VALIDATION OF MICROBIOLOGICAL

CONTROL MEASURES (“KILL-STEP”)

Wilfredo Ocasio, Ph.D.
October 2, 2020

1 Confidential - Eurofins. All rights reserved

AGENDA

• Couple of words about Eurofins

• Validation vs Verification
• Validation: Why? When? Who?
• What do we validate?
– Understanding the Product and Technology
• Validation Approach (How to Validate?)
• Case Study: Thermal processing of low-moisture foods (LMF)
– Definitions and public health importance
– Validation Approaches
– On-site validation of “kill step”

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 OUR COMPANY - EUROFINS

45,000
• Testing Services Employees

• Training and Consulting

• Auditing and Certification 47
Countries
• Product Design
• Research and Development
800 sites

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 FOOD SAFETY RESEARCH—WHO WE ARE AND WHAT WE DO
Antimicrobial Efficacy Cause of Spoilage
Challenge Studies Investigations
Studies
In-Plant
"We help food companies assess the Validation Studies
microbiological safety and regulatory
Thermal Death compliance of their products and/or Processing
Time Studies related processes by conducting Authority
Services
laboratory-based research studies
and on-site process validations." Regulatory
Compliance
Novel Technology
Evaluation/Validation Able to work with Clostridium botulinum….
Studies
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 Validation Overview

VALIDATION VS VERIFICATION

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 Food Protection Trends Nov/Dec 2014, pp. 410-425
• Gathering of 150
industry and academia
experts to share ideas
and perspectives on the
validation and
verification
requirements of PC rule.

6 Confidential - Eurofins. All rights reserved

VERIFICATION AND VALIDATION FSMA – PC RULE*
Validation:
“Obtaining and evaluating scientific and technical evidence that a
control measure, combination of control measures, or the food
safety plan as a whole when properly implemented, is capable of
effectively controlling the identified hazards.”
Verification:
“The application of methods, procedures, tests, and other
evaluations, in addition to monitoring, to determine whether a
control measure or combination of control measures is or has been
operating as intended and to establish the validity of the food
safety plan.”

*Federal Register/ Vol. 80 No. 180, September 17, 2015. Current Good and Manufacturing Practice,
Hazard Analysis and Risk-Based Preventive Controls for Human Foods
7
 Interrelationship among terms
Are the control measures the
Validation
right ones? Will they work?
“Initial”, “Efficacy”, “Research”

Are you staying within the critical

Monitoring limits?
“Real time”, “Continuous”

Verification: Do you say what you do?

Do you do what you say?
“Periodical”

8
Validation of Microbial Kill-Steps is: Validation of Microbial Kill-Steps is not:

Essential to Assure A Regulatory

Public Health Requirement
Easy to plan or Research and
execute Development
Planned with
To be Done Proper
Right by the Allotment of
Right Team Time and Cost
Assured to
Aligned with Inexpensive Demonstrate the
Commercial Needed “Kill”
Viability of
Product/Process

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 Validation Overview

WHY? WHEN? WHO?

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 Why validate?
 To demonstrate and document that the control measures in
place are sufficient to provide a microbiologically safe and
stable product
 Satisfy regulatory requirements (i.e., FSMA and other
pertinent regulations require validation of control
measurements on all product categories)
 Who is Responsible?
• FSMA PC Rule:
– “The Qualified Individual must do or oversee the validation of preventive
controls.”
• QI must have completed training equivalent to a standardized curriculum*
recognized by FDA or
• Be otherwise qualified through job experience
• ICMSF Microorganisms in Foods 8:
– “A team of experts is required for system validation because of the many skills
required such as engineering, microbiology, physical chemistry, etc.”
• 2013 Food Safety Summit**:
– “The establishment of food safety functions, including validation, is a team
effort.”
• Corporate Managers set policy and standards, define accountability and identify
the roles of all
• Supervisors and line workers implement the programs and ensure that functions
are effective and carried out properly

