Supplement

Parameters for Determining Inoculated Pack/Challenge

Study Protocols34

ADOPTED 20 MARCH 2009, WASHINGTON, D.C.

NATIONAL ADVISORY COMMITTEE ON MICROBIOLOGICAL CRITERIA FOR FOODS

NACMCF Executive Secretariat,* U.S. Department of Agriculture, Food Safety and Inspection Service, Office of Public Health Science,

Room 333 Aerospace Center, 1400 Independence Avenue S.W., Washington, D.C. 20250-3700, USA

MS 09-287: Received 2 July 2009/Accepted 7 August 2009

ABSTRACT

The National Advisory Committee on Microbiological Criteria for Foods developed guidelines for conducting challenge

studies on pathogen inhibition and inactivation studies in a variety of foods. The document is intended for use by the food

industry, including food processors, food service operators, and food retailers; federal, state, and local food safety regulators;

public health officials; food testing laboratories; and process authorities. The document is focused on and limited to bacterial

inactivation and growth inhibition and does not make specific recommendations with respect to public health. The Committee

concluded that challenge studies should be designed considering the most current advances in methodologies, current thinking on

pathogens of concern, and an understanding of the product preparation, variability, and storage conditions. Studies should be

completed and evaluated under the guidance of an expert microbiologist in a qualified laboratory and should include appropriate

statistical design and data analyses. This document provides guidelines for choice of microorganisms for studies, inoculum

preparation, inoculum level, methods of inoculation, incubation temperatures and times, sampling considerations, and

interpreting test results. Examples of appropriately designed growth inhibition and inactivation studies are provided.

SCOPE OF DOCUMENT

This document was prepared at the request of the

sponsoring agencies of the National Advisory Committee on

Microbiological Criteria for Foods (NACMCF). The

document is intended for use by the food industry, including

food processors, food service operators, and food retailers;

federal, state, and local food safety regulators; public health

officials; food testing laboratories; and process authorities.

The document is focused on and limited to bacterial

inactivation and growth inhibition. The document does not

consider toxigenic fungi or the inactivation of viruses.

INTRODUCTION AND STATEMENT OF CHARGE

Statement of Charge

Because of the many questions raised by regulatory and

industry users on the definition of potentially hazardous

food (PHF) or time/temperature control for safety (TCS)

food, the NACMCF is asked for its guidance to clarify these

issues.

1. What are the appropriate criteria that must be

considered for an inoculated pack/challenge study to

determine if a food requires time/temperature control

for safety (TCS)? For example, pathogen species/strain

selection, use of surrogate organism, number of

pathogen strains, inoculation level(s), incubation temperature(s), length of incubation/duration of study, food

product physical properties, etc.

2. What are the appropriate uses of mathematical growth

and inactivation models? Under what conditions can

these models be used as a substitute for inoculated pack/

challenge studies? Of the models currently available,

which ones are most suitable for use, and what are the

limitations of these models?

3. What are the limitations for applying the results of an

inoculated pack/challenge study conducted on one food

to another similar food?

4. Of the existing inoculated pack/challenge study protocols, e.g., those published by the American Bakers

Association, NSF International, and others, which are

most suitable for application to a wide variety of foods,

and what are the limitations of these protocols? Are there

existing protocols that are appropriate for specific foodpathogen pairs?

5. Develop a decision tree to aid in the design of an

appropriate inoculated pack/challenge study. Test or

‘‘desk check’’ the decision tree using the following five

foods: meat-filled puff pastry, (baked) cheese pizza,

* Author for correspondence. Tel: 202-690-6600; Fax: 202-690-6364;

E-mail: gerri.ransom@fsis.usda.gov.

{ Sponsored by the U.S. Department of Agriculture, Food Safety and

Inspection Service; U.S. Department of Health and Human Services,

Food and Drug Administration, and Centers for Disease Control and

Prevention; U.S. Department of Commerce, National Marine Fisheries

Service; and U.S. Department of Defense, Veterinary Service Activity.

This article may be reproduced without prior permission.

{ Mention of trade names or commercial products in this publication is

solely for the purpose of providing specific information and does not

imply recommendation or endorsement by the U.S. Department of

Agriculture and other NACMCF sponsoring agencies.

140

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chopped lettuce, cheese (blocks or slices), and lemon

meringue pie.

6. Identify the basic knowledge, skills, education, training,

experience, and abilities necessary for a multidisciplinary

work group or individual to be qualified to design,

conduct, and evaluate an inoculated pack/challenge study

and the pursuant results.

Background

The restaurant and retail food store industry, totaling

nearly 1.5 million establishments in the United States, and

their suppliers routinely use inoculation/challenge testing

to determine whether a specific food requires TCS. A food

establishment, including restaurants, retail food stores,

delis, caterers, and institutions or vending commissaries

that provide food directly to the consumer, is defined in

the U.S. Food and Drug Administration (FDA) Food Code

(117).

When laboratory testing is used to support a change in

how the product is handled in a food establishment (e.g.,

refrigerated to unrefrigerated holding, extending shelf life,

increasing ambient temperature storage, or eliminating the

need for date marking), the data are submitted to a state or

local regulatory agency or directly to the FDA in the form of

a variance application for approval. Food establishments or

manufacturers submitting laboratory data to support their

proposals must ensure the study is appropriate for the food

and pathogen of concern and incorporate the necessary

elements into the study to yield a valid design and

conclusion.

A variance from any provision in the FDA Food Code

must also show that no health hazard will result from the

modification or waiver and that product handling is under

appropriate control using a hazard analysis critical control

point (HACCP) plan. Examples of foods in which the need

for TCS was questioned include puff pastries with savory

meat, cheese, or vegetable fillings; churros (fried dough)

batter held unrefrigerated; sliced pasteurized processed

cheese held at ambient temperature for more than 4 h;

certain cheeses held unrefrigerated. State and local regulators who evaluate a variance application based on this

laboratory evidence need criteria to help them determine

whether the study was adequately designed and whether the

conclusions are valid.

The definition of PHF or TCS food was amended in the

2005 FDA Food Code (117); chap. 1, Definitions to include

pH and aw interaction tables, allowing the hurdle concept to

be used in the determination of whether TCS is necessary.

The two interaction tables, as well as a decision-making

framework, were developed by the Institute of Food

Technologists (IFT) and provided to the FDA in the report,

‘‘Evaluation and Definition of Potentially Hazardous

Foods’’ (53) (31 December 2001, IFT/FDA contract

no. 223-98-2333, task order no. 4). When the pH and aw

interaction tables and the decision-making framework are

insufficient to show that a food does not require TCS,

further product assessment using inoculation/challenge

testing is likely required.

The IFT report (53) with its recommendations to the

FDA left a number of unanswered questions regarding the

understanding and implementation of a product assessment

when based on pH and aw the producer is unable to

determine whether TCS is required. This need for

clarification was confirmed in a 2005 survey of stakeholders

conducted by the Conference for Food Protection (18).

THE COMMITTEE’S RESPONSE

Use and Limitations of This Document

The primary objective of this document is to provide

guidelines for challenge studies necessary to determine

whether a variance to TCS may be granted under the Food

Code. Secondarily, the guidelines presented in this document may be useful to laboratories conducting pathogen

inhibition and pathogen inactivation studies for a variety of

foods for evaluation of safety prior to introduction into

commerce. It may be useful to review the proposed study

with the appropriate regulatory agency to ensure the design

and methods are appropriate. Studies should be completed

under the supervision of and interpreted by an expert food

microbiologist (Table 1). One of the limitations of these

studies is the balance of statistical validity with practicality.

A certain amount of variability is expected with challenge

studies, which can affect the validity and interpretability of

results. However, because of resource constraints, variability is generally addressed through the use of worst-case

scenarios, which should provide conservative results.

Although this document encompasses a variety of sources,

individuals who conduct challenge studies must be aware of

the most current advances in methodologies and identification of new pathogens or regulatory concerns that may need

to be considered as well as pertinent statistical issues. This

document does not make specific recommendations with

respect to public health.

Types of Challenge Studies

There are several types of challenge studies that deal

with validation of food safety processing procedures,

product storage conditions, and shelf life. Shelf-life studies

focusing on product quality are not addressed in this report

because they are generally not related to food safety.

Nevertheless, many of the principles of food safety–related

challenge studies are applicable to quality shelf-life studies.

Food safety–related challenge studies differ according to the

objective of the study, such as a pathogen growth inhibition

study or a pathogen inactivation study or a combination of

the two, and depend on the type of product, production

process, and hazard analysis of the product.

Food safety–related challenge studies include the

following:

Pathogen growth inhibition studies. These studies are

used to evaluate the ability of a particular food product

formulation with a specific type of processing and

packaging to inhibit the growth of certain bacterial

pathogens when held under specific storage conditions

(time and temperature).

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Pathogen inactivation studies. These studies are used

to evaluate the ability of a particular food product

formulation, a specific food manufacturing practice, or their

combination to cause the inactivation of certain bacterial

pathogens. These studies may also be impacted by food

storage and packaging conditions and must account for

these variables.

Combined growth and inactivation studies. Combined studies may be used to evaluate the ability of a

particular food or process to inactivate certain bacterial

pathogens and to inhibit the growth of certain other

pathogenic bacteria or to achieve a level of inactivation

followed by inhibition of the growth of survivors or

contaminants introduced after processing.

Determining When a Challenge Study Is Needed

The first step in determining whether a challenge study

is needed is to describe the product and process, conduct a

hazard analysis to determine the significant biological

hazards, and assess what is known about the growth or

inactivation of these in the product (80). Consideration

should be given to potential routes of contamination,

intrinsic factors such as water activity (aw) and pH that

affect the likelihood of the product to support growth, the

use of processing technologies that destroy pathogens of

concern, and the historical record of safe use of the product

(53, 80). In 2000, the FDA requested the IFT to assemble a

scientific panel to examine the issue of determining when

foods required refrigeration for safety. In addressing their

charge, the panel defined these foods as TCS foods and

developed a framework for determining whether time and

temperature control is required for safety. This framework

included two tables (one for control of spores and one for

control of spores and vegetative cells) with aw and pH value

combinations that indicate when product assessment (e.g., a

microbiological challenge study) is needed (53). This

concept was subsequently adopted as the basis for defining

when foods need refrigeration or some other form of timetemperature control in the 2005 FDA model Food Code

(117). These aw and pH combinations are not specific to

individual pathogens; therefore, for specific foods where the

pathogen of concern is established, other pH and aw values

may define the need for refrigeration. Information on

parameters associated with control of growth of various

pathogens can be found in the literature, e.g., the

TABLE 1. Recommended minimum expertise needed for designing, conducting, and evaluating microbiological studiesa

Category Design Conductb Evaluate

Knowledge and

skills

Knowledge of food products and

pathogens likely to be encountered in

different foods. Knowledge of the

fundamental microbial ecology of

foods, factors that influence

microbial behavior in foods, and

quantitative aspects of microbiology.

Knowledge of processing conditions

and parameters. Knowledge of

statistical design of experiments.c

Knowledge of basic

microbiological techniques.

Ability to work using aseptic

technique, to perform serial

dilutions, and to work at

biosafety level 2 (114).

Knowledge of food products and pathogens

likely to be encountered in different

foods. Knowledge of the fundamental

microbial ecology of foods, factors that

influence microbial behavior in foods,

and quantitative aspects of

microbiology. Knowledge of statistical

analysis.c

Education and

training

Ph.D. in food science or microbiology

or a related field or an equivalent

combination of education and

experience.

B.S. in food science,

microbiology, or a related

field or an equivalent

combination of education and

experience. Appropriate

hands-on experience in food

microbiology is also

recommended.

Ph.D. in food science, microbiology, or a

related field or an equivalent

combination of education and

experience.

Experience Two years of experience conducting

challenge studies independently and

experience in design of challenge

studies under the guidance of an

expert food microbiologist.

Two years of experience

conducting challenge studies

is useful; however, close

supervision by an expert food

microbiologist may substitute.

Two years of experience conducting

challenge studies independently and

experience in evaluation of challenge

studies under the guidance of an expert

food microbiologist.

Abilities Ability to conduct literature searches.

Ability to write an experimental

protocol.

Ability to read and carry out an

experimental protocol. Ability

to perform microbiological

techniques safely and

aseptically.

Ability to analyze and interpret

microbiological data.

a State or local regulatory food programs that are presented an inoculation study in support of a variance request may not have expert food

microbiologists on staff to confirm the validity of the study. Options available to them include consulting with expert food microbiologists

in their state or local food laboratories or requesting assistance from FDA food microbiologists through their regional retail food

specialist.

b Working independently under the supervision of an expert food microbiologist. c It may be appropriate to consult with a statistician with applicable experience in biological systems.

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International Commission on Microbiological Specifications for Foods publication Microorganisms in Foods 5.

Characteristics of Microbial Pathogens (54). When the

intrinsic factors of a food are consistent with parameters that

are well recognized as controlling the growth of a pathogen,

microbiological challenge studies are not needed (91). For

example, there would be no need to assess whether a

product with a pH of 3.5 supported growth of Salmonella,

because this organism will not grow at pH values this low.

However, studies to determine whether Salmonella survives

at this pH or whether it is inactivated over time may be

warranted under some circumstances. It is important to use

expert food microbiologists and technologists to assess the

need for challenge testing (Table 1).

A challenge study may be needed to assess whether the

pathogen can grow in the product if properties such as pH,

aw, or their combination do not ensure pathogen control. For

more details on the use of pH and aw to control the growth

of bacterial pathogens, consult the Compendium of Methods

for the Microbiological Examination of Foods (90).

Determination of the need for a challenge study is referred

to as product assessment in the IFT and Food Code tables

(53, 117).

When growth inhibition occurs due to factors other than

or in addition to pH and aw, such as the addition of

preservatives including lactate and diacetate, the literature

may provide information relevant to the pathogen and food

product. However, it is necessary to ensure that the data are

applicable to the specific product and conditions of use. The

efficacy of an antimicrobial agent may be dependent on the

formulation of the product. For example, factors such as fat

content can decrease the efficacy of antimicrobial agents

such as nisin (36, 58) and sorbate (82, 98). Conversely, a

low pH may potentiate the activity of antimicrobials such as

sorbate and benzoate (39). These evaluations should be

done by expert microbiologists and food technologists with

knowledge of the characteristics and the mechanism of

action of microbial inhibitors.

It is not reasonable to expect that every individual food

product would need a microbiological challenge study.

Many food products for which the assessment tables

indicate that product assessment is needed have a long

history of safe use. However, safe history of a food product

is relevant only when all conditions remain the same. Even

apparently minor changes to a food product, process, or

packaging method may have a large impact on the safety of

the product. Moreover, changes in the ecology, physiology,

or genetic makeup of a pathogen may result in food safety

issues in products with a history of safety (31, 73, 84).

RESPONSE TO QUESTIONS

The Committee was asked by the supporting federal

agencies to answer six questions. The responses are

provided in order below.

1. What are the appropriate criteria that must be

considered for an inoculated pack/challenge study to

determine if a food requires time/temperature

control for safety (TCS)? For example, pathogen

species/strain selection, use of surrogate organism,

number of pathogen strains, inoculation level(s),

incubation temperature(s), length of incubation/duration of study, food product physical properties, etc.

General Factors to Consider When Designing a

Challenge Study

Standardization of methods is beneficial for comparing

results among different studies, but it is not possible to

develop a single protocol that is broadly applicable to a wide

variety of food types or even to one category such as fruits

and vegetables (12). Parameters that should be considered

when designing a microbial challenge study are outlined

below (12, 53, 80, 91, 122).

1.0. Obtaining expert advice and identifying a laboratory

2.0. Type of study

2.1. Growth inhibition studies

2.2. Inactivation studies

2.3. Combination studies

3.0. Factors related to the test product

3.1. Product preparation

3.2. Product variability

3.3. Competitive microflora

4.0. Target organism(s)

4.1. Identifying the pathogen(s) of concern

4.2. Use of surrogate organisms

4.3. Type and number of strains

5.0. Inoculum levels

5.1. Growth studies

5.2. Inactivation studies

6.0. Inoculum preparation

7.0. Method of inoculation

8.0. Storage conditions

8.1. Packaging

8.2. Storage and shipping

9.0. Sample considerations

9.1. Sampling

9.2. Sample analysis for target pathogens or toxins

9.3. Enumeration of indigenous microbial flora

9.4. Determination of physical parameters

10.0. Duration of study and sampling intervals

11.0. Interpreting test results

12.0. Elements to include in the report

1.0. Obtaining expert advice and identifying a

laboratory

Challenge studies must be designed and evaluated by

an expert food microbiologist. This expertise may or may

not reside within the staff of a testing laboratory. When it

does not, it is important to choose an advisor who can work

with the laboratory to conduct a proper study. Potential

sources of expertise include in-house experts, university

faculty, testing laboratories, and independent consultants.

Once a study design has been developed it may be

appropriate to consult with a statistician with applicable

experience in biological systems and have the design

reviewed by the regulatory body or intended recipient of

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the study. Suggested modifications can then be incorporated

before the study is executed.

Choosing a laboratory requires careful consideration

because not all laboratories have the expertise to design

challenge studies and the quality control procedures necessary

to produce valid results that will be accepted by the regulatory

authority or other reviewer. Laboratories may be certified by

various organizations and state or federal agencies for various

types of testing, e.g., water and waste water testing, ISO

17025, and Grade A dairy testing. However, these certifications do not necessarily qualify a laboratory to design and

conduct microbiological challenge studies. A laboratory

selected for challenge testing must be able to demonstrate

prior experience in conducting challenge studies. It is

necessary to ensure that personnel are experienced and

qualified (Table 1) to conduct the types of analyses needed

for the challenge studies and that these workers will follow

generally accepted good laboratory practices. Laboratories

conducting microbial challenge studies should use test

methods validated for the intended use. Some examples of

generally accepted methods are available in the most recent

editions of the references listed in Appendix A. In situations in

which approved methods are not available or applicable,

laboratories may consider using other widely accepted

methods, such as those that have been cited in peer-reviewed

journals. Failure to properly design the study and use valid

methods and appropriate controls may render the challenge

study unacceptable and require additional time and resources

to repeat the study. See the questions in Appendix B for

assistance in selecting a laboratory.

2.0. Type of study

Challenge studies are conducted for a variety of reasons.

The specific purpose of the study drives selection of bacterial

strains and inoculum level, choice of parameters tested, types

of analysis, and duration of the study as described below. For

example, studies evaluating growth inhibition should consider

bacterial species listed in Table 2, whereas the choice of

species for lethality or survival studies depends on the

selection of resistant strains relative to the process and

technology as well as compliance with regulations for specific

foods (e.g., FDA, U.S. Department of Agriculture, Food

Safety and Inspection Service [USDA-FSIS], and state laws

based on the Pasteurized Milk Ordinance).

2.1. Growth inhibition studies

The objective of a growth study may be to request

exemption (variance) from TCS or other requirements

defined by the Food Code, Pasteurized Milk Ordinance, or

TABLE 2. Potential pathogensa of concern for growth studies based on interaction of product pH and aw

b

aw values

pH values:

,3.9 3.9 to ,4.2 4.2–4.6 .4.6–5.0 .5.0–5.4 .5.4

,0.88 NGc NG NG NG NG NG

0.88–0.90 NG NG NG NG Staphylococcus aureus S. aureus

.0.90–0.92 NG NG NG S. aureus S. aureus L. monocytogenes

S. aureus

.0.92–0.94 NG NG L. monocytogenes Bacillus cereus B. cereus B. cereus

Salmonella Clostridium botulinum C. botulinum C. botulinum

L. monocytogenes L. monocytogenes L. monocytogenes

Salmonella Salmonella Salmonella

S. aureus S. aureus S. aureus

.0.94–0.96 NG NG L. monocytogenes B. cereus B. cereus B. cereus

Pathogenic E. coli C. botulinum C. botulinum C. botulinum

Salmonella L. monocytogenes L. monocytogenes C. perfringens

S. aureus Pathogenic E. coli Pathogenic E. coli L. monocytogenes

Salmonella Salmonella Pathogenic E. coli

S. aureus S. aureus Salmonella

Vibrio parahaemolyticus V. parahaemolyticus S. aureus

V. parahaemolyticus

.0.96 NG Salmonella Pathogenic E. coli B. cereus B. cereus B. cereus

Salmonella C. botulinum C. botulinum C. botulinum

S. aureus L. monocytogenes L. monocytogenes C. perfringens

Pathogenic E. coli Pathogenic E. coli L. monocytogenes

Salmonella Salmonella Pathogenic E. coli

S. aureus S. aureus Salmonella

V. parahaemolyticus V. parahaemolyticus S. aureus

V. vulnificus V. parahaemolyticus

V. vulnificus

a Campylobacter spp., Shigella, and Yersinia enterocolitica do not appear here because they are typically controlled when the pathogens

listed are addressed.

b Data are based on the PMP (106), ComBase predictor (50), ComBase database (49), or peer-reviewed publications (11, 17, 45). c NG, no growth; when no pathogen growth is expected, but formulation or process inactivation studies may still be needed.

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FDA, USDA-FSIS, national, state, provincial, or local

regulations. Other objectives may be to demonstrate safety

of a current formulation during extended shelf life under

normal refrigerated or ambient temperatures, to determine

whether formulation or processing changes are required if

the product is subjected to temperature abuse, or to

determine the effect of a modified formulation, process, or

packaging technology.

2.2. Inactivation studies

Inactivation studies may be used to determine whether

thermal processes provide adequate log reduction of a target

pathogen as defined by regulations or government policy

(e.g., FSIS requirement for a 5-log kill of Escherichia coli

O157:H7 in fermented dry sausage) (110). Inactivation

studies may also be used to determine whether nonthermal

technologies or combinations of pH, aw, preservatives, and

holding for specified times at specific temperatures prior to

release of product will provide sufficient lethality to render a

food product safe (e.g., 2-year aging of raw milk Parmesan

cheese or 3-day holding at room temperature to inactivate

Salmonella in mayonnaise).

2.3. Combination studies

Other studies involving both verification of inactivation

and evaluation of changes in the number of microorganisms

during extended storage combine concepts from both study

types above. For example, a manufacturer of processed meat

wishing to have a product line classified as Alternative 1 for

control of Listeria monocytogenes by FSIS regulation (9

CFR 430) (113) may undertake a study to demonstrate a 2-

log postlethality kill step of L. monocytogenes on ready-toeat meats by high pressure processing followed by growth

inhibition by product formulation during extended refrigerated storage. A producer of a cold-filled hot sauce with

pH 3.5 may wish to demonstrate a 5-log kill of acid-tolerant

Salmonella when held at 20uC (68uF) for 3 days as well as

no recovery or growth of the pathogen during ambienttemperature storage for 1 year.

3.0. Factors related to the test product

3.1. Product preparation

The product should be prepared under conditions most

conducive to growth or survival based on the intended

conditions of use and expected product variability. Consideration should be given to the physical properties (pH, aw,

etc.) of the prepared product and the impact that these

properties can have on the results of a challenge or

inactivation study. The product should be prepared so that

the critical physical properties are at the appropriate

minimum or maximum control limits intended for the

finished product (see section 3.2 below on product

variability).

Multicomponent products may take days to equilibrate

moisture, aw, or pH. Such products should generally be

inoculated prior to equilibration in regions of the product

that are considered the most permissive to growth, provided

these are areas reasonably likely to be contaminated. In

general, larger particles take longer to equilibrate. Studies to

determine growth, inactivation, or survival of a pathogen

present due to recontamination would involve inoculation of

product after equilibration.

3.2. Product variability

Knowledge of the manufacturing or production variability is needed to determine the appropriate test parameters for a challenge study. Variability within and among

lots should be determined by measuring formulation factors

such as pH, aw, etc. The greater the variability, the more

samples of product need to be evaluated, e.g., more

measurements need to be made to determine the upper or

lower control limits. When choosing an attribute such as pH

during the challenge test, that pH (including the uncertainty

in the measurement or manufacturing capability) becomes

the upper limit of the pH specification range for the product

subsequently manufactured.

Whenever possible, food from a commercial production

facility (manufacturing or food service kitchen or commissary) or manufactured in a laboratory that has pilot food

processing facilities should be used for the study. The food

produced in a pilot facility should be processed to mimic

conditions used during commercial operations (cooking

temperature and time, homogenization, hot fill, slicing, etc.).

