire, U.K.) containing L-cysteine (Fluka, Steinheim, Germany)
in accordance with the manufacturer's preparation instructions. Incubation of these plates took 3 days at 22 °C. Pseudomonads were enumerated on Pseudomonas agar (Oxoid, Hampshire, U.K.) containing
Pseudomonas CFC supplement (SR 0103), after incubation for 2 days at
22 °C. Microbiological enumeration was performed until the total
psychrotrophic viable count exceeded 10 8 cfu g −1.
2.8. pH and headspace composition (%O2, %CO2) measurements
pH was measured on a 5.0 g mixed shrimp sample by means of a
pH-electrode (InLab®427, Mettler Toledo GmbH, Schwerzenbach,
Switzerland) connected with a pH meter (SevenEasy, metler Toledo
67
GmbH). The product temperature during pH measurements was
5.0 ± 1.0 °C. Analysis of %O2 and %CO2 headspace composition of the
packaged samples occurred with the Checkmate® 9900 O2/CO2 (PBI
Dansensor A/S, Ringsted, Denmark).
2.9. Quantification of spoilage related VOCs by SIFT-MS
Selective ion flow tube mass spectrometry (Voice 200, Syft
Technologies™) analysis and data handling occurred according
to Noseda et al. (2012). 50 ± 0.5 g of each sample was packaged
in the high barrier PA/PE/EVOH/PE bags (Euralpack, Schoten,
Belgium), with 950.0 ± 5.0 mL of N2 gas using a Multivac A 300/42
packaging unit (Hagenmüller, Wolfert-schwenden, Germany)
(Noseda et al., 2012). Samples were measured in full scan mode
and in multiple ion mode (MIM). The full scan mode measures all
ionized masses as result of reactions with precursor ions H3O +,
NO +, O2+ in cps within the range of (m/z = 1–150). Full scans
were used to optimize the methods used to quantify the volatile
metabolites and to evaluate possible interference of unidentified
VOCs in the MIM scans. The targeted VOCs and the ions used for
their quantification with the MIM method are shown in Table 1.
The compounds were selected based on the preliminary screenings
of identically packaged samples with TD-GCMS analysis (Table 2),
preliminary research and on their occurrence as microbiological
spoilage metabolite found on fishery products according to
literature.
Table 1
Characteristic product ions of the volatile organic compounds analyzed with SIFT-MS, with the coinciding precursor ions, mass-to-charge ratio (m/z), branching ratio and reaction
rate coefficient (k).
Volatile compound
Alcohols
Ethanol
2-Propanol
Isobutyl alcohol
2,3-Butanediol
Aldehydes
Nonanal
Decanal
Ketones
Acetone
2-Butanone
2,3-Butandione
2-Pentanone
Sulfur compounds
Hydrogen sulfide
Methyl mercaptan
Dimethyl disulphide
Carbon disulfide
Dimethyl thioether
Amines
Ammonia
Methyl amine
Dimethyl amine
Trimethyl amine
Acids
Acetic acid
Esters
Ethyl acetate
Precursor
m/z
Branching ratio (%)
K
Characteristic product ion
H3O+
H3O+
H3O+
NO+
O2+
47
43
57
73
89
100
80
100
95
10
2.70E−09
2.70E−09
2.70E−09
2.40E−09
2.50E−09
C2H7O+
C3H7+
C4H9+
C4H9O+
C4H9O2+
O2+
H3O+
NO+
O2+
69
157
155
68
10
97
100
10
3.20E−09
3.90E−09
3.30E−09
3.20E−09
C5H9+
C10H21O +
C10H19O +
C5H8+
H3O+
NO+
NO+
NO+
NO+
59
88
102
86
116
100
100
100
65
100
3.90E−09
1.20E−09
2.80E−09
1.30E−09
3.10E−09
C3H7O+
NO + .C3H6O
NO + .C4H8O
C4H6O2+
NO + C5H10O
H3O+
O2+
H3O+
H3O+
NO+
O2+
NO+
35
34
49
95
94
76
62
100
100
100
100
100
100
100
1.60E−09
1.40E−09
1.80E−09
2.30E−09
2.40E−09
7.00E−10
2.20E−09
H3S+
H2S+
H3O + .CH4S
(CH3)2S2.H +
(CH3)2S2 +
CS2+
(CH3)2S+
H3O+
O2+
NO+
O2+
H3O+
H3O+
H3O+
18
17
31
31
46
58
60
100
100
100
65
100
10
90
2.70E−09
2.40E−09
8.20E−10
1.00E−09
2.10E−09
2.00E−09
2.00E−09
NH4+
NH3+
CH3NH2+
CH3NH2+
(CH3)2NH.H+
C3H8N+
(CH3)3 N.H+
NO+
90
100
9.00E−10
NO + .CH3COOH
H3O+
NO+
89
118
100
90
2.90E−09
2.10E−09
CH3COOC2H5.H +
NO + .CH3COOC2H5
68
B. Noseda et al. / International Journal of Food Microbiology 160 (2012) 65–75
Table 2
Volatile organic compounds identified with TD GC-MS in the headspace of MAP gray shrimp.
