emerging thermoduric / thermophillic bacteria and their inactivation

02/20/2013
/
THERMOPHILLIC BACTERIA AND
THEIR INACTIVATION BY
ULTRASONICATION
1
Sanjeev Anand
Associate Professor
Dairy Science Department
South Dakota state University
U.S. Dairy Industry Spore Seminar
EMERGING THERMODURIC
OUTLINE OF THIS TALK
02/20/2013
Common terms related to heat resistant bacteria
 Emerging thermoduric spore formers in the dairy
manufacturing environment
 Thermoduric progression during Cheddar cheese
manufacture – A case study
 Role ultrasonication can play to inactivate
thermoduric sporeformers and spores- A case
study

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THERMODURIC BACTERIA
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Organisms that survive thermal processing
treatments such as pasteurization
 The common genera considered thermoduric

U.S. Dairy Industry Spore Seminar
Bacillus
 Geobacillus
 Clostridium


More recently
Paenibacillus
 Anoxybacillus

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THERMODURICS THAT DO NOT SPORULATE

Micrococcus

Streptococcus

Microbacterium

Enterococcus

Coryneforms

Alcaligenes

Arthrobacter
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Lactobacillus
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
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THERMOPHILES
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
Organisms that tolerate and actively grow at
high temperatures
U.S. Dairy Industry Spore Seminar
45°C to 122°C / 113°F to 252°F
 Optimum growth for most at 55°C / 131°F
 Thermodurics may or may not be thermophilic

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THERMOPHILES OF CONCERN TO THE
DAIRY INDUSTRY





Ideally grow where
temperature reaches
40 to 65°C
U.S. Dairy Industry Spore Seminar

Anoxybacillus
flavithermus
Geobacillus
sterothermphilus
Bacillus licheniformis
Bacillus coagulans
Bacillus subtilis
Bacillus
sporothermodurans
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
Regeneration section
of heat exchangers
and in evaporators
 Result in high
numbers (up to 106
cfu/g) in the final
product

(Scott et al, 2007; Burgess et al, 2010, Tabit and Buys, 2010)
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WHY THERMODURIC THERMOPHILES ARE
DIFFICULT TO ELIMINATE?
02/20/2013
Form spores resistant to heat and chemicals
 Wide temperature growth range
 Exhibit a fast growth rate


U.S. Dairy Industry Spore Seminar

generation time 15-20 min
Readily form biofilms
Both spores and vegetative cells can attach to
stainless steel and fouled surfaces
 Foulant or biofilms may protect spores and vegetative
cells against CIP chemicals

(Flint et al, 1999 & 2001; Parker et al, 2001, Scott et al, 2007;
Burgess et al, 2010)
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BACILLUS SPOROTHERMODURANS
First detected as a high heat–resistant spore
(HHRS) in UHT milk
D140 and Z values of spores of 3.4–7.9 s and 13.1–
4.2°C, respectively
Strictly aerobic, can hydrolyze casein
 Contributes to the reduced quality of
commercially sterilized milk and milk products



Due survival and out growth of the spores to
unacceptably high levels
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02/20/2013

Non pathogenic
(Pettersson et al, 1996; Humer et al, 1998;
Vaerewijck et al, 2001, Tabit and Buys, 2010)
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BACILLUS LICHENIFORMIS
Facultative anaerobe of Bacillus subtilis group

Optimum growth temperature 30°C


Can grow from 15 to 55°C
Considered a common spoilage organism isolated
from raw milk

Some strains have been associated with toxin
production
(Mansour et al, 1999; Salkinoja-Salonen et al, 1999;
Burgess at al, 2010)
U.S. Dairy Industry Spore Seminar

Other members B. subtilis, and B. pumilus
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BACILLUS COAGULANS
A lactic acid forming species within genus Bacillus

Facultative anaerobic sporeformer
Some strains do not sporulate readily
 Optimum growth temperature 40 and 57°C
 Growth range 15°C-61°C