The product lots used for the challenge study should be

representative of normal production with the exception of

necessary adjustments to acidity, moisture, salt, aw, etc., to

yield the conditions most permissive to pathogen growth or

survival at each formulation control limit (worst-case

scenario based on knowledge of manufacturing variability).

Percent salt and moisture may be easier than aw to measure

and control by the producer for some products such as

processed meats, cheeses, and smoked seafood and,

therefore, may be used for control parameters in the

challenge study.

The target limits for moisture or aw will vary depending

on whether the objective of the study is to verify

inactivation or growth inhibition. For thermal inactivation

studies, lower moisture or aw levels should be used because

pathogens may have increased heat resistance under these

conditions (10, 24, 25, 38, 102). Similarly, increased solute

content has been shown to protect L. monocytogenes against

high hydrostatic pressure (43, 63). In contrast, for growth

challenge studies, targeting the upper limit of moisture or aw

is appropriate. For example, if the typical moisture range is

56 to 58%, a thermal inactivation study should be conducted

at no more than 56% moisture, but a growth challenge study

should be conducted at no less than 58% moisture.

When pH is one of the controlling factors, the food

should be prepared with the lowest amount of acid allowed

in the formulation so that the pH is at the upper range and

adjustment in the laboratory is not necessary. If the target

pH is 4.8 but the maximum pH observed in multiple

production batches is 5.0, a growth inhibition study or an

inactivation study should be conducted at a pH no lower

than 5.0. If pH adjustment is necessary and pH is adjusted

upward using sodium hydroxide, the titratable acidity prior

to pH adjustment should be measured and reported so it can

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be compared with that of the adjusted food product. If the

pH of the product needs to be reduced, it is important to use

the same acids that are predominant in the product.

Acidulants exert different degrees of antimicrobial

activity at the same pH. For example, acetic acid is the

most inhibitory for many microorganisms, followed by

lactic acid, with citric acid the least inhibitory (2, 3, 28, 30,

83). As a result, if the challenge study were conducted on a

product formulated with acetic acid (vinegar), the study may

not be valid for a reformulated product containing citric acid

(lemon juice) even if the final pH were the same. In some

cases, the number of challenge tests can be reduced for

multiple formulations having similar proximate analysis,

acidity, and aw, provided the formulation most permissive to

growth or survival is tested.

3.3. Competitive microflora

Competitive flora can affect the outcome of a challenge

study, particularly one determining growth of pathogens in a

food product. Inoculated product should contain typical

levels of competitive microflora, including starter cultures,

which may interfere with consistent growth of pathogens

during the study. The freshest product possible, i.e., within

the first 10% of its shelf life, should be used. For example, if

the shelf life is ,1 month, product should be used within 1

to 3 days of production. (For purposes of this document,

shelf life is defined as the time at a specified storage

temperature during which product quality is considered

acceptable for consumption. This assessment includes

acceptable flavor, appearance, and functionality based on

chemical changes or growth of spoilage microorganisms but

does not necessarily infer product safety by accepted

definitions in all countries.) Care should be taken during

the inoculation step to not introduce atypical spoilage

microorganisms that may inhibit pathogen growth. In rare

cases, naturally occurring bacteria can enhance growth or

survival of pathogens, potentially reducing the safety of the

product (19).

4.0. Target organism(s)

4.1. Identifying the pathogen(s) of concern

An expert food microbiologist should determine the

appropriate organisms for challenge testing. There are a

number of issues the microbiologist must consider,

including the specific product, the process used to prepare

it, and any pathogens that are epidemiologically or

ecologically relevant. There are a number of resources

available to assist in determining appropriate pathogen(s)

for a given food. Examples of assessments of the

appropriate challenge organism for specific food products

can be found in the IFT report on evaluation and definition

of potentially hazardous foods (53) (specifically, see Table

1, Table A, Table B, Table 4-1, and Table 6-1 in that report).

For easy reference, please refer to Appendix C.

Table 2 provides combinations of pH and aw values

that may allow growth of pathogens of concern based on

model predictions and published literature. This table may

be useful in selecting organisms for use in studies to assess

growth or inactivation by formulation. Although many

pathogens are listed for some pH and aw combinations, it

may not be necessary to evaluate each pathogen for a

specific food, because epidemiological attribution or

product characteristics may narrow the choice of appropriate

challenge organisms. For example, a seafood product might

be challenged with Vibrio or Salmonella because of

epidemiological attribution, whereas a pasteurized product

in which vegetative cells of pathogens have been eliminated

might be challenged with pathogenic sporeformers. L.

monocytogenes might be used if the study is designed to

determine growth or inactivation due to recontamination

with this organism in a ready-to-eat product.

The organism used for a challenge study to determine

inactivation due to product formulation may need to be

selected based on the resistance of the pathogen to the

bactericidal properties. For example, enterohemorrhagic E.

coli may be selected over Salmonella or Staphylococcus

aureus for a food with a pH of 4.3 and an aw of 0.98 because

it is generally considered to be more resistant to acid.

Ideally, when conducting a study to determine pathogen

growth in a food formulation, the fastest growing pathogen(s) likely to be present would be used. Predictive models

can be useful for determining which pathogen may grow

fastest under the conditions of the study. For example, if

predictive modeling demonstrates that Salmonella grows

better at a given pH and aw combination, then this pathogen

may be considered the best choice among the organisms of

concern for that product for use in a challenge study.

Although Table 2 is similar to Table B in the Food

Code (see Appendix D) and the IFT report (53), it is not

identical, and some explanation is required. First, Table 2 is

more extensive than Table B and includes both higher and

lower pH values and more defined categories for higher aw

values. Second, the IFT report (53) and the Food Code (117)

are specifically focused on foods that require temperature

control for safety, whereas the focus of this Table 2 is

broader. Finally, Table 2 considers time scales that may be

considerably longer than those typically of concern in retail

food safety. The table should not be interpreted to suggest

that a food falling within a particular pH and aw range needs

to be challenged with a pathogen, e.g., that high aw foods

with a pH of 3.9 need to be challenged with Salmonella.

Although Salmonella has been shown to grow at pH values

as low as 3.9, these studies have been done in laboratory

media under conditions ideal for growth, other than the pH

value. In foods, many factors interact to support or inhibit

pathogen growth. An expert microbiologist should use

Table 2 as a guideline to assess whether a challenge study

on a particular food with a specific pathogen is warranted.

Table 2 is useful for identifying appropriate pathogens

of concern for particular pH and aw combinations. However,

this information should not typically be used for the

selection of organisms in process inactivation (e.g., thermal

inactivation) studies. The choice of organism for these types

of studies should be based on the likelihood of pathogen

association with the specific food and pathogen resistance to

inactivation, as well as the public health objective of the

process and the intended use of the product. For example,

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nonproteolytic strains of Clostridium botulinum might be

selected as the appropriate target organism for some

refrigerated foods and L. monocytogenes for others,

depending on how likely it is that nonproteolytic C.

botulinum will be present, how long the product will be

held refrigerated, whether the product is ready-to-eat or will

be cooked prior to consumption, and other factors.

4.2. Use of surrogate organisms

Inoculation of foods with bacterial pathogens requires

adequate biological containment facilities and may require

governmental approval in the case of certain pathogens such

as C. botulinum. Therefore, in limited cases, nonpathogenic

surrogate organisms are especially useful for testing

specialized processing equipment in the plant, where the

introduction of the pathogen would pose an unacceptable

risk. Surrogates may also be useful for selecting the study

parameters before conducting the full study with the

pathogen. Care should be taken when using surrogates for

in-plant challenge studies because these organisms may

have adverse sanitary or regulatory implications should they

survive and contaminate the plant environment.

Surrogates are typically nonpathogenic proxies for the

pathogen of concern that have similar or more robust

survival capabilities under the conditions being studied.

Such proxies may include avirulent strains of pathogens,

where appropriate. The ideal surrogate should have the

following characteristics: nonpathogenic, inactivation characteristics and kinetics that can be used to predict those of

the target pathogen, similar susceptibility to injury,

reproducible growth, easy preparation of high-density

populations that are stable until used, easily enumerated

and differentiated, similar attachment capabilities, and

genetically stable (52).

Clostridium sporogenes PA3679 has proven to be an

excellent surrogate for C. botulinum when used in inoculated

pack studies to validate thermal processes for low-acid canned

foods. In certain cases, C. sporogenes may be suitable for

reducing the number of formulations to be verified using C.

botulinum because these organisms are culturally similar.

Formulations that support growth of C. sporogenes can be

excluded from further validation studies with C. botulinum.

However, C. sporogenes cannot be used as a direct substitute

to validate a product for inhibition of botulinum toxin

production (64). Other examples of surrogate-pathogen pairs

include Listeria innocua and L. monocytogenes (99) and

nonpathogenic E. coli and E. coli O157:H7 (26).

A surrogate that works well to predict the target

response for one type of process may not be an appropriate

surrogate in a different type of process. For example, the

heat resistance of various strains of C. botulinum spores did

not correlate with their resistance to high hydrostatic

pressure (71), so although C. sporogenes may be the

preferred surrogate for C. botulinum for canning processes,

another organism, such as Bacillus amyloliquefaciens may

be appropriate as a surrogate for C. botulinum for high

hydrostatic pressure studies (71, 86).

The choice of the surrogate needs to be justified, and

supporting documentation for its appropriate use for the

pathogen, food, and treatment being evaluated should be

incorporated into the final report. If no directly relevant

published comparison data are available, studies need to be

conducted to establish the validity of using a particular

surrogate-pathogen-process combination.

4.3. Type and number of strains

In order to account for variations in growth and survival

among strains, challenge studies should generally be

conducted using three to five bacterial strains either

individually or in combination (53, 75, 91). Where there

is considerable variability among strains or there is little

known about the growth of the organism in a particular food

product, as many as 10 strains may be used (e.g., some C.

botulinum or L. monocytogenes studies).

Generally, using an inoculum composed of multiple

strains (i.e., a cocktail) of a given pathogen is preferred

because it will help to encompass the variability among

organisms and may reduce the number of required tests.

Prior to use in the study, the strains selected should be

screened for antagonism that can be caused by production of

bacteriocins or other antimicrobial factors (53). Another

approach is to screen several strains in the food matrix under

investigation and determine which strain has the greatest

resistance, grows fastest, etc., and then to conduct the

challenge studies using that single strain (12, 91). Screening

parameters depend on the purpose of the challenge study,

e.g., to determine inactivation or growth characteristics in a

product. However, there are strains with atypical resistance,

e.g., the extremely high moist heat resistance of Salmonella

Senftenberg 775W (79). These strains may not be

appropriate for use in some studies because they are not

representative of strains reasonably expected to be present in

the applicable foods. Whether to use an individual strain or

cocktails of strains should be determined by an expert

microbiologist knowledgeable in food microbiology and

pathogen control.

Strains carrying markers such as antibiotic resistance or

green fluorescent protein may be useful for confirming that

the organisms recovered are the test organisms. When such

strains are used it is important to determine that they possess

the same characteristics as the parent strain without the

marker with respect to factors critical to the challenge study.

Furthermore, carriage of the resistance marker should be

verified to be stable under stressful conditions that may be

encountered during the challenge study.

Isolates should be appropriate for the food product

being challenged (53, 80, 91). This approach includes using

isolates from the food type, the food processing environment, and clinical specimens, as appropriate. Inactivation

studies should use strains that demonstrate tolerance to the

specific process for the product under consideration, such as

heat or high pressure processing (16, 24, 25, 71).

Biochemical characteristics, serology, genetic profile, virulence, or toxicity should be periodically reconfirmed as

appropriate. The test strains for growth challenge studies

should demonstrate robust growth in laboratory media or a

similar food without inhibitors under the conditions of the

study (e.g., temperature and atmosphere).

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5.0. Inoculum levels

The inoculum level used in the challenge study depends

on whether the objective of the study is to determine growth

or inactivation of a pathogen. It may be desirable to conduct

challenge studies using multiple inoculum levels to

determine the margin of safety in the process/formulation

(91).

5.1. Growth studies

When conducting studies to determine whether a

pathogen grows in a product, ideally the number of organisms

used should reflect the numbers normally expected in the

product. Typically, an inoculum level of between 2 and 3 log

CFU/g is used, even when this exceeds expected numbers,

because this level allows enumeration by direct plating (53,

91). Lower levels may be used if documentation of low levels

of natural contamination exists, because these lower levels

will more accurately represent the product’s ability to support

growth (91). When very low seeded populations (e.g., less

than 100 cells per sampling unit) are most appropriate,

consistent inoculation among individual samples may be

difficult to achieve. Calculating the level of organisms in the

product from the initial inoculum suspension, increasing

sample size (e.g., from 25 to 250 g) and the number of

replicate samples analyzed (e.g., from three to six samples),

and/or using enumeration methods such as the most probable

number (MPN) method will increase confidence in the

number of organisms in the inoculum.

The inoculum level may affect the apparent efficacy of

an antimicrobial or formulation combination to inhibit

microbial growth. If the inoculum populations are too high,

the factors inhibiting growth may be overwhelmed by the

inappropriate inoculum level, leading to the incorrect

conclusion that the formulation does not inhibit growth

(53, 80). In the case of sporeformers, germination and time

to observable growth or toxin production may be significantly reduced if high initial spore loads are used (69, 126).

In contrast, a high inoculum level of vegetative cells (e.g., 5

to 7 log CFU/g) in a growth study may also mimic the

population nearing stationary phase, which may result in an

apparent no-growth or low-growth observation.

5.2. Inactivation studies

When conducting inactivation studies, high numbers of

organisms are typically used, e.g., 6 to 7 log CFU/g (53, 91),

in order to quantify survivors and/or to document high

levels of inactivation. The target level of reduction, which

influences the inoculum level used, may depend on

regulations for specific food types, e.g., a 5-log reduction

of the appropriate pathogen in juice (21 CFR 120.24) (120),

4-log reduction for treatment of almonds (7 CFR 981) (105)

to inactivate Salmonella, and a 7-log reduction of

Salmonella in poultry products (9 CFR 381.150) (112).

Laboratories conducting inactivation studies in products that

are subject to regulations should be aware of the most

current requirements.

Inactivation studies may be conducted to assess the

lethality delivered by a specific process, e.g., the ability of

UV light to achieve a 5-log reduction of E. coli O157:H7 in

apple cider, or to determine inactivation of a pathogen over

time, e.g., the effect of preservatives on pathogen

inactivation during storage of a food product. In the former

case, relatively high inoculum levels are generally used, as

noted above. However, in the latter case, lower inoculum

levels consistent with expected pathogen contamination

levels might be used because preservatives would generally

not be expected to inactivate large numbers of pathogens,

depending upon the pH and other conditions. Studies might

also be important to determine survival or inactivation of a

pathogen in a product that is recontaminated.

Initial inoculum levels may affect the rate of die-off in

some foods (32, 95, 125), and this phenomenon needs to be

taken into consideration.

6.0. Inoculum preparation

Ideally, isolates from foods should be stored in a

manner to preserve the strain characteristics with respect to

survival, growth, resistance, etc. (e.g., frozen in glycerol or

freeze-dried). When reviving strains from the frozen or

lyophilized state, there should be one to two successive

transfers in a nonselective growth medium. Working

cultures, e.g., refrigerated slants, may be prepared and used

for a period of time (e.g., 7 to 30 days). The number of times

a culture is transferred to produce new working stock

cultures should be minimized to avoid genetic changes that

affect the phenotypic properties of the organism (91).

AOAC International guidelines for laboratories (8) indicate

there should be not more than five passages from the

primary reference material. In some instances even fewer

transfers may be appropriate, because organisms may

readily lose extrachromosomal elements such as plasmids

or other genetic markers and phage.

For challenge studies using vegetative cells, stationary

phase cells (18 to 24 h) grown on nonselective media under

conditions suitable for optimal growth of the specific

challenge culture should generally be used (53). However,

in certain instances it may be desirable to precondition or

adapt the culture to specific conditions that may be

applicable to the specific characteristics of the food product.

For example, for low pH foods it may be appropriate to acid

adapt cultures (34, 46, 65, 66), which can often be

accomplished by growing the culture in tryptic soy broth

with 1% glucose (14, 27). Cold adaptation at 7 to 8uC (44.6

to 46.4uF) for 7 days may reduce the lag phase for

pathogens (121), which may be important for assessing the

shelf life of refrigerated ready-to-eat products. Cold

adaptation may be more important for challenge tests of

foods with a short refrigerated shelf life, e.g., less than 7

days. Care should be taken to avoid habituation procedures

that cause cells to be more sensitive to the adverse

environment, e.g., simultaneous adaptation to cold and acid

conditions (95) or acid stressing cells prior to a heat

treatment (87).

For inactivation studies, cells that are grown at higher

than optimum temperatures may become more resistant to

heat than are cells grown at optimum temperatures (79, 96).

Increased heat resistance can also be observed with brief

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exposure to sublethal temperatures (heat shock) (15, 94,

123). For either inactivation or growth studies, adaptation of

cells should attempt to mimic the likely physiological state

of the organism at the time it contaminates the food.

Prior to use, cells should be washed (e.g., in buffer or

carrier medium) to remove nutrients or metabolites in the

spent medium that could have an impact on growth in the

test product. Cells should then be suspended in a carrier

(buffer or homogenized portion of the food) to inoculate the

food.

Composites containing multiple strains should have

approximately equal numbers of the individual strains. This

can be accomplished by previous experience enumerating

the strains under specific growth conditions or by using

turbidity measurements (e.g., optical density or McFarland

standards).

Spores of pathogens such as C. botulinum, Clostridium

perfringens, and Bacillus cereus can be prepared, washed,

and suspended in sterile water and frozen, preferably at

220uC (24uF) or below. As with vegetative cells,

composites should contain approximately equal numbers

of each strain. Spore suspensions can be enumerated to

determine the number of spores and then appropriate

volumes can be combined to prepare the inoculum.

Spore inocula are often heat shocked prior to use,

unless they are inoculated into the product immediately

prior to heating or processing. The decision on whether or

not to heat shock a spore inoculum will depend on the

expected state of the naturally occurring spores in the food

product and the conditions of use of that product. For

example, spores would not be heat shocked if the

challenge study is being conducted in a raw commodity

that will not be heated (e.g., raw reduced-oxygenpackaged fish). When it is desirable to have a mixture of

vegetative cells and spores, the suspension should not be

heat shocked.

It is important to verify the numbers of viable

organisms in the inoculum used. In addition to enumerating

the inoculum suspension itself, the inoculated food should

be enumerated to obtain a zero-time count. If the inoculum

level is low, an increased number of replicates of the

inoculum and/or product may be necessary. Rapid and

significant reductions in microbial populations are frequently observed when the food includes bactericidal ingredients

such as nisin or other commercial fermentation by-products

used for shelf-life extension. For example, a 0.5- to 2.5-log

reduction in L. monocytogenes was observed immediately

after inoculation in fresh soft cheese and in bologna and

ham containing lactic acid bacteria (LAB) fermentate or

nisin (35, 37).

A dry inoculum may be required for studies in lowmoisture foods or when added moisture needs to be avoided.

Inoculum can be prepared by freeze-drying (53, 80) or dried

on a product similar to the challenge food (53). For

preparation of a dehydrated inoculum, the organism may

require several days to months to stabilize (e.g., Salmonella

in skim milk powder) (59). As a result, viable populations of

the stabilized dried inoculum should be determined prior to

use.

7.0. Method of inoculation

Inoculation procedures for challenge studies are

described in the IFT report (53). As that report notes,

several critical considerations for the delivery of the

inoculum to the product include maintaining the intrinsic

or extrinsic characteristics of the product; simulating

contamination that could realistically occur under manufacturing or storage conditions; and ensuring that, where

appropriate, each of the unique interfaces of the product

components receive the inoculum.

Two factors important to maintaining the intrinsic

characteristics of the challenged product are minimizing

inoculum volume and matching the critical factors of the

food, such as pH and aw. Typically the inoculum volume

should be no more than 1% of the volume of the food and

when possible, less. Some methods that have been used to

minimize the inoculum volume include growing the

pathogen to high populations and concentrating by

centrifugation or growing the pathogen on a solid growth

medium and then harvesting a paste for use as the inoculum.

When challenging food products with reduced aw or pH, the

aw or pH of the diluent can be adjusted using a humectant or

acidulant similar to that contained in the food (53).

However, preliminary analysis should verify that modified

pH or aw of the buffer does not adversely affect viability of

the pathogens.

An important extrinsic factor is the package atmosphere

(see section 8.1. below: storage conditions, packaging).

Ideally, product should be first inoculated and then

packaged under an appropriate atmosphere that closely

duplicates the packaging system to be used during

commercial production. Alternatively, a common practice

is to use a needle to inoculate through the packaging using

some type of self-sealing rubber or silicon septum. Two

disadvantages of using the latter type of inoculation method

are long-term package integrity and inoculum distribution.

Also, when inoculating with a needle, culture should be

distributed over as large an area as possible to reduce the

concentration of cells, moisture, and/or nutrients in limited

areas. Package atmosphere (e.g., oxygen and carbon dioxide

in the headspace) should be monitored during the duration

of the study to assess the integrity of the package and to

ensure that the effect of changes in gas composition is

considered.

In general, the method of inoculation should place the

inoculum on or within the product in a manner that

realistically simulates potential contamination that might

occur during manufacture, preparation, shipment, or display

of the product. Liquid foods are inoculated by mixing the

inoculum throughout the product with agitation. In solid

foods, the inoculum may be mixed throughout a ground

product or applied on the surface by dipping, aerosolizing,

or spreading on the entire surface or on selected spots.

Dipping the product in a liquid inoculum or using an

aerosolized inoculum will allow organisms to be spread

over the entire surface of the product, including cracks and

crevices. However, if an aerosolized inoculum is used,

inoculation should be conducted in a biological safety

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cabinet to protect employees from the challenge organism.

Preliminary studies should be conducted to standardize the

amount of inoculum that contacts the product.

Many challenge products have multiple components or

layers. If contamination during assembly is possible, the

challenge inoculum should be applied to the various layers

or components. Unique growth conditions can exist at the

interfaces between components, such as the microenvironment between a pie crust and a pie filling. This area might

have a unique combination of factors that will allow growth,

so these areas should receive a portion of the inoculum. For

this reason, the food should not simply be homogenized and

inoculated. Other conditions of the microenvironment

should also be considered, such as fat-water emulsions,

microdroplets, or partitioning.

Inoculating a large batch prior to packaging or

inoculating individual samples can be valid depending on

the likely route of contamination, packaging considerations,

and practicality. Inoculating a single batch of product will

minimize the variability of the starting concentration as well

as create a less heterogeneous distribution of the pathogen if

the food can be mixed without destroying the product

integrity. This issue is particularly critical in growth or

inactivation studies in which documentation is needed to

meet a specific regulatory requirement (e.g., no more than a

1-log increase as evidence of growth inhibition of L.

monocytogenes in a deli salad or a 5-log reduction of E. coli

O157:H7 in juice). Dividing a large inoculated batch into

discrete portions for testing at each sampling interval

reduces the risk of contamination caused by repeatedly

resampling a large batch. Inoculating individual samples

may be more appropriate for studies representing postprocess contamination by contact (e.g., cooked frankfurters or

slices of cheese made with pasteurized milk) or when

production cannot be readily replicated in the laboratory

(e.g., filled pastries or individual packages with unique

atmosphere and packaging materials). Inoculation methods

that result in highly variable inoculum levels or uneven

distribution require a greater number of samples at each

sampling interval and potentially additional replicate

batches to be analyzed.

8.0. Storage conditions

8.1. Packaging

Product packaging for the challenge study should be

representative of typical commercial production. If the

commercial product is to be packaged under vacuum or a

modified atmosphere, the challenge study sample should be

packaged under the same conditions, including the use of

the exact gas mix used for modified atmosphere packaging,

use of packaging material of the same gas permeability, and

use of similar vacuum levels for vacuum-packaged product.

Specific modified atmospheres or vacuum packaging may

be inhibitory to some microorganisms but may stimulate

growth or toxin production by other microorganisms (53).

Care should be taken to ensure that headspace volume and

gas composition of the challenge study samples mimics the

commercial food product as closely as possible.

8.2. Storage and shipping

Storage temperatures used in the challenge study should

be representative of the expected temperature range that the

product will be exposed to during commercial distribution

and storage. For refrigerated foods, NACMCF recommends

that the studies be conducted at 7uC (44.6uF) to account for

expected consumer storage temperature in the United States

(75). Refrigeration studies may incorporate additional

temperatures (e.g., 4 to 6 or 10 to 12uC [39.2 to 42.8 or

50 to 53.6uF]) when a better understanding of the behavior

of the challenge organism is desired, such as with some

antimicrobial compounds whose inhibition of microbial

growth is temperature dependent (21, 91).

