By: Jeffrey L. Kornacki, Ph.D. President and Senior Technical Director
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1 Building World Class Microbiological Food Safety Systems for the Coming Storm How Many Samples to Test: Finished Product and Ingredient Testing Principles Or Where and How Raw Ingredient and Finished Product Testing Fit into the Big Picture-An Understanding of Microbiological Sampling Criteria By: Jeffrey L. Kornacki, Ph.D. President and Senior Technical Director Adjunct Faculty, Food Science Department, UGA Palm Springs, CA March 21, 2017 No electronic copies
2 Motivation for Sampling Criteria: Microbiological Risks Food safety (discussed previously) Food spoilage Disputes over results / specifications
3 Microbiological Risks-Food Spoilage Situations (ICMSF Sampling Categories 1-3)
4 Major Spoilage Microorganisms of Dairy Products Organism Activity Some Products Affected Pseudomonads LAB-Homo -Hetero Yeasts Molds Lipase, Protease production Acid, proteases Gas, proteases Fruity, yeasty, sweet flavors & gas Surface growth, colored spores. Proteases, Lipases Milk, cheese, butter Milk Cheese Cheese, butter, yogurt Cheese, butter, yogurt Sporeformers Coliforms Lipases, Proteases, gas, acid Acid, gas Milk, cheese Milk, cheese
5 Major Spoilage Microorganisms Dairy Products Pseudomonads and other gram (-), non-fermentative bacteria (milk, cheese, butter) Bacillus subtilis (milk) Lactic acid bacteria (milk) Yeast (cheese) Molds (cheese)
6 Potential Consequences of Spoilage (Loss of Shelf-Life) Loss of consumer confidence Loss of market share to others with better shelflife Perception of unsafe product Allegations of food poisoning Litigation
7 Disputes Related to Numbers Company A sells to company B B s specification: <10,000 APC/g A-tests show 7,000/g (a single sample) B-tests show 11,000/g (a single sample) What is the right number? Bacillus cereus in Infant Formula-Guideline Aerobic Plate Counts in oat bars specification <500/g, most in specification but sporadically tested at >100,000/g - Environmental contamination? - Laboratory contamination? - Localized area of the product? How many samples do I take?
8 A Bigger Picture of Food Safety Elements Preventive Controls Laws GMPs Sanitation SOPs HACCP Significance of post-process (post-ccp) contamination
9 Environmental contamination increases the risk of post-process contamination if the product is not biocidally treated in the end-use container High numbers usually required 1 x 10 9 cells per ml 10,000 # s of product ~200 cells per gram product
10 Microbial Growth Requirements Food (soil) Water Time
11 Microbial Growth Niches Operating practices (e.g. sanitation) e.g., torn hoses Maintenance / repair practices Design / fabrication of factory / equipment
12 Unsanitary Operating Practice Wheeled Vehicle Traffic: Raw to Finished
13 Unsanitary Maintenance/Repair Practices ILL fitting / protruding gasket at bottom of mix tank Unchanged gasket at bottom of mix tank
14 Unhygienic Designs Electrical Control Box Over Filling Apparatus Void Behind Conveyor Flights
15 Random Distribution
16 Non-Random Distribution
17 Sampling to Meet Microbiological Criteria Ingredients - Microbiological criteria Quantitative vs. qualitative assays What are appropriate specifications? Finished Product Routine: HACCP Verification Investigational: Scoping the problem Seeking the source (in-line tests) How many samples to take?
18 Microbiological Criterion: a yardstick to determine Food safety Shelf-life of perishable foods Suitability of a food or ingredient Adherence to GMPs
19 Microbiological Criteria: Other Benefits Ensures safety and quality of foods when appropriately used with other safety and quality systems (e.g. GMPs and HACCP) Elevates consumer confidence Provides regulators and industry with guidelines for the control of food processing systems Internationally accepted criteria advance free trade
20 Congressional Criticism of FDA The FDA's ability to guarantee the nation's food supply has been criticized by Congress and others following months of headline-grabbing food-borne illnesses, including E. coli contamination in spinach, Salmonella-tainted peanut butter and snack foods, and contaminated pet foods, toothpaste and other foods imported from China.
