International Journal of Food Microbiology 166 (2013) 270279

Contents lists available at ScienceDirect

International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Characterization of aerobic spore-forming bacteria associated with

industrial dairy processing environments and product spoilage
Genia Lcking a, Marina Stoeckel b, Zeynep Atamer b, Jrg Hinrichs b, Monika Ehling-Schulz c,
a
b
c

Department of Microbiology, Central Institute for Food and Nutrition Research, Technische Universitt Mnchen, 85350 Freising, Germany
Department Dairy Science and Technology, Institute of Food Science and Biotechnology, Universitt Hohenheim, 70599 Stuttgart, Germany
Functional Microbiology, Department of Pathobiology, University of Veterinary Medicine Vienna, 1210 Vienna, Austria

a r t i c l e

i n f o

Article history:
Received 12 March 2013
Received in revised form 3 July 2013
Accepted 7 July 2013
Available online 16 July 2013
Keywords:
Spore-forming bacteria
Dairy products
Dairy processing environment
Spore heat resistance
Food spoilage
Cytotoxic potential

a b s t r a c t
Due to changes in the design of industrial food processing and increasing international trade, highly
thermoresistant spore-forming bacteria are an emerging problem in food production. Minimally processed
foods and products with extended shelf life, such as milk products, are at special risk for contamination
and subsequent product damages, but information about origin and food quality related properties of highly
heat-resistant spore-formers is still limited. Therefore, the aim of this study was to determine the biodiversity, heat resistance, and food quality and safety affecting characteristics of aerobic spore-formers in the
dairy sector. Thus, a comprehensive panel of strains (n = 467), which originated from dairy processing environments, raw materials and processed foods, was compiled. The set included isolates associated with recent food spoilage cases and product damages as well as isolates not linked to product spoilage. Identication
of the isolates by means of Fourier-transform infrared spectroscopy and molecular methods revealed a large
biodiversity of spore-formers, especially among the spoilage associated isolates. These could be assigned to
43 species, representing 11 genera, with Bacillus cereus s.l. and Bacillus licheniformis being predominant. A
screening for isolates forming thermoresistant spores (TRS, surviving 100 C, 20 min) showed that about
one third of the tested spore-formers was heat-resistant, with Bacillus subtilis and Geobacillus
stearothermophilus being the prevalent species. Strains producing highly thermoresistant spores (HTRS, surviving 125 C, 30 min) were found among mesophilic as well as among thermophilic species. B. subtilis and
Bacillus amyloliquefaciens were dominating the group of mesophilic HTRS, while Bacillus smithii and
Geobacillus pallidus were dominating the group of thermophilic HTRS. Analysis of spoilage-related enzymes
of the TRS isolates showed that mesophilic strains, belonging to the B. subtilis and B. cereus groups, were
strongly proteolytic, whereas thermophilic strains displayed generally a low enzymatic activity and thus
spoilage potential. Cytotoxicity was only detected in B. cereus, suggesting that the risk of food poisoning by
aerobic, thermoresistant spore-formers outside of the B. cereus group is rather low.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Spore-formers are important contaminants in the dairy industry
because they can signicantly affect food quality and safety. However,
effective control of these bacteria in milk products and the processing
environment is still a difcult task, since knowledge about their origin
and food quality related characteristics, such as thermoresistance or
spoilage and toxic potential, is generally limited.
As spore-forming bacteria are ubiquitous in nature, they are also
present in many raw materials and dry ingredients of processed food.
For the dairy food production chain, the farm environment and raw

Corresponding author at: Functional Microbiology, Department of Pathobiology,

University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210 Vienna, Austria.
Tel.: + 43 125077 2460; fax: + 43 125077 2479.
E-mail address: monika.ehling-schulz@vetmeduni.ac.at (M. Ehling-Schulz).
0168-1605/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijfoodmicro.2013.07.004

milk, which holds spore counts up to 104 cfu/mL, are important contamination sources (Coorevits et al., 2008; Crielly et al., 1994;
Scheldeman et al., 2005; te Giffel et al., 2002). Moreover, the hydrophobic properties of endospores and their general resistance towards heat,
desiccation or disinfectants allow them to attach to processing equipment and survive cleaning procedures (Andersson et al., 1995; Ryu
and Beuchat, 2005; Simmonds et al., 2003). Generally, pasteurization
fails to effectively kill the heat-resistant endospores, limiting the possibilities of producing minimally processed foods or extending the shelf
life of pasteurized products. In addition, the food industry is increasingly
confronted with particular tolerant or resistant spore-formers, presumably due to the use of new ingredients and processing technologies in
connection with the production of new food products such as convenience foods (Heyndrickx, 2011). For instance, high contamination
rates with heat-resistant spore-formers have been reported from milk
powder, cocoa powder and gelatin extracts (De Clerck et al., 2004;
Lima et al., 2011; Rckert et al., 2004; Scott et al., 2007; Witthuhn et

