It is no secret that the food industry is under enormous pressure to find new ways to meet ever-increasing consumer demand for food and beverage products that are more “fresh” in appearance and taste, more flavorful and exotic, more conveniently produced and packaged to prepare and eat, with less preservatives but that travel further, last longer and are safe. Traditional retorting of products by food manufactures, which helps extend shelf life but results in a “canned taste” product has gone the way of the dinosaurs, as have conventional, more localized channels of distribution, in many cases.

With the formulation and production of these fresher, ready-to-eat items, newer issues pertaining to foodborne illness causing pathogens and spoilage have emerged.  Today, more than ever before, the onus is placed on food processors to develop effective ways of making products that are both safe and have a reasonable, perhaps even extended, shelf life.  Conducting a microbial challenge study can provide valuable information to food processors striving to meet these market challenges.

A microbial challenge study is used to simulate what happens to a product during processing, distribution and subsequent preparation and handling should it become contaminated. A properly carried out microbiological challenge study is performed by inoculating selected microorganisms into a food or formulation to determine if the organisms would present a potential health hazard or spoilage risk. Knowing when to perform a challenge study, how to design and conduct such a study, and how to interpret the results assist food processors in showing that their products are of high quality, stable, and safe in addition to assuring that they meet regulatory mandates or established finished product specifications.

Why Perform a Challenge Study?
Microbial challenge studies are being conducted more frequently by the food industry. The reasons for conducting a challenge study can be as varied as the food products studied, although the impetus to initiate such a study can be summed up in two words, “safety” and “quality.” A microbial challenge study helps the processor ensure the safety of any new or reformulated food product in which potentially hazardous microorganisms might be present in low or incidental numbers. It also helps to determine whether a food has the ability to “kill-off” any pathogens or toxins that may accidentally enter the product.

Historically, physical and chemical barriers, e.g., pH, salt, and water activity (a_w), have been relied upon to inhibit the growth of pathogens and extend product shelf life. The systematic reduction of these barriers in some products has raised safety concerns among regulators and demonstrated the need for processors to conduct challenge testing. Even if hazardous microorganisms are not expected to be present, performing a challenge study assists the food company in understanding how a product’s shelf life will be affected by an increased level of spoilage organisms due to accidental contamination.

The information that can be gleaned from challenge studies include proper product code dating and confirmation that changes in a product’s formulation, processing or packaging will inhibit microbial growth. The data can also be used as a basis for setting safety criteria at the critical control points (CCPs) in a food processing operation and finished product testing specifications.

When to Perform Challenge Studies
The decision whether to conduct a challenge study for a food must be based on an evaluation of a product’s susceptibility to pathogenic growth and spoilage. Does the product actually meet the shelf life expectation or goal set by the processor? Is the processor seeing spoilage in a product at the specified shelf life date, but due to changed circumstances in formulation, packaging, distribution, storage or preparation methods now requires an extended shelf life? Is there a certain essential risk associated with the product that must be addressed as per U.S. Food and Drug Administration (FDA) Food Code guidance? Depending on the processor’s answers to these questions— no, yes, and yes—a microbial challenge study should be considered.

The type of food product, processing methods and formulation of the product all play a role in this decision, as well. Some types of food products can be excluded from such studies, such as frozen foods because most microorganisms do not grow at extremely low temperatures and canned goods since the retort process these products undergo destroys harmful microorganisms. The products best suited for challenge study investigations are those that are stored at refrigeration or room temperatures (i.e., shelf stable) and are vulnerable to spoilage organisms and/or pathogenic growth. For pathogens, this includes any food that has a pH ≥ 4.6 or a_w ≥ 0.85 under the FDA Food Code. Food products typically included in studies range from shelf-stable salad dressings and condiments, refrigerated ready-to-eat (RTE) products like pasta entrees and deli salads, dairy products and juice drinks, to modified atmosphere packaged (MAP) products such as vegetables, meats, poultry and fish, bakery items (i.e., icings, non-fruit pies) stored at room temperature, confectionery and food formulated using new preservatives.

In addition, microbial challenge studies should be conducted when the processor requires useful information about specific processing protocols, particularly in facilities where raw materials are on the plant floor or when a heat or lethal step must be validated.

Finally, the formulation stage of a product is an ideal time to conduct a microbial challenge study. The data obtained about shelf stability, storage, preservatives and additives, and packaging help the processor to establish sound finished product specifications.

Challenge Study Design
The design of the challenge study is one of the most critical steps in conducting an effective and scientifically sound investigation. Unfortunately, there is no easy way to design these studies given the fact that there are so many ways of doing it. As a result, the possibility of generating inaccurate data can increase substantially depending on the number of variants involved, and processors should be aware of the potential problems this can cause. These problems generally occur when short cuts are taken at the design phase; for example, if the product to be challenged has a six- month shelf life but the study period allotted is only three months, or the product is tested for the wrong type of organism, or the wrong test method is used. Standardization of microbial challenge test design and methods is one way to reduce the incidence of these problems, and while there are few such standardized approaches currently available, the benefits of a universally accepted, scientifically based approach for specific products are many and should spur the development of more such standards.