21 Types of Microbiological Criteria: A way to make decisions Standards - Legal and mandatory (pathogen oriented -if reliable methods available) (Shell fish and E. coli) Guidelines advisory - regulators or industry use these to determine if hygienic provisions have been met (GMPs or CCPs) may include organisms of no public health significance Specifications purchase requirements set by industry Adapted from NRC An Evaluation of the Role of Microbiological Criteria for Foods and Food Ingredients. National Academic Press, Washington, D.C.
22 Factors to Consider Before Establishment of Microbiological Criteria Evidence for potential hazard (Epi, HA) Knowledge of natural or acquired microflora Ability of food to support microbial growth Effect of processing on microflora of food Potential for contamination and/or growth during processing, handling, storage, and distribution Health Status of consumers State that food is distributed (solid, liquid) Potential for abuse by the consumer
23 Factors to Consider Before Establishment of Microbiological Criteria (continued) Spoilage potential, utility, and GMPs Manner of preparation for ultimate consumption Reliability of assays for microbes Costs and benefits associated with application of the criterion (Cronobacter spp.)
24 The Microbiological Criterion: Essential Elements Identity of the food or ingredient, Contaminant of concern, Analytical method Sampling plan, Microbiological limits appropriate to the food and consistent with the sampling plan National Research Council An Evaluation of the Role of Microbiological Criteria for Foods and Food Ingredients. National Academic Press, Washington, D.C.
25 Microbiological Criterion: Organizations FAO/WHO: Codex Alimentarius International Food Standards Program ICMSF-International Commission for the Microbiological Specifications for Foods NACMCF-U.S. National Advisory Committee on Microbiological Criteria for Foods FDA (BAM Levels I, II, and III for Salmonella); zero-tolerance LM/25 g USDA ( Mega-Reg -carcasses E. coli, Salmonella); RTE Meats-LM, Salmonella negative in 25 g Other Governments: trade barriers
26 Sampling plans: Two Basic Types Attributes Sampling plans (ICMSF) Preferred when little knowledge of how a food processed, no past performance record) Widely used at ports of entry Variables Plans [requires knowledge of frequency distribution (commonly log 10 normal) distribution of bacteria]. Could be assumed if produced from a common source, under uniform conditions and have adequate micro data on product. MIL Standards
27 Where Angels Fear to Tread: Complete Discrimination Between Lots Proportion of Defective Sample Units Below 20% and above 20% Probability of accepting such a lot Proportion of defective sample units in the lot
28 Sampling Plans: How Much Testing is Enough? Lot based statistical sampling Probability of rejection/acceptance of contaminated lot. Lot- that quantity of product produced, handled, and stored within a limited time period under uniform conditions
29 ICMSF Sampling Plans- Terminology n = Number of sample units analyzed (chosen separately and independently) Greater n greater likelihood of rejecting a contaminated lot (greater stringency) c = Maximum allowable number of samples with unsatisfactory test results, e.g. positive for an organism, or a count above m Increasing c decreases stringency m = A microbiological criterion that separates good quality from defective quality
