ORIGINAL

O
Blackwell
Oxford,
International
IDTRIGINAL
Society
?1364-727X
of
UK RESEARCH
Dairy
Publishing
Journal
Technology
of
LtdDairy2007
Technology

O RI GI NA L
RESEA RCH The formation of thermophilic spores during the
manufacture of whole milk powder
S AR A A S C OTT,1,2 * J OHN D B ROOKS ,1 JAS NA R AKONJAC ,1
KYLIE M R WALKER 2 and S T E VE H F L INT 2
1
Massey University, Private Bag 11 222, Palmerston North, New Zealand and 2Fonterra Research Centre, Private Bag
11 029, Palmerston North, New Zealand

A survey was undertaken at a whole milk powder manufacturing plant to determine the origin and the
*Author for correspondence. E-mail: sara.scott@fonterra.com

rate of spore formation of thermophilic bacteria. Spores were generally not detected (< 10 cfu/mL) in
either the pasteurization process or the pasteurized milk directed into the powder plant. The predominant
sites of spore formation were the preheater plate heat exchanger and the evaporator. Spores began to be
detected approximately 9 h into an 18-h manufacturing run. Spore isolates were identified as
Anoxybacillus flavithermus and Geobacillus species. A. flavithermus predominated in the preheat
section, whereas a mix of both organisms was present in the later manufacturing stages.
Keywords Anoxybacillus flavithermus, Geobacillus, Milk powder, Spore formation, Thermophile.

their ability to form biofilms, as well as their fast

I N T RO D U C T I O N
During the manufacture of milk powder ther-
mophilic bacteria, that is, those bacteria that grow
between 40 and 65°C, are able to grow within the
regeneration sections of heat exchangers and in
evaporators. These sections of the manufacturing
plant are at ideal temperatures for thermophile
growth, typically operating from 45 to 75°C. The
growth of thermophiles results in high numbers
of bacteria (up to 106 cfu/g) being released into
the final product. Many studies have provided
evidence on the sites of vegetative growth during
the manufacture of milk powder but have had
difficulty establishing the site of sporulation (Stad-
houders et al. 1982; Murphy et al. 1999; Warnecke
2001).
The main thermophilic bacteria causing the
dairy industry concern are of the genus Bacillus or
those genera that formerly belonged to the Bacillus
genus. Thermophilic bacilli are not pathogenic.
However, after the powder is recombined and if the
conditions are suitable, their spores can germinate.
This may result in enzyme production, acid pro-
duction and consequential development of an
off-flavour in the product (Chopra and Mathur
1984; Chen et al. 2004). In addition, large numbers
*Author for of bacteria of any sort are usually unacceptable to
correspondence. E-mail:
most customers.
sara.scott@fonterra.com
The thermophilic bacilli are difficult to eliminate
© 2007 Society of from a manufacturing process because of the
Dairy Technology resistance of their spores to heat and chemicals,
growth rate (a generation time of approximately
15–20 min) and wide temperature growth range
(Etoa and Michiels 1988; Hill and Smythe 1994;
2004; Flint et al. 2001a; Parkar et al. 2003). It is
the development of spores that is of particular
concern to the dairy industry. Spores are able to
survive the low water activity and high temperature
of the drying process, the cleaning-in-place (CIP)
system and the long-term storage of the final
product. Vegetative cells are less likely to survive
under these conditions.
The process of thermophile contamination of
milk powder is understood to some extent. Initial
contamination of the milk powder plant probably
arises from low numbers of thermophiles present
in the raw milk that survive pasteurization,
although this is yet to be proven. However, in the
case of thermophiles the overall raw milk quality
has been shown to be unrelated to the quality of
the final milk powder product (Muir et al. 1986).
Thermophile contamination can also arise from
the reuse of by-products such as buttermilk and per-
meate from milk ultrafiltration, ingredients added
to the process such as lactose, and recycle loops in
manufacturing plants (Hill and Smythe 1994).
Both spores and vegetative cells are able to attach
to the stainless steel and fouled surfaces (Flint
et al. 2001a; Parkar et al. 2001). Once attached
to a surface, the spores probably germinate and
grow, with the vegetative cells forming a biofilm.
