Back to The Basics: Factors Influencing the Growth ... · Back to The Basics: Factors Influencing...
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Building World Class Microbiological Food Safety Systems for the Coming Storm
Back to The Basics: Factors Influencing the Growth,
Survival and Death of Microorganisms
By: Jeffrey L. Kornacki, Ph.D.
President and Senior Technical Director Kornacki Microbiology Solutions, Inc.
www.kornackifoodsafety.com Adjunct Assistant Professor, Food Science Department, UGA
Palm Spings, CA
March 20, 2017
No Electronic copies please
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“Our lives are inextricably woven with the lives of these creatures who we ignore until they cause us trouble”
Lynn Margulis and
Dorion Sagin Microcosmos, 1986
Kornacki Microbiology Solutions, Inc.
Thar she blows! Dead whale explodes Taiwanese street, shops showered after gases built up inside
Taiwan Apple Daily via Reuters Blood and guts litter this street in Tainan, Taiwan, after decomposing organs in the sperm whale in background caused it to explode. 11:48 ET Jan 29th, 2004
MSNBC staff and news service reports
No Electronic copies please
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General Principles of Food Microbiology: Groups of Significance to Foods
Five groups significant in foods Bacteria: e. g. Mycobacterium bovis-
tuberculosis, milk early 1900’s USA Fungi: yeasts, molds (e.g. Candida ? Aspergillus flavus) Rickettsia: e.g. C. burnetti, Q fever - milk Parasites: Cryptosporidium (300,000, 1993,
MKE, WI) Viruses: Hepatitis A, Noravirus, Noravirus-
like
No Electronic copies please
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General Principles of Food Microbiology: Microbes and Size Multiply in foods: Only Bacteria, fungi (focus) Entailments: spoilage, growth increases risk from
weaker pathogens and others, enrichment techniques can be done
Others: Not multiply in foods e. g. Rickettsia, parasites (viruses) Entailments: Enrichments cannot be done Require a concentration and/or amplification
(PCR) step
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Relative Size Relationships of Microorganisms
Micron (µm) •1/1,000,000 meter
Basketball (228,600 um; 9 inches)
Yeast (5µm)
Large Marble (25,000 µm; 1 in)
Listeria (0.5µm)
Head of Pin (1500 µm; 0.06 in)
Virus – 10-100 nm
Grape fruit (137,000 µm; 5.4 in)
Mold spore (3µm)
Kornacki, J. L. 2010. What Factors Are Required for Microbes to Grow, Survive and Die? Chapter 5. In, Principles of Microbiological Troubleshooting in the Industrial Food Processing Environment. Springer NY. Pp. 103-115.
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Listeria monocytogenes Growing on Stainless Steel – 800X (Grooves)
Image taken with an epiflourescent microscope of DNA stained biofilm Courtesy of Emeritus Professor Joseph Frank, University of Georgia
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Types of Microbes - Shape
Cocci: Staphylococcus aureus, Streptococcus pyrogenes
Rods: Bacillus cereus, Clostridium
perfringens, Listeria (short), Escherichia coli (short), Salmonella
Spiral: Campylobacter jejuni
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Types of Microbes – Cell Wall
Gram positive: Staphylococcus aureus Streptococcus pyrogenes Gram negative: E. coli, Salmonella Acid fast: Mycobacterium spp.
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Cell Walls and Staining
http://www.sp.uconn.edu/~terry/229sp03/lectures/structure.html
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Selected Gram Stain Reactions of Bacteria
Description English: microscopic image of a Gram stain of mixed Gram-positive cocci (Staphylococcus aureus ATCC 25923, purple) and Gram-negative bacilli (Escherichia coli ATCC 11775, red). Magnification:1,000.
Date 13 April 2010(2010-04-13) Source Y tambe's file Author Y tambe
Other versions
File:Staphylococcus_aureus_Gram.jpg, File:Escherichia_coli_Gram.jpg : microscopic image of a Gram stain of each bacteria
http://en.wikipedia.org/wiki/Gram_staining
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Spore Stain
http://www2.austin.cc.tx.us/microbugz/15sporestain.html
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Microbial Identifications Gram stain > 23 theoretical possibilities Gram positive or negative Rod or coccus shaped (or spiral) Spore or non-spore former Catalase (positive or negative) Oxidase (positive or negative) >25 possibilities: enough to get to Family and often
Genus level Other simple tests: gas, CHO fermentations
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“Since the days of cave man, the earth has never been a garden of Eden, but a Valley of Decision where resilience is essential to survival…To grow in the midst of dangers
is the fate of the human race”.