30 ICMSF Attributes Sampling Plans-Two Types Two Class Plans- Micro counts either above or below a pre-set concentration m Example: a plan with n=5, c=2, m=0 2 of 5 samples positive: Accept lot 3 or more of 5 samples positive: Reject lot Three Class Plans: Recognizes non-homogeneity of microbial populations
31 ICMSF Three Class Attributes Sampling Plans (continued) Creates three categories of food lots 1. Acceptable counts below m 2. Marginal counts > m but < M provided that the number of samples in this range is c or less 3. Unacceptable any lot with any sample > M
32 Attribute Plans (Black and White or Zones of Gray)
33 Three-class attributes plans Relative proportion of sample units in a lot 2 1 m 3 M 4 Mean log count acceptable Marginally acceptance defective
34 Complete Discrimination Between Lots Proportion of Defective Sample Units Below 20% and above 20% Probability of accepting such a lot Proportion of defective sample units in the lot
35 The Famous and Foreboding O/C Curve (the impact of n ) Ope rating characteristic curve s for sampling plans with n=5,10,30 and c=0 Probability of accepting n=5 n=10 n= Proportion of defective sam ple units in the lot
36 Operating Characteristic Curves: The Impact of c
37 ICMSF: International Commission for the Microbiological Specifications of Foods: Attributes Plans Food and hazard grouped into 15 risk categories Categories based upon 1. Degrees of health concern (e.g. organism or other toxic agent) and 2. Risk associated with the food itself (intrinsic factors) and extrinsic factors and conditions of use
38 ICMSF Attributes Sampling Plans Increasing Risk From Product or Environment Agent Conditions that Decrease Risk Conditions that do not change Risk Conditions that Increase Risk Spoilage n=5 Case 1 c=3 Case 2 c=2 Case 3 c=1 Increasing Microbial or Toxic Risk Indicator Case 4 c=3 N=5 Moderate Case 7 n=5, c=2 Case 5 C=2 Case 8 n=5 c=1 Case 6 C=1 Case 9 N=10 c=1 Serious C=0 Case 10 n=5 Case 11 n=10 Case 12 n=20 Severe C=0 Case 13 n=15 Case 14 n=30 Case 15 n=60 n=number of samples per lot; c=maximum number of samples allowed between m and M
39 ICMSF Suggested Sampling Plans for Combinations of Degrees of Health Concern and Conditions of Use (i.e., the 15 Cases ) Conditions in which food is expected to be handled and consumed after sampling in the usual course of events * Degree of concern relative Conditions reduce Conditions cause no Conditions may to utility and health hazard degree of hazard change in concern Increase concern Utility; general contamination, reduced shelf-life, incipient spoilage Increase shelf-life Case 1 Three class n=5, c=3 No change Case 2 Three-class n=5, c=2 Reduce shelf-life Case 3 Three-class n=5, c=1 Indicator, Low, indirect hazard Reduce hazard No change Increase hazard Case 4 Case 5 Case 6 Three-class Three-class Three-class n=5, c=3 n=5, c=2 n=5, c=1
40 ICMSF Sampling Plans for the 15 Cases (Continued) Conditions in which food is expected to be handled and consumed after sampling in the usual course of events * Degree of concern relative Conditions reduce Conditions cause no Conditions may to utility and health hazard degree of hazard change in concern Increase concern Moderate hazard; direct limited spread Case 7 Three-Class Case 8 Three-class Case 9 Three-class n=5, c=2 n=5, c=1 n=10, c=1