Contamination of the milk is likely to occur from

Vol 60, No 2 May 2007 International Journal of Dairy Technology 109

 Vol 60, No 2 May 2007
the biofilm, by sloughing (the release of fragments
of the biofilm) or shedding of individual cells. At
the end of a run, most of the thermophilic bacterial
cells will be removed or killed by CIP and the
exposure to sanitizer if used (Parkar et al. 2001).
However, foulant or biofilms may protect spores
and vegetative cells against CIP chemicals (Hinton
et al. 2003). There is evidence that viable bacterial
cells can remain attached to dairy manufacturing
surfaces following CIP (Austin and Bergeron
1995; Flint et al. 1999). This will result in ther-
mophilic cells remaining in the plant and seeding
the next manufacturing run.
In New Zealand, the main thermophilic bacteria
of concern to the dairy industry are Anoxybacillus
flavithermus and Geobacillus spp. (Flint et al.
2001b; Ronimus et al. 2003). These organisms
were formerly classified in the genus Bacillus.
Other bacilli also found in milk powder are the facu-
ltative thermophiles such as Bacillus licheniformis,
Bacillus coagulans and Bacillus subtilis, although
these are usually present at low levels (Crielly et al.
1994; Ronimus et al. 2003).
Traditionally, biochemical tests such as the API®
system (bioMérieux, Marcy l’Etoile, France) have
been used in the dairy industry to identify bacterial
contaminants. However, recently, polymerase chain
reaction (PCR)-based identification techniques
such as species-specific PCR, randomly amplified
polymorphic DNA (RAPD) profiling and partial
16S rDNA sequencing have provided evidence that,
in the past, A. flavithermus has been mistakenly
identified as Geobacillus stearothermophilus by
these traditional techniques (Flint et al. 2001b).
Ronimus et al. (2003) used RAPD profiling to
classify New Zealand thermophilic milk powder
isolates into seven groups. These included G.
stearothermophilus, three strains of A. flavithermus,
two strains of B. licheniformis, and B. subtilis.
Flint et al. (2001b), on the other hand, classified the
majority of New Zealand thermophilic milk powder
isolates as either Geobacillus thermoleovorans or
A. flavithermus using partial 16S rDNA sequen-
Figure 1 Schematic diagram of the evaporation and drying process. F–P indicate the points at which samples were taken.
110 © 2007 Society of Dairy Technology
cing. These results led to the development of 16S
rDNA primers specific for A. flavithermus and
Geobacillus groups, respectively. These primers
allowed identification of these two major ther-
mophilic contaminants by diagnostic PCR.
The identification of thermophilic dairy isolates
has focused mainly on the vegetative form rather
than on the spores. There is also little information
available about where and when sporulation of
thermophiles occurs in a milk powder manufactur-
ing plant. In this study, a survey was undertaken
at a whole milk powder manufacturing plant to
determine the site(s) and the rate of spore formation.
Spores isolated from product samples taken at dif-
ferent stages during whole milk powder manufac-
ture were identified using specific A. flavithermus
and Geobacillus rDNA primers.
M AT E R I A L S A N D M E T H O D S
Operation of the powder plant
This study was carried out at a New Zealand whole
milk powder factory. The raw milk treatment
(separation, pasteurization and standardization)
process was separate from the milk powder evap-
oration and drying process.
The raw milk treatment runs were 6–8 h in
length. The raw milk was preheated to 50°C by a
plate heat exchanger (PHE), and then separated
into skim milk and cream. At this point, ingredi-
ents, such as lactose, could be added to the skim
milk depending on the product to be manufactured.
The skim milk and cream were pasteurized
separately at 73°C for 15 s and 80°C for 15 s,
respectively. Following cream and skim milk past-
eurization, the two streams were mixed to achieve
the required composition (standardization) and
then stored at 4°C.
The milk powder manufacturing runs were
approximately 18 h in length. Figure 1 outlines the
evaporation and drying process in use at the time of
this study. This plant was operating at a flow rate of
approximately 40 000 L/h. Before evaporation,
 Vol 60, No 2 May 2007

Following heat treatment, the milk passed

Table 1 Sampling points in the raw milk treatment and the evaporation and drying
through a two-effect five-pass mechanical vapour
process
recompression falling film evaporator (Niro A/S
Sampling point Stage in process MKT, Soeborg, Denmark). The evaporator operated
at a temperature of 65°C in both effects. Effect 1
A Raw milk, before separation and after heating to 50°C had three passes, whereas effect 2 had two passes.
