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From: Food Quality & Safety magazine, December/January 2007

Coping with Shelf-Life

To help assure product safety and quality, microbiological shelf-life and challenge studies are essential R&D tools for food processors and manufacturers.

by Erdogan Ceylan, PhD

Raw or processed food products deteriorate during processing, distribution and storage. Unless it is sterile, such products will contain indigenous microflora and, in some cases, foodborne pathogens. Unfortunately, presence and growth of microorganisms or foodborne pathogens are not always differentiated by the quality changes in the product. To help assure product safety and quality, microbiological shelf-life and challenge studies are essential R&D tools for food processors and manufacturers.

Factors Affecting Product Shelf-life

Many factors influence shelf-life of food products. The shelf-life of food products is dependent on the interactive effects of intrinsic parameters (e.g. pH, water activity and preservatives) and extrinsic parameters (e.g. storage temperature, humidity level and gaseous environment), as well as the raw material quality and sanitary conditions applied during manufacturing. Microbiological and chemical changes, and physical deterioration during storage determine the shelf-life of a product. Microbiological changes occur as a result of microbiological growth of indigenous microflora. Chemical changes occur as a result of chemical reactions such as enzymatic degradation, non-enzymatic reactions and oxidation.

A complete shelf-life study is composed of microbiological, chemical and sensory evaluations, and determines the parameter that indicates the end of shelf-life. Shelf-life is defined as the period of time during which the quality of a food product remains acceptable for consumer consumption. At the end of shelf-life, the fresh quality of the food product is unacceptable or undesirable due to changes in taste, texture, aroma, color and appearance. Organoleptic qualities of the food begin to change as the spoilage bacteria, yeast or mold in the food grow. End of microbiological shelf-life may be determined based microbial counts. The number of organisms required to cause spoilage varies with the type of the organisms. Generally, however, it is accepted that 10 million bacteria per gram, 100,000 yeast per gram, or visible mold indicates the end of microbiological shelf-life.

A well-designed shelf-life study provides vital information on the microbiological, chemical and organoleptic changes in a product formulation during product storage. Shelf-life studies should be uniquely designed for each product. A number of variables must be considered when designing a shelf-life study. Storage temperature, relative humidity, types of analyses (i.e. microbiological, chemical or organoleptic analyses), method of analyses, sampling method, number of replications and duration of the study are some of these variables.

Critical Information Through Challenge Studies

In a challenge study, the stability of a product is determined against spoilage organisms or foodborne pathogens. If the count of the challenge organism does not increase during shelf-life storage, the product formulation is considered microbiologically stable against the challenge organism. Microbiologists define the increase as a 1-log (10 fold) increase in the count of the challenge organism.

A well-designed challenge study provides critical information on the microbiological stability of a product formulation against spoilage organisms or foodborne pathogens. Microbiological challenge studies also play an important role in determining whether a specific process is in compliance with the predetermined performance standards.

When conducting a microbiological challenge study, it is important to consider the key factors such as pH, water activity and preservative level that influence microbiological growth. These factors may be key in determining the stability of the product against challenge organisms during storage. Failure to consider the specific product parameters and environmental factors could result in faulty conclusions. Each key factor in the product formulation must be tested solely or in combination under worst-case conditions during its intended shelf-life. For example, the product must be challenged on the high side of the pH tolerance range. Conversely, the product must be challenged on the low side of preservatives within the tolerance range.

Water activity, temperature, gaseous environment and presence of antimicrobial compounds have a profound effect on the microbiological growth. These parameters are set at the time of product development. Changes in these parameters determine the shelf-life, and the microbiological stability and safety of the product.

Each microorganism has a minimum, optimum and maximum growth temperature. As the temperature increases from the minimum toward the optimum, the microbiological growth rate increases. The microbiological growth is fastest at the optimum temperature. As the temperature increases from the optimum toward the maximum, the microbiological growth rate decreases. As the storage temperature changes, not only the growth rate of the organisms but also the type of the spoilage organisms changes.

It is important to determine the shelf-life of the product at its intended shelf-life storage temperature. A few degree changes in storage temperature may have a significant effect on the length of the shelf-life. In the real world conditions during distribution and storage, the product temperature often fluctuates between low and high temperatures. It is, therefore, important to test the product under both commonly used target storage temperatures (e.g. 40°F for refrigerated storage and 75°F for ambient temperature storage) and abuse temperatures (e.g., 45° to 50° F for refrigerated storage and 85° to 90° F for ambient temperature storage).