41 ICMSF Sampling Plans for the 15 Cases (Continued) Conditions in which food is expected to be handled and consumed after sampling in the usual course of events * Degree of concern relative Conditions reduce Conditions cause no Conditions may to utility and health hazard degree of hazard change in concern Increase concern Serious Hazard; incapacitating but not usually life threatening, sequelae are rare, moderate duration Case 10 Two-class n=5, c=0 Case 11 Two-class n=10, c=0 Case 12 Two-class n=20, c=0 Severe hazard; for (a) the general population or (b) restricted populations, causing life threatening or substantial chronic sequelae or illness of long duration Case 13 Two-class n=15, c=0 Case 14 Two-class n=30, c=0 Case 15 Two-class n=60, c=0
42 ICMSF Attributes Sampling Plans- Microbial Toxin and Parasite Risk Ranking-Severe Hazards-General Population (Cases 13-15) Botulinum neurotoxin (C. botulinum, C. butyricum, C. barati): Brucella abortus, B. suis, B. melitensis Enterohemorrhagic E. coli O157:H7, O111:NM Salmonella typhi (serotypes paratyphi A, B and C) Shigella dysenteriae I Burkholderia cocovenans Vibrio cholerae (01 and 0139) Mycobacterium bovis Aflatoxins (from A. flavus, A. parasiticus) vcjd/bse Taenia solium
43 Severe Hazard-(Restricted Populations; Cases 13-15) Campylobacter jejuni serovar O:19 (& serotypes associated with Guillain-Barre Syndrome) Enteropathogenic E. coli (EPEC and ETEC)- infants C. perfringens type C (enteritis necrotans) protein deficient persons Clostridium botulinum (types A and B) (Infants) Enterobacter sakazakii (infants) Listeria monocytogenes (immunocompromised) Vibrio vulnificus (patients with liver disease) Hepatitis A (patients with liver disease) Cryptosporidium parvum (immunocompromised)
44 Serious Hazard (Incapacitating - Not Life Threatening; Sequelae Infrequent, Moderate Duration; Cases 10-12) Salmonella Enteritidis, typhimurium, and other serovars (Salmonellosis) Yersinia enterocolitica, pseudotuberculosis Shigella flexneri, boydii, sonnei (non-dysentery shigellosis) Listeria monocytogenes (general population) Hepatitis A (if not liver disease involved) Arcobacter butzleri, cryaerophilus Cryptosporidium parvum Cyclospora cayetanensis
45 Serious Hazard (Incapacitating - Not Life Threatening; Sequelae Infrequent, Moderate Duration; Cases 10-12) Cont. Trichothecene toxins (produced by Fusarium graminarium and related spp.) Zearalenone (produced by F. garaminarium and related spp.) Fumonisins (produced by F. moniliforme and related spp.) Ochratoxin A (produced by Penicillium verrucosum, Aspergillus ochraceus, and related spp., A. carbonarius and related spp.?)
46 Moderate (not usually life threatening; no sequelae; normally short duration; symptoms self limiting; can cause severe discomfort; cases 7-9) Bacillus cereus Clostridium perfringens (type A) Escherichia coli (EPEC, ETEC) Staphylococcal enterotoxins (Staphylococcus aureus) Vibrio cholerae non- 01 and non-0139 Vibrio parahemolyticus Small Round Structured Virus (SRSV; Norwalk) Biogenic amines (e.g. histamine)
47 Three Plans for Salmonella
48 FDA BAM Risk Categories Salmonella Sampling Category I (analogous to case 15 with ICMSF) Foods not normally subjected to a process lethal to Salmonella between. sampling and consumption and are intended for consumption by the aged, the infirm, and infants. 60 x 25 g samples or 4 x 375 g composites Category II (Analogous to case 14 with ICMSF) Foods not normally subjected to a process lethal to Salmonella between sampling and consumption 30 x 25 g samples or 2 x 375 g composites
49 FDA BAM RISK CATEGORIES Category III (Analogous to case 13 with ICMSF) Foods normally subjected to a process lethal to Salmonella between sampling and consumption. 15 x 25 g samples or 1 x 375 g composites The U.S. food industry adopted this routinely. Designed for investigations but - FDA may exceed this level in an actual investigation.