begin of shelf life
Alcohols
Ethanol
2-Propanol
Cyclopentanol
Cyclohexanol, 2-methyl-, cisAldehydes
Acetaldehyde
Benzaldehyde
Octanal
Nonanal
Decanal
Undecanal
Ethers
Ethyl ether
Isopentyl vinyl ether
Ketones
Acetone
2-Butanone
2-Pentanone
2,4-Octanedione
Esters
Ethyl acetate
Acids
Acetic acid
Sulfur compounds
Carbon disulfide
Dimethyldisulfide
2,4-Dithiapentaan
Thiourea
Hydrocarbons
Alkanes
Pentane, 2-methylPentane, 3-methylHexane
Cyclopentane, methylPentane, 2,2,4-trimethylButane, 2,2,3,3-tetramethylOctane
Hexane, 2,2,5-trimethylHeptane, dimethyl
Decane, 2,6,7-trimethylHeptane, 2,2,4,6,6PentamethylDecane, 2,6,8-trimethylDecane, 2,6,7-trimethylAlkenes
Benzene
Toluene
2-Ethyl,1-hexeen
4-Decene
Benzene, 1,3-bis(1,1-dimethylethyl)Amines
Trimethylamine
Terpenes
D-limonene
Others
Acetonitrile
Benzonitrile
Methylene chloride
Chloroform
Furan, tetrahydroBenzene, 1,4-dinitro-
early spoilage
50/0/50
50/10/40
50/30/20
50/50/0
50/0/50
x
x
x
x
x
x
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x
x
x
50/30/20
50/50/0
x
x
x
x
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x
x
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x
x
x
x
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50/10/40
x
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Underlined components have been included for quantification by means of the SIFT-MS methodology.
2.10. Statistical analysis
3. Results and discussion
Student's t-tests were used to assess significant (p b 0.05) differences between data points. The software used to explore statistical significances on the obtained results was S-Plus 8.0 for
Windows.
3.1. Identification of spoilage related VOCs by TD GC-MS
Table 2 represents the results of the screening with TD GC-MS
during the preliminary storage experiment. After 7 days of storage,
B. Noseda et al. / International Journal of Food Microbiology 160 (2012) 65–75
69
Table 3
Results of the microbiological analysis for the different investigated MAP gray shrimp conditions during storage at 4 °C. Values with a different superscript in one column show
statistical significance (p b 0.05).
% CO2 / %O2 / %N2
Time of storage (days)
0
0/0/100
0/10/90
0/30/70
0/50/50
50/0/50
50/10/40
50/30/20
50/50/0
0/0/100
0/10/90
0/30/70
0/50/50
50/0/50
50/10/40
50/30/20
50/50/0
0/0/100
0/10/90
0/30/70
0/50/50
50/0/50
50/10/40
50/30/20
50/50/0
Pseudomonas spp. log (cfu/g)
H2S producing bacteria log (cfu/g)
Lactic acid bacteria log (cfu/g)
3
a
3.8 ± 0.6
3.8 ± 0.6a
3.8 ± 0.6a
3.8 ± 0.6a
3.8 ± 0.6a
3.8 ± 0.6a
3.8 ± 0.6a
3.8 ± 0.6a
3.7 ± 0.3a
3.7 ± 0.3a
3.7 ± 0.3a
3.7 ± 0.3a
3.7 ± 0.3a
3.7 ± 0.3a
3.7 ± 0.3a
3.7 ± 0.3a
3.3 ± 0.6a
3.3 ± 0.6a
3.3 ± 0.6a
3.3 ± 0.6a
3.3 ± 0.6a
3.3 ± 0.6a
3.3 ± 0.6a
3.3 ± 0.6a
all samples packaged under the investigated MAP conditions were
rejected by >50% of the panel. In total 47 VOCs were identified, comprising alcohols, aldehydes, ether, ketones, esters, acids, hydrocarbons, sulfur containing volatiles and amines (trimethylamine).