B Skim milk, after pasteurization and cooling
Following evaporation, the concentrated milk
C Cream, after pasteurization and cooling
passed through a scraped surface preheater and then
D Standard milk, after final cooling
underwent homogenization before it was dried
E Ingredient
F Balance tank
(5 t/h) and packed.
G Heated milk, between the PHE and evaporator A CIP took place following every run. This took
H Evaporator pass 1 about 3 h and involved a water rinse, a 1.6% caustic
I Evaporator pass 2 wash followed by a water rinse and then a 0.9%
J Evaporator pass 3 nitric acid wash, and finished with a water rinse.
K Evaporator pass 4 Every five runs, parts of the evaporator and the DSI
L Evaporator pass 5 unit were manually cleaned to remove foulant
M Scraped surface heater build-up.
N Homogenizer
O Static fluid bed Sampling plan
P Vibrofluidizer The sampling points used to collect product during
PHE, plate heat exchanger. the different stages of both the raw milk treatment
and the evaporation and drying process are listed in
Table 1. The locations of the sampling points used
in the evaporation and drying process are also out-
lined in Figure 1. All liquid samples (approximately
8 mL) were taken from rubber septum sampling
points into 9 mL sterile vacuum tubes (Vacutte®
Greiner Labortechnik, Biolab, Auckland, New
Zealand) using sterile vacutainer needles. The
powder samples (from the static fluid bed and
vibrofluidizer) were taken by manually dipping a
sterile pottle into the powder.
End-of-run samples were taken from three raw
milk treatment runs, two standard whole milk
powder runs (named runs 1 and 2) and two instant
whole milk powder runs (named runs 3 and 4).
Samples were taken approximately 30 min before
the end of a raw milk treatment run (sampling
points A–E) and 1 h before the end of a milk powder
run (sampling points F–P).
Samples were also taken every 2 h throughout
two standard whole milk powder runs (runs 5 and
6). The samples were collected from the balance
tank through to pass 4 of the evaporator (sampling
points F–K). The first sample was taken approxi-
mately 1 h into the run, and then samples were
taken every 2 h thereafter.
The runs used for sampling were chosen at
random over a 3-month period from December 2003
to February 2004. All of the samples were frozen
at −20 °C for up to 48 h and defrosted rapidly at
Figure 2 Schematic diagram of the points at which foulant 55°C before plating.
samples were collected. (a) The DSI unit and (b) effect 1 of Foulant samples were collected during a manual
the evaporator. shutdown after a standard whole milk powder run.
Samples were scraped off aseptically from the DSI
the milk was heat-treated using both a PHE and unit (Figure 2a), the top orifice pan of the first and
direct steam injection (DSI). The PHE ranged in second pass of the first effect (Figure 2b) and a
temperature between 50 and 65°C and DSI raised structural pole within the separator body of the first
the milk temperature to approximately 95°C. effect (Figure 2b).

© 2007 Society of Dairy Technology 111

 Vol 60, No 2 May 2007
Table 2 Thermophilic bacterial counts of raw milk treatment process samples
Total thermophile Spore
count (cfu/mL) count (cfu/mL)
Raw milk (A) 10 < 10
Skim milk (B) < 10 < 10
Cream (C) 27 < 10
Standardized milk (D) < 10 < 10
Ingredient (E) < 10 < 10
Enumeration of bacteria
Ten grams of each powder or foulant sample was
reconstituted in 90 mL of 0.1% peptone water
(BBL®-Polypeptone-Peptone, Becton, Dickinson
& Company, North Ryde, NSW, Australia). Total
thermophile counts were determined using a pour-
plating technique. One millilitre of serial 10-fold
dilutions of each sample in 0.1% peptone water
was plated with Milk Plate Count Agar (MPCA,
Oxoid Ltd, Adelaide, Australia). Thermophilic
aerobic spores were similarly counted after a heat
treatment of the 10−1 diluted liquid sample or
reconstituted powder (in 0.1% peptone) at 100°C
for 30 min to eliminate the vegetative cells. Serial
dilutions of the heat-treated sample were prepared
in 0.1% peptone and 1 mL samples of the appro-
priate dilutions were pour-plated with MPCA +2%
starch (MPCA +S). All of the plates were prepared
in triplicate and incubated at 55°C for 48 h. The
final counts (cfu per gram or millilitre) were deter-
mined by counting those plates containing 20–300
colonies and multiplying the number of colonies
by the dilution factor. Plates of 1 mL undiluted
liquid milk samples were unable to be counted, as
it was too difficult to distinguish the colonies in the
opaque agar; therefore, those plates (prepared with
a 10−1 diluted sample) with less than 20 colonies
were still counted to give an approximate count.