50 Acceptance Criteria, Salmonella Product Category # of Units Tested with Negatives # of Units Tested with No More than One Positive Significance 95% Probability of One Organism or Less in I 60 (1500 g) 92 (2300 g) 500 g II 29 (725 g) 48 (1200 g) 250 g III 13 (325 g) 22 (550 g) 125 g
51 Don t it always seem to go that you don t know what you ve got till it s gone - Joni Mitchell
52 Finished Product Sampling: A Statistical Perspective
53 Finished Product Sampling: A Statistical Perspective
54 Suggestion for Setting m and M Pathogens-Clear cut Non-pathogens (e. g. APC): One approach: Track product history Determine mean count Set m equal to log of the mean Set M equal to 2 or 3 standard deviations above m depending with consideration of product rejection probabilities, and safety or spoilage
55 Variables Plans Variables Plans [requires knowledge of frequency distribution of microbescommonly assumed to be log 10 normal] Could be assumed if produced from a common source, under uniform conditions and have adequate micro data on product. MIL Standards Less often used
56 Variables Plans (assuming Log 10 normal distribution) Reject lot if µ + k 1 s > V (quality or safety limit) V = similar to M in the attributes plan (logs); Log 10 concentration related to quality or safety limits μ = average of log 10 of counts from n samples taken in a lot S = standard deviation of the log 10 counts k 1 = obtained from a table based upon the maximum proportion of samples p d to be allowed above V Then pick the probability of rejection P (from a table) of the lot with p d above V. Note: Samples taken and handled same way as with attributes plans
57 K 1 Values Calculated Using the Non-Central t-distribution- Safety/Quality Specification (Reject if µ + k 1 s > V*)
58 Comparison of three-class attributes plan and variables plan with standard deviation of concentration in lot assumed to be unknown a) three-class plan: n=5, c=1; m=5x10 4, M=10 5 b) variables plan: n=5, V=10 5
59 The Microbiological Criterion: Essential Elements Identity of the food or ingredient, Contaminant of concern, Analytical method Sampling plan, Microbiological limits appropriate to the food and consistent with the sampling plan National Research Council An Evaluation of the Role of Microbiological Criteria for Foods and Food Ingredients. National Academic Press, Washington, D.C.
60 Good vs. Bad Specifications: Qualitative Tests (Salmonella) Ingredient identity: Potato Flakes Contaminant of concern: Salmonella Analytical method: BAM Sampling Plan: BAM Category I (15 random, 25 gram samples composited into a 375 g sample) Microbiological Limits: Negative in 375 grams
61 Group Exercises: Good vs. Bad Specifications: Qualitative Tests (Listeria monocytogenes) Ingredient identity: Cheddar cheese Contaminant of concern: Listeria monocytogenes Microbiological Limits: Negative What is missing?
62 Sampling - Quantitative E. coli ( generic in cheese) What criteria would you use for a high moisture high ph cheese (cottage) What criteria would you use of a low moisture cheese (Parmesan)
63 Sampling- Staphylococcal Enterotoxin Staphylococcal Enterotoxin in Cheddar Cheese (ph 5.2) What is your specification? How would it change if the ph were 6.0?
64 Sampling Issues Regarding Concerns About the Accuracy of Positive Qualitative Data or Can I believe my positive pathogen result without a retest?
65 Retest Considerations Assume: Non-fat dry milk sporadically contaminated 1 of every 100 sub-samples in the lot Probability of finding this : 1/100 Probability of two positives (e.g. retests) positive: 1/100 x 1/100 = 1/10,000 What if contamination rate is 1/1000?
66 Other Retest Considerations-Die Off Between Samplings A example of a survival curve for Salmonella under dry conditions Salmonella cells/25 g analytic unit Days
67 Resolution of Sampling Concerns Related to Positive Qualitative Data: Laboratory Quality Assurance Sterility of media lots used Negative controls (validate positive results) Hand sample data Laboratory environmental records Use of rare positive controls, unique or otherwise labeled strains Tight records of all samples assayed and order of enrichments (if appropriate), samples transferred, plated, isolates picked, streaked and tube placements, etc. Tracking and comparison of isolates (positive control or other e.g. laboratory isolates) References: GLPs - United States Pharmacopea; ISO 17025, CDC Lab Manual: Quality Control in Microbiology, 1987 Kornacki, J.L Where do I start (beginning the investigation)? Chapter 6. In, J. L. Kornacki (Ed.) Principles of Microbiological Troubleshooting in the Industrial Food Processing Environment. Springer, New York. Pp