G. Lcking et al. / International Journal of Food Microbiology 166 (2013) 270279

al., 2011). Also, dehydrated components like herbs, spices or dried vegetables of canned ready-to-eat food products were shown to be important contamination sources of aerobic spore-formers (Oomes et al.,
2007; Postollec et al., 2012). Especially, the emergence of highly
heat-resistant endospores (HRS) even surviving ultrahigh temperature
treatment aimed at obtaining commercially sterile products, has increased the concerns, whether contaminated ingredients in combination with harsh food processing conditions might enhance the
adaptation and selection of extremely resistant spore producers
(Pettersson et al., 1996; Postollec et al., 2012).
Growth of spore-forming bacteria in dairy foods can negatively
affect both, product quality and product safety. Certain spore-formers
pose a risk of causing food poisoning by the production of toxins.
Among the aerobic spore-formers, Bacillus cereus is well known for its
potential to cause two types of food poisoning syndromes, an emetic
type by the production of the heat-stable cereulide and a diarrheal type
by the production of several heat-labile enterotoxins (Ehling-Schulz
et al., 2004; Ehling-Schulz et al., 2011; Stenfors Arnesen et al., 2008).
Infrequently, also other Bacillus species, such as Bacillus licheniformis,
Bacillus amyloliquefaciens and Bacillus pumilus have been reported to
produce toxic components, which may play a role in food poisoning
(Mikkola et al., 2004; Salkinoja-Salonen et al., 1999; Suominen
et al., 2001; for review see Ehling-Schulz and Messelhusser, 2013;
Logan, 2012). Moreover, contamination with spore-formers can
lead to microbial growth and premature spoilage of food products.
The production of microbial enzymes like proteases, lipases and
phospholipases can provoke changes in texture up to structural defects and typical off-avors. Well known are the bitty cream and
sweet curdling defects caused by the lecithinase- and proteolytic
activity of B. cereus, but also at-sour spoilage as well as bitter, fruity
or rancid off-avors due to other spore-formers have been described
for dairy products (Heyndrickx and Scheldeman, 2002; Huis in 't
Veld, 1996; Kalogridou-Vassiliadou, 1992; Meer et al., 1991). Food
spoilage and failure of food preservation, despite modern food technology and sterilization techniques, can lead to signicant economic
losses and/or reputational damage of food companies. With respect
to the growing discussion about food safety and security, a better
knowledge on origin, identity and food quality related characteristics might also help to improve control measures for spore-formers,
thereby contributing to the reduction of the food loss due to microbial
spoilage.
Although the diversity of spore-formers in raw milk and milk products has been studied in some detail, information on the spoilage and
toxigenic potential of highly heat-resistant spore-formers in the dairy
sector is generally lacking. Thus, the aim of this study was, beside
the determination of the biodiversity of aerobic spore-formers in the
dairy processing environment and food products, to screen for highly
thermoresistant spores and to decipher the food spoilage- and toxigenic
potential of the latter.
2. Materials and methods
2.1. Bacterial strain collection and growth conditions
In total, 467 aerobic spore-forming isolates were included in this
study: 379 food spoilage associated strains linked to cases of damage
were isolated from dairy products and industrial processing environments by 28 different dairies and food companies during a two-year
period. The origin of these contaminants was mainly dairy end products like pudding, milk or mixed milk drinks. Moreover, 10 isolates
originated from swab samples taken from the food processing environment (industrial equipment) and 93 isolates were obtained from
the dairy processing environment (Table 1).
This strain set was complemented by 88 spore-formers isolated from
98 food samples without visible trace of food spoilage, including raw materials, unprocessed intermediate products, dehydrated ingredients and

271

processed milk products (Table 2). For isolation of potentially heatresistant spore forming bacteria, foods were diluted with Ringer solution
(depending on the texture of the food matrix between 1:2 and 1:10) and
heated (20 min, 100 C) in an autoclave. After enrichment with BHI broth
(1:10) and incubation for 24 h, samples were plated on BHI agar and
cultivated at 30 C and 55 C, respectively, in order to gain mesophilic
as well as thermophilic isolates. All strains were routinely grown in
BHI broth (Merck) or on BHI agar plates supplemented with 1 mg/L
vitamin B12 and incubated at appropriate temperatures (30 C, 37 C
or 55 C) for 24 h. In addition, the isolates forming thermoresistant
spores (TRS) were tested for their growth and germination potential
on plate count skim milk agar (PC with 0.1% skim milk powder) at
10 C for 7 days.
2.2. Identication of isolates by FT-IR spectroscopy and partial
gene sequencing
All spore-formers were identied by Fourier-transform infrared
(FT-IR) spectroscopy as described previously (Oberreuter et al., 2002;
Wenning et al., 2008). In brief, strains were grown as lawns on tryptic
soy agar (TSA, Oxoid) for 24 h at 25 C or 55 C. A small amount of
cell material was suspended in 100 l sterile deionised water, applied
onto the ZnSe sample carrier and dried for 45 min at 40 C. Spectra
were recorded in transmission with a IFS 28B FT-IR spectrometer
(Bruker Optics) and analyzed using the OPUS software (version 3.1,
Bruker). Evaluation included a quality check of spectra and their identication by comparison with an existing reference database consisting of
about 850 spectra from 75 different spore-forming species and 13 genera. For univariate hierarchical cluster analysis (HCA) spectral regions
from 30002800 cm1, 18001500 cm1, 15001200 cm1, 1200
900 cm1 and 900700 cm1 with weight factor 1 and repro-level 30
were used. Spectral distances were calculated from second derivatives
of original spectra using the Ward's algorithm.
In case that FT-IR spectroscopy did not reveal unequivocal results,
species were determined by partial 16S rDNA sequencing as reported
previously (Fricker et al., 2011). Members belonging to the Bacillus
subtilis group were identied by partial sequencing of the gyrA gene,
using the oligonucleotide primers described by Chun and Bae (2000).
2.3. Spore production and thermal pre-screening
One colony from BHI agar was used to inoculate 3 mL BHI medium.
This pre-culture was incubated overnight at either 30 C (mesophilic
isolate), 37 C (Bacillus sporothermodurans) or 55 C (thermophilic

Table 1
Origin of spore-formers linked to food spoilage and cases of product damage.
Origin

