Characterization of spore forming organisms in fluid milk and milk powder plants in the Northeast
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Characterization of spore forming
organisms in fluid milk and milk powder
plants in the Northeast
Rachel Miller, Stephanie N. Masiello, David J. Kent, Alexis Andrus,
Nicole H. Martin, Kathryn J. Boor, and Martin Wiedmann
Milk Quality Improvement Program, Department of Food Science,
Cornell University
Overview
• Intro to sporeformers
• Case studies on sporeformer contamination in powder
plants
• Raw milk contamination patterns with sporeformers
– Psychrotolerant
syc oto e a t sporeformers
spo e o e s
– Mesophilic and thermophilic sporeformers
• Estimating the impact of raw milk spore counts on
whole milk powder spore counts
• Risk factors for spore former presence in raw milk
Sporeformers – classification
• Microbes and sporeformers can be classified by growth
temperatures:
– Psychrotolerant: Can grow at cold temperatures (around 6 C), but
optimum temperature is higher
– Mesophilic:
p Optimum
p ggrowth around 32 C; enumerated byy
“mesophilic spore count” (MSC)
– Thermophilic: Optimum growth around 55 C; enumerated by
“thermophilic
p spore
p count” ((TSC))
• Two General Classes of sporeforming bacteria: Bacilli and
Clostridia
Class Bacilli: Class Clostridia:
Notable Member: B.
B anthracis Notable Members:
‐ C. botulinum
Common in Milk: ‐ C. perfringens
‐ Psychrotolerant:
Psychrotolerant ‐ C.
C dificile
‐ Paenibacillus spp.
‐ B. weihenstephanensis Common in Milk:
‐ Mesophiles:
M hil ‐ Mesophiles:
M hil
‐ B. licheniformis ‐ C. tyrobutyricum
‐ B. pumilus ‐ C. beijerinckii
‐ B. sporothermodurans
h d ‐ C. butyricum
b
‐ B. subtilis ‐ C. sporogenes
‐ Thermophiles:
‐ Anoxybacillus flavithermus
‐ Geobacillus spp.Spores cause spoilage in dairy foods
Paenibacillus Bacillus sporo- Clostridium Anoxybacillus
Spoilage
spp some
spp., thermodurans tyrobutyricum flavithermus
flavithermus,
Organisms
Bacillus sp. (Thermophilic) C.butyricum Geobacillus sp.
Product Gas
G
Proteolysis, Proteolysis,
Quality Proteolysis formation,
Lipolysis Acid
Defects Butyric Acid
Adapted from M. Ranieri 5Spores not “just a quality issue”
Examples of microbiological
requirements for dairy powder
• Clostridium perfringens • Aerobic Plate CountPrevious Study: Thermophilic Spore‐ formers in Whole Milk Powder Processing Scott, SA, et. al, “The formation of thermophilic spores during the manufacture of whole milk powder.” Vol 60, No 2 May 2007 International Journal of Dairy Technology.
Detection of sporeformers
Highly Heat Specially Heat
Spore Pasteurization Resistant Spore Resistant Spore
80°C/12m
80 C/12m Treatment Treatment
100°C/30m 106°C/30m
Selects for
PSC
HHR‐MSC Anoxybacillus
6°C/10d
6 C/10d incubation
32°C/48h
/ flavithermus and
incubation
Geobacillus spp
MSC ST‐TSC
32°C/48h 55°C/48h
incubation incubation
HHR‐TSC
TSC 55°C/48h
55°C/48h incubation
incubation
Diversity of other methods are out there; no real standardizationIdentification of sporeformers
Isolate bacteria via plating
Amplify genetic target with
polymerase chain reaction (PCR), Compare to database to
followed by DNA sequencing characterize and identifyOverview
• Intro to sporeformers
• Case studies on sporeformer contamination in
powder plants
• Raw milk contamination patterns with sporeformers
– Psychrotolerant
syc oto e a t sporeformers
spo e o e s
– Mesophilic and thermophilic sporeformers
• Estimating the impact of raw milk spore counts on
whole milk powder spore counts
• Risk factors for spore former presence in raw milkNumerous studies have identified sources of spores in
both farm and processing plant environments
Plantt Environment
Pl E i t
- Incoming raw ingredients (Burgess
Farm Environment
et al., 2010)
- Bedding (Magnusson et al., 2007)
- Biofilm contamination ((Scott et al.,,
- Feed
F d (J(Julien
li ett al.,
l 2008)
2007)
- Soil (Vissers et al., 2007)
- Heat exchangers (Scott et al., 2007)
- Manure (Bagge et al., 2010)The spore populations of raw milk and
dairy powders are very different
Predominant spores in raw milk Predominant spores in dairy powders
- B. licheniformis (Coorevits et al. 2008) - Anoxybacillus sp. (Ruckert et al. 2004)
- B. pumilus (Sutherland et al. 1994) - B. licheniformis (Schedelman 2006)
- B.
