1 Module 1: Thermoduric Bacteria Thermodurics - Lloyd Metzger.pdf · SUMMARIZING Thermoduric thermophiles counts in the pasteurized milk increased with the run time Mid-day wash helped

Dairy ThermoduricsModule 1: Thermoduric Bacteria

Copyright © 2014. Sanjeev Anand. Dairy Science Department, South Dakota State University, SD. 57007.

1

What are thermodurics?

Organisms that survive thermal

processing treatments such as

industrial pasteurization

Some of these form spores, while

other are non-sporulating

The common sporulating genera

considered thermoduric include

Bacillus

Geobacillus

Clostridium

More recently

Paenibacillus

Anoxybacillus


2

What are spores?

Spores are bacterial adaptation to adverse conditions

They are dormant forms of bacterial cells

Thick outer covering consists of highly cross-linked polypeptides and peptidoglycan

Spore core (cytoplasm)- consists of necessary metabolic components and DNA

Allows resistance to extreme temperature such as pasteurization, cleaning and sanitation procedures


3

Carlin, 2010; Atrith, 2002; Mansur et al, 1998

SEM Bacillus speciesSom and Anand, 2012

Thermodurics that do not sporulate

Lactobacillus

Streptococcus

Enterococcus

Alcaligenes

Micrococcus

Microbacterium

Coryneforms

Arthrobacter


4

Growth temperature based

classification

Thermodurics can be divided in to three sub groups based on their

temperature of growth

Thermophilic

Mesophilic

Psychrotrotolerant


5

Thermoduric thermophiles

Organisms that tolerate and

actively grow at high

temperatures

45° to 122°C / 113° to 251.6°F

Optimum growth for most at

55°C/ 131°F

All Thermodurics may or may not

be thermophilic

Thermophiles of concern to the

dairy industry

Anoxybacillus flavithermus

Geobacillus stearothermophilus

Bacillus licheniformis

Bacillus coagulans

Bacillus subtilis

Bacillus sporothermodurans


6

(Scott et al, 2007; Burgess et al, 2010; Tabit and Buys, 2010)

Time it takes for a thermoduric thermophilic population to double

in size or the exponential rate of increase is relatively very short

Geobacillus – 25 minutes

Paenibacillus – 45 minutes

Anoxybacillus – 30 minutes

Thermoduric thermophiles grow rapidly


7

Thermoduric mesophiles

Thermoduric mesophiles in milk can also withstand industrial pasteurization

temperature

Optimally grow in the temperature range of 20 - 37ºC/ 68° - 98.6°F

In addition to many aerobic and anaerobic spore formers, thermoduric

mesophiles may also include some members of the genera Lactobacillus

and Streptococcus


8

Thermoduric psychrotolerant

Significant for the spoilage of refrigerated milk (7ºC/ 44.6°F or below)

< 10% of total bacteria counts (TBC) in raw milk

In general thermodurics are not psychrotolerant. Some exceptions are as

follows

Paenibacillus (B. polymyxa) was found in 66% of milk samples held at 5ºC/ 41°F for

3 weeks

Pseudomonas is the other predominate psychrotolerant species isolated from

milk, but it is not thermoduric

High levels of thermoduric psychrotolerant bacteria shorten shelf-life and

lowers quality of milk


(Ranieri and Huck, 2009; Wiedemann 2012)

9

Understanding the relationship of Standard

Plate Counts with Thermodurics

SPC: Standard Plate Counts are indicative of the total aerobic bacteria present in raw milk

A count of less than 1,000 cfu/mL is considered as good farming conditions, while counts under 10,000 cfu/mL is in general achievable

Several factors including environmental cross contamination, inadequate cleaning, milk residues, failure to rapidly cool and hold milk under 5°C/ 41°F can lead to high SPC

LPC: Lab Pasteurization Counts indicate the level of thermoduric bacteria (surviving 62.8°C/ 145°F for 30 min)

They are lower than SPC and in general an LPC count greater than 300 cfu/mL is considered high

In addition to raw milk, persisters in biofilms result in cross contamination of milk with thermodurics from inadequately clean contact surfaces


10

(Wallace, 2009)

Understanding relationship of SPC with

Preliminary Incubation Counts

PI: Preliminary Incubation Counts are mainly indicative of milk production

practices and farm conditions

Milk is held at 12.8°C/ 55°F for 18h prior to plating for obtaining PI. This

encourages rapid growth of psychrotolerant bacteria, which have the

ability to grow at refrigeration temperatures, and are mainly responsible for

limiting the shelf life of refrigerated milk

These counts are generally higher than SPC. Any increase greater than 3-4

folds is considered significant or PI Counts higher than 50,000/mL are

reflective of either mastitic animals or inadequate cooling of milk prior to

holding


11

(Wallace, 2009)

