Building World Class Microbiological Food Safety Systems for the Coming Storm

Transcription

1 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. Adjunct Assistant Professor, Food Science Department, UGA Palm Spings, CA March 20, 2017 No Electronic copies please

2 Kornacki Microbiology Solutions, Inc. 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 No Electronic copies please Thar she blows! Dead whale explodes Taiwanese street, shops showered after gases built up inside MSNBC staff and news service reports 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 29 th, 2004

3 General Principles of Food Microbiology: Groups of Significance to Foods Five groups significant in foods Bacteria: e. g. Mycobacterium bovistuberculosis, 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, Noraviruslike No Electronic copies please

4 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

5 Relative Size Relationships of Microorganisms Micron (µm) 1/1,000,000 meter Basketball (228,600 um; 9 inches) Grape fruit (137,000 µm; 5.4 in) Yeast (5µm) Mold spore (3µm) Large Marble (25,000 µm; 1 in) Head of Pin (1500 µm; 0.06 in) Listeria (0.5µm) Virus nm Kornacki, J. L 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

6 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

7 Types of Microbes - Shape Cocci: Staphylococcus aureus, Streptococcus pyrogenes Rods: Bacillus cereus, Clostridium perfringens, Listeria (short), Escherichia coli (short), Salmonella Spiral: Campylobacter jejuni

8 Types of Microbes Cell Wall Gram positive: Staphylococcus aureus Streptococcus pyrogenes Gram negative: E. coli, Salmonella Acid fast: Mycobacterium spp.

9 Cell Walls and Staining

10 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( ) 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

11 Spore Stain

12 Microbial Identifications Gram stain > 2 3 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) >2 5 possibilities: enough to get to Family and often Genus level Other simple tests: gas, CHO fermentations

13 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)

14 Approximate Temperature Ranges of Growth for Selected Microbial Categories Temperature ( 0 C) Minimum Optimum Maximum Psychrophilic Psychrotrophic Mesophilic Thermophilic Obligate Facultative

15 Types of Microbes Growth Temperature Relationships Psychrophiles NA Psychrotrophic Listeria monocytogenes, Yersinia enterocolitica Selected strains of Cronobacter (E. sakazakii 1 ) Entailments: refrigerated growth refrigerated foods cold environments cold enrichment Mesophilic Salmonella Thermophilic B. coagulans, C. thermosaccharolyticum 1 Gurtler, J. B., J. L. Kornacki, and L. R. Beuchat Enterobacter sakazakii: A coliform of increased concern to infant health. Int. J. Food Microbiol. 104:1-34.

16 Characterization Based Upon Optimum Growth Temperature

17 Minimum Growth Temperatures of Selected Microorganisms* Microorganism Aeromonas Clostridium Temperature o C 5 o C Genera include pyschrotrophs C. botulinum o C Enterobacter Genera include pyschrotrophs Escherichia coli 5-10 o C Lactobacillus 2 o C Leuconostoc 4 o C Listeria 1 o C Pseudomonas fluorescens 0-4 o C Salmonella 3-10 o C *Adapted from Kornacki, J. L. and D. A. Gabis Microorganisms and refrigeration temperature. Dairy, Food and Environmental Sanitation 10 (4):

18 Minimum Growth Temperatures of Selected Microorganisms (continued.)* Staphylococcus Yersinia enterocolitica Candida (Yeast) Saccharomyces Aspergillus Penicillium 5-10 o C 4 o C 0 o C 0-7 o C Genera include psychrotrophs Genera include psychrotrophs *Adapted from Kornacki, J. L. and D. A. Gabis Microorganisms and refrigeration temperature. Dairy, Food and Environmental Sanitation 10 (4): (now called Food Protection Trends)

19 Selective Properties of Foods Intrinsic vs. Extrinsic Intrinsic properties (water activity, ph, E h ) Extrinsic - temperature (cooking, smoking), drying, irradiation, high pressure, etc.)

20 Intrinsic Properties of Foods Influencing Microbes Intrinsic Factors 1 Water activity (a w ): Formulation salt in the moisture phase, dissolved solutes Dry vs. wet heat resistance 71.7 o C 15 seconds: fluid milk 7-9 log 10 cfu decline Salmonella 71.7 o C milk chocolate: many hours for 1Dvalue (one study; 70 o C, hours) 1 Goepfert, J. M., I. K. Iskander, and C. H. Amundson Relation of the heat resistance of salmonellae to the water activity of the environment. Appl. Microbiol. 19(3): Mitscherlich, E. and E. H. Marth Microbial Survival in the Environment. Springer-Verlag, New York. Table 6, Page 584.