Temperature changes may be incorporated into a

challenge study protocol if, for example, a manufacturer

distributes a refrigerated product under well-controlled

conditions for a portion of its shelf life, after which the

product may be subjected to elevated temperatures immediately prior to and during use (53). For shelf-stable

products, typical temperatures range from 24 to 35uC

(75.2 to 95uF) depending on expected storage room

temperatures (21). Humidity should also be considered as

a factor in storage conditions; for those products where the

moisture content can change in response to ambient

humidity conditions, the challenge study should be designed

to incorporate representative environmental humidity variation (80).

It is necessary to ensure that appropriate storage space

is available and that proper temperatures are maintained and

recorded throughout the study. Temperatures during storage

and transportation of commercially made products to the

laboratory should be monitored with continuous temperature recorders, data loggers, or periodic manual temperature

verification. Samples inoculated with pathogens should be

segregated and clearly labeled to prevent inadvertent human

consumption.

9.0. Sample considerations

9.1. Sampling

Sampling schemes for food microbiology experiments

are often dictated by common practice not solely based on

statistical design. The suggestions below reflect this

convention. The number of samples to be analyzed initially

and at each time interval during processing and/or storage

should be at a minimum two; however, analysis of three or

more samples is preferred. Replicates should be independent

trials using different batches of product and inoculum to

account for variations in product, inoculum, and other

factors. Generally, the number of samples and replicates

should be increased in situations of higher variability or

uncertainty. When the number of samples analyzed at each

time interval is only two, it is better for the study to be

repeated (replicated) more than two times. In studies with

three or more samples tested at each time interval, two

replicates are usually adequate. When analyzing samples for

botulinum toxin it is appropriate to select a greater number

of samples (e.g., five or more) per time point because of the

potential variability in toxin production among samples

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(74). For end-point lethality determination, 5 to 10 samples

per time interval may be appropriate. If supporting data

from other studies exist, the need for replication may be

reduced (91). Appropriate statistical experimental design

can improve the validity of the study. There are quantitative

methods for assessing the statistical quality of a study, e.g.,

power analysis. The study design may benefit from

consultation with a statistician familiar with food microbiology studies.

The sample preparation method should be selected

based on the type and properties of the food and the method

of inoculation, which depends on the food product and the

inoculation procedure (53, 91). In cases of solid foods

inoculated on their surface and in products where the

contamination is expected to be localized on their surface,

samples may be swabbed or sponged, washed or rinsed,

and/or agitated in a liquid buffer or diluent of known

volume. After thorough mixing, the rinsate is analyzed by

direct plating of appropriate dilutions onto appropriate

culture media (see section 9.2 below). The results can be

expressed per unit of surface area or per sample, especially

for items of irregular conformation. For example, surfaceinoculated frankfurters may be prepared for detection of L.

monocytogenes as whole links washed or rinsed with

diluents, and the results may be expressed per unit surface

area or whole link, if the links are of uniform size.

Alternatively, surface samples may be excised and

homogenized in diluent. The results may be expressed per

unit of surface area or per gram. For example, a spotinoculated leafy green may be sampled by cutting a surface

area surrounding and greater than that inoculated and the

sample can be homogenized or macerated to release

bacterial cells. Some foods, e.g., surface-inoculated whole

tomatoes or melons, may be sampled with a sterile cork

borer, extracting a defined section from an area of the

surface that was inoculated or treated.

Caution should be exercised when considering analysis

of composited samples in challenge studies. Compositing

multiple samples for pathogen enumeration eliminates

detection of variability among discrete samples and may

reduce sensitivity of the analysis. Furthermore, composited

samples may dilute toxins to less than detectable levels if

these toxins are present in only one of the multiple samples.

However, compositing samples before or pooling samples

after an enrichment procedure may be appropriate to

confirm absence of survivors in an inactivation study.

Pooling after enrichment can be used as a screening

procedure that will later allow determination of how many

original samples were positive. Compositing or pooling

approaches must be validated to assure sensitivity is not lost.

9.2. Sample analysis for target pathogens or toxins

The objective of sample preparation for microbial

analysis is to retrieve all microbial spores or cells of interest

(or toxin, where appropriate). Sample preparation should

provide conditions that will allow the metabolic activity to

lead to detectable colonies or other measurements indicating

activity and allow a measurement of survival or growth

levels. It is common to use a 1:10 initial dilution in

Butterfield’s phosphate buffer or buffered peptone water for

vegetative pathogens or spores. However, if the product has

a high salt or sugar content it may be necessary to modify

the dilution buffer to avoid shocking the cells. Enrichment

procedures for the target pathogen should be considered at

time points when levels of survivors are expected, or

previously determined, to be below the experimental limit of

detection by direct plating. Rapid detection methods that

have been validated (see Appendix A) are appropriate when

enumeration is not necessary.

Sample analysis must be done using methods that

permit the accurate and reproducible recovery of microorganisms. In all cases the amount of buffer or diluent used

must be defined and constant among samples, and this level

should be selected based on sample size, level of

contamination expected, and minimum level of detection

desired. The sample preparation protocol and washing,

rinsing, or blending time should be consistent, and the time

between sample processing and plating should be short and

constant for all samples. Sample preparation temperature

and time and conditions and variables involved in sample

preparation should be maintained constant to the extent

possible; these variables include volume or weight, surface

area, composition, and properties (e.g., pH) (12).

For growth studies, pathogens should be enumerated on

appropriate selective agar (see Appendix A). Inactivation

studies may result in injured cells, and direct plating of these

cells onto selective agar can overestimate the extent of

death. In such cases, samples should be prepared and tested

in ways that allow repair and recovery of injured organisms.

Recovery of injured cells can be enhanced by using

nonselective media such as tryptic soy agar (TSA) or plate

count agar overlaid with selective agar after 2 to 4 h of

incubation at optimum temperature (20, 40), by using

selective agar overlaid with nonselective agar (124), by

using agar underlay techniques (60, 61), or by replica

plating from a nonselective agar such as TSA to a selective

agar (100). Standard methods for extraction of C. botulinum

neurotoxins and S. aureus enterotoxins from foods can be

found in the references provided in Appendix A.

9.3. Enumeration of indigenous microbial flora

In addition to inoculated product, sometimes it is also

useful to test corresponding uninoculated control samples to

determine levels of background microflora surviving the

process or changes that occur during product shelf life (53,

91). Moreover, protocols for challenge studies to determine

growth inhibition or inactivation based on product formulation should consider and address potential effects of

naturally occurring microflora on the pathogens of concern.

In addition, spoilage and the end of shelf life are usually

associated with an increase in microbial populations. Thus it

is recommended that microbiological numbers such as

aerobic plate counts and number of spoilage organisms

typical for the product (e.g., LAB or yeasts and molds) be

obtained. Testing for these or other indicator microorganisms cannot substitute for pathogen testing. In addition, the

presence or absence of spoilage bacteria cannot be used as

an indicator of safety.

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LAB are expected in fermented or cultured food

products at relatively high populations (e.g., 6 log CFU/

g), but indigenous populations are low in most processed

foods. This group of bacteria is known to compete well with

low levels of pathogens for nutrients, can grow over a wide

range of temperatures, and can reduce the pH of the food

through acid production, and some strains can produce

bacteriocins that may inhibit some pathogens. Relying on

the presence of naturally occurring background levels of

LAB in foods is an unreliable method to control pathogens.

Conversely, competitive microflora may inhibit growth of

specific pathogens, and failure to account for this interaction

could lead to erroneous conclusions. Thus, it may be

important in some circumstances to monitor LAB growth

during the challenge study to determine if competition may

contribute to inhibition of pathogens during the trial.

Although they may be present, molds and yeasts may

not be initially visible on the food. Deamination of food

proteins by molds can produce ammonia and a localized

increase in pH that can increase the potential for pathogen

growth in that microenvironment (81). Populations of molds

and yeasts can be enumerated by using a variety of selective

plating media or by other validated procedures.

9.4. Determination of physical parameters

Food properties such as proximate composition (protein, fat, and moisture), pH, titratable acidity, aw, salt

content, and residual nitrite can influence the behavior of

pathogens. It may be important to measure these factors as

part of the challenge study. Some parameters, such as pH,

that may change during the study may need to be monitored

at appropriate points throughout the study in parallel with

microbial analysis. Sources of appropriate methods can be

found in Appendix A. The number of samples to be

analyzed is described in section 9.1 above.

Changes in pH can be an indicator of microbial

metabolism when microbial populations are not enumerated

or if growth is not significant. The pH of foods that are

homogeneous and likely have consistent pH throughout the

matrix can be measured on a representative sample. In

contrast, complex foods consisting of multiple discrete

components or ingredients may require multiple pH

measurements. For example, a sandwich may require

measuring the surface or interface pH of the components

in addition to a homogenized sample.

For obvious safety reasons, no sensory assessment

other than changes in appearance (phase separation,

turbidity, texture, and gas formation) should be performed

on challenge test samples. In some instances, the investigator should make a judgment if the product would be

considered ‘‘edible’’ based on visual and olfactory observations. Note that because pathogens or toxins may be

present, olfactory observations may constitute unacceptable

risk to the laboratory worker.

10.0. Duration of study and sampling intervals

Challenge studies should be conducted for at least the

intended shelf life of the product (21, 53, 122). For some

shelf-stable products this may mean holding products for a

year or longer. Ideally, products should be held for some

period beyond the end of the intended shelf life to account

for users who might consume the product past the end of the

declared shelf life and to add an additional margin of safety

(53). Depending on the shelf life of the product, this time

may be 25% (e.g., for products with shelf life of 3 to 6

months) to 50% (e.g., for products with shelf life of 7 to 10

days) longer than the intended shelf life of the food (53, 91).

This additional time may be important for recovery of cells

injured by heat or by antimicrobials in the product. For

some products that still have acceptable sensory properties

at the end of the intended shelf life, it may be important to

continue studies until overt spoilage occurs, as consumers

may consume the product as long as it does not appear

spoiled. Samples held under abuse conditions are unlikely to

last the full shelf life and are usually sampled for shorter

time periods (53). Samples, including controls, should be

analyzed initially after inoculation (in some cases, after a

short equilibration period) and then five to seven times over

the duration of the study (53). For long-shelf-life products, it

may be necessary to have more than seven sampling points.

The sampling interval should be determined based on

prior experience with similar products and in consideration

of the likely duration of survival or rate of growth or

inactivation. Depending on the product characteristics and

expected outcomes for products with a long shelf life, it may

be appropriate to test on a more frequent basis early in the

study (e.g., daily) and at longer intervals later in the study

(53).

A growth inhibition study may be ended when there is

greater than a 1- to 2-log increase in pathogen growth or

when toxin is detected in samples for two consecutive

sampling intervals (indicating growth of the pathogen of

concern) or there is gross spoilage such that the product is

no longer fit for consumption. Care should be taken in

making this determination, because spoilage and apparent

edibility are subjective.

When measuring pathogen inactivation, the study is

typically concluded when the pathogen is no longer

recovered from the product. However, in some cases (e.g.,

thermal death time studies) it may be important to take into

account the possibility of injured cells and to continue

incubation of samples until the end of product shelf life to

verify that injured cells do not recover and grow (91) or

produce toxin in the product over time. Alternatively,

attempts to recover the pathogen in noninhibitory enrichment media after a period of incubation in the product may

be used to verify the absence of survivors.

11.0. Interpreting test results

Interpreting the results of microbiological growth and

inactivation studies requires evaluation by expert microbiologists who will consider all relevant factors (53, 80, 91).

In determining whether a product supports growth of a

pathogen, it is rarely as simple as comparing final and initial

counts. Numbers from different sampling points may vary

due to inherent variation in sampling and enumeration

procedures, particularly when foods contain antimicrobial

compounds that limit growth. It may be difficult to

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determine if changes in numbers are real or due to analytical

variability. In addition, there may be an initial die-off in

some foods following inoculation, and if this die-off is

followed by growth that does not exceed the target inoculum

level, this growth may not be recognized. This issue may be

addressed by allowing a brief time (e.g., 2 h) for the

inoculum in the product to equilibrate prior to conducting

the initial count (53). Normal sample variation may result in

a spike at a sampling interval that may not be significant

(122); this variation can often be addressed through testing

of multiple samples. Graphical representation of the data to

examine trends may be useful in assessing whether actual

growth has occurred (53). This is particularly important in

cases where the data set contains one or more outlying data

points. The interpretation of inconsistent or highly variable

results is an important and complicated issue and should be

done by an expert microbiologist (see Table 1).

An increase in 1 log cycle over two or more time

intervals is generally considered significant by food

microbiologists (122). Smaller increases may be significant

depending upon the enumeration methods, number of

samples and replicates used, and the variability among data

points. Thus, in determining that a product does not support

growth of a pathogen, in general less than a 1-log increase

above the initial inoculum level throughout the intended

shelf life of the product and across replicate trials would be

an appropriate acceptance criterion (53, 91). This level

reflects the inherent variation that exists with enumeration

of microorganisms (53, 103).

Statistical methods can also be used to determine

whether differences in counts at specific sampling points

indicate true growth or are simply due to sampling and

measurement errors. Where the repeatability and reproducibility of the enumeration method have been determined

through validation studies and the standard deviation of

reproducibility can be calculated, a more precise determination of a significant difference may be made. For

example, Agence Franc ̧aise de Se ́curite ́ Santitaire des

Aliments (AFSSA) (13) recommends a 0.5-log CFU/g

increase between initial and final concentrations as

indicating that growth of L. monocytogenes has occurred.

This value is based on an estimation of measurement

uncertainty (55, 57), which is determined by doubling the

reproducibility standard deviation. It should be noted

however that the reproducibility standard deviation can

vary. Scotter et al. (92) conducted tests to validate the

International Organization for Standardization (ISO) method

for enumeration of L. monocytogenes in foods and found

that the reproducibility standard deviation ranged from 0.17

to 0.45 log CFU/g, depending on food product and level of

contamination. Thus, depending on the food, inoculum

level, and method of enumeration, a difference greater than

0.5 log CFU/g may (or may not) be an appropriate criterion.

It should also be noted that statistically significant

differences may not always be biologically relevant. An

expert microbiologist, using available data and past

experience, can best determine if the data represent a trend

of increasing numbers or are simply a product of the

variation seen in enumeration studies (91).

Where studies have been conducted with C. botulinum,

detection of toxins is measured rather than growth, as toxin

can be produced without an increase in number (47). No

toxin should be detected in the product over the duration of

the challenge study (53). In lieu of testing for Staphylococcus enterotoxins, limiting growth of S. aureus to less than 3

log CFU/g may be used (53). This limiting growth level was

based on the assumption that the initial population does not

exceed 3 log CFU/g and that a minimum of 6 log CFU/g is

needed to produce staphylococcal enterotoxins.

Where multiple formulations have been challenged,

growth or toxin production in one formulation but not in

another may provide useful data on the inhibitory properties

of the product with respect to pathogen growth. In this case,

the effect of formulation differences will help to identify

critical factors necessary to control pathogen growth or

toxin production. Similarly, if a product is produced by a

manufacturing process that encompasses the point of

‘‘failure,’’ this is an indication that the manufacturing

variability may be too great to assure the safety of a product

formulated in this manner.

For lethality experiments, log reductions should be

determined in replicate trials. The log reduction should meet

any existing regulatory performance standards that apply to

the food product. Where no performance standard exists, the

lowest log reduction achieved should exceed the expected

contamination level by an amount that incorporates a margin

of safety (a 2-log margin is often used) consistent with the

variability expected in the product and the process (91).

The discussion above indicates that universally acceptable rules for interpreting test results are not available and

points out the need for further consideration to produce clear

guidance on this subject.

12.0. Elements to include in the report

In order for others to assess the adequacy of a

challenge study, it is imperative that the study report

provide appropriate information, including an interpretation of the results. The report should begin with an

introduction that includes the purpose of the study and

reviews the data supporting the experimental design. The

report should include information characterizing the

product and process. The materials and methods should

be described as they would be in a scientific publication.

The results should include both raw and summarized data

and should be clearly presented. Any statistical design and

analysis of results should be thoroughly described. If

statistical analysis was not used, that fact should be clearly

stated and justified. A discussion should provide an

interpretation of the results and any limitations on the

applicability of the data. The conclusions should contain

any recommendations and should indicate the types of

changes in product formulation or processing that could

warrant a new challenge study.

2. What are the appropriate uses of mathematical

growth and inactivation models? Under what

conditions can these models be used as a substitute

for inoculated pack/challenge studies? Of the models

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currently available, which ones are most suitable for

use, and what are the limitations of these models?

Predictive food microbiology is a subdiscipline of food

microbiology that uses models (i.e., mathematical equations) to describe the growth, survival, or inactivation of

microbes in food systems. Mathematical growth and

inactivation models can always be used to help guide the

design of product assessments or challenge studies. In these

cases, the challenge studies will either substantiate (i.e.,

agree with or be conservative with respect to) the model

predictions or show those predictions to be invalid for the

specific product. An example of a conservative model

would be one that predicts a 2-log increase when the

challenge study shows a 1-log increase. Two ideal uses of

predictive models are for narrowing the choices for

treatments to be validated for safety and for choosing the

appropriate challenge microorganisms.

Intrinsic and extrinsic factors (pH, aw, temperature,

etc.) used as inputs for the model should be chosen with

care. The least restrictive parameters determined for the

range of processing conditions should be used. If the

conditions modeled suggest that growth could occur or

that there is limited lethality for the product or process,

then additional studies, product reformulation, or modification of target shelf life would be warranted. If there is

less confidence in the model, then limited challenge

studies may be warranted to verify the prediction from the

model (1).

Caution should be exercised when models alone are

used to make a decision. Use of models requires experience

and judgment, both in modeling and food microbiology.

When models alone are used to make a decision, those

models must be shown to be valid for the food in question

and should take into consideration lot-to-lot variation.

Validation may be based on published or unpublished data

for very similar or identical foods. The data should be

generated by a laboratory having personnel with the

appropriate knowledge, skills, and abilities in conducting

challenge studies (see Table 1) or be obtained from other

relevant published studies.

The two best-known multipathogen multifactor models

available today are the USDA Agricultural Research Service

Eastern Regional Research Center Pathogen Modeling

Program (PMP) (106) and the ComBase Predictor (50),

formerly known as the FoodMicroModel (Table 3). Both of

these modeling programs make predictions for a wide array

of foodborne pathogens and growth factors (temperature,

pH, etc.). Both programs are also based on data collected

primarily in laboratory media rather than foods and do not

always cover the full range of each growth parameter

(Tables 4 and 5). Elements of both models have been

TABLE 3. Examples of mathematical growth and inactivation models and their applicability to different foods

Model name Reference Applicability

American Meat Institute

Foundation Process Lethality

Determination Spreadsheet

4 The model provides meat processors with a science-based validation tool that can be

used to demonstrate the effectiveness of a specific heat process to destroy

microorganisms of concern.

ComBase Predictor 50 ComBase Predictor models are based on observations made in culture media and

comprise a set of 20 growth models, 7 thermal death models, and 2 nonthermal

survival models. Temperature, pH, and aw (usually as a function of NaCl) are the core

factors, but for some organisms, the effect of a fourth factor, such as CO2, nitrite, etc.,

is also featured.

Isothermal-Based Prediction Tool

(IBPT)

104 The software can be used to predict whether Salmonella, E. coli O157:H7, or S. aureus

will grow to a ‘‘level of concern’’ in raw beef and pork products.

Microbial Responses Viewer

(MRV) for ComBase (version

beta 1)

78 The MRV is a new database consisting of microbial growth–no growth data derived

from ComBase. The software allows the user to rapidly view growth–no growth

contour plots superimposed with actual ComBase data. Contours of any two of three

variables (temperature, pH, and aw) can be visualized while the third is held constant.

Opti.Form Listeria Control Model

2007

85 The model predicts Listeria growth for both uncured and cured cooked meat products.

The model will help to calculate the level of lactate and diacetate needed to control

Listeria in cured and uncured cooked meat and poultry products for their required

shelf life.

Pathogen Modeling Program

(PMP)

106 This predictive microbiology application was designed as a research and instructional

tool for estimating the effects of multiple variables on the growth, inactivation, or

survival of foodborne pathogens. Most of the models are based on experimental data

of microbial behavior in liquid microbiological media.

Perfringens Predictor 51 Perfringens Predictor provides a prediction of growth of C. perfringens during the

cooling of meats. This model is part of ComBase Predictor and may give more

accurate predictions than the C. perfringens model included in PMP (89, 97).

Seafood Spoilage and Safety

Predictor (SSSP, version 3.0)

77 Software includes models for relative rates of spoilage, models for growth of spoilage

bacteria in specific seafood, models to predict histamine formation by Morganella

spp., a model to predict the simultaneous growth of L. monocytogenes and lactic acid

bacteria in lightly preserved seafood, and a model to predict the growth boundary of

L. monocytogenes in lightly preserved seafood.

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validated (by both published and unpublished studies) to a

limited degree in different food systems.

There is also a wide array of computer models

developed in laboratory media and food systems that are

not part of PMP and ComBase. Examples of several models

are shown in Table 3. Some models published in the

scientific literature are not available in a user-friendly,

downloadable form. These models require some modest

modeling or spreadsheet manipulation skills on the part of

the user to produce a useful prediction.

Any discussion of modeling and validation of models

would be remiss if it did not also mention another tool that

is part of the ComBase Modeling Toolbox: the ComBase

browser (48). The ComBase browser provides access to

the ComBase database of microbial responses to food

environments. At the present time the database includes

more than 35,000 observations, of which more than

13,000 are from food and the balance (,22,000) are from

culture media. Researchers publishing microbial growth or

survival data are requested and encouraged to submit the

data to ComBase (49). The data contained in ComBase

may represent a useful source of published and unpublished data for validating models.

3. What are the limitations for applying the results of

an inoculated pack/challenge study conducted on

one food to another similar food?

Challenge studies on one product may sometimes be

applicable to other products. However, if there are significant

differences between the intrinsic properties of the product and

those of the food on which the challenge study was conducted,

the results of the challenge study may not be applicable. If the

challenge study is conducted using parameters or conditions

more conducive to growth or survival than those in the food

product under consideration, then additional challenge studies

may not be needed (76). For example, the results of a challenge

study for a specific pathogen in a product formulation with a

pH of 5.8 could be applied to a similar formulation where the

primary difference is a pH of 5.4. Nevertheless, an expert

microbiologist should make the determination of applicability

of one challenge study to additional products. The composition

of the two foods, e.g., protein content, carbohydrate source,

type of organic acid, fat, and moisture, should be considered in

determining the applicability of one study to another product.

Generally, the more similar the composition the more likely the

study will apply.

TABLE 4. Pathogen growth ranges used in ComBase and Pathogen Modeling Programsa

Pathogen

ComBase PMP

Temp (uC) pH

aw

(minimum)

Temp (uC) pH

aw

Minimum Maximum Minimum Maximum Minimum Maximum Minimum Maximum (minimum)

B. cereus

With CO2 5 34 4.9 7.5 0.974

Aerobic 5 42 4.7 7.5 0.97

Anaerobic 10 42 5.0 9.0 0.97

C. botulinum

(growth only)

Proteolytic 14 40 4.7 7.2 0.954 15 34 5.0 7.2 0.977

Nonproteolytic 4 30 5.1 7.5 0.974 5 28 5.0 7.0 0.977

C. perfringens 15 52 5 8 0.971 19 37 6.0 6.5 0.983

E. coli O157:H7

With CO2 10 30 4.5 7 0.961

Aerobic 5 42 4.5 8.5 0.97

Anaerobic 5 42 4.5 8.5 0.97

L. monocytogenes

With CO2 1 35 4.4 7.5 0.934

Aerobic 4 37 4.5 7.5 0.928

Anaerobic 4 37 4.5 8.0 0.97

S. aureus

(growth only)

Not specified 7.5 30 4.4 7.1 0.907

Aerobic 10 42 4.5 9.0 0.911

Anaerobic 12 42 5.3 9.0 0.872

Salmonella

With CO2 7 30 3.9 7.4 0.973

Aerobic 10 30 5.6 6.8 0.974

a Limits tested in ComBase (48) and the Pathogen Modeling Program (PMP) (106) do not necessarily represent limits for growth. See

Table 5 for growth limits.