Number of isolates

Pudding/dessert
Milk
Rice pudding
Soft or cream cheese
Mixed milk drinks
Protein powder
Cream
Mascarpone
Soy-based product
Yoghurt
Whole egg
Quiche
Vanilla sauce
Apple juice
Swab samples (industrial equipment)
Dairy processing environment
Total

74
40
34
34
24
16
13
11
9
7
6
5
2
1
10
93
379

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G. Lcking et al. / International Journal of Food Microbiology 166 (2013) 270279

Table 2
Origin of potentially heat-resistant spore-formers isolated from samples showing no
visible trace of spoilage.
Origin (food category and product)

Amount of contaminated Number

of isolates
food products/Total
amount

Raw materials and unprocessed components

10/12
Unheated intermediate products or
ingredients (of milk drink, rice pudding,
pudding, cheese)
Raw milk
1/1
Dehydrated ingredients
Milk powder (skim and whole milk)
8/11
Cocoa powder
8/13
Additives (binder, stabilizer, vanillin, poppy) 2/11
Spices (pepper, cinnamon, chili, ginger)
5/9
Dried herbs and mushrooms
2/5
Couverture
2/3
Processed foods
Rice pudding
4/10
Cheese
2/5
Yoghurt and curd
2/3
Mixed milk drinks
2/5
Pudding (chocolate, vanilla, semolina)
2/8
UHT milk
0/2
Total
50/98

27

4
13
11
5
8
4
2
5
3
3
2
1
0
88

isolate). For mesophilic isolates and B. sporothermodurans, 200 l of the

pre-culture was spread onto 2 SG agar plates (Nicholson and Setlow,
1990) and incubated at 30 C or 37 C, respectively for 35 days or
until N 80% free spores were observed as determined by phase contrast
microscopy. For thermophilic isolates, 200 l of the pre-culture was
spread onto a sporulation agar containing 3 g/L beef extract, 5 g/L peptone, 4 g/L yeast extract, 20 g/L agar and 0.1 mM MnCl2 and incubated
at 55 C for 7 days or until N80% free spores were obtained, respectively.
For isolation of heat-resistant spore-formers, one loopful of spores
from agar plates was suspended in 3 mL pre-cooled phosphate buffer
(2 mM KH2PO4 and 8 mM K2HPO4). The suspension was vigorously
vortexed and adjusted photometrically to an OD600nm of 0.15. Spore
samples were heated in glass tubes at 100 C for 20 min in an autoclave and afterwards immediately cooled on ice. Spore suspensions
were subsequently plated onto BHI agar and incubated for 2448 h
at 30 C, 37 C or 55 C, respectively. Spore-formers that survived this
treatment and grew well were classied as thermoresistant, while isolates that did not grow or grew poorly (5 colonies/plate) were excluded
from further experiments.

2.4. Heat treatment of spores

A previously described batch heating system was used for thermal
resistance studies at 110 C, 120 C and 125 C (Dogan et al., 2009;
Witthuhn et al., 2011). In brief, spore biomass from agar plates was
harvested by centrifugation (2218 g, 7 min, 2 C) and washed 5 times
with phosphate buffer. A thermal treatment step at 80 C for 10 min
was followed by 13 additional washing steps. Spore suspensions were
adjusted to a nal concentration of 35% ethanol. The suspensions were
kept at 2 C for 23 days in ethanol, then washed three times and stored
at 2 C in phosphate buffer. The prepared spore suspensions in phosphate
buffer contained about 105109 cfu/mL. Thermal treatments of spore suspensions in the batch system were performed in screw-capped stainless
steel tubes (50 mm 10 mm, wall thickness 2 mm) with a volume of
1.5 mL for 30 min at 110 C, 120 C and 125 C. After heating, the samples were diluted with Ringer solution and plated onto appropriate
solid media. Plates were incubated at 30 C or 55 C for 96 h. The number
of viable cells was determined before and after the heat treatment. Each
heating experiment was conducted three times, independently.

2.5. Screening for enzymatic activity

To determine the spoilage potential of isolates, the enzymatic activity
of proteases, lipases, phospholipases, -galactosidases and hemolysins
was determined using the following solid media: Skim milk agar,
tributyrin agar, egg yolk agar and X-Gal agar were prepared as described
previously (De Jonghe et al., 2010). Columbia agar with sheep blood
(Oxoid) was used to test the bacterial strains for -hemolysis. A single
colony of each strain was streaked on the different agars and plates
were incubated at the appropriate temperature (30 C, 37 C or 55 C)
for 24 to 48 h.
2.6. Cytotoxicity assays using Vero and HEp-2 cells
20 mL BHI medium was inoculated with an overnight pre-culture
(1:100 dilution) and cultures were incubated in 200 mL asks with
rotary shaking at 30 C, 37 C or 55 C, respectively. After 6 h of growth
5 mL samples of the cultures were harvested (10,000 rpm, 10 min) and
cell-free supernatants obtained by ltration (0.2 m pore size) was
used to test for heat labile toxins, such as the B. cereus enterotoxins. To
1 mL sample 10 l of a 0.1 M Na2-EDTA solution was added and cell cytotoxicity was determined employing a Vero cell bioassay described previously (Dietrich et al., 1999).
After 24 h of growth, additional culture samples (1 mL) were
removed and autoclaved (15 min at 120 C) to test the cultures for
the presence of heat stable cytotoxic compounds, such as the emetic
B. cereus toxin cereulide. For the latter test an established HEp-2
cell-based bioassay was used. Cytotoxicity was determined as described previously (Lcking et al., 2009).
3. Results
3.1. Biodiversity of spoilage associated and non-spoilage associated
aerobic spore-formers in dairy processing and food products
379 spore-forming strains linked to spoilage, which have been directly isolated by food companies or dairies, were typed by FT-IR
spectroscopy. Spectral reference libraries were used for species identication and in case that the spectral databases did not reveal an unequivocal result, partial 16S rDNA or gyrA sequencing was used for
determination of the species. Thus, the 379 isolates were assigned
to 43 different species, representing 11 genera (Table 3). Most of
the isolates (75%) belonged to the genus Bacillus, 9% to Geobacillus
and further 6% to Paenibacillus. The vast majority of the species
were mesophilic, while only seven strictly thermophilic species
were detected (representing 11% of the isolates). B. cereus s.l. was
the species group most frequently encountered: 23% of the isolates
belonged to this group of bacteria, which is well known not only for
its food spoilage but also for its food poisoning potential. Further
dominant species were the thermotolerant species B. licheniformis
(19%), the mesophilic species B. subtilis (10%) and the thermophilic
species Geobacillus stearothermophilus (8%) (Table 3). In addition to
the spoilage associated isolates, potentially heat-resistant aerobic
spore-forming strains were isolated from different raw materials,
food ingredients and processed milk products, which did not show
any visible traces of spoilage. These food samples (n = 98) were categorized into raw materials or unheated intermediate products (n = 13),
dehydrated ingredients (n = 52) and processed end products (n = 33)
(Table 2). After a selective heat step, aerobic spore-forming bacteria
were detected in about 50% of these food samples and a total of 88
isolates were collected. For the raw and unprocessed intermediate
products, 85% of the samples were contaminated, often simultaneously
with several different species from up to three genera. A high contamination rate (52%) was also found for dry ingredients, such as milk
powder, cocoa powder and spices. For processed products, e.g. rice
pudding, pudding, cheese or mixed milk drinks, a somewhat lower