Involved in coagulation and flat souring of
evaporated canned milk
(Nakamura et al, 1998; Vecchi and Drago, 2006;
Burgess et al, 2010; Vercammen et al, 2011)
U.S. Dairy Industry Spore Seminar

Initially considered a spore-forming Lactobacillus
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
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GEOBACILLUS (BACILLUS)
STEAROTHERMOPHILUS

Typically responsible for the flat-sour spoilage of
low-acid canned foods, including evaporated milk
(Head et al, 2008; Nazina et al, 2001; Dogan et al, 2009;
Burgess at al, 2009; Viedma et al, 2010)
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Grows between 37oC to as high as 76oC
 Spores of are extremely heat resistance with D121
value of 42s

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Aerobic or facultative anaerobic endosporeforming rods
 Optimum growth temperatures is 50 °C

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ANOXYBACILLUS FLAVITHERMUS
Obligate anaerobic or facultative aerobic

Recognized as a major contaminant in milk
powder
Normally present at low levels in raw milk
 May reach up to 105cfu/g levels in the final product
after 15–20h of plant operation
 Being thermophilic, rapidly form biofilms

(Ruckert et al, 2004; Heinin et al, 1982; Pikuta et al, 2000,
Burgess at al, 2010; Palmer et al, 2010)
U.S. Dairy Industry Spore Seminar
Optimum growth temperature is 60 and 65oC under
aerobic and anaerobic conditions, respectively
 Spores are very highly heat resistant

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
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PAENIBACILLUS SPECIES
Recognized as a new genus Paenibacillus in 1993

Facultative anaerobic or strictly aerobic
 Can grow from 10 to 40oC, the optimum being
37oC
 Recognized as an important fluid milk spoilage
organism


Found in both raw and pasteurized milk
(Daane at al, 2002; Huck at al, 2007)
U.S. Dairy Industry Spore Seminar
Since then, over 26 species have been identified
within the genus Paenibacillus
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
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SUMMARIZING
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U.S. Dairy Industry Spore Seminar
Several organisms represent thermodurics
 Thermodurics may or may not be sporeformers
 Spores, as well as, vegetative cells have thermal
resistance
 Thermodurics may be mesophiles, thermophiles
or psychrotrophs

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A STUDY UNDER PROGRESS IN OUR LAB
Commercial Cheddar cheese manufacturing
cycles scanned

Samples of milk, cheese, whey, and WPC collected
Microbial analysis for
Standard Plate Counts (SPC) on plate count agar at
35°C for 24h
 Thermoduric mesophiles (TM)
 Thermoduric thermophiles (TTh)

(Khilendra and Anand, 2013; unpublished)
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Raw Milk Counts
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6
4
3
SPC
2
TTh
TM
1
0
Start
Mid Day
End (22h)
U.S. Dairy Industry Spore Seminar
Log cfu / mL
5
20
3
2
1.5
1
0.5
0
Start
Pre midday wash
Thermoduric mesophiles
Post midday wash
End
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2.5
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Thermoduric Thermophiles increase during
long hours (22-24h) of milk pasteurization
(log cfu / mL)
Thermoduric thermophiles
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Cheddar cheese counts (2 days old)
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6
Log cfu/g
4
SPC
3
TM
2
TTh
1
0
Start
Pre midPost midday wash
day wash
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End
22
Cheddar cheese counts (30 days old)
4.5
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4
3
2.5
2
TTh
1.5
TM
1
0.5
0
Start
Pre midday wash
Post
mid-day
wash
U.S. Dairy Industry Spore Seminar
Log cfu/g
3.5
End
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THERMODURICS MAY CAUSE SLITS
Lactobacillus spp.
 Clostridium spp.