In 2000, NSF International introduced ANSI/NSF Standard 75 for Non- Potentially Hazardous Foods, which serves as an excellent example of the advantages of standardization that will benefit challenge study design and performance. The development of the standard was necessitated by increased regulatory scrutiny of bakery products such as soft pies and filled pastries that could present food safety risks when stored or displayed without refrigeration. NSF research indicates that in some cases, health inspectors were requiring the removal of certain products from non- refrigerator displays, resulting in lost sales. (Studies have shown a 40% increase in product movement when merchandise is stored in high-traffic, non-refrigerated display units.) The most significant problem identified was the lack of universally accepted methods to assure which bakery products were non- potentially hazardous, particularly with regard to assuring regulators that these products could be stored without temperature controls by retailers for a specified period of time.

ANSI/NSF Standard 75 provides test methods and evaluation criteria to allow for the determination that a product does not require refrigeration for safe storage and display. One of the advantages of this standardization is that it is geared to a specific type of product with a specific problem, which means that the manufacturer does not have to reinvent the method and study design wheel that others with similar products have already established. This standard contains requirements for baked goods that are intended to be held without temperature controls during transportation, holding, display, sale or use; are considered to be potentially hazardous; and are rendered non-potentially hazardous by formulation or through a manufacturing process, or both. Manufacturers that produce goods in the NSF Standard 75-specified food categories (specialty breads or pastries containing fresh, canned, frozen, or rehydrated vegetables or soft cheeses added prior to baking; bakery products including specialty breads or pastries filled or topped with cream, crème, custard or cheese after baking; products filled prior to baking such as pumpkin, sweet potato, custard or meringue pies; and components such as toppings, glazes, icings or fillings stored without temperature control prior to use in other products) now will potentially perform shelf life/microbial challenge studies in the same way, making it much easier for regulators to make a decision about the study’s validity.

When a processor determines that a challenge study is beneficial for a particular product, a number of factors must be considered for its design, including the selection of appropriate challenge organisms; the duration of the study; the level of inoculum; the storage temperature; and the packaging of samples.   

Selection of Appropriate Challenge Organisms. The pH, a_w, projected holding temperature and epidemiological history of a product are used to gauge its susceptibility to microbial growth. Ideal challenge organisms are typically those that have been isolated previously from similar foods that have experienced microbiological problems due to spoilage organisms and/or pathogenic organisms. Established measurements of minimum growth conditions for spoilage organisms—yeasts, molds and lactic acid bacteria—coupled with data on predominant spoilage organisms by food group are good indicators of the appropriate challenge organisms to be used (Tables 1 and 2).

The selection of appropriate pathogens for a challenge study involves a careful review of data regarding the incidence of foodborne illness, mortality and number of recalls associated with the product to be challenged. Published statistics and laboratory experience indicate that the top pathogenic organisms used in challenge studies are Salmonella, Listeria monocytogenes and E. coli O157:H7, followed by Clostridium botulinum, Staphylococcus aureus, Bacillus cereus and Clostridium perjfrigens (Table 3). Salmonella is of special concern because it is tuned to survive in foods under myriad conditions. Listeria monocytogenes is of concern because it grows in refrigerated conditions, and scientists are watching E. coli begin to adapt to new conditions in which they wouldn’t expect the pathogen to grow or survive. Reviewing recall data, such as a survey of FDA Class I-III recalls by microbiological agent between January 1999 and December 2000 which shows the top three pathogens of concern as Listeria (44%), Salmonella (26%) and yeast/mold (15%), also aids in the selection process.

Finally, attention must be paid to the food formulation or product component factors (pH, water activity, packaging and storage temperature) in the selection of appropriate pathogens for the study. With regard to the first two components—pH and water activity—the FDA Food Code provides some guidance. It defines a potentially hazardous food as “a food natural or synthetic that requires temperature control because it is in a form capable of supporting the growth of… infectious or toxigenic microorganisms the growth and toxin production of Clostridium botulinum… and the growth of Salmonella enteritidis.” This definition does not include “a food for which laboratory evidence demonstrates that the rapid and progressive growth of infectious or toxigenic microorganisms, or the growth of S. enteritidis in eggs or C. botulinum cannot occur, such as a food that has an a_w of (0.85 or less), or a pH of (4.6 when measured at 24°C) or less.” NSF International also publishes useful guidelines based on the minimum pH and minimum water activity of product components (Table 4).   

