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Chapter 3

Spores and Their Significance

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Learning Objectives• Understand how the canning industry overcomes the

challenge of spore heat resistance• Define the traits of low-acid canned foods and how they

must be processed• Understand the fundamental difference between a

bacterial spore and a vegetative cell• Correlate the unique properties of spores with the

challenge they present for food preservation• Compare a spore to a vegetative cell• Explain the physical chemical bases for spore heat

resistance• Compare the cycles of sporulation and germination with

the gain and loss of spore resistance characteristics2

What are spores?• Bacterial endospore – small, dormant, resistant derivative

of a bacterial cell that germinates under favorable growth conditions into a vegetative cell

• Spore – specialized cell form that can be used for dissemination, for survival in times of adverse conditions and / or for reproduction.

• Enable a cell to survive environmental stress- Heat - Freezing- High salt conditions- Drying- High acid conditions

• Spore-forming bacteria are found in foods grown in the soil and in animal products

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Spores in the Food Industry

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• Spore-forming bacteria and heat resistant fungi cause big problems in the food industry

• Toxin-producing spore-forming bacteria- Clostridium botulinum- Clostridium perfringens- Bacillus cereus

• Spoilage species- Alicyclobacillus- Geobacillus- Sporolactobacillus

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Sporeformers, Disease, and Food Spoilage

• Low-acid foods (>4.6 pH) packaged in cans, bottles, pouches or vaccum sealed containers are particularly vulnerable

• Diseases and spoilage caused by sporeformers are usually associated with thermally processed foods

• Heat kills vegetative cells but NOT spore-forming microbes

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Canning Industry• Early 1900s, US, appertization (heating foods

120°C) developed into canning industry as scientific principles were developed

• Prescott, Underwood, and Russell - spore-forming bacilli cause spoilage of thermally processed food

• Esty and Meyer – values for heat resistance of spores

• Ball – mathmatical foundation for commercial canning

rescued the US canning industry from near death in the 1940s due to botulism outbreaks

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Canning Industry

• Canned-food industry gave rise to- Quantitative thermal process- Understanding of spore heat

resistance- Aseptic processing- Implementation of HACCP concept

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Low-Acid Canned Foods• FDA and USDA

a food with a final pH of >4.6 and a water activity of >0.85

• Thermal process, facility, equipment, and formulations must be filed with the FDA and USDA before a canned product can be made

• Regulations are described in US Code of Federal Regulations (21 CFR, parts 108 to 114)

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Commercial Sterility• Uses heat to inactivate foodborne

pathogens and spoilage microoganisms that can grow in the food- Hermetically sealed – air tight containers- Shelf-stable product with negligible microbial

survival• Also produced by acidification or reduction

in the water activity of the food• Combine techniques to reduce heat and

improve product quality11

Inactivation of Clostridium botulinumspores

• Primary processing goal for low-acid food canning

• Most heat resistant pathogen• Severity of treatment depends on

- Class of food (uncured meat vs cured meat)- Spore content- pH and water activity- Storage conditions

• Foods with high spore loads (mushrooms, spices) require monitoring

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Values Used to Describe Thermal Inactivation of Spores

D value – time required for a 1-log reduction in viability at a given temperature

Represents the heat sensitivity of a microbe at a specific temperature

Typical survivor curve

D121oC = 6 min

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Values Used to Describe Thermal Inactivation of Spores

z value – temperature change required to alter the D value by a factor of 10

Expressed as degrees

Represents how heat sensitivity changes as temperature changes

Typical thermal resistance curve

Z = 13oF

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Canning Industry Process Design

Protects against:1. Public health hazard from C. botulinum

spores2. Spoilage from mesophilic spore-

formers3. Spoilage from thermophilic microbes in

containers stored in warm environments

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Canning Industry Process DesignGeneral requirements:• Low-acid canned foods

12D killing achieved with 3-6 min at 121oC (250oF)Inactivates C. botulinum spores with a D121oC of 0.21 min

and a z-value of 10oC (18oF)• Economic spoilage

5D killingInactivates mesophilic spores with D121oC of 1 min

• Foods distributed in warm climates12D killing – achieved with 20 min at 121oC (250oF)Inactivates spores from numerous spore-formers with a