*Training curriculum at http://www.iit.edu/ifsh/alliance/

**Food Protection Trends Nov/Dec 2014, pp. 410-425 12
 When to Validate?
 Initial validation is required prior to implementation of a control measure
or when changes occur that necessitate re-validation
 Examples of potential triggers for re-validation:
– Emergence of new pathogen or higher risk in product
– Monitoring or verification shows system failure
– Change in ingredient microbial load
– Change in product formulation
– Change in product intrinsic parameters
– Change in processing parameters or storage conditions
– Product category is linked to an outbreak
– Regulatory agency alerts
– New information from the scientific literature
– Consumer complaints
 Management of Change programs are essential in evaluating need for re-
validation

13
Validation Overview

WHAT DO WE VALIDATE?:
UNDERSTANDING THE PRODUCT AND TECHNOLOGY

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 UNDERSTANDING THE TECHNOLOGY AND PRODUCT

Validation is specific to the intended

product, process and process
parameters been evaluated.

Validation must be conducted using

a “worst case scenario” in terms of
critical process and product related
factors

15
 What do we validate?
 Control Measures
 Food or Beverage Sterilization
Note: Validation test
 Aseptic Zone Pre-sterilization parameters must represent
 Container Sterilization “worst case” condition.
 Maintenance of Sterility in Aseptic Chamber
 Control Measures Consist of Process Parameters (Critical
Factors, CCP’s)
 Temperature, Time, Concentration
 Process Parameters Contain Critical Limits
 Minimum Temperature = 121oC/250oF
 Minimum time = 3 min
UNDERSTAND THE TECHNOLOGY: HIGH PRESSURE PROCESS
Category Critical Factor Effect on Microorganism
Starting temperature of
More kill achieved as this
product and pressurization
factor increases
fluid
Time to reach target pressure
Undetermined
(ramp up)
More kill achieved as this
Target pressure factor increases
Process critical More kill achieved as this
factors Holding time at target pressure factor increases

Depressurization time Undetermined

More kill achieved as this
Storage temperature post-HPP
factor increases
More kill achieved as this
Storage time post-HPP factor increases (high acid
products)
More kill achieved as pH
pH
Product critical becomes more acidic
factors Less kill achieved as this
Water activity
factor decreases

17
HPP Process
85,000 psi for 3 min
Immediately or after 24 hr at 4C
UNDERSTANDING THE TECHNOLOGY: PULSED ELECTRIC FIELDS
• DC Power Supply Category Critical Factor Effect on Microorganism
– Converts Wall Power into More kill achieved as
Electrical field strength
High Voltage DC Power this factor increases
– Rated in Average Power Treatment time
(Watts) (combination of number More kill achieved as
• Pulse Modulator of pulses and pulse
width)
this factor increases
– Stores and Releases
Average Power in High Order of increasing
Process critical treatment efficiency:
Peak Power Pulses factors Pulse waveshape
– Key Parameters – Peak Oscillatory < Exponential
decaying < Squarewave
Voltage and Peak Current
• Treatment Chamber Order of increasing
Pulse polarization treatment efficiency:
– Applies Voltage Pulse to
Monopolar < Bipolar
Liquid
– Ensures Complete Treatment temperature
More kill achieved as
Treatment without Arcing this factor increases
– Multiple Treatment Less kill achieved as this
Conductivity
Chambers in Series factor increases
Ensure Complete Product critical More kill achieved as pH
Exposure to PEF pH
factors becomes more acidic
Less kill achieved as this Courtesy of Diversified Technologies, Inc.
Ionic strength
factor increases

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CRITICAL PROCESS PARAMETERS
Super Critical CO2 UV Radiation
Category Critical Factor Category Factor
Pressure UV wavelength(s)
Temperature Product flow profile
Treatment time
Geometric configuration of the
Process Critical
Agitation process system
Process Critical Factors
Factors Power
CO2 saturation and solubility
Distance between UV source and
Pressure cycles product