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4. Of the existing inoculated pack/challenge study

protocols, e.g., those published by the American

Bakers Association, NSF International, and others,

which are most suitable for application to a wide

variety of foods, and what are the limitations of these

protocols? Are there existing protocols that are

appropriate for specific food-pathogen pairs?

The committee agrees with an earlier assessment in the

IFT report (53) indicating that both the American Bakers

Association (ABA) and the National Sanitation Foundation

International (NSF) testing protocols suffer from significant

weaknesses. These are briefly highlighted below; for more

details, see Table 2 in the IFT report (53) comparing the

NSF, ABA, and expert panel’s protocols.

The NSF protocol provides test methods for determining

that a product does not require refrigeration for safety. The

NSF protocol lacks flexibility and is highly prescriptive in

specifying microbial strains and methods. It applies to a

limited number of products (breads and pastries with

vegetables or soft cheeses added prior to baking; bakery

products filled or topped with cream, cre`me, custard, or cheese

after baking; products filled prior to baking, such as pumpkin,

sweet potato, custard, or meringue pies; and toppings, glazes,

icings, or fillings stored without temperature control) and

excludes a number of products of potential concern (e.g.,

modified-atmosphere-packaged products, all products with a

pH , 4.6, and products stored without temperature control for

less than 24 h or more than 31 days). The aw and pH are the

only criteria for selection of challenge test organisms, with no

consideration of the process given the product in selecting

appropriate organisms. In addition, there is no consideration

given to challenge tests with C. botulinum, only with C.

perfringens. The recommendations would result in unnecessary and sometimes inappropriate challenge tests. There is no

consideration for the need to adapt the inoculum, and the

inoculum size is fixed for all products. The protocol does take

into consideration the need to inoculate different components

and interfaces of multicomponent products and requires

testing of duplicate samples per time point with multiple lots

of products. Overall, the protocol has significant limitations,

even for application to the intended products.

The ABA protocol (Industry Protocol for Establishing

the Shelf Stability of Pumpkin Pie) is even more limited in

scope (i.e., applies only to pumpkin pie intended for

distribution and display without refrigeration). The objective

of this protocol is to define the process that a manufacturer

can use to demonstrate the shelf stability of a pumpkin pie

product in accordance with the then current edition of the

FDA Food Code. This protocol is not an inoculated

challenge study but rather a method for validating a cooking

procedure (product reaches at least 82.2uC [180uF] at the

coolest point) with respect to the destruction of naturally

occurring microorganisms, both pathogenic and nonpathogenic. However, the absence of a pathogen in such a study

cannot be relied on to assess whether or not a pathogen

would grow if present in the product, because such a

pathogen may or may not have been present initially.

Additionally, monitoring the oxidation-reduction potential

in the product to ascertain whether C. botulinum would

grow and produce toxin is inadequate to make such a

determination. Thus, the protocol has significant limitations,

even for application to the intended product.

TABLE 5. Limits for growth when other conditions are near optimum based on references (54) and (116)

Pathogen Sourcea

Temp (uC) pH

aw

(minimum)

Maximum water

Minimum Maximum Minimum Maximum phase NaCl (%)

B. cereus FDA 4 55 4.3 9.3 0.92 10

ICMSF 4 55 5.0 8.8 0.93

C. botulinum (growth only,

proteolytic)

FDA 10 48 4.6 9 0.93 10

ICMSF 10–12 4.6 0.93 10

C. botulinum (growth only,

nonproteolytic)

FDA 3.3 45 5 9 0.97 5

ICMSF 3.3 5.0 0.97 5

C. perfringens FDA 10 52 5 9 0.93 7

ICMSF 12 50 5.5–5.8 8.0–9.0 0.97

Pathogenic E. coli FDA 6.5 49.4 4 9 0.95 6.5

ICMSF 7–8 44–46 4.4 9.0 0.95

E. coli O157:H7 ICMSF 8 44–45 4.5 Slow growth at 6.5,

no growth at 8.5

L. monocytogenes FDA 20.4 45 4.4 9.4 0.92 10

ICMSF 20.4 45 4.39 9.4 0.92

S. aureus (growth only) FDA 7 50 4 10 0.83 20

Aerobic conditions ICMSF 7 48 4 10 0.83

Anaerobic conditions ICMSF 5.0 0.90

Salmonella FDA 5.2 46.2 3.7 9.5 0.94 8

ICMSF 5.2b 46.2 3.8 9.5 0.94

a FDA, U.S. Food and Drug Administration (116); ICMSF, International Commission on Microbiological Specifications for Foods (54). b Most serovars will not grow below 7uC (44.6uF).

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The IFT expert panel report (53) is written to

encompass a wide variety of foods. The guidelines provide

a framework for determining whether foods need TCS. The

document also describes guidelines for challenge tests for

determining the ability of a food to support the growth of

one or more pathogens, but it does not address inactivation

challenge tests. The guidelines provide flexibility but result

in a potential for different interpretations as to what is

appropriate for specific food types. This makes it more

difficult for those reviewing and evaluating the data to

determine if the study itself was adequate, and thus the

reviewer may need to have technical expertise for the

assessment. This is a weakness inherent to any document

that is designed to apply to a broad range of food types.

Notermans et al. (80) developed a user’s guide to

microbial challenge testing for food safety and stability. The

document addresses selecting the appropriate microorganism, preparing the inoculum, inoculum size, inoculation

procedure, duration of the study, and sampling times. The

recommendations are generally consistent with those in this

NACMCF document, although less detailed. As with the

IFT expert panel report (53), technical expertise may be

required to interpret the adequacy of studies following these

guidelines.

Scott et al. (91) published guidelines for conducting L.

monocytogenes challenge tests for foods. Their article

covers guidelines for studies to evaluate both the ability of

a food to support the growth of L. monocytogenes and the

inactivation of the organism in a food. The article in large

part applies the recommendations in the IFT report (53) to

challenge studies involving L. monocytogenes and is thus

specific to a single organism. The protocols are also limited

to those food products in which growth or inactivation of L.

monocytogenes is a concern. The protocols in general are

consistent with those in the present document and are

appropriate for L. monocytogenes in refrigerated ready-toeat foods.

AFSSA, a European Union (EU) community reference

laboratory for L. monocytogenes, has recently published a

technical guidance document for conducting shelf-life

studies to determine compliance with microbiological

criteria for L. monocytogenes in ready-to-eat foods set

out in EC regulation No. 2073/2005 (13). Similar to Scott

et al. (91), the scope is limited to L. monocytogenes,

including information on how to conduct experiments of

the shelf life in naturally contaminated and artificially

contaminated ready-to-eat products. The document includes determination of shelf life in naturally contaminated

foods, called durability studies, which are not addressed in

this NACMCF document. The AFSSA document also

provides information on how to interpret the results

obtained against EU L. monocytogenes regulatory criteria

in ready-to-eat foods (no more than 100 CFU/g at end of

shelf life). The document does not address inactivation of

L. monocytogenes but does address many of the same key

points as this NACMCF document, such as taking into

account the product characteristics, batch variability, use

of multiple strains, adapting the challenge organisms, and

simulating natural conditions when inoculating product.

The protocol indicates that to assess growth potential

samples need be taken only initially and at the end of the

shelf life and that for homogeneous products enumeration

of only one sample is needed (three samples for

heterogeneous products) at each of these time points.

(More sampling times are recommended for studies

intended to assess maximum growth rate or lag time.)

The methods described in the AFSSA document are

appropriate for L. monocytogenes in refrigerated ready-toeat foods; however, the acceptance criteria differ from

those proposed here.

In several documents, NACMCF has provided guidance for conducting microbial challenge tests. In 1990,

NACMCF (74) made recommendations for extended-shelflife refrigerated cooked meat and poultry products that

included appendices on guidelines for thermal inactivation

studies using L. monocytogenes and for C. botulinum

inoculation studies. Those recommendations are generally

consistent with this NACMCF document. Although the

approaches used in the 1990 document are not specific to

refrigerated meat and poultry, they are specific for the

individual organism for which the guidance was developed.

The protocols are appropriate for their intended use.

In 2005, NACMCF (75) published an article on

considerations for establishing safety-based consume-by

date labels for refrigerated ready-to-eat foods; the appendix

to that document contained guidance for conducting

microbial challenge studies to validate the safety-based

use-by date label. This guidance was specific for L.

monocytogenes and is consistent with the guidance in this

NACMCF document. The protocol is appropriate for its

intended application (validation of a use-by date).

There are a number of good challenge test protocols

useful for specific purposes. This document and the IFT

report (53) are the most comprehensive, broad-based

documents that can be applied to assess the adequacy of

microbial challenge studies. Because these documents are

not specific to a food category, technical expertise may be

needed to assess the adequacy of the challenge study with

respect to appropriateness of the challenge organism,

storage temperatures, etc. However, a well-written report

should provide the rationale for many of the choices, thus

assisting in the review to determine study adequacy.

5. Develop a decision tree to aid in the design of an

appropriate inoculated pack/challenge study. Test or

‘‘desk check’’ the decision tree using the following

five foods: meat-filled puff pastry, (baked) cheese

pizza, chopped lettuce, cheese (blocks or slices), and

lemon meringue pie.

Due to the complexity of decisions needed, the

committee concluded that a decision tree could not be

developed. Instead, the committee developed a template

containing a series of questions to facilitate the design of an

appropriate challenge study. The template was validated

using five food products (Appendices E through J).

The examples in Appendices E through J were

developed to illustrate the thought processes that expert

microbiologists use in approaching the design of microbial

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 157

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challenge tests. These examples should not be considered

complete or accurate with respect to all parameters.

Moreover, other approaches to conducting the challenge

studies may be applied. The pass-fail criteria used in the

examples represent expert opinion and may need to be

verified with the appropriate regulatory agency.

6. Identify the basic knowledge, skills, education,

training, experience, and abilities necessary for a

multidisciplinary work group or individual to be

qualified to design, conduct, and evaluate an

inoculated pack/challenge study and the pursuant

results.

Refer to Table 1, Question 1, Section 1.0 and Appendix

B for this information.

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Bernard. 2001. Heat resistance of Salmonella typhimurium and

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meat and poultry products. Appendix A. Compliance guidelines for

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meat and poultry products. Appendix B. Compliance guidelines for

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of beef jerky. Food Microbiol. 22:423–431.

126. Zhao, I., T. J. Montville, and D. W. Schaffner. 2000. Inoculum size

of Clostridium botulinum 56A spores influences time-to-detection

and percent growth-positive samples. J. Food Sci. 65:1369–1375.

APPENDIX A. SOURCES OF ACCEPTED

LABORATORY METHODS*

N American Public Health Association. 2001. Compendium of

methods for the microbiological examination of foods, 4th ed.

F. P. Downes and K. Ito (ed.). American Public Health

Association, Washington, DC. (5)

N American Public Health Association. 2004. Standard methods

for the examination of dairy products, 17th ed. H. M. Wehr and

J. H. Frank (ed.). American Public Health Association,

Washington, DC. (6)

N AOAC International. 2007. Official methods of analysis, 18th

ed., rev. 2, W. Horwitz and G. Latimer, Jr. (ed.). AOAC

International, Gaithersburg, MD. (9)

N Health Canada. 2008. The Compendium of analytical methods,

vols. 1–5. Available at: http://www.hc-sc.gc.ca/fn-an/res-rech/

analy-meth/microbio/index-eng.php. Accessed 18 December

2008. (44)

N International Organization for Standardization. 2009. General

methods of tests and analysis for food products. ICS 67.050.

Listing of standards available at: http://www.iso.org/iso/

catalogue_ics_browse?ICS1~67&ICS2~050&. Accessed 15

June 2009. (56)

N U.S. Department of Agriculture, Food Safety and Inspection

Service. 1998. Microbiology laboratory guidebook, 3rd ed., B.

P. Dey and C. P. Lattuada (ed.) Available at: http://www.fsis.

usda.gov/Science/Microbiological_Lab_Guidebook/index.asp.

Accessed 18 December 2008. (107)

N U.S. Food and Drug Administration. 2001. Bacteriological analyticalmanual.Availableat:http://www.fda.gov/Food/ScienceResearch/

LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.

htm. Accessed 15 June 2009. (115)

APPENDIX B. CONSIDERATIONS FOR

SELECTING A LABORATORY

Note: The following questions may be useful in comparing

the capabilities of different laboratories. The questions are not

listed in order of importance. A negative response to one or more

of these questions does not necessarily disqualify a laboratory from

consideration. The most important considerations are associated

with qualifications of personnel for designing, conducting, and

evaluating challenge studies.

N Does the microbiologist in charge have experience performing

challenge studies including the food types you want to study? If

so, ask the laboratory to provide examples of the types of

challenge studies performed recently.

N What is the academic education and training of the microbiologist supervising the laboratory operations?

N What is the academic education and training of technicians

performing the laboratory experiments?

N Is the laboratory audited periodically or accredited by an

independent third party? If so, ask the laboratory to provide a

copy of certificates documenting the audit. If not, ask how the

laboratory ensures the quality of the processes and results, e.g.,

appropriate positive and negative controls, and a written,

implemented quality control system for the laboratory operations, including a corrective action plan. ISO17025 certification

is an example of a third party audit that would verify many of the

good laboratory practices that should be implemented. Accreditations and certifications do not necessarily qualify a laboratory

to design and conduct microbiological challenge studies. It is

important to confirm that the laboratory has the experience and

expertise necessary to perform the challenge studies

N Does the laboratory use approved, validated, or widely accepted

published methods for the requested analyses? If so, what are

the references for the methods used?

N Does the laboratory use certified reference materials (e.g.,

traceable positive controls) and standards (e.g., NIST calibrated

equipment), where applicable, to perform the requested tests?

N Does the laboratory use subcontractors to perform the analyses

in question? If so, how does the primary laboratory ensure the

subcontract laboratory produces valid results?

N If the protocol involves inoculation with a foodborne pathogen,

does the laboratory have appropriate biological safety containment and practices?

N Does the laboratory possess microbial strains that are appropriate

for the food to be challenged? How are the stocks maintained and

verified for purity and identity prior to the start of the study?

N If the protocol involves testing for a select agent (e.g.,

Clostridium botulinum or botulinum toxin), is the laboratory

approved to work with that particular agent? In the United

States, laboratories must be approved to work with each select

agent on which they perform tests or research.

* Dates of references current as of publication. Use most current version

available.

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APPENDIX D. FDA 2005 MODEL FOOD CODE

DEFINITIONS MOST RELEVANT

TO CHALLENGE STUDIES

The following definitions were extracted from the 2005 FDA

Food Code (117). Note: all paragraph and section references within

definitions refer to paragraphs and sections in the 2005 FDA Food

Code.

‘‘aw’’ means water activity, which is a measure of the free moisture

in the food that is available for microbial growth. It is the

quotient of the water vapor pressure of the substance divided by

the vapor pressure of pure water at the same temperature, and is

indicated by the symbol aw.

‘‘Consumer’’ means a person who is a member of the public, takes

possession of food, is not functioning in the capacity of an

operator of a food establishment or food processing plant, and

does not offer the food for resale.

‘‘Critical control point’’ means a point or procedure in a specific

food system where loss of control may result in an unacceptable

health risk.

Food establishment

(1) ‘‘Food establishment’’ means an operation that:

(a) stores, prepares, packages, serves, vends directly to the

consumer, or otherwise provides food for human consumption, such as a restaurant, satellite, or catered feeding

location; catering operation if the operation provides food

directly to a consumer or to a conveyance used to transport

people; market; vending location; conveyance used to

transport people; institution; or food bank; and

APPENDIX C. PATHOGENS OF CONCERN AND CONTROL METHODS FOR VARIOUS PRODUCT

CATEGORIES THAT MAY NEED A CHALLENGE STUDY (GROWTH INHIBITION, INACTIVATION,

OR COMBINATION) a

Product categoryb (examples)

Pathogens of concern

(alphabetical order)

Examples of process controlc (alone and in

combination, alphabetical order)

Meat and poultry: cooked (e.g., roast beef,

deli-style turkey, ham)

Clostridium botulinum and C. perfringens,

enterohemorrhagic E. coli, L.

monocytogenes, Salmonella,

Staphylococcus aureus

Cooling rate, heat treatment,d high pressure

processing, preservatives, storage timetemperature

Meat and poultry: dried and/or fermented

(e.g., fermented sausage, jerky, dry

cured ham)

C. botulinum, C. perfringens,

enterohemorrhagic E. coli, L.

monocytogenes, Salmonella, S. aureus

aw, drying, fermentation, heat treatment,

humidity, nitrites and other preservatives,

pH salting, storage time-temperature,

water-phase salt

Fish and seafood (e.g., smoked fish,

fresh oysters, pickled herring, pasteurized

crab meat)

B. cereus, C. botulinum, L. monocytogenes,

Salmonella, Shigella spp., S. aureus,

Vibrio cholerae, V. vulnificus, V.

parahaemolyticus

aw, drying, harvest site control, heat treatment,

high-pressure processing, nitrites, pH,

preservatives, salting, storage timetemperature, water-phase salt

Cultured dairy products, pH # 4.7

(e.g., yogurt, sour cream, buttermilk)

Enterohemorrhagic E. coli, Salmonella,

L. monocytogenes, S. aureus

Heat treatment, pH, preservatives, rate of acid

production, starter culture activity, storage

time-temperature

Cultured dairy products, pH . 4.7 to #5.4

(e.g., cottage cheese)

B. cereus, C. botulinum, enterohemorrhagic

E. coli, L. monocytogenes, Salmonella,

S. aureus

Heat treatment, hot fill, preservatives, storage

time-temperature

Cheese and cheese products (e.g., natural

Swiss cheese, processed cheese slices,

processed cheese spread)

C. botulinum, enterohemorrhagic E. coli,

L. monocytogenes, Salmonella, Shigella

spp., S. aureus

aw, emulsifiers, heat treatment, hot fill,

moisture content, pH, preservatives,

storage time-temperature

Butter and margarine (e.g., light salted

butter, whipped butter)

L. monocytogenes, S. aureus, Yersinia

enterocolitica

aw, heat treatment, moisture droplet size in the

water-in-oil emulsion, water-phase salt

Eggs and egg products (e.g., meringue, pooled

pasteurized egg yolks, sliced boiled eggs)

B. cereus, L. monocytogenes, Salmonella Heat treatment, preservatives, storage timetemperature

Fruits and vegetables (e.g., peeled carrots,

chopped lettuce)

B. cereus, C. botulinum, enterohemorrhagic

E. coli, L. monocytogenes, Salmonella,

Shigella spp., Y. enterocolitica

Heat treatment, storage time-temperature,

wash water sanitizers

Fats, oils, and condiments (e.g., garlic

in oil)e

B. cereus, C. botulinum, S. aureus,

Salmonella

aw, heat treatment, pH, preservatives, salt,

storage time-temperature

Acidified sauces, salad dressings, and

salsas

Enterohemorrhagic E. coli, Salmonella,

S. aureus

Heat treatment, pH, storage time-temperature,

titratable acidity

High aw syrups (e.g., light maple syrup) C. botulinumf Acidification (light syrups), aw, heat

treatment, preservatives

Confectionery products (e.g., chocolate products) Salmonella aw, heat treatment

Cereal grains and related products (e.g., fresh

pasta, cooked rice)

B. cereus, C. botulinum, Salmonella, S.

aureus

aw, heat treatment, pH, preservatives, storage

time-temperature

a Adapted from IFT report (53) Tables 4-1 and 6-1. b Combinations of products, storage in modified atmosphere, and use of novel preservatives or processes require special consideration. c Good agricultural practices where appropriate and good manufacturing practices and HACCP principles would help in reducing the

hazards.

d Heat treatment includes processes such as cooking, pasteurization, and other thermal processes intended to inactivate pathogens. e Only a concern in anoxic environments.

f Only a concern in light syrups and can be controlled by acidification.

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(b) relinquishes possession of food to a consumer directly

or indirectly through a delivery service such as home

delivery of grocery orders or restaurant takeout orders

or delivery service that is provided by common

carriers.

(2) ‘‘Food establishment’’ includes:

(a) an element of the operation such as a transportation

vehicle or a central preparation facility that supplies a

vending location or satellite feeding location unless the

vending or feeding location is permitted by the regulatory

authority; and

(b) an operation that is conducted in a mobile, stationary,

temporary, or permanent facility or location; where

consumption is on or off the premises; and regardless of

whether there is a charge for the food.

(3) ‘‘Food establishment’’ does not include:

(a) an establishment that offers only prepackaged foods that

are not potentially hazardous (time/temperature control for

safety) foods;

(b) a produce stand that offers only whole, uncut fresh fruits

and vegetables;

(c) a food processing plant including those that are located on

the premises of a food establishment;

(d) a kitchen in a private home if only food that is not

potentially hazardous (time-temperature control for safety

food) is prepared for sale or service at a function such as a

religious or charitable organization’s bake sale if allowed

by law and if the consumer is informed by a clearly visible

placard at the sales or service location that the food is

prepared in a kitchen that is not subject to regulation and

inspection by the regulatory authority;

(e) an area where food that is prepared as specified in

Subparagraph (3)(d) of this definition (above) is sold or

offered for human consumption;

(f) a kitchen in a private home, such as a small family

daycare provider; or a bed-and-breakfast operation that

prepares and offers food to guests if the home is owner

occupied, the number of available guest bedrooms does

not exceed 6, breakfast is the only meal offered, the

number of guests served does not exceed 18, and the

consumer is informed by statements contained in

published advertisements, mailed brochures, and placards

posted at the registration area that the food is prepared in a

kitchen that is not regulated and inspected by the

regulatory authority; or

(g) a private home that receives catered or home-delivered

food.

Food processing plant

(1) ‘‘Food processing plant’’ means a commercial operation that

manufactures, packages, labels, or stores food for human

consumption and provides food for sale or distribution to other

business entities such as food processing plants or food

establishments.

(2) ‘‘Food processing plant’’ does not include a food establishment.

‘‘HACCP plan’’ means a written document that delineates the

formal procedures for following the hazard analysis and critical

control point principles developed by The National Advisory

Committee on Microbiological Criteria for Foods.

‘‘Hazard’’ means a biological, chemical, or physical property that

may cause an unacceptable consumer health risk.

Packaged

(1) ‘‘Packaged’’ means bottled, canned, cartoned, securely

bagged, or securely wrapped, whether packaged in a food

establishment or a food processing plant.

(2) ‘‘Packaged’’ does not include a wrapper, carry-out box, or

other nondurable container used to containerize food with the

purpose of facilitating food protection during service and

receipt of the food by the consumer.

Potentially hazardous food (time/temperature

control for safety food)

(1) ‘‘Potentially hazardous food’’ (time/temperature control for

safety food) means a food that requires time/temperature

control for safety (TCS) to limit pathogenic microorganism

growth or toxin formation.

(2) ‘‘Potentially hazardous food’’ (TCS food) includes:

(a) an animal food that is raw or heat treated; a plant food that is

heat treated or consists of raw seed sprouts, cut melons, cut

tomatoes, or mixtures of cut tomatoes that are not modified

in a way so that they are unable to support pathogenic

microorganism growth or toxin formation or garlic-in-oil

mixtures that are not modified in a way that results in

mixtures that do not support pathogenic microorganism

growth or toxin formation; and

(b) except as specified in Subparagraph (3)(d) of this

definition (below), a food that because of the interaction

of its aw and pH values is designated as Product

Assessment Required (PA) in the Food Code Table A or

Table B of this definition:

(3) Potentially hazardous food (TCS food) does not include:

(a) an air-cooled hard-boiled egg with shell intact or an egg

with shell intact that is not hard boiled but has been

pasteurized to destroy all viable salmonellae;

(b) a food in an unopened hermetically sealed container that is

commercially processed to achieve and maintain commercial sterility under conditions of nonrefrigerated storage

and distribution;

(c) a food that because of its pH or aw value or the interaction of

aw and pH values is designated as a non-PHF and non-TCS

food in Table A or Table B of this definition;

(d) a food that is designated as Product Assessment Required

(PA) in Table A or Table B of this definition and has

undergone a Product Assessment showing that the

growth or toxin formation of pathogenic microorganisms

that are reasonably likely to occur in that food is

precluded due to

(i) intrinsic factors including added or natural characteristics of the food such as preservatives, antimicrobials,

humectants, acidulants, or nutrients;

(ii) extrinsic factors including environmental or operational factors that affect the food such as packaging,

modified atmosphere such as reduced oxygen

packaging, shelf life and use, or temperature range

of storage and use; or

(iii) a combination of intrinsic and extrinsic factors; or

(e) a food that does not support the growth or toxin formation of

pathogenic microorganisms in accordance with one of the

Subparagraphs (3)(a)–(3)(d) of this definition (above) even

though the food may contain a pathogenic microorganism or

chemical or physical contaminant at a level sufficient to

cause illness or injury.