Rene Dubos Mirage of Health
…And of bacteria (JLK)
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Approximate Temperature Ranges of Growth for Selected Microbial Categories
----------------------------------------------------------------------------------- Temperature (0C)
------------------------------------------------------- Minimum Optimum Maximum
----------------------------------------------------------------------------------Psychrophilic -15-0 10-20 20-22
Psychrotrophic --5-5 25-30 30-40 Mesophilic 5-25 25-40 40-50 Thermophilic 35-45 45-65 60-90 Obligate 40-45 55-65 70-90 Facultative 35-40 45-55 60-80
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Types of Microbes – Growth Temperature Relationships Psychrophiles – NA Psychrotrophic – Listeria monocytogenes,
Yersinia enterocolitica Selected strains of Cronobacter (E.
sakazakii1)
Entailments: refrigerated growth refrigerated foods cold environments
cold enrichment Mesophilic – Salmonella Thermophilic – B. coagulans, C. thermosaccharolyticum 1Gurtler, J. B., J. L. Kornacki, and L. R. Beuchat. 2005. Enterobacter sakazakii: A coliform of increased concern to infant health. Int. J. Food Microbiol. 104:1-34.
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Characterization Based Upon Optimum Growth Temperature
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Minimum Growth Temperatures of Selected Microorganisms*
Microorganism Temperature oC Aeromonas 5oC Clostridium Genera include pyschrotrophs
C. botulinum 3.3 – 10oC Enterobacter Genera include pyschrotrophs Escherichia coli 5-10oC Lactobacillus 2oC Leuconostoc 4oC Listeria 1oC Pseudomonas fluorescens 0-4oC Salmonella 3-10oC
*Adapted from Kornacki, J. L. and D. A. Gabis. 1990. Microorganisms and refrigeration temperature. Dairy, Food and Environmental Sanitation 10 (4):192-195.
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Minimum Growth Temperatures of Selected Microorganisms (continued.)*
Staphylococcus 5-10oC
Yersinia enterocolitica 4oC
Candida (Yeast) 0oC
Saccharomyces 0-7oC
Aspergillus Genera include psychrotrophs
Penicillium Genera include psychrotrophs
*Adapted from Kornacki, J. L. and D. A. Gabis. 1990. Microorganisms and refrigeration temperature. Dairy, Food and Environmental Sanitation 10 (4):192-195. (now called Food Protection Trends)
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Selective Properties of Foods Intrinsic vs. Extrinsic Intrinsic properties (water activity, pH, Eh) Extrinsic - temperature – (cooking, smoking),
drying, irradiation, high pressure, etc.)
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Intrinsic Properties of Foods Influencing Microbes Intrinsic Factors1
Water activity (aw): Formulation – salt in the moisture phase, dissolved solutes Dry vs. wet heat resistance 71.7oC – 15 seconds: fluid milk 7-9
log10 cfu decline Salmonella 71.7oC – milk chocolate: many hours for 1D- value (one study; 70oC, 12-17.5 hours) 1Goepfert, J. M., I. K. Iskander, and C. H. Amundson. 1970. Relation of the heat resistance of salmonellae to the water activity of the environment. Appl. Microbiol. 19(3):429-433. 2Mitscherlich, E. and E. H. Marth. 1984. Microbial Survival in the Environment. Springer-Verlag, New York. Table 6, Page 584.
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Limiting aw's of Selected Microbes Compared to Typical Food aw's aw Selected Food Microbe 0.98-1.00 Fresh Fish/Poultry
0.97 C. bot. type E 0.96 Some Ripened
Cheeses E. coli
0.95-1.00 Fresh Meats 0.93-0.96 Salmonella
0.92-0.95 B. cereus
0.90-0.98 C. botulinum
0.92 Listeria
0.90-0.94 Lactobacillus
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Limiting aw's of Selected Microbes Compared to Typical Food aw's
aw Selected Food Microbe 0.90 Maple Syrup Most Spoilage Bacteria
0.88 Most Spoilage Yeasts
0.84-0.92 S. aureus
0.83-0.87 Fermented Sausages
0.82-.94 Jelly
0.80-0.90 Aspergillus flavus
0.79-0.84 Fruit Juice Concentrates
0.8 Most Spoilage Molds
0.75-0.91 Jams
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Limiting aw's of Selected Microbes Compared to Typical Food aw's
aw Selected Food Microbe 0.69 Chocolate Candy
0.65-0.75 Some Cereals 0.61 Xerophilic Molds /
Osmophilic Yeasts
0.60-0.75 Syrups, Sugars 0.54-0.75 Honey
0.2 Dried Whole Milk
0.1-0.2 Some Cereals
Adapted from Jay 1992 and Banwart 1979, Ryser, 1999
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Approximate Minimum aw for Growth of Certain Groups of Microorganisms Important in Foods
Most spoilage bacteria 0.90-0.91 Most yeasts 0.87-0.94 Bacillus cereus 0.95 Osmophilic yeasts 0.60-0.78 Clostridium botulinum 0.90-0.98 Most molds 0.70-0.80 Type A 0.95 Xerophilic molds 0.60-0.70 Type B 0.94 Aspergillus 0.68-0.88 Type E 0.97 A. flavus 0.80-0.90 Enterobacter 0.95-0.98 Fusarium 0.80-0.92 Escherichia coli 0.94-0.97 Saccharomyces rouxii 0.62-0.81 Salmonella 0.93-0.96 Staphylococcus aureus 0.84-0.92 Vibrio parahaemolyticus 0.93-0.98 Halophilic bacteria 0.75
Adapted from Banwart, 1979
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Impact of aw on growth of Staphylococcus aureus
Banwart, G. J. 1979. Basic Food Microbiology, AVI Publishing.
