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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

Transcript of Back to The Basics: Factors Influencing the Growth ... Kornacki - Back to the... · Back to The...

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

“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

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

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

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.

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

Types of Microbes - Shape

Cocci: Staphylococcus aureus, Streptococcus pyrogenes

Rods: Bacillus cereus, Clostridium

perfringens, Listeria (short), Escherichia coli (short), Salmonella

Spiral: Campylobacter jejuni

Types of Microbes – Cell Wall

Gram positive: Staphylococcus aureus Streptococcus pyrogenes Gram negative: E. coli, Salmonella Acid fast: Mycobacterium spp.

Cell Walls and Staining

http://www.sp.uconn.edu/~terry/229sp03/lectures/structure.html

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

Spore Stain

http://www2.austin.cc.tx.us/microbugz/15sporestain.html

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

“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)

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

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.

Characterization Based Upon Optimum Growth Temperature

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.

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)

Selective Properties of Foods Intrinsic vs. Extrinsic Intrinsic properties (water activity, pH, Eh) Extrinsic - temperature – (cooking, smoking),

drying, irradiation, high pressure, etc.)

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.

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

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

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

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

Impact of aw on growth of Staphylococcus aureus

Banwart, G. J. 1979. Basic Food Microbiology, AVI Publishing.

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

Microbicidal Impact of Organic acids in the Un-

dissociated Form

pH scale

pH Growth Ranges for Selected Microbes

Adapted from Jay, J. M. 2005. Modern Food Microbiology, 7th Ed. Springer,, NY.

pH Scale

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

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

pH Values of Various Foods

Adapted from Jay, J. M. 2005. Modern Food Microbiology, 7th Ed. Springer,, NY

pH Values of Various Foods (continued)

Adapted from Jay, J. M. 2005. Modern Food Microbiology, 7th Ed. Springer,, NY

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.

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.

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

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.

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.

Spoilage of Raw Ground Beef Vs. Temperature

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

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

D-Value Illustrated: Hypothetical Survivor Curve of Microbe – D-value

Examples of Common Survivor Curves

Z-Value Illustrated: Hypothetical TDT VS.Temperature

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)

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

Biofilms Adherence Attachment – proteins, polysaccharides, glycoproteins Food residues More attachment Complex community More stress resistance (sanitizer, heat, etc.)

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

Thermal Destruction of Microbes: How can we deal with this confusion?

“Prediction is very hard, especially when it

is about the future.” – Yogi Berra

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.

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

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

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

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.

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

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.