Possible reasons for elevated raw milk counts

Counts Natural

Flora

Mastitis Dirty cows Dirty

equipment

Poor cooling

TBC > 10,000 √ √ √ √

TBC >100,000 √ √√ √√

High PIC

>50,000 or

>3X TBC

√ √√ √√

High LPC > 300 √ √√

Coliforms >100 √ √ √


12

(√ = possible; √√ = likely) (Murphy, 1997; Wallace, 2009; Greg and Ahmed 2011)

Troubleshoot high bulk milk counts


13

(Reinemann, 2011)

Dairy ThermoduricsModule 4: Persistence of Thermodurics in

Dairy Processing Plants


14

Why thermodurics are so difficult to eliminate

from the processing environment?

Resistant to thermal processing such as industrial pasteurization

Many form spores having greater resistance to heat and chemicals

Wide temperature growth range

Thermoduric thermophiles exhibit a faster growth rate

generation time 15-20 min

Readily form biofilms

(Flint et al, 1999 & 2001; Parker et al, 2001, Scott et al, 2007; Burgess et al, 2010; Hassan et al., 2010)


15

Persistence; The biofilm angle

Biofilms are congregation of sessile bacteria attached to a surface

Bound together with extracellular polymeric substance (EPS)

Proteins, lipids, and polysaccharides

Occur in areas of initial mineral deposits or organic matter buildup

Soiling within milking or processing equipment

Biofilms formed by thermoduric bacteria

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


16

(Costerton et al.,1994; Hassan et al., 2010, Anand et al., 2012)

Areas prone to thermoduric biofilms

Following are the major areas prone to biofilms in the processing plants

Pasteurizer heat exchange plates; regeneration section

Cream separators

Regeneration section of evaporators

Preheater of evaporation unit in spray drying

Membrane applications in milk and whey processing

Cream heater and recycle loops in butter plants

Remain ~45 - 60°C/ 113 - 140°F

Support growth of thermodurics, especially thermophiles

Other problem areas

Rubber seals, corners, hard to clean areas


17

(Scott, 2007; Burgess, et al., 2009)

Factors influencing biofilm formation

The ratio of planktonic (free-floating) cells to biofilm embedded cells is a

function of several interrelated factors

Types of bacteria

Surface energetics

Materials of construction

Microtopography of surfaces

Hydraulic factors

Biofilm chemistry


18

Stages of a typical biofilm formation

Stage I: (within 5-10 sec) surfaces are coated with milk proteinaceous

components and calcium phosphate

Stage II: (within 6-8 hours) primary colonization of bacterial cells with the

substratum

Extrapolysaccharides (EPS) appears to support this process

Stage III: (within 10-12 hours) irreversible biofilms

Formation of mature biofilms with embedded bacteria in the organic film matrix


19

2mo-old membrane showing long chains of cocci (A), 6mo-old membrane showing hill and valley structure (B),

Mushroom like structure (C), and Ruptured mushroom (D). (Hassan et al., 2010)

2-mo-old 6-mo-old

6-mo-old6-mo-old

AB

CD

SEM showing biofilm structures on used RO membranes


20

12mo-old membrane showing monolayer of rods and cocci connected by EPS (A), 14mo-old membrane showing

rods and cocci embedded in biofilm matrix (B), Distinct biofilm structures (C&D) (Hassan et al., 2010)

CD

BA

SEM showing different biofilm structures on aged membranes


21

What are some common cheese and

whey spoilages?

Spoilage of Cheddar cheese during storage

Flavor defects, bitterness, unclean flavors characteristic of atypical breakdown

of proteins

Production of biogenic amines in low fat and low salt cheese

Catabolism of amino acids involving deamination, decarboxylation,

desulphuration, oxidation, and reduction

Loss of functionality of whey proteins isolated and concentrated by

ultrafiltration

Gel strength and foaming stability

22


THERMODURICS MAY CAUSE SLITS

23

Causative organisms for these

slits in Cheddar cheese

samples

Lactobacillus spp.

Clostridium spp.

