Supplement bij veertiende jaargang, april 2006 - NVMM

of this spoilage without paying attention to differences in
spoilage potential between individual strains. Detailed
characterization of the strains causing spoilage was and
is often difficult or impossible due to a lack of methods
enabling sufficient resolution to distinguish between closely
related strains. This research was focused on developing a
novel genomics based approach called genomotyping
enabling very detailed and specific discrimination between
closely related strains of spoilage organisms.
Methods: Bacillus spoilage organisms were studied by
using a Smart Man Genomics approach. Genomic DNA
of relevant strains of Bacillus species (Bacillus subtilis,
Bacillus coagulans, Bacillus pumilis, Bacillus licheniformis,
Bacillus sporothermodurans and Bacillus cereus) was used
to construct genomic libraries which were spotted on
microarrays. Genomic DNA of about 50 Bacillus strains
was hybridized to these microarrays containing over 6000
spots and obtained hybridization results were analyzed.
Results: Analysis of obtained data resulted in selections
of biomarkers (DNA fragments) enabling the following
distinctions: 1) Unique markers for specific strains within
one species: 2) Markers specific for one species; 3) Markers
representing multiple but not all species; 4) Markers
present in all species studied. DNA sequences of representative
markers of all these groups have been determined
and will be presented. Also data on analysis of novel strains
will be presented.
Conclusions: Genomotyping enables both discrimination
between closely related strains of one species
and multiple species in one analysis. When based on
Smart Man Genomics this is a generic, flexible and cost
efficient approach for studying microbial spoilage. In the
forthcoming years genomotyping may result in a much
better understanding of specific spoilage causing bacterial
strains and thereby in improved possibilities for spoilage
control.
06.03
Bacterial spores in food processing; molecular detection,
identification and process survival analysis
S. Brul1,2 , L. Hornstra1 , M. de Haan1 , S.J.C.M. Oomes2 ,
A. van Zuylen4 , B.J.F. Keijser3 , F. Schuren3 , H. van der
Spek1 , R. Montijn3 1
Swammerdam Institute for Life Science, Molecular Biology
& Microbial Food Safety, Amsterdam, 2Unilever Research,
Advanced Food Microbiology, Vlaardingen, 3 TNO Quality
of Life, Department of Microbiology, Zeist, 4Unilever, UBF
Sourcing Unit, Oss
In the food processing industry extremely heat-resistant
bacterial endospores create problems due to their ability to
survive classical food preservation treatments (see among
others: Kort et al. AEM 2005:71:3556-64). The spores are
Ned Tijdschr Med Microbiol 2006; 4:Supplement
S33
able to subsequently germinate and form actively growing
vegetative cells that spoil foods and cause disease. With
genomic library micro-array hybridisation we identified
a number of unique genome fragements that correlated
with specific strains and their thermal stress resistance.
These genome fragments were converted into probes for
the Check-Points DNA-chip platform in order to perform
spore detection assays in real foods and food ingredients.
In addition, we showed with the sequenced Bacillus subtilis
laboratory strain that sporulation in the presence of a cocktail
of calcium, magnesium, iron, manganese and potassium
promotes thermal resistance of developing spores. This
observation correlated with an increased expression during
sporulation of genes encoding small acid soluble spore
proteins known to be important in the protection of spores
against a variety of stresses (see Oomes and Brul. IFSET
2004;5:307-16; O’Brien et al., 2006, proteomics manuscript
in preparation). Degradation patterns of specific molecular
markers upon spore thermal injury were found to be
predictive for the outgrowth behaviour of B. subtilis spores.
This finding was quantitatively measured and patented
(Keijser, 2005). Furthermore, the molecular program that
forms the basis of spore germination has been analysed
using genome-wide expression analysis. Here we observed
that genes involved in DNA-repair were transiently expressed
in germinating wild-type spores (Keijser et al., 2005,
paper in preparation). Of the genes that were specifically
expressed during spore germination individual knock-out
mutants were generated. Some of these mutants showed a
significantly delayed outgrowth but none were completely
perturbed in germination and outgrowth. Implications
of this for the inhibition of germination-specific damage
repair processes through mild combination preservation
techniques are discussed.
06.05
Spore germination of thermally injured Bacillus subtilis
spores
L.M. Hornstra1 , B.J.F. Keijser1 , S.J. Oomes2 , H. van der
Spek1 , M. de Haan1 , S. Brul1 1University of Amsterdam, Swammerdam Institute for
Life Sciences, Amsterdam, 2Research Center Unilever,
Microbiological Control, Vlaardingen
Over the years, the food industry has developed safe
food preservation methods to ensure healthy, safe and
tasty food products. Gradually, consumers demands
have changed to fresh-like foods were a minimum level
of processing has been applied to maintain positive
aspects of fresh food without compromising food
safety. Unfortunately, mild processing provides opportunities
for pathogenic and spoilage organisms, in
particular spore forming organisms as the spores are