G. Lcking et al. / International Journal of Food Microbiology 166 (2013) 270279

Table 3
Identication of spore-formers associated with dairy food processing and dairy foods.
Genus and species identication

Bacillus
Bacillus subtilis
Bacillus amyloliquefaciens
Bacillus exus
Bacillus sporothermodurans
Bacillus licheniformistt
Bacillus cereus group
Bacillus circulans
Bacillus coagulans
Bacillus pumilus
Bacillus oleronius
Bacillus simplex
Bacillus silvestris
Bacillus megaterium
Bacillus chitinolyticus
Bacillus rmus
Bacillus thermoamylovoranst
Bacillus massiliensis
Bacillus smithiit
Bacillus niabensis
Bacillus thermolactist
Geobacillus
Geobacillus stearothermophilust
Geobacillus pallidust
Geobacillus thermoleovoranst
Paenibacillus
Paenibacillus polymyxa
Paenibacillus amylolyticus
Paenibacillus woosongensis
Paenibacillus macerans
Paenibacillus glucanolyticus
Paenibacillus lactis
Paenibacillus lautus
Paenibacillus ginsengisoli
Paenibacillus barengoltzii
Paenibacillus taichungensis
Brevibacillus
Brevibacillus agri
Brevibacillus borstelensis
Brevibacillus parabrevis
Brevibacillus laterosporus
Lysinibacillus
Lysinibacillus fusiformis
Anoxybacillus
Anoxybacillus avithermust
Aneurinibacillus
Aneurinibacillus thermoaerophilust
Alicyclobacillus
Alicyclobacillus acidocaldariust
Alicyclobacillus tengchongensist
Virgibacillus
Virgibacillus halophilus
Virgibacillus pantothenicus
Sporosarcina
Sporosarcina globispora
Sporosarcina aquimarina
Sporosarcina contaminans
Sporolactobacillus
Sporolactobacillus terrae
Total
t
tt

Number of isolates
(associated to
spoilage)
39
11
1
21
71
89
18
2
13
3
5
2
2
1
2
2
2

Number of isolates
(not connected to
spoilage)
13
3
1
27
1
2
2
1

10
5
1
1

32
1

9
5
1

4
6
1
1
7
2
1
1
1
1
6
1
5
1
12
3

2
1
1
1
1
1
1
1
379

88

Thermophilic species.
Thermotolerant species.

contamination rate was observed: 36% of these samples were affected

(Table 2). Species identication of the 88 isolates revealed 18 different
species belonging to ve genera (Table 3). Likewise to the group of
spoilage associated isolates, Bacillus was the predominant genus in the
group of non-spoilage associated isolates (accounting for 76% of
the isolates). However, strains of B. licheniformis were detected
most frequently (accounting for 31% of the isolates), while B. cereus

273

was dominating the group of product damage associated isolates.