Duration of the shelf-life and challenge studies should match the target shelf-life of the product. It is desirable to test the product formulation beyond its intended shelf-life. Some regulatory agencies require testing the product formulation over product shelf-life storage plus one-third of the target shelf-life. The food product is sampled periodically to determine microbiological, chemical and organoleptic changes. The number of time points is decided based on reference studies.

The intended storage time is often divided into five to 12 time points. An excessive length of time between samplings may over- or under-estimate the changes in the product during storage. Significant changes in the product will be determined more accurately with high number of analyses. Triplicate samples from three different lots of product produced on different manufacturing dates should be used at each time point to improve the precision of testing.

In shelf-life studies, the type of the microbiological analyses and the plating procedures are selected based on the products’ characteristics or the types of the microorganisms known to be present in the product. In challenge studies, samples are analyzed for the counts of the challenge organism or its toxins. Background microflora may affect the survival of the inoculated challenge organisms. Thus, it is important to analyze the uninoculated control sample at each time point during its intended shelf-life.

Validated Methods

Reliability of the testing is directly related to the accuracy of the methods used. The use of validated methods from sampling to final data analyses is the key element for the precision of testing. Analyses of the samples should be performed using the methods described in the FDA Bacteriological Analytical Manual, AOAC, Compendium of Methods for the Microbiological Examinations of Foods, USDA Microbiology Laboratory Guidebook, or other validated methods.

The number of organisms inoculated to the food in a challenge study is normally higher than what would normally be present in the product as results of chance contamination. Die-off might occur after inoculation due to the abrupt changes (e.g. a low pH level) in the environment. If the initial inoculation level is too low, the incorrect conclusion can be made that the product formulation is stable. If the initial inoculation level is too high, the challenge organisms may overcome the inhibitory effect preservatives or hurdles. This may lead to the incorrect conclusion that the product formulation is stable. Typically, an inoculum level in the range of 102 to 104 cells per gram is used to observe either increases or decreases.

A number of strains of bacteria, yeast and mold are used in challenge studies as spoilage organisms. The ideal spoilage organisms are strains that have been isolated from similar products. The ideal foodborne pathogens are the strains that have been isolated from known foodborne outbreaks. Well-characterized strains of challenge organism from a reference culture collection such as the American Type of Culture Collection (ATCC) should be used. Strains of Bacillus cereus, Clostridium botulinum, Clostridium perfringens E. coli O157:H7, Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus are the commonly used pathogens. A summary of foodborne pathogens that may be used in challenge studies for different types of foods is given in Table 1.

A cocktail or mixture of multiple strains of the same organism should be used to challenge a food product in order to simulate the different origins of the strains entering the food products. The challenge organism is grown in media under optimal conditions. In certain studies, the challenge organisms may be adapted to certain conditions for the specific food formulations. For example, E. coli O157:H7 may be acid adapted for low pH products and Listeria monocytogenes may be cold adopted for refrigerated foods. Bacterial spore suspension should be heat shocked prior to inoculation to inactivate vegetative cells.

In microbiological studies, results only apply to those conditions tested. Any changes in product formulation such as an increase in the water activity level, storage temperature, and packaging conditions will invalidate the results of the shelf-life and challenge studies and require new studies. Results of the microbiological shelf-life and challenge studies are interpreted by use of pass or fail criteria. In shelf-life studies, the predetermined limits—10 million bacteria per gram, 100,000 yeast per gram or visible mold—indicate the end of shelf-life. In microbiological challenge studies growth or toxin product is used as determining factor.

A food formulation is considered stable in the absence of toxin formation or less than a 3-log increase over inoculum levels in Bacillus cereus and Staphylococcus aureus challenge studies. The absence of toxin formation is required in Clostridium botulinum studies, while a 3-log or less increase is the preferred criterion in Clostridium perfringens studies. Population growth is limited to less than a 1-log increase in E. coli O157:H7, Listeria monocytogenes, and Salmonella studies.

Proven Proficiency

Shelf-life and challenge studies should be conducted by skilled professionals with demonstrated proficiency using validated methods to ensure the experimental design evaluates the key factors and determined the product's potential for spoilage and food safety risks.

Dr. Erdogan Ceylan is director of research at Silliker Inc.’s Food Science Center in South Holland, Ill. He can be reached at



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