Sensors and Actuators B 106 (2005) 20–23

Early detection and differentiation of spoilage of bakery products

Rachel Needham a,∗ , James Williams b , Nikki Beales b , Phil Voysey b , Naresh Magan a
a Applied Mycology Group, Institute of Bioscience and Technology, Cranfield University, Silsoe, Bedfordshire MK45 4DT, UK
b Campden and Chorleywood Food Research Association, Chipping Campden, Gloucestershire GL55 6LD, UK

Available online 22 July 2004

Abstract

There is potential to differentiate between different types of bread spoilage using a Bloodhound BH-114 electronic nose. Microbial
spoilage caused by bacteria, yeast and fungi, and enzymic spoilage caused by lipoxygenase can de differentiated from one another and
from unspoiled bread analogues after 48 h using Cluster analysis, prior to signs of visible spoilage. Analysis of the bread analogues with gas
chromatography-mass spectrometry identified volatiles produced by the different spoilage types and unspoiled bread analogues. Microbial
analysis showed that the levels of each micro-organism used increased with time.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Electronic nose; Gas chromatography-mass spectrometry; Bread; Microbial spoilage; Physiological spoilage

1. Introduction ence of visible spoilage using an electronic nose and gas

Early detection of food spoilage is important because
of legislative and consumer pressure to reduce the use of
preservatives, particularly those based on organic acids, in
intermediate moisture bakery products [1]. Food spoilage
causes large economic loses due to waste of raw materials
or product. Microbial spoilage is the major problem caus-
ing deterioration of bread products but there is also a prob-
lem with physiological spoilage such as enzymic spoilage.
Mould spoilage accounts for between 1 and 5% of product
losses depending on the season, type of product being pro-
duced and the method of processing [2].
In the past, control of the safety of foods has mainly been
performed by product testing of both raw materials and end
products. The main problem with performing end product
testing is the high number of samples to be examined before
one can decide on the safety of a product batch, especially
when the micro-organisms are assumed to be heteroge-
neously distributed within the batch [3]. In the last decade
the rapid advances made in the development of electronic
nose technology has attracted much interest in applications
for the detection of spoilage micro-organisms [4].
The objectives of the study were early rapid detec-
tion and differentiation of major microbial contaminants
and physiological spoilage of bread before the pres-
∗ Corresponding author.
chromatography-mass spectrometry.
2. Materials and methods
2.1. Microbial species
Micro-organisms used for microbial spoilage were Bacil-
lus subtilis CRA 14160, Penicillium verrucosum Vmmope
20-07 and Pichia anomala NCPF 3357.
2.2. Bread analogue
Bread analogues (0.95 water activity, pH 5.7) were pre-
pared using a modified method of Patterson and Damoglou
[5]. Dough was prepared by mixing flour (500 g), margarine
(25 g), salt (2.5 g), baking powder (2.5 g), yeast extract
(2.5 g), glycerol (50 g) and water (300 ml). The preservative
calcium propionate was added at 2000 ppm of total dough.
Balls of dough weighing 30 g (for microbial spoilage)
and 15 g (for enzymic spoilage) were flattened into discs,
placed between two 15 cm2 squares of aluminium foil and
autoclaved at 121 ◦ C for 15 min. After autoclaving, whilst
still warm, analogues were sprayed with 60% isopropanol
before placing in Petri dishes. Bread analogues were subse-
quently incubated at 25 ◦ C in sealed containers containing
water/glycerol solutions to maintain the relative humidity.
After 24 h 30 analogues per treatment were randomly inoc-
ulated with 200 ␮l of spore/cell suspension to give approx-

