Instituto Valenciano de Microbiología

Masía El Romeral
Ctra. de Bétera a San Antonio Km. 0.3
46117 Bétera (Valencia)
Phone. 96 169 17 02
Fax 96 169 16 37
CIF B-96337217


Challenge test for foods and feed products - ISO/DIS 20976-1: 2016. Microbiology of the food chain — Guidelines for conducting challenge tests of food and feed products — Part 1: Challenge tests to study the growth potential, lag time and the maximum growth rate.

Test not accredited in our laboratory.

            The conservation effectiveness tests (Challenge) for food have as objectives to study the growth capacity, the latency time and the maximum growth rate of the inoculated microorganisms, in order to know their microbiological stability. The tests are carried out in the laboratory by inoculating one or several batches of a food with bacteria in a vegetative form or with spores. The species Listeria monocytogenes (vegetative bacteria) is used, and it is also possible to use any of the species that are considered adequate due to the characteristics of the food, including species such as Bacillus spp. (spores) It is recommended that strains that have been isolated from food be used and, if possible, a mixture of several strains of the same species, to eliminate the variability between strains. After the experimental inoculation of the food in the laboratory, the growth potential is studied by comparing the initial growth of the bacteria used in the test, with the growth obtained in the different sampling times. With the results obtained, the pertinent calculations are made.

The behavior of a microbial population in a food, i.e., its growth kinetics, depend on several characteristics of the food such as its water activity (aw: water activity), its pH, its concentration of preservatives, etc.; the physiological state of the bacteria; the conditions of conservation of the food (temperature, format of packaging and composition of the gaseous atmosphere in which it is); the food preparation process; as well as the interactions with the natural microorganisms present in the food.

It is important to determine the number of lots and the criteria for their selection. To determine the number of lots, the reproducibility of the food in the lines of production must be considered, especially in terms of the physical-chemical matrix of the food and its microbiological properties. It also depends on the degree of confidence required in the results to estimate the variability of the growth parameters. Therefore, a single guideline cannot be given to determine the number of lots under study. If the conservation efficacy test (Challenge) is carried out in a single batch, it allows only an estimation of the bacterial behavior for the same set of food characteristics. If between the different batches, due to the intrinsic characteristics of the food, it can be inferred that there may be variability among them in terms of microbial growth, it is necessary to study several batches in order to evaluate the variability of the bacterial response. Therefore, as general rule, it is recommended to study at least three batches, selecting representatives batches of the intrinsic variability of the matrix of the food studied. If a single batch is used, it must be justified and in this case, have chosen the one that presents more favorable conditions for the development of microorganisms, i.e., the worst scenario with the highest pH and water activity (aw), lowest concentration of preservatives, etc., so they must be informed by the applicant of the test.

In the laboratory, several units of each batch are used, which will depend on the variability in the physical-chemical and microbiological properties of the food. It should be taken in mind that a variability in inoculation is possible since the type and structure of the food matrix, as well as the inoculation procedure used, influence its distribution in the food. For this reason in the laboratory at least three units are inoculated per batch and per sampling point to determine the parameters previously referred to. From each of the inoculated units and batches should be sampled at different times of the test, which allow to obtain at least 8 or 10 times in which the counts of bacteria must be performed in order to prepare the curve of bacterial development and the corresponding calculations.