Identification of spore isolates
Approximately 10 spore-derived colonies isolated
from each product sample taken at the end of runs
5 and 6 were randomly picked from the surface of
the MPCA +S spread plates and identified using
PCR.
Specific 16S rDNA-derived primers (Flavo and
Levo) (Flint et al. 2001b), were used in combina-
tion with the universal primer Y1 (Young et al.
1991) to identify the spore-derived colonies. The
A. flavithermus primer Flavo is species specific.
However, the Levo primer is genus specific for
the Geobacillus group. The presence of a PCR
product, approximately 450 bp in length, with the
appropriate primer set identifies the isolate as
the organism corresponding to that primer set.
112 © 2007 Society of Dairy Technology
The products were analysed using agarose gel
electrophoresis.
R E S U LT S A N D D I S C U S S I O N
Thermophiles in the raw milk treatment
process
In order to determine whether the raw milk
treatment process is the predominant source of
thermophilic spore contamination, both total
thermophiles and thermophilic spores were
enumerated in milk samples taken from the end of
a raw milk treatment run (Table 2). This was repeated
for two additional milk treatment runs with similar
results (data not shown). Spores were not detected
in any of the milk samples (sampling points A–D),
demonstrating that this process was unlikely to be
a major source of spores in the final product.
There was no significant change (< 1 log cfu/mL)
in the total thermophile counts between the raw
milk (sample A) and standardized milk (sample D)
suggesting that no significant thermophile growth
occurred in the raw milk treatment plant. However,
the presence of vegetative cells at low counts
suggests that the pasteurized milk destined for
the milk powder plant is a source of low numbers
of vegetative thermophilic cells. Because of the
design of the plant, it was not possible to trace a
particular milk powder run to a milk treatment
run to determine the true relationship between the
thermophiles in the pasteurized milk and those of
the powder plant.
Our findings that the counts of vegetative cells
do not significantly increase during pasteurization
are in agreement with those of Reddy et al. (1975)
and Muir et al. (1986), who found that raw milk
treatment has very little influence on thermophile
numbers in milk destined for milk powder manu-
facture. This is probably because of the shorter run
times of raw milk treatment compared with the run
length of milk powder manufacture. In contrast to
these findings and our observations, Murphy et al.
(1999) have recorded thermophile counts in
pasteurized milk as high as 3500 cfu/mL. However,
as they did not examine the raw milk treatment
process, it is not possible to determine the reason
for this large discrepancy.
Location of thermophile growth and spore
formation sites during milk powder
manufacture
To determine where thermophiles grow and form
spores during milk powder manufacture, product
samples were taken at different stages throughout
the manufacturing process of standard whole milk
powder and instant whole milk powder. This was
repeated for each type of milk powder run. Figure 3
shows the total thermophile counts and the ther-
mophilic spore counts for the samples collected
Vol 60, No 2 May 2007
Figure 3 Thermophilic bacterial counts from samples taken
at different stages at the end of various whole milk powder
runs. Product samples were taken approximately 1 h prior to
the end of two standard whole milk powder runs (a) and two
instant whole milk powder runs (b). Each bar represents the
mean and standard deviation of triplicate counts. The powder
sample counts (O and P) were measured in log10 cfu/g and
the remaining sample counts (F–N) were measured in
log10 cfu/mL. A * indicates that no sample was taken.
during these four manufacturing runs. The total
thermophile plate count was always higher than the
thermophilic spore count, indicating that both
spores and vegetative cells were present.