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Ready-to-eat food

(1) ‘‘Ready-to-eat food’’ means food that

(a) is in a form that is edible without additional preparation to

achieve food safety, as specified under one of the

following parts of the Food Code: Paragraph 3-

401.11(A) or (B), Section 3-401.12, or Section 3-402.11,

or as specified in Paragraph 3-401.11(C); or

(b) is a raw or partially cooked animal food and the consumer

is advised as specified in the Food Code, Subparagraphs 3-

401.11(D)(1) and (2); or

(c) is prepared in accordance with a variance that is granted as

specified in the Food Code, Subparagraphs 3-401.11(D)

and (3); and

(d) may receive additional preparation for palatability or

aesthetic, epicurean, gastronomic, or culinary purposes.

(2) ‘‘Ready-to-eat food’’ includes:

(a) raw animal food that is cooked as specified in the Food

Code under Section 3-401.11 or 3-401.12, or frozen as

specified under Section 3-402.11;

(b) raw fruits and vegetables that are washed as specified in

the Food Code under Section 3-302.15;

(c) fruits and vegetables that are cooked for hot holding as

specified in the Food Code under Section 3-401.13;

(d) all potentially hazardous food (TCS food) that is cooked to

the temperature and time required for the specific food in

the Food Code under Subpart 3-401 and cooled as

specified under Section 3-501.14;

(e) plant food for which further washing, cooking, or other

processing is not required for food safety and from which

rinds, peels, husks, or shells, if naturally present, are removed;

(f) substances derived from plants such as spices, seasonings,

and sugar;

(g) a bakery item such as bread, cakes, pies, fillings, or icing

for which further cooking is not required for food safety;

(h) the following products that are produced in accordance

with USDA guidelines and that have received a lethality

treatment for pathogens: dry fermented sausages such as

dry salami or pepperoni; salt-cured meat and poultry

products such as prosciutto ham, country cured ham, and

Parma ham; and dried meat and poultry products such as

jerky or beef sticks; and

(i) foods manufactured as specified in 21 CFR Part 113

‘‘Thermally Processed Low-Acid Foods Packaged in

Hermetically Sealed Containers.’’

Reduced oxygen packaging

(1) ‘‘Reduced oxygen packaging’’ means:

(a) the reduction of the amount of oxygen in a package by

removing oxygen, displacing oxygen and replacing it with

another gas or combination of gases, or otherwise controlling

the oxygen content to a level below that normally found in

the atmosphere (approximately 21% at sea level); and

(b) a process as specified in Subparagraph (1)(a) of this

definition (above) that involves a food for which the

hazards Clostridium botulinum or Listeria monocytogenes

require control in the final packaged form.

(2) ‘‘Reduced oxygen packaging’’ includes:

(a) vacuum packaging, in which air is removed from a

package of food and the package is hermetically sealed so

that a vacuum remains inside the package;

(b) modified atmosphere packaging, in which the atmosphere

of a package of food is modified so that its composition is

different from air but the atmosphere may change over

time due to the permeability of the packaging material or

the respiration of the food (modified atmosphere packaging includes reduction in the proportion of oxygen, total

replacement of oxygen, or an increase in the proportion of

other gases such as carbon dioxide or nitrogen);

(c) controlled atmosphere packaging, in which the atmosphere

of a package of food is modified so that until the package is

opened, its composition is different from air, and continuous

control of that atmosphere is maintained, such as by using

oxygen scavengers, or a combination of total replacement of

oxygen, nonrespiring food, and impermeable packaging

material;

TABLE A. Interaction of pH and aw for control of spores in food that has been heat treated to destroy vegetative cells and then packageda

aw values

pH values:

#4.6 .4.6–5.6 .5.6

#0.92 Non-PHF, non-TCS food Non-PHF, non-TCS food Non-PHF, non-TCS food

.0.92–0.95 Non-PHF, non-TCS food Non-PHF, non-TCS food PA

.0.95 Non-PHF, non-TCS food PA PA

a PHF, potentially hazardous food; TCS food, time/temperature control for safety food; PA, product assessment required.

TABLE B. Interaction of pH and aw for control of vegetative cells and spores in food that has not been heat treated or has been heat

treated but not packageda

aw values

pH values:

,4.2 4.2–4.6 .4.6–5.0 .5.0

,0.88 Non-PHF, non-TCS food Non-PHF, non-TCS food Non-PHF, non-TCS food Non-PHF, non-TCS food

0.88–0.90 Non-PHF, non-TCS food Non-PHF, non-TCS food Non-PHF, non-TCS food PA***

.0.90–0.92 Non-PHF, non-TCS food Non-PHF, non-TCS food PA PA

.0.92 Non-PHF, non-TCS food PA PA PA

a PHF, potentially hazardous food; TCS food, time/temperature control for safety food; PA, product assessment required.

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(d) cook chill packaging, in which cooked food is hot filled

into impermeable bags that have the air expelled and are

then sealed or crimped closed, and the bagged food is

rapidly chilled and refrigerated at temperatures that

inhibit the growth of psychrotrophic pathogens; or

(e) sous vide packaging, in which raw or partially cooked

food is placed in a hermetically sealed, impermeable bag,

cooked in the bag, rapidly chilled, and refrigerated at

temperatures that inhibit the growth of psychrotrophic

pathogens.

‘‘Regulatory authority’’ means the local, state, or federal

enforcement body or authorized representative having jurisdiction over the food establishment.

‘‘Risk’’ means the likelihood that an adverse health effect will

occur within a population as a result of a hazard in a food.

‘‘Variance’’ means a written document issued by the regulatory

authority that authorizes a modification or waiver of one or more

requirements of this code if, in the opinion of the regulatory

authority, a health hazard or nuisance will not result from the

modification or waiver.

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 165

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APPENDIX E. FOOD PRODUCT CHECKLIST: MOZZARELLA SLICES

Evaluation of Mozzarella slices packaged under modified atmosphere packaging and stored at ambient temperatures for up to 2 wk

to enhance sales

Considerations Response Additional comments

1 Determine the purpose of the study

1.a Exempt from time/temperature

control for safety (no refrigeration

required)

NAa

1.b Variance from any regulatory

requirements (e.g., holding for

.4 h without temperature control)

Extended out-of-refrigeration storage of

modified-atmosphere or vacuum-packaged

Mozzarella slices for 2 wk; Food Code

variance.

1.c Validate lethality NA (used pasteurized milk in production of

cheese).

1.d Verify that formulation will inhibit

microbial growth in refrigerated

foods or under mild temperature

abuse

NA

2 Collect information regarding the product

2.a What are the ingredients? Cheese (pasteurized milk, salt, rennet, starter

cultures)

2.a.1 How consistent are the

ingredients from various sources,

lot to lot?

Ingredients same or similar lot to lot; pH,

moisture, salt can vary slightly but in

accordance with Standard of Identity (SOI)

as defined in 21 CFR 133.155–158 (119).

2.a.2 What are the pH, aw, and

proximate analysis (moisture,

salt, fat, protein, residual nitrite,

etc.) for product and/or

individual components?

pH 5.3–5.4; aw 0.96; proximate analysis at

end of production, 46–52% moisture, 1.0%

NaCl, 30% fat. Homogeneous throughout.

Note: aw is not measured or controlled

in typical production but is a function

of moisture and salt content; moisture

is limited by SOI. Starter culture

activity (acid development, measured

by pH) is a critical control point.

2.a.3 Do any of these values change

from preparation to

consumption?

Once the cheese is sliced and packaged, the

pH may increase from 5.4 to 5.9 during

refrigerated storage over a 3-mo period if

lactic acid bacteria starter cultures are killed

by heat used in molding.

The pH will not increase during the 2-

wk holding period at 23uC (73uF) at

retail.

2.a.4 If applicable, what are the

dimensions of cuts, pieces, etc?

NA

2.a.5 What is the normal microbial

load (species, etc.) at the

beginning and end of

production?

Microbial load: lactic acid bacteria starter

culture 7 log CFU/ml of milk; residual

cultures 2 log CFU/g. Reduction due to

heating at 70uC (158uF) during molding

step.

2.a.6 Is there likelihood that

contamination may be

internalized in or distributed

throughout individual

components?

Unlikely if produced under Good

Manufacturing Practices (GMPs), HACCP

using pasteurized milk; contamination by

nonsporeformers would be on the surface.

2.b What are the preparation steps?

2.b.1 Is the product an assembled

(multicomponent) product?

Product is not an assembled nor a

multicomponent product.

2.b.2 Is there a microbial reduction

step that is validated? What are

the parameters associated with

the microbial reduction step? Are

there different microbial

reduction steps for different

components?

Pasteurization is a validated heat inactivation

step for milk used to make the cheese; no kill

step for surface contamination of the cheese.

166 NACMCF J. Food Prot., Vol. 73, No. 1

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APPENDIX E. Continued

Evaluation of Mozzarella slices packaged under modified atmosphere packaging and stored at ambient temperatures for up to 2 wk

to enhance sales

Considerations Response Additional comments

2.b.3 Is there a potential for

recontamination?

Yes. Potential for recontamination during

slicing and packaging.

2.b.4 What is the variability in

parameters that affect lethality or

growth?

Little variability for lethality if prepared

under GMPs HACCP; growth potential can

vary depending on moisture and pH at the

end of production for high-moisture versus

low-moisture product.

2.b.5 How is the product packaged? After slicing or cutting, slices or blocks will

be vacuum packaged or modifiedatmosphere packaged with nitrogen–carbon

dioxide mix.

2.b.6 Is the product cultured or

fermented? Does it contain

starter culture intentionally

added?

Product made with starter culture but

populations reduced by heating at 70uC

(158uF) for molding step.

2.b.7 Does the product contain

antimicrobials (preservatives) or

other ingredients that might be

inhibitory, such as spices?

NaCl is present but not at inhibitory levels.

No antimicrobials are added to cheese, but

natamycin may be added to the surface of cut

or shredded cheese to inhibit mold.

2.c What are the storage conditions?

2.c.1 How will the product be

displayed for sale? Any changes

to packaging for display?

Slices or blocks will be packaged under

vacuum or modified atmosphere (nitrogen–

carbon dioxide mixture) for storage; product

may be displayed unrefrigerated for

increased sales but will otherwise be held

refrigerated to extend shelf life.

2.c.2 What temperatures (and times)

are expected during production,

preparation, and storage or

display?

During cheese production, milk will be

cultured and curd cooked at #40uC (104uF);

curd will be heated to 70uC (158uF) for

molding step; cheese cured at 3uC (37uF) for

up to 2 wk and distributed to retailers

typically at ,7uC (45uF); maximum storage

at 23uC (73uF) at retail for 2 wk.

Product quality will deteriorate rapidly

if temperature exceeds 23uC (73uF).

However, temperatures as high as

27uC (81uF), e.g., during

transportation, will have limited effect

on quality if the time does not exceed

4 h.

2.c.3 What potential is there for

storage or display at

temperatures greater than those

listed above in 2.c.2?

Product is unlikely to be stored at

temperatures greater than described;

temperatures exceeding 30uC (86uF) will

result in a significant decrease in product

quality (melting, fat separation).

2.c.4 Are there other hazards that may

be created by preparation,

storage, or display?

No, but molds may grow on the surface if

oxygen is present and when natamycin is not

used.

2.c.5 What is the estimated maximum

time from production to

consumption?

9 mo if stored at refrigeration temperatures;

2 wk for unrefrigerated storage.

2.c.6 What is the time to spoilage or

unacceptable quality?

2 wk unrefrigerated storage if held at 20–

23uC (68–73uF), shorter if temperatures

exceed 23uC; 9 mo refrigerated storage.

3 Determine if product assessment for growth or inactivation is needed

3.a Is a product assessment for growth

necessary based on pH and aw?

(See Appendix D, Tables A and B.)

If yes, also answer 4.e and 5.a.

Yes, product assessment required. Food

Code Table B is applicable because of

potential recontamination and survival of

spores. pH . 5.4 and aw 0.96.

3.b Is an inactivation study needed? If

yes, also answer 4.f and 5.b.

No, the purpose of this study is to determine

if pathogens likely to be present will grow in

the product if stored out of refrigeration;

milk has been previously pasteurized.

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 167

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APPENDIX E. Continued

Evaluation of Mozzarella slices packaged under modified atmosphere packaging and stored at ambient temperatures for up to 2 wk

to enhance sales

Considerations Response Additional comments

3.c Are there any regulations

applicable for lethality

(inactivation) or TCS (growth)?

Latest edition Food Code for TCS.

4 Determine pathogens of concern to include in the challenge study

4.a According to Table 2 and

Appendix C, which pathogens are

of concern? If food is not seafood,

Vibrio spp. may be excluded from

consideration.

Given a product pH of 5.4 and an aw of 0.96,

the pathogens of concern are B. cereus, C.

botulinum, pathogenic E. coli, L.

monocytogenes, Salmonella, S. aureus,

Vibrio parahaemolyticus, and V. vulnificus.

4.b Considering the ecology, product,

and epidemiological history, what

pathogens are reasonably likely to

occur? (Also see Appendix C.)

B. cereus spores survive pasteurization;

pathogenic E. coli, L. monocytogenes,

Salmonella, and S. aureus from

postprocessing handling.

Salmonella has been associated with

Mozzarella due to contamination during

production, not postprocess contamination;

illness associated with survival, not growth;

The most likely vegetative pathogens

to recontaminate the product are L.

monocytogenes and S. aureus. L.

monocytogenes is a more likely

pathogen to recontaminate the product

due to its ubiquity in the environment.

S. aureus is a likely contaminant from

workers’ hands.

no outbreak has been reported with B.

cereus, L. monocytogenes, or S. aureus (22).

Vibrio spp. were excluded from

consideration since seafood is not a

component.

C. botulinum was excluded from

consideration because the spores are

rare in the ecology of dairy products.

4.c What pathogens are likely to

recontaminate the product after

the inactivation step?

Recontamination can occur as indicated

above.

4.d Are there any baseline surveys that

indicate prevalence of pathogens

for the target product or a related

product?

No.

4.e For growth inhibition (TCS) studies:

4.e.1 Which pathogen(s) will grow the

fastest? Consider gram positive

versus gram negative; vegetative

microorganisms versus spore

formers. If food is not seafood,

Vibrio may be excluded from

consideration. Use a predictive

model or cite applicable

literature. Consider growth

potential through 1.5 times the

shelf life, if appropriate.

Please see 4.e.2, 4.e.3, and 4.e.4.

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APPENDIX E. Continued

Evaluation of Mozzarella slices packaged under modified atmosphere packaging and stored at ambient temperatures for up to 2 wk

to enhance sales

Considerations Response Additional comments

4.e.2 Predictive model At pH 5.4, aw 0.96, 27uC (80.6uF): PMP 7.0

version 1.1 predicts a 3-log S. aureus

increase within 29 h (22 h without lag)

under aerobic conditions; ComBase

Predictor predicts a 3-log S. aureus increase

within 18 h for the same conditions. For L.

monocytogenes, PMP predicts a 1-log

increase within 42 h for the same conditions

(7 h without lag); ComBase Predictor with

5,000 ppm of lactic acid predicts a 1-log L.

monocytogenes increase within 33 h for the

same conditions. PMP does not include B.

cereus predictions at aw 0.96 but ComBase

Predictor with 40% CO2 predicts a 3-log B.

cereus increase within 101 h.

Modeling was conservatively done at

the highest expected exposure

temperature.

Of the likely contaminants, L.

monocytogenes and S. aureus will grow

fastest at this aw and pH; S. aureus is

generally not a good competitor in

cheese made with starter cultures, but

starter cultures are reduced by heating

and molding step. If B. cereus growth

occurred, it would be at a slower rate

than that of L. monocytogenes or S.

aureus.

4.e.3 Compare choice with literature Stecchini et al. (101) indicated a 5-log

increase of L. monocytogenes during storage

at 5uC (41uF) for 21 days (pH and moisture

not reported).

4.e.4 Any further information on

growth or survival?

Data presented at the 2003 International

Association for Food Protection Annual

Meeting 2003 (29) on cheese shreds for L.

monocytogenes and Salmonella

demonstrated no growth on low-moisture

Mozzarella stored at 15uC (59uF) for 2 mo

(pH 5.0–5.5; 47% moisture; aw 0.965).

4.e.5 Based on the above analysis,

what challenge organisms are

chosen for growth inhibition

studies?

L. monocytogenes and S. aureus.

4.f If inactivation studies: NA

4.f.1 What is the lethal treatment?

(HPP, heat, acid, etc.)

4.f.2 Which microorganisms are most

resistant to the lethal treatment?

(HPP, heat, acid, etc.)

4.f.3 Will the lethality be delivered to

all areas of the product that may

contain the pathogen? Account

for all surface and internalized

contamination.

4.f.4 What is in the formulation that

may affect inactivation?

(Intrinsic factors may contribute

to lethality or resistance: aw,

moisture, salt, pH, fat, etc.)

4.f.5 Are there any data on pathogen

levels in the product?

4.f.6 Is there a regulatory requirement

or policy for log reduction for

this product? Cite requirement.

4.f.7 If there is no regulatory

requirement for log reduction,

use scientific basis for

determining acceptable reduction

(21, 76).

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 169

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APPENDIX E. Continued

Evaluation of Mozzarella slices packaged under modified atmosphere packaging and stored at ambient temperatures for up to 2 wk

to enhance sales

Considerations Response Additional comments

4.f.8 Based on the above analysis,

what challenge organisms are

chosen for inactivation studies?

5 Determine appropriate time and sampling intervals for challenge study

5.a For growth inhibition (TCS)

studies, use 1.25–1.5 times the

shelf life as testing time

14 days | 1.5 ~ 21 days.

5.a.1 Maximum time from production

to consumption

Maximum 9 mo if refrigerated; 21 days if

not refrigerated.

5.a.2 Actual time to spoilage or

unacceptable quality

Point of unacceptable quality: 21 days.

5.a.3 For growth inhibition studies,

determine appropriate sampling

intervals for microbial analysis;

use five to seven (preferred)

sampling intervals; fewer

sampling intervals should be

justified, e.g., using results from

similar products.

Sample at 0, 1, 2, 3, 4, 7, 14, 21 days. Based on predictive models, growth

could occur within 24–48 h at 27uC

(81uF); more than seven sampling

intervals are appropriate to ensure the

ability to identify minimum time to

growth.

5.b For inactivation studies determine

appropriate sampling points

considering the process and

formulation. Identify populations at

0 time and end of processing;

whenever possible, include

intermediate sampling intervals to

determine death curve.

NA

5.b.1 When inactivation treatments

may result in sublethal injury,

repair and growth of

microorganisms during product

shelf life should be considered

(21).

6 Determine inoculation, storage, and testing procedures

6.a Determine strains for use in study

(multiple strains for each species

are recommended; consider use of

appropriate food or clinical

isolates)

L. monocytogenes and S. aureus will be

tested individually using three-strain

mixtures. Each mixture will include isolates

from cheese or other dairy isolates or clinical

isolates.

6.b Determine if adaptation is

required for inoculum preparation

No adaptation necessary; product is low

acid, high aw at ambient temperatures.

6.c Determine method of inoculation

(surface, mixing, dipping, liquid,

dry, etc.)

Surface inoculation of individual 25-g slices;

two slices per package with inoculum on

inner surface between the two slices.

Using two slices per package with

inoculum in between will retain

moisture and provide a worst-case

scenario for growth.

6.d Determine size of inoculum

(populations, e.g., log CFU/g,

CFU per package, percentage of

inoculum vol/wt or vol/vol)

3 log CFU/g; 0.05 ml (50 ml) per package.

Each organism will be inoculated

independently (separate samples) to avoid

possible antagonistic effect between different

organisms.

Inoculum level is high considering

likelihood of contamination but will

allow enumeration by direct plating

and detection of growth and low levels

of inactivation by formulation during

storage; inoculum volume 1% of

sample size; preliminary data suggest

inoculum does not change pH and aw

appreciably.

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APPENDIX E. Continued

Evaluation of Mozzarella slices packaged under modified atmosphere packaging and stored at ambient temperatures for up to 2 wk

to enhance sales

Considerations Response Additional comments

6.e Determine packaging to be used Two inoculated slices will be used per

package unit; slices will be packaged with

60% nitrogen and 40% carbon dioxide

mixture and sealed; packaging material will

be gas-moisture impermeable.

Packaging is the same as commercial

product.

6.f Determine the incubation

temperature for growth inhibition

studies or temperature(s) for

thermal inactivation studies

23uC (73uF). 23uC (73uF) is the maximum

temperature to which the product will

be exposed without adverse changes in

product quality that would deter

purchase and consumption.

6.g Determine sampling method and

sample size

Entire sample (two slices) will be mixed in

the bag, and 25-g portions will be removed

for microbial analysis; sample will be

homogenized with equal volume of 0.1%

peptone buffer, and serial dilutions will be

plated on selective agar as appropriate per

FDA BAM methods (115).

6.h How many replicates are needed to

ensure confidence in data? Does

variability in proximate analysis or

production warrant more than two

or three replicate trials? Will

multiple variations of similar

formulations be tested? Has a

statistical design for choosing

formulations been used (block

design, central composite, etc.)?

Two replicate (unique production) lots using

highest moisture and pH combination;

triplicate samples per testing interval.

7 Determine other controls

7.a Is use of surrogates appropriate or

necessary? If so, justify.

Surrogates are not appropriate or necessary.

7.b Are uninoculated controls needed

to assess spoilage or competitive

microflora or for other purposes?

Uninoculated controls will be used to

monitor growth of molds or yeasts and other

spoilage microorganisms that can change pH

during testing interval and for proximate

analysis at the beginning of the study.

7.c What other controls are necessary

(including negative or positive

growth controls)?

Not required for this study; anticipate growth

if samples were held for sufficient time.

8 Determine pass-fail criteria

8.a What are the pass-fail criteria? No more than 1-log increase for L.

monocytogenes; no more than 3-log increase

for S. aureus.

A 1-log increase in L. monocytogenes

is considered significant growth, but

any detectable presence of L.

monocytogenes in a ready-to-eat food

renders the product adulterated.

8.b What are the limits for use of the

results?

Data apply only to Mozzarella with the

maximum moisture-pH-temperature-time

limits tested in this study.

a NA, not applicable.

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 171

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APPENDIX F. FOOD PRODUCT CHECKLIST: CHOPPED LETTUCE

Evaluation to determine the absence of measurable growth (,1 log) of pathogens of concern in chopped lettuce held out of refrigeration

for up to 8 h

Considerations Response Additional comments

1 Determine the purpose of the study

1.a Exempt from time/temperature

control for safety (no refrigeration

required)

NAa

1.b Variance from any regulatory

requirements (e.g., holding for

.4 h without temperature control)

Yes. The purpose of the study is to allow

chopped lettuce to be held out of temperature

control (at room temperature) for a period of

up to 8 h. This is a salad bar product

consumed on premises. Once the lettuce has

been removed from temperature control it

will be used or discarded within 8 h. Product

will not be re-refrigerated and offered for

service at a later time.

1.c Validate lethality NA

1.d Verify that formulation will inhibit

microbial growth in refrigerated

foods or under mild temperature

abuse

NA

2 Collect information regarding the product

2.a What are the ingredients? The single ingredient is heads of whole

Romaine lettuce that are chopped and

washed in water containing a wash water

sanitizer at concentrations specified on the

label.

2.a.1 How consistent are the

ingredients from various sources,

lot to lot?

Total plate count on the product varies

widely from batch to batch.

2.a.2 What are the pH, aw, and

proximate analysis (moisture,

salt, fat, protein, residual nitrite,

etc.) for product and/or

individual components?

The pH is estimated to be 5.8–6.2 (118).

Water activity in iceberg lettuce is 0.995–

0.998, and this is assumed to hold true for

Romaine (33). The product is very high in

water, with minimal amounts of salt, fat, and

protein.

2.a.3 Do any of these values change

from preparation to

consumption?

The pH is not likely to change. The aw may

decrease slightly as the product dries out, but

we have elected to ignore the impact this

drying would have on pathogen growth.

2.a.4 If applicable, what are the

dimensions of cuts, pieces, etc.?

