bacterial spores of importance in the dairy food system · •bacillus spp. (especially b....

Where the Spores Are

Bacterial Spores of Importance in the Dairy Food System

Rachel MillerMilk Quality Improvement Program

Cornell University

Outline

I. Introduction to bacterial sporesa. What are spores?

b. Why are they problematic in dairy?

II. Spores in specific commodities and recommendations for controlling spore levels

a. Fluid Milk

b. Cheese

c. Dairy Powders

What is a Bacterial Spore?

• Spore: DNA + protein coat

• Sporeformer: bacterium that forms a spore

McKenney et al. 2011 Nat Rev Micro

Why Form a Spore?

Persistence: Allows bacterial cell to survive harsh conditions

Why Form a Spore?

Persistence: Allows bacterial cell to survive harsh conditions

Resistance Condition Example of Processing Condition

High Temperature>175°F

HTST PasteurizationVat PasteurizationUHT Pasteurization

High Pressure HPP Treatment

Desiccation Freeze-drying

Chemical Sanitizers Acidic (HCl)Basic (NaOH)Peroxide (Peroxyacetic Acid)

UV Radiation UV Radiation

When are Spores Formed?

McKenney et al. 2011 Nat Rev Micro

•Found in many environments:• Soil •Recycled water• Feces• Feed

Where are Spores Found?

Leafly.com

Which Bacteria Form Spores?Two main classes:

Clostridia Bacilli

Oxygen Required for Growth?

No Yes/No

Key Spoilage Organisms

C. tyrobutyricumC. sporogenes

B. weihenstephanensisPaenibacillus spp.B. licheniformis

Pathogens C. botulinumC. difficile C. tetaniC. perfringens

B. anthracisB. cereus

Psychrotolerant = Growth @ 32-70°F (0-20°C)Mesophiles = Growth @ 85-100°F (30-40°C)Thermophiles = Growth @ 100-140°F (50°C)

How do we Test for Spores?

•Spore count tests:

Heat Treatment (kills bacterial cells)

Plating and incubation (allows spores to germinate to bacterial cells)

How do we Test for Spores?

•Spore count tests:

Heat Treatment (kills bacterial cells)

Plating and incubation (allows spores to germinate to bacterial cells)

PSC 6°C/10d

MSC 32°C/48h

TSC 55°C/48h

Spore Tests Currently Used in Industry

Spore Count MethodHeat

TreatmentIncubation

Spore Pasteurized (SP) 175°F (80°C) for 12 min

PSC: 43°F (6°C) for 10 daysMSC: 90°F (32°C) for 48 hrTSC: 131°F (55°C) for 48 hr

Highly Heat Resistant (HHR)

212°F (100°C)for 30 min

MSC: 90°F (32°C) for 48 hrTSC: 131°F (55°C) for 48 hr

Specially Thermoresistant Spore Enumeration (STSE)

223°F (106°C)for 30 min

TSC: 223°F (55°C) for 48 hr

Spores vary in their ability to withstand high temperatures

Spore Tests Currently Used in Industry

Spore Count MethodHeat

TreatmentIncubation

Spore Pasteurized (SP) 175°F (80°C) for 12 min

PSC: 43°F (6°C) for 10 daysMSC: 90°F (32°C) for 48 hrTSC: 131°F (55°C) for 48 hr

Highly Heat Resistant (HHR)

212°F (100°C)for 30 min

MSC: 90°F (32°C) for 48 hrTSC: 131°F (55°C) for 48 hr

Specially Thermoresistant Spore Enumeration (STSE)

223°F (106°C)for 30 min

TSC: 131°F (55°C) for 48 hr

Spores vary in their ability to withstand high temperatures

Lab Pasteurization Count

•NOT a spore test!

•145°F for 30 min

•Non-spore species that survive this test:• Staphylococcus spp. (e.g., S. epidermis)• Streptococcus spp. (e.g., S. thermophilus)• Enterococcus spp. • Lactobacillus spp. • Others

Spore Test MattersSporeformer SP

MSCSP TSC

HHR MSC

HHR TSC

STSE

Aeribacillus spp. ✓ ✓ ?

Anoxybacillus spp. ✓ ✓ ?

B. licheniformis ✓ ✓ ✓ ✓ ✓

B. cereus Group ✓

B. pumilus ✓

Geobacillus spp. ✓ ✓ ✓

Paenibacillus spp. ✓ ✓

Spore Test Matters

❖Spore tests are not a ‘one size fits all’ method

❖ Should be chosen to enumerate target bacterial spores of interest

Why are Spores Problematic in Dairy?

Sporeformers in the Headlines

Outline

I. Introduction to bacterial sporesa. What are spores?

b. Why are they problematic in dairy?