The consistent increase in both total ther-
mophile counts and spore counts from the balance
tank sampling point (F) to the pre-evaporator
sampling point (G) during instant whole milk
powder manufacture demonstrates that the preheat
section was one site of vegetative cell growth and
sporulation.
Further downstream in the milk powder process,
a noticeable increase in the total thermophile count
was observed between samples G and H for all of
the runs and samples H and I for run 4. A noticeable
increase in spore counts was also observed between
samples H and I (run 3) with a further increase
occurring during pass 2 (Figure 3b). This suggests
© 2007 Society of Dairy Technology
that vegetative cell growth and spore formation
occurred in the evaporator as well as the preheater.
These findings are consistent with previous studies,
which indicate that both thermophile growth
and sporulation can occur in both preheaters and
evaporators (Stadhouders et al. 1982; Murphy
et al. 1999; Warnecke 2001).
In evaporator pass 3, there was no noticeable
increase in either the total thermophile count or
the thermophilic spore count relative to pass 2,
indicating that vegetative growth and sporulation
did not occur in this stage of processing. During
evaporation, the total solids (TS) concentration of
the milk increased from 13% TS in the balance
tank to 48% TS when it exited the evaporator.
The absence of thermophile growth during passes
3, 4 and 5 probably relates to this concentration
increase. This observed trend is in agreement with
the finding of Lane (1982) that growth of the dairy
thermophile G. stearothermophilus was limited in
milk concentrate.
The thermophile counts recovered from the two
types of final whole milk powder products, standard
and instant, were similar. This is not surprising, as
the manufacturing processes used for these two
products were very similar. The only differences
were the addition of lecithin, vitamin A and vitamin
D to instant whole milk powder, and the operation
of the vibrofluidizer at 45–65°C, rather than at
ambient temperature, during the production of
instant whole milk compared with standard whole
milk powder.
Dynamics of thermophilic spore production in
a milk powder manufacturing plant
In the previous section, the preheater was identified
as the stage in which the most dramatic increase
in spore counts occurred during milk powder
processing. However, this conclusion was based on
the end-of-run samples. To monitor the dynamics
of spore formation, product samples were collected
every 2 h from the preheating and evaporation
sections of the milk powder plant throughout two
whole milk powder runs (runs 5 and 6). The spore
count results are presented in Figure 4.
Spores were detected (> 1 log cfu/mL) only after
approximately 9 h into both runs. At 18 h, spores
reached levels of up to 4.1 log10 cfu/mL in the evap-
orator pass samples, whereas none were detected in
the balance tank feeding the manufacturing process
(Figure 4). This confirms that spores were forming
within the milk powder manufacturing process and
were not a result of external contamination.
The rate of spore formation, the initial sites of
spore formation and the spore numbers in the
final product were different for the two runs. Spore
formation occurred at a faster rate during run 5
compared with run 6; they were detected after 11 h
in all of the samples except the pre-evaporator
113
 Vol 60, No 2 May 2007
Figure 4 Thermophilic spore counts of samples taken every 2 h over an 18-h run period of two standard whole milk powder
runs (runs 5 and 6). Each point represents the mean of triplicate counts.
Figure 5 Thermophilic bacterial counts of foulant that was
scraped aseptically from a milk powder manufacturing plant
after a standard whole milk powder run. Each bar represents
the mean and standard deviation of triplicate counts.
samples, and reached levels of 3 log10 cfu/mL
within 13 h. However, during run 6, spores were
detected in the passes 2 and 3 of the evaporator after
11 h, but they were not detected in pass 4 until 13 h
into the run and after 15 h in the preheat samples.
There are many factors that can vary between
different manufacturing runs and different milk
powder manufacturing plants. For example, the
milk quality (age, chemical and microbiological),
the temperature of the stored milk; the product
type; and the temperature regimes may be different.
Therefore, it is difficult to determine why the
rate of sporulation and the spore count differed
between runs 5 and 6.
To the authors’ knowledge this is the first time
the rate of sporulation has been successfully
monitored in a milk powder plant. A similar study
by Murphy et al. (1999), in a skim milk powder
pilot plant, found no notable difference in spore
numbers between the start of the run and the end of
114 © 2007 Society of Dairy Technology
the run or between preheater evaporator samples
and powder samples. However, total thermophile
counts increased throughout the run, and the
authors concluded that vegetative cell growth was
favoured over sporulation.