Among the thermophilic species, Bacillus thermoamylovorans (11%)
and G. stearothermophilus (10%) were found most frequently. Almost
40% of the isolates turned out to be strictly thermophilic and could be
assigned to eight different species, namely G. stearothermophilus,
Anoxybacillus avithermus, B. thermoamylovorans, Bacillus smithii,
Bacillus thermolactis, Geobacillus pallidus, Geobacillus thermoleovorans
and Aneurinibacillus thermoaerophilus (Table 3).
3.2. Screening for thermoresistant spore-formers
In order to characterize the spore-formers with respect to their heat
resistance, a thermal screening including all 467 strains was carried out.
Spores were produced on sporulation agar, which was successful for the
vast majority of isolates; only for 12% of the strains no spores were
detected by microscopy under the chosen conditions. The respective
spore suspensions were heated for 20 min at 100 C. As a result, spores
from 27% of the tested strains (n = 126) survived this heat treatment,
hereinafter referred to as thermoresistant spore-formers (TRS). The
remaining strains either did not grow at all (n = 330) or grew very
poorly (n = 11) after heating of their spore suspensions.
With respect to their food origin, the TRS isolates were detected in
all food categories analyzed: 14% were obtained from raw material
and unprocessed intermediate products, 23% from dehydrated ingredients and 56% from processed food (with and without visible spoilage)
(data not shown).
The thermoresistant spore-formers could be assigned to ve genera
and 17 different species. Half of the strains belonged to seven mesophilic
species and the other half to ten thermophilic or thermotolerant species.
The most dominant species producing heat resistant spores was B. subtilis
(26%), followed by G. stearothermophilus (17%) and B. licheniformis (11%).
Moreover, B. sporothermodurans (10%) and B. amyloliquefaciens (8%)
were quite frequently encountered among the TRS isolates (Fig. 1). To
determine the growth and germination potential at lower temperature,
all 126 TRS strains were tested for their growth behavior at 10 C on
plate count skim milk agar. Thirteen isolates (10%) were able to grow
under these conditions. Among them were isolates of the B. cereus
group (2) and B. simplex (2), which grew very well at 10 C. Furthermore, isolates of B. exus (1), B. amyloliquefaciens (4) and B. subtilis
(4) grew slowly under these conditions. Almost all isolates (96%)
were able to germinate and grow after placing them from 10 C back
to their preferred temperature at 30 C, 37 C or 55 C, respectively
(Fig. 1).
In addition, FT-IR spectroscopy was employed to generate metabolic ngerprints from 45 representative TRS isolates. Multivariate
statistical analysis of spectral data, using hierarchical cluster analysis
as an unsupervised method, revealed a high heterogeneity, thus
conrming the large biodiversity of this group of spore-formers. Although mesophilic and thermophilic isolates formed clearly separated
clusters, TRS strains of several species, such as B. thermoamylovorans
and B. amyloliquefaciens, were not grouped into dened clades (Fig. 2).
From the panel of TRS isolates (n = 126), 35 representative strains,
including four mesophilic and seven thermophilic/thermotolerant species, were chosen for further heat inactivation experiments at temperatures N100 C. Puried spore suspensions were heated for 30 min in a
batch-heating system using the following temperatures: 110 C, 120 C
and 125 C. The cell counts (log cfu/mL) before and after heating are
provided in Table 4. At 110 C, spores of eight strains (one mesophilic,
two thermotolerant and ve thermophilic strains) were completely
inactivated (log cfu/mL 1). The cell reduction of the other strains varied largely among the strains, ranging from 0 to 7 log units. At 120 C,
spores of 12 isolates were completely inactivated, while 15 survived
this heat treatment, showing spore count reduction of 3 to 7 log units.
Spores of eight strains, four mesophilic and four thermophilic ones,
even survived a heating in the batch system at 125 C for 30 min and
were designated as highly thermoresistant spore-formers (HTRS). The

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G. Lcking et al. / International Journal of Food Microbiology 166 (2013) 270279

35
spore survival at 100C for 20min

30

Number of isolates

germination after cold storage

vegetative growth at 10C

25
20
15
10
5
0

Fig. 1. Species distribution and growth properties of 126 TRS isolates, whose spores survived heat treatment at 100 C for 20 min. Vegetative growth was tested on plate count skim
milk agar at 10 C and germination potential was determined by placing the isolates back to 30 C, 37 C or 55 C, respectively, after a cold storage of 7 days.

HTRS isolates belonged to the species B. subtilis, B. amyloliquefaciens,

B. exus, B. smithii and G. pallidus. Strains B. subtilis F98 and B. smithii
F78 displayed with 4.5 log units the lowest cell count reduction under
the tested conditions (Table 4).

found to be cytotoxic towards Vero cells and none of the tested strains
showed cytotoxicity in the HEp-2 assay (Fig. 3).

4. Discussion
3.3. Spoilage and cytotoxic potential of thermoresistant spore-formers
To assess the potential risk of spoilage and food poisoning caused
by heat-resistant spore-formers, isolates were screened for enzymatic
activity and cytotoxicity. All identied TRS strains, including 63
mesophilic and 63 thermophilic or thermotolerant isolates, were tested
for their production of spoilage-related enzymes by a variety of selective media (Fig. 3). Although high or low enzymatic activity provides
no direct evidence for spoilage or no spoilage, it can serve as a rst indicator regarding the spoilage potential of food isolates. Fifty percent of
the tested strains showed proteolytic, and 23% of the tested strains
hemolytic activity. The vast majority of these strains were mesophilic,
belonging to B. subtilis, B. amyloliquefaciens and B. cereus group. However,
proteolytic activity was also found in 62% of the tested B. licheniformis
strains. Interestingly, when grown at 55 C (instead of 30 C), even
more B. licheniformis strains (92%) displayed a proteolytic phenotype
(data not shown). Lipolytic activity was detected in 28% of the
isolates, predominantly in B. licheniformis and strains of thermophilic species, such as B. thermoamylovorans, G. pallidus, A. avithermus
and A. thermoaerophilus. Furthermore, -galactosidase activity was
tested since this enzyme catalyzes the breakdown of lactose to glucose and galactose, resulting in signicant spoilage of dairy products.
-Galactosidase activity was found in 18% of the isolates, mainly belonging to thermophilic species. Phospholipase activity was only observed in B. cereus strains (Fig. 3). Besides the activity of secreted
enzymes, the cytotoxicity of the TRS strain set was determined using
two different bioassays: the HEp-2 cell assay for the detection of cytotoxic activity caused by heat stable toxins, such as the emetic B. cereus
toxin cereulide, and the Vero cell assay for the detection of heat labile
toxins, such as the B. cereus diarrheal associated enterotoxins. Although
all 126 heat-resistant spore-formers, representing 17 different species,
were tested for their cytotoxic potential, only one B. cereus strain was

