## 4 Discussion  
This study investigated the microbiome profiles of RTE spinach leaves washed with different sanitizers (ECAS, PAA) and compared with leaves washed with tap water and not washed at all (control), at three time points over 10 days (day 0, day 5, and day 10). Although a higher proportion of ASVs was found compared to previous studies [26, 27], their richness and evenness (alpha diversity) did not significantly differ among the types of sanitizer and the sampling points. We also found that the types of sanitizing washing, apart from a reduced heterogeneity over time, did not significantly  
Figure 3: The relative abundance (RA) ratio of (**a**) ASVs (17 ASVs with taxa identification in Table 4) and (**b**) taxa rank family (4) identified as significantly different in abundance on sampling days 5 and 10 by ANCOM analysis at a false discovery rate (FDR) of 0.05. The RA ratio is calculated as RA on day 5 or day 10 divided by RA on day 0. f-family. influence the community structure of the bacteria (beta diversity). ANCOM analyses identified that the composition of ASVs and families changed significantly over the sampling days. The number of ASVs identified (\(>\)2000) in the present study was much higher than that observed in the spinach leaf microbiome profiling studies by [PERSON] et al. [26] and [PERSON] et al. [27], who identified 673 and 190 operational taxonomic units respectively. In addition, 12 phyla were identified in this study compared to the four phyla observed by [PERSON] et al. [27] and 14 phyla detected by [PERSON] et al. [26]; however, the number of predominant phyla (\(n=4\)) and their relative proportions are similar in all three studies. In agreement with previous observations, the phylum Proteobacteria showed the highest total abundance on day 0, followed by phyla Bacteroidetes, Firmicutes, and Actinobacteria [3; 6; 26; 34]. The basal bacterial microbiome of RTE spinach leaves is therefore very similar to that of other minimally processed fruits and vegetables [3; 6; 27; 34; 60]. Our analyses also showed that the Shannon and Inverse Simpson diversity indexes and richness (ACE and Chao1) measures did not differ significantly among all spinach samples. Furthermore, the community composition of bacteria (beta diversity) for all types of washing did not differ significantly, indicating that ECAS treatments did not affect bacterial microbial diversity. This could be seen as a good outcome, since it has been suggested that the microbiome on fresh produce is not responsible for spoilage but acts as a natural biological barrier against spoilage organisms and pathogens [13; 14; 15]. On the other hand, a significant grouping of spinach microbial community structures was observed for sampling days and reduction over time of the heterogeneity of bacterial composition. The reduction in heterogeneity can be attributed to the reduction in the relative abundance of phylum Proteobacteria on day 5 and day 10, in accordance with the reduction observed by [PERSON] et al. [26] in RTE spinach leaves washed with chlorine and stored at 4 \({}^{\circ}\)C for a week. Moreover, the microbiome community on day 10 clustered distinctly due to a significant increase in the relative abundance of Bacteroidetes, similar to that observed by [PERSON] et al. [26] when the spinach leaves were stored at 4 \({}^{\circ}\)C for a week. ANCOM is a method based on compositional log-ratios to detect differences in relative abundance and has been used to detect taxa abundance in the spinach microbiome at ASV and family level. Taxa at ASV and family level for the different types of treatment were not significantly different, but differences in ASVs and family-related abundances were identified at different sampling days. ASVs identified as _Pseudomonadaceae_ and _Moraxellaceae_ families, and the order Bacillus (unclassified family) had a high relative abundance on day 5.