B cereus Group
G (D Jonghe
(De J h ett al.
l 2010) - Geobacillus
b ll sp. (Burgess
( et al.
l 2010))
- B. subtilis (De Jonghe et al. 2010)
- Paenibacillus sp. (Huck et al. 2007)Farm and Processing Plant Study sites
- 33 farms throughout NY State - 4 powder processing facilities
- Sampled
S l d bi-monthly
bi thl for
f 1 year - Sweet Whey/ Plant A
- NFDM/ Plant B
- Acid Whey/ Plant C
- WPC-80/ Plant D
- Sampled bi-monthly for 1 yearSampling scheme for powder plants
• April 2012 – March 2013
– 4 Plants Sampled Bimonthly
• 3 Time Points:
• Beginning (B)
• Middle (M)
• End (E)
– ~16-24 Hr Runs
16Raw
((wheyy Storage Crystal Powder
or milk) Tank(s) Tank(s) Silo
Raw Spray
Filtration Bagging
Tank(s) Dryer
Permeate
Separator Fitz Mill Storage
Silo(s)
Pasteurizer
Evaporator Sifter Ship
(HTST)Acronyms • PSC – Psychrotolerant Spore Count • MSC – Mesophilic Spore Count • TSC – Thermophilic Spore Count • SP – Spore Treatment • H or HHR – Highly Heat Resistant Spore Treatment • WIP – Work In Process
TS are the primary organism of concern among all
dairy powders tested
100%
90% 23% < Detection
29%
80%
70% Enrichment +
18%
60% 71% 77%
50% 95%
Di
Direct Pl
Plate +
33%
40%
30% 59%
20% 38%
10%
5% 3%
0% 2%
PSC MSC TSC
n=414 n=413 n=414
19The majority of sweet whey and NFDM samples
tested had detectable levels of TS and MS
Plant / Product TSC MSC HHR TSC HHR MSC
A / Sweet Whey 94.6 28.5 60.7 13.5
B / Nonfat Dry Milk 87.7 85.7 54.5 8.0
C / Acid Whey 18.1 22.2 0 0
D / WPC-80 16.0 12.3 2.3 4.7
Average 59.4 37.8 32.8 7.3
20Bacillus licheniformis is the most frequently
isolated sporeformer in raw milk
B. licheniformis
4% 2%
4%
Other
5%
Other Bacillus species
9%
B. pumilus
49% B. subtilis
11% B. cereus group
B. clausii
16% Paenibacillus spp.
n = 637Anoxybacillus sp. is the most prominent
p
sporeformer isolated from sweet whey/plant
yp A
2% 5% Anoxybacillus sp.
5%
Geobacillus sp.
15%
~ 75% NOT from
f Bacillus cereus Group
49%
raw milk
Bacillus
ac us licheniformis
c e o s
Bacillus subtilis
24%
Other
n = 417A number of different sporeformers are found
in WPC-80/plant
WPC 80/plant D
3%2% B ill lilicheniformis
Bacillus h if i
14% Geobacillus
33%
Bacillus thermoamylovorans
14%
Bacillus cereus Group
Other
Bacillus subtilis
17% 17%
Bacillus nealsonii
n = 185ount
Log Bacterial Co
Plant A/B representative – TSC
0.2 cfu/g
0% 0% 25%
25% 12 5%
12.5% 6 25%
6.25% 0% 0% 25% 0%
Raw WIP Finished
n=12 n=48 n=12DNA fingerprint patterns AT 1 AAATTCGCCTCATGCGATGCTAGGGCTATTAGCCTTATC AT 6 AAATTCGCCACATGCGATGCTAGGGCTCTTAGCCTTATC AT 169 AAGTTCGCCTCATGCGATGCTAGGACTATTAGCCTTATC
Initial molecular subtyping data – plant 2
Comparison with subtype
frequency in raw milk
Sample Cycle 1 Sample Cycle 2 Sample Cycle 3
Frequency of
AT B M E B M E B M E Total Isolation From
Raw Milk
1 4 2 4 5 4 3 5 5 3 35 21%
6 1 1 1 1 1 1 6 13%
9 2 1 1 4 1%
169 1 1 1 3 6%
351 2 1 3 1%
352 2 2 0%
353 4 1 1 1 3 1 2 1 14 0%
354 2 2 0%
355 1 1 0%
356 1 1 0%
357 1 1 0%
358 1 1 1 3 0%
359 1 1 2 0%
361 1 1 2%
376 1 1 0%
Total 79Location of samples positive for
Anoxybacillus (AT 353)
Raw Milk HTST
Preheater Spray Dryer
Receiving ((Past. & Regen)
g )
M‐1
SP5
M‐2
Evaporator
Raw Milk Silo Separator Sifter
(Effects 1‐4)
SP1
B‐1
B‐1 SP3
E‐1 B2
B‐2
Balance Tank Balance Tank M‐2 Evaporator
(Effects 5‐7) Collection Silo
(Whole) (Skim) E‐2
SP2
M‐3 B‐1
E‐3 M‐2
M‐2
Regenerator Preheater Dryer Feed Tank Bagging
E‐2
SP4 SP6
M‐3Location of samples positive for
