8/24/2016 Learning Objectives Food 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 Chapter 3 AN INTRODUCTION with the Spores and Their vation Significance Compare a spore to a vegetative cell Explain the physical chemical bases for spore heat resistance haRh空造Ka R.Mathe Compare the cycles of sporulation and germination with the gain and loss of spore resistance characteristics What are spores? The Life Cycle of an Endospore-Forming Bacterium vival in times of adw rse 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
8/24/2016 1 Chapter 3 Spores and Their Significance 1 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 characteristics 2 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 3 4
8/24/2016 Spore-forming bacteria and heat resistant fungi cause big problems in the food industry Toxin-producing spore-forming bacteria -Clostridium botulinum Spores in the Food Industry -Clostridium perfringens -Bacillus cereus ·Spoilage species Alicyclobacillus -Geobacillus Sporolactobacillus Sporeformers,Disease,and Food Canning Industry Spoilage Early 1900s,US,appertization(heating foods 120C)developed into canning industry as Low-acid foods (>4.6 pH)packaged in scientific principles were developed cans,bottles,pouches or vaccum sealed Prescott,Underwood,and Russell-spore-forming containers are particularly vulnerable bacilli cause spoilage of thermally processed food Diseases and spoilage caused by Esty and Meyer-values for heat resistance of sporeformers are usually associated spores with thermally processed foods Ball-mathmatical foundation for commercial Heat kills vegetative cells but NOT canning spore-forming microbes rescued the US canning industry from near death in the 1940s due to botulism outbreaks 2
8/24/2016 2 Spores in the Food Industry 5 • 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 6 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 7 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 8
8/24/2016 Canning Industry Low-Acid Canned Foods Canned-food industry gave rise to ·FDA and USDA Quantitative thermal process a food with a final pH of >4.6 and a -Understanding of spore heat water activity of >0.85 resistance Thermal process,facility,equipment,and Aseptic processing formulations must be filed with the FDA and USDA before a canned product can be Implementation of HACCP concept made Requlations are described in US Code of Federal Regulations(21 CFR,parts 108 to 114) Commercial Sterility Inactivation of Clostridium botulinum spores Uses heat to inactivate foodborne pathogens and spoilage microoganisms that Primary processing goal for low-acid food can grow in the food canning Hermetically sealed-air tight containers Most heat resistant pathogen Shelf-stable product with negligiblemicrobial Severity of treatment depends on survival -Class of food (uncured meat vs cured meat) Also produced by acidification or reduction -Spore content in the water activity of the food pH and water activity -Storage conditions Combine techniques to reduce heat and improve product quality Foods with high spore loads(mushrooms spices)require monitoring
8/24/2016 3 Canning Industry • Canned-food industry gave rise to - Quantitative thermal process - Understanding of spore heat resistance - Aseptic processing - Implementation of HACCP concept 9 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) 10 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 quality 11 Inactivation of Clostridium botulinum spores • 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 12
8/24/2016 Values Used to Describe Thermal Values Used to Describe Thermal Inactivation of Spores Inactivation of Spores 10 D value-time required Typical survivor curve zvalue-temperature 103 for a 1-log reduction D121c=6 min change required to in viability at a given alter the Dvalue by a Z=13f temperature factor of 10 101 Represents the heat Expressed as degrees sensitivity of a 0 Represents how heat microbe at a specific sensitivity changes as temperature temperature changes 481216 emperature(F Heating Time(min] Canning Industry Process Design Canning Industry Process Design General requirements: Protects against: Low-acid canned foods 1.Public health hazard from C.botulinum 12D killing achieved with 3-6 min at 121C(250F) spores Inactivates C.botulinum spores with a Di2roc of 0.21 min and a z-value of 10C (18F) 2.Spoilage from mesophilic spore- ·Economic spoilage formers 5D killing 3.Spoilage from thermophilic microbes in Inactivates mesophilic spores with D:2tc of 1 min containers stored in warm environments Foods distributed in warm climates 12D killing achieved with 20 min at 121C (250F) Inactivates spores from numerous spore-formers with a of 3-4 min
8/24/2016 4 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 13 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 14 Canning Industry Process Design Protects against: 1. Public health hazard from C. botulinum spores 2. Spoilage from mesophilic sporeformers 3. Spoilage from thermophilic microbes in containers stored in warm environments 15 Canning Industry Process Design General 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 killing Inactivates mesophilic spores with D121oC of 1 min • Foods distributed in warm climates 12D killing – achieved with 20 min at 121oC (250oF) Inactivates spores from numerous spore-formers with a D121oC of 3-4 min 16
8/24/2016 Aseptic Processing Aseptic Processing Case example-Aseptie fiing system Improves the quality of low-acid canned foods sa Reduces energy.packaging material,and distribution costs Clean room ·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 一00000000000000001d presterilized containers 3.Container is hermetically sealed in a sterile Storage and refrigeration are not required for aseptic technique environment Preserve Longer shelf life than conventional retorting process Bacteriology of Sporeformers of Public Health Significance Clostridium botulinum Principle microbial hazard Three species cause foodborne illness heat-processed vacuum- Clostridium botulinum packed foods Clostridium perfringens minimally processed refrigerated foods Bacillus cereus Other sporeformers may be associated Genus Clostridium -Gram-positive with food and food spoilage anaerobic spore-forming bacilli energy by fermentation 5
8/24/2016 5 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 17 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 illness Clostridium botulinum Clostridium perfringens Bacillus cereus • Other sporeformers may be associated with food and food spoilage 19 Clostridium botulinum • Principle microbial hazard - heat-processed vacuumpacked foods - minimally processed refrigerated foods • Genus Clostridium - Gram-positive - anaerobic - spore-forming bacilli - energy by fermentation 20