The survival of thermophiles in foulant
In order for thermophiles to grow in the plant there
must be an initial source of inoculum. One possible
source is bacteria trapped in foulant that remains in
the plant between CIPs and may be only partially
removed during manual cleaning (Hinton et al.
2003). Accumulation of foulant can occur for a
variety of reasons (Jeurnink et al. 1996). In areas
where process temperatures are greater than 65°C,
for example in the separator and the DSI unit,
foulant will gradually build up. In this study, the
steam did not mix very well with the milk in the
DSI unit, resulting in an accumulation of layers of
burnt milk. Fouling can also occur in areas of flow
recirculation, for example under the orifice pans.
To determine if foulant was a possible source of
inoculum, foulant samples were taken during a
shutdown of the milk powder plant and the total
thermophile and thermophilic spore counts were
determined. Foulant was taken from the DSI unit,
the orifice pans from passes 1 and 2 of the evaporator,
and a support pole in the separator of evaporator
effect 1.
The total thermophile count and the thermophilic
spore count were similar for all of the samples,
suggesting that the bacteria present in these
samples were predominantly in their spore form
(Figure 5). Foulant collected from the evaporator
had thermophile counts that were at least 2 log
higher than those for the foulant from the DSI unit.
This was expected given that the DSI unit was at
a temperature of 95°C. Spores would be able to
survive this temperature and possibly be deposited
on the DSI unit surface but there would be no
vegetative cell growth.
Although there is evidence that thermophile
attachment and growth occur faster on foulant than
on un-fouled stainless steel (Hinton et al. 2003), to
the authors’ knowledge, this is the first study that
Vol 60, No 2 May 2007
Figure 6 Identification of Anoxybacillus flavithermus
from the powder plant isolates by species-specific PCR.
The gel represents only a fraction of analysed samples.
Lanes: 1, DNA size standard; 2–11, end-of-run pass 2
evaporator spore isolates; 12, blank (no template); 13,
Geobacillus thermoleovorans DSM 5366 (negative control);
14, A. flavithermus DSM 2641 (positive control);
15, DNA size standard.
Figure 7 Identification of Geobacillus spp. from the powder
plant isolates by genus-specific PCR. The gel represents only
a fraction of analysed samples. Lanes: 1, DNA size standard;
2, Geobacillus thermoleovorans DSM 5366 (positive
control); 3, A. flavithermus DSM 2641 (negative control);
4, blank (no template); 5–14, end-of-run pass 2 evaporator
spore isolates; 15, DNA size standard.
has provided evidence that foulant is a source of
thermophile contamination in a full-scale milk
powder plant.
The identification of thermophilic spores
A PCR assay, designed by Flint et al. (2001b), was
used to determine whether thermophilic spore
contamination in the milk powder manufacturing
plant was caused by A. flavithermus and/or the
Geobacillus group. The presence of a PCR product
of approximately 450 bp in length with the appro-
© 2007 Society of Dairy Technology
priate primer set, as described in the materials
and methods section, identified the isolate as the
organism corresponding to that primer set.
Examples of these PCR products run on electro-
phoresis gels are shown in Figures 6 and 7. The
spore-derived isolates used for these PCR assay
examples originated from run 6, pass 2 of the
evaporator. Three of these isolates were identified
as A. flavithermus and seven were identified as
Geobacillus spp.
Approximately 10 spore-derived colonies were
isolated and identified from each sample. All of the
isolates from samples collected at the end of runs
5 and 6 were identified as either A. flavithermus or
Geobacillus spp. (data not shown); therefore, no
further identification was necessary. These two
organisms have frequently been isolated from New
Zealand milk powders, and have also been found
overseas (Flint et al. 2001b; Warnecke 2001;
Ronimus et al. 2003; Rueckert et al. 2004).
Figure 8 shows the proportion of thermophilic
spores identified as either A. flavithermus or Geo-
bacillus spp. throughout the different stages of run
5. Anoxybacillus flavithermus predominated in
the preheating section of the milk powder process
whereas there was a mix of the two types of
organisms in the evaporation and drying stages of
the process. In run 5 the Geobacillus group did not
appear until the second pass of the evaporator.