68 Retest Considerations Related to Quantitative Data
69 Retest Considerations Related to Quantitative Data (Incidence VS Count) Individual cells 100 cells/clump Lot A=100 kg Prevalence =5 positive 1 kg sample units Concentration=5 cells/100 kg Lot B=100 kg Prevalence =5 positive 1 kg sample units Concentration =500 cells/100 kg
70 Retest Considerations Related to Quantitative Data Similar Issues to Qualitative but dealing with more cells Clumps Non-homogeneous distribution Die off
71 Resolution Increase homogeneity: mixing, blending, homogenizing Retests on same day use same method of sample preparation and analysis Increase n Example: B. cereus in non-fat dry milk for infant formula n=50 VS n=5
72 In Line Sampling- A Problem Solving Tool Flow diagram/plant tour Select key sampling points How many samples to take (invasive pathogen approach) Note: 20 x 375 (each 375 g from 15 x 25g) 15 x 20 = 300 samples (~ 95 % confidence of finding a lot with 1 % contamination)
73 In-Line Sampling: An Example Rendered Animal Proteins RAP flow diagram
74 Rendered Animal Protein Production (Later Stages) Expellers I * Surge hopper I * Grinding I *** Sifting I Silo **** I Load Out System ***** * Places where 10 x 375 g samples taken * No. Positives found
75 Environmental Contamination of Processed Foods Environmental contamination potential exists when products are not biocidally treated in the final container Most Processed food are contaminated after processing from growth niches in the environment
76 Microbial Growth Niches Operating practices (e.g. sanitation) Maintenance/repair practices Design/fabrication of factory/equipment
77 Environmental Sampling A most powerful tool if done properly
78 Assessments of Process and Hygiene through Product- A Routine Statistical Approach to Sampling
79 Hypothetical examples of using data from an assay for a microbiological Indicator to verify the effectiveness of a food safety system 1. System under control Log cfu/g Lot Number Log cfu/g Lot Number 2. Lack of control due to excess variability 3. Loss of control due to gradual process failures Log cfu/g Lot Number Log cfu/g Lot Number 4. Loss of control due to abrupt process failures Log cfu/g Lot Number 5. Loss of control due to a reoccurring, transitory failure
80 Summary and Conclusions Microbes are frequently not homogeneously distributed in foods Sampling of ingredients and finished products is a key (but not the only) element in ensuring food safety Sampling of ingredients and finished products alone will never be enough to guarantee food safety Microbiological criteria should be based upon sound statistical principles which consider the risk associated with the analyte and its interaction with the product Attributes sampling plans such as FDA BAM and ICMSF exist and can be referenced
81 Summary and Conclusions (cont.) Reliance upon retests to validate previous product positives is unreliable Principles of Statistical Process Control can be applied to testing lots for indicator organisms as part of a finished product QC program
82 References International Commission for the Microbiological Specifications of Foods (ICMSF) Microorganisms in Foods 7: Microbiological Testing in Food Safety Management. Kluwer Academic Plenum Publishers. New York. Smoot, L. Michelle, M. D. Pierson Indicator Microorganisms and Microbiological Criteria, Chapter 4. In, M. Doyle, L. R. Beuchat, and T. J. Montville (eds.), Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington, D. C. Taylor, M. T., J. N. Sofos, P. Bodnaruk, and G. R. Acuff Sampling Plans, Sample Collection, Shipment, and Preparation for Analysis, Chapter 2. In, Yvonne Salfinger and Mary Lou Tortorello (eds.), Compendium of Methods for the Microbiological Examinations of Foods, 5 th ediition. American Public Health Association, Washington, D.C.
83 Other References NACMCF Response to Questions Posed by the Department of Defense Regarding Microbiological Criteria as Indicators of Process Control or Insanitary Conditions Adopted 10JUN2015, Washington, DC. cd a28e-b c967/nacmcf-report-process-control pdf?MOD=AJPERES Miller, J. M Quality Control in Microbiology. In, CDC Laboratory Manual. U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control. Atlanta, GA Kornacki, J. L Where do I start (Beginning the investigation), Chapter 6. In, J. L. Kornacki (Ed.), Principles of Microbiological Troubleshooting in the Industrial Food Processing Environment. Springer, New York. Pp American Society for Quality Assurance ANSI/ISO In, American National Standard: General Requirements For the Competence of Testing and Calibration Laboratories. Quality Press, Milwaukee, WI.
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