## 3 Results and Discussion
### Industrial-Scale Application of a Rosin Acid-Based Product in the Extraction Area  
In the 1990s, AGRANA company voluntarily stopped using formaldehyde to combat microorganisms in the production process. As an alternative, hop acid- or rosin acid-based products were used instead [18; 58]. Previous studies have demonstrated a significant reduction in microbial metabolites, such as lactic acid, acetic acid, butyric acid, glucose and fructose, due to the application of a rosin acid-based product in the extraction area, but no influence on microbial counts was detected [13]. These culture method-based results could be confirmed with a culture-independent method, flow cytometry. No significant difference in the total number of bacteria in the samples was detected in the samples with and without rosin acid application in the industrial or laboratory batch trials. The alpha diversity expressed as the Simpson and Shannon indices increased significantly (\(p\) = 0.0110 and \(p\) = 0.0064) when only samples from the extraction tower and countercurrent mixer were included in the statistical analyses, where the product was dosed. Furthermore, the dosing also influenced the increasing diversity at all sampling points, which also included samples from juice purification (Table 4).  
In some samples, only the relative abundance of the microorganism shifted, but in others, additional bacterial species were detected. In tower juice and in the cassette-juice mixture, the alpha diversity also increased due to the occurrence of additional bacteria belonging to _Actinobacteria_, such as _Arthrobacter_ and _Proteobacteria_. These bacteria may have been detected because some of them were gram-negative. Rosin acid-based products are not effective against Gram-negative bacteria [59]. Thus, the dosing of the rosin acid-based product caused a shift in the bacterial community composition.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Respenses**} & \multirow{2}{*}{\(N\)} & \multirow{2}{*}{\(n\)} & **Processing Periods (\(p\)-Values \({}^{*}\))** & **Contrast Analysis: Significant Difference between** & **Treatments (\(p\)-Values \({}^{*}\))** & **Sampling Points (\(p\)-Values \({}^{*}\))** \\ \hline (log10) Bacterial Count & 78 & 2 & 0.0101 & November and January & 0.7794 & \textless{}0.0001 \\ Simpson Index & 70 & 2 & 0.1506 & & 0.0559 & \textless{}0.0001 \\ Shannon Index & 70 & 2 & 0.1528 & & 0.0244 & 0.0006 \\ OTU Quantity & 70 & 2 & 0.0584 & November and January & 0.0007 & \textless{}0.0001 \\ \hline \hline \end{tabular} \(N\), complete cases; \(n\), replicates of measurements; no significant interactions between the model effects with the exception of treatment-sampling points at OTU. \({}^{*}\), Effects were considered as significant at \(p\) \textless{}0.05.  
\end{table}
Table 4: Statistical evaluation of significant differences of bacterial count, based on flow cytometric analyses, alpha diversity expressed as Simpson and Shannon index and OTU quantity, based on Illumina sequencing based on MiSeq analyses. Samples of extraction area and juice purification with or without treatment (adding of rosin acid-based product in extraction area) were statistically analyzed; calculations are based on repeated measures analysis and contrast analysis with Bonferroni correction at a significant level of 5%.