U.S. Dairy Industry Spore Seminar
Causative organisms
for these slits in
Cheddar cheese
samples
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Counts during whey processing
(at the end of 22h continuous cycle)
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9
8
Log cfu/g
6
5
SPC
4
TM
3
TTh
2
1
0
Whey
Past.
Whey
Whey
permeate
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WPC
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SUMMARIZING
Thermoduric thermophiles counts in the
pasteurized milk increased with the run time
Post mid-day cheese samples also showed higher
thermoduric thermophiles
 Thermoduric mesophiles did not show significant
variations during the entire run
 Thermoduirc counts of whey concentrates were
below log 2.0 even at the end of a 22h long run
 Ripening studies are in progress
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U.S. Dairy Industry Spore Seminar
Mid-day wash helped to some extent in reducing
their counts
 Post mid-day run resulted in a greater build up of
thermoduric thermophiles
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A STUDY RECENTLY COMPLETED IN OUR
LAB
Ultrasonication parameters standardized in skim
milk
Low frequency (20-100kHz)
 500 W, 20 kHz, VC 505 (Sonics & Materials, Inc., Newtown,
CT, USA)


U.S. Dairy Industry Spore Seminar
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02/20/2013
Previously reported thermoresistant strains of
Bacillus, Geobacillus, and Anoxybacillus selected
 Propagated as vegetative cells
 Spores prepared and harvested
 Inactivation study

Ultrasonication followed by pasteurization
(Som and Anand, 2012; unpublished)
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INACTIVATION OF THERMODURIC
VEGETATIVE CELLS BY ULTRASONICATION
Anoxybacillus flavithermus
Bacillus coagulans
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U.S. Dairy Industry Spore Seminar
Log reductions
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6
4
3
2
1
0
Pasteurization
Ultrsonication
Ultrsonication + Pasteurization
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INACTIVATION OF THERMODURIC SPORES
BY ULTRASONICATION
80
U.S. Dairy Industry Spore Seminar
70
60
50
40
30
20
10
0
02/20/2013
% reductions in spore counts
Ultrasonication
B. licheniformis
Ultrsonication + Pasteurization
B. coagulans
Ultrsonication+higher heat
G. sterothermophilus
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After Ultrasonication
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Higher magnifications
Ultrasonication +
pasteurization
No structural changes in spores were observed after ultrasonication alone or a
combined ultrasonication and pasteurization
SEM images of B. licheniformis spores
U.S. Dairy Industry Spore Seminar
Lower magnifications
Before Ultrasonication
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SUMMARIZING
Pasteurization followed by ultrasonication can
effectively inactivate thermoduric spores up to 65.7 % in
the case of G. stearothermophilus spores
 A combination of higher heat treatment at 80oC for 1
min and ultrasonication can further enhance the
inactivation to 75.3%
U.S. Dairy Industry Spore Seminar

Thermodurics such as A. flavithermus and B.
coagulans can be inactivated to a higher extent
by ultrasonication and it’s combination with
pasteurization
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
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ON-GOING RESEARCH

An on-line ultrasonicator (1000 watts) with a
frequency of 20kHz is being procured from Hielscher
USA, Inc.
The process has the potential to also improve the
microbial quality of other dairy products such as
fluid milk, and cheese
U.S. Dairy Industry Spore Seminar
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02/20/2013
This prelim data on the effectiveness of
ultrasonication was generated during a Discovery
project
 We are currently engaged in testing the
combination of ultrasonication and
pasteurization in reducing counts of thermoduric
bacteria and spores in non fat dried milk
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ACKNOWLEDGEMENT
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U.S. Dairy Industry Spore Seminar
Graduate students: Khilendra Bhanduriya,
Laura Mealy, Som Nath Khanal
 Undergraduate researchers: Kaylee King, Kim
Buehner, Megan Huegli, and Eric Flynn
 Dr. Lloyd Metzger, Cheese trials; Dr. K.
Muthukumarappan, Ag. Biosystem Engg.,
ultrasonication equipment
 DRI and MDFRC: Research funding

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