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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
growth rate (a generation time of approximately
During the manufacture of milk powder ther- 15–20 min) and wide temperature growth range
mophilic bacteria, that is, those bacteria that grow (Etoa and Michiels 1988; Hill and Smythe 1994;
between 40 and 65°C, are able to grow within the 2004; Flint et al. 2001a; Parkar et al. 2003). It is
regeneration sections of heat exchangers and in the development of spores that is of particular
evaporators. These sections of the manufacturing concern to the dairy industry. Spores are able to
plant are at ideal temperatures for thermophile survive the low water activity and high temperature
growth, typically operating from 45 to 75°C. The of the drying process, the cleaning-in-place (CIP)
growth of thermophiles results in high numbers system and the long-term storage of the final
of bacteria (up to 106 cfu/g) being released into product. Vegetative cells are less likely to survive
the final product. Many studies have provided under these conditions.
evidence on the sites of vegetative growth during The process of thermophile contamination of
the manufacture of milk powder but have had milk powder is understood to some extent. Initial
difficulty establishing the site of sporulation (Stad- contamination of the milk powder plant probably
houders et al. 1982; Murphy et al. 1999; Warnecke arises from low numbers of thermophiles present
2001). in the raw milk that survive pasteurization,
The main thermophilic bacteria causing the although this is yet to be proven. However, in the
dairy industry concern are of the genus Bacillus or case of thermophiles the overall raw milk quality
those genera that formerly belonged to the Bacillus has been shown to be unrelated to the quality of
genus. Thermophilic bacilli are not pathogenic. the final milk powder product (Muir et al. 1986).
However, after the powder is recombined and if the Thermophile contamination can also arise from
conditions are suitable, their spores can germinate. the reuse of by-products such as buttermilk and per-
This may result in enzyme production, acid pro- meate from milk ultrafiltration, ingredients added
duction and consequential development of an to the process such as lactose, and recycle loops in
off-flavour in the product (Chopra and Mathur manufacturing plants (Hill and Smythe 1994).
1984; Chen et al. 2004). In addition, large numbers Both spores and vegetative cells are able to attach
*Author for of bacteria of any sort are usually unacceptable to to the stainless steel and fouled surfaces (Flint
correspondence. E-mail:
most customers. et al. 2001a; Parkar et al. 2001). Once attached
sara.scott@fonterra.com
The thermophilic bacilli are difficult to eliminate to a surface, the spores probably germinate and
© 2007 Society of from a manufacturing process because of the grow, with the vegetative cells forming a biofilm.
Dairy Technology resistance of their spores to heat and chemicals, 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 cing. These results led to the development of 16S
of the biofilm) or shedding of individual cells. At rDNA primers specific for A. flavithermus and
the end of a run, most of the thermophilic bacterial Geobacillus groups, respectively. These primers
cells will be removed or killed by CIP and the allowed identification of these two major ther-
exposure to sanitizer if used (Parkar et al. 2001). mophilic contaminants by diagnostic PCR.
However, foulant or biofilms may protect spores The identification of thermophilic dairy isolates
and vegetative cells against CIP chemicals (Hinton has focused mainly on the vegetative form rather
et al. 2003). There is evidence that viable bacterial than on the spores. There is also little information
cells can remain attached to dairy manufacturing available about where and when sporulation of
surfaces following CIP (Austin and Bergeron thermophiles occurs in a milk powder manufactur-
1995; Flint et al. 1999). This will result in ther- ing plant. In this study, a survey was undertaken
mophilic cells remaining in the plant and seeding at a whole milk powder manufacturing plant to
the next manufacturing run. determine the site(s) and the rate of spore formation.
In New Zealand, the main thermophilic bacteria Spores isolated from product samples taken at dif-
of concern to the dairy industry are Anoxybacillus ferent stages during whole milk powder manufac-
flavithermus and Geobacillus spp. (Flint et al. ture were identified using specific A. flavithermus
2001b; Ronimus et al. 2003). These organisms and Geobacillus rDNA primers.
were formerly classified in the genus Bacillus.
Other bacilli also found in milk powder are the facu-
M AT E R I A L S A N D M E T H O D S
ltative thermophiles such as Bacillus licheniformis,
Bacillus coagulans and Bacillus subtilis, although Operation of the powder plant
these are usually present at low levels (Crielly et al. This study was carried out at a New Zealand whole
1994; Ronimus et al. 2003). milk powder factory. The raw milk treatment
Traditionally, biochemical tests such as the API® (separation, pasteurization and standardization)
system (bioMérieux, Marcy l’Etoile, France) have process was separate from the milk powder evap-
been used in the dairy industry to identify bacterial oration and drying process.
contaminants. However, recently, polymerase chain The raw milk treatment runs were 6–8 h in
reaction (PCR)-based identification techniques length. The raw milk was preheated to 50°C by a
such as species-specific PCR, randomly amplified plate heat exchanger (PHE), and then separated
polymorphic DNA (RAPD) profiling and partial into skim milk and cream. At this point, ingredi-
16S rDNA sequencing have provided evidence that, ents, such as lactose, could be added to the skim
in the past, A. flavithermus has been mistakenly milk depending on the product to be manufactured.
identified as Geobacillus stearothermophilus by The skim milk and cream were pasteurized
these traditional techniques (Flint et al. 2001b). separately at 73°C for 15 s and 80°C for 15 s,
Ronimus et al. (2003) used RAPD profiling to respectively. Following cream and skim milk past-
classify New Zealand thermophilic milk powder eurization, the two streams were mixed to achieve
isolates into seven groups. These included G. the required composition (standardization) and
stearothermophilus, three strains of A. flavithermus, then stored at 4°C.
two strains of B. licheniformis, and B. subtilis. The milk powder manufacturing runs were
Flint et al. (2001b), on the other hand, classified the approximately 18 h in length. Figure 1 outlines the
majority of New Zealand thermophilic milk powder evaporation and drying process in use at the time of
isolates as either Geobacillus thermoleovorans or this study. This plant was operating at a flow rate of
A. flavithermus using partial 16S rDNA sequen- approximately 40 000 L/h. Before evaporation,