Type of SC-CO2 system Solids contenta

pH
Absorption constant at treatment
Solution buffering capacity Product wavelength
Product critical Critical Factors
factors Water content
Turbidity
Fat content
Additives Transmissivity

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 Microbiological Hazards

VALIDATION APPROACH – HOW TO

VALIDATE?
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THE TWO ELEMENTS OF VALIDATION
• There are two elements to validation
1. Scientific or technical support for the control measure
2. Initial in-plant demonstration proving that the control measure can
be performed as expected
• Both elements must be completed, however, in some cases
element 2 may serve both purposes
• Element 2 is often confused with verification

USDA FSIS. 2013 Compliance Guideline HACCP Systems Validation.

http://www.fsis.usda.gov/shared/PDF/HACCP_Systems_Validation.pdf

22
VALIDATION MAY BE ACCOMPLISHED BY:
• Collecting and evaluating scientific and technical information
– Generally-held historical knowledge
– Published “safe harbor” processes
– Regulatory (performance standards) Data must match the
– Published processing guidelines specific
– Peer-reviewed scientific literature or technical data product/technology
– Previous validation studies being validated
– Scientifically-developed mathematical models
• Conducting validation studies
– Collecting scientifically-valid experimental data on-site
– Challenge studies, TDT’s and/or inoculated packs in the lab
– Operational data and surveys
• Collecting data at establishment
(using tools of monitoring and/or verification during a defined a validation period)

Control of Salmonella and Other Bact Pathogens. 2017 WileyBlackwell ed.

 How to Validate?

LOW-MOISTURE FOODS (LMF)

THERMAL PROCESS CASE STUDY
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 THERMAL PROCESS VALIDATIONS – DEFINITIONS
• Target Organism: Pathogenic organism(s) of significance
in a given food product.
• Surrogate Organism: Non-pathogenic organism which
mimics resistance of target organism and is appropriate
for use in validation work.
• Process Lethality: Measure of process’ ability to destroy
the target organism, normally expressed as “log
reduction”.
• Kill-Ratio: Mathematical correlation between the
destruction of surrogate and target organisms.
• D-value: The time in minutes at a constant temperature
necessary to destroy 90% of the microorganisms
present.
• Z value: Change in temperature necessary to bring about
a 10-fold (1-log) change in the D value.

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LOW MOISTURE FOODS (LMF)
• Low Moisture Foods (FDA, Codex) – Foods with a water activity of 0.85
or below.
• Terms (Low Moisture Foods and Low Water Activity Foods) have been
used interchangeably but…
– Moisture – The amount of water present in the product
– Water activity (aw) - The ratio of the vapor pressure of water in a food matrix
compared to the that of pure water (aw = 1.0)
• Water activity is a measure of the amount of water that is chemically
“bound” and hence biologically unavailable.
– aw <0.6 – no microbial growth;
– aw <0.91 – most pathogenic bacteria inhibited (except for S. aureus, 0.85)
– aw <0.75 – yeast and mold are inhibited
– Most LMF have aw in 0.4 – 0.85 range

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LMF FOODS AND PROCESSES
Examples of LMF Examples of LMF Processes
• Dried Fruits and Vegetables • Drying (dry pasta)
• Cereal based products (breakfast cereals) • Extrusion (dried pet food)
• Confections (chocolates and hard candies)
• Dry roasting (almonds)
• Peanut and other nut butters
• Dried protein powders (dry milk, hydrolyzed • Superheated steam (spices and condiments)
vegetable protein powder) • Baking (cookies, pastries)
• Egg powders • Processes prior to drying:
• Teas and herbs
– Pasteurization (milk for drying)
• Spices and condiments – Blanching (fruits and vegetables)
• Flour
• Vacuum Microwave (dried fruits and
• Snacks Big Challenge:
vegetables)
• Dried pet foods Most legacy equipment used for
• Syrups
this application not designed as • Radiofrequency, RF (spices, milk powder, egg
a microbial control measure.
white powder)