The second product component, storage temperature, is a factor in cases where a product is likely to allow for the growth of psychrotrophic pathogens, such as Listeria monocytogenes, non-proteolytic Clostridium botulinum, Yersinia enterocolitica, Bacillus cereus and Aeromonas hydrophilia.  

The third product component—the type of packaging in which a product is stored—also significantly influences the selection of challenge organisms. Packaging determines the amount of oxygen and other gases available for microbial growth in a food. A product packaged in modified atmosphere or under vacuum may be susceptible to growth by anaerobic organisms (e.g., Clostridium botulinum) or microaerophilic spoilage organisms (e.g., lactic acid bacteria). In addition, products stored in an aerobic environment (bakery goods, fresh meats, or produce in a retail display case) are more susceptible to growth by aerobic organisms, such as Pseudomonas and molds. Facultative anaerobes, such as Salmonella and E. coli O157:H7, can grow with or without oxygen and must be considered for challenge studies encompassing all types of packaging. While packaging is often analyzed for oxygen and gas headspace and MAP packages can be prepared with any gas type or ratio with specialized equipment, these measurements can affect the outcome of a microbial challenge study and thus, it is recommended that manufacturers provide the actual finished product in its package for the study.

Following the selection of appropriate challenge organisms, specific strains of the organisms are chosen for the study. In general, using composite cultures of at least five strains of an organism will provide more information about the susceptibility of the product to an organism than an individual strain. For instance, a syrup product may be resistant to one yeast, Sactharomyces cerevisiae, but susceptible to the growth of Zygosactharomyces rouxii. The use of multiple strains in a composite increases the confidence that the challenge study reflects the susceptibility of the product in real-life situations. For spoilage organisms, isolates from the formulation being analyzed best represent the typical product.

Level of Inoculum. If the objective of the study is to determine product stability, 10,000 colony forming units per gram (cfu/g) is usually an appropriate level of inoculum to use. Microbes exhibit a period of “no growth” in most susceptible foods, and some challenge organisms may die immediately after being added to the test sample. Given that the detection limit of the plating methods used to enumerate organisms is 10 cfu/g, accurate counts may be compromised if low inocula levels are used. The use of 10,000 cells per gram will prevent the erroneous conclusion that a product is stable in cases in which the sample had a low initial inoculation level and die-off occurred. If the aim is to demonstrate a significant reduction in organisms, as with the validation of a heat step or Juice processing, one to 10 million organisms per gram are typically inoculated into the product.

Duration and Number of Analyses. A challenge study should be conducted for the length of a product’s expected shelf- life. The number and timing of analyses is based on studies conducted with similar products. The product should be evaluated at intervals, with a minimum of five to seven data points included in the study in order to record significant changes in counts. If the shelf-li1 is counted in days, tests should be conducted daily. If the product has a shelf life measured in weeks or months, the test intervals should be once per week, at minimum.

Storage Conditions of Samples. If a product is expected to be held at refrigeration temperature, challenge study samples should also be held at refrigeration temperatures. The same holds true if the product is expected to be held at room temperature. A product prone to temperature abuse (e.g., stored in hot warehouses or held in substandard refrigerated retail cases) should also be challenged at those temperatures.

Packaging of Samples, Relative Humidity. Again, test samples should be placed in the same packaging as the final product will be delivered to retailers to ensure that the challenge study results reflect as accurately as possible real-world conditions. However, if the formulation being tested is expected to be shelf stable, then it is advisable to use sterile containers. Although the disadvantage is that the study is not simulating typical storage conditions, the advantage is that sample variation is eliminated.

Analyzing the Samples and Data Interpretation
Deciding which type of culture media to use is based on whether or not background microflora is present in the product sample. The use of a non-selective versus selective agar is always preferred because it allows for the recovery of injured but viable cells. However, if there is background microflora in the product, a selective agar, with or without non-selective underlay, must be used in order to isolate the challenge organism from the organisms already present in the product.

The product sample is then inoculated using aseptic technique. It is important that the product be inoculated at all appropriate surface and internal points where microbial growth can potentially occur. Figure 1 illustrates the points of inoculation for a chocolate meringue pie, including those points where filling and crust and filling and meringue interface. Such interface points should be included as inoculation points in the challenge study.

The interpretation of collected data from the challenge study is based on an evaluation of trends. An increase of one log (from 10,000 to 100,000) is generally considered significant if the increase is noted at two or more intervals. Normal sample variation may lead to a spike in counts at one time interval, and need not be significant. If the counts of organisms stay the same or decrease during the study, the product is considered to be microbiologically stable against challenge with those organisms for that time period. Microbiologically stable organisms that survive, but do not grow, may represent potential health hazards if present at significant levels.

Challenge Studies in the Real World
The following case studies taken from actual challenge studies illustrate some of the top reasons that food processors have these studies conducted, as well as concepts discussed earlier.