These results were used for the selection of the VOCs quantified
with SIFT-MS. The compounds that were selected for quantification
with SIFT-MS are underlined in Table 2.
3.2. Impact of O2 (with and without CO2) on the microbiological growth
and metabolite production in MAP gray shrimp
3.2.1. Microbiological analysis
Fig. 1A and B and Table 3 show the results of the microbiological enumerations during the shelf life experiment. The initial psychrotrophic
count of 5.5±0.2 log cfu g−1 indicates a high but still acceptable initial
quality of the peeled shrimp. On-board handling and on-board storage
partly explains this high number, but also handling and contact with
work surfaces prior to and after the mechanical peeling step significantly
influenced this initial bacterial number. Fig. 1A represents the microbiological growth on the shrimp samples packaged under the four MAP conditions without CO2. As can be seen, the presence of O2 in the packages
clearly stimulated microbiological growth on the shrimp product. After
3 days of storage the limit of 7.0 log cfu g−1 was exceeded for these samples and after 5 days 8.0 log cfu g−1 was exceeded. In the absence of O2
(MAP condition 0/0/100), microbiological growth was significantly impeded on day 5 (p b 0.01) while it was not yet at day 3 (p=0.12). From
these results, one could conclude that a CO2 deficient gas mixture should
also lack O2 in order to delay microbiological growth in shrimp stored at
4 °C. Fig. 1B shows the total psychrotrophic count of the four MAP conditions in the presence of 50% CO2. No significant differences in microbiological counts were observed between MAP condition 0/0/100 and
50/0/50. This result was expected since Gram-negative bacteria are
generally sensitive for anaerobic conditions and certainly for the presence of CO2. The presence of CO2 probably changed the dominating
spoilage bacteria to CO2 tolerant bacteria i.e. Gram-positive bacteria
like lactic acid bacteria or to Gram-negative Photobacterium phosphoreum
species. (Lopez-Caballero et al., 2002; Mejlholm et al., 2005). Interestingly, the addition of 30% and 50% O2 to a CO2 enriched atmosphere
appeared to induce an additional inhibitory effect on microbiological
5
abc
6.1 ± 0.5
6.3 ± 0.0a
5.6 ± 0.2b
6.7 ± 0.2c
3.6 ± 0.1d
3.9 ± 0.5de
3.2 ± 0.3e
b3.00
6.1 ± 0.1a
6.4 ± 0.2ac
5.5 ± 0.0b
5.6 ± 0.1b
6.7 ± 0.2c
6.7 ± 0.2c
6.5 ± 0.2c
6.1 ± 0.5c
3.1 ± 0.5a
4.3 ± 0.0b
4.9 ± 0.1c
4.3 ± 0.0b
5.3 ± 0.1e
5.2 ± 0.1e
5.5 ± 0.2e
4.8 ± 0.1c
7
a
6.7 ± 0.1
7.6 ± 0.4b
7.2 ± 0.2b
6.9 ± 0.0c
3.5 ± 0.2d
4.2 ± 0.5d
3.6 ± 0.2d
3.0 ± 0.2e
6.7 ± 0.2a
7.6 ± 0.1b
7.3 ± 0.3b
7.3 ± 0.3b
7.8 ± 0.0c
7.8 ± 0.0c
7.1 ± 0.5b
7.0 ± 0.4b
3.9 ± 0.2a
5.5 ± 0.1b
5.9 ± 0.3bc
5.7 ± 0.0c
5.5 ± 0.4bc
5.9 ± 0.4bc
6.6 ± 0.4d
6.1 ± 0.1d
a
7.6 ± 0.1
8.1 ± 0.1b
7.9 ± 0.2ba
7.6 ± 0.1a
4.2 ± 0.1d
4.6 ± 0.5de
4.6 ± 0.2e
3.5 ± 0.4f
7.6 ± 0.0a
8.8 ± 0.2b
8.3 ± 0.0c
7.9 ± 0.3ad
8.2 ± 0.3de
8.5 ± 0.1e
7.7 ± 0.2a
7.0 ± 0.1f
4.7 ± 0.1a
5.8 ± 0.0b
6.0 ± 0.0c
6.1 ± 0.1cd
6.3 ± 0.2d
6.8 ± 0.0e
6.8 ± 0.1e
6.7 ± 0.1e
10
12
–
–
–
–
4.7 ± 0.2a
5.1 ± 0.2a
5.0 ± 0.1a
3.6 ± 0.1b
–
–
–
–
8.8 ± 0.1a
9.1 ± 0.0b
8.8 ± 0.1a
7.1 ± 0.0c
–
–
–
–
7.5 ± 0.1a
7.5 ± 0.2a
7.6 ± 0.0a
6.8 ± 0.1b
–
–
–
–
–
–
–
4.0 ± 0.2a
–
–
–
–
–
–
–
7.6 ± 0.2a
–
–
–
–
–
–
–
7.5 ± 0.3a
growth. This observation was found to be applicable for all microbial
group examined, with some deviations for LAB counts (Table 3).