II. Spores in specific commodities and recommendations for controlling spore levels

a. Fluid Milk

b. Cheese

c. Dairy Powders

Fluid Milk – HTST Pasteurized

• Primary sporeformers of concern:• Growth at refrigeration temperatures

• Paenibacillus spp.• Growth at 40-45°F

• Grows in milk

Photo: wikipedia.com



• Paenibacillus spp.• Growth at 40-45°F

• Grows in milk

• Bacillus spp. (especially B. weihenstephanensis)• Some grow at 40-45°F

• Some able to grow in milk, but not all

• New species B. wiedmannii a new pathogenic species



• Paenibacillus spp.

• Bacillus spp. (especially B. weihenstephanensis)

• Associated defects:• Proteolysis (protein break-down)

• Lipolysis (degrade lipids)

• “Bitty cream”

• Recommended spore test:

• SP-MSC

• SP-TSC (maybe)

• Primary sources of spores:

• Raw milk

• Some in-plant biofilm contamination

Fluid Milk – HTST Pasteurized Sporeformer SP

MSCSP TSC

HHR MSC

HHR TSC

STSE

B. cereus Group ✓

Paenibacillus spp. ✓ ✓(?)

Fluid Milk – UHT Pasteurized • Primary sporeformer of concern:

• B. sporothermodurans

• Associated Defects:

• Proteolysis (protein break-down)


• Recommended spore tests:

• HR-MSC

• Other: 212°F for 30-40 min

• Primary source of spores

• Raw milk

Sporeformer SP MSC

SP TSC

HHR MSC

HHR TSC

STSE

Bacillussporothermodurans

✓ ✓

Fluid Milk – UHT Pasteurized

Dairy Powders

• Primary sporeformers of concern:

survive high heat treatments• Aeribacillus spp.• Anoxybacillus spp.• Bacillus licheniformis• Geobacillus spp.

• Associated defects• Sensory defects in reconstituted products• Proteolysis• Lipolysis

Dairy PowdersSporeformer SP

MSCSP TSC

HHR MSC

HHR TSC

STSE

Aeribacillus spp. ✓ ✓ ?

Anoxybacillus spp. ✓ ✓ ?

B. licheniformis ✓ ✓ ✓ ✓ ✓

Geobacillus spp. ✓ ✓ ✓

• Recommended spore tests: Depends on downstream use

• Sources of spores in dairy powders:• It’s complicated….

Dairy Powders

Raw Milk is Often Implicated as an Important Source of Spores

Predominant spores in RAW MILK- B. licheniformis- B. pumilus- B. cereus Group - B. subtilis - Paenibacillus sp.

Predominant spores in DAIRY POWDERS- Anoxybacillus sp. - B. licheniformis - Geobacillus sp. - B. cereus Group

Image sources: Google images

Spore Populations Among Bulk Tank and Raw Milk and Dairy Powders are Significantly Different

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

A

B

C

D

E

Proportion of isolates

Sou

rce

Aeribacillus pallidus Anoxybacillus sp. B. cereus slB. cf. aerophilus B. clausii B. coagulansB. licheniformis B. pumilus B. safensisB. species B. subtilis sl B. thermoamylovoransGeobacillus species Lysinibacillus sp. Other

Acid Whey

BT Raw Milk

Sweet Whey

WPC-80

NFDM

Dairy Powders

•Recommended control strategies• Requires low spore counts in raw

(concentration effect)• Prevention of biofilm formation

(contamination effect)

Watterson et al. 2014

Cheese

• Primary sporeformers of concern:

Anaerobic (growth without oxygen) • Clostridium spp. (especially C. tyrobutryicum)

• Associated defects:• Proteolysis (protein break-down)


• Late-blowing

Image Source: Google Images

CheeseSporeformer SP

MSCSP TSC

HHR MSC

HHR TSC

STSE

Clostridium spp. ✓ ✓

• Recommended spore tests:• SP with incubation under anaerobic conditions

• Primary sources of spores:• Prevent contamination in raw milk (silage linked to

C. tyrobutyricum contamination)

Recommended Control Strategies

• Controlling spore load in raw milk• Proper cow hygiene• Avoid silage if late-blowing is a concern

• Controlling in-plant contamination• Cleaning and sanitation to prevent biofilm

formation

• Determine spore populations in product, build spore tests around that

Take Home Messages

•Spores are difficult to inactivate

•Prevention of contamination is key to controlling levels

•Spore tests are not a ‘one size fits all’• Select method based on target spore

populations

Thank you

Rachel [email protected]

bacterial spores of importance in the dairy food system · •bacillus spp. (especially b....

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