## 4 Discussion
While elevate E seemed to inhibit the proliferation of presumptive _Enterobacteriaceae_ in the samples incubated at 25 \({}^{\circ}\)C, the eluate A showed a remarkable bactericidal effect from day 1 of incubation, decreasing the CFU/mL levels below the detection limits after 7 days. On the other hand, at 4 \({}^{\circ}\)C, the effect on presumptive _Enterobacteriaceae_ (as well as the other bacterial groups) was markedly reduced. The lower antibacterial effects observed for the partially purified preparations in the assays carried out at 4 \({}^{\circ}\)C could be explained by a lower diffusion of the antimicrobials in the food matrix. Another possible explanation could be that the antimicrobial(s) only act on actively growing cells. Previous studies have shown that the activity of some bacterioxins may be energy-dependent, as in the case of nisin [38] or microcin L [39]. In another example, the killing activity of enterocin AS-48 against _Staphylococcus aureus_ CECT 976 in culture medium decreased markedly for the incubation temperatures of 10 \({}^{\circ}\)C or 4 \({}^{\circ}\)C compared to 37 \({}^{\circ}\)C [40]. Regarding the impact of _Paenibacillus_ and its partially-purified extracts on bacterial diversity, the results obtained in the present study indicate that inoculation with the UJA2219 strain had no remarkable impact on the microbiota of the puree. These results agree with those obtained in the culture-dependent experiments in which the inoculated strain failed to inhibit proliferation of the different bacterial groups analyzed. Regarding treatments with culture extracts, the observed effect for the most representative genera of the samples (_Pseudomonas_ and _Enterobacteriaceae_) was different depending on the treatments: in the case of the genus _Pseudomonas_ the eluate with NaCl (E) makes the population increase in relative abundance almost twice while the treatment with the eluate of acetonitrile (A) decreases it to undetectable limits. In the case of _Enterobacteriaceae_, it was the treatment with the NaCl eluate (E) that had the greatest effect, reducing the relative abundance of this group by half, while the eluate from the purification with acetonitrile (A) did not have such a marked effect on this population. These results seem to be contradictory with those obtained by the culture-dependent approach (according to which eluate A had a remarkable effect on the population of presumptive _Enterobacteriaceae_). However, it should be taken into account that the selective medium used (EMB) also supports the growth of _Pseudomonas_[41] and, while _Pseudomonas_ are non-fermentative, they may be difficult to differentiate from the true fermentative _Enterobacteriaceae_ in mixed growth. Therefore, the results could be explained assuming that the counts obtained on EMB agar as presumptive _Enterobacteriaceae_ include, at least in part, _Pseudomonas_. An additional effect of treatment with eluate A was an increase in the relative abundance of _Enterococcus_. While neither eluate A or B had antibacterial activity on the test strain _E. faecalis_ S-47, the increase in the relative abundance of _Enterococcus_ in samples treated with eluate A could be linked to the observed decrease in the relative abundance of _Pseudomonas_. It could be speculated that the _Pseudomonas_ population acts as a competitor against enterococci. In addition to competition for nutrients, _Pseudomonas_ may produce antibacterial peptides, such as pyocins, lectin-like bacteriocins and modified microcircins [42]. These resultsillustrate the complex effects of incorporating antimicrobial substances into food systems and how the culture-independent methods such as next-generation sequencing provide new insights on the complexity of microbial interactions in foods [43]. Furthermore, the observed differences in the effects of partially purified culture extracts on bacterial diversity could also be interpreted considering that _Paenibacillus_ UJA2219 may produce different antimicrobial substances.