Heads arrive whole and are chopped into

pieces about 5 by 5 cm.

2.a.5 What is the normal microbial

load (species, etc.) at the

beginning and end of

production?

No published data are available on incoming

heads of lettuce. Internal company data on

the lettuce after chopping shows the

following trends, based on several years of

sample collection, where sample size was

25 g.

Data presented here were collected

after the lettuce had been washed

and cut.

Total aerobic plate counts are normally

distributed with a mean of 5.5 log CFU/g

and a standard deviation of 1.5 log CFU/g. S.

aureus has been found in 1 of 50 samples,

Salmonella in 1 of 200 samples, B. cereus in

1 of 10 samples. E. coli is generally absent,

but 1 of 20 samples had .2 log MPN/g. L.

monocytogenes was not detected in tests of

more than 200 samples.

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APPENDIX F. Continued

Evaluation to determine the absence of measurable growth (,1 log) of pathogens of concern in chopped lettuce held out of refrigeration

for up to 8 h

Considerations Response Additional comments

2.a.6 Is there likelihood that

contamination may be

internalized in or distributed

throughout individual

components?

Published laboratory data show that

internalization in fresh cut lettuce is possible

(93). The extent to which this happens under

real world conditions is not clear.

2.b What are the preparation steps? Receive lettuce from vendor, store in cooler

until use, remove from cooler, remove and

discard outer leaves, cut off bottom end,

separate remaining leaves and wash them in

water containing wash water sanitizer at

label concentrations, spin to remove excess

water, chop into pieces approximately 5 by

5 cm.

2.b.1 Is the product an assembled

(multicomponent) product?

The product is not assembled and is not

multicomponent.

2.b.2 Is there a microbial reduction

step that is validated? What are

the parameters associated with

the microbial reduction step? Are

there different microbial

reduction steps for different

components?

The wash step has been shown to result in a

1- to 2-log reduction in aerobic plate count.

The microbial reduction reported here

is not considered in the design of the

challenge study for this product.

2.b.3 Is there a potential for

recontamination?

There is a slight potential for

recontamination. Lettuce is hand chopped in

a food service kitchen environment. Data on

actual product (see above) indicate that S.

aureus may contaminate the product but that

L. monocytogenes does not represent a

significant risk. Employees receive annual

food safety training, and managers are

certified by accredited Food Managers

Certification testing. Standard procedures are

in place to prevent cross-contamination of

this product during preparation.

2.b.4 What is the variability in

parameters that affect lethality or

growth?

The product does vary due to normal

biological variation. The pH and aw values

are very permissive to growth, so variability

is unlikely to influence pathogen growth.

2.b.5 How is the product packaged? The product is not packaged but may be

placed in plastic bins and covered with

plastic wrap for refrigeration prior to display.

2.b.6 Is the product cultured or

fermented? Does it contain

starter culture intentionally

added?

No.

2.b.7 Does the product contain

antimicrobials (preservatives) or

other ingredients that might be

inhibitory, such as spices?

There are no antimicrobials, preservatives, or

other inhibitory ingredients.

2.c What are the storage conditions?

2.c.1 How will the product be

displayed for sale? Any changes

to packaging for display?

Display in open containers on salad bar.

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 173

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APPENDIX F. Continued

Evaluation to determine the absence of measurable growth (,1 log) of pathogens of concern in chopped lettuce held out of refrigeration

for up to 8 h

Considerations Response Additional comments

2.c.2 What temperatures (and times)

are expected during production,

preparation, and storage or

display?

Product is stored below 5uC (41uF) prior to

preparation. Preparation takes approximately

2 h per batch and takes place at room

temperature (21.1uC, 70uF). Product may

either be covered with plastic wrap and

refrigerated after preparation or placed at

room temperature for sale and consumption.

The 8 h starts from the time of

preparation unless the product will be

rapidly cooled to 5uC (41uF) within

4 h after preparation, in which case

the 8 h starts when the chopped

product is removed from

refrigeration.

2.c.3 What potential is there for

storage or display at

temperatures greater than those

listed above in 2.c.2?

The restaurant is climate controlled. Our data

show that the room temperature is usually

21.1uC (70uF) but can in some cases increase

to 23.9uC (75uF) for short periods of time.

2.c.4 Are there other hazards that may

be created by preparation,

storage, or display?

Recontamination by the consumer during

serving is possible, but sneeze guards and

tongs are used, as per normal Food Code

practice.

2.c.5 What is the estimated maximum

time from production to

consumption?

The maximum amount of time the product

will be out of temperature control is 8 h.

2.c.6 What is the time to spoilage or

unacceptable quality?

The product is overtly spoiled after 24 h at

room temperature.

3 Determine if product assessment for growth or inactivation is needed

3.a Is a product assessment for growth

necessary based on pH and aw?

(See Appendix D, Tables A and B.)

If yes, also answer 4.e and 5.a.

Yes, product assessment is required

according to Food Code Table B.

3.b Is an inactivation study needed? If

yes, also answer 4.f and 5.b.

No.

3.c Are there any regulations

applicable for lethality

(inactivation) or TCS (growth)?

The Food Code defines this product as

requiring temperature control for safety.

There are no requirements for lethality on

this product.

4 Determine pathogens of concern to include in the challenge study

4.a According to Table 2 and

Appendix C, which pathogens are

of concern? If food is not seafood,

Vibrio spp. may be excluded from

consideration.

Based on pH and aw, B. cereus, C.

botulinum, C. perfringens, L.

monocytogenes, pathogenic E. coli,

Salmonella, S. aureus, Shigella spp., and

Y. enterocolitica should be considered.

4.b Considering the ecology, product,

and epidemiological history, what

pathogens are reasonably likely to

occur? (Also see Appendix C.)

Product testing shows that B. cereus and S.

aureus are present. Epidemiological data

would suggest E. coli O157:H7 as the

primary concern, followed by Salmonella

and Shigella. C. botulinum and C.

perfringens were excluded based on the

nature of the finished product (loosely

packed chopped leaves). Although L.

monocytogenes will grow on chopped lettuce

(62), L. monocytogenes was excluded based

on lack of epidemiological evidence (41), as

was Y. enterocolitica and B. cereus.

4.c What pathogens are likely to

recontaminate the product after

the inactivation step?

See response to 2.b.3.

4.d Are there any baseline surveys that

indicate prevalence of pathogens

for the target product or a related

product?

See response to 2.a.5.

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APPENDIX F. Continued

Evaluation to determine the absence of measurable growth (,1 log) of pathogens of concern in chopped lettuce held out of refrigeration

for up to 8 h

Considerations Response Additional comments

4.e For growth inhibition (TCS)

studies:

4.e.1 Which pathogen(s) will grow the

fastest? Consider gram positive

versus gram negative; vegetative

microorganisms versus spore

formers. If food is not seafood,

Vibrio spp. may be excluded from

consideration. Use a predictive

model or cite applicable

literature. Consider growth

potential through 1.5 times the

shelf life, if appropriate.

See response to 4.e.2 and 4.e.3.

4.e.2 Predictive model A temperature of 21uC (69.8uF), pH 6.2, and

aw 0.995 were assumed for the following

predictions:

When typical lag time values are assumed,

ComBase Predictor shows a 1-log increase

after 6.5 h (E. coli O157:H7), 8.2 h

(Salmonella), 9.4 h (S. aureus), 12 h (L.

monocytogenes), and 18.5 h (Shigella). PMP

7.0 predicted a 1-log increase (including lag)

in 9.9 h (E. coli O157:H7), 8.3 h

(Salmonella), 9.1 h (S. aureus), 9.5 h (L.

monocytogenes), and 15.9 h (Shigella).

When lag time is assumed to be zero,

ComBase Predictor shows a 1-log increase

after 3.4 h (E. coli O157:H7), 3.6 h

(Salmonella), 4.1 h (S. aureus), 4.6 h (L.

monocytogenes), and 10 h (Shigella). PMP

shows a 1-log increase (excluding lag) after

3.6 h (E. coli O157:H7), 3.0 h (Salmonella),

5.6 h (S. aureus), 3.2 h (L. monocytogenes),

and 6.7 h (Shigella).

4.e.3 Compare choice with literature Literature data (four growth rates) for E. coli

O157:H7 growth in cut iceberg lettuce were

extracted from published studies (1, 23, 67).

The four data points were fit to a simple

literature-based model, and growth rate at

21uC (69.8uF) was estimated.

The literature-based model predicted about

0.86-log increase in E. coli O157:H7 after

8 h at 21uC (69.8uF). Note that this

prediction considers only growth rate and

neglects lag time.

4.e.4 Any further information on

growth or survival?

No.

4.e.5 Based on the above analysis,

what challenge organisms are

chosen for growth inhibition

studies?

Results from the modeling and epidemiology

show E. coli O157:H7 and Salmonella to

represent the greatest risk. Also, modeling

results presented above demonstrate that the

growth of the two organisms is similar.

Challenge studies will be done with E. coli

O157:H7 due to the greatest epidemiological

link to illness.

The ComBase modeling analysis

above shows that the product could

be of questionable safety when held

at room temperature for 8 h.

Literature-based model suggests that

the 8-h holding might be acceptable

based on a ,1-log growth.

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 175

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APPENDIX F. Continued

Evaluation to determine the absence of measurable growth (,1 log) of pathogens of concern in chopped lettuce held out of refrigeration

for up to 8 h

Considerations Response Additional comments

A challenge study was justified in

order to provide a more conclusive

answer. The study will be designed to

identify the period of time the growth

remains below 1 log CFU/g.

4.f If inactivation studies: NA

4.f.1 What is the lethal treatment?

(HPP, heat, acid, etc.)

4.f.2 Which microorganisms are most

resistant to the lethal treatment?

(HPP, heat, acid, etc.)

4.f.3 Will the lethality be delivered to

all areas of the product that may

contain the pathogen? Account

for all surface and internalized

contamination.

4.f.4 What is in the formulation that

may affect inactivation?

(Intrinsic factors may contribute

to lethality or resistance: aw,

moisture, salt, pH, fat, etc.)

4.f.5 Are there any data on pathogen

levels in the product?

4.f.6 Is there a regulatory requirement

or policy for log reduction for

this product? Cite requirement.

4.f.7 If there is no regulatory

requirement for log reduction,

use scientific basis for

determining acceptable reduction

(21, 76).

4.f.8 Based on the above analysis,

what challenge organisms are

chosen for inactivation studies?

5 Determine appropriate time and sampling intervals for challenge study

5.a For growth inhibition (TCS)

studies, use 1.25–1.5 times the

shelf life as testing time

Assuming the product is to be held for 8 h,

the product should be tested for 8 | 1.5 ~

12 h.

5.a.1 Maximum time from production

to consumption

8 h. See comment to 2.c.2.

5.a.2 Actual time to spoilage or

unacceptable quality

Prior data indicate 24 h at room temperature

results in an unacceptable product.

5.a.3 For growth inhibition studies,

determine appropriate sampling

intervals for microbial analysis;

use five to seven (preferred)

sampling intervals; fewer

sampling intervals should be

justified, e.g., using results from

similar products.

Test at 0, 2, 4, 6, 8, 10, 12 h. If cost is an issue, a fewer number of

time points could be evaluated (e.g.,

0, 3, 6, 9, and 12 h).

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APPENDIX F. Continued

Evaluation to determine the absence of measurable growth (,1 log) of pathogens of concern in chopped lettuce held out of refrigeration

for up to 8 h

Considerations Response Additional comments

5.b For inactivation studies determine

appropriate sampling points

considering the process and

formulation. Identify populations at

0 time and end of processing;

whenever possible, include

intermediate sampling intervals to

determine death curve.

NA

5.b.1 When inactivation treatments

may result in sublethal injury,

repair and growth of

microorganisms during product

shelf life should be considered

(21).

6 Determine inoculation, storage, and testing procedures

6.a Determine strains for use in study

(multiple strains for each species

are recommended; consider use of

appropriate food or clinical

isolates)

A cocktail of marked strains will be used. E.

coli O157:H7 strains will be a combination

of human isolates and food isolates

associated with sporadic illnesses or

outbreaks where leafy greens were

implicated.

In order to easily enumerate the E.

coli O157:H7 amid a high natural

background population, the selected

strain will be modified to express an

appropriate marker (e.g., antibiotic

resistance, green fluorescent protein).

6.b Determine if adaptation is

required for inoculum preparation

Adaptation of inoculum not needed. (42)

6.c Determine method of inoculation

(surface, mixing, dipping, liquid,

dry, etc.)

Chopped leaves will be spot inoculated on

both uncut surface and cut edges, briefly air

dried in a biosafety cabinet, and then stored

at refrigeration temperature until the

following day.

Dip inoculation would add excess

moisture that is difficult to remove

without a salad spinner. Salad

spinners used to remove moisture

from inoculated lettuce generate

potentially dangerous aerosols in a

laboratory, and it is difficult to

decontaminate the spinner. The

lettuce is refrigerated at 5uC (41uF)

after inoculation to duplicate the

temperature profile of the restaurant

lettuce.

6.d Determine size of inoculum

(population, e.g., log CFU/g, CFU

per package, percentage of

inoculum vol/wt or vol/vol)

Spot inoculum (approximately 10 ml for a

10-g sample) will be applied in multiple

(four or more) spots. The target final

concentration will be 3 log CFU/10-g

sample.

6.e Determine packaging to be used Samples will be stored in loosely sealed

plastic containers.

6.f Determine the incubation

temperature for growth inhibition

studies or temperature(s) for

thermal inactivation studies

Although the product is typically held at

21uC (70uF), the product will be incubated at

25uC (77uF) to represent the worst-case

condition.

6.g Determine sampling method and

sample size

Each 10-g sample will be combined with

90 ml of 0.1% peptone and homogenized for

1 min at high speed prior to dilution and

plating onto appropriate selective media.

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 177

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APPENDIX F. Continued

Evaluation to determine the absence of measurable growth (,1 log) of pathogens of concern in chopped lettuce held out of refrigeration

for up to 8 h

Considerations Response Additional comments

6.h How many replicates are needed to

ensure confidence in data? Does

variability in proximate analysis or

production warrant more than two

or three replicate trials? Will

multiple variations of similar

formulations be tested? Has a

statistical design for choosing

formulations been used (block

design, central composite, etc.)?

Two replicate trials will be conducted, and

three samples will be analyzed at each time

point and plated in duplicate. Each trial will

use fresh lettuce from a different batch and

fresh inoculum and will be conducted on a

different day.

7 Determine other controls

7.a Is use of surrogates appropriate or

necessary? If so, justify.

The use of surrogates is not appropriate or

necessary.

7.b Are uninoculated controls needed

to assess spoilage or competitive

microflora or for other purposes?

Uninoculated controls (one) will be sampled

at each time point. They will be plated on

tryptic soy agar and on the selective agar

used for the study. The visual appearance of

the control lettuce will be described at each

time point.

7.c What other controls are necessary

(including negative or positive

growth controls)?

The concentration of E. coli O157:H7 will be

determined in the freshly prepared inoculum

as well as the freshly inoculated lettuce at

time 0.

8 Determine pass-fail criteria

8.a What are the pass-fail criteria? Less than a 1-log increase for E. coli

O157:H7 at the end of study (12 h).

8.b What are the limits for use of the

results?

Results are applicable to similarly prepared

Romaine and iceberg lettuce. These data do

not apply to finely chopped or shredded

Romaine and iceberg lettuce, which are

likely to support more rapid growth.

Given the results of this study, it may

not be necessary to conduct full

studies on other leafy greens, but

some study is needed before data can

be more widely applied.

a NA, not applicable.

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APPENDIX G. FOOD PRODUCT CHECKLIST: MEAT-FILLED PASTRY

Evaluation of display of fully cooked meat-filled pastry for up to 12 h at room temperature

Considerations Response Additional comments

1 Determine the purpose of the study

1.a Exempt from time/temperature

control for safety (no refrigeration

required)

NAa Not a shelf-stable product.

1.b Variance from any regulatory

requirements (e.g., holding for

.4 h without temperature control)

Want to hold a fully cooked meat product up

to 12 h at room temperature (assuming

consumption within 2 h after purchase).

Discarded if not served within 12 h.

1.c Validate lethality NA Processed in state or federally

inspected food processing

establishment meeting regulatory

cook and cool requirements.

1.d Verify that formulation will inhibit

microbial growth in refrigerated

foods or under mild temperature

abuse

NA

2 Collect information regarding the product

2.a What are the ingredients? Ready-to-eat product that contains cooked

ground beef, spices, salt, pastry dough.

2.a.1 How consistent are the ingredients

from various sources, lot to lot?

Highly consistent lot to lot. Product specifications in place,

produced at a food processing

establishment under GMPs.

2.a.2 What are the pH, aw, and

proximate analysis (moisture, salt,

fat, protein, residual nitrite, etc.)

for product and/or individual

components?

Beef filling: pH 6.2, aw 0.97.

Pastry dough: pH 7.0, aw at interface 0.97;

aw at exterior surface 0.75.

If this were an inactivation study,

percent fat content may be important;

not relevant for this growth study.

2.a.3 Do any of these values change

from preparation to consumption?

No change of pH. The exterior of the pastry

may increase above aw 0.75 the longer the

product is held.

Potential for aw to increase on external

surface if condensate forms between

the package and pastry surface.

2.a.4 If applicable, what are the

dimensions of cuts, pieces, etc?

NA Component dimensions consistent

with product specifications.

2.a.5 What is the normal microbial load

(species, etc.) at the beginning and

end of production?

Vegetative pathogens are inactivated during

cooking. There is a potential for sporeforming pathogens to survive cooking. There

is a potential for low levels of

microorganisms (aerobic plate count of up to

2 log CFU/g).

Fully cooked at processing

establishment.

2.a.6 Is there likelihood that

contamination may be internalized

in or distributed throughout

individual components?

Yes, spores surviving the cooking process

could be distributed throughout the product.

Internal and external vegetative

pathogens are destroyed by cooking

process. However, vegetative

pathogens could be introduced on

external surfaces during handling or

packaging.

2.b What are the preparation steps?

2.b.1 Is the product an assembled

(multicomponent) product?

Yes. See product ingredients and

description above.

2.b.2 Is there a microbial reduction step

that is validated? What are the

parameters associated with the

microbial reduction step? Are

there different microbial reduction

steps for different components?

Yes. Adequate lethality and cooling to result

in a ready-to-eat product (meets all

regulatory requirements for cooking and

cooling). One cook and cool process for the

multicomponent product.

Achieving minimum internal

temperature of 73.9uC (165uF),

resulting in at least a 6.5-log reduction

of Salmonella for lethality, see (108);

for proper cooling, see (109).

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 179

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APPENDIX G. Continued

Evaluation of display of fully cooked meat-filled pastry for up to 12 h at room temperature

Considerations Response Additional comments

2.b.3 Is there a potential for

recontamination?

Yes, L. monocytogenes is a potential

recontaminant.

Although product is individually

wrapped, vegetative pathogens could

be introduced on external surfaces

during handling or packaging.

Control of this potential L.

monocytogenes postlethality

contamination is managed per 9 CFR

430 (113).

2.b.4 What is the variability in

parameters that affect lethality or

growth?

Limited variability in production of cooked

product due to controls in a regulated food

processing establishment. Limited variability

during refrigerated distribution and storage

up to the time of display for sale.

2.b.5 How is the product packaged? Individually hand wrapped in the inspected

establishment in a clear plastic wrap.

Wrapped pastries are placed in labeled

boxes.

Provides protection from moisture and

air.

2.b.6 Is the product cultured or

fermented? Does it contain starter

culture intentionally added?

No.

2.b.7 Does the product contain

antimicrobials (preservatives) or

other ingredients that might be

inhibitory, such as spices?

No. Low level of spices and salt would

not likely be inhibitory to pathogen

growth.

2.c What are the storage conditions?

2.c.1 How will the product be displayed

for sale? Any changes to

packaging for display?

Product will remain individually wrapped.

2.c.2 What temperatures (and times) are

expected during production,

preparation, and storage or

display?

Delivered refrigerated at or below 5uC

(41uF) to the retail establishment and kept

refrigerated until moved out for display.

Held at room temperature for display to

customers. Displayed for up to 12 h at room

temperature: 24uC (75uF). The product is

expected to be consumed or refrigerated

within 2 h of purchase.

2.c.3 What potential is there for storage

or display at temperatures greater

than those listed above in 2.c.2?

Higher temperatures are possible if product

is heated and displayed under a heat lamp.

A separate study may be required for

product stored under a heat lamp.

2.c.4 Are there other hazards that may

be created by preparation,

storage, or display?

No.

2.c.5 What is the estimated maximum

time from production to

consumption?

7 days (refrigerated). Labeled use-by date is 7 days after

production.

2.c.6 What is the time to spoilage or

unacceptable quality?

10 days (refrigerated) or 2 days at ambient

temperatures.

Product is to be discarded after 12 h

of ambient display but may continue

to have an acceptable appearance and

odor at the end of the display period.

Storage under a heat lamp may lead

to unacceptable organoleptic quality.

3 Determine if product assessment for growth or inactivation is needed

3.a Is a product assessment for growth

necessary based on pH and aw? (See

Appendix D, Tables A and B.) If

yes, also answer 4.e and 5.a.

Yes, for beef filling, pH 6.2, aw 0.97. The outer pastry component with an

aw of 0.75 does not require product

assessment for growth.

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APPENDIX G. Continued

Evaluation of display of fully cooked meat-filled pastry for up to 12 h at room temperature

Considerations Response Additional comments

3.b Is an inactivation study needed? If

yes, also answer 4.f and 5.b.

No.

3.c Are there any regulations applicable

for lethality (inactivation) or TCS

(growth)?

Yes, maximum 4-h holding time limit when

there are no temperature controls for safety

(Food Code).

4 Determine pathogens of concern to include in the challenge study

4.a According to Table 2 and Appendix

C, which pathogens are of concern?

If food is not seafood, Vibrio spp.

may be excluded from

consideration.

B. cereus, C. botulinum, C. perfringens, L.

monocytogenes, pathogenic E. coli,

Salmonella, S. aureus, V. parahaemolyticus,

and V. vulnificus.

4.b Considering the ecology, product,

and epidemiological history, what

pathogens are reasonably likely to

occur? (Also see Appendix C.)

C. perfringens, C. botulinum, and B. cereus. Vegetative cells are not a concern due

to USDA FSIS validated cooking

process. Postprocess contamination

would be limited to the outside of the

pastry shell, which has very low aw

and would not support growth.

Standard GMPs will also reduce

likelihood of pathogen

recontamination.

4.c What pathogens are likely to

recontaminate the product after the

inactivation step?

Recontamination of meat filling is not likely

because it is encased within a pastry shell.

4.d Are there any baseline surveys that

indicate prevalence of pathogens for

the target product or a related

product?

No, not for meat-filled pastry products.

4.e For growth inhibition (TCS) studies:

4.e.1 Which pathogen(s) will grow the

fastest? Consider gram positive

versus gram negative; vegetative

microorganisms versus spore

formers. If food is not seafood,

Vibrio may be excluded from

consideration. Use a predictive

model or cite applicable literature.

Consider growth potential through

1.5 times the shelf life, if

appropriate.

B. cereus based on predictive models (see

4.e.2).

4.e.2 Predictive model Predictive models were used to gauge

comparative growth of C. perfringens, C.

botulinum, and B. cereus in the meat filling.

The PMP predicts a 1-log increase of C.

perfringens in approximately 32 h based on

pH 6.2, aw 0.983 (lowest aw in program),

and 37uC (highest temperature in program).

ComBase Predictor predicts a 1-log increase

in approximately 13 h, assuming pH 6.2, aw

0.971, and 37uC (98.6uF).

All modeling included the lag phase.

This was considered appropriate given

that spore-forming organisms require

both germination and outgrowth.

Predictive models estimate that B.

cereus will grow faster than C.

perfringens and that both organisms

would grow faster than C. botulinum.

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 181

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APPENDIX G. Continued

Evaluation of display of fully cooked meat-filled pastry for up to 12 h at room temperature

Considerations Response Additional comments

The PMP predicts growth of C. botulinum in

.10 days at 26.7uC (estimated room

temperature of 80uF) based on pH 6.2, aw

0.977 (lowest aw in program). ComBase

predicts a lag time for proteolytic C.

botulinum of about 2 days, assuming pH 6.2,

aw 0.97, and 37uC (98.6uF), and a slightly

shorter lag time for nonproteolytic C.

botulinum at 30uC (86uF) and aw 0.974 (the

least permissive conditions allowed by the

model).