Similar results were obtained for run 6 (data not
shown) except that the Geobacillus group was
detected in all of the evaporator passes but not in
the preheater.
To examine whether the foulant could be a
source of either A. flavithermus or Geobacillus
spores, these samples were analysed as described
above to determine the identity of the thermophilic
spores. Figure 9 shows an example of PCR products
amplified from spore-derived isolates originating
from the pass 1 pan foulant. The isolates from all of
the foulant samples were identified as Geobacillus
spp. No A. flavithermus spores were detected. These
results provide further evidence that Geobacillus
spp. predominates in the evaporator. Anoxybacillus
flavithermus found in the evaporator milk samples
probably originated from cells shedding from
biofilm or foulant material in the preheat section.
Although there is evidence that certain bacterial
species can predominate in different sections of
milk powder manufacturing plant equipment
(Langeveld et al. 1995; Hinton et al. 2001; Warnecke
2001), this appears to be the first study in which
thermophilic spores have been identified throughout
the different stages of milk powder manufacture.
CONCLUSIONS
This study has demonstrated that the appearance of
high numbers of thermophilic spores in whole milk
115
 Vol 60, No 2 May 2007
Figure 8 Relative representation of Geobacillus spp. and
Anoxybacillus flavithermus spores in a milk powder
manufacturing plant.
Figure 9 Identification of Geobacillus spp. in the foulant
samples using genus-specific PCR. Lanes: 1, DNA size
standard; 2, Geobacillus thermoleovorans DSM 5366
(positive control); 3, Anoxybacillus flavithermus DSM 2641
(negative control); 4, blank (no template); 5–14, spore isolates
from the evaporator pass 1 pan foulant; 15, DNA size
standard.
powder is a result of sporulation occurring within
the manufacturing plant. The raw milk used is not
a contributory factor although vegetative cell
growth during milk treatment (separation, pasteur-
ization and standardization) may contribute to low
levels of thermophiles in the milk powder manu-
facturing process. The predominant site of both
116 © 2007 Society of Dairy Technology
vegetative cell growth and sporulation appeared to
be the preheat section of the evaporator, although
sporulation also occurred in the evaporator itself.
The major thermophilic spore contaminants were
identified as A. flavithermus and Geobacillus spp.
using primers specific for these organisms.
A. flavithermus dominated in the preheat section,
whereas there was a mix of the two types in the
later stages of the manufacturing process.
One possible source of thermophilic bacteria
was foulant that remained in the manufacturing
plant after a CIP. In foulant samples obtained from
the DSI unit and the evaporator, the numbers of
spores recovered were as high as 7 log10 cfu/g. The
predominant thermophilic spore type isolated from
the foulant was identified as Geobacillus spp.
Knowledge of where these spores form and their
identity will help the dairy industry design strategies
to prevent their formation.
A C K N OW L E D G E M E N T S
The authors would like to thank Bruce Hill and
Betty Smythe for their valued discussion, Katherine
Sparkes for her excellent laboratory work, the
Pahiatua factory staff and Tuan Truong for helping
with this study, and the Technology for Industry
Fellowship for funding this work.
REFERENCES
Austin J W and Bergeron G (1995) Development of bacterial
biofilms in dairy processing lines. Journal of Dairy
Research 62 509–519.
Chen L, Coolbear T and Daniel R M (2004) Characteristics of
proteinases and lipases produced by seven Bacillus sp.
isolated from milk powder production lines. International
Dairy Journal 14 495–504.
Chopra A K and Mathur D K (1984) Isolation, screening and
characterization of thermophilic Bacillus species isolated
from dairy products. Journal of Applied Bacteriology 57
263–271.
Crielly E M, Logan N A and Anderton A (1994) Studies on
the Bacillus flora of milk and milk products. Journal of
Applied Bacteriology 77 256–263.
Etoa F X and Michiels L (1988) Heat-induced resistance of
Bacillus stearothermophilus spores. Letters in Applied
Microbiology 6 43–45.
Flint S H, van den Elzen H, Brooks J D and Bremer P J (1999)
Removal and inactivation of thermo-resistant streptococci
colonising stainless steel. International Dairy Journal 9
429–436.