Lopez-Caballero et al. (2002) reported similar results where P.
phosphoreum was involved in the spoilage of MAP deepwater shrimp
(Parapenaeus longirostris). The growth of this specific spoilage organisms
is known to be affected by the O2 concentration in the atmosphere
(Lopez-Caballero et al., 2002). According to Debevere and Boskou
(1996) and Dalgaard et al. (1997) oxygen in MAP actively reduces the
growth rate of the SSO P. phosphoreum.
An increase of the O2 concentration in the presence of an CO2
enriched atmosphere, inhibited the growth of H2S producing bacteria
(Table 3). These H2S producing bacteria are often associated with bacteria from the genus Shewanella. Whether the dominating flora were
Shewanella species or Photobacterium species, high oxygen concentrations in combination with high carbon dioxide concentrations
appeared to have an inhibitory effect on the microbiological flora.
3.2.2. pH and headspace composition (%O2, %CO2) measurements
The evolution of the pH and headspace O2 and CO2 concentrations
during storage are shown in Figs. 2A, B, and 3A, B, C and D respectively. The initial pH of untreated gray shrimp was found to be 8.3 ± 0.1.
An initial pH decrease of ca. 0.7 can be observed due to dissolving of
CO2 in the water phase of the gray shrimps. For the MAP conditions
0/10/90, 0/30/70, 0/50/50 the headspace %O2 is decreasing as microbiological growth is augmenting, which is coinciding with a simultaneous increase of %CO2. For the MAP conditions 50/10/40, 50/30/70
and 50/50/0, an initial increase in the O2 concentration was observed
as the result of the dissolution of CO2 into the product. Here, although
the same volume of O2 was present in the samples packaged in MA's
without CO2, the %O2 did not decrease to the same extent at the end
of the experiment. In the 50/50/0 samples, no decrease was observed
at all. These results indicate that in the presence of 50% CO2, aerobic
respiration seemed to be suppressed.
3.2.3. Evolution of spoilage related VOCs with SIFT-MS
The effect of O2 on the metabolism of the microbial flora present
on the modified atmosphere packaged gray shrimp was investigated
by monitoring the presence and quantities of typical VOCs related to
fish spoilage in the headspace (HS) of the packaged products.
70
A
B. Noseda et al. / International Journal of Food Microbiology 160 (2012) 65–75
10
log (cfu/g)
9
8
0/0/100
0/10/90
7
0/30/70
0/50/50
6
5
0
2
4
6
8
10
12
time of storage (days)
B
10
log (cfu/g)
9
8
50/0/50
50/10/40
3.2.3.3. HS levels of ketones. Four ketones were monitored in the headspace of the packages over time. Significant increase in headspace
levels were only observed for acetone in the shrimp packaged in
MAs containing oxygen (Fig. 5A, B). Fig. 5A shows that acetone was
produced in aerobic conditions. The higher the oxygen concentration
in the packaging, the higher acetone production. A similar trend was
observed in Fig. 5B in the presence of CO2, however, here the concentrations of acetone produced were reduced. The results were
analogous to those of the GC-MS results. The source of the acetone
metabiosis may be various (Hausinger, 2007). The most known
is acetone fermentation. Many anaerobic bacteria from the class
Clostridia produce acetone during fermentation by decarboxylation
of acetoacetate. But our results suggest an aerobic metabiosis. Literature describes also alternative pathways which might explain our
results. A plausible route is suggested by Nemecek-Marshall et al.
(1999). Some marine Vibrionaceae of the genera Vibrio, Photobacterium
and Shewanella, but also Pseudomonaceae have the possibility for an
aerobic L-Leucine dependent acetone formation (Hausinger, 2007;
Massey et al., 1976; Nemecek-Marshall et al., 1999). Gray shrimp
are known to be a rich dietary source of Leucine which makes this
hypothesis plausible.