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Intrinsic Properties of Foods Influencing Microbes pH effects Dissociated vs. un-dissociated organic
acids – general principles Lactic acid bacteria in salad dressing Same pH, different acids, big problems
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Microbicidal Impact of Organic acids in the Un-
dissociated Form
pH scale
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pH Growth Ranges for Selected Microbes
Adapted from Jay, J. M. 2005. Modern Food Microbiology, 7th Ed. Springer,, NY.
pH Scale
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Limiting pH Values Reported for Selected Organisms
pH value
Microorganism
6.0 Aeromonas hydrophila
5.0 Clostridium botulinum Group II
Clostridium perfringens
Pseudomonas fragi
Shigella sonnei
4.9 Bacillus cereus
4.6 Clostridium botulinum Group I
4.5 E. coli O157:H7
Adapted from Jay, J. M. 2005. Modern Food Microbiology, 7th Ed. Springer,, NY
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Limiting pH Values Reported for Selected Organisms (cont.)
pH Microorganism 4.3 Lactococcus lactis 4.1 Listeria monocytogenes 4.05 Salmonella sp. 4.0 Staphylococcus aureus 3.6 Gluconobacter spp. 3.34 Lactobacillus plantarum 3.16 Lactobacillus brevis 3.0 Penicillium roqueforti 2.0 Alicylobacillus acidocaldarius
1.8 Zygosaccharomyces bailii
Adapted from Jay, J. M. 2005. Modern Food Microbiology, 7th Ed. Springer,, NY
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pH Values of Various Foods
Adapted from Jay, J. M. 2005. Modern Food Microbiology, 7th Ed. Springer,, NY
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pH Values of Various Foods (continued)
Adapted from Jay, J. M. 2005. Modern Food Microbiology, 7th Ed. Springer,, NY
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Microbial Oxygen Relationships
Aerobic growth in Fluid Thioglycolate broth Anaerobic growth in Fluid Thioglycolate broth Micro-aerobic growth in Fluid Thioglycolate broth Anaerobic growth in Fluid Thioglycolate broth
Kornacki, J. L. 2010. What Factors Are Required for Microbes to Grow, Survive and Die? Chapter 5. In, Principles of Microbiological Troubleshooting in the Industrial Food Processing Environment. Springer NY. Pp. 103-115.
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Microbial Relationships to AirMicrobial Relationships to Air
Strict Strict MicroaerobeMicroaerobe Facultative StrictFacultative Strict
Aerobe Anaerobe Aerobe Anaerobe AnaerobeAnaerobe
Kornacki, J. L. 2010. What Factors Are Required for Microbes to Grow, Survive and Die? Chapter 5. In, Principles of Microbiological Troubleshooting in the Industrial Food Processing Environment. Springer NY. Pp. 103-115.
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Effect of Food ORP (Eh) on Growth of Selected Microbes Oxidation Reduction (ORP) or Red/Ox potential (Eh) Dissolved oxygen – Seafood and C.
botulinum – FDA requirements Processes that reduce the Red/Ox potential Cooking, smokehouse, oven treatments
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Extrinsic Factors Influencing Microbial Growth and Survival in Foods - Temperature
Growth rate and lag phase Fungal growth assay: processed cheese
example (Yousef and Marth1, Meulenkamp, et al.)
Lag phase, growth rate vs. temperature
1Yousef , A. E. and E. H. Marth. 1987. Quantitation of growth of mold on cheese. J Food Prot. 50:337–341.
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Example of Microbial Growth Curve
Kornacki, J. L. 2010. What Factors Are Required for Microbes to Grow, Survive and Die? Chapter 5. In, Principles of Microbiological Troubleshooting in the Industrial Food Processing Environment. Springer NY. Pp. 103-115.