The PMP predicts a 1-log increase in B.

cereus in 5 h at pH 6.2, aw 0.97, and 37uC

(98.6uF) under aerobic conditions and

approximately 12 h under anaerobic

conditions. ComBase predicts a 1-log

increase in approximately 14 h at pH 6.2, aw

0.97, and 37uC (98.6uF) with 0% CO2.

4.e.3 Compare choice with literature Spices are an ingredient in the meat filling.

B. cereus is a known contaminant of spices

(88).

4.e.4 Any further information on growth

or survival?

No.

4.e.5 Based on the above analysis, what

challenge organisms are chosen

for growth inhibition studies?

B. cereus.

4.f If inactivation studies: NA

4.f.1 What is the lethal treatment?

(HPP, heat, acid, etc.)

4.f.2 Which microorganisms are most

resistant to the lethal treatment?

(HPP, heat, acid, etc.)

4.f.3 Will the lethality be delivered to all

areas of the product that may

contain the pathogen? Account for

all surface and internalized

contamination.

4.f.4 What is in the formulation that

may affect inactivation? (Intrinsic

factors may contribute to lethality

or resistance: aw, moisture, salt,

pH, fat, etc.)

4.f.5 Are there any data on pathogen

levels in the product?

4.f.6 Is there a regulatory requirement

or policy for log reduction for this

product? Cite requirement.

4.f.7 If there is no regulatory

requirement for log reduction, use

scientific basis for determining

acceptable reduction (21, 76).

4.f.8 Based on the above analysis, what

challenge organisms are chosen

for inactivation studies?

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APPENDIX G. Continued

Evaluation of display of fully cooked meat-filled pastry for up to 12 h at room temperature

Considerations Response Additional comments

5 Determine appropriate time and sampling intervals for challenge study

5.a For growth inhibition (TCS) studies,

use 1.25–1.5 times the shelf life as

testing time

1.5 | 14 h (target shelf life plus up to 2 h in

the hands of the consumer) ~ 21 h.

5.a.1 Maximum time from production to

consumption

Refrigerated up to 7 days, 12 h at room

temperature and 2 h to consumption after

leaving the store. May or may not be heated

prior to consumption.

5.a.2 Actual time to spoilage or

unacceptable quality

Expected to have 7-day shelf life in

refrigerator. At room temperature product

may appear to be spoiled after 2 days.

5.a.3 For growth inhibition studies,

determine appropriate sampling

intervals for microbial analysis;

use five to seven (preferred)

sampling intervals; fewer

sampling intervals should be

justified, e.g., using results from

similar products.

Times 0, 14, and 21 h. Due to the very short shelf life of this

product and the fact that predictive

models estimated limited growth in

the time frames of the study (e.g.,

approximately 1 log unit of growth),

sampling times were set at 0, 14 (the

end of the target shelf life), and 21 h

(1.5 times the target shelf life).

5.b For inactivation studies determine

appropriate sampling points

considering the process and

formulation. Identify populations at

0 time and end of processing;

whenever possible, include

intermediate sampling intervals to

determine death curve.

NA

5.b.1 When inactivation treatments may

result in sublethal injury, repair

and growth of microorganisms

during product shelf life should be

considered (21).

NA

6 Determine inoculation, storage, and testing procedures

6.a Determine strains for use in study

(multiple strains for each species

are recommended; consider use of

appropriate food or clinical isolates)

Use at least three strains of B. cereus. This should include a composite of

clinical strains from foodborne illness

outbreaks as well as isolates from

food.

6.b Determine if adaptation is required

for inoculum preparation

Adaptation not required.

6.c Determine method of inoculation

(surface, mixing, dipping, liquid,

dry, etc.)

Injection of inoculum into the meat filling

through the pastry.

Inoculum spores mixed with meat

filling supplied by manufacturer.

6.d Determine size of inoculum

(populations, e.g., log CFU/g, CFU

per package, percentage of

inoculum vol/wt or vol/vol)

2–3 log CFU/g not to exceed 0.1% of filling

volume.

Inoculum size is verified with time 0

sample of filling.

6.e Determine packaging to be used Plastic cellophane wrap.

6.f Determine the incubation

temperature for growth inhibition

studies or temperature(s) for

thermal inactivation studies

Incubate at 30uC (86uF). This represents a reasonable maximum

ambient temperature.

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APPENDIX G. Continued

Evaluation of display of fully cooked meat-filled pastry for up to 12 h at room temperature

Considerations Response Additional comments

6.g Determine sampling method and

sample size

Duplicate filled pastries will be sampled

from each of the three replicate lots at each

time point. Each sample in its entirety will be

blended or stomached in a 1:10 dilution of

buffer. Duplicate plate counts will be run for

each sample.

6.h How many replicates are needed to

ensure confidence in data? Does

variability in proximate analysis or

production warrant more than two or

three replicate trials? Will multiple

variations of similar formulations be

tested? Has a statistical design for

choosing formulations been used

(block design, central composite,

etc.)?

Three replicate production lots are to be

tested, preferably lots made with separate

batches of ingredients or on separate days.

If different formulations, three

replicates per formulation.

7 Determine other controls

7.a Is use of surrogates appropriate or

necessary? If so, justify.

Surrogates are not appropriate or necessary.

7.b Are uninoculated controls needed to

assess spoilage or competitive

microflora or for other purposes?

An uninoculated control is needed for each

replicate lot to monitor for natural

contamination.

7.c What other controls are necessary

(including negative or positive

growth controls)?

Not required for this study; anticipate growth

if samples were held for sufficient time.

8 Determine pass-fail criteria

8.a What are the pass-fail criteria? No more than a 3-log increase of B. cereus. The 3-log increase level selected for

B. cereus is based on the increase

suggested in the IFT report (53).

Some regulatory agencies may

consider a lower log increase to be

appropriate.

8.b What are the limits for use of the

results?

These results cannot be applied to pastries

held at higher than ambient temperatures,

e.g., holding under a heat lamp.

a NA, not applicable.

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APPENDIX H. FOOD PRODUCT CHECKLIST: LEMON MERINGUE PIE

Validate that the formulation of lemon meringue pie will inhibit pathogen growth under nonrefrigerated conditions

Considerations Response Additional comments

1 Determine the purpose of the study

1.a Exempt from time/temperature

control for safety (no refrigeration

required)

Exempt from time/temperature control after

opening.

Labeled shelf life of 3 days.

1.b Variance from any regulatory

requirements (e.g., holding for

.4 h without temperature control)

NAa

1.c Validate lethality NA

1.d Verify that formulation will inhibit

microbial growth in refrigerated

foods or under mild temperature

abuse

NA

2 Collect information regarding the product

2.a What are the ingredients? Pie crust: flour, shortening, water, salt.

Filling: water, sugar, modified food starch,

corn syrup solids, margarine, lemon juice

solids, high fructose corn syrup, sodium

citrate, agar agar, potassium sorbate, natural

flavor, locust bean gum, artificial color

(FD&C yellow no. 5).

Meringue: unpasteurized egg whites, sugar,

cream of tartar.

2.a.1 How consistent are the ingredients

from various sources, lot to lot?

Very consistent, same or similar lot to lot.

2.a.2 What are the pH, aw, and

proximate analysis (moisture, salt,

fat, protein, residual nitrite, etc.)

for product and/or individual

components?

Baked crust: pH 6.2, aw 0.45.

Cooked filling: pH 4.2, aw 0.88.

Meringue: pH 4.6, aw 0.93.

Values are after baking.

2.a.3 Do any of these values change

from preparation to consumption?

No.

2.a.4 If applicable, what are the

dimensions of cuts, pieces, etc?

NA

2.a.5 What is the normal microbial load

(species, etc.) at the beginning and

end of production?

After baking, aerobic plate count of ,10

CFU/g.

2.a.6 Is there likelihood that

contamination may be internalized

in or distributed throughout

individual components?

Yes, during slicing the contamination may

occur along the sliced edge of all three

components (crust, filling, and meringue).

2.b What are the preparation steps? Mix dough, sheet, form, bake. Cook the

filling to set the starch, fill the baked crust,

cool to ambient temperature, spread

meringue evenly over filling and bake. Cool

to ambient temperature, package.

Product is prepared in a commercial

manufacturing facility, cooled to room

temperature, packaged, and shipped at

ambient temperature.

2.b.1 Is the product an assembled

(multicomponent) product?

Yes.

2.b.2 Is there a microbial reduction step

that is validated? What are the

parameters associated with the

microbial reduction step? Are

there different microbial reduction

steps for different components?

All three components have heat inactivation

steps.

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APPENDIX H. Continued

Validate that the formulation of lemon meringue pie will inhibit pathogen growth under nonrefrigerated conditions

Considerations Response Additional comments

2.b.3 Is there a potential for

recontamination?

Yes, contamination may occur after opening

and slicing.

2.b.4 What is the variability in

parameters that affect lethality or

growth?

Low variability.

2.b.5 How is the product packaged? Paperboard box or plastic dome over an

aluminum pie plate.

2.b.6 Is the product cultured or

fermented? Does it contain starter

culture intentionally added?

No.

2.b.7 Does the product contain

antimicrobials (preservatives) or

other ingredients that might be

inhibitory, such as spices?

Sodium citrate and potassium sorbate in the

filling.

2.c What are the storage conditions?

2.c.1 How will the product be displayed

for sale? Any changes to

packaging for display?

Refrigerated or ambient, no change to

packaging.

2.c.2 What temperatures (and times) are

expected during production,

preparation, and storage or

display?

Cooled to ambient temperature after baking,

shipped and displayed at ambient

temperatures 20–35uC (68–95uF) until the

end of labeled shelf life of 3 days.

Unacceptable quality at 5 days.

2.c.3 What potential is there for storage

or display at temperatures greater

than those listed above in 2.c.2?

Unlikely.

2.c.4 Are there other hazards that may

be created by preparation,

storage, or display?

No. However, hazards may be introduced

during slicing and serving.

2.c.5 What is the estimated maximum

time from production to

consumption?

3 days.

2.c.6 What is the time to spoilage or

unacceptable quality?

5 days.

3 Determine if product assessment for growth or inactivation is needed

3.a Is a product assessment for growth

necessary based on pH and aw? (See

Appendix D, Tables A and B.) If

yes, also answer 4.e and 5.a.

Yes, according to Table B, a product

assessment is required for the meringue

component but not the crust or the filling.

3.b Is an inactivation study needed? If

yes, also answer 4.f and 5.b.

Yes, a separate inactivation study is being

conducted on the meringue.

3.c Are there any regulations applicable

for lethality (inactivation) or TCS

(growth)?

Yes, purpose of study is to get a variance

from need for time/temperature control for

safety.

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APPENDIX H. Continued

Validate that the formulation of lemon meringue pie will inhibit pathogen growth under nonrefrigerated conditions

Considerations Response Additional comments

4 Determine pathogens of concern to include in the challenge study

4.a According to Table 2 and Appendix

C, which pathogens are of concern?

If food is not seafood, Vibrio spp.

may be excluded from

consideration.

From Appendix C, pathogens of concern in

egg products are Salmonella and Listeria.

From Table 2, for pH 4.6 and aw 0.94, L.

monocytogenes and Salmonella would be the

organisms of concern.

4.b Considering the ecology, product,

and epidemiological history, what

pathogens are reasonably likely to

occur? (Also see Appendix C.)

Salmonella and L. monocytogenes are

known to be in retail and food service

environments.

4.c What pathogens are likely to

recontaminate the product after the

inactivation step?

Salmonella and L. monocytogenes.

4.d Are there any baseline surveys that

indicate prevalence of pathogens for

the target product or a related

product?

There are studies documenting the presence

of Listeria in the retail deli environment

(68).

4.e For growth inhibition (TCS)

studies:

4.e.1 Which pathogen(s) will grow the

fastest? Consider gram positive

versus gram negative; vegetative

microorganisms versus spore

formers. If food is not seafood,

Vibrio may be excluded from

consideration. Use a predictive

model or cite applicable literature.

Consider growth potential through

1.5 times the shelf life, if

appropriate.

See 4.e.2.

4.e.2 Predictive model The PMP indicates that L. monocytogenes

will not grow, but the model does not go

below pH 5.6 for Salmonella, so growth rate

under pH and aw conditions in meringue is

unknown.

4.e.3 Compare choice with literature Literature shows Salmonella and L.

monocytogenes growth can occur at pH 4.6;

most of these studies were in laboratory

media with high aw.

4.e.4 Any further information on growth

or survival?

No.

4.e.5 Based on the above analysis, what

challenge organisms are chosen

for growth inhibition studies?

Salmonella. Since we are unable to determine the

likelihood of growth of Salmonella

from predictive models or from the

literature, this organism was chosen

for a challenge study.

4.f If inactivation studies: NA

4.f.1 What is the lethal treatment?

(HPP, heat, acid, etc.)

4.f.2 Which microorganisms are most

resistant to the lethal treatment?

(HPP, heat, acid, etc.)

4.f.3 Will the lethality be delivered to all

areas of the product that may

contain the pathogen? Account for

all surface and internalized

contamination.

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APPENDIX H. Continued

Validate that the formulation of lemon meringue pie will inhibit pathogen growth under nonrefrigerated conditions

Considerations Response Additional comments

4.f.4 What is in the formulation that

may affect inactivation? (Intrinsic

factors may contribute to lethality

or resistance: aw, moisture, salt,

pH, fat, etc.)

4.f.5 Are there any data on pathogen

levels in the product?

4.f.6 Is there a regulatory requirement

or policy for log reduction for this

product? Cite requirement.

4.f.7 If there is no regulatory

requirement for log reduction, use

scientific basis for determining

acceptable reduction (21, 76).

4.f.8 Based on the above analysis, what

challenge organisms are chosen

for inactivation studies?

5 Determine appropriate time and sampling intervals for challenge study

5.a For growth inhibition (TCS) studies,

use 1.25–1.5 times the shelf life as

testing time

3 days | 1.5 ~ 4.5 days (round to 5 days).

5.a.1 Maximum time from production to

consumption

3 days.

5.a.2 Actual time to spoilage or

unacceptable quality

5 days.

5.a.3 For growth inhibition studies,

determine appropriate sampling

intervals for microbial analysis;

use five to seven (preferred)

sampling intervals; fewer

sampling intervals should be

justified, e.g., using results from

similar products.

Sample at time 0, then at days 1, 2, 3, 4,

and 5.

5.b For inactivation studies determine

appropriate sampling points

considering the process and

formulation. Identify populations at

0 time and end of processing;

whenever possible, include

intermediate sampling intervals to

determine death curve.

NA

5.b.1 When inactivation treatments may

result in sublethal injury, repair

and growth of microorganisms

during product shelf life should be

considered (21).

NA

6 Determine inoculation, storage, and testing procedures

6.a Determine strains for use in study

(multiple strains for each species

are recommended; consider use of

appropriate food or clinical isolates)

A mixture of at least five strains of

Salmonella isolated from eggs or egg

products and including at least one

Salmonella Enteritidis strain isolated from

clinical or egg samples associated with

outbreaks.

6.b Determine if adaptation is required

for inoculum preparation

Not necessary for this study.

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APPENDIX H. Continued

Validate that the formulation of lemon meringue pie will inhibit pathogen growth under nonrefrigerated conditions

Considerations Response Additional comments

6.c Determine method of inoculation

(surface, mixing, dipping, liquid,

dry, etc.)

Inoculate the cut face of the meringue for

single slices of pie by distributing 25 ml of

liquid inoculum from the filling-meringue

interface to the surface of the meringue; a

noninhibitory dye will be added to inoculum

to facilitate identification of the sampling

area.

A preliminary study should be

conducted to ensure that the dye is

not inhibitory to Salmonella unless

previously documented in the

scientific literature.

6.d Determine size of inoculum

(populations, e.g., log CFU/g, CFU

per package, percentage of

inoculum vol/wt or vol/vol)

Target 2–3 log CFU per site for each slice.

6.e Determine packaging to be used Packed in a ventilated plastic container that

prevents contamination of the slice but

allows exchange of air.

6.f Determine the incubation

temperature for growth inhibition

studies or temperature(s) for

thermal inactivation studies

35uC (95uF).

6.g Determine sampling method and

sample size

For each sample, the entire slice

(approximately 100 g) will be placed in a

sterile plastic sampling bag. The sample will

be homogenized with an equal volume of

0.1% peptone buffer, and serial dilutions

will be plated on appropriate Salmonella

selective agar using the FDA BAM method

(7).

6.h How many replicates are needed to

ensure confidence in data? Does

variability in proximate analysis or

production warrant more than two or

three replicate trials? Will multiple

variations of similar formulations be

tested? Has a statistical design for

choosing formulations been used

(block design, central composite,

etc.)?

Three replicate trials, pies made from

different batches of ingredients for each trial,

triplicate slices per trial. Separate slices will

be assayed for each sampling interval (n ~ 9

for each sampling interval).

Three replicate trials with three

samples at each interval were chosen

because of the inherent variability of

inoculating individual slices for each

sampling time interval.

7 Determine other controls

7.a Is use of surrogates appropriate or

necessary? If so, justify.

No surrogates are necessary.

7.b Are uninoculated controls needed to

assess spoilage or competitive

microflora or for other purposes?

Yes. An uninoculated pie for aerobic plate

counts and counts of yeasts and

molds.

7.c What other controls are necessary

(including negative or positive

growth controls)?

NA

8 Determine pass-fail criteria

8.a What are the pass-fail criteria? Must show ,1-log growth of Salmonella

throughout the 5-day testing period.

8.b What are the limits for use of the

results?

Would be applicable only to meringue pies

with very similar pH and aw in both the

filling and the meringue.

a NA, not applicable.

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APPENDIX I. FOOD PRODUCT CHECKLIST: MERINGUE TOPPING

Evaluation of the adequacy of thermal inactivation of pathogens of concern in meringue topping for lemon meringue pie

Considerations Response Additional comments

1 Determine the purpose of the study

1.a Exempt from time/temperature

control for safety (no refrigeration

required)

NAa

1.b Variance from any regulatory

requirements (e.g., holding for

.4 h without temperature control)

NA

1.c Validate lethality Validate lethality of meringue topping heat

treatment (baking).

1.d Verify that formulation will inhibit

microbial growth in refrigerated

foods or under mild temperature

abuse

NA

2 Collect information regarding the product

2.a What are the ingredients? Pie crust: flour, shortening, water, salt.

Filling: water, sugar, modified food starch,

corn syrup solids, margarine, lemon juice

solids, high fructose corn syrup, sodium

citrate, agar agar, potassium sorbate, natural

flavor, locust bean gum, artificial color

(FD&C yellow no. 5).

Meringue: unpasteurized egg whites, sugar,

cream of tartar.

2007 supplement to the 2005 Food

Code specifies that pasteurized egg

white be used for meringue. This is

an example illustrating an

inactivation challenge study and

could potentially be used to obtain

a variance for the use of

unpasteurized egg whites.

2.a.1 How consistent are the ingredients

from various sources, lot to lot?

Very consistent, same or similar lot to lot.

2.a.2 What are the pH, aw, and

proximate analysis (moisture, salt,

fat, protein, residual nitrite, etc.)

for product and/or individual

components?

Baked crust: pH 6.2, aw 0.45.

Cooked filling: pH 4.2, aw 0.88.

Raw meringue: pH 4.6, aw 0.93.

2.a.3 Do any of these values change

from preparation to consumption?

aw may decrease for the meringue at the

exposed surface.

2.a.4 If applicable, what are the

dimensions of cuts, pieces, etc?

NA

2.a.5 What is the normal microbial load

(species, etc.) at the beginning and

end of production?

Before cooking: aerobic plate count ,1,000

CFU/g.

After baking: aerobic plate count ,10

CFU/g.

2.a.6 Is there likelihood that

contamination may be internalized

in or distributed throughout

individual components?

Salmonella may be present in unpasteurized

egg whites used for meringue topping.

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APPENDIX I. Continued

Evaluation of the adequacy of thermal inactivation of pathogens of concern in meringue topping for lemon meringue pie

Considerations Response Additional comments

2.b What are the preparation steps? Mix dough, sheet, form, bake. Cook the

filling to set the starch, fill the baked crust,

cool to ambient temperature, spread

meringue evenly over filling and bake. Cool

to ambient temperature, package.

Product is prepared in a commercial

manufacturing facility, cooled to

room temperature, packaged and

shipped at ambient temperature.

2.b.1 Is the product an assembled

(multicomponent) product?

Yes.

2.b.2 Is there a microbial reduction step

that is validated? What are the

parameters associated with the

microbial reduction step? Are

there different microbial reduction

steps for different components?

Purpose of this study. All three components

(crust, filling, meringue) have heat

inactivation steps, but the crust gets heat

treated twice, the filling gets heat treated

twice and there is an added inactivation due

to the pH, and the meringue gets heat treated

once.

2.b.3 Is there a potential for

recontamination?

Very unlikely, controlled through GMPs at

the commercial manufacturing facility.

2.b.4 What is the variability in

parameters that affect lethality or

growth?

Low variability.

2.b.5 How is the product packaged? Paperboard box or plastic dome over an

aluminum pie plate.

2.b.6 Is the product cultured or

fermented? Does it contain starter

culture intentionally added?

No.

2.b.7 Does the product contain

antimicrobials (preservatives) or

other ingredients that might be

inhibitory, such as spices?

Sodium citrate and potassium sorbate in the

filling.

2.c What are the storage conditions?

2.c.1 How will the product be displayed

for sale? Any changes to

packaging for display?

Refrigerated or ambient, no change to

packaging.

2.c.2 What temperatures (and times) are

expected during production,

preparation, and storage or

display?

Crust cook: 85uC (185uF) final temperature,

15 min total cooking time in 176.7uC

(350uF) nonhumidified oven.

The cook time for the meringue is

based on the time required to

achieve the characteristic browning.

Filling set: 90.6uC (195uF) for 10 min.

Meringue set: 15 min total cooking time in a

preheated 176.7uC (350uF) oven.

2.c.3 What potential is there for storage

or display at temperatures greater

than those listed above in 2.c.2?

NA; purpose of this study is to validate

microbial reduction.

2.c.4 Are there other hazards that may

be created by preparation,

storage, or display?

No.

2.c.5 What is the estimated maximum

time from production to

consumption?

NA

2.c.6 What is the time to spoilage or

unacceptable quality?

NA

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APPENDIX I. Continued

Evaluation of the adequacy of thermal inactivation of pathogens of concern in meringue topping for lemon meringue pie

Considerations Response Additional comments

3 Determine if product assessment for growth or inactivation is needed

3.a Is a product assessment for growth

necessary based on pH and aw? (See

Appendix D, Tables A and B.) If

yes, also answer 4.e and 5.a.

NA

3.b Is an inactivation study needed? If

yes, also answer 4.f and 5.b.

Yes.

3.c Are there any regulations applicable

for lethality (inactivation) or TCS

(growth)?

2007 supplement to the 2005 Food Code

specifies that pasteurized egg white be used

for meringue.

4 Determine pathogens of concern to include in the challenge study

4.a According to Table 2 and Appendix

C, which pathogens are of concern?

If food is not seafood, Vibrio spp.

may be excluded from

consideration.

From Appendix C, pathogens of concern in

egg products are B. cereus, Salmonella, and

L. monocytogenes.

4.b Considering the ecology, product,

and epidemiological history, what

pathogens are reasonably likely to

occur? (Also see Appendix C.)

B. cereus spores would be expected to

survive the heat treatment but would not

grow out due to the aw of meringue.

Salmonella is more prevalent and present in

higher numbers than L. monocytogenes in

unpasteurized liquid egg products.

Salmonella has been associated with

numerous products containing undercooked

egg ingredients, including meringue pie (72).

For this study, we are concerned

with pathogen survival, not growth;

therefore, a pathogen with a low

infectious dose was chosen as the

challenge organism.

4.c What pathogens are likely to

recontaminate the product after the

inactivation step?

NA The objective of the study is to

evaluate inactivation and not

recontamination.

4.d Are there any baseline surveys that

indicate prevalence of pathogens for

the target product or a related

product?

In FSIS (111) risk assessments regarding

eggs and egg products, Salmonella estimates

in unpasteurized liquid whole egg product

were 0 and 100 cells per ml, on average, in

pooled product from multiple eggs (see Fig.