7
50/30/20
50/50/0
6
5
0
2
4
6
8
10
12
time of storage (days)
Fig. 1. Total psychrotrophic counts of gray shrimp samples, packaged in MAP conditions Δ 0/0/100, □ 0/10/90, 0/30/70, ■ 0/50/50 (% CO2/O2/N2) in A and ▲ 50/0/50,
○ 50/10/40, 50/30/20, ● 50/50/0 (% CO2/O2/N2) in B, during storage at 4 °C. Error
bars represent standard deviations.
3.2.3.1. HS levels of alcohols. Four alcohols (listed in Table 1) were
monitored in the headspace of the packages with SIFT-MS during
storage. Of these compounds, relevant evolutions in the headspace
concentration during microbiological growth were observed only for
ethanol (Fig. 4A, B). Once total psychrotrophic counts exceeded 8.0
log cfu g −1, ethanol was significantly produced in anaerobic conditions (Fig. 4A). The same effect, but even more pronounced, is observed in the samples packaged in MA's with CO2 (Fig. 4B). In the
atmosphere 50/0/50, anaerobic ethanol production (fermentation)
is stimulated. This was also observed in the 50/10/40 samples, at
the moment oxygen got depleted (Fig. 3C). In the presence of elevated O2 concentrations the balance shifts towards an aerobic
metabolism.
3.2.3.2. HS levels of aldehydes. Concentrations of nonanal and decanal
did not significantly increase during the whole storage period.
A
3.2.3.4. HS levels of acids and esters. Fig. 5C, D, E and F show the evolution of acetic acid and ethyl acetate in the headspace of the packages
during storage. Acetic acid increases in relation to microbiological
growth were found to be comparable for all the studied MAP conditions. Also for the measured ethyl acetate concentrations, a significant
increase was observed once the psychrotrophic counts exceeded 8 log
cfu g −1. A significantly higher ethyl acetate production was observed
in the samples packaged in MA's with CO2, compared to the other
samples. These findings coincided with the GC-MS results. In fish,
ethyl acetate is thought to be formed from mono-amino, monocarboxylic acids (Whitfield, 1998) or by esterification of alcohols with
carboxylic acids, presumably ethanol and acetic acid. Interestingly,
the production of ethyl acetate seemed not to be influenced by the
presence of oxygen, while the production of ethanol, a possible intermediary metabolite in the production of ethyl acetate was influenced
by the presence of oxygen.
3.2.3.5. HS levels of sulfur compounds. The sulfur compounds monitored in this research were hydrogen sulfide, methyl mercaptan,
dimethyl sulfide, dimethyl disulfide and carbon disulfide. Sulfur
compounds are known to have a low OT, which means that a small
concentration increase may cause a significant sensorial deviation.
According to Devos et al. (1990) the OTs for hydrogen sulfide, methyl
mercaptan, dimethyl sulfide, dimethyl disulfide and carbon disulfide
are 27, 2, 6, 48 and 297 μg/m 3, respectively. Bacterial formation of
sulfur compounds are the result of the breakdown of the sulfur
containing amino acids cysteine and methionine. Hydrogen sulfide
can be considered as a fish spoilage associated compound, evolution
B
9
8.5
pH
8.5
pH
9
8
8
7.5
7.5
7
7
0
2
4
6
8
10
12
time of storage (days)
Fig. 2. pH evolution of the gray shrimp samples, packaged in MAP conditions Δ 0/0/100, □ 0/10/90,
20, ● 50/50/0 (% CO2/O2/N2) in B, during storage at 4 °C. Error bars represent standard deviations.
0
2
4
6
8
10
12
time of storage (days)
0/30/70, ■ 0/50/50 (% CO2/O2/N2) in A and ▲ 50/0/50, ○ 50/10/40,
50/30/
B. Noseda et al. / International Journal of Food Microbiology 160 (2012) 65–75
B
80
70
60
50
40
30
20
10
0
% CO2 in headspace
% O2 in headspace
A
0
2
4
6
8
10
80
70
60
50
40
30
20
10
0
12
0
2
% CO2 in headspace
% O2 in headspace
D
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
4
6
8
10
12
time of storage (days)
time of storage (days)
C
71
80
70
60
50
40
30
20
10
0
0
12
time of storage (days)
2
4
6
8
10
12
time of storage (days)
Fig. 3. Evolution of the % O2 and % CO2 in the headspace of the packaged gray shrimp samples with MAP conditions Δ 0/0/100, □ 0/10/90, 0/30/70, ■ 0/50/50 (% CO2/O2/N2) in
respective A and B and ▲ 50/0/50, ○ 50/10/40, 50/30/20, ● 50/50/0 (% CO2/O2/N2) in respective C and D. Error bars represent standard deviations.