0925-4005/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2004.05.032
 R. Needham et al. / Sensors and Actuators B 106 (2005) 20–23
imately 1 × 106 CFU g-1 or left uninoculated as controls.
For enzymic spoilage 150 ␮l of lipoxygenase at 50 mM was
added. Analogues were placed in sealed containers at 25 ◦ C
containing water/glycerol solutions. Samples were divided
into three pieces after 0, 24, 48, 72, and 96 h and analysed
with an electronic nose, gas chromatography-mass spec-
trometry (GC-MS) and microbial populations enumerated.
2.3. Microbiological analysis
A 10 g sample was aseptically weighed and a 1:10 dilu-
tion performed using Maximum Recovery Diluent (LABM
025762) (MRD). A decimal dilution series was then per-
formed using MRD and 1 ml pour plates prepared. For the
bacteria examined, a total aerobic count was performed us-
ing Plate Count Agar (PCA, Lab M lab 149). The plates
were allowed to set, inverted and incubated at 30 ◦ C ± 1 ◦ C
for 48 h, and all resultant colonies counted. For the yeast and
fungi examined, Rose-Bengal Chloramphenicol Agar (Ox-
oid, CM549, SR078) (RBCA) was used. The pour plates
were allowed to set, then inverted and incubated at 25 ±
1 ◦ C for 5 days and all resultant yeast and filamentous fungi
counted.
2.4. Electronic nose sampling
Samples were analysed using the method of Keshri and
Magan [6]. Samples were placed in 500 ml capacity (25 cm
× 15 cm) sample bags (BDH, UK), inflated with filtered air
and sealed before being left for 1 h at room temperature to
equilibrate. The headspace of the sample was analysed us-
ing a Bloodhound BH-114 electronic nose (Bloodhound sen-
sors Ltd, Leeds, UK) and normalised divergence responses
analysed using XLSTAT version 3.4 (Microsoft Excel add-in
programme). Principle component analysis (PCA), discrimi-
nant function analysis (DFA) and cluster analysis (CA) were
applied to differentiate between spoilage types.
Fig. 1. Cluster analysis dendrogram of microbial and enzymic spoilage of bread analogues based on their volatile profiles after 48 h growth at 25 ◦ C on
0.95 water activity bread analogues.
21
2.5. Extraction of volatiles by headspace solid phase micro
extraction (SPME)
Sample (5 g) was placed into a 20 ml vial, and sealed.
The vial was equilibrated at 75 ◦ C for 15 min with agitation.
The headspace of the vial was then sampled for 15 min at
75 ◦ C (with agitation) using a DVB/Carboxen/PDMS coated
SPME fibre. Volatiles adsorbed onto the fibre were analysed
by thermal desorption at 250 ◦ C in the injector port of a
GC-MS.
2.6. GC-MS analysis of volatiles
Analyses were carried out on a Varian 3800 gas chro-
matograph (GC) and Varian Saturn 2000 ion trap mass spec-
trometer (MS) via a CTC Combi-Pal autosampler. A 25 m
× 0.25 mm fused silica with DB-5 stationary phase column
was used with a helium carrier gas flow rate of 1 ml min−1 .
The desorption temperature was 250 ◦ C with a column tem-
perature of 2 min at 50 ◦ C, then 5 ◦ C min−1 up to 250 ◦ C. The
MS analysis mode used was SCAN 29–350 m/z. Spectral
matching with the Wiley library of mass spectral data tenta-
tively identified peaks. Peak areas were normalised against
the peak area of an internal standard, phenol-D6 , added im-
mediately prior to analysis.
3. Results and discussion
Using cluster analysis (95% confidence level) it was pos-
sible to differentiate between microbial and physiological
(lipoxygenase) spoilage after 48 h (Fig. 1). It was also pos-
sible to distinguish spoiled bread from unspoiled, control
bread analogues. It was not possible to differentiate between
types of microbial spoilage with only the filamentous fun-
gal spoilage caused by Penicillium verrucosum being distin-
guishable. It has however been shown previously [7] that it
is possible to differentiate between bacteria, yeasts and fila-
22 R. Needham et al. / Sensors and Actuators B 106 (2005) 20–23

Fig. 2. Comparison of CFUs for microbial spoilage of bread analogues over time.