Case Study 1: Deli Salad. A national food chain prepares a deli salad with a shelf life of 28 days for retail sale and would like to determine the safety of the product.   

Listeria monocytogenes is selected as the challenge organism for this refrigerated product. The duration of the challenge study is set at 28 days (the expected shelf life). The deli salad is inoculated and tested every three days. As shown in Figure 2, the product was stable until Day 15, after which the Listeria counts increased dramatically. With this data, the food processor could then make the decision whether or not to reformulate the product or whether to set a shelf life of 15 days.

Case Study 2: Pie Safety. A pie company would like to hold their pumpkin pies in grocery stores at ambient temperature for five days during the Thanksgiving rush. Is that safe to do?

The study was conducted using NSF Standard 75. In order to make a decision about which organisms with which to challenge, the pH and water activity of the different pie components were measured. In this case, the pH and aw of the crust are 5.45 and 0.397, respectively; the pH and aw of the filling are 4.81 and 0.94 1, respectively. These numbers indicated that the pumpkin pie should be challenged with four organisms: Staphylococcus aureus, Salmonella, Listeria and Bacillus cereus. Following the determination of the challenge organisms, the standard was followed. The product was tested for 1.3 times the five-day shelf life. Figure 3 shows that this pie met the requirements of the standard; in other words, the organisms did not increase by a log, which is generally considered a problem. The pie company now had a green light to safely hold its pies for five days at room temperature.

Case Study 3: Salad Dressing Stability. A company produces a Ranch salad dressing and would like to store the product at room temperature for six months. Is the product safe and stable?   

A wide variety of shelf-stable products that are highly acidic or have vinegar components (salad dressings, mustards, ketchups, barbeque sauces, and so on) undergo microbial challenge testing to determine whether spoilage organisms like yeasts, molds and lactic acid bacteria will grow during the expected shelf life. In this case, the expected shelf life of the salad dressing was six months and the duration of the study was set for this amount of time. Although the temptation to accelerate the shelf life study can exist, it is important to restate that challenge studies be conducted for the length of the expected shelf life. In the case of high-acid products like salad dressings, experience has shown that yeast and lactic acid bacteria may not start to grow until 12-16 weeks into the study. Initial counts may decrease to less than 10 cfu/g, only to increase again to more than a log difference later in the study. Figure 4 shows yeast beginning to grow in the product at approximately Week 12, followed by a significant increase in yeast counts in the next few weeks. The study demonstrated that the salad dressing was not stable for the expected six-month shelf life. Again, the processor could then make the decision whether or not to reformulate the product to extend the shelf life or whether to set a shelf life of three months.

Case Study 4: Preservative. A client is developing a diced potato product and would like to determine which preservative system is best to prevent the growth of lactic acid bacteria.

Challenge studies are well suited to discover whether or not a preservative system will work in a product to reduce spoilage and extend shelf life. Since the standard spoilage organism in potato products is lactic acid bacteria, the processor wanted to try four different preservatives to see which would prevent the organism’s growth. One control (untreated) product and four products treated with the preservatives, were tested. As shown in Figure 5, the control and three of the treated products spoiled very quickly, most by Week 4 or 5. The preservative used in Treatment 4 proved very effective, in fact doubling the shelf life of the product when compared to the other treatments.

Case Study 5: Specifications. A client wants to see what specification they should use to get a shelf life of seven days for an orange juice type product.

In this case, an orange juice type product was inoculated with yeast at 1,000 cfu/mL, 100 cfu/mL and 10 cfu/mL to determine the time (in days) to spoilage. At 1,000 cfu/mL, the time to spoilage was four days; at 100 cfu/mL, the time to spoilage was five days; and at 10 cfu/mL, the time to spoilage was more than seven days. The processor was able to set specifications for the product at 10 cfu/mL.

Conclusion
Challenge studies can provide a wealth of valuable information about the safety and quality of products to food processors. Through challenge studies, processors can ascertain and extend product shelf life, prevent the release of microbiologically unstable products and protect brand names by avoiding costly recalls. Although challenge studies require the finished product to be available for effective testing, making the decision to conduct a challenge study early in the formulation stage of a product is advantageous. During product development, the laboratory can often conduct preliminary testing to predict how close the food manufacturer is to the desired shelf life, safety and quality goals.

Michael S. Curiale, Ph.D., is Director of Silliker Laboratories Corporate Research Center in South Holland, IL. The author or coauthor of more than 40 refereed research articles, Curiale has written extensively on a broad range of food safety subjects including food- borne pathogens, shelf-life and challenge studies and rapid detection methods.

Ellen M. Vestergaard, M.S., is a project manager at Silliker Laboratories Corporate Research Center, and is responsible for the design and management of shelf life and challenge studies. Vestergaard has an extensive background with state-of-the-art analytical methods for spoilage organisms and foodborne pathogens. Both authors can be reached via info@silliker.com.