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LMF: VALIDATION IS ESSENTIAL TO PUBLIC HEALTH
• Pathogens will not grow in low-moisture foods
– Misconception of low-risk due lack of growth
• Pathogens may survive for extended periods
– 12-months in almond kernels, aw = 0.4 @ -19, 4 and 24oC *
• Low-infection dose = high risk even in absence of growth
– Salmonella: 1-3 CFU/g in chocolate outbreak; 0.04-0.45 CFU/g potato chips
outbreak**
• Pathogens demonstrate higher thermal resistance in low water activity
environments (Podolak et al., 2010)
– Fat/oil, solids, and other intrinsic properties of a food product can protect/shield
microorganisms from thermal treatment (i.e., chocolate products, Hiramatsu et al.,
2005)
• Salmonella is most prevalent but others pathogens have caused outbreaks
(i.e., E. coli O157:H7 in cookie dough in 2009)

*Journal of Food Protection 2012, 75(8) 1394-1403

**Control of Salmonella and Other Bact Pathogens. 2017 WileyBlackwell ed.
28 Confidential - Eurofins. All rights reserved
EXAMPLES OF OUTBREAKS RELATED TO LMF IN THE USA
YEAR PRODUCT PATHOGEN
2009 Peanut butter Salmonella Typhimurium
2009 Raw cookie dough E. coli O157:H7
2011 In-shell hazelnuts E. coli O157:H7
2012 Dried pet food Salmonella Infantis
2012 Turkish pine nuts Salmonella Enteritidis
2013 Tahini sesame paste Salmonella Montevideo
2014 Nut butter Salmonella Braenderup
2015-2016 Flour E. coli O121, E. coli O26
2018 Tahini (2 outbreaks) Salmonella Typhimurium
2018 Dried Coconut Salmonella Typhimurium
2018 Puffed wheat cereal Salmonella Mbandaka
2019 Flour E. coli O26
2019 Pig Ear Dog Treats Salmonella

www.CDC.gov
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 LOW MOISTURE FOOD PROCESS VALIDATION
• Thermal process validation Study roadmap

Final Result:
Phase I: Phase II: Phase III:
Determination of the
Product / Process Microbial Kinetics In-Process Validation
level of reduction of the
Assessment (Building Studies (Developing the (Executing the Validation
target organism delivered
the Foundation) Tools) Study)
by the process
PHASE I: PROCESS/PRODUCT ASSESSMENT - BUILDING THE FOUNDATION
• Understand Regulatory Requirements
• Define Individual Responsibilities
• Target Organisms
– Salmonella, Escherichia coli O157:H7
• Surrogate Organism
– Enterococcus faecium (NRRL B-2354) common surrogate for Salmonella
• Understand the process:
– Equipment being validated
– Manner of heat application
– Temperature and time (equipment settings versus actual product
temperature)
– Order of introduction of ingredients
– Depth and distribution of product on conveying belt
– Other extrinsic parameters (relative humidity, air flow, etc.)
• Understand the product:
– Intrinsic properties (pH, aw, moisture, fat/oil, solids, added or natural
antimicrobials, dielectric properties, etc.)
– Physical properties (piece size, shape, surface structure, surface area)
PHASE I: PROCESS/PRODUCT REVIEW CONT.
Example:

Zone Zone Zone

Product 1 2 3 Product
Entering Exiting
Dryer Dryer
160-170ºF; 180-190ºF; 170-180ºF;
30 min 60 min 60 min

Note: Heat resistance of Salmonella is greater at

aw = 0.9 aw = 0.4
lower water activities.