D121oC of 3-4 min16

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Aseptic Processing• Improves the quality of low-acid canned foods• Reduces energy, packaging material, and

distribution costs• Three steps

1. Product is commercially sterilized outside of the container and achieves uniform temperature – quick high temp treatment

2. Product is cooled and transferred to presterilized containers

3. Container is hermetically sealed in a sterile environment

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Aseptic Processing

• Storage and refrigeration are not required for aseptic technique• Preserves food quality• Longer shelf life than conventional retorting process 18

Bacteriology of Sporeformers of Public Health Significance

• Three species cause foodborne illnessClostridium botulinumClostridium perfringensBacillus cereus

• Other sporeformers may be associated with food and food spoilage

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Clostridium botulinum• Principle microbial hazard

- heat-processed vacuum-packed foods

- minimally processed refrigerated foods

• Genus Clostridium- Gram-positive- anaerobic- spore-forming bacilli - energy by fermentation

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Clostridium botulinum• Produce antigenically distinct toxins A-G that

are strain specific• Two groups

- Type Imesophilesheat-resistant sporesproteases cause spoilage

- Type IInon-proteolyticspores are less heat-resistantgrow at low temperatures (botulism hazard) 21

Clostridium perfringens• Widespread in soils and intestines of humans and

certain animals• Grows rapidly in protein-rich food such as meats

that are cooked and then temperature abused (40-140oF)

• Enterotoxin production during growth at 40-140oF• Other toxins lead to gas gangrene• Ubiquitous distribution in food and

food environments• Spores survive cooking• Very fast growth rate in warm foods – 6-9 min

doubling time at 43-45oC (110oF) 22

Clostridium perfringens• Two classes of heat sensitive spores

• Both survive cooking of foods• Both stimulated to germinate by heat

shock• Both cause diarrheal foodborne illness

D-value Z-value

Heat-resistant D90oC (D194oF) 15-145 min

9-16oC (16-29oF)

Heat-sensitive D90oC (D194oF) 3-5 min 6-8oC (11-14oF)

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Bacillus cereus• Toxins

- heat-labile enterotoxin that causes diarrhea

- Heat-stabile toxin causing vomiting• Must grow to a high number (> 106 / g) to

cause illness

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Bacillus cereus• Spores located central to subterminal in

vegetative cell• Germination occurs within the range of

8-30oC (46.4-86oF)• Heat resistance of D95oC (D203oF) 24 min• Spores survive cooking

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Heat Resistance of C. botulinum Spores

• Most important sporeformers for public health safety of canned foods

• Heat resistance - decreases at pH lower than 5 or higher than 9- increases with lower water activity - increases with high salt or sugar concentrations- increases if spores are coated in oil

• Inactivation depends on spore concentration, pH, and food type

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Spoilage by Sporeformers• Low-acid foods are treated with heat

sufficient to kill C. botulinum but not more resistant spores

• Acid foods are not heated enough to kill all spores; however, most sporeformers do not grow under acid conditions

• Cured meats and hams are not heated enough to kill spores and must be refrigerated to prevent spoilage by nonpathogenic sporeformers

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Spoilage by Sporeformers• Spores naturally found in food and the cannery

environment contribute to spoilage• Dry ingredients contain high spore levels

- spices- sugars- starches- flours

• Spores accumulate in the food-processing plant- thermophilic spores on heated equipment- saccharolytic clostridia in plants for sugar-

rich foods 28

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Spoilage by Sporeformers

• Spoilage sporeformers are controlled by- monitoring raw foods to limit the

initial spore load- thermal processing for storage and

distribution conditions- cooling products rapidly- chlorination of cooling water- good manufacturing practices

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Spoilage by Sporeformers• Heat resistant fungi cause spoilage of

acidic foods particularly fruit products

• Heat kills most filamentous fungi and yeast

• Heat resistant fungi produce thick-walled ascospores that survive >85oC for 5 min

• Some produce mycotoxins• Prevent fungal growth by manipulation

of water activity and oxygen and use of antimycotic agents

Talaromyces ascospores

Neosartorya ascospora

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Spore Biology

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Spore Structure• Biochemically, structurally, and physiologically distinct from

vegetative cells• Exosporium

- outermost spore layer- varies in size among species

• Spore coats - under the exosporium- protects cortex from lytic

enzymes• Outer membrane

- keeps small molecules from permeating the spore

• Cortex - peptidoglycan structurally similar to vegetative cell except

for the presence of diaminopimelic acid- dehydrates the core- plays a role in resistance