The identication and characterization of a comprehensive collection (n = 379) of spoilage associated spore-forming isolates, provided
by various food companies and dairies, revealed a very large taxonomic
diversity covering as much as 43 species from 11 different genera with
growth capacities over a wide temperature range. Ninety percent of the
strains were assigned either to Bacillus, Geobacillus or Paenibacillus,
which is in line with previous reports listing these spore-forming genera as the prominent ones in the dairy sector (Coorevits et al., 2008;
Huck et al., 2007; Ivy et al., 2012; Scheldeman et al., 2006). Thus, our
study emphasizes the importance of not only psychrotolerant but also
mesophilic and thermophilic spore-formers in the dairy processing
environment. The presence of aerobic spore-formers in dairy food products is often associated with the reduction of shelf life and food spoilage,
as soon as external conditions are favorable for bacterial growth. For instance, although only a small subset of the TRS strains isolated during
this study were able to grow on skim milk agar at low temperature, almost all of the isolates germinated and grew at higher temperatures,
even after a pre-incubation step at 10 C for 7 days.
For the spoilage associated isolates, members of the B. subtilis group
and the B. cereus group were identied most frequently, indicating that
these contaminants are the most important spoilage organisms. B. cereus
group strains are, due to the toxin production capacity of certain strains,
not only a food quality but also a food safety problem and more effective
control measures are needed (Ehling-Schulz et al., 2011; Stenfors
Arnesen et al., 2008). Only 11% of the spoilage associated isolates were
strictly thermophilic, underpinning the minor role of thermophilic species in the context of spoiled dairy products. However, almost 40% of
the spore-formers isolated from food samples without visible trace of
spoilage turned out to be strictly thermophilic, demonstrating that thermophilic species are common contaminants of various food products
and ingredients.

G. Lcking et al. / International Journal of Food Microbiology 166 (2013) 270279

275

Fig. 2. Hierarchical cluster analysis of FT-IR spectral data derived from a representative subset of thermoresistant spore-formers (n = 45). Spectral distances were calculated from
second derivatives of spectra using the Ward's algorithm.

The thermal screening for isolates producing heat-resistant spores

showed that 27% of the 467 isolates included in this study could be
assigned to the group of thermoresistant spores (TRS) surviving a heat
treatment at 100 C for 20 min. When a subset of TRS was tested at
higher temperatures, 23% of spores even survived a heat treatment of
30 min at 125 C (HTRS). All detected species among the group of TRS,
except Bacillus simplex, A. avithermus and Alicyclobacillus acidocaldarius,
have also been found in heat treated samples (100 C for 30 min) from
dairy farms (Scheldeman et al., 2005), emphasizing that the primary
production and raw milk are important but not the sole contamination
sources for spore-formers in the dairy production chain. For instance,
27% of the TRS and even 75% of the HTRS were derived from non-dairy
related ingredients, such as cocoa powder and spices. The latter groups
of ingredients turned out to be important yet underestimated contamination sources in dairy production that require special attention and
further research. Very recently, Lima et al. (2012) reported on the
occurrence of heat-resistant spore-formers (surviving heating at 110 C
for 5 min) in samples taken from different stages of a cocoa powder production line. In particular, spores of B. subtilis subsp. subtilis were found
to be able to survive the cocoa powder production chain (Lima et al.,

2012) and, as shown by our current work, may subsequently enter the
dairy processing environment (see Tables 2 and 4, respectively).
Among the TRS and the HTRS isolates, the amount of mesophilic
and thermophilic/thermotolerant strains was identical in our study,
illustrating the production of highly heat-resistant spores to be common
for both types. Nevertheless, some of the spoilage relevant genera, such
as Paenibacillus, Brevibacillus and Lysinibacillus were not found among
the TRS isolates and only two out of the 90 tested B. cereus group strains
were recovered after 20 min at 100 C. In total, the spores of two thirds
of the 467 spore-formers, mainly isolated from processed milk products,
were sensitive towards this heat treatment and are presumably not able
to survive common food processing procedures. Instead, they may present post-heat treatment contaminants, e.g. due to recontamination occurring during lling or packing of foods. However, it cannot be excluded
that the spore resistance properties of these strains might have been
originally higher and got lost during cultivation and sporulation under
laboratory conditions. Similar observations were made in previous studies (Huemer et al., 1998; Kort et al., 2005; Lima et al., 2011; Scheldeman
et al., 2006). It is well known that the heat resistance of spores is
inuenced by many external factors, such as the sporulation, maturation

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G. Lcking et al. / International Journal of Food Microbiology 166 (2013) 270279

Table 4
Thermal screening: Survival of spores after heating for 30 min at 110 C, 120 C and 125 C, respectively.
Strain

Origin

Cell count (log cfu/mL) before and after heating at different temperatures:
110 C

120 C

Initial
Mesophilic
B. subtilis F46
B. subtilis F59
B. subtilis F68
B. subtilis F98
B. subtilis H244c
B. amyloliquefaciens F55
B. amyloliquefaciens F62
B. amyloliquefaciens F85
B. amyloliquefaciens F45PL
B. amyloliquefaciens H298c
B. exus F56
B. exus F5PL
B. circulans H291c
Thermotolerant
B. licheniformis F72
B. licheniformis F2
B. licheniformis F14c
Thermophilic
B. thermoamylovorans F23
B. thermoamylovorans F34c
B. thermoamylovorans F37
B. thermoamylovorans F41c
B. thermoamylovorans F42
B. thermoamylovorans F61
B. thermoamylovorans F12
B. smithii F64
B. smithii F78
B. smithii F93
G. stearothermophilus F4c
G. stearothermophilus F52
G. stearothermophilus F74
G. pallidus F40c
G. pallidus F43c
G. pallidus F8c
A. avithermus F26c
A. avithermus F48c
A. thermoaerophilus F18c
a
b
c