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PHASE I: PRODUCT/PROCESS REVIEW CONT.
• Identify of critical control points/limits
– Examples:
• Temperature
• Time / belt speed
• Sterilant concentration
• Relative humidity
• Air flow rate
• Product formulation, pH, water activity, 16
moisture 14
Surrogate

• Set test parameters based on critical 12

Target

limits 10

– Worst-case parameters (critical limits)

Log D-value
8

• Conduct preliminary temperature 6

mapping (cold spot) 4

• Can temperature data be obtained in 2

the product itself? 0

145 150 155 160 165 170 175
Temperature (°F)

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 PHASE II: MICROBIAL KINETICS (TDT) DEVELOPING THE TOOLS

Thermal Death Time (TDT) Study:

• Specific to the product and organism (e.g. Listeria
monocytogenes in rice flakes with aw = 0.4)
• Result = D- and z values
– Surrogate should have equal or greater heat resistance
• Characteristics of potential surrogate organisms: Enterococcus faecium
– Non-pathogenic
– Stable
– Easy to grow to high levels
– Easily differentiated
– Shows similar and quantifiable resistance as compared to the target
pathogen in the context of the specific technology and food matrix
– Allows for comparison between the heat resistance of a surrogate and a
target pathogen (“kill-ratio”)

34
PHASE II: MICROBIAL KINETICS (TDT) DEVELOPING THE TOOLS

TDT Protocol Development Process:

• Follow NACMCF* guidelines
• Preparation of organism(s)
– Growth conditions can impact thermal resistance
• Inoculation method for selected “worst case” product
– Prevent changing intrinsic product parameters (pH, aw, etc.)
– Use of sand, chalk, spray (atomizer), freeze-dried culture
• Heat treatment method
– Water bath, Oil bath
• Recovery / enumeration method for target and surrogate
organisms
– Avoid use of selective media

*National Advisory Committee for the Microbiological Criteria For Foods. Parameters for Determining
Inoculated Pack/Challenge Study Protocols. 2010 J. Food Protection Vol. 73 No. 1 PP. 140-202
35
 PHASE II: MICROBIAL KINETICS (TDT)
Example Survival Graph: Results from One TDT Study
8.0
8.0 165ºF
165ºF
7.0
7.0
170ºF
170ºF
6.0
6.0
CFU/g
Log CFU/g

175ºF
175ºF
5.0
5.0
180°F
180°F
Survival Log

4.0
4.0
190°F
Survival

190°F
3.0
3.0

2.0
2.0

1.0
1.0

0.0
0.0
00 10
10 20
20 30
30 40
40 50
50 60
60 70
70 80
80 90
90
Treatm
Treatm ent
ent tim
tim ee (m
(m in)
in)

36
PHASE II: MICROBIAL KINETICS (TDT)
Thermal resistance comparison
1.6
Salmonella
1.4
y = -0.0486x + 9.1968 Surrogate
R2 = 0.997
1.2

1 Surrogate IS NOT
Log D-value

0.8
appropriate for
y = -0.0267x + 5.288
2
R = 0.9916
Salmonella
0.6

0.4
Surrogate IS
0.2 appropriate for
0 Salmonella
155 160 165 170 175 180 185 190
Treatment Temp. (°F)
 Phase III: In-Process Validation
 Preparation of inoculated products with surrogate organism
– Inoculation should not change intrinsic properties
– Need a high starting inoculum level
– Inoculum should be stable (product or ingredient should not be
antimicrobial)
– Entry point to the process should reflect the worst case possibility of
contamination
 Conduct the on-site process validation using inoculated worst case product
(with surrogate)
– Worst case processing conditions
– Inoculated product is subjected to the process (cold spot)
– Insertion and recovery of inoculated samples from process is critical
 Collect all test parameter data
 Enumerate surviving number of surrogate organisms in processed product

38
PHASE IV: PROCESS LETHALITY CALCULATION
Final calculations:
• Surrogateinitial – Surrogatefinal = log
reduction of surrogate
• Use kill ratio between surrogate and
target pathogenic organism to calculate
the log reduction of the target
pathogenic organism
– Kill ratio determined by TDT studies
– Kill ratio x log reduction of surrogate = log
reduction of target pathogenic organism

39
• Wilfredo Ocasio
• wilfredoocasio@EurofinsUS.com
• mobile: +1.925.980.8431

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