Challenge test for foods and feed products (ISO 20976-1: 2019. Part 1: Growth potential, lag time and the maximum growth rate).

The conservation efficacy tests (Challenge) for foods have the objective of studying the growth capacity, the latency time and the maximum growth rate of the inoculated microorganisms, in order to know their microbiological stability. The assays aim at evaluating growth potential or growth kinetics (lag time and maximum growth rate). Growth potential studies serve to validate the shelf life of a food at a microbiological level and assess whether the food, under specific conditions, allows the growth of the inoculated microorganism; while growth kinetics studies assess whether intrinsic characteristics (pH, aw...etc.) or extrinsic characteristics (temperature, gas composition...etc.) can exert an impact on the behavior of the inoculated microorganism. In the study of growth kinetics, the lag time and the maximum growth rate are determined, the latter can be used to determine the shelf life of the food.

The behavior of a microbial population in a food depends on various 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, packaging format and composition of the gaseous atmosphere in which it is found); the food preparation process; as well as the interactions with the natural microorganisms present in the food.

The tests are performed in the laboratory by inoculating one or several batches of a food with bacteria in a vegetative and spore form. The strains used in the test must be those that the manufacturer considers appropriate for the characteristics of the food, for example, Listeria monocytogenes, Salmonella spp. or species such as Bacillus spp. (spores). It is recommended that strains that have been isolated from food be used and, to estimate growth potential, if possible, a mixture of several strains of the same species, to account for variability between strains. To estimate growth rate, only one strain should be used per assay.

It is important to determine the number of lots and the criteria for their selection. To determine the number of batches, the variability of the production process must be considered. If the variability of the characteristics of the food between the different batches (for example, the physicochemical or microbiological properties) is large enough to cause differences in the behavior of the microbiological growth, it is necessary to study different batches, at least 3 batches. If the preservation efficacy test (Challenge) is performed on a single batch, it only allows an estimation of bacterial behavior for the same set of food characteristics. Therefore, as a general rule, it is recommended to study at least three batches, selecting representative batches of the intrinsic variability of the food matrix studied. If a single batch is used, it must be justified: to assess the impact of a new formulation, use a batch that presents more favorable conditions for the development of microorganisms, that is, the worst scenario (for example, with the highest pH and water activity , lower concentration of preservatives, etc.), or have previously shown that the variability between batches is not significant and must be reported by the applicant for the test. According to the standard, three units of one batch are inoculated in the laboratory for each sampling time, if only one batch is received, or one sample for three different batches for each sampling time. Sampling must be carried out from each of the inoculated units and batches at different times of the test, allowing a minimum of 5 points to be obtained for the estimation of growth potential and 8 for the growth kinetics test in which the counts must be carried out. of bacteria to be able to elaborate the bacterial development curve and the corresponding calculations.

In addition, control foods must be used in the test, not inoculated with the test microorganism, which allow the level of natural contamination of the food to be determined. For this, the same number of units are analyzed as for the test with the inoculated samples (one unit from 3 different batches) that are only analyzed at two times, initial and final test time.

The ISO 20976-1 standard does not establish a criterion to determine whether the food allows the growth of the test microorganism, but this criterion must be established prior to the tests in accordance with the objective of the test, for example: the food allows the growth if a growth potential greater than 0.5 log CFU/g is observed.