8/24/2016 Clostridium botulinum Clostridium perfringens Produce antigenically distinct toxins A-G that Widespread in soils and intestines of humans and are strain specific certain animals ·Two groups ws rapidly in protein-rich food such as me Type I recooked and then temperature abused(40. mesophiles Enterotoxin production during growth at 40-140F heat-resistant spores Other toxins lead to gas gangrene proteases cause spoilage Ubiquitous distribution in food anc Type II food environments non-proteolytic Spores survive cooking spores are less heat-resistant Very fast growth rate in warm foods-6-9 min grow at low temperatures(botulism hazard) doubling time at 43-45C (110F) Clostridium perfringens Bacillus cereus ·Toxins Two classes of heat sensitive spores heat-labile enterotoxin that causes D-value Z-value diarrhea Heat-resatom)() Heat-stabile toxin causing vomiting Heat-sensitive Daopc (D140F)3-5 min 6-8-C (11-14F) Must grow to a high number (>106/g)to cause illness Both survive cooking of foods Both stimulated to germinate by heat shock Both cause diarrheal foodborne illness 6
8/24/2016 6 Clostridium botulinum • Produce antigenically distinct toxins A-G that are strain specific • Two groups - Type I mesophiles heat-resistant spores proteases cause spoilage - Type II non-proteolytic spores are less heat-resistant grow 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) 23 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 24
8/24/2016 Bacillus cereus Heat Resistance of C.botulinum Spores Most important sporeformers for public health Spores located central to subterminal in safety of canned foods vegetative cell Heat resistance Germination occurs within the range of decreases at pH lower than 5 or higher than 9 8-30C(46.4-86℉) increases with lower water activity .Heat resistance of D9sc(D2030F)24 min increases with high salt or sugar concentrations Spores survive cooking -increases if spores are coated in oil Inactivation depends on spore concentration,pH, and food type Spoilage by Sporeformers Spoilage by Sporeformers Spores naturally found in food and the cannery Low-acid foods are treated with heat environment contribute to spoilage sufficient to kill C.botulinum but not more resistant spores Dry ingredients contain high spore levels Acid foods are not heated enough to kill all ·spices spores:however,most sporeformers do sugars not grow under acid conditions -starches Cured meats and hams are not heated -flours enough to kill spores and must be Spores accumulate in the food-processing plant refrigerated to prevent spoilage by -thermophilic spores on heated equipment nonpathogenic sporeformers saccharolytic clostridia in plants for sugar- 27 rich foods
8/24/2016 7 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 25 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 26 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 27 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 sugarrich foods 28
8/24/2016 Spoilage by Sporeformers Spoilage by Sporeformers Heat resistant fungi cause spoilage of Spoilage sporeformers are controlled by acidic foods particularly fruit products monitoring raw foods to limit the initial spore load Heat kills most filamentous fungi and yeast thermal processing for storage and Heat resistant fungi produce thick- distribution conditions walled ascospores that survive >85C cooling products rapidly for 5 min -chlorination of cooling water Some produce mycotoxins good manufacturing practices Prevent fungal growth by manipulation of water activity and oxygen and use Talaromyces of antimycotic agents Exosporium Spore Structure outermost spore layer varies in size among species Spore coats Spore Biology ·under the exosporium protects cortex from lytic enzymes Outer membrane Cortex or eemcelcp dehydrates the core plays a role in resistance 8
8/24/2016 8 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 29 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 thickwalled 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 30 Spore Biology 31 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 32
8/24/2016 ·Germ cell wall structure similar to Spore Structure Spore Biology vegetative cells Macromolecules ·Inner membrane Small acid-soluble proteins -complete membrane present only in spores major role in resistance cells Amino acid and nucleic acid biosynthesis ·Core enzymes are present only in vegetative cells contains DNA,ribosome,enzymes Small molecules in the core are different to cells contains dipicolinic acid and divalent cations Diaminopimelic acid small acid soluble proteins bound to DNA Ions are immobile due to no free water low water content responsible for dormancy and pH is 1-1.5 units lower resistance few high energy compounds Spore Biology Spore Biology Resistance Dormancy Survives very long periods without nutrients Metabolic dormancy Metabolically dormant -Resistance to heat,radiation,chemicals,and dessication no detectable metabolism core dehydration major cause-low water content small acid-soluble proteins no enyzme activity √impermeability -25-40 million year spore resurrected from amber DNA different to modern Bacillus species
8/24/2016 9 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 resistance 33 Spore Biology Macromolecules - 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 cells Small 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 35 Spore Biology Resistance • 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 amber DNA different to modern Bacillus species 36
8/24/2016 Spore Biology Spore Biology Heat Resistance Heat Resistance(cont'd) Huge problem for the food industry DNA damage does not occur with wet heat Withstand 100C for several minutes -protected by small acid-soluble proteins Quantified as D,value-time in minutes at a DNA damage does occur with dry heat temperature (t)needed to kill 90%of a population Low core water content is a major factor Extended survival of spores at elevated cortex creates and maintains dehydration temperatures is paralleled with longer survival water-driven chemical reactions inhibited time at lower temperatures low water stabilizes macromolecules Sporulation Spores are made in response to Cycle of Sporulation and environmental stress or nutrient depletion Germination Genes are expressed at specific times and places during sporulation Morphological and biochemical changes occur that are distinct to the vegetative cell 10
8/24/2016 10 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 37 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 38 Cycle of Sporulation and Germination 39 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 40