Bacillus licheniformis (AT 1)
Raw Milk HTST
Preheater Spray Dryer
Receiving ((Past. & Regen)
g ) B1
B‐1
E‐1
E‐1 SP5
B‐2
B‐2
B2
B‐2 M‐2
Evaporator
Raw Milk Silo M‐2 Separator
(Effects 1‐4)
E‐2 Sifter
SP1 B‐3 B‐3
M‐3 B‐1 SP3
M‐3
E‐3 M‐1 E3
E‐3
Balance Tank Balance Tank B‐2 Evaporator
(Effects 5‐7)
M‐1 Collection Silo
(Whole) (Skim) M‐2
SP2 E‐1
B‐2 B‐1
M‐2 E‐1
E‐2
Regenerator Preheater Dryer Feed Tank B‐3 Bagging
E‐2
SP4 M‐3 SP6
B‐3
E‐3 M‐3
E‐3Powder Plants Summary
and Conclusions
• In some plants spore counts increase as production runs proceed
– Consistent with published findings from Australia & New Zealand
• Some
S spore fformers may persist
i t in
i processing
i liline over ti
time
– Likely in locations that cannot be sanitized (niches? biofilms?)
• Some spore formers appear to represent introduction from raw
material
• Molecular subtyping will help us identify the organisms and their
sources / niches.
– Will facilitate identification of target equipment and equipment areas for
(costly) interventions
• There is negligible correlation between MSC and TSC counts, nor is
there a correlation between MSCH and TSCH
– Monitoring for one type of sporeformer will not provide insights into
potential problems with other typesOverview
• Intro to sporeformers
• Case studies on sporeformer contamination in powder
plants
• Raw milk contamination patterns with sporeformers
– Psychrotolerant
syc oto e a t sporeformers
spo e o e s
– Mesophilic and thermophilic sporeformers
• Estimating the impact of raw milk spore counts on
whole milk powder spore counts
• Risk factors for spore former presence in raw milkPresence of psychrotolerant
sporeformers in raw milk
• Collected bulk tank raw milk samples from 108
farms, heat treated to eliminate vegetative
cells, incubated at 6 C for 21 day
– Any bacterial growth was confirmed as
sporeformer
p
– 40 samples contained low enough levels of
psychrotolerant sporeformers so that no growth
was observed over 21 daysHigh farm = > 20,000 cfu/mL at 21 days Low farm = ≤ 100 cfu/mL at 21 days post pasteurization post pasteurization
Psychrotolerant Sporeformer Counts by
MonthPsychrotolerant Sporeformer Counts by
MonthMesophilic and thermophilic spore‐
formers In raw milk
• Study Design:
– 33 Farms in NY state
– Sampling: April 2012-March 2013
• Bulk tank samples collected bi
bi-monthly
monthly from each
farm
– Questionnaire administered at each sampling
• Frequency of milking
• Type of bedding
• Type of teat dip usedIn raw milk, MS are more prevalent than TS
100% 4%
26%
38%
Peercent off Samplles
75%
< -1.5 log CFU/mL
50%
(n=1198)
74% -1.5 to 1.0 log CFU/mL
25% 57% > 1.0 log CFU/mL
0%
MSC TSC
Test Type
- All BT raw milk samples had MS
- Not all samples had detectable levels of TSMS level is significantly different between farms
D6
D3
C9
C6
C3
Farm
B8
Apr-May
B5
Jun-Jul
B2 A S
Aug-Sep
A7 Oct-Nov
Dec-Jan
A4
Feb-Mar
A1
0 5 10 15 20
Cumulative MSC (log CFU/mL)SCC and SPC versus TSC
SCC vs
vs. Log TSC Log
g SPC vs. Log
g TSC
2.5 2.5
2 2
Log TSC
C
Log TSC
C
1.5 1.5
1 1
0.5 0.5
0 0
0 200 400 600 800 1000 0 2 4 6
SCC X 1000 Log SPCCorrelating raw milk spore counts to
WMP spore counts: a calculation
Ex: sample with spore count of 50 spores/mL milk
Step 1: “Convert”
Convert to powder
50 spores x 7.8 mL milk = 390 spores/g powder
mL milk g powder
p
In theory, this milk would produce WMP with 2.6 log spores/gMSC requirements‐ An example
Farm H1
Spores/mL 501 583 459 440
