## 3 Results and Discussion
### Microbial Diversity on Endive Lettuce along the Washing Process Chain
Since the TVC after storage returned to almost the same level as the unwashed and stored endive raw material, it is of special interest which microorganisms predominate the microbial community. The analysis of the culturable microbial community of entive raw material before storage revealed 77% not identified microorganisms (43 sub-clusters), and the identified fraction was composed of _Herbspirillum huttiense_, Moraxellaceae, Pseudomonas fluorescens_, Rhizaceae Sporidiobolaceae, _Stentrorophonomas_ sp., and Yersiniaceae (Figure 2A). At the end of the washing process the microbial community of entive lettuce shifted. The amount of not identified microorganisms increased to 93% (36 sub-clusters) and the identified microorganisms belonged to Arthroformataceae Porphyromonadaceae, and Staphylococcusaceae. After seven days of storage, none of the microorganisms obtained from the entive raw material could be identified (nine sub-clusters) by MALDI ToF MS using the SARAMISTM database (Figure 2B). After washing with PTW for 180 s in the pre-bath and subsequent storage of the end product, all identified microorganisms belonged to the family Staphylococcusaceae and 89% (14 sub-clusters) could not be identified. Staphylococcusaceae were also found in all other samples tested in different abundances. Members of the family Staphylococcusaceae can be human pathogens, e.g., _Staphylococcus aureus_, which is able to produce enterotoxins which can cause staphylococcal food poisoning [13]. However, the microorganisms could only be classified to the family level; thus, no statement can be made about the possible pathogenicity of the grown microorganisms. Nevertheless, since members of the family Staphylococcusaceae can even grow at refrigerator temperatures [26] and they seem to be less sensitive to the treatment with PTW, their occurrence should be monitored with care. Directly after cutting, cell exudates have to be removed because they can act as a good reservoir for bacterial growth; hence, an application of sanitizers directly after cutting might enhance microbial inactivation. The application of PTW directly after cutting in the pre-rinsing step for 10 s (trial B) or 30 s (trial C) showed reductions of TVC of 0.6 log units and 1.0 log units, respectively. After storage, the microbial reduction was lower than that for the samples after washing with water only. The application of PTW for 10 s in the pre-rinsing step led only to minor changes of the culturable microbial community after treatment, which might also be the result of the colony sampling. Furthermore 87% of the analyzed microorganisms obtained from the entive raw material could not be identified  
Figure 1: Culturable microbial community structure of entive lettuce (identified colonies only) along the washing process chain. The washing was conducted without the addition of sanitizers (trial A) and the samples were analyzed after washing (**A**) and after storage for seven days at 2 °C (**B**). Sampling positions: (1) entive raw material; (2) entive after pre-bath; (3) entive after pre-rinsing; (4) entive after first wash; (5) entive after second wash; (6) entive after final rinsing. [17 sub-clusters) and, after the washing process, the amount of not identified microorganisms increased to 92% (10 sub-clusters). Members of the family Moraxellaceae were found before and after washing, whereas members of the family Staphylococcusaceae were not found after washing (Figure 3A). However after storage at 2 \({}^{\circ}\)C, members of the family Staphylococcusaceae were again detected in the unwashed and washed entire lettuce. Additionally, members of the family Pseudomonasaceae were detected in higher amounts in all samples tested (Figure 3B). The amount of not identified microorganisms after storage was 50% for the entire raw material (nine sub-clusters) and 71% for the washed entire lettuce (nine sub-clusters).