Figure 1 Schematic diagram of the evaporation and drying process. F–P indicate the points at which samples were taken.

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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).

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The products were analysed using agarose gel


Table 2 Thermophilic bacterial counts of raw milk treatment process samples
electrophoresis.
Total thermophile Spore
count (cfu/mL) count (cfu/mL) R E S U LT S A N D D I S C U S S I O N
Raw milk (A) 10 < 10 Thermophiles in the raw milk treatment
Skim milk (B) < 10 < 10
process
Cream (C) 27 < 10
In order to determine whether the raw milk
Standardized milk (D) < 10 < 10
treatment process is the predominant source of
Ingredient (E) < 10 < 10
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
Enumeration of bacteria results (data not shown). Spores were not detected
Ten grams of each powder or foulant sample was in any of the milk samples (sampling points A–D),
reconstituted in 90 mL of 0.1% peptone water demonstrating that this process was unlikely to be
(BBL®-Polypeptone-Peptone, Becton, Dickinson a major source of spores in the final product.
& Company, North Ryde, NSW, Australia). Total There was no significant change (< 1 log cfu/mL)
thermophile counts were determined using a pour- in the total thermophile counts between the raw
plating technique. One millilitre of serial 10-fold milk (sample A) and standardized milk (sample D)
dilutions of each sample in 0.1% peptone water suggesting that no significant thermophile growth
was plated with Milk Plate Count Agar (MPCA, occurred in the raw milk treatment plant. However,
Oxoid Ltd, Adelaide, Australia). Thermophilic the presence of vegetative cells at low counts
aerobic spores were similarly counted after a heat suggests that the pasteurized milk destined for
treatment of the 10−1 diluted liquid sample or the milk powder plant is a source of low numbers
reconstituted powder (in 0.1% peptone) at 100°C of vegetative thermophilic cells. Because of the
for 30 min to eliminate the vegetative cells. Serial design of the plant, it was not possible to trace a
dilutions of the heat-treated sample were prepared particular milk powder run to a milk treatment
in 0.1% peptone and 1 mL samples of the appro- run to determine the true relationship between the
priate dilutions were pour-plated with MPCA +2% thermophiles in the pasteurized milk and those of
starch (MPCA +S). All of the plates were prepared the powder plant.
in triplicate and incubated at 55°C for 48 h. The Our findings that the counts of vegetative cells
final counts (cfu per gram or millilitre) were deter- do not significantly increase during pasteurization
mined by counting those plates containing 20–300 are in agreement with those of Reddy et al. (1975)
colonies and multiplying the number of colonies and Muir et al. (1986), who found that raw milk
by the dilution factor. Plates of 1 mL undiluted treatment has very little influence on thermophile
liquid milk samples were unable to be counted, as numbers in milk destined for milk powder manu-
it was too difficult to distinguish the colonies in the facture. This is probably because of the shorter run
opaque agar; therefore, those plates (prepared with times of raw milk treatment compared with the run
a 10−1 diluted sample) with less than 20 colonies length of milk powder manufacture. In contrast to
were still counted to give an approximate count. these findings and our observations, Murphy et al.
(1999) have recorded thermophile counts in
Identification of spore isolates pasteurized milk as high as 3500 cfu/mL. However,
Approximately 10 spore-derived colonies isolated as they did not examine the raw milk treatment
from each product sample taken at the end of runs process, it is not possible to determine the reason
5 and 6 were randomly picked from the surface of for this large discrepancy.
the MPCA +S spread plates and identified using
PCR. Location of thermophile growth and spore
Specific 16S rDNA-derived primers (Flavo and formation sites during milk powder
Levo) (Flint et al. 2001b), were used in combina- manufacture
tion with the universal primer Y1 (Young et al. To determine where thermophiles grow and form
1991) to identify the spore-derived colonies. The spores during milk powder manufacture, product
A. flavithermus primer Flavo is species specific. samples were taken at different stages throughout
However, the Levo primer is genus specific for the manufacturing process of standard whole milk
the Geobacillus group. The presence of a PCR powder and instant whole milk powder. This was
product, approximately 450 bp in length, with the repeated for each type of milk powder run. Figure 3
appropriate primer set identifies the isolate as shows the total thermophile counts and the ther-
the organism corresponding to that primer set. mophilic spore counts for the samples collected