mentous fungi in the early phases of spoilage after only 24 h
incubation at 25 ◦ C. Microbiological analysis of the samples
showed the CFUs of the micro-organisms increased with
time but did not differ greatly between type of microbial
spoilage (Fig. 2).
GC-MS analysis identified 59 volatiles from the differ-
ent samples some of which were produced by all spoilage
types and others which were only produced by individual
types. Due to the high number of volatiles identified, for
data analysis, volatiles previously found to be important

Fig. 3. Comparison of volatiles produced by microbial and enzymic spoilage of bread analogues detected by GC-MS after 24 h incubation at 25 ◦ C.
 R. Needham et al. / Sensors and Actuators B 106 (2005) 20–23
in spoilage [8,9] were taken to observe differences in the
volatiles produced (Fig. 3). After 24 h P. anomala was the
only spoilage type to produce pentanol. Methylbenzene was
the only volatile analysed present in greater amounts in con-
trols when compared to spoiled treatments.
4. Conclusions
There is potential for use of an electronic nose to differ-
entiate between types of bread spoilage before visible signs
of spoilage occur. Enzymic spoilage caused by lipoxygenase
could be distinguished from microbial spoilage using clus-
ter analysis after 48 h. GC-MS analysis showed differences
in the levels and types of volatiles produced by the different
spoilage types.
Acknowledgements
This work has been carried out as part of the EU project
(Contract no. QLK1-2000-01763) “Rapid detection of mi-
crobial contaminants in food products using electronic nose
technology”. http://www.e-nose.net.
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Biographies
Rachel Needham received a BSc (Hons) in Microbiology in 1998 and
an MSc in Applied Microbiology and Biotechnology in 2000 from the
University of Wolverhampton. She is currently a PhD student in the Ap-
plied Mycology group at Cranfield University at Silsoe using electronic
nose technology to detect spoilage of bread.
Nikki Beales received a BSc (Hons) in Applied Biology in 1998 from
the University of the West of England and a MSc in Applied Microbi-
ology and Biotechnology with distinction in 2001 from the University
of Wolverhampton. She is currently a Higher Research Officer in the
Preservation and Spoilage group in the Microbiology department at
Campden and Chorleywood Food Research Association and is a part
time PhD student at Wolverhampton University using food ingredients as
natural antimicrobials. Her key areas of responsibility include challenge
testing and shelf life analyses, and research including the RA project:
food ingredients as natural antimicrobials.
Dr Phil Voysey studied Microbiology at the University of Surrey and a
PhD at the University of Bristol. For 8 years he was a Research/Senior
Microbiologist at the Flour Milling & Banking Research Association
(FMBRA), and for a further 3 years the Group Microbiology Labora-
tory Manager at Northern Foods, Nottingham. Currently he is a section
Leader in the Microbiology Department at Campden & Chorleywood
Food Research Association (CCFRA) and his duties involve; managing a
UKAS-accredited media preparation laboratory; organising and running
microbiology training courses and the CCFRA Microbiology Proficiency
Scheme; answering Cereals & Milling Microbiology enquiries; managing
Microbiological Risk Assessment projects; and acting as the Technical
Secretary for the Statistics & Sampling Working Party.
Dr. James Williams works at Campden and Chorleywood Food Research
Association (CCFRA) in the chemistry department. He is a research
chemist specialising in GC-MS and analysis of food samples.
Prof. Naresh Magan FIBiol is Head of the Applied Mycology Group
at Cranfield University, Silsoe and Academic Dean of the Faculty of
Medicine and BioSciences. He runs an internationally recognised re-
search group specialising in early diagnosis and control of spoilage
microorganisms and their toxins from entering the human and animal
food chains. He currently co-ordinates EU wide programmes of re-
search on electronic nose technology and the Mycotoxin Prevention
Cluster. His research interests include forensic mycology, electronic nose
systems, ecophysiology of spoilage microorganisms, biopesticides and
bioremediation strategies using microorganisms.