3-45 from the FSIS risk assessment). In

addition, estimates in unpasteurized liquid

egg white product were less than 10 cells per

ml, on average, in pooled product from

multiple eggs (see Fig. 3-44 from the FSIS

risk assessment). On some occasions MPNs

of Salmonella can exceed 1,000 CFU/ml for

both unpasteurized liquid whole egg and

liquid egg white products, but these events

appear to be rare. The FSIS identified

Salmonella Enteritidis in some but not all

samples collected and analyzed. Thus, the

Salmonella Enteritidis levels used in the

inactivation study described below would

represent a worst case.

For an inactivation study,

quantitative levels are more

important than qualitative

prevalence, as levels help estimate

the amount of kill necessary to

protect public health.

4.e For growth inhibition (TCS)

studies:

NA

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APPENDIX I. Continued

Evaluation of the adequacy of thermal inactivation of pathogens of concern in meringue topping for lemon meringue pie

Considerations Response Additional comments

4.e.1 Which pathogen(s) will grow the

fastest? Consider gram positive

versus gram negative; vegetative

microorganisms versus spore

formers. If food is not seafood,

Vibrio may be excluded from

consideration. Use a predictive

model or cite applicable literature.

Consider growth potential through

1.5 times the shelf life, if

appropriate.

4.e.2 Predictive model

4.e.3 Compare choice with literature

4.e.4 Any further information on growth

or survival?

4.e.5 Based on the above analysis, what

challenge organisms are chosen

for growth inhibition studies?

4.f If inactivation studies:

4.f.1 What is the lethal treatment?

(HPP, heat, acid, etc.)

Heat.

4.f.2 Which microorganisms are most

resistant to the lethal treatment?

(HPP, heat, acid, etc.)

Sporeformers would be most heat resistant

but have been eliminated as a risk due to

inability to grow in the product. Salmonella

Enteritidis is the most appropriate challenge

organism.

4.f.3 Will the lethality be delivered to all

areas of the product that may

contain the pathogen? Account for

all surface and internalized

contamination.

Yes.

4.f.4 What is in the formulation that

may affect inactivation? (Intrinsic

factors may contribute to lethality

or resistance: aw, moisture, salt,

pH, fat, etc.)

Relatively high sugar content of the

meringue will reduce the aw, potentially

leading to increased heat resistance.

4.f.5 Are there any data on pathogen

levels in the product?

See 4.d.

4.f.6 Is there a regulatory requirement

or policy for log reduction for this

product? Cite requirement.

No.

4.f.7 If there is no regulatory

requirement for log reduction, use

scientific basis for determining

acceptable reduction (21, 76).

Using 2-log reduction as worst case and

building in a 2-log margin of safety, the

target reduction is 4-log units.

4.f.8 Based on the above analysis, what

challenge organisms are chosen

for inactivation studies?

Salmonella Enteritidis.

5 Determine appropriate time and sampling intervals for challenge study

5.a For growth inhibition (TCS) studies,

use 1.25–1.5 times the shelf life as

testing time

NA

5.a.1 Maximum time from production to

consumption

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APPENDIX I. Continued

Evaluation of the adequacy of thermal inactivation of pathogens of concern in meringue topping for lemon meringue pie

Considerations Response Additional comments

5.a.2 Actual time to spoilage or

unacceptable quality

5.a.3 For growth inhibition studies,

determine appropriate sampling

intervals for microbial analysis;

use five to seven (preferred)

sampling intervals; fewer

sampling intervals should be

justified, e.g., using results from

similar products.

5.b For inactivation studies determine

appropriate sampling points

considering the process and

formulation. Identify populations at

0 time and end of processing;

whenever possible, include

intermediate sampling intervals to

determine death curve.

Times 0 and 15 min. Sampling at more than three time

points would allow a D-value to be

calculated that may be of use in

further defining the cook process

but is not in the current study

design.

5.b.1 When inactivation treatments may

result in sublethal injury, repair

and growth of microorganisms

during product shelf life should be

considered (21).

NA

6 Determine inoculation, storage, and testing procedures

6.a Determine strains for use in study

(multiple strains for each species

are recommended; consider use of

appropriate food or clinical isolates)

A mixture of at least five strains of

Salmonella isolated from eggs or egg

products and including at least one

Salmonella Enteritidis isolated from clinical

or egg samples associated with outbreaks.

6.b Determine if adaptation is required

for inoculum preparation

Not necessary for this study.

6.c Determine method of inoculation

(surface, mixing, dipping, liquid,

dry, etc.)

Will mix concentrated, washed inoculum

into the egg whites before beating to a

meringue. Finished meringue will be

weighed and spread evenly over the surface

of a cooked lemon filling that has been

cooled to room temperature. The

concentration of Salmonella in the uncooked

finished meringue will be determined as

described below.

Because of the potential for aerosols,

beating of egg whites should be

done in a biological safety cabinet.

The assumption is also made that

the filling will not be chilled below

ambient temperature prior to

application of the meringue.

6.d Determine size of inoculum

(populations, e.g., log CFU/g, CFU

per package, percentage of

inoculum vol/wt or vol/vol)

A final target level of at least 4 log CFU per

pie.

6.e Determine packaging to be used NA

6.f Determine the incubation

temperature for growth inhibition

studies or temperature(s) for

thermal inactivation studies

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APPENDIX I. Continued

Evaluation of the adequacy of thermal inactivation of pathogens of concern in meringue topping for lemon meringue pie

Considerations Response Additional comments

6.g Determine sampling method and

sample size

The sample size will be the whole meringue

from a single pie. The whole meringue will

be enriched for Salmonella using the BAM

method (7). In addition, at T0 one pie will be

used to determine the initial number of

Salmonella recovered in the meringue prior

to baking by removing the meringue from

the pie, mixing thoroughly, and taking three

10-g samples of the meringue for

enumeration.

6.h How many replicates are needed to

ensure confidence in data? Does

variability in proximate analysis or

production warrant more than two or

three replicate trials? Will multiple

variations of similar formulations be

tested? Has a statistical design for

choosing formulations been used

(block design, central composite,

etc.)?

There will be three replicate trials. Each trial

will consist of three inoculated baked pies

plus one T0 unbaked pie; thus, a total of 12

pies will be needed for the study.

7 Determine other controls

7.a Is use of surrogates appropriate or

necessary? If so, justify.

No surrogates are appropriate.

7.b Are uninoculated controls needed to

assess spoilage or competitive

microflora or for other purposes?

NA

7.c What other controls are necessary

(including negative or positive

growth controls)?

Temperature will be verified in several

places in the oven during baking.

8 Determine pass-fail criteria

8.a What are the pass-fail criteria? Must achieve .4-log reduction within

15 min in a 176.7uC (350uF) oven.

Based on nondetection of Salmonella

upon enrichment of the meringue.

8.b What are the limits for use of the

results?

Limitations of this study include the volume

and depth of the meringue on the pie. The

temperature of the filling may impact the

results. The data will apply for longer but not

shorter cook times at the oven temperature

indicated. These data could apply to other

types of filling.

a NA, not applicable.

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 195

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APPENDIX J. FOOD PRODUCT CHECKLIST: BAKED CHEESE PIZZA

Evaluation of baked cheese pizza held out of refrigeration for up to 8 h

Considerations Response Additional comments

1 Determine the purpose of the study.

1.a Exempt from time/temperature

control for safety (no refrigeration

required)

NAa

1.b Variance from any regulatory

requirements (e.g., holding for .4 h

without temperature control)

Holding of baked cheese (sliced) pizza

without refrigeration for up to 8 h.

1.c Validate lethality NA Assembled at another facility (central

commissary) and held refrigerated

until baked at retail store.

1.d Verify that formulation will inhibit

microbial growth in refrigerated

foods or under mild temperature

abuse

NA

2 Collect information regarding the product

2.a What are the ingredients? Pizza crust: flour, salt, shortening.

Cheese: pasteurized milk, salt, rennet,

starter cultures.

Tomato sauce: canned tomato paste, water,

oregano, basil, garlic.

2.a.1 How consistent are the ingredients

from various sources, lot to lot?

Ingredients same; relatively consistent

composition of sauce and cheese but

preparation of pizza may vary considerably

with respect to amounts of ingredients.

2.a.2 What are the pH, aw, and proximate

analysis (moisture, salt, fat, protein,

residual nitrite, etc.) for product

and/or individual components?

Proximate analysis: 10% protein, 8% fat,

1% salt, 46–49% moisture

pH: crust, 6.8; sauce, 4.5; cheese, 5.4

aw: crust, 0.70; sauce, 0.98; cheese, 0.95–

0.96.

2.a.3 Do any of these values change from

preparation to consumption?

No.

2.a.4 If applicable, what are the

dimensions of cuts, pieces, etc?

NA

2.a.5 What is the normal microbial load

(species, etc.) at the beginning and

end of production?

Microbial load: ,100 CFU/g after baking;

primarily spore-forming microorganisms

potentially including B. cereus, C.

perfringens, and C. botulinum.

2.a.6 Is there likelihood that

contamination may be internalized

in or distributed throughout

individual components?

Yes, each of the components is likely to

contain pathogenic bacterial spores.

2.b What are the preparation steps?

2.b.1 Is the product an assembled

(multicomponent) product?

Yes. Product consists of a thin crust

covered with sauce and topped with a layer

of cheese.

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APPENDIX J. Continued

Evaluation of baked cheese pizza held out of refrigeration for up to 8 h

Considerations Response Additional comments

2.b.2 Is there a microbial reduction step

that is validated? What are the

parameters associated with the

microbial reduction step? Are there

different microbial reduction steps

for different components?

No further kill step after pizza is baked.

Baking has been validated to eliminate all

vegetative bacterial pathogens.

2.b.3 Is there a potential for

recontamination?

Yes, there is potential for recontamination

once the pizza cools from the baking

process and is handled by food service

workers.

Due to the large surface area, the

pizza is expected to cool to room

temperature rapidly after baking;

therefore, growth of C. perfringens

is not a concern.

2.b.4 What is the variability in parameters

that affect lethality or growth?

Little variability in parameters that affect

lethality if baked to an endpoint of visual

doneness.

2.b.5 How is the product packaged? Not packaged. Trays containing pizzas are

shipped from commissary to food service

establishment.

2.b.6 Is the product cultured or

fermented? Does it contain starter

culture intentionally added?

No.

2.b.7 Does the product contain

antimicrobials (preservatives) or

other ingredients that might be

inhibitory, such as spices?

NaCl is present but not at inhibitory levels.

No antimicrobials are added.

2.c What are the storage conditions?

2.c.1 How will the product be displayed

for sale? Any changes to packaging

for display?

Held in an enclosed display cabinet where

the maximum temperature is 30uC (86uF).

2.c.2 What temperatures (and times) are

expected during production,

preparation, and storage or

display?

Only the baked product holding

temperature is relevant; in this instance,

30uC (86uF) for up to 8 h at retail.

2.c.3 What potential is there for storage

or display at temperatures greater

than those listed above in 2.c.2?

There is the possibility that product will be

held at temperatures as great as 40uC

(104uF), but quality deterioration would

occur in less than 8 h.

2.c.4 Are there other hazards that may be

created by preparation, storage, or

display?

L. monocytogenes contamination from the

environment may occur; handling can

result in contamination with S. aureus.

2.c.5 What is the estimated maximum time

from production to consumption?

Maximum 8 h store display; 2 h from sale

to consumption (total of 10 h).

2.c.6 What is the time to spoilage or

unacceptable quality?

Product is of acceptable quality for the

duration of the study, even though it may

appear to be dried out. Little is known

about unacceptable quality parameters for

pizza and what consumers may determine

to be of unacceptable quality. In

accordance with general food safety

practices, food should be consumed or

refrigerated within 2 h of purchase.

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 197

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APPENDIX J. Continued

Evaluation of baked cheese pizza held out of refrigeration for up to 8 h

Considerations Response Additional comments

3 Determine if product assessment for growth or inactivation is needed

3.a Is a product assessment for growth

necessary based on pH and aw? (See

Appendix D, Tables A and B.) If yes,

also answer 4.e and 5.a.

Yes, product assessment required; Food

Code Table B is applicable because of

potential recontamination and survival of

spores; pH . 5.0 and aw . 0.92 in parts of

the product and product not protected from

recontamination.

Multicomponent product. Crust has

low aw, but aw will be increased by

moisture from sauce. Sauce also

lowers the pH of the crust. Moisture

loss of product occurs over time.

3.b Is an inactivation study needed? If

yes, also answer 4.f and 5.b.

No, the purpose of this study is to

determine if pathogens likely to be present

will grow in the product if stored out of

refrigeration.

3.c Are there any regulations applicable

for lethality (inactivation) or TCS

(growth)?

Latest edition of the Food Code for TCS.

4 Determine pathogens of concern to include in the challenge study

4.a According to Table 2 and Appendix

C, which pathogens are of concern?

If food is not seafood, Vibrio spp.

may be excluded from consideration.

Based on a measured pH of 5.4 and a

maximum aw of 0.96 for cheese, the

organisms of concern are B. cereus, C.

botulinum, pathogenic E. coli, L.

monocytogenes, Salmonella, and S. aureus.

Vibrio spp. were excluded from

consideration since seafood is not

involved.

Based on a measured pH of 5.3 and a

maximum aw of 0.98 at the cheese-sauce

interface, same organisms as above.

Based on a measured pH of 5.0 and a

maximum aw of 0.97 at the sauce-crust

interface, same organisms as above.

4.b Considering the ecology, product,

and epidemiological history, what

pathogens are reasonably likely to

occur? (Also see Appendix C.)

B. cereus and C. botulinum spores survive

baking; L. monocytogenes and S. aureus

may be present from postprocessing

handling.

No known illnesses have occurred from

consumption of cheese pizza. However,

illnesses due to E. coli O157:H7 were

associated with frozen pepperoni pizza,

although the cause of the outbreak was

undetermined (70).

Pathogenic E. coli and Salmonella

are inactivated during adequate

baking. They are also not likely to

be present in the environment and

therefore recontamination of the

cheese pizza with these organisms

is unlikely.

C. botulinum was excluded from

consideration because of the aerobic

conditions and the reduced pH

levels and because spores of B.

cereus are more common and likely

to grow faster.

4.c What pathogens are likely to

recontaminate the product after the

inactivation step?

Study is designed to determine safety if

recontamination should occur.

4.d Are there any baseline surveys that

indicate prevalence of pathogens for

the target product or a related

product?

No.

4.e For growth inhibition (TCS) studies:

4.e.1 Which pathogen(s) will grow the

fastest? Consider gram positive

versus gram negative; vegetative

microorganisms versus spore

formers. If food is not seafood,

Vibrio may be excluded from

consideration. Use a predictive

model or cite applicable literature.

Consider growth potential through

1.5 times the shelf life, if appropriate.

No one organism was determined to grow

faster. See 4.e.2.

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APPENDIX J. Continued

Evaluation of baked cheese pizza held out of refrigeration for up to 8 h

Considerations Response Additional comments

4.e.2 Predictive model Cheese surface at pH 5.4, aw 0.96, 27uC

(80.6uF): PMP 7.0 version 1.1 predicts a 3-

log increase in S. aureus within 29 h (22 h

without lag) under aerobic conditions;

ComBase Predictor predicts a 3-log

increase in S. aureus within 18 h for the

same conditions. For L. monocytogenes,

PMP predicts a 1-log increase within 42 h

for the same conditions (7 h without lag);

ComBase Predictor with 5,000 ppm of

lactic acid predicts a 1-log increase within

33 h for the same conditions. PMP does

not include B. cereus predictions at aw

0.96, but ComBase Predictor with 40%

CO2 predicts a 3-log increase in B. cereus

within 101 h.

For the sauce-crust interface (pH 5.0, aw

0.97), PMP predicts a 3-log increase in S.

aureus within 34 h (approximately 23 h

without lag) under aerobic conditions;

ComBase Predictor predicts a 3-log

increase in S. aureus within 23 h

(approximately 16 h without lag) for the

same conditions. For L. monocytogenes,

PMP predicts a 1-log increase within 52 h

for the same conditions (approximately 9 h

without lag); ComBase Predictor with

5,000 ppm of lactic acid predicts a 1-log

increase in 34 h (within 13 h without lag)

for the same conditions. PMP predicts a 3-

log increase in B. cereus in approximately

21 h (8.5 h without lag); ComBase

Predictor with 40% CO2 predicts a 3-log

increase in B. cereus in 85 h (in 41 h

without lag) for the same conditions.

For the cheese-sauce interface (pH 5.3, aw

0.98), PMP predicts a 3-log increase in S.

aureus within 22 h (approximately 16 h

without lag) under aerobic conditions;

ComBase Predictor predicts a 3-log

increase in S. aureus within 15 h (10 h

without lag) for the same conditions. For L.

monocytogenes, PMP predicts a 1-log

increase within 22 h for the same

conditions (approximately 5 h without lag);

ComBase Predictor with 5,000 ppm of

lactic acid predicts a 1-log increase in 19 h

(within 8 h without lag) for the same

conditions. PMP predicts a 3-log increase

in B. cereus in approximately 15 h (10 h

without lag); ComBase Predictor with 40%

CO2 predicts a 3-log increase in B. cereus

within 42 h (21 h without lag).

4.e.3 Compare choice with literature

4.e.4 Any further information on growth

or survival?

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 199

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APPENDIX J. Continued

Evaluation of baked cheese pizza held out of refrigeration for up to 8 h

Considerations Response Additional comments

4.e.5 Based on the above analysis, what

challenge organisms are chosen for

growth inhibition studies?

L. monocytogenes, S. aureus,

and B. cereus.

Modeling results suggest that L.

monocytogenes, S. aureus, and B.

cereus are all likely candidates for a

challenge study, and none could be

completely excluded from

consideration based on modeling

alone.

4.f If inactivation studies: NA

4.f.1 What is the lethal treatment? (HPP,

heat, acid, etc.)

4.f.2 Which microorganisms are most

resistant to the lethal treatment?

(HPP, heat, acid, etc.)

4.f.3 Will the lethality be delivered to all

areas of the product that may

contain the pathogen? Account for

all surface and internalized

contamination.

4.f.4 What is in the formulation that may

affect inactivation? (Intrinsic factors

may contribute to lethality or

resistance: aw, moisture, salt, pH,

fat, etc.)

4.f.5 Are there any data on pathogen

levels in the product?

4.f.6 Is there a regulatory requirement or

policy for log reduction for this

product? Cite requirement.

4.f.7 If there is no regulatory requirement

for log reduction, use scientific

basis for determining acceptable

reduction (21, 76).

4.f.8 Based on the above analysis, what

challenge organisms are chosen for

inactivation studies?

5 Determine appropriate time and sampling intervals for challenge study

5.a For growth inhibition (TCS) studies,

use 1.25–1.5 times the shelf life as

testing time

10 h | 1.5 ~ 15 h.

5.a.1 Maximum time from production to

consumption

Maximum 10 h.

5.a.2 Actual time to spoilage or

unacceptable quality

NA

5.a.3 For growth inhibition studies,

determine appropriate sampling

intervals for microbial analysis; use

five to seven (preferred) sampling

intervals; fewer sampling intervals

should be justified, e.g., using

results from similar products.

Sample at 0, 4, 8, 10, 15 h.

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APPENDIX J. Continued

Evaluation of baked cheese pizza held out of refrigeration for up to 8 h

Considerations Response Additional comments

5.b For inactivation studies determine

appropriate sampling points

considering the process and

formulation. Identify populations at 0

time and end of processing; whenever

possible, include intermediate

sampling intervals to determine death

curve.

NA

5.b.1 When inactivation treatments may

result in sublethal injury, repair and

growth of microorganisms during

product shelf life should be

considered (21).

6 Determine inoculation, storage, and testing procedures

6.a Determine strains for use in study

(multiple strains for each species are

recommended; consider use of

appropriate food or clinical isolates)

Multistrain mixtures for L. monocytogenes,

S. aureus, and B. cereus will be used. Each

pathogen composite will be tested

individually (i.e., inoculate one set of

samples with L. monocytogenes composite,

inoculate a different set of samples with S.

aureus composite, etc.).

6.b Determine if adaptation is required

for inoculum preparation

No. Although sauce pH is low, L.

monocytogenes comes from the

environment and would not be

adapted to acid. Adaptation is not a

concern for S. aureus or B. cereus.

6.c Determine method of inoculation

(surface, mixing, dipping, liquid, dry,

etc.)

Slice an entire pizza into 16 individual

slices (approximately 75 g each). (Assume

that the pizza is approximately 1,200 g.)

Individual slices of pizza will be inoculated

on the surface and the sliced edge with S.

aureus, L. monocytogenes, or B. cereus.

Each replicate will require 10

inoculated slices (2 for each

sampling time interval) and 5

control slices for each organism

tested.

6.d Determine size of inoculum

(populations, e.g., log CFU/g, CFU

per package, percentage of inoculum

vol/wt or vol/vol)

Not less than 2 log CFU/g for L.

monocytogenes, S. aureus, or B. cereus,

surface inoculated, including the cut

surface, delivered by spot inoculation

(several 50-ml spots).

As noted above, each organism will be

inoculated independently to avoid possible

antagonist effect between different

organisms.

Inoculum level is high considering

the likelihood of contamination but

will allow enumeration by direct

plating and detection of growth and

low levels of inactivation by

formulation during storage;

inoculum volume no more than 1%

of sample size; preliminary data

suggest inoculum does not change

pH and aw appreciably.

6.e Determine packaging to be used Product is not packaged during typical

display but should be protected from the

environment during the study by placing in

a cardboard or plastic pizza container with

a loose fitting lid.

6.f Determine the incubation

temperature for growth inhibition

studies or temperature(s) for thermal

inactivation studies

Incubated at 30uC (86uF). Maximum temperature product will

be exposed without adverse changes

in product quality that would deter

purchase and consumption.

6.g Determine sampling method and

sample size

Analyze an entire slice of pizza

(approximately 75 g).

Slices will be tested for S. aureus,

B. cereus, and L. monocytogenes

according to methods provided in

Appendix A.

J. Food Prot., Vol. 73, No. 1 GUIDANCE FOR INOCULATED PACK/CHALLENGE STUDY PROTOCOLS 201

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APPENDIX J. Continued

Evaluation of baked cheese pizza held out of refrigeration for up to 8 h

Considerations Response Additional comments

6.h How many replicates are needed to

ensure confidence in data? Does

variability in proximate analysis or

production warrant more than two or

three replicate trials? Will multiple

variations of similar formulations be

tested? Has a statistical design for

choosing formulations been used

(block design, central composite, etc.)?

Three replicate (unique production) lots

(i.e., three whole pizzas) per organism

tested; duplicate samples (slices) per testing

interval.

Greatest variability likely occurs in

the production of different lots of

pizza.

7 Determine other controls

7.a Is use of surrogates appropriate or

necessary? If so, justify.

No surrogates used.

7.b Are uninoculated controls needed to

assess spoilage or competitive

microflora or for other purposes?

Uninoculated controls will be used to

monitor other spoilage microorganisms that

can change pH during testing interval.

7.c What other controls are necessary

(including negative or positive growth

controls)?

8 Determine pass-fail criteria

8.a What are the pass-fail criteria? No more than a 1-log increase for L.

monocytogenes; no more than a 3-log

increase for S. aureus or B. cereus.

A 1-log increase in L.

monocytogenes is considered

significant growth, but note that L.

monocytogenes detectable in 25 g of

a ready-to-eat food would render the

product adulterated.

Maximum 3-log increase selected for

S. aureus and B. cereus are based on

increases suggested in the IFT report

(53).

Some regulatory agencies may

consider a lower log increase to be

actionable.

8.b What are the limits for use of the

results?

These results are applicable only to cheese

pizza with tomato sauce and not to pizza

containing meat or vegetable toppings.

Minor variations in the amount of

cheese or tomato sauce are not likely

to have a significant impact on

growth of the test organisms.

a NA, not applicable.

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doi:10.4315/0362-028X.74.4.522

ERRATUM

In the article ‘‘Parameters for Determining Inoculated Pack/Challenge Study Protocols,’’ by the National Advisory Committee on

Microbiological Criteria for Foods, which appeared in the Journal of Food Protection, 73(1):140–202, there are additions that need to

be made to Table 2 on p. 144.

For an aw value of .0.96 and a pH value of 4.2–4.6, L. monocytogenes should be added to the list of potential pathogens so that it

includes Pathogenic E. coli, Salmonella, S. aureus, and L. monocytogenes.

d

The footnote designator d should be added to all three L. monocytogenes entries in the pH 4.2–4.6 column and the following

footnote added: d L. monocytogenes should not be used if the pH of the product is ,4.4.

522

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