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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
Figure 3 Thermophilic bacterial counts from samples taken instant whole milk compared with standard whole
at different stages at the end of various whole milk powder
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
Dynamics of thermophilic spore production in
mean and standard deviation of triplicate counts. The powder a milk powder manufacturing plant
sample counts (O and P) were measured in log10 cfu/g and In the previous section, the preheater was identified
the remaining sample counts (F–N) were measured in as the stage in which the most dramatic increase
log10 cfu/mL. A * indicates that no sample was taken. 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
during these four manufacturing runs. The total every 2 h from the preheating and evaporation
thermophile plate count was always higher than the sections of the milk powder plant throughout two
thermophilic spore count, indicating that both whole milk powder runs (runs 5 and 6). The spore
spores and vegetative cells were present. count results are presented in Figure 4.
The consistent increase in both total ther- Spores were detected (> 1 log cfu/mL) only after
mophile counts and spore counts from the balance approximately 9 h into both runs. At 18 h, spores
tank sampling point (F) to the pre-evaporator reached levels of up to 4.1 log10 cfu/mL in the evap-
sampling point (G) during instant whole milk orator pass samples, whereas none were detected in
powder manufacture demonstrates that the preheat the balance tank feeding the manufacturing process
section was one site of vegetative cell growth and (Figure 4). This confirms that spores were forming
sporulation. within the milk powder manufacturing process and
Further downstream in the milk powder process, were not a result of external contamination.
a noticeable increase in the total thermophile count The rate of spore formation, the initial sites of
was observed between samples G and H for all of spore formation and the spore numbers in the
the runs and samples H and I for run 4. A noticeable final product were different for the two runs. Spore
increase in spore counts was also observed between formation occurred at a faster rate during run 5
samples H and I (run 3) with a further increase compared with run 6; they were detected after 11 h
occurring during pass 2 (Figure 3b). This suggests in all of the samples except the pre-evaporator

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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.