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Spoilage of Raw Ground Beef Vs. Temperature
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Extrinsic Factors Influencing Microbial Growth and Survival in Foods Heat
Heat resistance of Microbes Spores > gram positive (non-
sporeformers) > gram negatives Pasteurization: vegetative cells not spores
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Microbial Heat Resistance Parameters TDT’s and phantom TDT curves D-values Time at a given temperature to reduce population 10 X z- values Temperature change that changes D-value 10X 12 D The proverbial “bot” cook F-values Fo @ a given temperature – retorting (F121oC = 3 min for 12 D) Total TDT curve - reproduced from an organisms z and one D-value Lethality of a process – data needed: z, D, and heat penetration data
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D-Value Illustrated: Hypothetical Survivor Curve of Microbe – D-value
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Examples of Common Survivor Curves
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Z-Value Illustrated: Hypothetical TDT VS.Temperature
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Biofilms Add Greater Complexity to the Thermal Process Calculation
“Growth on surfaces offers numerous advantages to microorganisms and therefore biofilms are the predominant growth form of microorganisms in natural environments”
- Joseph Frank, Professor, Food Science Department and Center for Food
Safety, University of Georgia (personal communication)
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Biofilms: Advantages to the Organism Access to nutrients under low nutrient
conditions that predominate in nature Protection from environmental or host
stress Ease of genetic exchange Jefferson, K. K. 2004. What drives bacteria to produce a biofilm? FEMS
Microbiol. Ltr. 236:163-173
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Biofilms Adherence Attachment – proteins, polysaccharides, glycoproteins Food residues More attachment Complex community More stress resistance (sanitizer, heat, etc.)
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Native Biofilms – Some Examples
1Mariani, et. al. 2007. Biofilm ecology of wooden shelves used in ripening the french raw milk smear cheese Reblochon de Savoie. J. Dairy Sci. 90:1653-1661. 2Donlan, R. M. Biofilms: Microbial life on surfaces. See http://bcbsma.medscape.com/viewarticle/441355_print 3Slide Provided Courtesy of Dr. Amy Wong, Food Research Institute, Madison, WI July, 2008
Ripening Shelves During Cheese Manufacture1
-Steel Surface in Industrial Water System2
Acridine Orange Stained Biofilm-Catch Pan Below Condensor Unit– RTE Meat Plant3
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Thermal Destruction of Microbes: How can we deal with this confusion?
“Prediction is very hard, especially when it
is about the future.” – Yogi Berra
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How can we deal with this confusion? Gompertz and other equations Probability of total destruction at a given
time vs. temperature1,2
1Chmielewski and Frank. 2004. A predictive model for heat
inactivation of L. monocytogenes biofilm on stainless steel. J. Food Prot. 67(12):2712-2718.
2 Chmielewski and Frank. A predictive model for heat inactivation of Listeria monocytogenes biofilm on buna-N rubber. LWT-Food Science and Technology 39(1):11-19.
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Is Freezing and Effective Way to Kill Bacterial Pathogens? Freezing and Thawing Ice crystal formation and freezing rates Experience with C. perfringens in dry
product Trichinella not withstanding
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Effects of Extrinsic Variables on Intrinsic Properties of Foods Carbonation and pH Cooking, irradiation on ORP (Eh), free
radicals, rancidity Drying on aw
Enterobacteriaceae on dried dog chews
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Relative Humidity and Surface Growth % R.H. and aw
aw x 100 = % R. H. As % R.H. increases growth increases on
foods and soiled environmental surfaces
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Yersinia survival on FRP:Temperature effect
A A A A A
A
A AB A
B B
0
2
4
6
8
0 3 6 9 12 15
Days after attachment
Log
cfu/
coup
on
4 C10 C
Microbial Survival in the Presence of Condensate (high humidity)
Source: Adapted from Allan, Yan, and Kornacki. 2004. Surface material, temperature, and soil effects on the survival of selected foodborne pathogens in the presence of condensate. J. Food Prot. 67(12):2666-2670. See also, Allan, Yan, Genzlinger, and Kornacki. 2004. Temperature and biological soil effects on the survival of selected foodborne pathogens on a mortar surface. J. Food Prot. 67(12):2661-2665.
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Effects of Heterogeneous Microflora Restricted microflora Common in foods Selective environments Salmonella serotypes unique to certain
factories, foods Cronobacter spp. (E. sakazakii) – infant formula and I.f. factories Selective media/enrichments for recovery Processed vs. raw foods
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Summary/Conclusion
Dynamic relationship: Microbes, foods, environments (including
people) Need to understand how these affect
specific food pathogens, and thereby influence testing, and survival in the food matrix and the environment.