# Domestic Use Simulation and Secondary Shelf Life Assessment of Industrial _Pesto alla genovese_
## 3 Results and Discussion
### Microbiological Analyses
Even if the approach does not allow to follow growth kinetics, the general trend of counts observed in both scenarios allows to state that _pesto alla genovese_, during the period after opening, has high microbial stability and that the contamination by environmental microbes occurring upon opening and use, is not followed by exponential growth at any storage temperature. In the first scenario of P2, very low TMB counts were observed, with the highest count of 104 CFU/g for sample E at 5 d after the first opening. As for P1, also P2 showed lower TMB counts during storage, thus confirming the high stability of this product category. In the second scenario of P2, higher TMB counts were observed compared to S1, 793 CFU/g being the maximum values recorded for sample E after 11 d of household storage. Despite the higher degree of use in S2, consisting of 3 openings with product withdrawal, TMB counts remained at very low levels for any of the household conditions. This suggests that neither the household environment contamination nor the storage temperature, even in the worst scenario, can significantly affect the microbiological quality of _pesto alla genovese_. The highest TMB count observed amongst all samples was 964 CFU/g, which is more than three orders of magnitude lower than the maximum acceptable microbial growth in food of 10\({}^{6}\)[20] and even lower than the threshold of 10\({}^{7}\) CFU/g, which indicates spoiled foods [21]. For the sake of completeness, also the maximum recorded counts were subjected to comparison by ANOVA, which highlighted significant differences among treatments, without any clear indication on the effect of the scenario on the microbial load. Significant changes were not found on the TMB counts amongst the scenarios and the type of pesto used (\(p\) = 0.8). On the other hand, despite significant changes during storage time were found (\(p\) < 0.05), it should be noted that in most of the cases the microbial load observed 20 d after opening was not significantly different from the one detected at day 5. The _Closridium_ growth in S1P1 was observed only at day 20 of storage at a very small extent (5 CFU/g) in three samples out of five (A, B, C). In S2 it was slightly higher than the one observed in S1, with a maximum of 55 CFU/g after 16 d in sample D.  
In both Pesto sauces P1 and P2, higher _Closridium_ counts were observed in the second scenario, possibly because of the higher number of openings and the longer exposure to the environment (jar kept uncapped), which could have increased the probability of contamination. Interestingly, in P1 it was noticed a higher _Closridium_ growth than the one observed on P2, in which only one sample (A at t\({}_{16}\)) showed Clostridia. The different pH and a\({}_{w}\) values of the pesto sauces, however, do not influence the degree of contamination by _Closridium_, but the germination of spores that are already present. The different degree of _Closridium_ contamination between the two pesto sauces could be due to the household environment. To corroborate this hypothesis, _Closridium_ counts for S2P1 were only observed in household D, suggesting that this environment is the source of contamination during the openings of the jars. The selective parameters (pH and a\({}_{w}\)) of both pesto sauces are able to prevent the germination of _Closridium_ spores, and the _Closridium_ counts observed are a consequence of the germination induced by the thermal shock. The limiting pH for germination of _Cl. botulinum_ spores is 4.8-5.0 [22] hence, the very low pH of P2 (4.18) avoids the germination of any _Clostridium_ spore. On the other hand, the pH of P1 (5.64) alone could allow spore germination, but this event is unlikely thanks to the very low \(\mathrm{a_{w}}\) value (0.9046), taking into account that in optimum conditions (30-40 \({}^{\circ}\)C and pH 7.0) the \(\mathrm{a_{w}}\) limiting _Clostridium_ spores' germination is 0.94-0.97 and by decreasing the pH of the medium, the limiting \(\mathrm{a_{w}}\) increases [22].