Milk powder
Poppy (baking ingredient)
Chocolate puddingb
Ginger mixture
ESL chocolate milka
Curry powder
Cocoa powder
Wild garlic spice
Cocoa powder
Unknowna
Kafr lime leaves
Unknowna
Chocolate desserta
b

6.00
8.19
7.28
5.56
7.96
9.77
6.19
7.91
7.87
8.43
7.11
7.97
6.01

0.02
0.02
0.24
0.12
0.17
0.34
0.07
0.14
0.50
0.11
0.14
0.49
0.23

After heating

Initial

2.98
5.96
4.22
1.97
8.19
2.03
5.19
2.91
4.37
2.40
5.23
b1
1.02

6.11
8.19
7.26
5.48
7.79
8.61
7.07
8.00
7.95
8.12
7.40

0.21
0.40
0.05
0.14
0.25
0.05
0.08
0.68
0.81
0.41
0.31

0.21
0.02
0.00
0.33
0.01
0.37
0.02
0.01
0.06
0.05
0.07

Initial

After heating

0.20

8.28 0.08

b1

0.10
0.15

5.71 0.04
7.71 0.08

1.25 0.11
b1

8.00
8.06
8.12
8.01

0.02
0.09
0.05
0.01

1.44 0.17
1.58 0.08
b1
1.51 0.01

0.19
0.24

7.03 0.11
5.71 0.21

1.26 0.00
1.11 0.10

0.86
0.10
0.51

7.37 0.00
6.07 0.02
7.92 0.10

b1
b1
1.14 0.27

0.00

7.87 0.14

1.13 0.32

1.80 0.29

8.78 0.04

b1

b1
1.96
b1
2.33
1.95
b1
b1
2.44
1.86
2.18
1.00

5.67 0.05

b1

9.06 0.06

b1

0.14

7.68 0.05

b1

0.12

7.80 0.19

b1

0.30
0.34

8.05 0.00
7.18 0.03

b1
b1

6.65
5.58
7.34
5.74
7.22
5.96
7.92
7.79
7.85

1.47
1.17
b1
b1
1.11
1.07
1.03
b1
1.66

7.68 0.01
8.94 0.21
7.87 0.08

b1
1.83 0.30
b1

Cheese spreada
Chocolate drinkb
Cocoa powder
Cocoa powder
Cocoa powder
Poppy (baking ingredient)
Adjusted milk (for rice pudding)b
Dessert with curd
Caraway spice
Cream puddingb
Milk powder
Rice pudding
Cheese spreadb
Cocoa powder
Cocoa powder
Raw milk
Cheese spreada
Milk powder
Cheese spreada

8.70
6.62
7.30
7.24
7.90
6.12
6.86
6.26
5.77
7.26
5.92
7.40
6.14
7.95
7.79
7.99
6.70
5.80
8.78

3.15
b1
3.28
b1
2.13
3.53
b1
2.68
4.22
6.76
4.44
7.28
5.55
4.58
2.29
4.73
b1
b1
3.53

0.12
0.05
0.00
0.11
0.04
0.05
0.15
0.29
0.18
0.08
0.07
0.02
0.12
0.06
0.13
0.07
0.05
0.06
0.04

After heating

0.21

Cheese spread
Milk powder
Adjusted milk (for rice pudding)b

125 C

0.34
0.25
0.11
1.13
0.14
0.22
0.04
0.25
1.24

0.69

0.01
0.08
0.04
0.06
0.24
0.01
0.10
0.13
0.12

8.78 0.04

0.19
0.09
0.00
0.30

Spoiled food product.

Intermediate product before heat treatment.
Data from Witthuhn et al. (2011).

and recovery conditions as well as the composition of the heating media

(Cazemier et al., 2001; Coroller et al., 2001; Hornstra et al., 2009; Melly
et al., 2002; Sanchez-Salas et al., 2011). Thus, high thermoresistance
seems not to be a predetermined, constant spore property, but can be
triggered in some spores in a heterogenous manner by several environmental conditions. This is in accordance with our own results from the
thermal screening at higher temperatures (110 C125 C), revealing
that spores of both thermophilic and mesophilic isolates of several
species were highly heat-resistant in a strain-dependent manner. The
most heat-resistant spores in our study belonged to strains of B. subtilis,
B. amyloliquefaciens, B. exus, B. smithii and G. pallidus. The production
of highly thermoresistant endospores was reported previously for
B. subtilis and B. amyloliquefaciens strains isolated from various
food products like cocoa powder, canned soup ingredients or ropy
bread (Ahn et al., 2007; Lima et al., 2011, 2012; Oomes et al.,
2007). This indicates that the latter two species are generally abundant
in processed foods, presumably due to their potential of forming extremely heat-resistant spores.
Besides the spore heat resistance, also the spoilage potential of the
analyzed TRS isolates turned out to be a strain-specic trait within a
species. Main producers of spoilage enzymes were clearly found
within in the mesophilic species B. subtilis, B. amyloliquefaciens and
B. cereus. All tested strains belonging to these species showed strong