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
Figure 5 Thermophilic bacterial counts of foulant that was burnt milk. Fouling can also occur in areas of flow
scraped aseptically from a milk powder manufacturing plant recirculation, for example under the orifice pans.
after a standard whole milk powder run. Each bar represents To determine if foulant was a possible source of
the mean and standard deviation of triplicate counts. inoculum, foulant samples were taken during a
shutdown of the milk powder plant and the total
thermophile and thermophilic spore counts were
samples, and reached levels of 3 log10 cfu/mL determined. Foulant was taken from the DSI unit,
within 13 h. However, during run 6, spores were the orifice pans from passes 1 and 2 of the evaporator,
detected in the passes 2 and 3 of the evaporator after and a support pole in the separator of evaporator
11 h, but they were not detected in pass 4 until 13 h effect 1.
into the run and after 15 h in the preheat samples. The total thermophile count and the thermophilic
There are many factors that can vary between spore count were similar for all of the samples,
different manufacturing runs and different milk suggesting that the bacteria present in these
powder manufacturing plants. For example, the samples were predominantly in their spore form
milk quality (age, chemical and microbiological), (Figure 5). Foulant collected from the evaporator
the temperature of the stored milk; the product had thermophile counts that were at least 2 log
type; and the temperature regimes may be different. higher than those for the foulant from the DSI unit.
Therefore, it is difficult to determine why the This was expected given that the DSI unit was at
rate of sporulation and the spore count differed a temperature of 95°C. Spores would be able to
between runs 5 and 6. survive this temperature and possibly be deposited
To the authors’ knowledge this is the first time on the DSI unit surface but there would be no
the rate of sporulation has been successfully vegetative cell growth.
monitored in a milk powder plant. A similar study Although there is evidence that thermophile
by Murphy et al. (1999), in a skim milk powder attachment and growth occur faster on foulant than
pilot plant, found no notable difference in spore on un-fouled stainless steel (Hinton et al. 2003), to
numbers between the start of the run and the end of the authors’ knowledge, this is the first study that

114 © 2007 Society of Dairy Technology


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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
Figure 6 Identification of Anoxybacillus flavithermus further identification was necessary. These two
from the powder plant isolates by species-specific PCR. organisms have frequently been isolated from New
The gel represents only a fraction of analysed samples. Zealand milk powders, and have also been found
Lanes: 1, DNA size standard; 2–11, end-of-run pass 2 overseas (Flint et al. 2001b; Warnecke 2001;
evaporator spore isolates; 12, blank (no template); 13, Ronimus et al. 2003; Rueckert et al. 2004).
Geobacillus thermoleovorans DSM 5366 (negative control); Figure 8 shows the proportion of thermophilic
14, A. flavithermus DSM 2641 (positive control); spores identified as either A. flavithermus or Geo-
15, DNA size standard. 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
Figure 7 Identification of Geobacillus spp. from the powder the foulant samples were identified as Geobacillus
plant isolates by genus-specific PCR. The gel represents only spp. No A. flavithermus spores were detected. These
a fraction of analysed samples. Lanes: 1, DNA size standard; results provide further evidence that Geobacillus
2, Geobacillus thermoleovorans DSM 5366 (positive spp. predominates in the evaporator. Anoxybacillus
control); 3, A. flavithermus DSM 2641 (negative control); flavithermus found in the evaporator milk samples
4, blank (no template); 5–14, end-of-run pass 2 evaporator
probably originated from cells shedding from
spore isolates; 15, DNA size standard.
biofilm or foulant material in the preheat section.
Although there is evidence that certain bacterial
has provided evidence that foulant is a source of species can predominate in different sections of
thermophile contamination in a full-scale milk milk powder manufacturing plant equipment
powder plant. (Langeveld et al. 1995; Hinton et al. 2001; Warnecke
2001), this appears to be the first study in which
The identification of thermophilic spores thermophilic spores have been identified throughout
A PCR assay, designed by Flint et al. (2001b), was the different stages of milk powder manufacture.
used to determine whether thermophilic spore
contamination in the milk powder manufacturing
CONCLUSIONS
plant was caused by A. flavithermus and/or the
Geobacillus group. The presence of a PCR product This study has demonstrated that the appearance of
of approximately 450 bp in length with the appro- high numbers of thermophilic spores in whole milk

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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
Figure 8 Relative representation of Geobacillus spp. and Sparkes for her excellent laboratory work, the
Anoxybacillus flavithermus spores in a milk powder Pahiatua factory staff and Tuan Truong for helping
manufacturing plant.
with this study, and the Technology for Industry
Fellowship for funding this work.

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