of its concentration are presented in Fig. 6A and B. Although the hydrogen sulfide concentration increased during storage under both
aerobic and anaerobic MAP conditions, Figure A suggests that for anaerobic conditions, the onset of hydrogen sulfide production occurred
at lower microbiological numbers in comparison with the onset of
production under aerobic conditions. However, ultimately all samples
ended with high final concentrations ranging between 200–300 μg/
m 3. In the samples packaged in MA's with CO2 (Fig. 6B), no significant
increases were observed for hydrogen sulfide during storage. Evolutions in methyl mercaptan concentrations are shown in Fig. 6C and
D. Fig. 6C shows that a significant increase in concentration occurred
in the samples packaged in MA's without CO2 for(conditions 0/0/100,
0/10/90, 0/30/70 and 0/50/50) between 8 and 9 log cfu g −1. Once the
limit of 9 log cfu g −1 is reached, relatively high concentration increases from 7 ± 1 μg/m 3 at day 0 to 370 ± 38 μg/m 3 and 159 ±
30 μg/m 3 were observed for MAP conditions 50/10/40 and 50/0/50,
respectively (Fig. 6D). Methyl mercaptan can be a final end product
of the breakdown of methionine, but can also be an intermediate
product of the metabiosis of dimethylsulfide and dimethyldisulfide.
Dimethylsulfide concentrations increased significantly under the anaerobic conditions 0/0/100 and 50/0/50. Increases were observed
from 8 log cfu g −1 on. When its OT is taken into consideration, this
compound might play an important role in causing sensorial deviations. In the headspace of the samples packaged in MA's with O2, a
B
8000
ethanol
g/m3
g/m3
A
significant dimethylsulfide concentration increase was observed
only for condition 50/10/40 after approximately 9 log cfu g −1, when
the O2 in the headspace was depleted. Dimethyldisulfide concentrations remained for all investigated packaging conditions under the
OT (Fig. 6G and H), indicating that this compound has a lower contribution to the off-odors produced. Production, even here, also appears
to be favored in anaerobic storage conditions. Carbon disulfide increased significantly for the MAP conditions 0/0/100, 0/10/90, 0/30/
70 and 0/50/50 between 8 and 9 log cfu g −1. As can be seen in
Fig. 6I, this compound plays an important role in the spoilage of aerobically stored gray shrimp. Yet no significant increases were observed in the samples packaged in a MA with 50% CO2 (Fig. 6J).
These results indicate that sulfur compounds in general play an
important role in the spoilage of gray shrimps. Especially, in aerobically stored gray shrimp, hydrogen sulfide, methyl mercaptan and
carbon disulfide play an important role. This was already observed
for other crustaceae by Chinivasagam et al. (1998) who stated that
amines and sulfides were the major components causing off-odors.
Besides this, in anaerobic (0/0/100) stored gray shrimp samples, the
production of sulfides occurred earlier at lower microbiological
counts. Modifying the atmosphere by addition of 50% CO2 inhibited
hydrogen sulfide and carbon disulfide production. The more oxygen
that is added in the presence of 50% CO2, the lower the concentrations
methyl mercaptan and dimethyldisulfide that are produced probably
ethanol
15000
HS concentration
6000
HS concentration
20000
4000
2000
0
5
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
10000
5000
0
5
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
Fig. 4. Headspace concentrations (μg/m3) of ethanol (A and B) in MAP gray shrimp in relation to the microbiological growth, with Δ 0/0/100, □ 0/10/90,
(% CO2/O2/N2) and ▲ 50/0/50, ○ 50/10/40, 50/30/20, ● 50/50/0 (% CO2/O2/N2). Error bars represent standard deviations.