proteolytic- and most of them even hemolytic activity. Such high

rates of enzymatic activities are in accordance with the large amount
(almost 40%) of B. subtilis, B. amyloliquefaciens and B. cereus isolates
included in this study, which have been isolated from food spoilage
cases. Even if no direct correlation between the enzymatic activity
and spoilage can be drawn in general, our results demonstrated that
more strains from spoilage associated samples were proteolytic
(58%) than strains from non-spoilage associated foods (40%) (data
not shown). The high spoilage potential of these species is also emphasized by a previous work, analyzing spore-formers isolated from
raw milk for spoilage associated enzyme activities (De Jonghe et al.,
2010).
Moreover, about half of the B. licheniformis isolates, which were frequently isolated from spoiled products, were proteo- and lipolytic. We
could not detect any enzymatic activity for the original HRS organism
B. sporothermodurans and the proteolytic activity of obligate thermophilic spore-formers was low. However, lipase- and -galactosidase
activity was detected in 34% and 38% of the thermophilic isolates,
respectively, demonstrating their potential for lactose fermentation.
Interestingly, a high rate of -galactosidase activity was recently
reported for psychrotolerant Paenibacillus spp. isolated from uid
milk production- and processing environments (Ivy et al., 2012).
This might be a hint that -galactosidase activity is connected to

mesophilic:

cytotoxic potential
(heat stable)

277

cytotoxic potential
(heat labile)

phospholipolysis

-galactosidase

lipolysis

Species (amount of isolates)

hemolysis

proteolysis

G. Lcking et al. / International Journal of Food Microbiology 166 (2013) 270279

B. subtilis (33)
B. sporothermodurans (12)
B. amyloliquefaciens (10)
B. cereus (2)
B. flexus (2)
B. circulans (2)
B. simplex (2)

thermotolerant:

B. licheniformis (13)

thermophilic:

G. stearothermophilus (21)
B. thermoamylovorans (8)
G. pallidus (5)
A. flavithermus (3)
A. thermoaerophilus (4)
B. smithii (5)
G. thermoleovorans (2)
B. thermolactis (1)
A. acidocaldarius (1)

> 75 % of isolates positive

15-49 % of isolates positive

50-75 % of isolates positive

< 15 % of isolates positive

Fig. 3. Screening for spoilage and toxigenic potential. Different agar plates (skim milk-, blood-, tributyrin-, X-Gal and egg yolk-agar) and two bio-assays (employing HEp-2 and Vero
cells) were used to determine enzyme activities linked to food spoilage and the cytotoxic potential of 126 TRS spore-formers (for details see Material and methods).

specic ecological niches in food production, although it cannot be

excluded that the mesophilic species also possess -galactosidase activity at temperatures not yet tested. For instance, Hu et al. already described a cold active -galactosidase from a Planococcus sp. isolate (Hu
et al., 2007).
In contrast to the other enzyme activities, lecithinase activity and
the production of cytotoxic substances were exclusively found in
B. cereus isolates. For none of the other 124 TRS isolates, belonging
to 16 different spore-forming species, cytotoxic components could
be detected, proposing that the risk of food poisoning caused by
aerobic, spore-forming contaminants outside the B. cereus group is
in general rather low. A low rate of cytotoxicity among (non-B. cereus
group) Bacillus isolates (8 out of 333) was also observed in a previous
study (From et al., 2005). Nevertheless, some studies reported on the
presence of B. cereus-like toxins in several non-B. cereus group Bacillus
species using PCR detection methods, immunological or cell culture
based assays (Beattie and Williams, 1999; De Jonghe et al., 2010;
Phelps and McKillip, 2002; Rowan et al., 2003; Taylor et al., 2005),
while further characterization of the detected bioactive substances
is often missing and their possible role in food poisoning uncertain.
The structure and mechanism of some toxic, mainly surfactin-like compounds produced by B. licheniformis, B. mojavensis, B. amyloliquefaciens,
B. subtilis or B. pumilus have been analyzed, but their association with
food poisoning is still under debate (Apetroaie-Constantin et al., 2009;
From et al., 2007a, 2007b; Mikkola et al., 2000, 2007).
In summary, this study provides a comprehensive insight into
food quality and food safety relevant characteristics of spoilage- and
non-spoilage associated aerobic spore-forming bacteria in the dairy
processing sector. The food processing environment as well as food
ingredients, such as cocoa powder and spices, were identied as

important contamination sources emphasizing the multiple realms

of entrance of spores in dairy products, which require more attention.
Signicant differences in the spore heat resistance and spoilage potential were observed not only between different species, but also between different strains within a species. These heterogenous interand intraspecies resistance proles of genetically closely related
spore-formers should be studied in more detail to (i) improve mathematical models in predictive microbiology (Postollec et al., 2012)
and to (ii) develop novel risk related diagnostic tools (Ehling-Schulz
and Messelhusser, 2013). Most importantly, our study indicates
that the spoilage potential of mesophilic spore-formers producing
thermoresistant spores is much higher than the spoilage potential
of TRS isolates belonging to strictly thermophilic species, although
the latter are frequently found in foods not associated with spoilage.
Highly heat-resistant spore-formers, which produced spores surviving
125 C for 30 min, were found among mesophilic as well as among
thermophilic isolates. Compared to the spoilage potential, the toxigenic
potential of aerobic TRS isolates was, outside the B. cereus group, negligible. It can therefore be assumed that highly heat-resistant aerobic
spore-formers are rather a food quality problem than a food safety
issue.

Acknowledgments
This research project (AiF 16012N) was supported by the
German Ministry of Economics and Technology (via AiF) and the
FEI (Forschungskreis der Ernhrungsindustrie e.V., Bonn). We thank
Romy Wecko and Gertrud Huith for excellent technical support and
Martina Fricker for helpful advice regarding spore production.

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G. Lcking et al. / International Journal of Food Microbiology 166 (2013) 270279

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