21 Limiting a w 's of Selected Microbes Compared to Typical Food a w 's a w Selected Food Microbe Fresh Fish/Poultry 0.97 C. bot. type E 0.96 Some Ripened Cheeses Fresh Meats E. coli Salmonella B. cereus C. botulinum 0.92 Listeria Lactobacillus

22 Limiting a w 's of Selected Microbes Compared to Typical Food a w 's a w Selected Food Microbe 0.90 Maple Syrup Most Spoilage Bacteria 0.88 Most Spoilage Yeasts S. aureus Fermented Sausages Jelly Aspergillus flavus Fruit Juice Concentrates 0.8 Most Spoilage Molds Jams

23 Limiting a w 's of Selected Microbes Compared to Typical Food a w 's a w Selected Food Microbe 0.69 Chocolate Candy Some Cereals 0.61 Xerophilic Molds / Osmophilic Yeasts Syrups, Sugars Honey 0.2 Dried Whole Milk Some Cereals Adapted from Jay 1992 and Banwart 1979, Ryser, 1999

24 Approximate Minimum a w for Growth of Certain Groups of Microorganisms Important in Foods Most spoilage bacteria Most yeasts Bacillus cereus 0.95 Osmophilic yeasts Clostridium botulinum Most molds Type A 0.95 Xerophilic molds Type B 0.94 Aspergillus Type E 0.97 A. flavus Enterobacter Fusarium Escherichia coli Saccharomyces rouxii Salmonella Staphylococcus aureus Vibrio parahaemolyticus Halophilic bacteria 0.75 Adapted from Banwart, 1979

25 Impact of a w on growth of Staphylococcus aureus Banwart, G. J Basic Food Microbiology, AVI Publishing.

26 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

27 Microbicidal Impact of Organic acids in the Undissociated Form ph scale

28 ph Growth Ranges for Selected Microbes ph Scale Adapted from Jay, J. M Modern Food Microbiology, 7 th Ed. Springer,, NY.

29 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 Modern Food Microbiology, 7th Ed. Springer,, NY

30 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 Lactobacillus plantarum 3.16 Lactobacillus brevis 3.0 Penicillium roqueforti 2.0 Alicylobacillus acidocaldarius 1.8 Zygosaccharomyces bailii Adapted from Jay, J. M Modern Food Microbiology, 7th Ed. Springer,, NY

31 ph Values of Various Foods Adapted from Jay, J. M Modern Food Microbiology, 7th Ed. Springer,, NY

32 ph Values of Various Foods (continued) Adapted from Jay, J. M Modern Food Microbiology, 7th Ed. Springer,, NY

33 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 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

34 Microbial Relationships to Air Strict Microaerobe Facultative Strict Aerobe Anaerobe Anaerobe Kornacki, J. L 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

35 Effect of Food ORP (E h ) on Growth of Selected Microbes Oxidation Reduction (ORP) or Red/Ox potential (E h ) Dissolved oxygen Seafood and C. botulinum FDA requirements Processes that reduce the Red/Ox potential Cooking, smokehouse, oven treatments

36 Extrinsic Factors Influencing Microbial Growth and Survival in Foods - Temperature Growth rate and lag phase Fungal growth assay: processed cheese example (Yousef and Marth 1, Meulenkamp, et al.) Lag phase, growth rate vs. temperature 1 Yousef, A. E. and E. H. Marth Quantitation of growth of mold on cheese. J Food Prot. 50:

37 Example of Microbial Growth Curve Kornacki, J. L 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

38 Spoilage of Raw Ground Beef Vs. Temperature

39 Extrinsic Factors Influencing Microbial Growth and Survival in Foods Heat Heat resistance of Microbes Spores > gram positive (nonsporeformers) > gram negatives Pasteurization: vegetative cells not spores

40 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 F a given temperature retorting (F 121 o C = 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

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

42 Examples of Common Survivor Curves

43 Z-Value Illustrated: Hypothetical TDT VS.Temperature

44 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)

45 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 What drives bacteria to produce a biofilm? FEMS Microbiol. Ltr. 236:

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

47 Native Biofilms Some Examples Ripening Shelves During Cheese Manufacture 1 1 Mariani, et. al Biofilm ecology of wooden shelves used in ripening the french raw milk smear cheese Reblochon de Savoie. J. Dairy Sci. 90: Steel Surface in Industrial Water System 2 2 Donlan, R. M. Biofilms: Microbial life on surfaces. See 3 Slide Provided Courtesy of Dr. Amy Wong, Food Research Institute, Madison, WI July, 2008 Acridine Orange Stained Biofilm- Catch Pan Below Condensor Unit RTE Meat Plant 3

48 Thermal Destruction of Microbes: How can we deal with this confusion? Prediction is very hard, especially when it is about the future. Yogi Berra

49 How can we deal with this confusion? Gompertz and other equations Probability of total destruction at a given time vs. temperature 1,2 1 Chmielewski and Frank A predictive model for heat inactivation of L. monocytogenes biofilm on stainless steel. J. Food Prot. 67(12): 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.

50 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

51 Effects of Extrinsic Variables on Intrinsic Properties of Foods Carbonation and ph Cooking, irradiation on ORP (E h ), free radicals, rancidity Drying on a w Enterobacteriaceae on dried dog chews

52 Relative Humidity and Surface Growth % R.H. and a w a w x 100 = % R. H. As % R.H. increases growth increases on foods and soiled environmental surfaces

53 Microbial Survival in the Presence of Condensate (high humidity) Yersinia survival on FRP: Temperature effect Log cfu/coupon B A A A A A A A A B A B 4 C 10 C Days after attachment Source: Adapted from Allan, Yan, and Kornacki Surface material, temperature, and soil effects on the survival of selected foodborne pathogens in the presence of condensate. J. Food Prot. 67(12): See also, Allan, Yan, Genzlinger, and Kornacki Temperature and biological soil effects on the survival of selected foodborne pathogens on a mortar surface. J. Food Prot. 67(12):

54 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

55 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.