24

A STUDY UNDER PROGRESS IN OUR LAB

Commercial Cheddar cheese manufacturing cycles scanned

Samples of milk and cheese analysed for

Standard Plate Counts (SPC) on plate count agar at 35°C for 24h

Thermoduric mesophiles (TM)

Thermoduric thermophiles (TTh)

25(Khilendra and Anand, 2013; unpublished)

26

0

1

2

3

4

5

6

StartMid Day

End (22h)

Log c

fu/ m

L

Raw Milk Counts

SPC

TM

TTh

27

0

0.5

1

1.5

2

2.5

3

StartPre mid-

day washPost mid-

day washEnd

Thermoduric Thermophiles increase during

long hours (22-24h) of milk pasteurization

(log cfu / mL)

Thermoduric mesophiles Thermoduric thermophiles

28

0

1

2

3

4

5

6

7

StartPre mid-

day washPost mid-

day washEnd

Lo

g c

fu/g

Cheddar cheese counts (2 days old)

SPC

TM

TTh

SUMMARIZING

Thermoduric thermophiles counts in the pasteurized milk

increased with the run time

Mid-day wash helped to some extent in reducing their counts

Post mid-day run resulted in a greater build up of thermoduric

thermophiles

Post mid-day cheese samples also showed higher thermoduric

thermophiles

Thermoduric mesophiles did not show significant variations during

the entire run

29

Incidence of spore formers in milk

powders

The common bacilli constituting 92% of total bacterial population in milk

powders include the following

Geobacillus stearothermophilus

Bacillus licheniformis

Anoxybacillus flavithermus


30

(Ruckert et al., 1992)

Bacteria present in raw milk are concentrated an estimated 10 times as milk

powders are processed

Spore counts of generally less than 50 cfu/ mL in raw milk would thus become

approximately 500 cfu/ g even if no significant growth occurred during powder

processing

It is thus Important to start with high quality raw milk

Milk powder related issues

(McGuiggan et al., 2002; Rückert et al., 2004, 2006; Kim et al., 2009)


31

Cross contamination and build-up

during powder manufacture

Cross contamination does occur during manufacture of milk powders

Favorable growth conditions within segments of the processing line support

thermophilic growth during longer manufacturing cycles and formation of

resistant biofilms

Thermal operating conditions (between 45 to 75°C/ 113 to 167°F) may result

in a build up of about 5 logs of thermophiles during processing

Bypassing the pre-heater reduced the growth of bacteria in the evaporation

stage and ultimately reduced the numbers in milk powder

Limiting the production cycle to 12 hours substantially reduced thermophilic

counts in the milk powder


32

(Murphy et al., 1999; Scott et al., 2007)

Control of thermoduric sporeformers

Control in processing plant

Physical removal

Control biofilm formation

Inactivation


33

Spore reduction in plants

Spore removal by centrifugation (Bactofuge)

Spores have higher density (1.2 to 1.3 gr/l) than vegetative cells

Anaerobic spores reduce by 98%

Aerobic spores reduce by 95%

Inclusion of bactofuge may extend the shelf life of pasteurized milk by up to 3

days

However, waste from this process may be as high as 5% of the raw material


34

(Kilcast and Subramaniam, 2000)

Spore removal by microfiltration

(Bactocatch)

Cross flow microfiltration using a 1.0 micron ceramic membrane can

reduce spore by 4-5 log cycles

High retention levels (> 99.98%) have been observed for spore-formers, such as

Bacillus cereus, using double layer MF membranes

Combined microfiltration and pasteurization can reduce spores by 5-6 log

cycles


35

(Olesen and Jensen, 1989; Saboya and Maubois, 2000)

Thermal inactivation of spores

High heating UHT (150 - 200°C/ 302 - 392°F) for a fraction of second can

inactivate most of the heat resistant spores

Thermal shock and activation process

Heat shock is also the most likely mechanism of thermophilic spore activation

and subsequent germination

Germination may result in enzyme and acid production and consequential

development of off-flavors in the product


36

(Scott, 2007; Burgess, 2010)

Credits

Conceptualization, content, and analysis : Sanjeev Anand, Ph.D., Associate

Professor, Dairy Science Department, South Dakota State University, SD

57007.

Email: [email protected]. Phone: (605) 688-6648.

Contributions from my M.S. students form some parts of the overall content

of the Modules (I-VI): Mallika Avadhanula, Diwakar Singh, Som Nath Khanal,

Sowmya Marka, and Kim Buehner, Dairy Science Department, South

Dakota State University, SD 57007.

Copyright © 2014. Sanjeev Anand. Dairy Science Department, South

Dakota State University, SD. 57007.

Project supported by: Midwest Dairy Foods Research Center (MDFRC).


37

mailto:[email protected]

Documents

1 Module 1: Thermoduric Bacteria Thermodurics - Lloyd Metzger.pdf · SUMMARIZING Thermoduric thermophiles counts in the pasteurized milk increased with the run time Mid-day wash helped