0/30/70, ■ 0/50/50
72
B. Noseda et al. / International Journal of Food Microbiology 160 (2012) 65–75
B
2000
acetone
g/m3
g/m3
A
HS concentration
1000
500
0
500
250
0
5
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
5
acetic acid
8
9
10
400
acetic acid
300
HS concentration
HS concentration
7
D
200
150
100
50
0
5
6
7
8
9
200
100
0
10
5
E
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
psychrotrophic microbiological counts log cfu/g
F
20
ethyl acetate
g/m3
g/m3
6
psychrotrophic microbiological counts log cfu/g
g/m3
g/m3
C
600
ethyl acetate
450
HS concentration
15
HS concentration
acetone
750
1500
HS concentration
1000
10
5
0
5
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
300
150
0
5
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
Fig. 5. Headspace concentrations (μg/m3) of acetone (A and B), acetic acid (C and D) and ethyl acetate (E and F) in relation to the microbiological growth on gray shrimp packaged in
MA with Δ 0/0/100, □ 0/10/90, 0/30/70, ■ 0/50/50 (% CO2/O2/N2) and ▲ 50/0/50, ○ 50/10/40, 50/30/20, ● 50/50/0 (% CO2/O2/N2). Error bars represent standard deviations.
because the selected micro flora do not produce these VOCs or the
metabolism of the micro flora was altered resulting in a diminished
production of these VOCs.
3.2.3.6. HS levels of amines. Debevere and Boskou stated in 1996 that
the addition of oxygen in packaged cod fillets actively reduced microbiological TMA-N and TVB-N production. This finding was also clearly observed for microbiological trimethylamine (TMA) and dimethylamine
(DMA) production in this study. Fig. 7A describes the TMA concentration evolutions for the conditions (0/0/100, 0/10/90, 0/30/70, 0/50/50)
during storage in relation to the microbiological growth. For the MAP
condition 0/0/100, a significantly higher concentration of 3900 ±
936 μg/m 3 was reached between 8 and 9 log cfu g −1compared to the
concentrations achieved in the samples packaged in MA's containing
O2. These concentrations remained on average below 1000 μg/m3. A
similar evolution is observed for dimethylamine yet in a lower concentration range (Fig. 7C). It is indeed known that O2 actively inhibits the
trimethylaminoxide (TMAO) reducing bacteria, but also exerts an inhibitory effect on the TMAO-reductase activity (Debevere and Boskou,
1996). Fig. 7B and D reveal the concentration evolutions for TMA and
DMA, respectively in the headspace of samples packaged in MAs with
50% CO2 combined with 0, 10, 30 and 50 % O2. Here increase in TMA is
clearly observed for the samples packaged with 0 % O2. This increase
is observed from total psychrotrophic counts of approximately 8 log
cfu g−1. This extent of TMA increase was found to be significantly
lower in the samples packaged MAs with O2. In addition, TMA production also appeared to start at higher microbiological counts, ca. 9 log
cfu g−1, when O2 was present. A similar trend was observed for
dimethylamine, which originates from dimethylamineoxide (DMAO).
Ammonia, an amino acid breakdown product, significantly increased
in all samples during storage (Fig. 7E and F). In the absence of CO2,
high concentrations were reached in the range of 2000 to 4000 μg/m3.
Ammonia production appeared to start at a lower total psychrotrophic
count in samples packaged in 100% CO2 compared to the samples packaged in the MAs containing O2. In the samples packaged in MAs with
50% CO2 (Fig. 7F), a significant increase in the concentration of ammonia
during storage was also observed, but the concentrations were much
lower when compared to those produced under the conditions shown
in Fig. 7E. These results indicate that amines played an important role
in the spoilage process of this fishery product. Furthermore, the high
product pH enhances their effect. Minor amine production may already
cause significant amine headspace concentrations (Noseda et al., 2010).
Even in the presence of CO2, TMA remained a key spoilage metabolite, which indicates that the spoilage micro-organisms or at least
one species of them was able to reduce TMAO. The hypothesis that
P. phosphoreum and/or Shewanella spp. were involved in the spoilage
process is supported by this result.