Flint S, Palmer J, Bloemen K, Brooks J and Crawford R
(2001a) The growth of Bacillus stearothermophilus on
stainless steel. Journal of Applied Microbiology 90 151–
157.
Flint S H, Ward L J H and Walker K M R (2001b) Functional
grouping of thermophilic Bacillus strains using ampli-
fication profiles of the 16S−23S internal spacer region.
Systematic and Applied Microbiology 24 539–548.
Hill B M and Smythe B W (1994) Progress in understanding
the behaviour of thermophilic bacteria during milk
Vol 60, No 2 May 2007
powder manufacture. In Milk Powders for the Future II,
pp 19–26. Palmerston North, New Zealand: New Zealand
Dairy Research Institute.
Hill B M and Smythe B W (2004) Thermophilic spores in
milk powder destined for use in UHT and retort sterilisation
processes. In Proceedings of New Zealand Microbiological
Society Conference. Palmerston North, New Zealand: New
Zealand Microbiological Society.
Hinton A R, Trinh K T, Brooks J D and Manderson G J (2003)
Thermophile survival in milk fouling and on stainless
steel during cleaning. Transactions of the Institution of
Chemical Engineers 80 299–304.
Hinton A R, Trinh K T, Manderson G and Brooks J D (2001)
Thermophile growth in the preheating section of a milk
powder pilot plant. In Proceedings of 6th World Congress
of Chemical Engineering. Melbourne, Australia 23rd–27th
September 2001.
Jeurnink T J M, Walstra P and de Kruif C G (1996) Mech-
anisms of fouling in dairy processing. Netherlands Milk
and Dairy Journal 50 407–426.
Lane A N (1982) Thermophiles in milk powder. Postgraduate
Diploma in Dairy Science and Technology Dissertation.
Massey University, Palmerston North, New Zealand.
Langeveld L P M, van Montfort-Quasig R, Weerkamp A H,
Waalewijn R and Wever J S (1995) Adherence, growth
and release of bacteria in a tube heat exchanger for milk.
Netherlands Milk and Dairy Journal 49 207–220.
Muir D D, Griffiths M W, Phillips J D, Sweetsur A W M and
West I G (1986) Effect of the bacterial quality of raw milk
on the bacterial quality and some other properties of low-
heat and high-heat dried milk. Journal of the Society of
Dairy Technology 39 115–118.
Murphy P M, Lynch D and Kelly P M (1999) Growth of
thermophilic spore-forming bacilli in milk during the
© 2007 Society of Dairy Technology
manufacture of low-heat powders. International Journal
of Dairy Technology 52 45–50.
Parkar S G, Flint S H, Palmer J S and Brooks J D (2001)
Factors influencing attachment of thermophilic bacilli to
stainless steel. Journal of Applied Microbiology 90 901–
908.
Parkar S G, Flint S H and Brooks J D (2003) Physiology of
biofilms of thermophilic bacilli—potential consequences
for cleaning. Journal of Industrial Microbiology and Bio-
technology 30 553–560.
Reddy P C, Atwal J S and Srinivasan R A (1975) Studies on
the survival of thermophilic bacteria during manufacture
and storage in dried milks. Indian Journal of Dairy
Science 28 289–295.
Ronimus R S, Parker L E, Turner N, Poudel S, Ruckert A and
Morgan H W (2003) A RAPD-based comparison of
thermophilic bacilli from milk powders. International
Journal of Food Microbiology 85 45–61.
Rueckert A, Ronimus R S and Morgan H W (2004) A RAPD-
based survey of thermophilic bacilli in milk powders from
different countries. International Journal of Food Micro-
biology 96 263–272.
Stadhouders J, Hup G and Hassing F (1982) The conceptions
index and indicator organisms discussed on the basis of
the bacteriology of spray-dried milk powder. Netherlands
Milk and Dairy Journal 36 231–260.
Warnecke F (2001) The ecology of thermophilic bacilli of
milk powder processing plants. M. Phil. Thesis. Univer-
sity of Waikato, Hamilton, New Zealand.
Young J P W, Downer H L and Eardly B D (1991) Phylogeny
of the phototrophic rhizobium strain BTAi1 by
polymerase chain reaction-based sequencing of a 16S
rRNA gene segment. Journal of Bacteriology 173 2271–
2277.
117