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Spore Structure• Germ cell wall- structure similar to

vegetative cells• Inner membrane

- complete membrane- strong permeability

barrier- phospholipid content

similar to vegetative cells

• Core- contains DNA, ribosome, enzymes- contains dipicolinic acid and divalent cations- small acid soluble proteins bound to DNA- low water content responsible for dormancy and

resistance33

Spore BiologyMacromolecules

- Small acid-soluble proteins - present only in spores- major role in resistance- bind DNA altering its resistance to damage by

chemicals, enzymes, UV light- Amino acid and nucleic acid biosynthesis

enzymes are present only in vegetative cellsSmall molecules in the core are different to cells

- Diaminopimelic acid- Ions are immobile due to no free water- pH is 1-1.5 units lower- few high energy compounds 34

Spore Biology

Dormancy• Metabolically dormant

- no detectable metabolism- major cause - low water content- no enyzme activity

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Spore BiologyResistance

• Survives very long periods without nutrients- Metabolic dormancy- Resistance to heat, radiation, chemicals, and

dessication core dehydration small acid-soluble proteins impermeability

- 25-40 million year spore resurrected from amberDNA different to modern Bacillus

species 36

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Spore Biology

Heat Resistance• Huge problem for the food industry• Withstand 100oC for several minutes• Quantified as Dt value – time in minutes at a

temperature (t) needed to kill 90% of a population

• Extended survival of spores at elevated temperatures is paralleled with longer survival time at lower temperatures

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Spore Biology

Heat Resistance (cont’d)

• DNA damage does not occur with wet heat- protected by small acid-soluble proteins

• DNA damage does occur with dry heat

• Low core water content is a major factor- cortex creates and maintains dehydration- water-driven chemical reactions inhibited- low water stabilizes macromolecules

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Cycle of Sporulation and Germination

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Sporulation

• Spores are made in response to environmental stress or nutrient depletion

• Genes are expressed at specific times and places during sporulation

• Morphological and biochemical changes occur that are distinct to the vegetative cell

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Unequal cell division

Mother engulfs the forespore

Endospore develops inside the mother cell 41 42

Activation• Dormant spores resume metabolism when they

germinate• Nutrients induce germination• Spores germinate more completely if induced

prior to nutrient exposure• Spore activation

- sublethal heat shock (10 min at 80oC)- changes induced are unkown- reversible- releases some dipicolinic acid

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Germination• Most attributes are lost within 20-30

min of encountering a germinant• Germinants trigger germination

- unknown mechanism- amino acids- sugars- salts- dipicolinic acid

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Events in spore germination

Requires biosynthetic reactions and nutrients

Amino acids, sugars, salts, DPA

Excretes 30% of dry weight

Core water content same as cells

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Outgrowth• Not distinct from spore germination• About 25 min after start of germination until

first cell division• Requires the synthesis of new amino acids,

nucleotides and small molecules• Germination occurs for about 60 min before

DNA replication is initiated• Volume of the outgrowing spore increases• Requires synthesis of cell wall and membrane

components46

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Summary• Spores are unique life forms that are

resistant to many stresses• Heat resistance of spores in food is measured

as their D value – the number of minuets at a given temperature required to kill 90% of the spores

• The z value is the number of degrees that it takes to change the D value by a factor of 10.

• Low-acid canned foods are those with a pH of >4.6 and water activity of >0.85

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Summary• Commercial sterilization is achieve through

the application of a 12D botulinum cook• Many sporeformers cause economic spoilage• Sporulation is the process by which a

vegetative cell produces a spore• Germination and outgrowth enable the spore

to resume life as a vegetative cell• Spore cortex is a unique form ofpeptidoglycan

that contributes to spore resistance.• Dipicolinic acid and small acid-soluble proteins

contribute to spore resistance. 49

Summary• Low water content of a spore helps make it

resistant• Sporulation, germination, and outgrowth are

parts of the spore’s life cycle.

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