B. Noseda et al. / International Journal of Food Microbiology 160 (2012) 65–75
B
400
g/m3
g/m3
A
hydrogen sulfide
200
100
0
5
6
7
8
9
10
5
0
5
10
psychrotrophic microbiological counts log cfu/g
D
40
methyl mercaptan
20
10
0
5
500
6
7
8
9
10
methyl mercaptan
0
10
5
g/m3
F
500
dimethylsulfide
400
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
200
dimethylsulfide
150
HS concentration
g/m3
HS concentration
9
125
300
200
100
0
100
50
0
5
5
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
4
g/m3
H
g/m3
G
dimethyldisulfide
20
dimethyldisulfide
15
HS concentration
HS concentration
3
2
1
0
5
6
7
8
9
10
5
0
5
10
psychrotrophic microbiological counts log cfu/g
I
J
20000
carbon disulfide
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
g/m3
g/m3
8
250
psychrotrophic microbiological counts log cfu/g
120
carbon disulfide
90
HS concentration
15000
HS concentration
7
375
HS concentration
HS concentration
30
E
6
psychrotrophic microbiological counts log cfu/g
g/m3
g/m3
C
hydrogen sulfide
15
HS concentration
HS concentration
300
20
73
10000
5000
0
5
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
60
30
0
5
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
Fig. 6. Headspace concentrations (μg/m3) of hydrogen sulfide (A and B), methyl mercaptan (C and D), dimethylsulfide (E and F), dimethyldisulfide (G and H) and carbon disulfide (I and J) in
MAP gray shrimp in relation to the microbiological growth, with Δ 0/0/100, □ 0/10/90, 0/30/70, ■ 0/50/50 (% CO2/O2/N2) and ▲ 50/0/50, ○ 50/10/40, 50/30/20, ● 50/50/0 (% CO2/O2/N2).
Error bars represent standard deviations.
74
B. Noseda et al. / International Journal of Food Microbiology 160 (2012) 65–75
B
5000
g/m3
trimethylamine
4000
HS concentration
HS concentration
g/m3
A
3000
2000
1000
0
5
6
7
8
9
5000
3000
2000
1000
0
10
5
psychrotrophic microbiological counts log cfu/g
D
g/m3
50
dimethylamine
40
30
20
10
0
5
6
7
8
9
g/m3
4000
3000
2000
1000
0
5
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
8
9
10
dimethylamine
40
30
20
10
0
5
HS concentration
g/m3
HS concentration
ammonia
7
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
F
5000
50
10
psychrotrophic microbiological counts log cfu/g
E
6
psychrotrophic microbiological counts log cfu/g
HS concentration
HS concentration
g/m3
C
trimethylamine
4000
500
ammonia
400
300
200
100
0
5
6
7
8
9
10
psychrotrophic microbiological counts log cfu/g
Fig. 7. Headspace concentrations (μg/m3) of trimethylamine (A and B), dimethylamine (C and D) and ammonia (E and F) in MAP gray shrimp in relation to the microbiological
growth, with Δ 0/0/100, □ 0/10/90, 0/30/70, ■ 0/50/50 (% CO2/O2/N2) and ▲ 50/0/50, ○ 50/10/40, 50/30/20, ● 50/50/0 (% CO2/O2/N2). Error bars represent standard deviations.
4. Conclusion
The findings described above show that the presence of O2 in the
headspace of packaged gray shrimp can have a positive as well as a negative effect on the shelf life of the product. The presence of O2 in the atmosphere enhanced microbiological growth and although O2 had an
inhibitory effect on the production of some VOCs (e.g. trimethylamine
and ethanol), one could conclude that O2 had no added value in the
preservation of gray shrimp in a CO2 free atmosphere. However, in
case of the use of high O2 concentrations in combination with high
CO2 concentrations, different observations were made. With these gas
combinations, an inhibitory effect of O2 on the microbiological growth
was clearly observed, most probably because the dominating CO2 tolerant micro-organisms were inhibited by O2. Based on earlier published
research, one could hypothize that O2 sensitive bacteria dominated in
the microbial flora in the sample packaged in MAs with CO2. In the samples packaged in atmospheres with CO2 and O2, the O2 was not consumed
by the microorganisms growing on the samples. Moreover, metabolite
production was inhibited by the O2 present in the CO2 packaged samples.
The production of some sulfur compounds such as methyl mercaptan,
dimethylsulfide and dimethyldisulfide appeared to be inhibited by O2,
and the production of trimethylamine and dimethylamine appeared to
be initiated only at higher microbiological counts in the presence of O2,
resulting in a better sensorial evaluation of the fishery product at the
end of the shelf life. Based on these findings, we could propose a modified
atmosphere containing a high O2 and a high CO2 concentration (e.g. 50%
CO2/50%O2) as an improved gas combination to prolong the shelf life of
cooked and peeled C. crangon stored at 4 °C.
Acknowledgement
The Ghent University ‘Geconcerteerde Onderzoeks Actie’ (GOA
project) ‘Fast and convenient mass spectrometry-bases real-time
monitoring of volatile organic compounds of biological origin’ of the
Flemish government is gratefully acknowledged for the support in
this research through instrumentation credits and by financial
means. We thank Lore Vandermeersch and Karlien De Roo (ENVOC)
for the excellent technical assistance in the identifications of the volatile organic compounds with TD-GC-MS.
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