Log spores/g WMP 36
3.6 37
3.7 36
3.6 35
3.5
Given a customer requirement of 2.9 log MSC spores/g powder
=> 4/4 samples from farm H1 would result in WMP above specification
Farm L1
Spores/mL
Spores/m 0 10
0 0 20.4
0.4
Log spores/g WMP 0 1.9 0 2.2
Given a customer requirement of 2
2.9
9 log MSC spores/g powder
=> 0/4 samples from farm L1 would result in WMP below specificationDistribution of MSC and TSC – NYS MSC TSC
Distribution of MSC and TSC –
NYS vs.
vs a selected milkshed
MSC TSC
• 98.6% of individual cooperative bulk tank raw milk samples would meet
WMP MSC and TSC specifications of 2
2.9
9 and 2
2.3
3 log spores/g
spores/g, respectivelyOverview
• Intro to sporeformers
• Case studies on sporeformer contamination in powder
plants
• Raw milk contamination patterns with sporeformers
– Psychrotolerant
syc oto e a t sporeformers
spo e o e s
– Mesophilic and thermophilic sporeformers
• Estimating the impact of raw milk spore counts on
whole milk powder spore counts
• Risk factors for spore former presence in raw milkHow to identify “risk factors” for high
levels of sporeformers in raw milk
• For a sufficient number of farms, determine (i) spore
counts in
i raw milk
ilk ((e.g., TSC, MSC)) and
d (ii) perform
f
survey of factors that are hypothesized to be associated
with higher spore counts (“questionnaire”)
( questionnaire )
– Silage quality, bedding, cleanliness of cows, etc.,
• Perform statistical analysis to identify factors that are
significantly associated with higher spore countsTABLE
A 11. S
Summary off ffarm management practicesi iincluded iin data analyses
Cow Hygiene Score assigned from 1:4 (1 representing cleanest) based on the presence
of dirt and manure
Milki Routine
Milking R ti P
Preparation
ti off udder,
dd gloves
l worn dduring
i milking,
ilki type
t off teat
t t
disinfectant used (spray, dip), problem cows segregated during milking,
problem cows milked last
General Number of cows milked
milked, average production per cow
Management
Environment Housing type, bedding type, frequency of bedding change, ventilation in
housing area (air flow),
flow) frequency of pen cleaning,
cleaning use of recycled water
Sanitation Type of bulk tank sanitizer used, milking units sanitized between groups,
frequency of bulk tank cleaningA “one size fits all” approach may not be appropriate
g
for targetingg MS and TS in bulk tank raw milk7.00
6.00
5.00
4.00
og cfu/mL orr g
3.00 MSC
TSC
2 00
2.00
lo
HHR TSC
STSE
1.00
0.00
‐1.00
SampleSummary
- C
Currentt specifications
ifi ti for
f mesophilic
hili and
d th
thermophilic
hili spores
in dairy powders represent an important hurdle
y
- Systems approach
pp ((farm and p
processinggpplant)) is needed to
produce high quality powders (particularly WMP) that fulfill
stringent customer specs
- Focusing on processing alone may not be sufficient
- Focus on reducing raw milk SCC and SPC will not necessarily
reprove raw milk spore counts
- While specific risk factors for high thermophilic and mesophilic
spore counts remain to be defined, there are ample potential
spore sources in the dairy farm environment
- Udder and milking hygiene and related factors are likely to
be criticalAcknowledgments
• Dairy Research Institute • Nicole Martin
• Kathryn Boor
• NYS Milk Promotion • Mark Jandricic
Advisory Board • Stephanie Masiello
• p y
Steve Murphy
• Matt Ranieri
• Rob Ralyea
• Alexis Andrus
• Rachel Miller
• Nancy CareyYou can also read
